Clinical Molecular Diagnostics by Shiyang Pan 2021

Shiyang Pan
Jinhai Tang
Editors
Clinical Molecular
Diagnostics
sachyhoc.com
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Clinical Molecular Diagnostics
Shiyang Pan • Jinhai Tang
Editors
Clinical Molecular Diagnostics

Editors
Shiyang Pan Jinhai Tang
Department of Laboratory Medicine Department of General Surgery
The First Affiliated Hospital of Nanjing The First Affiliated Hospital of Nanjing
Medical University Medical University
Nanjing Nanjing
China China
ISBN 978-981-16-1036-3 ISBN 978-981-16-1037-0 (eBook)

https://doi.org/10.1007/978-981-16-1037-0
© People’s Medical Publishing House Co. Ltd. 2021

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Foreword
With the rapid development of research on genomics and proteomics, many disease-associated
biomacromolecules have been identified and put into clinical practice. In recent years, with the
rise of the concept of precision medicine and wide application of next-generation sequencing
(NGS) and digital PCR in clinical laboratories, molecular diagnostics has gained further atten-
tion. So far, it has been widely used in disease diagnosis, guidance for individual therapy, and
evaluation of treatment efficiency and prognosis. Therefore, in this era of increasing attention
on molecular diagnostics, it is urgent to help clinical chemists, doctors, and medical students
worldwide to acquire the information regarding the emerging molecular diagnostic items in
short order. Thus, Clinical Molecular Diagnostics emerges at the right moment.
For more than 20 years, Prof. Pan, the editor-in-chief of Clinical Molecular Diagnostics,
has committed to the identification of novel molecular biomarkers, and has established several
outstanding techniques in molecular diagnostics including quantitative detection of plasma
DNA and methylation of tumor suppressor genes. Specially, he identified a novel tumor marker
named tumor specific protein 70 (SP70) and established the corresponding detection methods
which have been applied in the clinical laboratory measurement. Prof. Tang is an accomplished
breast surgeon, having not only done much pioneering work in breast cancer diagnosis, surgi-
cal treatment, and molecular marker screening, but also made great achievements in the
research on the drug resistance mechanisms of breast cancer. In addition, this book is also the
brainchild of many experts active in molecular diagnosis, molecular medicine research, and
clinical medicine from different universities or hospitals. I believe that under the elaborate
organization by Prof. Pan, this book will manifest the latest advances in molecular diagnostics
from the perspective of clinical practice.
The first distinguishing feature of this reference book is its clinical application. In addition
to traditional molecular biological techniques, the book includes brand-new perspectives
requiring greater recognition and understanding from clinical practice in the hope of serving
as a source of inspiration for readers who run into clinical problems. Besides, the book has a
wide coverage, including basic introduction, molecular markers, and methodological tech-
niques of molecular diagnostics for infectious, genetic, or autoimmune diseases, cancers, and
other rare diseases. Many novel concepts, such as companion diagnostics and precision medi-
cine, can be found in this reference book. Thus, Clinical Molecular Diagnostics is not only a
tool for clinical chemists but also a guideline for clinical diagnosis and therapy. Since the end
of 2019 and still not resolved at this moment, the outbreak of COVID-19 has posed a serious
threat to human health. Considering the requirement and necessity of insight into the basic
characteristics of 2019-nCoV as well as nucleic acid detection, authors of this book specially
added corresponding contents to supply theoretical and practical support to health workers all
over the world.
It is both a privilege and an honor to have been invited to take part in the edition of such an
exceptional book. To observe and comment on its birth and growth is both rewarding and
stimulating. I will be truly proud to know that clinical chemists, doctors, and medical students
v
vi Foreword
use this text as a primary source. Maintaining the highest standards of quality while providing
crucial contemporary information that is both concise and readable, this volume will be a suit-
able tool for medical professionals.
Chinese Academy of Engineering Hongbing Shen

Nanjing Medical University,
Nanjing, China
Coordinators
Xuan Cao Huanyu Liang Li Wang Shuxian Yang

Ying Chen Jingping Liu Lin Wang Mengyao Yu
Hongmei Ding Liang Ma Ming Wang Litao Zhang
Ming Gu Shuxian Miao Yaman Wang Nannan Zhang
Wenjing Guo Wenwen Shang Rongrong Wang Wei Zhang
Bin Hu Meiyan Shu Xiaohong Xia Xiang Zhang
Jiahong Jiang Kankan Su Erfu Xie Yiting Zhang
Huanyu Ju Mengya Shan Mengxiao Xie Yuanda Zhang
Kara Pan Dan Wang Li Xue Zheng Zhang
Jie Li
vii
Preface
With the advancement of research in human genomics and proteomics, a large number of bio-
logical macromolecules related to diseases have been discovered and applied to the clinic.
Molecular diagnosis has been widely used in the field of diseases diagnosis, individualized
treatment, efficacy monitoring, and predicting prognosis, becoming an indispensable assistant
of doctors. With the rapid development of molecular diagnosis, the broad masses of medical
workers need to know a growing number of molecular diagnostic techniques; at the same time,
the growing molecular inspection team also needs to keep up with the development of technol-
ogy. This book of clinical molecular diagnostics is aimed at solving the presenting problems
during application of the emerging molecular medicine research results to the clinic.
The committee of Clinical Molecular Diagnostics is composed of more than 30 active
experts in molecular diagnosis, molecular medicine research, and clinical medicine. This book
includes four parts. The basic principles and techniques are mainly introduced in the first part.
It lays a good foundation of theoretical knowledge for medical workers and also provides guid-
ance for researchers engaged in molecular diagnosis transformation research. The second part
focuses on the research and application achievements of molecular diagnostic markers in
recent years and reevaluation of markers which have been used in clinic for many years. In
addition, prospect and value assessment of more new markers are introduced. We hope that it
can bridge the gap between basic research and clinical application of molecular markers. The
third part, Molecular Detection Technology, introduces a series of emerging technologies such
as next-generation sequencing, digital PCR, and liquid biopsy, opening the door for medical
workers to understand the rapid developing molecular diagnostic technology. The fourth part
mainly focuses on molecular diagnosis of common clinical diseases; this part systematically
expounds the pathogenesis, clinical manifestations, and routine basis of diagnosis and treat-
ment based on the clue of diagnosis and treatment path.
Laboratory diagnosis plays an important role in clinical precision medicine. There are mil-
lions of active biological molecules that are related to the occurrence and development of dis-
eases. However, less than one percent of the molecular markers have been identified and
applied in the clinic. With the rapid development of molecular detection and diagnostic tech-
nology, liquid biopsy technologies such as CTC and ctDNA detection have emerged. Although
these novel technologies have been gradually applied in clinical practice, they are still in the
initial stage, and the scope of technology application should be established to avoid overuse.
This book is a collection of the latest research results in clinical molecular diagnostics, which
can help strengthen the mutual understanding between laboratory research and clinical prac-
tice. It hopes to serve as a trigger and to lay a foundation for further research in the field of
clinical molecular diagnosis. Thanks for your efforts.
Nanjing, China Shiyang Pan

Jinhai Tang
ix
Acknowledgments
During the compilation of Clinical Molecular Diagnostics, we aim to produce a handbook for
molecular diagnosis used by clinic. It is also of great value for scholars who are devoted to
clinical molecular diagnostics. To facilitate reading and using in everyday work, we have
included numerous illustrations as well as concise summary tables.
This book has received great support from many units and experts. We are grateful to the
National Natural Science Foundation of China, National Clinical Research Center of
Laboratory Medicine, National Key Clinical Department of Laboratory Medicine, Key
Laboratory for Laboratory Medicine of Jiangsu Province of China, Nanjing Medical University,
and First Affiliated Hospital of Nanjing Medical University. We appreciate the help of Prof.
Hong Shang, the Chairman of Chinese Medical Doctor Association Laboratory Physicians
Branch, and Prof. Chengbin Wang, the Chairman of the Chinese Society of Laboratory
Medicine. The book contains illustrations provided by many scholars who have offered good
advice and valuable points of view. We thank them for their contributions.
xi
Contents
Part I Principles of Clinical Molecular Diagnostics
1 Molecules of Disease and Their Detection Methods �� 3

Lutao Du and Chuanxin Wang
2 Assay Performance Evaluation�� 11
Lixin Wang and Zhiyun Shi
3 Establishment of Biological Reference Interval�� 27
4 Ethics: Informed Consent, Patient Privacy �� 39
Qinghe Meng and Xu Qian
5 Bioinformatics�� 45
Chenglu He and Yong Duan
6 Report and Consultation �� 61
Yongqing Tong
7 Factors Associated with Variation�� 91
Xue Qin
8 Quality Control and Quality Assurance�� 97
Gaowei Fan and Qingtao Wang
9 Precision Medicine �� 115
Yingping Cao and Xianjin Zhu
Part II Molecular Biomarkers and Signals from Diseased Functional Organ
10 Molecules in Body Systems �� 123

Shiyang Pan and Xincen Duan
11 Molecules in Signal Pathways �� 139
Shiyang Pan and Wei Zhang
12 Endocrine and Metabolism �� 155
Shichang Zhang and Xiaoting Chen
13 Immune System�� 167
Xiuru Guan
14 Lipoproteins�� 179
Changmin Wang and Zhiwei Li
15 Transport and Carrier Proteins�� 195
xiii
xiv Contents
16 Coagulation and Fibrinolysis�� 207

Hong Wang and Yun Ling
17 Cardiovascular System�� 221
Haitao Ding and Juan He
18 Pregnancy �� 229
Xianzhang Huang and Enyu Liang
19 Urine�� 241
Bingfeng Zhang and Qing Li
20 Bone�� 253
Lieying Fan
21 Cancer �� 261
Wenling Zhang, Yumei Huang, and Jian Xu
22 Translation Research of Novel Biomarker�� 285
Shiyang Pan and Yuexinzi Jin
Part III Advanced Molecular Diagnostic Techniques
23 Next-Generation Sequencing (NGS)�� 305

Min Wang
24 Digital PCR�� 329
Min Wang and Xianping Li
25 Biosensor�� 345
Lei Zheng and Ye Zhang
26 Microfluidic Chip�� 357
Xueen Fang
27 Liquid Biopsy �� 377
Jianyu Rao, Weibo Yu, Teresa Kim, and Thomas Lee
28 Molecular-Targeted Imaging�� 395
Fang Wang, Jian Xu, and Wenying Xia
29 Fluorescence In Situ Hybridization�� 405
Min Hu and Weimin Wu
30 Circulating DNA Quantification�� 413
Min Hu and Zeyou Wang
31 DNA Methylation Detection Techniques�� 427
Shiyang Pan and Jiexin Zhang
Part IV Disease Presentation and Clinical Laboratory Procedures
32 Immunological Disorders�� 439

Hong Mu, Chunlei Zhou, Ling Fang, Feng Xie, Yan Zhang,
and Huanhuan Chen
33 Liver Diseases �� 463
Qishui Ou, Hong Mu, Chunlei Zhou, Zhaojing Zheng, and Juan Geng
34 Pancreatic Diseases�� 493
Lu Yang, Huanyu Ju, Yuan Mu, and Chunrong Gu
Contents xv
35 Digestive Tract Disease�� 511

Genyan Liu, Yuqiao Xu, Shiyang Pan, Weijuan Song, Jia Wang,
Fei Jin, Zhenzhen Cai, Yi Zhang, and Xiang Qian
36 Kidney Diseases�� 553
Zhaojing Zheng, Juan Geng, Ye Jiang, Meijuan Zhang, Ruixia Yang,
Gaoxia Ge, Huaguo Xu, and Xiaojie Zhang
37 Cardiovascular Disease�� 583
Zhou Zhou and Yahui Lin
38 Lung Disease�� 595
Liang Ming, Ting Sun, Haitao Ding, Juan He, Wenjuan Wu, Min Zhang,
Simin Yang, Huaguo Xu, Fang Ni, Shiyang Pan, Qun Zhang, and Yongping Lin
39 Blood Disorders�� 641
Zhuang Zuo, Cheng Cameron Yin, Lixia Zhang, Lin Wang, and Zhen Ren
40 Endocrine and Metabolic Diseases �� 665
Hong Yuan, Jingyuan Zhao, Erfu Xie, Lujiang Yi, Zhaojing Zheng,
and Juan Geng
41 Neurological Disease�� 717
Jie Wu, Yutong Zou, Yingchun Xu, Mengxiao Xie, Zhaojing Zheng,
and Juan Geng
42 Reproductive Organ Cancer �� 751
Jinhai Tang, Xiangjun Cheng, Jieshi Xie, Zheng Cao, Yanhong Zhai,
and Boyan Song
43 Prenatal Diagnosis and Preimplantation Genetic Diagnosis�� 769
Chengcheng Liu, Xiaoting Lou, Jianxin Lyu, Jian Wang, and Yufei Xu
44 Transplant Matching �� 801
Binwu Ying and Lijuan Wu
45 Paternity Testing�� 813
Binwu Ying and Juan Zhou
Appendixes�� 821
About the Editors and Contributors
About the Editors
Shiyang Pan Prof. Shiyang Pan has studied lung cancer over
20 years, during which time he has found novel biomarkers for
NSCLC and authored more than 100 peer-reviewed reports. Dr.
Pan is the editor-in-chief of Clinical Molecular Diagnostics, which
was published in the end of 2013 by People’s Medical Publishing
House (PMPH), and has served on the editorial board of the
Chinese Journal of Laboratory Medicine. Dr. Pan is the director of
the Laboratory Medicine Department of Nanjing Medical
University and the National Key Clinical Department of Laboratory
Medicine. He is the chairman of the Jiangsu Society of Laboratory
Medicine, vice chairman of the Chinese Society of Laboratory
Medicine and IFCC committee member of Molecular Diagnostics,
and he has served on review committees for the NSFC and Health
Administration of China.
Jinhai Tang Prof. Tang is Full Professor of General Surgery, the

director of Jiangsu Province Hospital (the First Affiliated Hospital
with Nanjing Medical University), and the vice principal of Nanjing
Medical University. Prof. Tang is vice president of China Hospital
Association (CHA) and vice chairman of the Breast Disease
Training Committee, Chinese Medical Doctor Association (CMDA).
Prof. Tang is proficient in the diagnosis and treatment of breast can-
cer especially in breast conserving surgery. He is one of the active
advocates and promoters of the standardized comprehensive diag-
nosis and treatment of breast cancer in China. He has achieved a
series of innovative achievements in breast cancer diagnosis, surgi-
cal treatment, and molecular marker screening and mechanism
research related to breast cancer prognosis. In recent years, Prof.
Tang has focused on the research on drug resistance mechanism of
breast cancer. In the past five years, he is the author of over 100
articles. Several articles have been published in top journals, such as
Chemical Society Review, Journal of Controlled Release, and Blood
Reviews. Moreover, Prof. Tang has been engaged in hospital man-
agement for many years and is the initiator of hospital manage-
ment—airport two-way service platform theory.
xvii
xviii About the Editors and Contributors
Hongbing Shen Prof. Shen is an academician of the Chinese

Academy of Engineering. He is the principal of Nanjing Medical
University, director of Jiangsu Province Key Laboratory of
Biomarkers and Prevention of Malignant Tumors, director of the
Collaborative Innovation Center for Personalized Medicine in
Tumors, and director of the International Joint Research Center of
“Environment and Human Health.” He is mainly committed to the
molecular and epidemiological studies of the environment and
tumors, and has made outstanding achievements in the early diagno-
sis of tumors, genetic susceptibility and recurrence, and molecular
markers of prognosis. He has received the National Outstanding
Youth Science Foundation, the National “863” Project, the National
“973” Project, the National Natural Science Foundation’s Key
Projects, and the Innovation Research Group of the Fund Committee.
Currently, he has published more than 400 SCI papers. He has
received a series of awards including Outstanding Youth Fund,
Changjiang Scholars Special Professor of the Ministry of Education,
Special Government Allowance Specialist of the State Council,
National Leading Talent of 10 Million Projects, and Ho Leung Ho
Lee Foundation Science and Technology Progress Award.
Editorial Board
Yongtong Cao Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing,

People’s Republic of China
Liang Chen Department of Thoracic Surgery, The First Affiliated Hospital of Nanjing
Medical University, Nanjing, Jiangsu, People’s Republic of China
Ming Chen Department of Clinical Laboratory Medicine, Southwest Hospital, Third Military
Medical University (Army Medical University), Chongqing, People’s Republic of China
Wei Chen Department of Clinical Laboratory, The First Affiliated Hospital, Xi’an Jiaotong
University, Xi’an, Shanxi, People’s Republic of China
Yu Chen Department of Laboratory Medicine, The First Affiliated Hospital, College of
Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China
Wei Cui Department of Laboratory Medicine, National Cancer Center/National Clinical
Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing, People’s Republic of China
Erhei Dai Department of Clinical Laboratory Medicine, Hebei Medical University, The Fifth
Hospital of Shijiazhuang, Hebei, People’s Republic of China
Shengmiao Fu Central Laboratory, Hainan Provincial People’s Hospital, Haikou, Hainan,
Weiling Fu Department of Clinical Laboratory, Southwest Hospital,The First Affiliated
Hospital of the Third MilitaryUniversity, Chongqing, People’s Republic of China
Chunfang Gao Department of Laboratory Medicine, Shanghai Eastern Hepatobiliary Surgery
Hospital, Shanghai, People’s Republic of China
Yuhua Gong Department of Clinical Laboratory, The Third People′s Hospitel of Zhengjiang,
Zhengjiang, Jiangsu, People’s Republic of China
About the Editors and Contributors xix
Ming Guan Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical

College, Fudan University, Shanghai, People’s Republic of China
Wei Guo Department of Laboratory Medicine, Zhongshan Hospital, Fudan University,
Shanghai, People’s Republic of China
Xiaolin Guo Department of Laboratory Medicine, The First Affiliated Hospital of China
Medical University, Shenyang, Liaoning, People’s Republic of China
Xiaoke Hao Clinical Laboratory Medicine Center of PLA, Xijing Hospital, Fourth Military
Medical University, Shanxi, People’s Republic of China
Yingyong Hou Department of Pathology, Qingpu Branch of Zhongshan Hospital, Fudan
University, Shanghai, People’s Republic of China
Zhibin Hu Department of Epidemiology, Center for Global Health, School of Public Health,
Nanjing Medical University, Nanjing, Jiangsu, People’s Republic of China
Shan Huang Guizhou Nursing Vocational College, Guiyang, People’s Republic of China
Yong Ji Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing
Mei Jia Department of Clinical Laboratory, Peking University People’s Hospital, Beijing,
Xuemei Jia Department of Gynecology, Women’s Hospital of Nanjing Medical University,
Nanjing Maternity and Child Health Care Hospital, Nanjing, Jiangsu, People’s Republic of
China
Li Jiang Department of Laboratory Medicine, Sichuan Provincial People’s Hospital,
University of Electronic Science and Technology of China, Chengdu, Sichuan, People’s
Republic of China
Hui Kang Department of Laboratory Medicine, The First Affiliated Hospital of China
Medical University, Shenyang, Liaoning, People’s Republic of China
Xiaopeng Lan Institute for Laboratory Medicine, Fuzhou General Hospital, PLA, Fuzhou,
Fujian, People’s Republic of China
Jinming Li National Center for Clinical Laboratories, Beijing Hospital, Beijing, People’s
Republic of China
Li Li Department of Laboratory Medicine, Shanghai General Hospital, Shanghai JiaoTong
University School of Medicine, Shanghai, People’s Republic of China
Shijun Li Department of Clinical Laboratory, the First Affiliated Hospital of Dalian Medical
University, Dalian, Liaoning, People’s Republic of China
Yan Li Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan,
Hubei, People’s Republic of China
Yongzhe Li Department of Clinical Laboratory, Peking Union Medical College Hospital,
Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People’s
Republic of China
Pu Liao Chongqing Center for Clinical Laboratory, Chongqing, People’s Republic of China
Alex J. Liu Mayo Clinic(MayoBlvd.Phoenix), Rochester, USA
Shuye Liu Clinical Laboratory Department of Tianjin Third Central Hospital, Tianjin,
xx About the Editors and Contributors
Wenen Liu Department of Clinical Laboratory, Xiangya Hospital of Central South

University, Changsha, People’s Republic of China
Yong Liu Department of Clinical Laboratory, Shengjing Hospital of China Medical
University, Shengyang, Liaoning, People’s Republic of China
Zhijuan Liu Department of Laboratory Medicine, Tibet Autonomous Region People’s
Hospital, Tibet, People’s Republic of China
Hui Lu Department of General Surgery, The First Affiliated Hospital of Nanjing Medical
University, Nanjing, Jiangsu, People’s Republic of China
Xinxin Lu Department of Clinical Laboratory, Beijing Tongren Hospital, Capital Medical
University, Beijing, People’s Republic of China
Wanshan Ma Department of Laboratory Medicine, Shandong Provincial Qianfoshan
Hospital, Shandong University, Jinan, Shandong, People’s Republic of China
Xiaoling Ma Department of Laboratory Medicine, Anhui Provincial Hospital, Anhui Medical
University, Hefei, Anhui, People’s Republic of China
Hua Niu Department of Clinical Laboratories, The First People’s Hospital of Yunnan
Province, Kunming, Yunnan, People’s Republic of China
Baishen Pan Department of Laboratory Medicine, Zhongshan Hospital, Fudan University,
Lin Peng Department of Otorhinolaryngology Head and Neck Surgery, Tianjin First Central
Hospital, Tianjin, People’s Republic of China
Jianping Ren Department of Clinical Laboratory, Shanxi Provincial Hospital of Traditional
Chinese Medicine, Taiyuan, Shanxi, People’s Republic of China
A. Xiangren Department of Laboratory Medicine, People′s Hospitel of Qinghai Province,
Xining, Qinghai, People’s Republic of China
Baoen Shan Research Center, the Fourth Hospital of Hebei Medical University, Shijiazhuang,
Hebei, People’s Republic of China
Han Shen Department of Laboratory Medicine, Nanjing Drum Tower Hospital, Nanjing
University Medical School, Nanjing, Jiangsu, People’s Republic of China
Lisong Shen Departments of Clinical Laboratory and Urology, Xinhua Hospital, Shanghai
Jiaotong University School of Medicine, Shanghai, People’s Republic of China
Zuojun Shen Department of Clinical Laboratory, Division of Life Sciences and Medicine,
The First Affliated Hospital of USTC, University of Science and Technology of China, Hefei,
Anhui, People’s Republic of China
Hongbin Shen Department of Epidemiology, School of Public Health, Nanjing Medical
Haixiang Su Department of Laboratory Medicine, Gansu Provincial Cancer Hospital,
Lanzhou, Gansu, People’s Republic of China
Guirong Sun Department of Laboratory Medicine, The Affiliated Hospital of Qingdao
University, Qingdao, Shandong, People’s Republic of China
Jianrong Su Department of Clinical Laboratory, Beijing Friendship Hospital, Capital Medical
Ziyong Sun Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China
About the Editors and Contributors xxi
Aiguo Tang Department of Laboratory Medicine, The Second Xiangya Hospital, Central

South University, Changsha, Hunan, People’s Republic of China
Zhihua Tao Department of Laboratory Medicine, The Second Affiliated Hospital of Zhejiang
University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China
Yaping Tian Laboratory of Translational Medicine, Beijing Key Laboratory of Chronic
Heart-Failure Precision Medicine, Chinese PLA General Hospital, Beijing, People’s Republic
of China
Chengbin Wang Department of Clinical Laboratory Medicine, the First Medical Center,
Chinese PLA General Hospital, Beijing, People’s Republic of China
Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous Region,
Urumqi, Xinjiang, People’s Republic of China
Hualiang Wang Department of Molecular Biology, Shanghai Centre for Clinical Laboratory,
Hui Wang Department of Clinical Laboratory, Peking University People’s Hospital, Beijing,
Jianzhong Wang Clinical Laboratory, Peking University First Hospital, Beijing, People’s
Republic of China
Lanlan Wang Department of Laboratory Medicine, West China Hospital, Sichuan University,
Chengdu, Sichuan, People’s Republic of China
Lin Wang Department of Clinical Laboratory, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China
Peichang Wang Clinical Laboratory of Xuanwu Hospital, Capital Medical University,
Beijing, People’s Republic of China
Department of Laboratory Medicine, Zhujiang Hospital, Southern Medical University,
Guangzhou, People’s Republic of China
Qingtao Wang Department of Clinical Laboratory, Beijing Chaoyang Hospital, Capital
Medical University, Beijing, People’s Republic of China
Xiaozhong Wang Department of Clinical Laborotory, The Second Affiliated Hospital of
Nanchang University, Nanchang, Jiangxi, People’s Republic of China
Yuming Wang Department of Clinical Laboratory, Second Affiliated Hospital of Kunming
Medical University, Kunming, Yunnan, People’s Republic of China
Guoqiu Wu Center of Clinical Laboratory Medicine, Zhongda Hospital, Southeast University,
Nanjing, Jiangsu, People’s Republic of China
Yong Wu Department of Medicine Clinical Laboratory, The Third Xiangya Hospital of
Central South University, Changsha, Hunan, People’s Republic of China
Xinyou Xie Department of Clinical Laboratory, Xiasha Campus, Sir Run Run Shaw Hospital,
Zhejiang University School of Medicine, Hangzhou, Zhejiang, People’s Republic of China
Bin Xu Center of Clinical Laboratory Science, Jiangsu Cancer Hospital, Nanjing, Jiangsu,
Guobin Xu Department of Clinical Laboratory, Peking University Cancer Hospital and
Institute, Beijing, People’s Republic of China
Wei Xu Department of Clinical Laboratory, The First Hospital of Jilin University
Changchun, Jilin, People’s Republic of China
xxii About the Editors and Contributors
Wenrong Xu Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School
of Medicine, Jiangsu University, Zhengjiang, Jiangsu, People’s Republic of China
Xiaohong Xu Department of Clinical Lab, Zhejiang Cancer Hospital, Hangzhou, Zhejiang,
Yuanhong Xu Department of Clinical Laboratory, The First Affiliated Hospital of Anhui
Medical University, Anhui Medical University, Hefei, People’s Republic of China
Zekuan Xu Department of General Surgery, The First Affiliated Hospital of Nanjing Medical
Di Yang Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical
Jun Yu Department of Neurology, University of Florida College of Medicine, Gainesville,
FL, USA
Bingchang Zhang Department of Microbiology, Clinical Laboratory, Provincial Hospital
Affiliated to Shandong University, Jinan, Shandong, People’s Republic of China
Guoxin Zhang Department of Gastroenterology, The First Affiliated Hospital of Nanjing
Hua Zhang Department of Clinical Laboratory, Guizhou University, Guizhou Provincial
People’s Hospital, Guiyang, Guizhou, People’s Republic of China
Man Zhang Clinical Laboratory Medicine, Beijing Shijitan Hospital, Capital Medical
Xin Zhang Clinical Laboratory, Hospital of Xinjiang Production and Construction Corps,
Urumqi, Xinjiang, People’s Republic of China
Yunli Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Liaoning
Medical University, Jinzhou, Liaoning, People’s Republic of China
Zhan Zhang Department of Clinical Laboratory, The Third Affiliated Hospital of Zhengzhou
University, Zhengzhou, Henan, People’s Republic of China
Zhihong Zhang Department of Pathology, the First Affiliated Hospital of Nanjing Medical
Xin Zhao Department of Clinical Laboratory, Beijing hospital, Beijing, People’s Republic of
China
Qin Zhou The Ministry of Education Key Laboratory of Clinical Diagnostics, School of
Laboratory Medicine, Chongqing Medical University, Chongqing, People’s Republic of China
Yiwen Zhou Department of Clinical Laboratory Medicine, Shenzhen Hospital, Southern
Medical University, Shenzhen, Guangdong, People’s Republic of China
Contributors
Zhenzhen Cai Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Yingping Cao Department of Clinical Laboratory, Fujian Medical University Union Hospital,
Fuzhou, Fujian, People’s Republic of China
Zheng Cao Department of Laboratory Medicine, Beijing Obstetrics and Gynecology Hospital,
Capital Medical University, Beijing, People’s Republic of China
About the Editors and Contributors xxiii
Huanhuan Chen Department of Laboratory Medicine, The First Affiliated Hospital of

Xiaoting Chen Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Xiangjun Cheng Department of Laboratory Medicine, The First Affiliated Hospital of
Haitao Ding Inner Mongolia People’s Hospital, Huhhot, Inner Mongolia Autonomous
Region, People’s Republic of China
Lutao Du The Second Hospital of Shandong University, Shandong, Jinan, People’s Republic
of China
Xincen Duan Department of Laboratory Medicine, Zhongshan Hospital of Fudan University,
Yong Duan Department of Clinical Laboratory, The First Affiliated Hospital of Kunming
Gaowei Fan Department of Clinical Laboratory, Beijing Chaoyang Hospital, Capital Medical
Lieying Fan Department of Clinical Laboratory, Shanghai East Hospital, School of Medicine,
Tongji University, Shanghai, People’s Republic of China
Ling Fang Department of Laboratory Medicine Center, China-Japan Union Hospital of Jilin
University, Changchun, Jilin, People’s Republic of China
Xueen Fang Department of Chemistry and Institutes of Biomedical Sciences, Fudan
Gaoxia Ge Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Juan Geng Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of
Medicine, Shanghai, People’s Republic of China
Chunrong Gu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Xiuru Guan The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang,
Chenglu He Department of Clinical Laboratory, The First Affiliated Hospital of Kunming
Juan He Inner Mongolia People’s Hospital, Huhhot, Inner Mongolia Autonomous Region,
Min Hu Department of Laboratory Medicine, The Second Xiangya Hospital of Central South
University, Changsha, Hunan, People’s Republic of China
Xianzhang Huang Department of Laboratory Medicine, The Second Affiliated Hospital of
Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of
China
Yumei Huang Department of Laboratory Medicine, Hunan Cancer Hospital and Affiliated
Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan,
xxiv About the Editors and Contributors
Ye Jiang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Fei Jin Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical
Yuexinzi Jin Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Huanyu Ju Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Teresa Kim Department of Pathology and Laboratory Medicine, University of California at
Los Angeles, Los Angeles, CA, USA
Thomas Lee Department of Pathology and Laboratory Medicine, University of California at
Qing Li Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical
Xianping Li Department of Laboratory Medicine, The Second Xiangya Hospital of Central
Zhiwei Li Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous
Region, Urumqi, Xinjiang, People’s Republic of China
Enyu Liang Department of Laboratory Medicine, The Second Affiliated Hospital of
Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of
China
Yahui Lin Center of Laboratory Medicine, Beijing Key Laboratory for Molecular Diagnostics
of Cardiovascular Diseases, State Key Laboratory of Cardiovascular Disease, National Center
for Cardiovascular Diseases & Fuwai Hospital, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, People’s Republic of China
Yongping Lin Department of Clinical Laboratory, The First Affiliated Hospital of Guangzhou
Medical University, Guangzhou Medical University, Guangzhou, People’s Republic of China
Yun Ling Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Chengcheng Liu Department of Laboratory Medicine, The First Affiliated Hospital of
Genyan Liu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Xiaoting Lou Key Laboratory of Laboratory Medicine, Ministry of Education of China,
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou,
Zhejiang, People’s Republic of China
Jianxin Lyu Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou
Medical College, Hangzhou, Zhejiang, People’s Republic of China
Qinghe Meng Department of Laboratory Medicine, The University of Texas MD Anderson
Cancer Center, Houston, TX, USA
Liang Ming Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou
Hong Mu Tianjin First Central Hospital, Tianjin, People’s Republic of China
About the Editors and Contributors xxv
Yuan Mu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Fang Ni Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Qishui Ou The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian,
Shiyang Pan Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Xiang Qian Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Xu Qian Institute of Cancer and Basic Medicine, Chinese Academy of Sciences, Beijing,
Department of Laboratory Medicine, Cancer Hospital of the University of Chinese Academy
of Sciences, Beijing, People’s Republic of China
Department of Laboratory Medicine, Zhejiang Cancer Hospital, Hangzhou, Zhejiang, People’s
Republic of China
Xue Qin Department of Clinical Laboratory, First Affiliated Hospital of Guangxi Medical
University, Nanning, Guangxi, People’s Republic of China
Jianyu Rao Department of Pathology and Laboratory Medicine, University of California at
Zhen Ren Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Zhiyun Shi Department of Medical Experimental Center, General Hospital of Ningxia
Medical University, Yinchuan, Ningxia Hui Autonomous Region, People’s Republic of China
Boyan Song Department of Laboratory Medicine, Beijing Obstetrics and Gynecology
Hospital, Capital Medical University, Beijing, People’s Republic of China
Weijuan Song Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Ting Sun Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou
Jinhai Tang Department of General Surgery, The First Affiliated Hospital of Nanjing Medical
Yongqing Tong Department of Clinical Laboratory, Renmin Hospital of Wuhan University,
Wuhan, Hubei, People’s Republic of China
Changmin Wang Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur
Autonomous Region, Urumqi, Xinjiang, People’s Republic of China
Chuanxin Wang The Second Hospital of Shandong University, Shandong, Jinan, People’s
Republic of China
Fang Wang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Hong Wang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
xxvi About the Editors and Contributors
Jia Wang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Jian Wang Department of Molecular Genetic Diagnostics, Shanghai Children’s Medical
Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of
China
Lin Wang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Lixin Wang Department of Medical Experimental Center, General Hospital of Ningxia
Medical University, Yinchuan, Ningxia Hui Autonomous Region, People’s Republic of China
Min Wang Department of Laboratory Medicine, The Second Xiangya Hospital of Central
Qingtao Wang Department of Clinical Laboratory, Beijing Chaoyang Hospital, Capital
Medical University, Beijing, People’s Republic of China
Zeyou Wang Department of Laboratory Medicine, The Second Xiangya Hospital of Central
Jie Wu Department of Laboratory Medicine, Peking Union Medical College Hospital,
Lijuan Wu Department of Laboratory Medicine, West China Hospital, Sichuan University,
Weimin Wu Department of Laboratory Medicine, The Second Xiangya Hospital of Central
Wenjuan Wu Shanghai East Hospital South Branch Affiliated to Tongji University, Shanghai,
Wenying Xia Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Erfu Xie Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Feng Xie Department of Laboratory Medicine Center, China-Japan Union Hospital of Jilin
University, Changchun, Jilin, People’s Republic of China
Jieshi Xie Department of Laboratory Medicine, Beijing Obstetrics and Gynecology Hospital,
Capital Medical University, Beijing, People’s Republic of China
Mengxiao Xie Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Huaguo Xu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Jian Xu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Yingchun Xu Department of Laboratory Medicine, Peking Union Medical College Hospital,
Yufei Xu Department of Molecular Genetic Diagnostics, Shanghai Children’s Medical
Center, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of
China
About the Editors and Contributors xxvii
Yuqiao Xu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Lu Yang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Ruixia Yang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Simin Yang Shanghai East Hospital South Branch Affiliated to Tongji University, Shanghai,
Lujiang Yi Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Cheng Cameron Yin Department of Hematopathology, The University of Texas MD
Anderson Cancer Center, Houston, TX, USA
Binwu Ying Department of Laboratory Medicine, West China Hospital, Sichuan University,
Weibo Yu Department of Pathology and Laboratory Medicine, University of California at Los
Angeles, Los Angeles, CA, USA
Hong Yuan Dalian Municipal Central Hospital, Dalian, People’s Republic of China
Yanhong Zhai Department of Laboratory Medicine, Beijing Obstetrics and Gynecology
Hospital, Capital Medical University, Beijing, People’s Republic of China
Bingfeng Zhang Department of Laboratory Medicine, The First Affiliated Hospital of
Jiexin Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Lixia Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Meijuan Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Min Zhang Shanghai East Hospital South Branch Affiliated to Tongji University, Shanghai,
Qun Zhang The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s
Republic of China
Shichang Zhang Department of Laboratory Medicine, The First Affiliated Hospital of
Wei Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Wenling Zhang Department of Medical Laboratory Science, The Third Xiangya Hospital,
Central South University, Changsha, Hunan, People’s Republic of China
Department of Medical Laboratory Science, Xiangya School of Medicine, Central South
University, Changsha, Hunan, People’s Republic of China
Xiaojie Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
xxviii About the Editors and Contributors
Yan Zhang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing
Ye Zhang Department of Laboratory Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, Guangdong, People’s Republic of China
Yi Zhang Department of Clinical Laboratory, Qilu Hospital of Shandong University, Jinan,
Shandong, People’s Republic of China
Yutong Zou Department of Laboratory Medicine, Peking Union Medical College Hospital,
Jingyuan Zhao The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning,
Lei Zheng Department of Laboratory Medicine, Nanfang Hospital, Southern Medical
University, Guangzhou, Guangdong, People’s Republic of China
Zhaojing Zheng Shanghai Children’s Medical Center, Shanghai Jiao Tong University School
of Medicine, Shanghai, People’s Republic of China
Chunlei Zhou Tianjin First Central Hospital, Tianjin, People’s Republic of China
Juan Zhou Department of Laboratory Medicine, West China Hospital, Sichuan University,
Zhou Zhou Center of Laboratory Medicine, Beijing Key Laboratory for Molecular
Diagnostics of Cardiovascular Diseases, State Key Laboratory of Cardiovascular Disease,
National Center for Cardiovascular Diseases & Fuwai Hospital, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing, People’s Republic of China
Xianjin Zhu Department of Clinical Laboratory, Fujian Medical University Union Hospital,
Zhuang Zuo Department of Hematopathology, The University of Texas MD Anderson Cancer
Center, Houston, TX, USA
Part I
Principles of Clinical Molecular Diagnostics
Molecules of Disease and Their
Detection Methods 1
Lutao Du and Chuanxin Wang
The birth and development of molecular biology is an 1.1 Overview

important event and has become a leading discipline in the
natural science. It drives the research of life science to a new In the past, diagnosis of diseases often relied on clinical
stage and has produced vast knowledge in clinical sciences. experience, and the disease was judged by summarizing the
Molecular diagnosis is based on the theory of molecular laws from signs and symptoms. With the development of
biology, and the studies include the changes of the exis- molecular analyses, the various proteins, enzymes, hor-
tence, structure, or expression regulation of endogenous or mones, and lipids contained in our body fluids can be
exogenous biological macromolecules and macromolecular detected by molecular biological detection methods. In
systems in human body using the techniques and methods of addition to the above substances, nucleic acid has become a
molecular biology, so as to provide information and popular molecular target for detection in recent years,
decision-making basis for the prevention, prediction, diag- which have greatly expanded the role of clinical laboratory
nosis, treatment, and outcome of diseases. The development in various areas. Molecular diagnosis refers to the diagnosis
of molecular biology can be roughly divided into three or auxiliary diagnosis of diseases by detecting substances
stages: In the 1950s, it was determined that protein is the such as DNA, RNA, or protein and provides more direct
main basic material of life, and DNA is the material basis of evidence for the disease by analyzing the presence, varia-
biological inheritance, which laid a theoretical foundation tion, or expression of genes or proteins. At present, there
for the development of molecular biology. In the early are many molecular diagnostic methods, which are mainly
1970s, Watson and Crick proposed that DNA double helix divided into nucleic acid detection methods and protein
structure and base complementary pairing are the basic detection methods.
ways of nucleic acid replication and genetic information
transmission, laying the most important foundation for
understanding the relationship between nucleic acid and 1.2 Molecular Mechanism of Diseases
protein and its role in life. After the 1970s, with the emer-
gence of PCR technology, the launch of the human genome The occurrence and development of human diseases such as
project, and the rapid development of high throughput leukemia, malignant tumors, diabetes, neurodegenerative
sequencing technology and mass spectrometry technology, diseases, cardiovascular and cerebrovascular diseases, and
humans began to deeply understand the nature of life and hypertension are all related to the abnormal structure, func-
make great strides in the field of molecular diagnosis. This tion, and interaction of proteins and their complexes. The
chapter will introduce the main methods of molecular diag- nature of disease is protein dysfunction, which is caused by
nosis, including nucleic acid detection and protein detec- changes in protein quality and quantity. Protein production
tion. It can be assured that molecular has a bright future, but process is shown in Fig. 1.1. The molecular mechanism of
the road will be difficult and tortuous. diseases mainly includes changes in gene structure, gene
expression caused by cell regulatory factors or other factors,
foreign pathogenic genes, post-translation processing, and
degradation of proteins. Since proteins are the main mole-
cules performing life functions, errors in the transcription,
L. Du · C. Wang (*) translation, degradation, and interaction of various mole-
The Second Hospital of Shandong University,
Shandong, Jinan, People’s Republic of China cules related to protein synthesis can lead to the occurrence
e-mail: cxwang@sdu.edu.cn of diseases.
© People’s Medical Publishing House Co. Ltd. 2021 3

S. Pan, J. Tang (eds.), Clinical Molecular Diagnostics, https://doi.org/10.1007/978-981-16-1037-0_1
4 L. Du and C. Wang
DNA methylation
DNA
Pre-rRNA m7GPPPN PolyA

Pre-mRNA
snoRNA
Pre-miRNA
rRNAs
Nuclear
m7GPPPN PolyA m7GPPPN PolyA membrane
Nucleus
Ribosome
Cytoplasm
miRNA
mRNAs
m7GPPPN PolyA m7GPPPN PolyA m7GPPPN PolyA
Noncoding RNA
Inhibition of
Protein isoforms protein synthesis
Fig. 1.1 Protein production process
1.3 Nucleic Acid Detection Methods large amount of a target fragment is amplified using DNA
polymerase and a pair of specific primers in vitro conditions.
The common techniques for nucleic acid detection include PCR technology is a cyclic reaction that is programed under
nucleic acid amplification technology, sequencing technol- suitable conditions, so the amplification products are rising
ogy, nucleic acid hybridization technology, chip technology, exponentially in theory. The most advantage of PCR is the
and biosensing technology. large increase of amplification products.
The PCR reaction system mainly includes primers, Taq
DNA polymerase, dNTPs, template DNA, buffer, and other
1.3.1 Nucleic Acid Amplification Technology components. Classical PCR reaction process requires dena-
turation, annealing, and extension. The denaturation tem-
Nucleic acid amplification technology refers to a process in perature is usually selected at 95 °C, the annealing
which a nucleic acid of a specific sequence (DNA or RNA) temperature is determined according to the Tm value of the
is selectively multiply replicated in vitro, while other genes primer, and the extension generally requires 72 °C. Double-
are not affected. The most commonly used technique for stranded DNA is denatured into a single strand at a tem-
nucleic acid amplification is polymerase chain reaction perature of 95 °C in vitro, and then the primer could
(PCR). PCR technology refers to a technique in which a combine with the denatured single-strand DNA under the
1 Molecules of Disease and Their Detection Methods 5
principle of complementary pairing at a low temperature of nucleic acid molecules. This technology randomly dis-
(usually Tm-5 °C). Finally, at 72 °C, the dNTPs are contributes the nucleic acid template into a large number of
tinuously added to the 3′-OH of the primer to synthesize reaction units for amplification reaction, and when the
DNA. Each of these three thermal reaction processes is reaction is finished, the positive signals of each reaction
called a cycle [1]. unit are sequentially counted to realize quantitative analy-
At present, there are dozens of technologies derived sis of the nucleic acid molecules. In view of the unique
from PCR technology, including nested PCR, reverse tran- advantages of digital PCR technology, it is especially suit-
scription PCR (RT-PCR), quantitative real-time PCR (qRT- able for applications that cannot be distinguished by Ct
PCR), droplet digital PCR (ddPCR), etc. Nested PCR is a values, e.g., copy number variation, mutation detection,
variant of polymerase chain reaction (PCR) that uses two gene relative expression research (such as allelic imbal-
pairs (rather than one pair) of PCR primers to amplify a ance expression), second-generation sequencing results
complete fragment. The first pair of PCR primers amplify- validation, miRNA expression analysis, single cell gene
ing fragments is similar to normal PCR. The second pair of expression analysis, and the like [5].
primers, called nested primers (because they are inside the
first PCR amplified fragment), binds inside the first PCR
product such that the second PCR amplified fragment is 1.3.2 Sequencing Technology
shorter than the first amplification. The advantage of nested
PCR is that if the first amplification produces an erroneous Sequencing technology can quickly and accurately acquire
fragment, the probability of primer pairing and amplifica- the genetic information of organisms, which has been of
tion on the wrong fragment for the second time is extremely great significance for life science research. For each organ-
low. Therefore, the amplification of nested PCR is very ism, the genome contains genetic information for the entire
specific and is generally applied to detection of microbes organism. Sequencing technology can truly reflect the
such as viruses and tumor genes [2]. Reverse transcription genetic information on genomic DNA, and thus reveal the
PCR is a widely used variant of the PCR. In RT-PCR, an complexity and diversity of the genome comprehensively,
RNA strand is reverse transcribed into a complementary playing a very important role in life science research.
DNA, and then amplification is performed by PCR using Sequencing technology can be traced back to the 1950s [6].
this complementary DNA as a template. Transcription of a As early as 1954, there had been reported the sequencing
single strand RNA into complementary DNA (cDNA) is techniques, that is, Whitfeld and colleagues [7] used a chem-
referred to “reverse transcription” and is accomplished by ical degradation method to detect a polyribonucleotide
an RNA-dependent DNA polymerase (reverse transcrip- sequence. The chemical degradation method invented by
tase). Subsequently, another strand of DNA is completed Sanger and colleagues in 1977 marked the birth of the first
by a deoxynucleotide primer and a DNA-dependent DNA generation of sequencing technology. Since then, the second
polymerase. Exponential amplification of RT-PCR is a generation of sequencing technology has been produced,
very sensitive technique for detecting very low copy num- including Roche’s 454 technology, Illumina’s Solexa tech-
ber RNA. RT-PCR is widely used in the diagnosis of nology, and ABI’s SOLiD technology [8]. Recently, Helicos’
genetic diseases and can be used to quantitatively monitor single-
molecule sequencing (SMS) technology, Pacific
the content of certain RNAs [3]. When quantitative analy- Biosciences’ Single Molecule Real Time (SMRT) sequenc-
sis is performed by PCR after completion of the reverse ing technology, and nanopore single-molecule sequencing
transcription process, qRT-PCR or ddPCR techniques are technology being studied by Oxford Nanopore Technologies
also used for quantitative analysis as technology advances. Company are the third-generation sequencing technologies.
Quantitative analysis is more sensitive and more accurate. The advantage of the third-generation sequencing technol-
QRT-PCR is a method of measuring the total amount of ogy is that it achieves the inherent continuity of the DNA
product after each PCR cycle with a fluorescent chemical polymerase, a reaction that can measure thousands of bases
in a DNA amplification reaction. Quantitative analysis of a [9]. And the accuracy of the third-generation sequencing
specific DNA sequence in a sample to be tested can be cal- technology is very high, reaching 99.9999%. In addition, it
culated using an internal reference or an external refer- can directly measure the sequence of RNA, greatly reducing
ence. Depending on the fluorescent dye used, it is further the systematic error caused by reverse transcription in vitro.
divided into the SYBR Green I method and TaqMan probe The third-generation sequencing technology is currently
method [4]. DdPCR is a new technique for the absolute used for genome sequencing, methylation studies, and muta-
quantification and amplification of nucleic acid molecules. tion identification. Nowadays, sequencing technology is
Compared with traditional PCR, digital PCR has better moving toward high- throughput, low-cost, and long-read
reproducibility of results and more accurate quantification length (Table 1.1).
6 L. Du and C. Wang
Table 1.1 Characteristics and applications of each sequencing technology

Sequencing type Advantages Disadvantages Application
The first generation of (1) Fast speed, low cost, and Only one single sequence can be (1) STR paternity test, forensic
sequencing technology accurate positioning measured at one time identification
(2) Accurate measurement is about (2) SNaPshot sequencing
800 bp, and the longest is technology
1000–1500 bp
The second generation of (1) It can measure multiple (1) Each detected segment is (1) Whole genome resequencing
sequencing technology sequences at one time, and the limited to 250–300 bp (WGRS), looking for individual
amount of data is large (2) Since the splicing is performed mutations, InDel, CNV, SV, etc.,
(2) By accumulating DNA by overlapping regions of the with a large amount of demand data
fragments by the database, the sequence, some sequences may be (2) Whole exome sequencing
sequencer can detect all attached detected many times (1%–1.5%) (WES), capture all exons in the
DNA sequence information at one (3) Because PCR enrichment is genome for sequencing, and obtain
time used, a small number of sequences high sequencing depth (50 X–150
(3) Small fragments need to be cannot be amplified, resulting in X)
spliced into growing fragments by loss of information, and there is a (3) Target sequencing, capturing and
bioinformatics analysis probability that a mismatch base enriching the target region of
will be introduced in the PCR interest for sequencing
(4) Transcriptome sequencing
(RNA-Seq), which studies
sequencing at the transcriptional
level, including mRNA, lncRNA,
microRNA, and the like
The third generation (1) Fast speed, one performance test (1) High cost (1) Genomic sequencing
sequencing technology 10–40 kb (2) The error rate is relatively (2) Methylation research
(2) No longer need to assemble high, about 15% (3) Mutation identification (SNP
(3) Single molecule sequencing, no (3) DNA polymerase-dependent detection)
need for PCR amplification in the activity
sequencing process (4) The bio-information analysis
(4) Expand application: RNA software is not rich enough
sequence, methylated DNA
sequence
1.3.3 Nucleic Acid Hybridization Technology 1.3.4 Chip Technology
Nucleic acid hybridization technology is one of the basic At present, the chip technology for nucleic acid detection is
techniques of molecular biology. In recent years, it is being mainly gene chip technology. Gene chips are also known as
widely used in molecular biology, and its detection process DNA chips, DNA arrays, cDNA chips, DNA microarrays, and
has the advantages of specificity, sensitivity, and rapidity. oligonucleotide arrays. Gene chip technology is an advanced
The basic principle of hybridization is the annealing of two technology combination of multidisciplinary integration of
single-stranded nucleic acids with base complementation to molecular biology, microelectronics, physics, chemistry, and
form a double strand. The hybridization partners for diag- computer science. It utilizes hydrogen bonding between com-
nostic purposes are viral probes of known sequence and viral plementary bases of nucleic acid double strands to form a
nucleic acids in the sample to be tested, which are detected stable double-stranded structure, and the detection of the sam-
by specific methods after hybridization. If there is a hybrid- ple is achieved by detecting the fluorescent signal on the sin-
ization signal, it indicates the presence of viral nucleic acid gle strand of interest. Now, matured chips include a chip for
in the sample, which in turn demonstrates the presence of detecting gene mutations and an expression gene chip for
viral infection. The viral nucleic acid to be tested can be detecting gene expression levels of cells. Gene chips are the
extracted from pathological tissues or extracted from puri- most matured and first commercialized products in biochip
fied virions. After extraction, it can hybridize to the probe on technology. The gene chip is developed based on the principle
the membrane (solid phase hybridization), or hybridize of nucleic acid probe complementary hybridization. Nucleic
directly in the hybridization solution of the test tube (liquid acid probe refers to a synthetic sequence in which a detectable
phase hybridization). In addition, viral nucleic acids can be substance is attached, and the probes are used to identify spe-
hybridized directly on tissue sections or cell smears (in situ cific genes in a mixture of nucleic acids based on the principle
hybridization) [10]. of base complementation.
The gene chip technology mainly includes four basic These signals are converted into electrical or optical signals
technical steps: (i) Chip preparation – mainly adopts surface by transducers, and then processed by the signal processing
chemistry method or combinatorial chemistry method to and amplification system, and displayed or recorded on the
treat solid phase matrix such as glass piece or silica gel, and instrument. It can be divided into enzyme sensor, microor-
then arrange DNA fragments or protein molecules in a spe- ganism sensor, immune sensor, tissue sensor, and cell sensor
cific order on the substrate. Human gene chips containing 1 according to the different biological sensitive materials.
million DNA probes are currently being prepared. (ii) Based on the transducer, the biosensor can be classified into
Biological sample preparation and processing: biological electrochemical biosensor, photometric biosensor, mediator
samples are a mixture of biological molecules, and generally biosensor, semiconductor biosensor, and piezoelectric crys-
cannot react directly with the chip except for a few special tal biosensor. Now biosensing technology is mainly used in
samples. The sample is subjected to specific biological treat- portable diagnostic instruments, blood glucose and oxygen
ment, and information molecules such as proteins or DNA monitoring, and continuous metabolite monitoring and is
and RNA are obtained and labeled to improve the sensitivity widely used in clinical examination, monitoring, and reha-
of the detection. (iii) The reaction between biomolecules and bilitation evaluation. At present, non-invasive and minimally
chip: the reaction between biomolecules and the chip is a invasive detection has become an important development
critical step in chip detection. By selecting appropriate reac- direction of biomedical sensing technology [12].
tion conditions to optimize the process, the mismatch ratio is
reduced, thereby obtaining a signal that best reflects the
nature of the organism. (iv) The detection and analysis of the 1.4 Protein Detection Methods
chip signal: now the most common method used to detect the
chip signal is to put the chip into a chip scanner, and obtain At present, common molecular biotechnologies for protein
the relevant biological information by collecting the fluores- detection include spectrum technology, protein chip technol-
cence intensity and position of each reaction point and ana- ogy, labeled immunoassay technology, and mass spectrome-
lyzing the image by relevant software [11]. try technology.
1.3.5 Biosensing Technology 1.4.1 Spectrum Technology
Biosensors are products based on sensors that are infiltrated The analysis of the radiation energy spectrum emitted by
and integrated by disciplines such as biology, medicine, elec- matter or the change of the energy spectrum caused by the
trochemistry, optics, thermodynamics, and electronics. It is a interaction between radiant energy and matter is called spec-
selective small analytical device consisting of a bioactive tral analysis. A photochemical instrument that separates
substance as a sensitive component with a suitable trans- complex color light into a spectrum is called a spectrometer.
ducer. Biosensor technology is characterized by rapidity, There are many types of spectrometers, in addition to spec-
sensitivity, specificity, and simplicity and has broad applica- trometers used in the visible range, as well as infrared spec-
tion prospects in the field of molecular biology detection. trometers and ultraviolet spectrometers. According to the
The basic components of a biosensor include a sensor, a working principle of modern spectroscopy instruments, it
transducer, and a detector with molecular recognition capa- can be divided into two categories: classic spectrometers and
bilities. With molecular recognition capability, biological new spectrometers. The classic spectrometer is an instru-
component (such as enzymes, antigens, antibodies, nucleic ment based on the principle of spatial dispersion; the new
acids, etc.) or the organism itself (such as cells, organelles, or spectrometer is an instrument based on the modulation prin-
tissues) can be used as sensitive material. A membrane struc- ciple. Classic spectrometers are slit spectroscopy instru-
ture formed by immobilized sensitive material is a sensor of ments. The modulation spectrometer is non-spatial
the biosensor. spectroscopic, which uses a circular aperture to let the light
The working principle of the biosensor is to produce bio- enter. According to the principle of splitting of the dispersive
chemical reaction with the biosensing material in biosensor components, spectroscopic instruments can be divided into
and the test substance in sample through the molecular rec- prism spectrometer, diffraction grating spectrometer, and
ognition function, and generate signals such as ions, protons, interference spectrometer. At present, the most commonly
gases, light, heat, and mass changes. Under certain condi- used spectroscopy techniques are UV-Vis spectroscopy,
tions, the magnitude of the signal is quantitatively related to infrared spectroscopy, fluorescence spectroscopy, and
the amount of the substance being tested in the sample. Raman spectroscopy [13].
8 L. Du and C. Wang
1.4.2 Protein Chip Technology 1.4.4 Mass Spectrometric Technique
Protein chip refers to the immobilization of a large number Mass spectrometry is an analytical method that analyzes the
of protein molecules on a carrier surface in a pre-set arrange- mass-to-charge ratio (m/z) of ions of a sample. The sample
ment to form a microarray. Based on the principle of spe- needs to be ionized firstly, and then the ions are separated by
cific binding between protein molecules, a microfluidic the mass-to-charge ratio (m/z) through using the different
biochemical analysis system is constructed to achieve accu- motion behavior of ions in the electric or magnetic field.
rate, rapid, and large information detection of biomolecules. Qualitative and quantitative results of the sample can be
Although Roger Ekin described the technical principles of obtained by mass spectrometry and related information of
protein chips in his environmental material theory in 1980s, the sample. Early mass spectrometers were mainly used for
microchip detection technology did not receive great atten- isotope determination and inorganic elemental analysis.
tion until significant achievements in the field of genome They were used for organic matter analysis since the 1940s.
and proteomics research. With just one experiment on one Gas chromatography-mass spectrometers appeared in the
plate, the possibility of measuring thousands of cell biology 1960s, which greatly expanded the application field of mass
parameters provides the perfect solution for building pro- spectrometers. Since then, mass spectrometers have become
teomic comprehensive assay tools. The DNA chip has a an important instrument for organic matter analysis. The
good hybridization system and can analyze the entire tran- application of computers has led to a dramatic change in
scription system of the cell by a single reaction test. Since mass spectrometry, making its technology more mature and
there is no absolute correspondence between mRNA and more convenient. Some new mass spectrometry techniques
proteome in cells, solving the problem of differences have emerged since the 1980s, such as fast atom bombard-
between genomic and proteomic studies requires other ment ionization sources, matrix-assisted laser desorption
high-throughput techniques that can directly analyze the ionization sources, electrospray ionization sources, atmo-
properties of the detected proteins. In the past few years, spheric pressure chemical ionization sources, liquid
different high-throughput analysis and detection technology chromatography- mass spectrometry, inductively coupled
platforms have been established, and the development of plasma mass spectrometer, Fourier transform mass spec-
micro-chip technology has exceeded the DNA chip technol- trometer, etc. These new ionization techniques and new mass
ogy, and there are reports on protein chip detection based on spectrometers have made significant progress in mass
a large number of different samples. Protein chip technol- spectrometry.
ogy is mainly used in gene expression screening, antigen Because mass spectrometry has the advantages of high
and antibody detection, biochemical reaction detection, and sensitivity, low sample consumption, fast analysis speed,
drug screening. This has the advantage of enabling rapid and simultaneous separation and identification, it has been
detection of small amounts of crude biological samples widely used in various fields such as chemistry, chemical
(serum, urine, body fluids, etc.) [14]. engineering, materials, environment, geology, energy,
medicine, criminal investigation, life science, and sports
medicine [17].
1.4.3 Labeled Immunoassay
Labeled immunoassay technology is a general term for a large 1.5 Future Trends
class of ultra-sensitive, high-specific detection technologies.
Because of its many unique advantages, it has been widely used The rapid development of molecular biology technology,
in basic medicine and clinical fields. Their basic principles are especially omics sequencing technology, has raised the entire
the same, and the signals emitted by the final measurement dif- medical science research to the molecular level with rapid
fer only by the difference of the markers. Although there are progress and impact on disease prevention, diagnosis, and
more than ten methods reported in the literature, there are some therapy. Therefore, the significance of molecular biotechnol-
applications that have been widely used: radioimmunoassay ogy is not only reflected in pure scientific value, but more
(RIA), enzyme immunoassay (EIA), fluorescence immunoas- importantly, it will be important with another aspect to the
say (FIA), time-resolved fluoroimmunoassay (TRFIA), and disease evaluation by its application to personalized
chemiluminescence immunoassay (CLIA) [15, 16]. medicine.
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1. Tatipally S, Srikantam A, Kasetty S. Polymerase chain reaction
11. Pongor L, Kormos M, Hatzis C, et al. A genome-wide approach
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to link genotype to clinical outcome by utilizing next generation
nosis: a systematic review. Trop Med Infect Dis. 2018;3:107.
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2. Zhang X, Zhang Y, Liu X, et al. Nested quantitative PCR approach
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for urinary cell-free EZH2 mRNA and its potential clinical applica-
12. Bhalla N, Jolly P, Formisano N, et al. Introduction to biosensors.
tion in bladder cancer. Int J Cancer. 2016;139:1830–8.
Essays Biochem. 2016;60:1–8.
3. Bachman J. Reverse-transcription PCR (RT-PCR). Methods
13. Tonannavar J, Deshpande G, Yenagi J, et al. Identification of

Enzymol. 2013;530:67–74.
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4. Jozefczuk J, Adjaye J. Quantitative real-time PCR-based analysis
spectral analysis. Spectrochim Acta A Mol Biomol Spectrosc.
of gene expression. Methods Enzymol. 2011;500:99–109.
2016;154:20–6.
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14. Feng Y, Wang B, Chu X, et al. The development of protein chips
reaction (dPCR) and its emerging biomedical applications. Biosens
for high throughput screening (HTS) of chemically labeling small
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15. Zhao L, Wang D, Shi G, et al. Dual-labeled chemiluminescence
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Assay Performance Evaluation
2
In vitro diagnostic reagents are special items used by medi- ument EP15-A3 “User Precision Verification and Deviation
cal institutions to diagnose diseases and provide clinical Estimation” to meet different needs.
parameters for diagnosis and treatment. The quality of the The current EP5-A2 document “Evaluation of Precision
tests directly affects the clinical test results. This is linked to Performance of Quantitative Measurement Methods;
the positioning of the nature of the disease, the mastery of Approved Guideline-Second Edition” has been widely used
the disease severity, the choice of treatment methods, and the for precision performance evaluation, and this document will
judgment of prognosis. Nowadays, a large number of indi- be detailed in this chapter [1, 2].
vidualized in vitro detection reagents are gradually entering
the market, and the scale is expanding. To ensure the quality
of individualized diagnostic reagents and assure the effec- 2.1.1 Terminology and Definitions
tiveness of the detection system, we need to verify the detec-
tion performance of these systems. • Precision (measurement) Closeness of independent test
results obtained under specifying conditions.
• Imprecision The degree of dispersion of each independent
2.1 Precision measurement under specific conditions.
• Repeatability conditions Independent test results are
Precision refers to the closeness of independent measure- obtained in a short period of time by the same operator
ment results obtained under specifying conditions. Usually and the same instrument using the same method on the
we can only quantitatively measure the inconsistency of same test substance.
measurement results, that is, imprecision. The time-related • Repeatability The closeness of the results is obtained by
precision components are mainly reproducible (in-batch pre- continuous measurement of the same test object under the
cision), inter-assay precision, intra-day precision, daytime same detection conditions.
precision, and indoor precision. • Reproducibility conditions The test results are obtained
Precision performance is one of the basic analytical per- by different operators using the same method on different
formance of the detection system. It is likewise the basis for instruments to measure the same test items.
other methodological evaluations. If the precision is poor, • Reproducibility The closeness of the results obtained
additional performance evaluation experiments cannot be per- from the same test object under varying detection
formed. The US National Clinical and Laboratory Standards conditions.
Institute (CLSI) issued EP05-A3 document “Evaluation of • Run In the interval where the authenticity and precision of
Precision of Quantitative Measurement Procedure; Approved the detection system are stable, it usually does not exceed
Guideline-Third Edition” for a thorough introduction to pre- 24 h or less than 2 h.
cision performance evaluation, designed to provide a ref- • Sample Derived from one or more parts of the population,
erence for precision performance evaluation experiments. which provides information about the population, usually
Simultaneously, the committee issued another guidance doc- as the basis for the conclusion of the population. Note:
For example, collecting a small amount of serum from a
large amount of serum.
L. Wang (*) · Z. Shi • Intermediate precision conditions The measurement
Department of Medical Experimental Center, results are obtained by measuring the same test item on
General Hospital of Ningxia Medical University, Yinchuan,
Ningxia Hui Autonomous Region, People’s Republic of China the same instrument using the same test method under

12 L. Wang and Z. Shi
d ifferent operating conditions. It should be noted that (1) tion protocols. It is utilized to verify and evaluate the pre-
there are four elements of the operating conditions, time, cision performance of the instrument used or the self-built
calibration, operator, and instrument, and (2) the factors detection system. However, if the manufacturer’s evaluation
that change the operating conditions need to be clari- should include this factor, it should also include variables
fied. In this precision evaluation, they are usually called such as unusual locations and equipment.
“between-run,” “within-day,” “between-day,” “within- The scheme adopts the 2 × 2 × 20 experimental method,
device,” and “within-laboratory.” that is, 2 batches are tested every day, and 2 batches are tested
• Intermediate precision Precision under intermediate pre- in each batch for 20 days, and 80 valid data are obtained. The
cision conditions. program also provides a more intuitive and practical experi-
mental record form. After the experimenter has completed
the experiment and obtained enough data, the batch, batch,
2.1.2 O
verview of the Precision Evaluation day, and total imprecision can be obtained through a simple
Process (Fig. 2.1) calculation. In addition, after the experimenter obtains the
imprecision data, if it is greater than the manufacturer’s dec-
2.1.3 Features of the EP5-A2 Program laration, the chi-square test can still be used to judge whether
there is a meaningful difference. If there is no noteworthy
The program provides an experimental guide for evaluat- difference, it is still acceptable.
ing quantitative measurement methods and instrument pre-
cision performance and should be the most comprehensive
and statistically significant in the current precision evalua- 2.1.4 E
P5-A2 Experimental Protocol
and Requirements
Need for Precision Evaluation 2.1.4.1 Experimental Preparation

Reagents and Calibrators The same batch of reagents and
calibrators should be used throughout the evaluation process.
Assess Sources of Variation Because the experiment does not independently evaluate cer-
tain variability factors, the addition of these variability can
more accurately reflect the test performance.
Select and Optimize Study Protocol Experimental Sample

Matrix Select a matrix similar to a clinical sample as much
as possible. Stable, serum matrix controls are typically used.
Perform Preliminary Checkout
Concentration It is recommended to use two concentra-
tions. Try to choose a concentration that is close to the manu-
facturer’s stated performance or a concentration close to the
Complete Experimental Steps
“medical decision level.”
2.1.4.2 Experimental Method

Check Data Integrity The entire experiment should collect 20 days of usable data,
using 2 concentrations of experimental samples, 2 batches per
day, 2 replicates per batch, and 80 acceptable data for each
Analyze Data concentration. Evaluation experiments should be structured
into several gradual stages according to requirements, and
each stage should take the necessary quality control measures
to detect outliers. The method should be tested for acceptabil-
Summarize Results
ity of all previous data every 5 days after the start of the method
familiarization phase to assure the validity of the results.
Instrument familiarity stage In order to avoid problems
End in the actual instrument performance evaluation process, the
operator should be proficient in the instrument’s operating,
Fig. 2.1 Steps in the evaluation of precision for quantitative measure- maintenance, sample preparation, calibration, and testing
ment procedures procedures. This phase can be carried out after the training
2 Assay Performance Evaluation 13
period provided by the manufacturer or at the same time. should be cleared. At the same time, the batch of experimen-
There is no requirement to collect data at this stage until the tal data should be removed and re-run a batch. Target values,
operator can operate the instrument correctly. warning limits, and loss of control limits for all acceptable
Method Familiarization Because some of the steps in the data are calculated every 5 days. If the previously acceptable
evaluation experiment are rarely used in routine measurements, results are now unacceptable, the batch is rejected, and the
in order to prevent these unfamiliar steps from affecting the experiment is continued until a total of 40 batches of valid
results of the evaluation experiment, it is necessary to practice data are obtained for 20 days.
the method several times before performing the evaluation.
The formal experiment was performed in two batches
per day, and the test was repeated twice in each batch. Each 2.1.5 D
ata Collection, Processing,
batch was isolated by at least 2 h, and four data per day were and Statistical Analysis
obtained for each concentration. This phase typically lasts
5 days and gets data. For complex instruments, the method 2.1.5.1 Experimental Data Record
and familiarity can be extended appropriately. If the data In order to facilitate data management and statistical pro-
in this stage pass the acceptability test, it will be counted cessing, each batch of acceptable data can be filled in a form.
together with the data of the subsequent experimental stage. The record form can be changed according to the user’s situ-
At the end of the familiar phase of the method, a prelimi- ation, as long as it is convenient to use.
nary precision evaluation experiment is required. The usual
practice is to continuously measure 20 times (2 concentra- 2.1.5.2 Outlier Test
tions) using the same quality control as the precision test,
and then calculate the standard deviation and coefficient of Daytime Outliers The routine quality control program can
variation of the consequences. If a significant difference is detect batch or daytime outliers. The data of the out-of-
found from the expected results, the manufacturer must be control batch should be deleted after finding the cause, and
contacted, and the follow-up experiment terminated until the then re-run a batch.
problem is resolved. The data for this phase can also be used
to determine intra-assay outliers in the familiar phrase of the Intra-group Outlier The standard deviation obtained by
method and in subsequent experiments. the preliminary precision evaluation experiment was used as
After the experimental phase of the method, the experi- the criterion for judging the outliers of the repeated measure-
ment still required to last for 15 days. The experimental ment results in the batch. If the absolute value of the repeated
method is the same as the method familiarization stage. measurement is more than 5.5 standard deviations, then the
Record the experimental data and recalculate the quality batch of data is rejected. After finding the outliers, find the
control limits in a series of QC charts every 5 days, and ver- cause and repeat the batch analysis. If more than 5% of the
ify the acceptability of all data. If a batch is rejected because data is rejected, the reason cannot be found. Then consider
of quality control or operational difficulties, it is necessary to that the performance of the instrument is not stable enough;
re-run a batch of experiments after finding and correcting the you should contact the manufacturer.
cause. If possible, add at least 10 patient specimens to each
batch to simulate the actual procedure. 2.1.5.3 Repeatability Estimate
Repeatability evaluation uses the following formula:
2.1.4.3 Quality Control
X Xij 2
1 2 2
A routine quality control procedure must be performed in j 1 j 1 ij 1
the precision evaluation experiment. Use at least one appro- Sr ,
4I
priate concentration of quality control sample in each batch
of measurement. If two or more concentrations of quality where
control are routinely utilized, this should also be the case in
this experiment. I = total number of days (generally 20)
At the end of the method familiarization phase, a pre- j = run number within-day (1 or 2)
liminary quality control chart should be established, and Xij1 = result for replicate 1, run j on day i
the target value x and standard deviation (s) should be Xij2 = result for replicate 2, run j on day i
computed using the first 5 days of quality control data. Since
the preliminary estimate has lower statistical performance, Two results are required for each batch when using the
±3 s is used as the warning limit. Use ±4 s as the limit of above formula. If there is only one batch of results with no
loss. Subsequent QC data is depicted in the figure. If there more than 10% of evaluation days during the experiment, the
is runaway data, the cause should be found and the QC data statistical calculation of the results is still valid.
Otherwise, the number of experimental days should be • Total error The combination of determination errors that can
increased until the requirements are satisfied [1, 2]. affect the accuracy of the analysis results, including random
errors and systematic errors, is estimates of inaccuracy.
2.2 Accuracy
2.2.2 Features of the EP9-A2 Program
The accuracy performance is part of the important analyti-
cal properties of a detection system or method. The impor- The program is mainly used to evaluate the bias between
tance of the method performance evaluation experiment is the two measurement methods of the same test item and
second only to the precision evaluation experiment, which determine whether the bias is within an acceptable range.
is the experimental basis for the subsequent analytical mea- Usually the new method is called the experimental method,
surement range, analysis sensitivity, and biological reference and the method compared with it is called the comparison
interval evaluation. method. The comparison method can be a reference method,
At present, there are many options for the evaluation of or a method declared by the manufacturer or a conventional
the accuracy of performance. This section mainly introduces method currently used by the user, and the accuracy of the
the EP9-A2 document issued by the National Association of latter should have been confirmed.
Clinical and Laboratory Standards Institute (CLSI) in 2002, The program detects 8 samples per day for 5 days and sta-
namely, “Method Comparison and Bias Estimation Using tistically processes the data detected between the two meth-
Patient Samples; Approved Guideline-Second Edition,” ods, and the bias between the analytical methods is acceptable.
which is important for the specification of methodological The experimental process is relatively simple, with an empha-
comparison experiments. It also introduced another guid- sis on statistical processing. The scheme performs an outlier
ance document, EP15-A2, “User Verification of Performance test and a constant evaluation of the bias of two different cal-
for Precision and Accuracy; Approved Guideline-Second culations and four graphs, and then performs an appropriate
Edition” to meet the need of different laboratories [3, 4]. range test of the X value. Based on the results obtained above,
the expected bias was calculated by linear regression and the
residual method, and compared with the allowable bias; the
2.2.1 Definitions whole result has higher statistical efficiency.
• Trueness The complete expression is the measurement

accuracy, which is the degree of consistency between the 2.2.3 E
P9-A2 Experimental Protocol
mean and the true value of a large number of test results. and Requirements
It is also a qualitative concept and can only be described
by the degree. It is usually expressed by the “bias” statis- 2.2.3.1 Experimental Preparation
tic, which is the opposite of accuracy. This concept has
eliminated the effect of imprecision. If there is still a bias, Sample Preparation
it means that there is a systematic error, so it is different • Source Collect and process fresh patient samples accord-
from accuracy. ing to the protocol.
• Accuracy The complete expression should be the mea- • Storage If possible, avoid storing specimens, and measure
surement accuracy, which is the degree of consistency them on the same day of collection; otherwise, select stor-
between the test result and the measured true value, which age conditions and time according to the stability of the
is related to the accuracy and precision of the measure- components to be tested.
ment. Accuracy is a qualitative concept rather than a • The number of samples should be analyzed at least 40
quantitative one, and can only be defined as good or bad. specimens, which are a little better. Each sample must be
An estimate that measures accuracy from the reverse is of sufficient quantity for both methods to be tested in
“deviation.” duplicate. If the required sample size is not obtained from
• Bias Measurement bias refers to the difference between one patient, two (no more than two) patient specimens
the expected value and the acceptable value of the mea- with the same medical history and approximately the
surement result. In general, using the accepted (deter- same concentration of the test substance can be mixed.
mined or referenced) method and the evaluated method, • The concentration should be evaluated within the clini-
the dispersion between the repeated test values of the cally meaningful range, i.e., within the medically deter-
sample is expressed in the unit of measurement or per- mined level. It should normally be distributed from the
centage. That is, the difference between the average and reference range, below the reference range, and as far as
the reference value. possible within the analytical measurement range.
Comparison Method Selection ence material to determine whether it is consistent with the
It has been mentioned above that the methods currently used manufacturer’s declaration or other specified performance
in the laboratory, the methods declared by the manufacturer, requirements.
and the accepted reference methods are all comparable
methods. The comparison method should have the follow- 2.2.4.1 Comparison of Patient Sample Results
ing characteristics with respect to the experimental method to Those of Another Procedure
and has better precision than the experimental method; it is Obtain 20 patient samples, the concentration of which
not interfered by known interfering substances; the same should be distributed throughout the linear range. Do not
unit as the experimental method is used; the result is trace- use samples that exceed the linear range. Some concentra-
able. In addition, the analytical measurement range of the tions are not easy to obtain, and the same disease specimens
comparison method is at least the same as the experimental can be mixed (not more than 2). The collected specimens
method before it can be used for comparison. should be stored until there is a sufficient amount of
specimens.
2.2.3.2 Experimental Method These 20 specimens were measured by test and com-
parative procedures. These measurements can be completed
Instrument Familiarity Stage In order to avoid problems on the same day, and it can also last for 3–4 days, and 5–7
in the actual instrument performance evaluation process, the samples can be measured every day. The latter conclusion is
operator should be proficient in the instrument’s operating more reliable than the former. Each analytical method should
procedures, maintenance procedures, sample preparation be completed within 4 h. If the specimen is stored, it should
methods, and calibration and testing procedures. It is usually be measured within 1–2 h after reconstitution.
5 days, which can be shorter for very simple instruments and Each method is guaranteed by quality control procedures.
longer for complex instruments. Regular quality control pro- Any batch that is rejected due to quality control or opera-
cedures should be established at this stage. tional difficulties should be retested after the problem is
corrected.
Formal Experiment Eight samples were measured daily Experimental data processing calculates the difference in
using two methods, and each sample was repeatedly mea- results between the two methods for each specimen.
sured twice for a total of 5 days. In the repeated measure-
Test procedure result i –
ment of the sample, the first measurement sequence is Individual sample bias bi .
Comparison procedure result i
specified, and the second detection is performed in the
reverse order. For example, the samples can be performed
Individual sample bias in percent = %bi .
in the following order: 1, 2, 3, 4, 5, 6, 7, 8 and 8, 7, 6, 5, 4,
3, 2, 1. The concentrations in the sequence should be
Test procedure result i
arranged as randomly as possible. The reverse order of the Comparison procedure result
second specimen can reduce the effect of cross-contamina- %bi 100 i
.
tion and drift on the average value of replicated specimens. Coomparison procedure result i

Daily samples should be measured within 2 h to ensure
analyte stability.
Plot the bias or percentage bias of the results of the two
2.2.3.3 Quality Control methods for each specimen: the horizontal axis represents
Regular quality control procedures should be established the comparison method, and the vertical axis represents the
prior to formal testing. Any method that occurs out-of- percentage bias. Check the bias graph to see if the differences
control should be re-measured until the required number of in the results of the samples within the concentration range
samples is reached. tested between the two methods are relatively consistent. If
they are consistent, use the following average bias to com-
pare with the manufacturer’s statement; If the bias or per-
2.2.4 Simple Accuracy Evaluation Plan centage bias is not consistent within the concentration range,
the data should be divided into several parts, and each part
EP15-A document of CLSI provides two procedures to independently calculates the average bias. If the bias shows a
verify accuracy: one is to use patient specimens for meth- gradual change in the concentration, the average bias cannot
odological comparisons, similar to the EP9-A2 document, be calculated. More data is needed to confirm the accuracy
but with experimental time, number of samples, number of of the method.
repetitions, and statistical processing. The former is simple; Calculate the bias and/or percent between the two
the other is to calculate the recovery rate by testing the refer- procedures.
are available from the National Institute of Standards and

I I
b
i 1 i
%bi
b %b i 1
. Technology (NIST) and CAP. Their target values repre-
n n
sent the mean of the method. Of course, the reference sub-
Calculate the standard deviations of the bias and/or bias in stance should have been clearly evaluated as suitable for
percent. the method. In addition, the target value of the reference
substance may be related to the reagent lot number. If the
b b
I 2
i reagent lot number is changed, the target value of the spe-
Sb i 1
. cific method may not be suitable for the new lot number
n 1

of reagents. The manufacturer should be consulted for
advice on the correctness of the method and the applica-
%b %b
I 2
S %b
i 1 i
. ble reference material.
n 1 • A reference obtained from an ability comparison test.

These substances are set by a large number of laboratories
If the estimated or percentage bias is less than the manufac- and a number of representative reagents and system
turer’s stated or percentage bias, the laboratory proves that calibrators.
the bias is consistent with the statement, and no further sta- • Accuracy confirmation or quality control provided by the
tistical analysis is required. manufacturer. These substances are specifically designed
If the bias or percentage bias is greater than the manufac- for use in analytical systems, but are generally not appli-
turer’s stated bias or percentage bias, the following steps can cable to methods from another manufacturer. Here we
be used to perform a statistical test of the difference. mainly talk about authenticity control products. Of
Assuming a false rejection rate is α, usually choose course, we think that it is also feasible to use calibrators
α = 1% or α = 5%. with different lot numbers, because it has partially elimi-
Determine the values of tα,n−1, where n represents the num- nated the matrix effect with the reference method, and it
ber of patient specimens. For example, if α = 1%, n = 20, is currently commonly used for calibration verification.
t0.01,19 = 2.539, other values of tα,n−1 can be obtained from the • Interlaboratory quality assessments are analyzed by
t .s b numerous laboratories, and their mean values can be used
statistics book; Calculation of bias verification value,
n to assess consistency. Of course, if there are a sufficient
+ β, where β is the bias value declared by the manufacturer. number of laboratories in the same group, the average
value is reliable. For a reliable mean, a minimum of 10
Calculate the verification limits for bias as laboratories within the same methodology group is
required. The quality evaluation may take a sample value
t sb t sb
and , from a small number of reagent batches. For a single lab-
n n oratory using a new batch of reagents, this method may
affect the reliability of the target value.
where β is the manufacturer’s claimed value of bias. • Substances provided by third parties that have been val-
Calculate the verification limits for percent bias as ued in a number of different ways. These substances are
similar to the ability to compare experimental objects or
t s% b t s% b
and . regional property controls. Usually, fewer laboratories are
n n involved in calculating the mean of the same group, and

the results have less reliable target values. In addition, a
relatively small number of reagents with different lot
2.2.4.2 Method of Setting Reference Materials numbers are sampled, which also affects the reliability of
The accuracy evaluation can be verified by measuring the the specified target value.
recovery rate or deviation of the reference material by using • The analyte concentration may be fixed to a predeter-
the abovementioned commonly used comparison test. Of mined concentration. For example, the pressure of a part
course, the reference materials mentioned here are not lim- of the gas in the blood may be fixed at a predetermined
ited to reference materials derived from reference methods value by tonometry.
or decisive methods, and may have multiple sources.
Procedure for Demonstration of Accuracy
Sources of Reference Materials with Reference Materials
• Fresh frozen human serum or some other body substance • Select the material most suitable for this method. A mini-
without ingredients. Analytes for such materials have mum of two levels is required. Although five levels were
been valued with reference or deterministic methods and selected to simulate capacity comparison experiments,
more levels are suitable for fully evaluating the entire values assigned or verified by experiments of national or
measurement range. The level chosen should be represen- international organizations; (3) according to the survey
tative of the minimum and maximum measurement ranges value or verification value obtained by the scientific or
of the method. The user should pay attention that the engineering organization’s cooperative experiment; (4)
selected level value may represent a good precision level when none of the above is available, the expected value
value of the method. can be determined quantitatively, such as the mean value
• Prepare samples according to the manufacturer’s instruc- measured by a special group.
tions. The analytes should be thoroughly mixed before • Alpha (α) error/Type I error/False positive The possibility
use, and each sample measured in duplicate. of rejecting invalid assumptions. No analyte is present in
• Calculate the mean x and standard deviation (SD) of the sample, but the result is positive, which is the possibil-
the test results at each concentration. ity of a false positive.
• The results are compared to the set requirements, such as • Beta (β) error/Type II error/False negative Possibility of
the ability to compare the acceptance criteria of the exper- falsely accepting invalid assumptions. There is a certain
imental organizer or the total allowable error of the amount of analyte in the sample, but the result is negative,
medical. which is the possibility of false negatives.
• Blank Samples that do not contain the analyte to be
detected, or samples whose concentration is at least one
2.3 Sensitivity order of magnitude lower than the lowest level of the
analyte.
The minimum analyte concentration detectable by a detec- • Limit of blank (LoB) The maximum test result observed
tion system or method is referred to as analytical sensitivity for a blank sample under the specified probability condi-
or detection limit. For items of particularly low concentrations. (1) LoB is not the actual concentration detected, but
tion, determining the limits of detection is important for the to ensure that the positive signal at the actual concentra-
diagnosis or treatment monitoring of the disease. For exam- tion becomes the detection limit; similarly, LoB is the
ple, elevated cTn is an essential basis for the diagnosis of lowest value expected for a sample containing an analyte
acute myocardial infarction. All of them explicitly require equal to the LoD level under the specified possibility con-
the laboratory to determine the low limit of detection (LoD) ditions. (2) This is the same as the “lower limit of detec-
and the variation at low concentrations; another example is tion,” which is obtained by the prescribed measurement
prostate-specific antigen (PSA), which is an important indi- procedure and can give the lowest detection result with a
cator to monitor the recurrence of patients after treatment. certain measurement uncertainty. Also called “critical
For a long time, the clinical requirements clearly report the value.”
minimum amount of PSA. Negative and positive nucleic • Limit of detection (LoD) The minimum amount of ana-
acid test reports also require that the minimum amount of lyte in the sample can be detected under specified prob-
nucleic acid that can be detected should be equivalent to how ability conditions, but may not be quantified to an exact
many viruses. Therefore, determining the detection limit of a value. Also called “lower limit of detection,” “minimum
detection system or method is one of the important tasks of detectable concentration.” Sometimes used to indicate
the laboratory [5]. “sensitivity.”
• Limit of quantitation (LoQ)/Lower limit of quantitation
With specified acceptable precision and accuracy, the
2.3.1 Definitions minimum amount of analyte in a sample can be quantita-
tively determined. Also known as “determined lower
In 2004, the Clinical and Laboratory Standards Institute limit” and “lower limit of detection ranges.”
(CLSI) issued the EP17-A document, the “Protocols
for Determination of Limits of Detection and Limits of
Quantitation; Approved Guideline,” which recommended 2.3.2 Discussion of Several Common Terms
the use of blank limit detection limits and quantitative tests.
The limits are used to indicate the sensitivity performance of • The lowest limit is the “blank limit” (LoB), which is the
the detection system or method. The concepts and terminol- maximum value that you would expect to see for a series
ogy are now described as follows: of analyte-free samples. It should be noted that LoB is an
observed test result, and all other limits refer to the actual
• Accepted reference value A widely recognized reference concentration of the analyte.
value, which is derived from the following: (1) theoretical • The second lowest limit is the “detection limit” (LoD),
or established values based on scientific principles; (2) which refers to the actual concentration of the analyte.
The observed result at this concentration is just greater Therefore, the detection method must be cleared: What
than LoB, so it is called “detected.” is the amount of analyte that can be detected? The standard
• The “limit of quantitation” (LoQ) is the lowest actual con- curve starts from zero. Is the reported amount of analyte
centration of the analyte at which the analyte can be available from zero? This is the problem involved in the
detected. At the same time, the uncertainty of the observed lower limit of detection (LLD). The statistics show that if
test results is lower than or equal to the quality target set the fluctuation of the blank response is subject to the normal
by the laboratory or manufacturer. Uncertainty goals (or distribution, the probability of the blank response Xblank of
bias and imprecision) must be consistent with LoQ or be each single test is 95%:
feasible for the laboratory.
X blank 2·Sblank X blank X blank 2·Sblank , which is
• The lower end of the measurement range (LMR) is the
lowest level that meets the qualifications. These qualifica- | X blank X blank | 2·Sblank .
tions include all specified properties of the method, such
as bias and imprecision, uncertainty, and other common Therefore, if the reaction response of the test sample is
properties. greater than the blank mean, but the difference between
• The lower end of the linear range (LLR) is the lowest con- the blank mean and the 2Sblank or the 3Sblank, only the
centration that has a linear relationship between the response is the response of the single test of the blank
response of the method and the true concentration. This sample. There is no analyte present in the sample, or the
also requires that the laboratory set nonlinear error goals analyte is zero.
that must be consistent with all regulations regarding It should be noted that the detection system that directly
linearity. reads the concentration unit will report zero for the detection
below zero, and its distribution is not normal. Therefore,
The above limits have a relationship of LoB < LoD ≤ LoQ, the calculated mean and standard deviation cannot accu-
and other limits have no fixed relationship with each other. rately represent the true condition of the detection limit. If
The relative positions of these limits are determined by the the detection response can be expressed by an initial value
set quality targets, nonlinear errors, and set characteristics. such as absorbance, fluorescence intensity, luminescence
intensity, etc., the Sblank is effective at this time. Therefore,
the initial value should be used to calculate the mean and
2.3.3 L
ower Limit of Linear Range (LLR), standard deviation, and then converted to concentration
Biological Limit of Detection (BLD), units.
and Functional Sensitivity (FS) Note: When the sample response is proportional to
the amount of analyte in the sample, the detection limit
2.3.3.1 Lower Limit of Linear Range (LLR) is processed and understood with reference to the above
Always make a blank sample for each test. The detection description. If the sample detection response is negatively
method is often calibrated to the zero point with a blank proportional to the amount of analyte in the sample (i.e.,
response, and then the reaction response of each test sam- when the slope of the response curve is negative), the direc-
ple is the relative response of the analyte after subtracting tion of the data algebra when processing and understanding
the blank sample response. However, the amount of blank the lower limit is the opposite of the above description.
response also fluctuates. If the blank test is repeated multiple
times, the average level of the blank response and the dis- 2.3.3.2 Biological Limit of Detection (BLD)
creteness index of all blank responses to the blank mean are A response signal greater than the lower limit of detec-
indicated by the blank (response) mean and standard deviation (LLD) indicates that there is an analyte in the sample,
tion. When determining method performance or plotting a but the method does not correctly report the quantitative
standard curve, the blank response is often expressed as a result. Because of the range of such low concentrations or
blank mean. other magnitude, the response of a single test sample is
In actual work, only one blank is made at a time, and each less reproducible. A sample with a concentration just equal
blank response has a 50% probability, which is greater than to the detection limit has only a 50% probability of being
or less than the blank mean. When the blank response is less detected, and as the analyte concentration in the sample
than the blank mean, the response to the same sample (with- increases, the probability of being detected increases. So
out the blank response) seems to reflect a little more ana- how many detection responses can be used to report quan-
lyte, and the detection method seems to be more sensitive. titative results well?
When the blank response is greater than the blank mean, it In principle, repeated tests are performed on samples that
seems that the analyte that was previously detectable is now are slightly above the detection limit (certainly not in the
undetectable. empty sample), and the sample detection response after sub-
tracting the blank response is expressed as mean and stan- 2.3.3.4 Experimental Precautions
dard deviation. According to the normal distribution law, the Two different types of samples are typically prepared. One
response of a single test sample has a probability of 95% or is a “blank” sample, i.e., contains no analyte, and the analyte
99.7%, and the mean value of the response is 2 or 3 times concentration is zero; the other is a “detection limit” sample,
the standard deviation of the response. A single-detection which contains a low concentration of analyte. It is gener-
response with a larger mean value can be sure that there ally necessary to prepare several “detection limit” samples
is an analyte, that is, the analyte can be detected; however, with a concentration 1 to 4 times higher than LLD. Blank
if the single-detection response with a smaller mean value and “detection limits” samples are repeatedly tested by the
crosses the detection low limit (the upper limit of the blank detection system to calculate their respective mean and stan-
response), it means that the test method cannot distinguish dard deviation. Different detection limits are estimated from
whether it is blank or analyte by one sample test. Therefore, blank and “detection limit” sample data.
the 95% or 99.7% single-detection minimum of these sam-
ple detection responses is also larger than the lower limit of Blank Sample
detection (LLD), so that it can be quantitatively reported. In One blank sample is used as a blank, and the other is used
a plurality of samples near the detection limit, the minimum to prepare a detection limit sample. The ideal blank sample
analyte concentration (or other magnitude) that meets such should have the same matrix as the patient sample being
conditions is the biologic limit of detection (BLD) of the tested. A “zero concentration” calibrator in a series of cali-
detection system or method. brators of the detection system or a sample-specific dilution
The biologic limit of detection is defined as follows: the containing no analyte is often used as a blank. For some
minimum response that a sample may have in a single test is items, samples of patients who have no disease after surgery,
just greater than the blank detection low limit response, and such as non-PSA serum from patients with prostate tumors,
the analyte concentration or other quantity contained in the can be used as blank samples.
sample is the lower limit of the biological detection. In the
measurement, the detection system or method can quantita- Detection Limit Sample
tively report the minimum concentration of the analyte or When the sensitivity performance of the method is con-
other quantitative values by detecting the lower limit plus 2 firmed, the analyte is added to the blank sample to prepare a
times or 3 times the standard deviation of the detection limit. detection limit sample. The amount of analyte added should
The specific measurement method for biological limit of be the manufacturer’s stated limit of detection. When estab-
detection (BLD) is: lishing the detection limit, it is necessary to prepare several
95%probability : BLD LLD 2 s detection limit sample. detection limit samples, and their concentration should be
within the range of 1–4 times the expected detection limit
99.7%probability : BLD concentration. There is no specific regulation on the number
LLD 3 s detection limit sample of repeated tests, but it is often recommended to do 20 times,
which meet the requirements of repeated tests for clinical
This definition more fully represents the detection limit of tests. Manufacturers often recommend doing 10 times. In
the actual sample, such as: What concentration is different order to reduce expenses, the laboratory often adopts 10
from zero or no analyte? When confirming the manufactur- times.
er’s stated BLD, the concentration of the test limit sample
should be the same as the manufacturer’s instructions. Time Required for the Experiment
If the detection limit is mainly known from the reproducibil-
2.3.3.3 Functional Sensitivity (FS) ity of blank samples, it is often done in batch or short-term
Functional sensitivity (FS) refers to the average concentra- experiments. If the detection limit is mainly known from the
tion of a sample with a corresponding detection limit when repeatability of the “detection limit” sample, it is advisable
the CV is 20% during the day. This is the limit of the low- to perform a longer experiment, which represents the day-
est concentration or other amount that the detection system time detection performance. Actually do 10 tests (10 days).
or method can quantitatively report the analyte. In order to
estimate FS, multiple detection limits are used to determine
the precision performance at low concentrations. The cor- 2.3.4 L
imits of Blank, Limits of Detection,
responding concentration of or closest to 20% CV is selected and Limits of Quantitation
as the value of the lowest or other quantitatively quantifiable
value. When verifying the FS declared by the manufacturer, In 2004, CLSI published the EP17-A document “Protocols
the concentration of the test limit used should be the same as for Determination of Limits of Detection and Limits of
the manufacturer’s instructions. Quantitation; Approved Guideline,” which is applicable
to all quantitative inspection items (even if the results are reagents, quality control products, operating procedures,
reported qualitatively), especially for medical tests where inspectors, etc.) and the final output of the instrument, and is
the level of decision is very low (e.g., close to 0). This pro- an important instrument performance index. The evaluation
gram is not just suitable for clinical laboratories but also for of the measurement range helps to identify sources of error
in vitro diagnostic test manufacturers. in many aspects, such as methodological principles, instru-
ments, calibrators, reagents, operating procedures, and qual-
2.3.4.1 Overview ity control plans. For the multi-point calibration test item
The classical method of determining the limits of detection is (generally five levels, if not possible, including at least three
usually to repeatedly test the blank sample and the very low different concentration levels of high, medium, and low),
concentration series of samples for a certain test item, manu- analysis and measurement range evaluation experiment is
facturer, or method to obtain the respective series of results. generally not required. At this time, analytical measurement
The test result of the blank sample is used to determine the range of the detection system is determined by the high and
critical point of a response, and the value above the criti- low values of the calibrator.
cal point is considered to contain the analyte in the sample, The College of American Pathologists (CAP) began pro-
which is a positive result. The value of this critical point in viding linear validation of some clinical testing programs in
combination with the standard deviation of the low concen- 1988 and used polynomial regression analysis to determine
tration sample is used to determine the analyte concentration linearity for data returned by each laboratory. The high-value
that is very likely to exceed the expected maximum blank liner verification products provided by some manufacturers
value. can meet the needs of clinical laboratory evaluation of ana-
lytical measurement range. When the manufacturer does not
2.3.4.2 General Method for Determining provide a commercial linear verification product, the labo-
the Limits of Blank (LoB) ratory can determine the analytical measurement range of
The discrete distribution of the blank sample and the low the method by selecting a high concentration of the patient
concentration sample test results is due to random measure- sample, and after various dilutions or preparations, compar-
ment errors. Signals with blank measurements close to zero ing the expected value with the measured value [6].
can occur inside the instrument, and many instruments auto-
matically convert negative signals to zero or less positive
values, outputting only non-negative results. Assuming that 2.4.1 Definitions
a value exceeds the percentile of the 95% distribution of the
true blank sample value, the display is significantly different • Allowable difference/Allowable error In a detection sys-
from the blank measurement. When the observed value of a tem, the amount of analysis deviation from various fac-
sample exceeds this limit, the analyte concentration in the tors that the user can afford and meet the medical
sample exceeds zero. requirements. Generally, the range of the allowable error
Using this limit, a 5% probability that the value given by of a single test is expressed by the allowable error of the
the real blank sample is considered to be the presence of the target value measured by the sample.
analyte, which is a false positive due to a type I error (or • Analyte Measurable components are called analytes.
alpha error). At the same time, it is observed that the detec- • Analytical result The final result of a measurement on a
tion value of the sample containing the low concentration test specimen; the result is usually in concentration or
of the analyte will be lower than this limit. If there is no activity units.
detectable analyte in the sample, we will make a type II error • Bias The difference between the expected value of the test
(or beta error) or false negative. Therefore, how to determine result and the acceptable reference value.
the position of the low concentration sample and the blank • Input variable The given value that is used as a reference
detection zone is critical, and the method developer should (independent variable) and against which the output vari-
reasonably set the value and the beta value according to the able is compared; the input variable is represented by X
relative cost of controlling the two types of errors. and its value is plotted along the X-axis.
• Intercept/Y intercept The point where a function inter-
sects an axis; the value of a variable, when the value for
2.4 Analytical Measurement Range the other variables is zero; the y-intercept is the value of
the y-variable when the x-variable has a value of zero.
The analysis of the measurement range, that is, the lin- • Least squares regression Refers to a statistical method
ear detection range of the quantitative detection item, is a that minimizes the sum of the squares of the distances of
performance of a constant ratio between the signals of the each data point from the longitudinal distance (vertical to
entire detection system (including instruments, calibrators, the X-axis direction) to the assumed regression line.
• Linear equation An equation representing a linear rela- • Trueness Consistency of the mean and acceptable refer-
tionship. A typical linear equation is: Y = a + bX, where X ence value of repeating test results. Note: Accuracy is
and Y are independent and dependent variables, b is the usually expressed in terms of statistical bias. It is a mea-
slope, and a is the Y-intercept. sure of inaccuracy.
• Linear range Refers to the range that covers the accept-
able linear relationship of the detection system, and the
nonlinear error is less than the set standard. 2.4.2 EP6-A Protocol and Requirements
• Linearity When testing samples, the ability to obtain the
analyte content can be directly proportional to the rela- 2.4.2.1 Experimental Requirements
tionship within a certain range.
• Linear regression A statistical calculation that results in Device Familiarization Period
the parameters that describe the assumed linear relation- Laboratory personnel who are familiar with the analysis
ship between values of an independent and a dependent procedures for instrument evaluation must be very familiar
variable wherein the independent variable is known with the operation of the instrument, quality control and cali-
exactly. bration methods, and the correct collection of samples. The
• Measurement error/Error of measurement The difference training provided by the manufacturer is very useful. The
between the measurement result and the true value (or equipment must be installed and used in the laboratory for a
recognized the reference value). period of time, and laboratory personnel can understand and
• Measurement procedure A set of operations, described operate the instrument correctly.
specifically, used in the performance of particular mea-
surements according to a given method. Note: Formerly, Duration of the Experiment
the term analytical method was used in this document. All experimental data should be collected in as short a time
• Measuring range A combination of measured values that as possible. If possible, a single analytical test should be
allows the error of the measuring instrument to be within completed within days.
a specified range. Use of the term “reportable range” in
previous CLSI documents. Specimen of the Experiment
• Output variable The dependent variable, the value of Number of Samples
which is compared to the input (independent) variable. Five measurement points are the minimum requirements
• Polynomial regression Multiple least squares regression when using a polynomial regression method to evaluate and
of different orders: analyze the measurement range. More measurement points
can more accurately evaluate linearity, and the analytical
Y a b1 x first-order polynomial or linear fit measurement range obtained is wider.
Y a b1 X b2 X 2
seccond-order polynomial , and When the validity of the analytical measurement range is
confirmed in the laboratory, 5–7 samples are required, and
Y a b1 X b2 X 2 b3 X 3 third-order polynomial
each sample is repeatedly measured twice.
When verifying the declared range or improved method,
• Repeatability (Measuring instrument) Under the same 7–9 samples are required, and each sample is repeatedly
measurement conditions, the same sample is repeatedly measured 2–3 times; when establishing the analytical mea-
measured in a short period of time. The ability of a measurement range, 9–11 samples are required, and each sample
suring instrument to repeatedly give very similar indica- is repeated 2–4 times. Manufacturers hope to have more
tions, and the repeatability can be quantitatively expressed measurement points (20–30% wider than the expected ana-
by the discrete characteristics of the indications. lytical measurement range) so that “inflection points” can be
• Standard deviation of y about a regression/Standard error detected and a wider analytical measurement range can be
of regression (SDy,x) determined. According to the degree of imprecision, each
• Measure the mean Y and the independent variables in the concentration level is measured 2–4 times.
newspaper model. The standard error of the difference Samples with high and low concentration are accurately
between the Y values corresponding to X. proportioned to samples of different concentrations at equal
• Testing system Includes the entirety of the testing process, intervals, but the same interval is not necessary. As long as
including instrument, sample, personnel, reagents, sup- the correlation between the samples is known, samples with
plies, and procedures; it also includes such attributes special concentrations are also acceptable. Sample prepara-
(method characteristics) as sample type, testing tempera- tion requires a sufficient sample volume to meet the required
ture, humidity, etc. sample dilution and measurement.
Matrix Effects Analyte Range

The type of sample used to verify the analytical measure- The selected analytical measurement range should include
ment range should be similar to that used in clinical test- or be equal to the minimum and maximum concentration
ing, and all samples should be free of interference factors ranges declared by the manufacturer. If the declared ana-
(such as hemolysis, jaundice, lipemia, etc.) calibrated by the lytical measurement range is not consistent with the selected
manufacturer. concentration range, you can choose a suitable sample con-
The ideal sample type is a patient’s sample, and all sample centration for new experiments, or round off the end point to
concentrations are prepared from samples of two analytical narrow the linear range appropriately (five or more samples).
concentrations that are close to the expected upper and lower When evaluating the analytical measurement range, there
analytical measurement range (or lower measurement limit). are several important concentrations to be aware of: the low-
Because the final concentration of the measured patient est analytical concentration or the lower limit of the ana-
sample represents a validated analytical measurement range, lytical measurement range, different medically determined
high and/or low value concentrations need to be adjusted to levels, the highest analytical concentration, or the upper limit
achieve the desired range. of the analytical measurement range.
In general, patient samples are diluted with a diluent rec-
ommended by the manufacturer or confirmed by the labo- Sample Preparation and Value Assignment
ratory. Non-recommended diluents, such as saline or other If the high and low concentrations are unknown, each tube
diluents, may affect the test results due to matrix effects. At must be numbered to determine its relative concentration.
this time, use the minimum amount of diluent. For equidistant concentration, each tube can be assigned an
When patient samples at high concentration levels integer (such as 1, 2, 3, 4, 5, etc.), that is, the concentration of
are not readily available, they can be prepared by add- each tube can be unknown before analysis. When verifying
ing analytes to patient samples at low concentration lev- the analysis measurement range, the measurement averages
els. When the analyte is free of interfering substances, the of the high and low values are used. If the concentrations of
added analyte need not be of high purity. When interfering the intermediate analysis tubes are not equally spaced, the
substances are present in the analyte, the source, purity, correlation between adjacent tubes must be known. It is also
expected impact, etc. of the analyte must be mentioned in possible to first prepare a middle concentration tube with
the report. If a concentrated solution containing the ana- high and low concentration, and then use the low and middle
lyte is added to the patient sample, add as little as possible concentration and high and middle concentration to distrib-
(less than 10% of the total volume in principle) and record ute the concentration of other tubes.
the solvent used.
Low-concentration samples can be collected directly 2.4.2.2 Analytical Sequence
from patient samples, or the pretreatment of dialysis, heat Before the analysis, the instrument calibration and qual-
treatment, chromatography, etc. can be used to reduce the ity control must be in very good condition. The analytical
concentration of analytes to the required concentration. sequence should be random, but if there is significant carry-
Note that the pre-treatment method chosen should not alter on contamination or drift, choose the order in which sub-
the physical or chemical properties of the analyte or matrix. sequent samples have the least impact. Or insert at least 2
Low-level samples can be used to prepare high-level analyte low-value samples after high-value samples with significant
samples. They can also be used to dilute high-value samples carryover contamination, but the results of the 2 low-value
or to prepare intermediate-level samples with high-value samples are not included in the statistics.
samples.
When using an aqueous solution of an analyte as a sample, 2.4.2.3 Preliminary Data Check
matrix effects may affect the response curve and interpreta- Preliminary data check data can be conveniently recorded in
tion of the results. Although high-purity analytes minimize a worksheet or a computer table program. The measurement
interference effects, slightly lower-purity materials are also results must first be comprehensively evaluated for accept-
acceptable. A large number of conventional clinical chemis- ability and effectiveness. Detailed results evaluation must
try methods are calibrated using aqueous materials, and the gradually refer to the following models:
target value can be determined by weighing.
(a) Check the data for extreme obvious differences or errors,
Selection of Materials Used to Supplement Samples and if analysis or technical problems are found and cor-
Caution must be taken when selecting additives, and the use rected, repeat the entire experiment.
of ingredients from other species can also have some effects. (b) If no discernible analysis or technical result discrepancies
The instrument or reagent manufacturer’s recommendations or errors are found, visually inspect all results for potential
also help determine the appropriate material. outliers in each analysis. Take the measurement result as
the Y value, and calculate the concentration or relative Determination of the Linear Range
concentration as the X value for the X − Y coordinate Polynomial linear evaluation first assumes that the data
graph. In the graph, each Y value has a corresponding X points are nonlinear, and on the premise that the random
value. The mean points of repeating determination of each error is small, it is assumed that the data points completely
concentration level are on the graph. These points are con- fall within a straight line or a curve. Regardless of whether
nected manually or by computer, and the approximate the optimal curve is a straight line, it does not affect (within
deviation of each point from the straight line is observed. the linear range) reliable results obtained by interpolating
This makes it easy to find outliers, obvious transcription between experimental data points at other points.
errors, or instrument malfunctions. Polynomial regression methods are used to evaluate
(c) If necessary, arrange the test data of each group in the nonlinearities, which is why polynomials are chosen. This
order of test time to check whether there is drift or trend method has two parts: The first step is to determine whether
change. If any obvious deviations are found, after correct- fitting the data with a nonlinear polynomial is better than
ing the errors, the entire batch of data must be replaced. linear. The second step is to judge whether the difference
To avoid uncorrected errors, select the “good” data in the between the optimal nonlinear model and the linear fit is
repeated measurement results for replacement. less than the allowable deviation of the method when the
Experimental data can find real problems in data points of the nonlinear polynomial fit are better than
operation. linear.
(d) Observe the difference between the measurements at Evaluation of linearity requires at least 5 samples of dif-
each concentration level. In the linear model, the slopes ferent concentrations, and each level is repeated twice. First
of the segments are approximately equal, and the increas- you need to know the concentration or the proportional rela-
ing or decreasing trend indicates nonlinearity. tionship between the solutions. Different concentrations can
(e) When a measured value (Yi) of a given concentration sig- be equally spaced or unequal (but you need to know the rela-
nificantly deviates from another Y value, a visual inspec- tionship between them). For example, the coverage range
tion can determine that it is an outlier. Outliers should be of 5 concentration levels is 20–100 mmol/L, and the other
deleted from the data. concentrations are 40, 60, and 80 mmol/L at equal intervals.
(f) If two or more unexplainable outliers are found, the per- The X value can be represented by 20, 40, 60, 80, and 100
formance of the detection system should be suspected. (or 1, 2, 3, 4, and 5).
Find the cause of the problem and ask the manufacturer Next, perform the polynomial regression analysis for
for assistance if necessary. first-, second-, and third-order polynomials. This can be
(g) Visual inspection of the X − Y scatter plot is very impor- done with a variety of commercial statistical software such
tant for subsequent linear evaluation. It can easily find as Excel, SPSS, and SAS (Table 2.1).
nonlinearity, or whether the measurement range is too The linear polynomial model is a straight line, which is
narrow or too wide, and can also choose a more suitable the optimal equation to determine whether a certain method
statistical analysis method for subsequent statistical is linear. The quadratic polynomial model represents a kind
analysis. of parabolic response curve, which has two types: an increas-
ing trend (curve up) or decreasing trend (curve down). The
Outlier Inspection cubic polynomial model represents an “S” shaped response
Outlier means that a single test result visually or statistically curve that is nonlinear at both ends of the measurement
deviates from other test results. It is only applicable to the range.
evaluation of a single repeated measurement result, and is The regression coefficient is represented by bi. In the qua-
not applicable to multiple repeated determinations or mea- dratic polynomial model, b2 is a nonlinear coefficient. In the
surement averages of a concentration level. The presence of cubic polynomial model, b2 and b3 are nonlinear coefficients.
outliers indicates nonlinearity or systematic errors. Outlier Calculate the standard deviation of the slope of each nonlin-
checks can identify the source of the error (a transcription ear coefficient (can be calculated by the regression program),
error, system instability, etc.), or speculate on the cause of and then perform t test to determine whether the nonlinear
the error. Outliers refer to a model that is not fit to other coefficient is statistically significant, that is, whether the
data and can be calculated by statistical methods. However,
in most cases, outliers can be found by visual inspection of
detected and expected values. A single outlier can be deleted Table 2.1 Polynomial regression equation expressions
without replacement in the data set. If more than one outlier Exponent Equation
appears, the detection system may have too low precision Linear Y = aX + b
and should be deleted according to standard methods. In this Square Y = aX2 + bX + c
case, the cause must be found and corrected. Cubic Y = aX3 + bX2 + cX + d
coefficient is significantly different than zero. The coeffi- clinically acceptable error, which is clinically acceptable. If
cients b0 and b1 in the linear polynomial model do not need any point DLi exceeds the set target, it means that the point
to be analyzed because they do not reflect nonlinearity. The may be nonlinear. At this time, the following two methods
test is calculated as following, for b2 and b3: are used for processing:
bi
t= • Try to find the cause of nonlinearity (in sample prepara-
SE i tion, disturbing substances, instrument calibration, etc.).

• Observe the scatter plot of the measured and expected
The calculation formula of degrees of freedom is values to determine whether the nonlinearity is at the ends
df = L·R – Rdf. L is the number of samples with different or in the middle of the concentration range of the analysis.
concentrations prepared, R is the number of repeated detec- If it is at both ends, try to round off the DLi, the maximum
tion, and Rdf is the degree of freedom occupied by regres- concentration point, and perform statistical analysis
sion analysis. In the above example, in the cubic polynomial again, which will reduce the linear range.
regression, L = 5, R = 2, Rdf = 4, df = 5·2 − 4 = 6. Check the
t-value table (both sides, α = 0.05). If there are no nonlinear EP6-A emphasizes that any user needs to determine their
coefficients b2 or b3 (p > 0.05), it is considered that there is a own linearity requirements, or a nonlinear tolerance range,
linear relationship. When the precision is good, the analysis which should be based on the needs of the laboratory cus-
is completed. If the nonlinear coefficient b2 of the quadratic tomer and the characteristics of the method used.
polynomial model, or any of b2 or b3 of the cubic polynomial Factors to be considered in setting the error target: The
model is compared with 0, and there is a significant differ- linear target is derived from the biased target, so it should
ence (p < 0.05), then the set of data is nonlinear. It should be a small child or equal to the biased target, and the biased
be noted that this is only statistically significant, only the target should be less than or equal to the measurement error.
nonlinearity is detected, and it does not represent how much
it affects the patient’s test results. Considerations for Random Error
Linear evaluation should also consider the impact of random
Degree of Nonlinearity errors (evaluated by repeatability in this protocol). Random
When nonlinearity is detected, the regression standard error errors originate from random variation (variation of the
(Sy·x) is calculated to determine the most appropriate qua- analysis system), which may lead to low nonlinear evalua-
dratic or cubic polynomial model. Sy·x is a measure of the tion ability. Repeatability is best evaluated by the collective
difference between the mean and the corresponding value of variance of all repeated measurements of the L samples. It
the model, so the smaller the Sy·x, the more suitable the model is a variation of the total measurement mean that does not
is for the data set. depend on the concentration of the analyte and is expressed as
The deviation from linearity (DL) at each concen- SDr (or CVr relative error). If the SDr between samples with
tration can be calculated by the following formula: different concentrations is approximately equal, the impre-
DLi = p(Xi) − (b0 + b1Xi). The value of x ranges from x1 to cision between the samples with different concentrations is
xS, and p(xi) is the value of the optimal polynomial regres- relatively constant and can be expressed by SDr. If the dif-
sion model at xi. Therefore, DLi is the integer value of the ference at high concentrations is large, the magnitude of the
quadratic polynomial model and the linear polynomial (lin- imprecision may change in proportion to the concentration
ear) model at each different concentration, or the difference value, and CVr should be used at this time. The magnitude of
between a cubic polynomial model and a linear polynomial the imprecision can be calculated by analysis of variance, and
(linear) model. This is a measure of the difference between the square root of the variance is the magnitude of the error.
the nonlinear model and the linear model, at each of the con- When the measurement is repeated twice, the difference
centrations measured. DLi should be the same as the target between the two measurements can be easily calculated
unit for comparison. If you want to convert to a percentage, using the following formula:
divide each DLi by the concentration value (known value) or
r ri 2
L 2
the measured average (relative concentration) and multiply i1
SDr i 1
by 100%. 2 L

Compare DLi at each concentration level with the set error
range. If DLi is less than the preset error, even if a statisti- ri1 and ri2 are the actual results of the two measurements of
cal nonlinearity is detected, because the nonlinearity error is this method, or the percentage of the mean (but pay atten-
less than the set target, the patient results are processed in a tion to the unit of each dilution concentration). If a per-
linear manner, and the introduced error does not exceed the centage value is used, CVr must be used instead of SDr.
L is the number of samples and the number of repeating used for gene amplification testing and include EDTA,
measurements is 2. sodium citrate anticoagulation whole blood, bone marrow,
If the number of repeated measurements is more than 2 serum, plasma, sputum, cerebrospinal fluid, urine, and secre-
times, the random error should be calculated by analysis of tions. In general, if the amount of the sample is sufficient
variance, and the formula is as follows: for the culture of the pathogen, the amount is also abundant
2
for nucleic acid extraction and subsequent amplification

L R
i 1
rij ri
j 1
detection.
SDr ,
L R 1
2.5.1.3 Sampling and Transport Containers
where R = number of replicates at each level (j = 1,…,R); When taking samples such as blood, disposable sterile,
L = number of levels (i = 1,…,L); closed containers such as vacuum blood collection tubes
ri = average result at level i. should be used. Whole blood and bone marrow specimens
Compare Sr with the set target of imprecision (concentra- must be anticoagulation, and the preservatives, anticoagu-
tion unit or percentage unit). If Sr exceeds the set target, the lants, and related reagent materials used for sampling should
precision may be too low to be used to truly and reliably not interfere with nucleic acid amplification and detection
evaluate the linear relationship. At this time, the instrument processes. EDTA and citrate are the preferred anticoagu-
or operation process should be checked to find the cause of lants, and heparin anticoagulation cannot be used because
the faint imprecision, and the experiment should be repeated heparin is a potent inhibitor of the Taq enzyme and is dif-
after correction. If the performance of the method is the ficult to remove in subsequent nucleic acid extraction steps.
same as the previous evaluation of repeatability, the number Blood samples for clinical amplification of RNA
of repeated measurements is doubled (4 times), which can (such as HCV RNA) are best not to use serum samples.
reduce the standard deviation of the mean by about 40%. Anticoagulation is recommended. Anti-coagulation with
EDTA should be used, and plasma should be separated as
soon as possible (within 3 h) to avoid RNA degradation. If
2.5 Variation Factors of Pre-analysis anticoagulation is not performed, serum must be separated
within 1 h after the blood draw. Glassware should be treated
2.5.1 Collection, Transport, and Preservation with high pressure before use, because glassware often con-
of Nucleic Acid Test Specimens tains RNase that is not easily deactivated, preferably heat
sterilized. Baking at 250 °C for more than 4 h can perma-
Since the collection, transport, and preservation of speci- nently inactivate RNase.
mens have a great influence on DNA and RNA, quality con-
trol before PCR analysis is very important for the accuracy 2.5.1.4 Anti-pollution in Specimen Collection
and validity of the assay results. The collection of specimens should use disposable materials,
The clinical gene amplification testing laboratory shall and the transport containers should also be sterile and closed
establish standard operating procedures for the collection of disposable devices. When using a non-closed sampling sys-
various clinical specimens according to the testing require- tem, such as urine, secretions, and callus sampling, care must
ments, and train the clinical specimen collection personnel. be taken to prevent contamination from the sampler’s dander
The time of specimen collection should be in the process of or secretions.
the development of the patient’s disease, and false negative Disposable gloves must be worn during sampling. The talc
results may occur in the specimen collection too early or too in the gloves has an inhibitory effect on PCR. Powder-free
late. gloves or pre-wash gloves should be used. In addition, the
use of disposable masks and disposable caps is also an effec-
2.5.1.1 Preparation of Specimen Collection Site tive measure to prevent contamination, and saliva and air
The cleaning and disinfection of the specimen collection site contamination specimens can be avoided.
can remove contaminated microorganisms or other debris,
but should be moderate, so the preparation of the specimen 2.5.1.5 Evaluation of Sampling Quality
collection site should be carried out by trained personnel. The quality of specimen collection can be evaluated in sev-
eral ways, namely, the composition of the cells, the number
2.5.1.2 Type and Collection of Specimens of cells of the desired type, and the total amount of nucleic
The type and collection of specimens should be based on acids. Evaluation methods include macroscopic observation,
the pathogen being tested. Clinical specimens are commonly microscopic observation, and chemical analysis.
References 4. Clinical and Laboratory Standards Institute. EP09-A2 document:

method comparison and bias estimation using patient samples;
approval guideline. 2nd ed. Wayne, PA: CLSI; 2002.
1. Clinical and Laboratory Standards Institute. EP05-A3 document:
5. Clinical and Laboratory Standards Institute. EP17-a document: pro-
evaluation of precision of quantitative measurement procedure;
tocols for determination of limits of detection and limits of quantita-
approval guideline. 3rd ed. Wayne, PA: CLSI; 2014.
tion; approval guideline. Wayne, PA: CLSI; 2004.
2. Clinical and Laboratory Standards Institute. EP05-A2 docu-
6. Clinical and Laboratory Standards Institute. EP6-a document:
ment: evaluation of precision performance of quantitative mea-
evaluation of the linearity of quantitative measurement proce-
surement methods; approval guideline. 2nd ed. Wayne, PA:
dures: a statistical approach; approved guideline. Wayne, PA:
CLSI; 2004.
CLSI; 2003.
3. Clinical and Laboratory Standards Institute. EP15-A2 document:
user verification of performance for precision and trueness; approval
guideline. 2nd ed. Wayne, PA: CLSI; 2005.
Establishment of Biological Reference
Interval 3
According to the International Organization for physiological or pathological conditions, taking into account
Standardization ISO 15189/5.5.5, the biological reference the source of the sample. This document is prepared for the
interval should be reviewed periodically. If the laboratory determination of any type of reference interval as it takes
has reason to believe that a particular reference interval is no into account the selection of appropriate reference individu-
longer applicable to the reference population, it should inves- als and the effects of various factors prior to analysis. This
tigate and, if necessary, take corrective measures. When the document also points out that the establishment of the multi-
laboratory changes the test procedure or the pre-test proce- center reference interval can be used to integrate the data of
dure, the biological reference interval should also be re- different sub-centers to establish a reference interval if the
evaluated if applicable. Therefore, in actual clinical measurement method of the analyte has better traceability,
applications, in addition to determining the reference range thereby reducing the burden of collecting 120 reference indi-
for new test methods and test items, it is necessary to periodi- viduals in each laboratory. The Robust statistical method
cally review the test methods and items that have been introduces Robust, a modern statistical method. When the
applied to the clinic or verify them with appropriate proce- number of reference individuals is limited (minimum 20
dures. If not applicable, it must be re-established. Therefore, cases), a reference interval with confidence can also be
the establishment of a reliable biological reference interval established. Now, more and more laboratories are currently
for upcoming and ongoing testing projects is one of the using commodity test kits or test systems, and the reference
essential tasks that need to be accomplished in pre-clinical value data provided by the manufacturer or other laborato-
and clinical applications. ries can be used directly and requires clinical laboratory
validation.
3.1 Biological Reference

3.1.1 Definitions and Terms
The biological reference interval is intended to guide the
clinical laboratory, diagnostic instrument reagent manufac- Before the establishment of the biological reference interval,
turer, and clinical laboratory personnel to determine the we must understand the definition of some of the following
extent to which a test result is at a certain level. In 2008, the terms. The definitions of these terms were proposed by the
American Society for Clinical and Laboratory Standards EPTRV chapter of the International Federation of Clinical
published the “Guidelines for How to Define, Establish, and Chemists (IFCC) and the International Hematology
Verify Participation Approvals for Clinical Laboratories- Standardization Committee (IFSH) and have been widely
Third Edition (CLSI C28-A3).” The document emphasizes recognized by the World Health Organization (WHO) and
that the selected sample set should be derived from a healthy other organizations around the world.
population when determining the reference value or refer-
ence interval. Similar methods can be applied to the estab- • Reference individual According to the clearly defined
lishment of reference values in other types such as selection (incorporation) criteria, the individuals
participating in the experiment are selected, and the
corresponding reference values can be obtained from the
L. Wang (*) · Z. Shi test. Normally, the criteria for “healthy people” are diffi-
Department of Medical Experimental Center,
General Hospital of Ningxia Medical University, Yinchuan, cult to formulate, but exclusion criteria for unhealthy fac-
Ningxia Hui Autonomous Region, People’s Republic of China tors can be established.

• Reference population A group of all reference to individu- 3.1.2 Clarifications

als. Note: The reference group usually has an unknown
number of members, so it is a hypothetical entity; the ref- For different purposes, the reference value can be for a rela-
erence group may have only one member. tively healthy population, or it can be related to other physi-
• Reference sample group Inadequate number of persons ological or pathological conditions. In any case, the reference
selected to represent the reference population. In a statis- value allows us to compare the observed data with the rele-
tical sense, a certain number of reference individuals are vant reference data from the identified test population. This
a sampling group in the reference population, and the comparison can be used as part of the decision-making pro-
establishment process of the reference interval is a spe- cess regarding the meaning of observations and the state of
cific study of the reference sample group. the subject.
• Reference value A value or measurement obtained by Reference values are all values observed or measured on
observing or measuring a certain number of reference all reference individuals in a reference sample group. The
individuals of a particular type. The value obtained by reference interval is usually between the reference limit of
performing a project on a reference individual is the value the determined percentile. That is, the reference interval will
of the individual, and a set of reference values can be refer to the set of observation interval values obtained by
obtained from the reference sample group. The reference defining a specific percentage (e.g., 95%) in the reference
value reflects the fluctuation state of the functional indica- sample group or the predicted reference population.
tor data of human anatomy, physiology, and biochemistry
in a healthy state.
• Reference distribution The distribution of reference 3.2 stablishment of Biological Reference
E
values. Interval
• Reference limit The threshold value obtained from the ref-
erence distribution is used to describe the position of a 3.2.1 P
rotocol Outline for Obtaining
partial reference value, such as less than or equal to the Reference Values and Establishing
upper limit, greater than or equal to the upper limit. The Reference Intervals
reference values are arranged from small to large. For
most analytes, the lower limit of 2.5% of the value distri- 3.2.1.1 New Analyte or Analytical Method
bution can be referenced, with a high limit of 97.5%. When testing new analytes or using new analytical methods,
When only the one-sided reference limit has clinical sig- the following procedures must be used to develop reference
nificance, it can be determined that 5% or 95% is the ref- values and to make reference intervals:
erence limit.
• Reference interval The interval between, and including, • According to the medical literature, establish the corre-
two reference limits. According to the distribution char- sponding biological variability and analysis of interfering
acteristics of the reference value and the clinical use substances inventory (if it is a new analyte, the role of
requirements, an appropriate statistical method is literature is not great, pre-experimental research on inter-
selected for the inductive analysis to determine a part of fering substances is required).
the reference distribution as the reference interval. The • Establish selection (or exclusion) and grouping criteria to
commonly determined percentage ranges between 2.5% identify reference individuals.
and 97.5%. Clinical test results are considered “nor- • When conducting a reference interval study, reference
mal” if they are within a defined reference interval and individuals must complete a questionnaire and sign a
are considered “abnormal” beyond the reference written informed consent.
interval. • According to the results of questionnaire surveys and
• Observed value (patient laboratory test result) A value other corresponding health assessments, potential refer-
obtained by observing or measuring a particular type of ence individuals were classified.
sample from a subject (such as a patient). This value can • According to the expected reference extreme value, the
be used clinically to compare with reference value, refer- corresponding number of reference individuals is
ence distribution range, reference limit, or reference determined.
interval. • Select the appropriate individuals for specimen collec-
tion, and correctly collect and process biological speci-
Figure 3.1 demonstrates the relationship between the mens in accordance with the usual operation methods of
terms defined. patient specimens.
3 Establishment of Biological Reference Interval 29
Fig. 3.1 Relationship
between terms
• Under certain conditions, according to the operation the reference limits and reference intervals (if appropri-
method of conventional patient specimens, the ate, the reference intervals can be ranked).
corresponding analysis methods are used to analyze the • Record all the steps and procedures above and keep them
specimens to obtain reference values. on file.
• Review the reference values, prepare histograms, and
evaluate the distribution of the data. The above operation process is based on a priori sampling
• Identify and eliminate possible erroneous data and method that first selects reference individuals, determines
outliers. reference values, and then conducts laboratory inspections.
• Organize and analyze reference values. For example, In practice, when the inspection group is a potential refer-
choose a method of judgment and estimation to estimate ence individual who is expected to be healthy, completing
the questionnaire and collecting samples are often performed from the reference sample is the first step in selecting refer-
simultaneously. Once an exclusion is found, the individual’s ence individuals. Each institution or researcher has its own
analytical measurements should be cancelled. health standards. Before further experiments, these standards
When selecting a reference individual, it is important to need to be defined in what state a person is considered
distinguish between control and uncontrollable biological healthy. The first step in selecting a reference individual is to
variation. Some factors can be controlled by standardized establish a standard that excludes non-healthy individuals
procedures during the preparation of the collection of refer- from the included reference sample group; of course, each
ence individuals. Other factors, such as age and gender, can institution or researcher may have different understanding of
be used as relevant statistical criteria for classification. health standards, but these standards should be defined
Additional sources of variation should be fully considered before analysis. Assessing the health of a candidate reference
when defining the selection criteria for a reference individual may require multiple tests, including medical his-
individual. tory investigations, physical examinations, and/or certain
Small-scale studies are most appropriate using a priori laboratory tests. The health standard used as the reference
method. Clinical consultations and examinations are required value study should be clearly described and recorded, and at
for potential reference individuals, and they are selected by least each reference individual should be investigated and
laboratory methods to determine whether they meet the spec- recorded in the form of a health assessment questionnaire in
ified inclusion criteria. If the conditions are met, samples are order to evaluate the health status of the included reference
collected using standard procedures for analysis. individuals.
The corresponding a posteriori method requires the same
series of operational procedures, except that the order of 3.2.2.1 Exclusion Criteria
some steps is different. It is built on a large number of medi- Exclusion criteria should be described in detail, and individ-
cal examination individuals and test data. For this method, a uals who are supposed to meet the exclusion criteria among
large-scale sample laboratory test is performed first, and then supplemental reference individuals must be excluded from
the data is summarized and a reference value is established. the replacement reference sample. Examples of some poten-
It also needs to take into account the specific individuals and tial exclusion criteria may be noted in Table 3.1.
their respective detection values when making reference val-
ues, but only after the tests are performed. 3.2.2.2 Partitioning Criteria
The grouping standard has the characteristics of grouping
3.2.1.2 Previously Measured Analyte reference samples from all selected reference individuals
For analytes that have established a valid biological refer- according to their respective classification methods. The
ence interval, under appropriate circumstances, valid refer- most common grouping criteria are age and gender. In addi-
ence intervals established by other authoritative laboratories tion, it is important to emphasize that exclusion criteria in
or manufacturers can be converted, and the reference interval one study may become grouping criteria in other studies [1].
of this laboratory can be obtained without conducting a new
reference interval study. 3.2.2.3 Selection of Reference Individuals
In this process, reference can be made to the NCCLS doc- The reference individuals for determining the reference
ument EP9-A2, using patient samples for method compari- interval for a relatively healthy population need not necessar-
son and bias estimation, and verifying the comparability of ily be young adults; they can be very similar to the patient
the analytical detection system. It may be necessary to make population being medically evaluated. In fact, the
a brief assessment to validate the transferred reference Subcommittee objected to the clear “gold standard” concept
interval. of young and healthy adults in general, and suggested that
Table 3.1 Examples of possible exclusion criteria and partitioning

3.2.2 Selection of Reference Individuals factors
Exclusion criteria Partitioning factors
This section offers guidance and suggestions on how to
Alcohol abuse Age
select reference individuals from a reference population to
Fasting or nonfasting Sex
form a reference sample group. It mainly discusses two dif- Obesity Race
ferent sample extraction techniques (speculative method and Genetic factors Geographic location
induction method). It will also discuss the definition of Pregnancy Diet
exclusion and grouping and give a sample survey form. Abnormal blood pressure Smoking
Therefore, how to define health is the first problem faced Vitamin abuse Period of the menstrual cycle
in any research, and how to exclude non-healthy individuals Recent illness Exercise
the reference interval for age factors could in many cases be Table 3.2 Sample questionnaire
more clinically applicable. Some changes in laboratory test All information will be kept strictly confidential and used only for
results due to changes in age cannot be used to assess health disease diagnosis.
status, e.g., cholesterol or endocrine changes in elderly Subject ID Sample ID Phone
patients. The reference individual should not be an inpatient Address
or clinical patient unless absolutely necessary. Studies such Age Sex: (M) (F) Race
Height Weight Occupation
as pediatrics or the elderly may be necessary.
Do you think you are in good health? (Y) (N)
Transcendental and posterior methods are two common
Do you exercise regularly? (Y) (N)
methods for selecting reference individuals from a reference If be, how many hours of exercise a week?
population. When sampling using a priori method, clear How much activity? (light) 1, 2, 3, 4, 5, 6, 7,
exclusion and grouping criteria need to be established before 8, 9, 10 (heavy)
selecting reference individuals. It is more appropriate for Do you have high blood pressure? (Y) (N)
Are there any hereditary diseases in your (Y) (N)
methods that have been studied in detail and developed labo-
family?
ratory procedures. After developing the method, a compre- If yes, describe
hensive search of the literature is needed to identify various Have you been feeling sick recently? (Y) (N)
known biological variations. Diverse information from the If so, when?
literature was subsequently transformed into exclusion and For what reason?
Have you been hospitalized recently? (Y) (N)
grouping criteria for the current study. After the standard is
If yes, why?
established, a questionnaire is usually conducted during the When?
admission process to exclude certain individuals from the Have you ever taken any medicine (Y) (N)
sampling process and exchange selected individuals into cor- prescribed by a doctor?
responding subclasses. Before collecting a blood sample, all If yes, what?
Do you take any vitamin? (Y) (N)
the above processes need to be completed. In the process, it
If yes, what?
is necessary to select an appropriate reference individual to Have you taken any analgesics like aspirin (Y) (N)
meet the statistical requirements. recently?
During a posteriori sampling, corresponding exclusions If yes, what?
and groupings are also required, but the order of execution is Have you taken any cold or allergy medicine (Y) (N)
recently?
not the same (e.g., it is a questionnaire after sampling and
If yes, what?
analysis testing). A posteriori method is important for labo- When?
ratory operations that are new or have not been thoroughly Are you taking any diet pills? (Y) (N)
studied and have very little information in the literature. Do you use tobacco? (Y) (N)
Since we did not know the subgroup grouping criteria at the If yes, when begin?
beginning, when using this method to conduct a question- How many a day?
Do you eat a special diet? (Y) (N)
naire, it is necessary to include more detailed content than in If yes, please describe
the prior survey method. Do you drink alcoholic beverages? (Y) (N)
If yes, what?
3.2.2.4 Sample Questionnaire How much a day?
This document takes Table 3.2 as an example. In order to For women
protect the privacy of respondents, it is important that the Are you still menstruating? (Y) (N)
If have, last menstruation time?
information and test results involved in the questionnaire are If not, are you on hormone replacement
kept confidential. There are some changing factors such as therapy?
name, address, and contact phone number, which must be Are you still breastfeeding? (Y) (N)
fully considered. In this way we can more easily get in touch Are you pregnant now? (Y) (N)
with the reference individual if they find that there is some If yes, what is your due date?
Are you using oral or other methods of (Y) (N)
abnormal trend in the reference individual. There is no doubt
contraception?
that with a positive medical diagnosis, we have the obliga-
tion and responsibility to inform the person or his or her doc-
tor. In such cases, laboratories should establish an appropriate laboratory, so that the laboratory can decide whether to fol-
medical evaluation and confidentiality notification mecha- low up in case of any problems in the test.) Of course, anony-
nisms. But sometimes using anonymous surveys may be a mous investigations have more unpredictable problems.
better way to obtain certain required data, but then a number- Especially in the case of sampling research using speculative
ing system is used. (Note: As an anonymous reference indi- methods, another possible change must be paid attention to,
vidual, it is the responsibility to keep in touch with the that is, through exclusion and grouping, to classify issues
that will definitely affect the study such as the disease state. Table 3.3 Pre-analytical factors for consideration
Questions aimed at revealing information about the state of Specimen
the disease known to affect the test under study should be Subject preparation Specimen collection processing
included. Fasting and Environmental Specimen
nonfasting conditions transportation
The laboratory shall obtain written informed consent of
Rest time before Body posture (lying and Specimen storage
each reference individual in a timely manner. The consent collection standing)
shall clearly state that all laboratory personnel have the right Sampling time Sampling time Specimen
to obtain samples and to use relevant laboratory test data and pretreatment
investigation information to determine the reference interval. Drug factors Sampling site
The questionnaire and informed consent usually conducted Stress Sampling technique
at the same time. The questionnaire, informed consent, and Physiological Additives and blood
period collection tube
the nature of the study must be reviewed by the institution’s
Internal Review Board or Human Subjects Committee.
3.2.4 Analysis of Reference Values
3.2.3 Pre-analytical and Analytical The reference interval refers to the interval between two
Considerations numbers (inclusive), that is, the interval between all numbers
between the highest and lowest limit values. The individual
The analysis results obtained from the reference group must test values drawn from the reference population can be eval-
reflect all the variables before and during the analysis that uated with a specific percentage (usually 95%), which means
can affect the test results. Therefore, all pre-analytical influ- that 95% of these test values belong to this interval. For most
encing factors, including the preparation of the test subject, analytes, values below the lower limit and above the upper
sample collection and processing, the analysis methods, and limit are considered to be distributed outside the 2.5th and
instrument operation conditions must be carefully specified. 97.5th percentiles, respectively. In some cases, it makes
And whether it is for patients or for research reference indi- sense to have only one reference limit, usually the high limit,
viduals, they must be implemented equally. the 97.5th percentile.
It is important to control pre-analytical factors. In other The two commonly used statistical methods to determine
words, the factors that influence clinical decision-making these reference limits are nonparametric and parametric pro-
must be minimized. Therefore, for a specific analyte, refer- grams. These steps are described in detail in the EPTRV file
ence intervals for each group should be established. by Solberg [2–5]. The non-parametric methods do not require
These pre-analysis scenarios provide the basis for our special mathematical table to evaluate the probability distri-
grouping, and different reference intervals need to be deter- bution of the observed reference value. In practice, there are
mined in different situations. Of course, if the laboratory and parameter methods, hypothetical reference observations, or
clinician can control certain situations before the analysis, some values converted from mathematics according to a
there is no need to determine the reference interval in these Gaussian (i.e., “normal”) distribution curve. Because the ref-
different situations. However, in some emergencies, a pre- erence values of most analytes do not follow the Gaussian
determined standardization situation may not be applicable arrangement, they need to be converted into some measure-
at all. Therefore, it is important to understand such as how to ment units when using the parametric method, that is, they
interpret the results reasonably when the test sample deviates are “normalized.” Of course, you should choose the most
from standardized test conditions. appropriate conversion form (such as logarithmic form,
Generally speaking, there are two kinds of influencing power form, or some other original scale function) according
factors before analysis, named biological factors and meth- to your needs. Then check on this new scale whether the ref-
odological factors. Biological factors include metabolic and erence value really follows the Gaussian distribution. This
hemodynamic causes. Potential cell destruction processes involves the corresponding comprehensive statistical theory
(from physical exercise to venipuncture) must be considered. and related computer programs.
Subjects taking drugs that lead to induced enzyme produc- The non-parametric method is much simpler, as long as
tion should be excluded. Pre-analytical methodological fac- the order of reference data increases from small to large. In
tors involve the collection and treatment of samples. The addition, the first consideration for the establishment of a
contents to be considered include specimen collection tech- reliable reference interval is to select the appropriate refer-
nology, additives, and the order of filling the tubes (for blood ence subjects, and the number of tests should be sufficient to
samples). Table 3.3 summarizes the critical factors to be con- prevent errors before analysis, and to evaluate the reference
sidered regarding subject preparation. interval from the observation data without statistical meth-
ods. Therefore, non-parametric methods are recommended, is not particularly prominent when using the nonparametric
although parametric methods using statistical and computer method). It is possible that most reference values can meet
technology should be convenient for laboratories, if needed. this requirement, but one or two reference values may show
different probability distributions. When these values are in
3.2.4.1 Minimum Number of Reference Values the middle of other values, it is difficult to distinguish them
Only when at least n = (100/P) – 1 observations are obtained unless the person performing the biochemical analysis hap-
can the two percentiles of a distribution be distinguished pens to know that these observations represent an atypical
from P%. The main reason for this is that the non-parametric distribution or that they are caused by a problem with an
method is only based on the rank of the observations (in algorithm or program. But usually these reference values are
order of magnitude) and ignores the detection values. For not in the range of the retained test results, and it is easy to
example, if 9 observations are randomly taken from a certain identify them as “outliers” that require special attention.
population, only 9 percentile estimates can be obtained based Unless we have learned that outliers are abnormal obser-
on the 9 ranks when the ranks are arranged in order of vations (e.g., due to analysis errors or errors in quality con-
magnitude. The minimum observation value accounts for trol before object analysis), we should keep these irrelevant
10% of the total non-parametric evaluation, and the maxi- items instead of deleting them. When the extreme values are
mum observation value represents for 90%. Therefore, deleted, the reference limits evaluated using the non-
according to the formula, an example of 9 observation results parametric method and at least 120 observations change only
[9 = (100/P) − 1, where P = 10.0] indicates that if the non- slightly or not change at all.
parametric method of the population decile is required to There are many statistical methods available for testing
obtain the results, the minimum sample size required is outliers. Most test methods assume that the reference values
defined as 10% of the population’s percentiles. conform to a Gaussian distribution. In addition, if the outli-
Similarly, when the 2.5th percentile is evaluated from the ers are the extremum of individual test, it is possible to hide
5th percentile, or the 95th percentile is evaluated from the the outliers in non-extreme conditions. In the reference value
97.5th percentile (i.e., P = 2.5), the minimum number of tests evaluation, Dixon proposed a test method, namely, the ratio
should be 39.Using non-parametric methods, the minimum D/R, where D is the difference between the observed extreme
sample observation will be the 2.5th percentile of the popula- value (maximum or minimum) and the second largest (or
tion, and the maximum sample observation will be the 97.5th minimum) observed value, and R is the range between all
percentile of the population. observed values including the extreme value.
Of course, it is not advisable to determine the 95% refer- Reed and colleagues suggested taking the value range 1/3
ence interval for non-parametric evaluation entirely based on as the intercept value, that is, if the observed value D is equal
the extremes of the observed values. This is not normal and to or greater than 1/3 of the range R, the observed extreme
does not represent the true percentile value of the population. value can be deleted [6]. When the sample size is 120, this
Reed and colleagues [6] suggested that at least 120 observa- standard is too conservative, as pointed out by Reed and col-
tions should be obtained from each group of reference leagues. In other words, it may be impossible to exclude out-
objects. Its advantage is that we can use the non-parametric liers that do not match the distribution of the remaining
method to calculate the 90% confidence limit of each refer- observations. If there is no evidence that the outliers are
ence extreme value. At the 95% confidence limit, 153 refer- anomalous observations, and they are assumed to be not
ence values are needed to evaluate the reference extremes at completely consistent with the Gaussian distribution, then
the same percentile, and at the 99% confidence limit, 198 the 1/3 law of D/R can be applied, especially when the refer-
reference values are needed. ence interval is determined by using non-parametric meth-
It may be difficult to collect 120 individuals from certain ods. Therefore, we support this test method and cut-off
groups when reference individuals (e.g., newborns, children, values when testing the statistical significance of outliers in
elderly) are not readily available. If possible, sufficient refer- the observed reference values.
ence individuals of similar age are allowed to be studied. No When there are two or three outliers on the same side of
matter how many reference values are obtained, the data still the distribution (i.e., all outliers are maxima or minima), then
need to be analyzed according to non-parametric methods the 1/3 law (or a method similar to the D/R rule) cannot be
and reported in appropriate percentiles. used to indicate the minimum extreme value. The outliers are
statistically significant and will mask other outliers with less
3.2.4.2 Treatment of Outlying Observations extreme values. In this case, we should use the 1/3 law to
An important inherent assumption in the evaluation of refer- calculate the minimum extreme outliers and assume that it is
ence limits is that all reference values measured are homoge- the only outlier. If this method can be used to reject this out-
neous observations. All other values come from the same lier, other outliers will naturally be excluded. If the minimum
probability distribution (even so, the form of the distribution extremum cannot be rejected, then all extremums can be
accepted, or a test that includes all outliers can be used. This This can be done in two steps. First, approximately 60 sam-
test is called the zone method. After rejecting any outliers, ples are taken experimentally in each subgroup. When there
we also need to test the remaining data to find additional are two subgroups (e.g., men and women or two age groups),
outliers. the standard significance test can be used to test the statisti-
cal significance of the difference between the groups.
3.2.4.3 Partitioning of Reference Values x1 x2
Before acquiring and analyzing the subject specimen, the z 1
,
possibility of establishing independent intervals for the sub- s12 s22 2
groups in the subject needs to be considered. Only on the

n1 n2
basis of clinical utility and/or physiological can independent
reference intervals be established for gender or age groups. where x1 and x2 are the mean of the observations of the two
Of course, for new analytes, we do not have the necessary subgroups, s12 and s22 are the observed variance, and n1 and
information to determine these issues. However, if these con- n2 are the parameter numbers of each subgroup, respectively.
ditions are met, then at least 120 subjects per gender, age, or Assume that each industry group has at least 60 objects.
other subgroup need to be sampled. Since the z-test is essentially a non-parametric test, it can be
In general, we do not assume that as long as the difference used directly on the original data without considering
between the observed mean values of the two subgroups is whether the values conform to the Gaussian distribution.
statistically significant (5% or 1% probability level), then However, if the data is highly skewed and only requires a
each subgroup has an independent reference interval. simple transformation, such as a logarithmic transformation,
However, whether the observed difference is of clinical sig- then a numerical distribution form similar to a Gaussian dis-
nificance or not, as long as the sample size is large enough, tribution can be generated, and then we can use the z-test for
the significant statistical test can be performed. Sinton and the transformed data.
colleagues suggested that independent reference intervals The calculated statistic z should be compared with a “crit-
need to be established only if the difference between the ical” value
mean values is at least equal to 25% of the 95% reference
z 3 naverage / 120 3 n1 n2 / 240
1/ 2 1/ 2
interval of all samples [7]. .

However, research by the Expert Group has shown that
smaller differences between subgroups can lead to situations In addition, the larger standard deviation, for example, s2,
where the proportion of exceeding the upper limit of the ref- should be checked to see whether it exceeds 1.5s1, or, equiva-
erence extreme (ungrouped) or lower than the lower limit of lently, whether s2/(s2 − s1) is less than 3.
the reference extreme in each subgroup is not 2.5 on each For example, suppose at the end of the first stage of col-
side % [8]. This situation indicates that the sensitivity and lecting samples, the reference value of each group is 60.
specificity of a subgroup are significantly different from the Then, if the calculated z value exceeds z* = 3(60/120)½ = 2.12,
overall sensitivity and specificity of the population, which or if the larger standard deviation exceeds 1.5 times the
hinders our use of laboratory results as part of the diagnostic smaller standard deviation, each group should continue to
procedure. sample and extend the reference individual to 120. Repeat
However, this also happens when the subgroups have the the z-test and standard deviation comparison. If the average
same mean and a standard deviation ratio of 1.5 or more. In number of reference individuals in each group is 120, then
this case, the wider distribution (subgroup) will significantly z* = 3. If the average number of reference individuals in each
exceed both sides of the narrower distribution. However, this group exceeds 120, the threshold value of the test statistic z
ratio rarely occurs. In the vast majority of cases, the standard will be greater than z* = 3.
deviation of the subgroups is roughly the same, although the For example, if the average number of reference individu-
difference of the mean values is statistically significant. After als in each group is 500, the threshold value z* = 6.12. At this
considering these studies, the expert group made the follow- time, if the z value exceeds z*, or the larger standard deviation
ing recommendations. First, before the actual sampling of exceeds 1.5 times the smaller standard deviation, then no mat-
the reference object, the possibility of establishing a refer- ter what the z value is, it is assumed that the difference between
ence interval for the subgroup of the analyte needs to be con- the two reference intervals has clinical practical significance,
sidered. At the same time, the physiological information and the reference interval of each group must be calculated. If
associated with each analyte and the potential use of each this is not the case, then only the total reference interval of the
subgroup interval in clinical work need to be evaluated. two groups of reference individuals needs to be calculated.
If the evaluation results indicate that there are differences In order to compare the mean of 3 or more groups, it is
between the groups and have clinical significance, at least recommended to use analysis of variance (ANOVA) for
120 reference objects need to be sampled for each subgroup. statistical processing. However, the statistical difference

between the means of all groups depends on the difference possible outliers, the previously proposed Reed 1/3 rule can
between the mean of the two groups or the difference be used here. Any significant outliers should be removed and
between the mean of one group and the rest of the groups. replaced with new patient specimens until 20 test results are
Therefore, the F-test of analysis of variance must be obtained without outliers.
performed a pairwise comparison between the means at the The receiving laboratory may use the 95% reference limit
same time (such as Tukey’s test). This can ensure that all reported by the manufacturer or laboratory when not more
these tests maintain a high probability of detecting true dif- than two of the values of the 20 test subjects (10% of the test
ferences at the probability level of 0.05. It must be noted, results) exceed the result limit of the initial report. If more
however, that if any difference between the two means is sta- than three test results exceed the reference limit, then 20 ref-
tistically significant, it must be retested with a more stringent erence specimens similar to the first one need to be collected
z-test. without any outliers. If the result of exceeding the manufac-
turer’s or experimental report’s reference limit is within two,
the receiving laboratory may use the manufacturer’s or pro-
3.3 erification of Biological Reference
V vide a reference interval as the laboratory reference limit.
Interval However, if more than three results exceed this limit, the user
should recheck the analysis steps used, taking into account
For the same or comparable analytical systems, there are the possible differences in biological characteristics between
three basic methods that can be used to assess the acceptabil- the two groups of samples taken, and whether the receiving
ity of the reference interval conversion process. laboratories should develop their own reference intervals
By examining factors related to the initial reference value according to holistic research guidelines. This method
study, a more subjective assessment of the acceptability of requires the receiving laboratory to use comparable or identi-
the conversion can be made. When conducting this assess- cal analytical methods to test the 20 selected subjects. If less
ment process, the geographic and demographic factors of the than two cases are exceeded, the manufacturer’s or labora-
reference population need to be fully explained for the tory’s reference limit can be acceptable. This method can be
review. In addition, the detailed steps prior to and during the statistically verified through a binomial distribution.
analysis, the performance of the analysis, all reference val- It is also possible to assess and determine the acceptabil-
ues, and the method of evaluating reference interval need to ity of conversions by accepting laboratories to test with more
be explained. If the laboratory staff judges that these factors reference individuals (n = 60) in their own target population
are consistent with the operation of the receiving laboratory and comparing them with large-scale initial research refer-
and the test subject population, no confirmatory study is ence values. Here, the pre-analytical and analytical factors of
required in the laboratory, and the reference interval can be the preliminary reference value study must be consistent
directly converted. with the operating procedures of the receiving laboratory. In
In addition, the user or receiving laboratory may wish to addition, if the geographic location and demographic factors
verify the reference interval conversion by the manufacturer of the two groups of people are significantly different, and
or other laboratories. A small number of reference individu- the reference values will be different, the conversion of the
als (n = 20) in their own test population can be tested in the reference interval cannot be performed.
laboratory, and these reference values can be compared with The selection of reference individuals and the acquisi-
large initial studies. Here, the analysis of the initial reference tion of reference values follow the discussion in this chap-
value study and the pre-analysis factors must be consistent ter. After taking appropriate data to check and remove
with the operations of the receiving laboratory. In addition, if outliers, a comparison of the reference value of the smaller
it is known that the geographic location and demographic sample with the reference value of the relatively larger
factors between the two groups are known to be significantly original sample reported by other laboratories is performed.
different, and the reference values are different, the reference See if there is a possibility of grouping in the reference
interval cannot be converted. When conducting a transforma- population as a whole, and then process both sets of refer-
tion validation study, you must select reference individuals ence values in the same way. If this assessment does not
and obtain reference values in accordance with the above find a significant and significant difference (grouping dif-
guidelines. These 20 individuals can represent the healthy ference) between the reference values reported by other
population of the receiving laboratory and meet the corre- laboratories and the reference values of the receiving labo-
sponding exclusion and grouping criteria. According to the ratory’s concise test, then the reported reference interval
corresponding requirements, after testing 20 specimens, the can be invoked. However, if significant differences are
test results need to be tested to ensure that they can represent found in accordance with the packet agreement, further
a result group with the same statistical significance, and to sampling is required for comparison, or full-scale reference
ensure that none of the results are outliers. In order to test for value studies are used directly.
Others say that there may be a more general solution that samples, and the overall size of the subgroups. The pre-
can be used to solve the problem of calling the reference analysis situation of the study should be recorded, as well as
interval between different analysis systems. Reference val- details of the analytical method, non-stick density and inac-
ues can be called as long as the “real” biological reference curacy, and statistical methods used in the analysis. For sub-
distribution is determined. In order to obtain a “true” biologi- group reference values where the number of subjects is
cal distribution status data, we need to consider the color insufficient (newborns, etc.), the percentiles and number of
effects of local methodological bias and imprecision to observations should be reported, and sometimes all values
adjust or modify the reference value distribution reported by and full ranges need to be reported.
other laboratories. The receiving laboratories make reverse
adjustments to their respective analysis systems to obtain
reference distribution data. It is believed that as long as the 3.4.2 Manufacturer Presentation
mathematical relationship between the two methods is prop-
erly described, even if the units of measurement are differ- Manufacturers of laboratory equipment, especially manufac-
ent, this scheme can guide the call of reference intervals turers of data management systems, should be able to pro-
between different methodologies. However, such a scheme vide the ability to print reference intervals and patient-related
has not been fully explored, and further research is needed in demographic information. Manufacturers of quantitative
the future. diagnostic test equipment and reagents should be able to pro-
vide the corresponding reference interval information (oper-
ating manual and packaging instructions) on the product
3.4 escription of Biological Reference
D label. In this appraisal, the manufacturer needs to refer to this
Interval document and make users aware of the basic standards of
reference sample size, quality control of various factors
3.4.1 Laboratory Presentation before and during the analysis, and statistical processing
methods, which must meet the requirements of this docu-
Each clinical quantitative test result is accompanied by a cor- ment. If the test has been thoroughly studied and has a well-
responding reference interval. Reports that include multiple known subgroup factor, the manufacturer should provide a
test results should highlight results beyond the reference reference interval for each subgroup. Manufacturers should
interval in some way. The reference interval used should indicate whether differences between subgroups have been
reflect the subgroup grouping, which is important for tested for the most common grouping factors, such as gen-
laboratory-specific reference populations. Labeling the der, age, fasting/nonfasting, date time, pregnancy, and
patient’s results in the report is very useful to reflect the rela- posture.
tionship between the results and the reference interval. The The geographic distribution of the reference population
term “reference interval” should be used instead of normal, used by the manufacturer must be similar to that of the sales
regular, or expected. You can also choose to print High or market population. It is important to recognize that there are
Low next to the results. differences among subgroups of reference individuals in dif-
Printing the reference interval of all subgroups in advance ferent regions, which are manifested not only by regional
can lead to confusion in the report results. A good solution is differences but also by environmental, dietary, and ethnic
that the computer or instrument prints only the reference factors. In order to support the conversion process, it is nec-
interval corresponding to a specific patient. In most cases, essary to prepare the details of the combined items in the
the reference interval for a subgroup can be determined reference interval for query.
based on the age and gender of the patient. Any report that
uses a subgroup reference interval requires the grouping of
patients in the title of the report or in the patient demograph- References
ics section.
A document detailing the reference population and refer- 1. Clinical and Laboratory Standards Institute. C28-A2 document:
ence interval study should be provided to all laboratory ser- how to define and determine reference intervals in the clinical labo-
ratory; approved guideline. 2nd ed. Wayne, PA: CLSI; 2000.
vice users. This laboratory will need to be updated after any
2. Solberg HE, Petit Clerc C. Approved recommendation (1988) on
changes affecting the use of the reference interval by the the theory of reference values. Part 3. Preparation of individuals and
laboratory. A memo describing the change in the reference collection of specimens for the production of reference values. Clin
interval should be sent to all users of the laboratory slaugh- Chim Acta. 1988;177:S3–11.
3. Solberg HE, Stamm D. IFCC recommendation: the theory of refer-
ter. This should include the number and geographic factors
ence values. Part 4. Control of analytical variation in the produc-
of the reference individuals, the health assessment criteria tion, transfer and application of reference values. J Automat Chem.
used, the exclusion and grouping criteria for the reference 1991;13:231–4.
4. Solberg HE, Stamm D. International Federation of Clinical

6. Reed AH, Henry RJ, Mason W. Influence of statistical method
Chemistry (IFCC) IFCC recommendation. The theory of reference used on the resulting estimate of normal range. Clin Chem.
values. Part 4. Control of analytical variation in the production, 1971;17:275–84.
transfer and application of reference values. Ann Biol Clin (Paris). 7. Sinton TJ, Cowley DM, Bryant SJ. Reference intervals for calcium,
1991;49:487–90. phosphate, and alkaline phosphatase as derived on the basis of
5. Solberg HE. Statistical treatment of reference values in labora- multichannel-analyzer profiles. Clin Chem. 1986;32(1 Pt 1):76–9.
tory medicine: testing the goodness-of-fit of an observed distribu- 8. Harris EK, Boyd JC. On dividing reference data into subgroups to
tion to the Gaussian distribution. Scand J Clin Lab Invest Suppl. produce separate reference ranges. Clin Chem. 1990;36:265–70.
1986;184:125–32.
Ethics: Informed Consent, Patient
Privacy 4
Qinghe Meng and Xu Qian
4.1 Overview chapter will review and discuss the key ethical issues includ-
ing informed consent and patient privacy during the perfor-
Molecular diagnostics has been dependent on genetic or mance of such testing. We hope these discussions will lead to
genomic testing or other high-throughput omic technolo- future standard principles in the course of medical practice.
gies and progressed significantly by rapid advances of novel
technologies and bioinformatics during the last decade of
intensive research. It is now has been moved on to the clini- 4.2 Informed Consent
cal implication in the diagnosis, individual-level treatment
regimens, prognosis, as well as risk assessment and disease 4.2.1 Challenges
prevention [1–3]. In the meanwhile, consumer genomics
companies who provide the direct-to-consumer (DTC) test- The fundamental basis of informed consent represents the
ing services are rapidly emerging [4]. Notably, ethical issue, physician-patient interaction where the patient should be
an important topic of debate crossing the fields such as soci- fully respected and informed such as the procedure, benefits,
ology, psychology, and legal issues, is naturally arising and potential risks, and be able to understand and make a consen-
should be carefully considered while the exciting achieve- sus on it [7]. Informed consent also extends to the autonomy
ment has been made [5, 6]. For example, given by the certain and confidentiality that are protected by certain laws and reg-
features of genetic or genomic information which differenti- ulations. Clinically, genetic and genomic testing are tools that
ated it from other medical data, a by-product of genetic test- can identify a set of genes that are strongly linked to treat-
ing and secondary or incidental findings of genomic testing able diseases of individuals being sequenced or genotyped.
are beyond the scope of traditional informed consent. Also, For this purpose, the informed consent for genetic or genomic
the acquired data for biobank repositories and accompanying testing would include comprehensive information that will be
databases would make the sensitive information of individual delivered to a patient after testing, benefits and the potential
patient to be potentially identifiable. Thus, a comprehensive risks, and also an agreement on such a testing [8].
review of these issues and the establishment of a new protocol However, the complexity of genetic/genomic testing and
or procedure adapted to the current laws and regulations in the expanded usage pose challenges to traditional informed
the field of molecular diagnostics are urgently needed. This consent [1, 9, 10]. Such issues like right to know or not to
know, expected results or un-expected results, and interpret-
ing the results of uncertain significance with patients are
Q. Meng (*)
Department of Laboratory Medicine, The University of Texas MD critical at this point of time. For example, in a recent paper,
Anderson Cancer Center, Houston, TX, USA two cases are presented, and one of them has no significant
e-mail: qhmeng@mdanderson.org medical problems, but has a likely pathogenic JAK2-V617F
X. Qian mutation from genomic sequencings in a clinical trial among
Institute of Cancer and Basic Medicine, healthy individuals [11]. Another case is gene sequenc-
Chinese Academy of Sciences, Beijing, ing reveals an 85delAG mutation in the BRCA1 gene in a
patient with bone marrow aplasia which may significantly
Department of Laboratory Medicine, Cancer Hospital of the increase the risk of developing breast, ovarian, and hemato-
University of Chinese Academy of Sciences, Beijing,
People’s Republic of China logic malignancies [11]. Obviously, the use of genomic test-
ing for screening would generate uncertain significances that
Department of Laboratory Medicine, Zhejiang Cancer Hospital,
Hangzhou, Zhejiang, People’s Republic of China may go beyond the anticipation of each individual with raised

40 Q. Meng and X. Qian
worry, distress, and misunderstanding. In the second case, ance. The Belmont Report, published in 1979 by the US
the incidental finding or the secondary finding is common as “National Commission for the Protection of Human Subjects
seen in many studies [12, 13]. Currently, NCCN guidelines of Biomedical and Behavioral Research,” outlines three
for breast and ovarian cancer recommend a new standard for core principles for the ethical conduct: respect for persons,
BRCA testing that detects BRCA1/2 pathogenic mutation beneficence, and justice. These three principles are basics
detected by tumor profiling on any tumor type in the absence of current practice guidelines. For example, the American
of germline mutation analysis. However, there are no further Association of Clinical Chemistry (AACC) recommends ten
discussions for collateral detection of genetic risk variants principles of ethical conduct as follows:
among the individuals with or without family history. Thus,
like the authors recommended, a comprehensive counseling 1. Uphold standards of professionalism, be honest in all
prior to genetic or genomic testing is of importance to possi- professional endeavors, and maintain a high level of per-
bly explain the potential broaden findings and the limitations. sonal integrity.
Several challenges exist for a comprehensive counseling. 2. Avoid scientific and professional misconduct including,
First, the spectrum of genetic variants is broadening, and but not limited to fraud, fabrication, plagiarism, conceal-
interpretation for certain novel variant remains challeng- ment, inappropriate omission of information, and mak-
ing. However, these comprehensive results should be vocal- ing false or deceptive statements.
ized. Physicians who lack genetic knowledge may have low 3. Report any health care professional who engages in
confidence, and even geneticists may not be knowledgeable fraud or deception or whose deficiency in character or
when the diagnosis and follow-up clinical decision-making competence jeopardizes patient care or other personnel.
are involved. There is consensus on actionable clinical con- 4. Maintain a high level of quality in the product(s) of my
dition that should be returned to patients. Thus, who and professional endeavors, including validity and reliability
how to give an effective counselor would need the coordi- of test results, interpretive opinions, publications, and
nation of physicians, geneticist, and public health workers. scientific research.
This way, molecular pathologists may take a role. Second, 5. Respect the privacy and confidentiality of protected
it is necessary to explore the patient’s hope in a counsel- health information encountered during the course of my
ing. Notably, despite the significance of targeted therapy, professional activities in accordance with legal and ethi-
the response rate to these therapies varies. Additionally, the cal obligations.
identification of germ-line variants implies that the fam- 6. Continuously strive to augment my professional qualifi-
ily members of the patient may also be at potential risk. cations, knowledge, and skills, and present them
These issues lead to a discussion on the “right to know” accurately.
and “right not know” where the privacy of each individual 7. Promote the safety and welfare of patients, employees,
should be concerned. Third, there is uncertain significance co-workers, colleagues, the public, and the environment.
when genetic information with incomplete certainty is 8. Avoid, or promptly disclose and work to resolve, actual
concerned [14]. Also, there are limitations for the technol- or potential conflicts of interest.
ogy itself that may result in false-positive or false-negative 9. Encourage open and honest discussion among physi-
results [15]. Thus, regarding to the updated outcomes when cians, other healthcare providers and/or facility manag-
technology advances, the genomic data storage may be nec- ers regarding disclosure to patients of information about
essary for future reanalysis [8]. However, currently there medical errors, if such information is material to any
is no standard protocol to address this issue. Furthermore, patient’s well-being.
there are disparities regarding the racial and ethnic minori- 10. Comply with relevant laws and seek to change them
ties by which the reference library is established. Further when they are contrary to the best interests of the patient.
development of a dataset at population level would mini- (https://www.aacc.org/membership/ethic-guidelines.
mize this effect, and thus the longevity of genomic data of Assessed on March 19, 2019)
each individual would require processes to achieve consent
over a long time period [16]. In particular, the Health Insurance Portability and
Nevertheless, it is important to make the informed con- Accountability Act of 1996 (HIPAA) Privacy Rule,
sent fulfilling the ethical, legal, and social considerations, the Clinical Laboratory Improvement Amendments of
and new consent models remain to be established for both 1988 (CLIA) (42 CFR 493), and the American Medical
clinical laboratories and consumer genomics companies. Association (AMA) code of medical ethics (https://www.
ama-assn.org/delivering-care/ethics/code-medical-ethics-
genetics-reproductive-medicine, assessed on March 19,
4.2.2 Regulations and Recommendations 2019) have clear guidance on ethic issues that may fulfill the
requirement being related to genetic/genomic testing. On
We herein illustrated some common regulations and rec- Feb 2014, the Department of Health and Human Services
ommendations that we can follow as central practice guid- (HHS) promulgated a joint of the Office of Civil Rights
4 Ethics: Informed Consent, Patient Privacy 41
(OCR)/Medicare & Medicaid Services (CMS) rule, revis- order tests, receive test results, or both. This authorized per-
ing both the CLIA laboratory requirements and the HIPAA son should provide informed consent before a testing (42
Privacy Rule. As an example, the following recommenda- CFR §493.1241[a]). The following factors are commonly
tions will help laboratories to develop informed consent included in the consent:
forms per CLIA requirements (42 CFR §493.1241[a] and
1291[f]), particularly in relation to genetic testing provided 1 . A general description of the test.
directly to consumers: 2. The interpretation of results, including positive and nega-
The laboratory that initially accepts a test request (regardless of tive results, as well as potential non-informative results or
whether the laboratory performs the testing on-site or refers the false-positive or false-negative results.
patient specimens to another laboratory) is responsible for veri- 3. Data assessment. How and to whom test results will be
fying that the test requestor is authorized by state laws and regu- released and under what circumstances results can be dis-
lations to do so. Laboratories that receive patient specimens
from multiple states or have specimen collection sites in multi- closed (i.e., to a licensed healthcare provider, 42 CFR
ple states should keep an updated copy of the requirements of §493.1291[f]).
each state regarding authorized persons and review test requests 4. The person requesting testing has the opportunity of con-
accordingly. sulting healthcare professionals pretest and on any addi-
Although referral laboratories might be unable to verify that
the person submitting the original test request qualifies as an tional testing.
authorized person, the test results may only be released to per- 5. What specimen will be tested, residual specimen storage,
sons authorized by state laws and regulations to receive the and the option for residual specimen after the test is
results, the persons responsible for using the test results, and the complete.
referring laboratory.
6. The individual’s or authorized person’s signature.
In accordance with these regulations, the American
College of Medical Genetics and Genomics (ACMG) has Represented consent forms for genetic testing are illus-
established a series of proposals on informed consent for trated in Table 4.1 [20, 21].
clinical genome-scale sequencing [17–19]. In general, the
limitations for the interpretation of the test results, the main-
tenance of the patient privacy, the implications for family 4.3 Patient Privacy and Confidentiality
members, and the return of the results with clinical utility
should be included. Specifically, 56 genes are required to The patient’s right to privacy and confidentiality is defined
being examined, and the report of its mutations (incidental as the limitations to access and use of sensitive personal
or secondary findings) is also required regardless of the pri- information, and reform group health insurance, respec-
mary test indication. tively. In the USA, patient privacy is protected by certain
Notably, for those tests being related to research study, laws, including the Americans with Disabilities Act (Public
the HHS secretary’s Human Research Protection advisory Law 101- 336) and the HIPAA (Public Law 104-191).
committee issued recommendations on July 22, 2015, However, with the rapid growth of services for genetic or
regarding the HIPAA and CLIA issues on returning of genomic testing provided by clinical laboratories or DTC
individual test results: “HHS should clarify that research- companies, the protection of genetic or genomic informa-
ers who identify clinically actionable information from a tion of an individual is somehow obligated to the current
research test conducted in a non-CLIA-certified labora- privacy rules.
tory be able, without legal penalty, to refer a subject to a
CLIA-certified laboratory for additional testing.” In addi-
tion, it is also suggested that written policies and proce- 4.3.1 Challenges
dures should be made by the institutional review boards to
review plans for returning results. These recommendations Regarding genetic and genomic testing, what is known
emphasize the importance of returning of test results for is about privacy and confidentiality of patients as well as
each individual. information-sharing practices at a time when numbers of
patients being identified as at risk of a heritable condition
are increased. On the contrary to most medical informations
4.2.3 W
hat Should Be Included which are relevant only to an individual, genetic or genomic
in the Informed Consent? information can have direct, substantial implications for third
parties and public health genomics [9, 22, 23]. This raises
The informed consent currently used in the clinic is simi- the questions such as patient’s family members who seem to
lar to those for research purpose (https://escholarship. be susceptible to genetic conditions (familial data sharing)
umassmed.edu/psych_cmhsr/119/. Assessed on March 19, should be notified or not and how should the genomic data be
2019). CLIA regulations define an authorized person as a kept, being accessible for personal or commercial use [24].
person authorized by state laws or regulations who may In general, respecting a person’s autonomy reflects roughly
Table 4.1 Informed consent form for molecular genetic testing
INFORMED CONSENT FORM FOR MOLECULAR GENETIC TESTING
For requesting genetic testing, the following points I have been counseled and understand that:
1. My healthcare provider wants me to have a test for________________________________.
Patients are required to give informed consent prior to having a genetic test. This should occur after discussing with my hea
2.lth care provider, or consulting geneticist, or genetic counselor, the purpose of the test, its predictive or diagnostic capability, and its
relative benefits and potential drawbacks.
3. DNA testing requires a blood sample, buccal swab, muscle or skin biopsy, all of which have risks associated with obtaining the
sample. Additional samples may be needed if the sample is damaged in shipment or inaccurately submitted.
4. The genetic tests offered by the Molecular Diagnostics Laboratories are performed to identify mutations (gene changes) that
may cause or predispose to disease. Targeted tests are performed when a family gene change is known; mutation identification or
full sequence tests are performed to search for an unknown gene change.
5. Genetic tests can be offered to confirm or rule out a diagnosis, to test for a disease before symptoms develop, to determine
carrier status or for prenatal diagnosis. My health care provider will tell me about why he/she would like to order genetic testing.
6. A negative genetic test for a disease, in many cases, will not completely rule out that disease. I may still have or be a carrier for
that disease. My health care provider will use my health and family history to interpret what the negative result means for me.
7. A positive result may mean that I have or ampredisposed to developing a genetic disease, though the actual degree of which
may vary and is often less than 100%. I may consult with my health care provider or ask to be referred to a genetic professional
to discuss any additional testing or counselingthat may be helpful.
8. In order to perform accurate prenatal testing, samples from the affected individual, parents, or additional family members may
be required.
9. DNA-based studies performed are specific to the condition indicated above. The accuracy of genetic testing is limited by the
methods employed, the clinical diagnosis, and the nature of the specific condition for which testing is requested. In some cases,
the test will detect an abnormality, called a mutation, in the gene. In other cases the test is unable to identify an abnormality
although an abnormality may still exist. This event may be due to the current lack of knowledge of the complete gene structure or
an inability of the current technology to identify certain types of changes (mutations) in a gene. These tests represent the newest
service currently available for clinical laboratory testing, however, improvements will be made as scientific knowledge advances.
As with any complex genetic test, there is always a small possibility of a failure or error in sample analysis. Extensive measures
are taken to avoid these errors. The methods are not 100% accurate due to the possibility of rare genetic variations in the DNA of
an individual or due to the complexity of the testing itself. A low error rate, estimated to be approximately 1 in 1000 samples, is
thought to exist in any laboratory.
10. Results will only be released to a licensed healthcare provider, to those allowed access to test results by law, and to those
whom I authorize in writing.
11. I understand my specimen will be destroyed within 60 days of collection. If my sample is retained beyond 60 days of
collection, it will be de-identified.
YOU MUST CHECK ONE. ƶ
ƶI explicitly agree that my sample may be retained beyond 60 days of collection for the purposes of quality control or research;
the retained sample will be de-identified.
ƶI do not wish for my specimen to be retained; destroy within 60 days of collection.
The risks, benefits and limitations of DNA testing have been explained to me. I have read and will receive a copy of this consent
form.
______________________________ __________________________
Patient Signature Date Print name Date
Physician/Counselor/Clinician Statement:
I have explained DNA testing to the patient/parent/guardian.

The consent form and limitations of genetic testing were reviewed with the patient/guardian.
I accept responsibility for pre-test and post-test genetic counseling.
_____________________________ __________________________
Clinician Signature Date Print name Date

4 Ethics: Informed Consent, Patient Privacy 43
that patients should be allowed to decide whether and to mation and confers rights on individuals, including the rights
whom to disclose their personal health information. When to access to all protected health information maintained in the
it comes to disclosing patients’ personal health information, individual’s “designated record set” [HIPAA Privacy Rule and
physicians and clinical lab should: Definitions, 45 C.F.R. § 160.103 (2015); 45 C.F.R. § 164.524
(2015)]. Under the HIPAA Privacy Rule, CLIA requires
1. Restrict disclosure to the minimum necessary informa- laboratories to establish procedures and protocols to protect
tion; and the confidentiality of patient information, including genetic
2. Notify the patient of the disclosure, when feasible. testing-related information throughout all phases of the
Physicians may disclose personal health information laboratory-controlled testing process (42 C.F.R §493.1231).
without the specific consent of the patient (or autho- Laboratories that perform molecular genetic testing should
rized surrogate when the patient lacks decision-making establish and follow procedures and protocols that include
capacity): defined responsibilities of all employees to ensure appropriate
3. To other health care personnel for purposes of providing access, documentation, storage, release, and transfer of confi-
care or for health care operations; or dential information and prohibit unauthorized or unnecessary
4. To appropriate authorities when disclosure is required by access or disclosure. AMA also requires the ethical principle
law. of confidentiality that information shared by a patient with a
5. To other third parties situated to mitigate the threat when physician in the course of treatment is not shared with oth-
in the physician’s judgment there is a reasonable proba- ers (Code of Medical Ethics Opinion 3.2.1 https://www.ama-
bility that: assn.org/delivering-care/ethics/confidentiality). In addition,
(a) The patient will seriously harm him/herself. the Genetic Information Non-discrimination Act (GINA) has
(b) The patient will inflict serious physical harm on an clear descriptions to protect people with potential risk genetic
identifiable individual or individuals. profile from discrimination in employment and health insur-
ance decisions. However, the GINA also has some shortcom-
However, there is a degree of variation and not all situations ings as mentioned elsewhere [27].
require specific consent (https://bioethicsarchive.george-
town.edu/pcsbi/sites/default/files/PrivacyProgress508_1.
pdf. Privacy and Progress in Whole Genome Sequencing. 4.3.3 How to Protect Patient Privacy?
Presidential Commission for the Study of Bioethical Issues.
October 2012. Accessed on April 30). Braverman reviewed Respect for persons is one of the principle ethical conducts.
several cases which were addressed regarding the physi- Importantly, patients have the right to determine to whom,
cian’s duty to remind family members of genetic test results when, and to what extent their individual and private health
in clinical practice. And there is little consensus on judicial information is revealed in the clinical practice. This informa-
decisions in these cases [25]. Regarding data sharing, in a tion includes, but is not limited to, medical diagnosis, treat-
survey asking “which one do you think needs the strongest ment plans, medications, and genetic information. Notably,
protection for data sharing?,” the most frequent answer was any entity protected by HIPAA is responsible for protecting
that the protection of family sharing data should be strength- patient privacy and securing patient’s protected health infor-
ened, rather than their own personal data [23]. A recent mation (PHI) and other confidential information. However,
investigation on clinical genetics services from 16 focus breaches of patient’s privacy can happen in a number of ways
groups with 80 healthcare professionals has shown that a [28]. It is suggested that an education for patients, physi-
familial approach to confidentiality has not been accepted or cians, insurers, and other healthcare professionals would be
adopted as a standard [26]. It is noteworthy that the anonym- necessary to improve privacy protection and safeguard data
ity of individuals is also challenged by the diversity of DTC security. We illustrated an example on how to protect patient
personal genome testing (for a detailed discussion, please go privacy by establishing steps as follows:
to review Braverman and colleagues [25]). Hence, additional
measures beyond traditional policies of privacy are neces- 1. Proper PHI Disposal: Proper PHI disposal and other con-
sary to safeguard the potential use of personal and familial fidential information, whether in paper or electronic for-
data sharing of genetic or genomic information. mat, is a requirement of HIPAA. PHI in paper form should
never be thrown into regular trash cans. Electronic PHI
must be appropriately stored or deleted, erased, and refor-
4.3.2 Regulations and Recommendations matted if no longer needed.
2. Proper disclosure of PHI: Disclosures of PHI without the
In the USA, the HIPAA Privacy Rule establishes standards to patient’s authorization is regarded as a violation of the
protect the privacy of individually identifiable health infor- Privacy Rule under HIPAA.
3. Using IT technology and security to protect patient

7. Appelbaum PS. Clinical practice. Assessment of patients’ compe-
tence to consent to treatment. N Engl J Med. 2007;357:1834–40.
privacy. 8. Robson ME, Bradbury AR, Arun B, et al. American Society of
4. Develop a formal safety management process, including Clinical Oncology Policy statement update: genetic and genomic
the development of policies and procedures, internal testing for cancer susceptibility. J Clin Oncol. 2015;33:3660–7.
audits, emergency plans, and other safeguards to ensure 9. Bunnik EM, Schermer MH, Janssens AC. The role of disease char-
acteristics in the ethical debate on personal genome testing. BMC
compliance by medical staff. Med Genomics. 2012;5:4.
5. Develop policies for verifying access authorization,
10. Korngiebel DM, Zech JM, Chappelle A, et al. Practice implica-
device control, and processing guests. tions of expanded genetic testing in oncology. Cancer Invest.
6. Develop and provide documentation, including instruc- 2019;37:39–45.
11. Marron JM, Joffe S. Ethical considerations in genomic testing for
tions on how your medical office can help protect PHI hematologic disorders. Blood. 2017;130:460–5.
(e.g., unregister it before leaving your computer). 12. Nishimura AA, Shirts BH, Dorschner MO, et al. Development of
clinical decision support alerts for pharmacogenomic incidental
Additionally, genetic or genomic medicine is one of the findings from exome sequencing. Genet Med. 2015;17:939–42.
13. Dorschner MO, Amendola LM, Turner EH, et al. Actionable,

few disciplines in which the privacy and beneficence of the pathogenic incidental findings in 1,000 participants’ exomes. Am J
patient should be balanced and fully addressed in the clini- Hum Genet. 2013;93:631–40.
cal practice. This necessitates the development of informed 14. Punj S, Akkari Y, Huang J, et al. Preconception carrier screening by
consent for proper use (e.g., long-term data storage and genome sequencing: results from the clinical laboratory. Am J Hum
Genet. 2018;102:1078–89.
analysis) as the main tool for protecting the autonomy of the 15. Manrai AK, Funke BH, Rehm HL, et al. Genetic misdiagnoses and
patient. More importantly, patient or their family members the potential for health disparities. N Engl J Med. 2016;375:655–65.
have the right to recall the use and sharing of their personal 16. Ohno-Machado L, Farcas C, Kim J, et al. Genomes in the cloud:
information. balancing privacy rights and the public good. AMIA Jt Summits
Transl Sci Proc. 2013;2013:128.
17. Directors ABo. Points to consider for informed consent for genome/
exome sequencing. Genet Med. 2013;15:748–9.
4.4 Conclusion 18. Directors ABo. ACMG policy statement: updated recommenda-
tions regarding analysis and reporting of secondary findings in
clinical genome-scale sequencing. Genet Med. 2015;17:68–9.
With the development of genetic and genomic technologies 19. American Society of Human Genetics Board of Directors, American
and the rapid expansion of clinical applications, clinicians College of Medical Genetics Board of Directors. Points to consider:
are facing the challenge in management and contextualiza- ethical, legal, and psychosocial implications of genetic testing in
tion of patients’ genetic or genomic information. This will children and adolescents. Am J Hum Genet. 1995;57:1233–41.
20. National Heart Lung, Blood Institute Working Group, Fabsitz RR,
increase pressure on suppliers to understand the practical and et al. Ethical and practical guidelines for reporting genetic research
ethical complexity of genetic and genomic testing. In consid- results to study participants: updated guidelines from a National
eration of all strict legal policy and regulatory rules, as well Heart, Lung, and Blood Institute working group. Circ Cardiovasc
as the nature of genetic or genomic medicine, protocols and Genet. 2010;3:574–80.
21. McGuire AL, Beskow LM. Informed consent in genomics and
procedures should be developed, implemented, and followed genetic research. Annu Rev Genomics Hum Genet. 2010;11:
in the clinical practice. 361–81.
22. Molster CM, Bowman FL, Bilkey GA, et al. The evolution of pub-
lic health genomics: exploring its past, present, and future. Front
Public Health. 2018;6:247.
References 23. Takashima K, Maru Y, Mori S, et al. Ethical concerns on shar-
ing genomic data including patients’ family members. BMC Med
1. Shendure J, Findlay GM, Snyder MW. Genomic medicine-progress, Ethics. 2018;19:61.
pitfalls, and promise. Cell. 2019;177:45–57. 24. Bunnik EM, Janssens AC, Schermer MH. Informed consent in
2. Boycott KM, Hartley T, Biesecker LG, et al. A diagnosis for all direct-to-consumer personal genome testing: the outline of a
rare genetic diseases: the horizon and the next frontiers. Cell. model between specific and generic consent. Bioethics. 2014;28:
2019;177:32–7. 343–51.
3. Directors ABo. Clinical utility of genetic and genomic services: a 25. Braverman G, Shapiro ZE, Bernstein JA. Ethical issues in contem-
position statement of the American College of Medical Genetics porary clinical genetics. Mayo Clin Proc Innov Qual Outcomes.
and Genomics. Genet Med. 2015;17:505–7. 2018;2:81–90.
4. Allyse MA, Robinson DH, Ferber MJ, et al. Direct-to-consumer 26. Dheensa S, Fenwick A, Lucassen A. Approaching confidentiality
testing 2.0: emerging models of direct-to-consumer genetic testing. at a familial level in genomic medicine: a focus group study with
Mayo Clin Proc. 2018;93:113–20. healthcare professionals. BMJ Open. 2017;7:e012443.
5. Ramos E, Weissman SM. The dawn of consumer-directed testing. 27. Prince AE, Roche MI. Genetic information, non-discrimination,
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6. Directors ABo. Direct-to-consumer genetic testing: a revised posi- Couns. 2014;23:891–902.
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Bioinformatics
5
Chenglu He and Yong Duan
5.1 Overview 5.1.2 Research Categories
5.1.1 Concept and Background Current research areas of bioinformatics mainly include the
following aspects: (1) integration and management of bioin-
With advances in technology, human understanding of DNA formatics data; (2) processing and analyzing of bioinformat-
was gradually established in the 1950s. The large-scale and ics data; (3) bioinformatics analysis methods and tools; and
high throughput sequencing technologies have been emerging (4) application of bioinformatics in clinical medicine, mainly
since the 1990s, deepening our understanding of the genetic including genome comparison, analysis of transcriptome
material of life. As a result, these technologies generate mas- data, and prediction of protein structure and function.
sive amounts of biological data beyond human analytical The challenges faced by bioinformatics are various, mainly
capabilities. The growth of the data is explosive. It is a huge as follows: how to integrate human intelligence with bioinfor-
challenge for current knowledge to try to store and manage as matics analysis software perfectly; how to integrate bioinfor-
much as 100,000 human genes and to find useful information. matics with clinical medical data effectively; how to effectively
How we make use of information is a difficult problem. The develop the potential value of bioinformatics in clinical medi-
solutions to these problems involve many interdisciplinary cine; and how to protect intellectual property rights.
challenges. Besides modern biology, mathematics, statistics,
and computer science are all required. The intersection of
these areas led to the emergence of bioinformatics. 5.1.3 C
ommon International Bioinformatics
Bioinformatics is a new field formed by the intersection Centers
of biology, computer science, and applied mathematics. To
achieve the objective, we should reveal the biological signifi- National Center for Biotechnology Information It is part
cance of data through the acquisition, processing, storage, of the National Library of Medicine, which is responsible for
retrieval, and analysis of biological laboratory data. It is one sharing data and develops analytic software of bioinformat-
of the major frontiers and core areas of life and natural sci- ics databases at the molecular level. The tasks include medi-
ences in the twenty-first century. The focus of research is the cal issues, developing and promoting bioinformatics
biological information on structural and functional informa- databases, data storage, exchange, and standardization of
tion, which is mainly represented by genomics and pro- biological nomenclature. Common databases include
teomics, or more precisely, from the information encoded in GenBank, PubMed, etc. (Website: https://www.ncbi.nlm.
the nucleic acid and protein sequences. nih.gov/pubmed/) [1].
European Institute of Bioinformatics It is the DNA and

RNA sequence database of the European Molecular Biology
Laboratory and provides query services for the database
(Website: http://www.ebi.ac.uk/) [1].
National Institute of Genetics, Japan It is a Japanese

C. He · Y. Duan (*) DNA database (DDBJ). It is mainly responsible for the
Department of Clinical Laboratory, The First Affiliated Hospital of retrieval services of human genetic information from data-
Kunming Medical University, Kunming, Yunnan,
People’s Republic of China bases (Website: http://www.ddbj.nig.ac.jp) [1].

46 C. He and Y. Duan
5.1.4 Common Bioinformatics Database including the expression profiles of microRNA in different
tissues. It supports predicting target genes through microRNA
With the rapid development of biotechnology and successful and analyzing related microRNA through RNA (website:
development of the Human Genome Project, a wide variety http://www.microrna.org/).
of databases have been established. They can be broadly
classified into two categories: primary databases, which are LncRNA Database It is a functional RNA molecule that is
mainly responsible for the storage of raw data, and second- transcribed from a non-coding sequence between protein-
ary databases, where relevant information is processed based encoding genes and transcribed with a length of more than
on primary databases, such as protein structure sequences. 200 nucleotides. Up to now, more than 3500 lncRNAs have
With the popularity of the Internet, most of these databases been found in mammalian genomes and involved in the
can be accessed or downloaded over the Internet. The gen- body’s physiological processes through regulation of gene
eral database retrieval method is as follows: expression and are closely related to various disease types.
Typical databases are as follows:
Genome Data The genome database is an essential part of LncRNA Base provides interaction information of long
the molecular bioinformatics database. The main body is the non-coding RNA (lncRNA), pseudogene, and circular RNA
model organism genome database, which is one of the most (circRNA) and the information on the competing endoge-
important human genome databases (GDB). Besides, there nous RNA (ceRNA) regulatory network. Pan-Cancer expres-
are the nematode genome database ACEDB and the yeast sion profiles and interaction networks of 14 cancer types
genome database SGD. The GDB database provides tabular (>6000 samples) were constructed. This regulatory interac-
genomic structure data, including gene units, PCR sites, tion network information is based on high-throughput CLIP-
cytogenetic markers, EST, repeat fragments, etc. In addition, Seq experimental data (Website: http://starbase.sysu.edu.
it can display genome maps, including cytogenetic markers, cn/).
linkage maps, overlap group maps, and transcription maps, ChIP Base provides a comprehensive identification and
as well as providing allele polymorphism databases. Also, annotation of expression profiles and transcriptional control
GDB database includes hypertext links with other network of long non-coding RNAs. The high-throughput RNA-seq
information resources such as GenBank and EMBL, OMIM, identification of lncRNA, expression profile, and transcrip-
MedLine, and so on (website: http://www.gdb.org). tion factor binding sites identified by ChIP-Seq assay were
integrated (Website: http://rna.sysu.edu.cn/chipbase/).
MicroRNA Database It is a kind of endogenous non-
coding RNA with 20–25 nucleotides in length found in CircRNA Database It is a type of circular RNA molecule
eukaryotes. Mature microRNAs are generated by cleaving that exists in almost every organism. Currently, thousands of
long primordial transcripts through a series of nucleases and circRNA have been identified in humans and other model
then assembled into RNA-induced silencing complexes with organisms. Studies have shown that circRNA acts as a sponge
target according to the degree of complementation by base molecule for miRNA or RNA-binding proteins and can play
pairing. It recognizes the different orientations of the silenc- an important role in physiological and disease processes. As
ing complex either degrade the target mRNA or suppress circRNA research increases, known circRNA data informa-
translation of the target mRNA. Typical databases include tion is growing rapidly. Common databases are as follows:
the following.
The microRNA database is a comprehensive database • CircRNA Base Human (hg19), mouse (mm9), and infor-
that provides information on published miRNA sequence mation on collecting circRNA from multiple species,
data, annotations, and predicted gene targets. It is one of the including C. elegans (ce6), Drosophila melanogaster
most important public databases for storing miRNA infor- (dm3), Spearfish (latCha1), and Coelacanth (Website:
mation. This database retrieves the known miRNA and tar- http://www.circbase.org/).
get information. The database is also used for other miRNA • Circ Net database provides identification of new cir-
information queries, such as fewer target relationship pre- cRNA; integration of circ RNA-microRNA-RNA interac-
dictions, and miRBase database URLs (website: http:// tion network; expression level of circRNA subtypes;
www.mirbase.org). genomic annotation of circRNA subtypes; and sequence
The TargetScan database is primarily used to predict of circRNA subtypes (Website: http://circnet.mbc.nctu.
miRNA target gene databases by searching stored 8mer and edu.tw/).
7mer sites that match each miRNA seed region (website:
http://www.targetscan.org). Protein Database It is a protein information database
The microRNA database provides predictive information established in May 2005. The content includes qualitative
about microRNA target in human, mouse, rat, and Drosophila, descriptions such as molecular structure, sample source,
5 Bioinformatics 47
expression vector, host, chemical analysis method, and information, is an automated and comprehensive database
molecular structure composition. There are many commonly of human genes, genome maps, proteins, and diseases. It
used protein databases, but Uniprot is considered to be the covers several databases of genetic analysis data, integrat-
most comprehensive protein database with the most compre- ing literature information, expression, and function to orga-
hensive annotation information. Other protein databases nize comprehensive information in terms of the locations,
include PDB (Protein Data Bank). pathways, mutations, homologous genes, sources, refer-
Uniprot (Universal Protein) integrates the data of Swiss- ences, diseases, etc. There are also cardiovascular biomedi-
Prot, TrEMBL, and PIR-PSD. It is the most informative and cal databases (Cardio) and immune disease-related
resource-rich, free of charge, available at http://www.uni- databases (FIMM and IDR).
prot.org/.
PDB is a structural database containing proteins, nucleic
acids, and other biological macromolecules. It is an impor- 5.2 Biological Sequence Analysis
tant resource in structural biology research (Website: http://
www.rcsb.org/). Traces of human evolution over hundreds of millions of
years are often reflected in biological sequences. When the
SNP Database A Single Nucleotide Polymorphism (SNP) origins are the same, different sequences might have specific
refers to a single base-pair mutation in a DNA sequence or structural, compositional, and functional similarities.
an alteration of A, T, C, or G in the DNA sequence. In other Once an unknown new sequence is obtained, biological
words, it means one or more of the specific localization sites sequences analysis software is often used to analyze whether
in the genome. It is the most common genetic variation in it has similar structure or composition with other genes, or
human beings, which accounts for more than 90% of all whether it encodes a protein with other function. The most
known polymorphisms. SNPs are widely present in the commonly used analysis software programs are BLAST,
human genome, with an average of one base pair per 500– FASTA, and multi-sequence analysis software cluster. The
1000 bases. The polymorphism of SNP only involves the software compares unknown new sequences with sequence
variation of a single base, which can be caused by the transi- data and draws corresponding conclusions.
tion of a single base, the insertion, or deletion of a base. But
usually, the SNP does not include the latter two cases.
The Single Nucleotide Polymorphism Database dbSNP 5.2.1 Sequence Analysis
(http://www.ncbi.nlm.nih.gov/SNP/) was established by
NCBI in collaboration with the National Human Genome Data Format Because the sequence analysis software only
Research Institute. It is a resource bank for single base sub- recognizes specialized input data format, it is necessary to
stitution, short insertion, and deletion of polymorphisms. arrange the data sequence according to the specific data
dbSNP was developed to complement and assist GenBank, sequence format before data analysis. These formats are
which contains nucleotide sequences from any organism. It readable texts that describe the sequence with identification
should be noted that the database will stop receiving SNP and related information in a particular order and form.
submissions from non-human species on September 1, FASTA is a commonly used format for a single sequence.
2017, and stop SNP queries from non-human species on The FASTA format begins with a greater than sign (>), fol-
November 1, 2017. However, all previous SNP data of non- lowed by the identifier (>sp | p42261.2 | GRIA1_HUMAN)
human species can still be downloaded in FTP of dbSNP of the sequence, and then the description information of the
database. sequence. Following a line break is sequence information.
Spaces, line breaks, and empty lines are allowed in the
Disease Database The most common human diseases, sequence until the next greater than sign indicates the end of
such as tumors, cardiovascular diseases, and immune- the sequence, as shown in Fig. 5.1. When we need to convert
related diseases, are complex diseases. Complex diseases, a sequence to a format such as PIR, we can use ReadSeq and
unlike single-deficient genetic diseases, do not conform to other tools for the conversion.
Mendel’s Law. The development of the diseases is a com-
plicated biology process caused by genetic material changes Sequence Retrieval Before sequence analysis, we need to
and external environmental changes that are involved. retrieve the database to obtain the sequence of interest. The
There are several corresponding databases to study differ- Entrez of NCBI is the most commonly used platform (https://
ent disease types, such as TCGA, the tumor-related data- www.ncbi.nlm.nih.gov/nuccore), as shown in Fig. 5.2.
base related to the molecular mutation map associated with Entrez includes nucleic acids, proteins, and Medline Abstract
cancer development and development, COSMIC, gene databases and has established perfect links among these
fusion, and SNP. GeneCards, including gene expression three databases. Therefore, protein products and related lit-
Fig. 5.1 FASTA format
Fig. 5.2 NCBI Home Page
erature can be queried from a DNA sequence, and each entry masker.org) is designed explicitly for genome repeat
has the information, which gives information close to the sequence processing with the study of the genome, non-
query entry. coding RNA, and other related fields.
Repeat Sequence Processing Repeat sequence is multiple Conceptual Translation It is a way of predicting whether
copies of gene sequences. In the biological genome, it can be the DNA product encodes or how long the amino acid
divided into three categories according to the frequency of sequence is encoded by a given DNA sequence that based on
repetition. (1) A highly repetitive sequence is a sequence the genetic code and relied on gene recognition software.
repeated at hundreds to millions of times, usually less than ORF Finder (http://ncbi.nlm.nih.gov/gorf/gorf.html) is com-
10 short fragments of nucleotide residues. For example, sat- monly used for gene recognition. It has an excellent ability to
ellite DNA in heterochromatin accounts for about 10% of the identify open reading frames of prokaryotes. Of course, there
genome in mice. (2) Moderate repeats are repeats ranging is still a lack of software for 100% prediction results. It is
from ten to hundreds of copies, accounting for about 20% of necessary to select appropriate analysis software according
mice, such as the rRNA gene and tRNA gene. (3) Mild repeat to the specific advantages of different software programs so
sequence refers to some sequences that repeat 2–10 times, that the analysis can predict unknown genes comprehen-
such as the tRNA gene. Repeat Masker (http://www.repeat- sively and accurately.
5 Bioinformatics 49
Analysis of Nucleic Acid Sequence Characteristics In the

process of searching for genes, prokaryotic genomes are
Inputs multiple sequences by Clustal
relatively simple compared with eukaryotic genome because
they do not contain introns. In other words, the eukaryotic
coding region is more complicated because it is often divided
into different exons depending on the non-coding region. For
example, the 5′ non-coding region of many eukaryotic genes
Calculate the distance between sequences by
has a CpG island structure and plays a role in transcriptional
fast alignment of sequence pairs and obtain a
regulation. But CpG islands are rare in the coding region. So distance matrix
how do we study CpG islands within the non-coding regions?
The region distribution mismatch is used to predict the pro-
moter region of a gene. In addition, since sequence analysis
revealed the sequence of junction regions between exons and
introns, those sequences are frequently stored according to Constructing a Guide Tree by Adjacency Method
the GT-AG rules in online analysis software such as ORF
(http://ncbi.nlm.nih.gov/gorf/gorf.html for the open reading
frame of the gene of interest).
5.2.2 Multiple Sequence Analysis Progressive alignment of multiple sequences

based on the boot tree
When we analyze unknown DNA, we often need to know

whether there is a similarity between unknown DNA and Fig. 5.3 Cluster multi-sequence alignment process
known biological sequence to determine the biological prop-
erties of the sequence. The unknown DNA sequence is then
compared with multiple sequences from a group of known gous macromolecules of different species are compared
homologous but different species of origin, in order to deter- (assuming that the structures of these macromolecules are
mine the size of homology between the sequence and other known), the difference (amino acid or nucleotide substitu-
sequences. On the one hand, we can understand the genetics. tion number) is only proportional to the independent evolu-
On the other hand, it can be used to describe the distances tion time of the compared organisms after mutation from the
between homologous genes and to analyze molecular evolu- common ancestors. This difference is used to determine the
tion. Clustal is a common tool for multi-sequence alignment. position in the re-evolution of the compared species, and a
It is an offline multi-sequence alignment tool based on pro- phylogenetic tree is thus created. The common method used
gressive alignment. The simple principles are as follows: to build evolutionary trees is agglomerative hierarchical
first, the distance matrix is constructed by the alignment of clustering. This method represents different classes with a
multiple sequences, inferring the relationship between the distance matrix. The algorithm merges any two closest
two sequences. Then, the phylogenetic tree is generated by classes into a combined partition at each loop and then
calculating the distance matrix, weighting the closely related merges all classes step by step into only one large class. It
sequences. Next, starting from the closest two series, gradu- should be noted that the evolutionary tree is only an intuitive
ally introduce adjacent sequences, and reconstruct align- way to represent the evolutionary relationship. It cannot
ments until all sequences are added as shown in Fig. 5.3. guarantee that it can genuinely reflect the real changes in the
evolutionary process. We need to integrate other means for
comprehensive analysis.
5.2.3 Molecular Polygenetic Tree
Assuming that the rate of evolution of biological macromol- 5.2.4 Comparative Genomics
ecules is relatively constant, the amount of evolutionary
change in macromolecules is positively correlated with the Comparative genomics is a discipline based on genome map-
time it takes for macromolecular evolution. In other words, ping and sequencing, which compares known genes and
the evolutionary change of macromolecules is a function of genome structures to understand gene function, expression
evolutionary time, so it can be used as an indicator to mea- mechanism, and species evolution. Using the coding
sure the genetic relationship between different evolutionary sequence and structural homology between model organism
units (such as species). If the primary structures of homolo- genome or human genome, we can clone human disease
genes, reveal gene function and molecular mechanism of dis- resent basic information and to share gene chip data. In 2016,
eases, and elucidate the evolutionary relationship of species an article published in Nature Protocols called transcript-
and internal structure of the genome. level expression analysis of RNA-seq experiments with
Through bioinformatics, we compare the genome HISAT, StringTie, and Ballgown introduced the analysis
sequences with similar evolutionary relationships; find new method of transcriptome data, mainly using the following
genes, non-coding RNAs and regulatory sequences, etc.; and three software programs: (1) HISAT (https://ccb.jhu.edu/
try to use the features formed by evolutionary selection to software/hisat/index.shtml) – this software uses a large num-
reach various aspects of their sequences, for example, the ber of FM indexes to cover the entire genome, enabling a
similarity of the sequence, the location of the gene, the length rapid comparison of RNA-seq reads with genomes, com-
and number of the coding region of the gene, the proportion pared to software speeds such as STAR Fast. (2) StringTie
of the non-coding region in each genome, and more. For (http://ccb.jhu.edu/software/stringtie) – this software is
instance, when compared with the human genome, the num- capable of applying transcript neural network algorithms and
ber of bases in the mouse is roughly the same, but the num- optional de novo assembly for transcript assembly and pre-
ber of genes is far apart. At the same time, the genetic dicting expression levels. Compared with programs such as
structure and activity of human beings are far different from Cufflinks, StringTie achieves more complete and accurate
those of mice. A little bit unveiled the superiority of human gene reconstruction and better predicts expression levels. (3)
beings on the road to evolution. Ballgown (https://github.com/alyssafrazee/ballgown) is a
gene expression analysis in R language. The tool can predict
the differential expression of genes and transcripts using the
5.3 Transcriptomics Data Analysis results of RNA-seq experimental data (StringTie, RSEM,
Cufflinks). However, Ballgown does not detect differential
5.3.1 Gene Expression Profile Analysis exons well, and DEXSeq, rMATS, and MISO can make up
for the above deficiencies. The detailed analysis steps are as
Gene expression process is also part of the genetic informa- follows: (1) Use HISAT to match the read fragments to the
tion for an organism. It reflects various functional states of reference genome and annotate, but HISAT will still detect
the gene and is regulated by factors such as time and space. the clipping sites not listed in the annotation file. (2) The
Therefore, it is necessary to study the gene as well as the reads on the alignment will be presented to StringTie for
inseparable expression aspects of the research gene. With the transcript assembly, and StringTie will assemble each sam-
development of biochip technology, gene expression research ple separately while estimating the expression level of each
has entered a high-throughput era, enabling the simultaneous gene and isoform during assembly. (3) All transcripts are
study of the expression of thousands of genes. presented to StringTie’s merge function to merge, and then
The gene chip data is derived from the fluorescence inten- create a transcript of all the samples, to facilitate the next
sity signal of the hybridization points extracted from the step of comparison. (4) The merged data is once again pre-
high-density hybrid dot matrix of the gene chip. After sys- sented to StringTie. In addition to using the merge data to
tematic collection, processing, analyzing, screening with re-estimate the transcript abundance, StringTie can addition-
valid data, and aggregation of relevant gene expression pro- ally provide data on the number of transcripts reads to the
files, the cells at the hybridization point are finally integrated. next Ballgown. (5) Ballgown obtained all transcripts and
Learn information, extract functional gene expression pro- their abundance from the previous step and classified them
files, and lay the foundation for further research. However, in according to experimental conditions [2].
the face of such a huge amount of data on gene chips, we
extract effective data, link the hybrid information of thou- Genome Annotation The genome annotation is a hotspot
sands of gene points with organic life activities, and charac- of current functional genomics research using bioinformatics
terize the characteristics and laws of life and the function of methods and tools to perform high-throughput annotation of
genes. The way to clarify is bioinformatics that is worth con- the biological functions of all genes in the genome. The
sidering. In particular, the problem begins primarily with the research of genome annotation includes two aspects: gene
following aspects: recognition and gene function annotation. The core of gene
recognition is to determine the exact location of all genes in
Raw Data Processing Gene chip data is generally stored in a genome-wide sequence. The prediction of new genes from
text format, and the data appears in a matrix. Each column genomic sequences is mainly based on a combination of
represents a different experiment. Each row represents the three methods: (1) analysis of mRNA and EST data to obtain
expression profile of each gene or probe under different results directly; (2) indirect evidence from known genes and
experimental conditions. The standard format is used to rep- protein sequences through similarity alignment; and (3) De
5 Bioinformatics 51
novo prediction based on various statistical models and aglo- • Fold Change Fold analysis is the fastest method applied to
rithms. High-throughput functional annotation of predicted the analysis of gene chip data, classifying the ratio of
genes can be annotated with new annotations using annota- gene chips from large to small, the ratio being the ratio of
tion information from known functional genes: (1) sequence cy3/cy5, also called R/G value. In general, there is no sig-
database similarity search; (2) sequence motif search; and nificant difference in gene expression in the range of 0.5
(3) clustering of orthologous group (COG). With the acceler- to 2.0, and gene expression is thought to change signifi-
ated rate of microbial genome-wide sequencing, it is neces- cantly outside the range. Because the experimental condi-
sary to develop an efficient, comprehensive genome tions are different, this threshold range must be adjusted
annotation system with a Web interface. In recent years, according to the confidence interval. The information
there have been some such tools in the world, such as the obtained after processing is output in different formats
Java-based microbial genome database (JMGD) interface. depending on different requirements, such as column
Although JMGD provides a good graphical interface pro- charts, pie charts, and dot charts. The advantage of this
gram, it does not have automatic genome annotation. The method is that the necessary chips can save research costs.
Protein Extraction, Description, and Analysis Tool The downside is that the conclusions are too simple, and
(PEDANT) developed by the German National Center for it is difficult to find clues of high-level functionality. In
Environmental and Health Research is a large-scale genomic addition to genes with very large fold changes, the reli-
analysis system that integrates many genomic functional ability of other small changes is valuable. This method
information and structural information. PEDANT annota- may be suitable for preliminary experiments or screening.
tions are powerful and versatile, but there is no easy-to-use Also, the multiple values are arbitrary and may be inap-
graphical interface and a strong hardware system support. At propriate. For example, if a differentially expressed gene
present, the complete sequencing of microbial genomes is is screened twice, no one will be selected, the sensitivity
usually done independently by small and medium laborato- will be zero, and many differentially expressed genes may
ries. It is necessary to develop and integrate a genomic infor- also appear. The results led to the belief that the multiple
mation annotation system based on Linux system with a free screening methods were blindly speculated.
database management system, free software, and public • T-test Another method of differential gene expression
database resources. analysis is a t-test, where two samples for comparison are
considered different if t exceeds a criterion selected based
Differential Gene Expression Analysis Differential gene on confidence. However, t-tests are often limited by sam-
refers to a gene that expresses a significant difference in ple size. Due to the high cost of gene chips, it takes time
RNA level at different levels of environmental stress, time, to repeat experiments. Small sample microarray experi-
space, etc. Changes in structure, gene mutation, or methyla- ments are very common, but small samples can lead to
tion under different factors lead to the differential expression unreliable mutation estimation. To overcome this short-
of genes. One of the purposes of the gene expression profil- coming, researchers have proposed a regulated t-test
ing chip experiment is to find differentially expressed genes based on the existence of a correlation between gene
between two samples, usually using the ratio of the signal in expression levels and mutations. Bayesian conditional
the experimental group and the control group as a measure of probabilities (Bayes’ theorem) statistical methods can
the difference in gene expression between the two states, in a supplement the estimation of the variability of any gene
two-color fluorescence system. The ratio of Cy5/Cy3 is used by detecting the expression levels of other genes adjacent
to measure the difference in gene expression, also known as to the same chip. This method is superior to the simple
the expression difference value. In a short oligonucleotide t-test and corrects the multiple analysis of the standard
chip such as Affymetrix, a single-color fluorescent labeling deviation of gene expression.
method is used, and the experimental group and the control • Nonparametric Analysis Because the microarray data
group are respectively detected by two chips, and the differ- have “noise” interference and do not satisfy the hypothe-
ence value is the signal ratio of the two chips. It is worth sis of a normal distribution, it may be risky to use t-test
noting that the noise and some factors of the chip itself and and regression model for screening. The non-parametric
the characteristics of the biology itself bring great trouble to test does not require data to fulfill the assumption of par-
the screening of differentially expressed genes. It is neces- ticular distribution, so it is feasible to use the non-para-
sary to set a criterion for the differentially expressed genes. metric method to filter variables, although extensive. At
This screening criterion is called the differentially expressed present, in addition to the traditional non-parametric t-test
gene threshold. According to different research purposes, and Wilcoxon rank-sum test, some new non-parametric
gene chip data analysis methods mainly include the follow- methods are also applied to the analysis of gene expres-
ing categories: sion profile data, such as empirical Bayes method and sig-
nificance analysis of microarray (SAM). The disadvantage area) represents a graph of a collection and its relation-
of the parametric method is that there are hypothesis tests ships. It can be used to represent the approximate rela-
for the analysis data. For example, changing the variation tionship between multiple data sets, but also to perform
in the sample can significantly affect the results of the set operations. There are many tools for drawing Venn
analysis, and the transformation of the same data (such as diagrams, such as the online drawing tool VENNY2.1
logarithm) can also have a significant impact on the (http://bioinfogp.cnb.csic.es/tools/venny/index.html).
results of the analysis. The nonparametric method is more • Volcano Plot Volcano Plot (Fig. 5.5) is a type of image
effective for this situation, but it is less sensitive to express used to show differences between groups because most of
data analysis than the parametric method. the gene expression is none or very small from a global
• Regression Analysis Some simple parametric analysis perspective when the organism changes. Only a small part
methods currently used are based on data transformation of the gene expression has undergone significant changes.
(such as logarithm) to achieve a normal distribution as a Therefore, volcano maps are commonly used in RNA
premise, or an estimated empirical distribution. However, expression profiling and chip data analysis and are most
these two methods may be unreasonable for gene expres- commonly used to analyze differential expression of
sion data. The non-parametric method ignores the distri- genes and can also be applied in other omics. The essence
bution of data, and the parameter method misjudges the of the volcano map is a plus version of the scatter plot,
distribution of data. Regression analysis of gene expres- which contains two important concepts: (1) significant,
sion profiles is a statistical method that can handle the that is, p-value, the difference test p-values of the two sets
linear dependence of multiple genetic variables. So the of samples, with negative log-log10 (p-value) conversion
researchers proposed using regression analysis of gene as the ordinate; (2) with log2 (Fold Change) as the hori-
expression profiling data or using the cross-variable (Cox) zontal axis, you can get the volcano map. Using certain
regression method to analyze gene expression profile data screening conditions (such as Fold Change greater than 2
for patient survival prediction. times, significant P value less than 0.05), you can filter out
significantly differentially expressed genes for follow-up
Graphics With the exponential growth of gene sequence, studies. There are many tools for drawing volcano maps,
the graphical visualization method of gene sequence has such as GraphPad Prism.
become an important means to study gene sequence. It is • Bar Chart (Fig. 5.6) is a statistical report graph of the
more helpful for us to classify genes and analyze their evolu- expression level that is visualized by the length of the
tionary relationship by using graphical expression method. rectangle. The data distribution is represented by a series
The graphical types of common gene sequences mainly of vertical stripes with different heights. It usually shows
include the following: one variable with one or more values (different times or
• Venn Diagram (Fig. 5.4) It was first used by Venn in

70
down:842 up:639
1880 in the article “On the Graphical and Mechanized
Performance of Propositions and Reasoning” in the form
60
of a fixed-position cross-ring. A closed curve (internal

50
male female
–log10 (p value)
40
30
20
359 1365 116

10
0
–6 –4 –2 0 2 4
log2 (fold-change)
Fig. 5.4 Venn Diagram Fig. 5.5 Volcano Plot

5 Bioinformatics 53
sis (ORA) was only for a group of genes, and the develop-
200
up-regulated ment of high-throughput omics data made functional class
scoring (FCS) come into being. The better improvement and
down-regulated
understanding of biological pathways and complex networks
have led to the development of pathway topology (PT) and
network topology (NT) based methods. Gene functional
150
149
132 enrichment analysis mainly includes KEGG analysis, GO
analysis, signal pathway analysis, and drug target analysis.
number of genes
Kyoto Encyclopedia of Genes and Genomes (KEGG) It

100
is a database that integrates information on genomic, bio-

107
chemical, and systemic functions to reveal the genetic and
biochemical blueprints of life phenomena. It is a manually
79
created knowledge base on knowledge of system functions.
It is formed using a computable form of capture and organi-
50
zation of experimental knowledge. In addition, KEGG has

powerful graphics capabilities that use graphics to introduce
numerous metabolic pathways and relationships between
pathways. Genomic information is stored in the GENES
database, including complete and partially sequenced
0
male female genomic sequences; functional information is stored in the

PATHWAY database, including graphical cellular biochemi-
Fig. 5.6 Bar Chart
cal processes such as metabolism, membrane transport, sig-
naling, cell cycle, and concomitant sub-pathways
different conditions) for smaller dataset analysis. The bar information; the LIGAND database includes information
graph can also be arranged horizontally or in multiple about chemicals, enzyme molecules, enzyme reactions, and
dimensions. There are many drawing tools for histograms, more. By connecting with other large bioinformatics data-
such as EXCEL. bases in the world, KEGG can provide researchers with more
extensive biological information. KEGG provides Java’s
graphical tools for accessing genomic maps, comparing
5.3.2 Functional Enrichment Analysis genomic maps and operational expression maps, as well as
other tools for sequence comparison, graph comparison, and
With the rapid development of high throughput sequencing pathway computation. KEGG established the KEGG
technology and the wide application of related technologies, Orthology (KO) System to develop and facilitate cross-
biomedical related research fields have entered the post- species annotation processes by linking molecular network
genome era in which large-scale omics data has grown expo- related information to the genome.
nentially. On the one hand, this has enabled biomedical
research to shift from the analysis of individual genes to the Gene Ontology Analysis Today’s biologists waste too
research at the system level, which has an important impetus much time and energy searching for biometric information.
to reveal the basic molecular mechanisms of biomedicine. This situation boils down to the reason of the confusing bio-
On the other hand, such a large amount of data also pose a logical definitions: not only is it difficult for computers to
huge challenge to extraction and analysis of such huge accurately find definitions which randomly changes over
amount of data. In order to discover patterns from complex time and changes because of multiple human factors; even if
omics data, researchers often perform enrichment analysis of it is completely handled by humans, it is still almost impos-
gene functions, expecting to discover biological pathways sible to do. The Gene Ontology (GO) project is the result of
that play a key role in biological processes, thereby revealing efforts to align functional descriptions of gene products in
and understanding the basic molecular mechanisms of bio- various databases. The project was originally initiated by the
logical processes. The enrichment analysis of gene function integration of three model biological databases in 1988:
has become a routine means of functional omics data analy- FlyBase (Drosophila), Saccharomyces Genome Database
sis, and with the development of high-throughput omics (SGD), and the Mouse Genome Database (MGD). Since
data, such as the change from gene chip data to RNA-seq then, GO has grown and expanded, and now contains dozens
data, a series of corresponding analytical methods have been of databases of animals, plants, and microbes. Gene Ontology
developed. The earliest developed over-representation analy- can be divided into three parts: molecular function, biologi-
cal process, and cellular component. The GO analysis calcu- the protein involved in the pathway. The result obtained by
lates the hypergeometric distribution relationship between the chip is the change in the amount of mRNA expression
these differential genes and one or several specific branches encoding these proteins. From mRNA to protein expression,
of the GO classification based on the selected differential microRNA regulation, translational regulation, post-
genes. The GO analysis returns a p-value for each GO with translational modification (such as glycosylation, phosphor-
the presence of the differential gene. The p-value indicates ylation), protein transport, and other processes, there is often
that the differential gene is enriched in the GO. The GO anal- no linear relationship between mRNA expression and pro-
ysis can be suggestive on the experimental results. Through tein expression. Therefore, a change in mRNA does not nec-
GO analysis of differential genes, we can find GO classifica- essarily mean a change in the amount of protein expressed. It
tion entries that enriched for differential genes. It can help to should also be noted that in certain pathways, such as the
understand which gene function is related to which differen- EGF/EGFR pathway, cells can regulate this pathway by
tially expressed gene in different samples. altering the degree of protein phosphorylation (regulating
protein activity) while maintaining the same amount of pro-
Molecular Function Molecular function describes activity tein. Therefore, the results of the chip data pathway analysis
in molecular biology, such as catalytic activity or binding need to be supported by following protein function experi-
activity. GO molecular functions define functions rather than ments, such as Western blot/ELISA, IHC (immunohisto-
the whole molecule, and do not specifically indicate the spe- chemistry), overexpression, RNAi (RNA interference),
cific temporal and spatial information of these functions. knockout (gene knockout), transgene (genetically modified),
Molecular function mostly refers to the function of a single and so on.
gene product, and a small part of it also refers to the function
of a complex formed by this gene product. The definitions of
the function include catalytic activity, transport activity, 5.3.3 Timing Analysis
binding activity, etc., and more narrow definitions include
adenylate cyclase activity or bell-shaped receptor binding The gene regulatory network is a continuous and complex
activity. dynamic system. The regulation between genes is a dynamic
event that changes with time and environment. The genomic
Biological Process Biological pathways are multi-step pro- DNA microarray provides researchers with a good tool for
cesses with an ordered structure of molecular functions. For understanding the regulatory network. The time-series gene
example, cell growth and maintenance and signal transduc- expression data contains abundant gene regulation informa-
tion are relatively extensive. Some more specific examples tion. It is conceivable that the changes in genes located
include transport such as pyrimidine metabolism or alpha upstream of the gene regulatory network should be in front
glycosyl groups. Biological processes are not exactly equal of the downstream genes. When the expression of upstream
to biological pathways. Therefore, GO does not include genes (such as transcription factor TF) changes, this change
complex mechanisms or path dependencies. will spread along with the gene regulatory network. When
the expression level of each gene at different times is mea-
Cellular Component The intracellular location refers to the sured, important information about the order of regulation
organelle or gene product group where the gene product is between genes and the regulatory targets can be derived
located (e.g., rough endoplasmic reticulum, nucleus or ribo- from such time-series data. In general, time-series gene
some, proteasome, etc.). expression data contain a larger amount of information that
is derived from the gene regulatory network than static gene
Pathway Analysis Pathway analysis calculates the hyper- expression data of the same size. Time series analysis is a
geometric distribution of selected differential genes. It method of analyzing the development process, direction,
returns a p-value for each path with a differential gene, and a and trend of time series and predicting the possible future
small p-value indicates that the differential gene is enriched targets in the time domain. This method uses the principle
in the pathway. The pathway analysis is suggestive on the and technique of time series analysis in probability and sta-
experimental results. Pathway analysis of differential genes tistics, uses the data correlation of the time series system,
can be used to find pathway entries with enriched differential establishes the corresponding mathematical model, and
genes, and to find out which differential genes may be describes the timing state of the system to predict the future.
involved in the change of pathways of different samples. For genomics, on the one hand, time series analysis is a
Unlike GO analysis, the results of pathway analysis are more method of gene clustering. For example, for transcriptome
indirect because pathways are interactions between proteins, data analysis, finding genes that are significant at different
and changes in pathways can be caused by changes in the points is a frequently used clustering method. Commonly
amount of protein involved in the pathway or the activity of used analytical methods include Short Time-series
5 Bioinformatics 55
Expression Miner (STEM), which is a clustering method methods, etc.). Between the correlations, WGCNA built a
different from the expression change. It is characterized by bridge between sample characteristics and changes in gene
the ability to derive important information about the order expression [3].
of regulation between genes and the regulatory targets. It is
generally believed that the time-series gene expression data
contains a larger amount of information for deriving the 5.3.5 Analysis of Transcriptional Regulation
gene regulatory network than the same size of static gene
expression data; on the other hand, the time series analysis, The regulation of gene expression in eukaryotes is an
due to the addition of time factors, can change over time. extremely complex process that requires a precise interaction
Changes in gene expression in the life system are also of a series of transcriptional regulators and DNA sequences.
dynamic and related to the surrounding time points. And If these non-coding transcriptional regulatory abnormalities
this relationship can be pre-calculated (called the expected such as mutations can cause many diseases, screening analy-
value), and then compared with the expected value of the sis and functional exploration of these transcriptional ele-
experimental data. ments are necessary for gene expression regulation and
biological function research. A commonly used analysis
software is TRRUST (https://www.grnpedia.org/trrust/),
5.3.4 Gene Co-expression Network Analysis which is a database that records the regulatory relationships
of transcription factors, including not only the target genes
Gene co-expression network analysis is a network diagram corresponding to transcription factors but also the regulatory
based on the similarity of gene expression data, with differ- relationships between transcription factors. Currently, the
ent colored dots representing genes with similar expression database only stores regulatory information related to
profiles. Genes are linked to form a network. The construc- humans and mice. There are also many software programs
tion of the co-expression network is conceptually simple and such as AnimalTFDB, HumanBase (https://hb.flatironinsti-
intuitive. Through the similarity of gene expression, the pos- tute.org/), and Dire (https://dire.dcode.org/).
sible interactions of gene products can be analyzed, so as to
understand the inter-gene interactions and find the core
genes. The core gene is an important hub and plays a key role 5.4 Protein Structure Analysis
in the network module.
Weighted gene co-expression network analysis (WGCNA) 5.4.1 Protein Structure Prediction
is a commonly used method for co-expression network anal-
ysis. It first assumes that the gene network obeys the scale- Protein is the embodiment of life activities, and its structure
free distribution and defines the adjacency function with the determines its function. Proteins composed of linear amino
gene co-expression correlation matrix and gene network acids need to be folded into specific spatial structures to
structure. Then it computes the distinct coefficients of the have corresponding physiological activities and biological
different nodes and builds a system clustering tree accord- functions. The analysis of the spatial structure of proteins is
ingly. The different branches of the cluster tree represent dif- important for understanding the function of proteins, the
ferent gene modules, and the degree of co-expression of execution of functions, the interaction between biological
genes in the module is high, while the degree of co-expression macromolecules, and the development of medicine and
of genes belonging to different modules is low. If certain pharmacy (such as the design of drug targets). In order to
genes always have similar expression changes in a physio- understand protein function more quickly, you can’t just
logical process or different tissues, then we have reason to wait for the results of protein determination, especially
believe that these genes are functionally related and can be before researching unknown proteins. There are obvious
defined as a module. When the gene module is defined, we advantages by predicting protein structure first. The study of
can use these results to do a lot of further work, such as protein structure and function has a long history. Due to its
screening the core genes of the module, correlating traits, complexity, the prediction of its structure and function is
modeling metabolic pathways, and establishing genetic complicated both in methodology and in basic theory.
interaction networks. WGCNA can quickly extract gene co- Statistical methods have been successfully applied to the
expression modules associated with sample features from prediction of protein secondary structure. The empirical
complex data (N multi-packets) for subsequent analysis. parameter method proposed by Chou and Fasman is the
Simply put, it calculates the expression correlation between most prominent example. The method statistically analyzed
genes, clusters genes with expression correlation into a mod- the secondary structure distribution characteristics of vari-
ule, and then analyzes the module and sample characteristics ous amino acids, and obtained corresponding parameters
(including clinical features, surgical methods, treatment (Pα, Pβ, and Pt) and used for prediction. In general, the
structure of a protein is divided into four levels: 1 primary is pGenThreader (http://bioinf.cs.ucl.ac.uk/psipred/) and
structure: protein sequence; 2 secondary structure: α-helix Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.
and β-sheets mode; 3 tertiary structure: residues in space cgi?id = index). With the development of technology, it is
layout; 4 four-level structure: interaction between proteins. believed that there will be more and more accurate new
In recent years, another layer of protein structure between methods for the prediction of protein structure.
the secondary and tertiary structures, the so-called protein
fold, has proven to be very useful. “fold” describes a hybrid
combination of secondary structural elements. The tech- 5.4.2 Protein-Protein Interaction
nique for predicting protein secondary structure based on
sequence or multiple sequence alignment has been rela- The completion of the sequencing of the human genome
tively mature, but the prediction of tertiary structure is quite marks the advent of a new era of biological research, the
difficult. Often for tertiary structure predictions, this can post-genomic era. The genome-wide sequence information
only be done by homology alignment with known structural is not enough to explain and speculate on various life phe-
protein sequences. A number of related databases have been nomena of cells. Protein is the ultimate performer of cell
established for protein structure prediction. This method is activity and function. The complex interaction between pro-
the most accurate method for predicting the tertiary struc- teins determines the complexity of organisms. The hottest
ture. But this method does not always work, because about research topics have gone back to the protein. Interacting
80% of known protein sequences cannot find similar protein proteins in the body’s cells can reveal the function of pro-
sequences of known structure. Protein structure prediction teins at the molecular level and are essential for the regula-
mainly includes the following aspects: (1) protein physico- tion of growth, development, differentiation, apoptosis, and
chemical properties and primary structure analysis – analy- understanding of biological regulation mechanisms, provid-
sis of protein PI, MW, amino acid composition, extinction ing important theoretical basis for exploring the mechanisms
coefficient, and stability coefficient often uses online analy- of major diseases treatment, prevention, and new drug
sis software such as Expasy (https://Web.expasy.org/prot- development. Therefore, understanding and elucidating the
param/). Expasy is also commonly used to analyze the mechanism of interaction between proteins is of great sig-
hydrophilicity and hydrophobicity of proteins; the Tmpred nificance, and it is a hotspot in the cross-research of life sci-
method is commonly used to analyze the transmembrane ences and other disciplines in the post-genome era. Common
region of proteins. It is based on the statistical analysis of analysis software includes STRING (https://string-db.org/)
the Tmbase database to predict protein transmembrane and BioGrid (https://thebiogrid.org/).
regions and transmembrane direction (http://www.ch.emb-
net.org/software/TMPRED_form.html); signal peptide pre-
diction method SignalP (http://www.cbs.dtu.dk/services/ 5.4.3 Protein Function Prediction
SignalP), which is a prediction of the secretory signal pep-
tide, rather than the protein involved in intracellular signal- Proteins are the most essential and versatile macromolecules
ing, is more than 90% accurate. (2) Common methods for in the body, and the understanding of their function is critical
protein secondary structure prediction include CFSSP to the development of medical science. With the develop-
(http://cho-fas.sourceforge.net/) and PSIPRED (http://bio- ment of the post-genome era, a large number of protein
inf.cs.ucl.ac.uk/psipred/). (3) Protein domain prediction – sequences with unknown structures and functions have
common methods include InterPro (http://www.ebi.ac.uk/ emerged in the NCBI database, and these protein sequences
interpro/scan.html) and Pfam (http://pfam.xfam.org/). (4) have already become a research hotspot. Commonly used
Commonly used methods for protein tertiary structure pre- protein function prediction software is Discovery Studio
diction include two methods. The rationale for the protein (http://www.discoverystudio.net/), a software for profes-
structure homology modeling (also known as homology sional life science molecular model of a new generation of
modeling) is that the tertiary structure of the protein is more molecular modeling and simulation environment. It is mainly
conservative than the primary structure of the protein. The used for protein structure-function research and drug discov-
other is folding recognition. The basic principle is to iden- ery. The main functions of DS include protein characteriza-
tify the similar folding type from the protein structure data- tion (including protein-protein interaction), homology
base and the sequence to be tested, and then to realize the modeling, etc. DS can be applied to life science research
spatial structure prediction of the sequence to be tested. The fields, bioinformatics, structural biology, new drug discov-
number of protein folding types in nature is limited. Many ery, etc. In the past 10 years, protein function prediction
proteins, although enjoying low sequence similarity, may methods have been continuously improved, and it is believed
still have the same folding type, which is the theoretical that future protein function predictions will become more
basis for folding recognition. The commonly used software and more accurate.
5 Bioinformatics 57
5.5 Bioinformatics and Precision genes will undergo local mutations. For example, some
Medicine patients can have thalassemia and albinism at birth because
these patients are born with a lack of enzymes. The third is
With the rapid development of genomics, bioinformatics, that patients can have acquired genetic mutations, such as
and precision medicine, bioinformatics and medical infor- patients with hypertension and diabetes. These patients have
matics have grown more and more cross-integrated. Data found a wide range of genes in the process of genetic testing
analysis of multiple molecular groups must be combined and found a large number of genes in the process of gene
with cells, tissues, organs, individuals, and even populations sequencing. Single nucleotides exhibit a polymorphic distri-
[4]. Information can accurately predict the occurrence and bution. Nowadays, there are some new genomics indicators
development of complex diseases and predict the applicable that can provide people with diagnostic information, such as
population of drug therapy, radiotherapy, chemotherapy, and genomic DNA sequences, proteomics, enzymology, etc.
immunotherapy. Therefore, the field of research and applica- These substances involve omics information, so they can
tion of bioinformatics technology become even broader. accurately and widely reflect the nature of the disease.
Features can help medical staff to find out the cause in time
to improve the accuracy of the drug in drug treatment.
5.5.1 B
ioinformatics and Precision Medical
Diagnosis 5.5.1.2 Detection of Pathogenic Microorganisms
Identification of Bacteria At present, there are mainly two
Precision medicine is a very systematic project that analyzes identification methods in the identification of bacteria,
the state of human disease and then combines clinical treat- namely, phenotypic identification method and molecular
ment to provide people with the best diagnosis and treat- genetic identification method [8]. However, with the contin-
ment. Nowadays, people’s awareness of health is improved. uous development of bioinformatics technology,
Therefore, the Chinese government is more dedicated to the bioinformatics technology can have a new identification

prevention and treatment of major diseases. Various medical method, which has higher resolution and is called multi-site
reforms are also underway to improve the understanding of sequence analysis. This method is more convenient and
risk mechanisms, and research and treatment programs for faster than the two traditional methods of bacterial identifica-
many diseases are underway. Big data combines digital tion with the help of bioinformatics techniques for DNA
imaging, systems biology, and information science to pro- sequencing. Another method of identification is called
vide patients with a good treatment basis for each patient. matrix-assisted laser desorption/ionization time-of-flight
The molecular profile and the information characteristics of mass spectrometry, which is a novel microbial identification
the mutation are analyzed to develop a personalized treat- method developed by the application of bioinformatics tech-
ment plan [5]. nology. It mainly identifies bacteria by the characteristics of
bacterial protein fingerprints. In the process of identifying
5.5.1.1 Genetic Testing bacteria, the bacteria were identified by analyzing the pris-
A number of researchers such as Xiaoqiang Qiu [6] and Feng tine data of bacteria by Spectra Bank database and SPE-
Shen [7] suggest that genetic testing can prevent diseases CLUST cluster analysis software in bioinformatics
through genetic testing and genetic screening. This technol- technology.
ogy has a huge impact on human health. In many cases, the
occurrence and development of human diseases are strongly Identification of Virus A new type of virus identification
related to gene mutations and have a strong connection with technology developed by bioinformatics technology is called
gene expression. In human genes, it contains a lot of chemi- genomic barcode technology. The genomic barcode technol-
cal information, so the genetic testing of the human body can ogy mainly uses a segment of the DNA of the virus, and then
improve health management to a certain extent. Related quickly and accurately identifies the species through this
studies have shown that there are about 4000 diseases that fragment. A new type of virus identification technology,
are closely related to genetic factors. In patients with heart genomic barcode technology can identify and classify vari-
disease, diabetes, and cancer, their genes can change. Related ous viruses. Compared with traditional single-gene sequence
research and analysis show that there are three main reasons technology, the new genomic barcode technology can dis-
for the occurrence of a disease caused by genetic changes. play information that cannot be expressed by single gene
First, under the influence of the environment, people can sequences. Another technique for the identification of viruses
have genetic mutations, which will produce cancer cells is high throughput sequencing, which enables the sequenc-
under the interference of chemical substances. The second is ing of millions of DNA molecules at a time. In the identifica-
the congenital defect of the gene. Under this defect, people’s tion of viruses, GLC Genomic Workbench software is
commonly used to analyze high throughput sequencing and cular diseases are very high, becoming the first killer of
generated DNA data. Meanwhile, some strategy for novel human health. According to related statistics, there are more
gene function of pathogen will rise subsequently [9]. than 70 million stroke patients in China, and more than 90%
of them need lifelong treatment. Moreover, cardiovascular
Identification of Fungi The application of bioinformatics and cerebrovascular diseases can lead to various complica-
technology in fungal identification is mainly realized by tions, such as elevated blood pressure, which can lead to
gene chip technology. Gene chip technology analyzes the death and disability. In the prevention of stroke, it is gener-
information of these genes efficiently and accurately. ally necessary to pay attention to the diagnosis of H-type
Therefore, gene chip technology has a very good prospect in hypertension, i.e., hypertension with elevated homocysteine
the identification of pathogenic organisms. The most com- (≥10 μmol/L). In the process of human cell metabolism, a
monly used analysis software in gene chip technology is kind of intermediate product is produced. If the product is
Arraypro. Arraypro analysis software can analyze not only very high in the blood of the human body, it will cause the
the image of genes but also process various data of genes. In blood pressure of the human body to rise. Under the influ-
the process of research and identification of human skin ence of genetic factors, the level of folic acid in patients
pathogenic fungi, biologists have identified the fungus by decreases, which directly leads to the development of hyper-
PCR microarray method with bioinformatics, and then ana- tension and stroke. According to relevant research, there are
lyzed the gene data using Arraypro software and Prism soft- 300 million hypertensive patients in China, and two-thirds of
ware. The accuracy rate is as high as 90% [10]. patients have H-type hypertension. In the primary prevention
study of stroke in China, H-type hypertension was deeply
Identification of Parasites The identification of parasites in explored, and the big data method was adopted to enable
pathogenic biology can be characterized by proteomics in patients to use antihypertensive drugs accurately.
bioinformatics to identify the protein composition of various
life stages of parasites. The protein map of parasites in differ- Cancer Treatment Cancer is a common type of disease.
ent life stages is important components for parasites identifi- Related studies have shown that the cause of cancer is the
cation. Biologists identified multiple new genes by proteomic mutations of cells. The gene imprinting and tumor markers
analysis during the proteome analysis of Leishmania don- of cancer patients can be analyzed by means of big data. In
ovani. Through the research of bioinformatics technology, the process of genetic testing, risk can be larger, and genes
the protein profiles of Leishmania donovani at different are directly identified to interfere with the susceptibility of
developmental stages can be studied. Through this analysis, individual tumors. Big data has been comprehensively
the drug targets against Leishmania donovani can be studied, applied in the treatment, chemotherapy, targeting of diseases,
and corresponding resistant drugs can be studied, promoting and biological treatment. It has also begun to make bold
the development of biopharmaceutics. attempts in the treatment of cancer, to a certain extent. To
With the wide application of bioinformatics technology in reduce mortality, related studies have also analyzed patients
multidisciplinary fields, bioinformatics technology has been with advanced cancer, the patient’s expected life span is
applied in many fields of pathogenic biology research. In the extended, and the patient’s life cycle is extended to give
future, bioinformatics will inevitably be in the field of patho- patients new hope that they can wait for new drug research to
genic biology research and play an important role. But it cure their disease.
requires constant exploration of this field. The application of
bioinformatics technology in pathogenic biology can not
only promote the development of pathogenic biology but 5.5.3 Bioinformatics and the Future
also improve the early diagnosis rate of difficult diseases of Precision Medicine
caused by rare pathogen infections and provide more reliable
data for an accurate diagnosis. It has important significance With the development of biotechnology such as high
for the survival and development of the human population. throughput sequencing technology, genome, transcriptome,
epigenetic group, proteome, metabolome, microbiome, and
other multi-omics sequencing data can be quickly produced
5.5.2 B
ioinformatics and Precision Medical within a few days and widely applied to the research and
Prevention and Treatment clinical applications of precision medicine; these large and
complex data not only constitute the key information needed
Prevention and Treatment of Cardiovascular and for precision medicine, but also they grow faster than any
Cerebrovascular Diseases In recent years, the number of other types of data in the field. At present, the precision med-
patients with cerebrovascular diseases in China has increased icine program has been launched in many countries.
year by year, and the morbidity and mortality of cerebrovas- Scientists from all over the world are working to collect
5 Bioinformatics 59
genomic information of 100,000 and millions of people to ulation level. Therefore, the translational biomedical infor-
build an authoritative and new population health big data matics is particularly important in the context of today’s big
knowledge system. In this era, as a populous country, China’s data and precision medicine and also gives birth to the more
precision medicine urgently needs to establish a genetic advanced technology such as next-generation gene sequenc-
database belonging to China. In 2016, the Ministry of ing technology (NGS) mentioned in Morganti’s published
Science and Technology of China listed “Accurate Medical articles [12].
Research” as a major research project. The Chinese Academy The new paradigm for translational biomedical informat-
of Sciences also launched the Chinese Population Accurate ics will fully integrate molecular data and clinical medical
Medical Research Program in 2016. It is expected to com- data, especially the fine clinical phenotypic information, to
plete the DNA samples of 4000 volunteers and the collection predict disease development and individualized therapeutic
of various phenotypic data within 4 years. In this regard, we effects through precision and modeling. It will lead to the
can foresee that with the development of large cohort development of medicine with surprising results. In the face
research, multi-group big data represented by genomes will of the advent of the aging society, we saw the trend of turn-
continue to be generated. But these big data itself cannot pro- ing from clinical treatment to health management. The appli-
duce value. Its value lies in the disease phenotype and other cation of translational biomedical informatics will play an
information that can be further applied to medical and health important role in predicting the trend of diseases.
decisions with means such as association rule mining, data In short, we believe that the integration and transforma-
mining, and knowledge discovery. Therefore, promoting the tion of bioinformatics will make precision medicine become
transformation of big data into knowledge and building an more mature and perfect in revealing the mysteries of life. It
integrated platform system for the research and application will play an increasingly important role to promote biology
of big gene data is a critical step in the field of precision and medicine to enter a new realm.
medicine to the clinic. It can be applied to early screening,
molecular typing, and individual diseases. It also involves
multiple clinical decision-making scenarios, such as chemo- References
therapy, efficacy prediction, and monitoring. The develop-
ment and research of multi-group precision medicine big 1. Pan S. Clinical molecular diagnostics. Beijing: People’s Medical
Publishing House; 2013.
data also faces challenges such as data sharing and security 2. Pertea M, Kim D, Pertea GM, et al. Transcript-level expression
privacy issues, integrated analysis of multidimensional analysis of RNA-seq experiments with HISAT, StringTie and
omics data, standardization of biometric data analysis meth- Ballgown. Nat Protoc. 2016;11:1650–67.
ods, and interpretation of standardization. Therefore, more 3. Wang M, Ji Z, Wang S, et al. Mechanisms to protect the pri-
vacy of families when using the transmission disequilibrium test
national policy support, scholars in the fields of IT and artifi- in genome-wide association studies. Bioinformatics (Oxford,
cial intelligence, and experts in various fields are needed to England). 2017;33:3716–25.
develop industry standards and ultimately promote the devel- 4. Ma W. Medical molecular biology. Beijing: Higher Education
opment of precision medicine to automation, intelligence, Press; 2008.
5. Chen J, Lin Y, Shen B. Informatics for precision medicine and
standardization, and simplification. healthcare. Adv Exp Med Biol. 2017;1005:1–20.
Around 2008, the international academic community pro- 6. Qiu X. The change of clinical research thinking in the era of big data
posed the concept of translational bioinformatics, which and precision medicine. Chin J Cancer Prev Control. 2017;9:85–9.
aims to apply the data, models, and software developed in 7. Shen F, Cheng Z. Clinical research for hepatocellular carcinoma:
opportunities and challenges in the age of precision medicine and
bioinformatics to the issues of translational medicine. big data. Chin J Pract Surg. 2016;36:599–602.
Furthermore, Chowkwanyun et al. [11] also published a 8. Wang J, Cheng Y, Chen C. Application of bioinformatics in hospital
paper promoting Precision Medicine in public health. infection. Chin J Nosocomiol. 2014;16:4158–60.
Conceptually, bioinformatics is mainly a new discipline 9. Du X, Huang J. Strategy for pathogen novel gene function research.
Chin J Zoonoses. 2016;32:665–9.
formed by the development of the human genome project 10. Wu J, Wang P, Liu Y. Metagenome and detection of animal micro-
and multi-molecular omics technology, while traditional bio- bial pathogens. Chin J Comp Med. 2014;9:72–7.
medical informatics is much broader in connotation than 11. Chowkwanyun M, Bayer R, Galea S. “Precision” public health-
bioinformatics. It is not only the molecular level, but also the between novelty and hype. N Engl J Med. 2018;379(15):1398–400.
12. Morganti S, Tarantino P, Ferraro E, et al. Complexity of genome
image data analysis at the cell and tissue level, the clinical sequencing and reporting: next generation sequencing (NGS) tech-
electronic medical record analysis at the individual level of nologies and implementation of precision medicine in real life. Crit
the patient, and the epidemiological data analysis at the pop- Rev Oncol Hematol. 2019;133:171–82.
Report and Consultation
6
Yongqing Tong
6.1 Overview fundamental source of biodiversity. Nongenetic variation is

caused by the environment and mainly includes variations
Accurate test results depend on high-quality specimens. The caused by external factors such as light and water sources.
collection of specimens plays a very important role in ensur- The mutations in the human genome are mainly divided
ing the accuracy of test results. Without qualified specimens, into three categories: the first type of variation is single
accurate results cannot be detected. Therefore, the first step nucleotide polymorphism (SNP); the second type of varia-
in comprehensive quality control is to ensure high-quality tion is a small deletion or insertion (Indel), which refers to
specimens. In the quality control work before molecular the insertion or deletion of a small fragment sequence occur-
diagnostic analysis, proper processing during specimen ring at a certain position in the genome, and the length is
collection is the key to ensuring sample integrity, checking usually less than 50 bp; the third type of variation is a large
qualitative detection of nucleic acids, and accurate quantita- structural variation, which is more than one type, including
tive detection. Improper handling may result in degradation insertion or deletion of long fragment sequences of 50 bp
of the nucleic acid, or false negatives or low levels of detec- or longer, chromosomal inversion, sequence translocation
tion (in quantitative testing). In addition, specimen collection within chromosomes or between chromosomes, copy num-
must also comply with the requirements of the appropriate ber variation, And some more complex forms of variation,
biosafety guidelines. This chapter focuses on pre-acquisition etc. In order to distinguish from SNP mutations, the second
considerations and acquisition procedures for common types and third types of variation are often referred to as genomic
of specimens in clinical molecular diagnostics. structural variations (SV). Compared with SNPs, SV has a
greater impact on the genome and can be used to explain the
characteristics of human population diversity. Some rare and
6.2 Genetic Variation and Description identical structural mutations are often associated with the
occurrence of diseases (including some cancers) and even
Genetics and variation [1] are the basis of species formation pathogenic causes.
and biological evolution. The genetic material deoxyribonu- Genetic variation is an academic naming of genetically
cleic acid (DNA) is transmitted to the offspring through the altered sequence variations in DNA nucleotide sequences in
parent, so that the offspring present similar traits to the previ- cells. The Human Genome Variation Society (HGVS) has
ous generation, keeping the species relatively stable. At the established a systematic method for naming gene mutations.
same time, there are differences between the parents and the Specific gene mutation naming methods can be found at http://
offspring and offspring, which are not exactly the same. This www.HGVS.org/varnomen. HGVS gene mutation naming
phenomenon is called variation. Variants are classified into guidelines are constantly updated as needed. This article is
heritable and nongenetic variations [2]. Genetically mutated based on the v15.11 version updated in February 2016 [3].
is a variation caused by changes in hereditary material, includ- The naming of human genes mainly includes gene
ing genetic mutations, genetic recombination, and chromo- names and gene symbols. According to the Human Gene
somal variation (Fig. 6.1). Among them, genetic mutation Nomenclature Committee (HGNC) guidelines for human
is the fundamental source of new biological genes and the gene naming (updated 2016), the naming of human genes
should follow six basic principles: first, the uniqueness
of each gene’s symbol; second, the gene symbol is an
Y. Tong (*) abbreviation of the gene name, generally no more than 6 let-
Department of Clinical Laboratory, Renmin Hospital of Wuhan
University, Wuhan, Hubei, People’s Republic of China
ters; third, the gene symbol should be the Latin alphabet or it

62 Y. Tong
Inherited
Father has mutation in all cells Father has germline

and transmits it to his child. Father has mosatic mutation
that affects germline and mosatic mutation.
Child is heterozygous in every cell. Child is heterozygous
somatic cells. Child is
heterozygous in every cell. in every cell.
De novo
Father has mutation in a

Mutation occurs in zygote
single sperm cell and
within first few cell
transmits it to the child.
divisions. Child is
Child is heterozygous in every
heterozygous in every cell.
cell.
Somatic
Child has mosatic mutation that

occurs early in postzygotic Child has mosatic mutation
development and is present in a that occurs later in
percentage of his cells. development and affects
fewer cells(e.g skin cells).
Fig. 6.1 Inherited diagram of some of the ways mutations
is combined with Arabic numerals; fourth, the gene symbol case Latin letters or their combination with Arabic numerals
should not contain punctuation; fifth, the gene symbol should (except C#, ORF# symbols). No Roman numerals are used
not end with G; sixth, the gene symbol does not involve other (the Roman numbers used in the past are to be changed to
species. equivalent Arabic numerals); ② gene symbols are applied in
Gene full name naming rules: ① the beginning of the name italics when writing. But in the directory exception; ③ Greek
applies lowercase letters, but there are three exceptions, that letters are not used as gene symbols. All Greek letters used in
is, the name of the disease, the phenotype, or the initials of the past should be converted to Latin letters; ④ gene names
the initials; ② if there is an alias, it should be included in the prefixed with Greek letters should be converted to equivalent
name, and brackets; ③ if it is the name of another species, it Latin letters and placed at the end of the gene symbol, genes
must be written at the end and marked with brackets. Gene with similar properties can be arranged in alphabetical order;
symbol naming rules: ① human gene symbols are upper- ⑤ punctuation not be used (except for HIJA immunoglobulin
6 Report and Consultation 63
and T cell receptor gene symbols can be used with sub-words); ber or the gene symbol recommended by the Human Genism
⑥ gene symbols usually do not represent selective transcripts, Organization (HUGO) Gene Nomenclature Committee,
but when a group has multiple small coding sequences to form the location of the mutation, and the type of variation. For
a variety of different large gene products, these small coding example, “NG_007938.1:g.12083G>A”, “NG_007938.1” is
sequences can be represented by different symbols; ⑦ the rep- the nucleic acid sequence acceptance number and version,
resentation of tissue specificity or molecular weight should be “g.12083” indicates the position in the nucleic acid sequence,
avoided. ⑧ Certain letters or combinations of letters should and “G > A” indicates that the original base is G The variant
be avoided as the pre- and post-suffix of the gene symbol to base is A. The HUGO gene symbol is used to describe, for
try to give a specific meaning; ⑨ the symbol of the oncogene example, “GJB2:c.76A>C”. Such a naming method facili-
corresponds to the retrovirus homologous oncogene, but the tates finding the location in the gene sequence in which it is
gene symbol does not have a “v-” or “c-” prefix. In addition, located. If the mutation occurs only in a sequence or gene,
for some customary gene names, the previous forms, such as the nucleic acid sequence or gene symbol can be omitted
survivin and p53, can be used in lowercase, italic represent after the first occurrence, but if there are different sequences
genes, standard bodies represent proteins; BCL-2 or Bcl-2, or genes that mutate, each description needs to be written.
c-Myc, mixed case, italics represent genes and standard bod-
ies represent proteins.
6.2.3 Types of Variant Sequences
6.2.1 The Level of Sequence Variation To avoid confusion you need to indicate the sequence type
when describing sequence variations. g represents the genomic
The goal of HGVS’s norm is to make all variant descriptions sequence, c represents the coding DNA, m represents the
unique, stable, meaningful, memorable, and unambiguous. mitochondrial sequence, r represents the RNA sequence, and
All mutations occur eventually with changes in DNA levels p represents the protein sequence. For example, g.476A>T,
that cause changes in the corresponding RNA or protein lev- c.76A>T, m.8993T>C, r.76a>u, p.Lys76Asn.
els. Therefore, the most basic rule in describing the mutation
is that when the first description of the mutation occurs, the
DNA level variation must be written, and the corresponding 6.2.4 E
xpression of Variant Types Specific
RNA and protein variations can be described in parenthe- Abbreviations Are Used to Describe
ses, for example, “c.78G>C(p. Trp26Cys).” The four bases Different Types of Sequence Variations
AGCT involved in DNA variation need to be capitalized,
while the agcu in RNA needs to be lowercase. Amino acids in ① “>” indicates base substitution (DNA and RNA levels);
protein level are recommended with a three-letter abbrevia- e.g., g.123456G>A, r.123c>u. Substitution at the protein
tion because single-letter abbreviations are ambiguous (e.g., level is described as p.Ser321Arg.
Ala, Arg, Asn, and Asp, all of which begin with the A letter, ② “_” is used to define the range of the variant base, e.g.,
Gln, Glu, and GLy, all of which begin with the G letter). “c.76_78delACT” indicates the deletion of the 76–78 base
When several variations are expressed at the same time, they (ACT) of the coding DNA.
should be listed. Variations are clearly expressed at the three ③ “del” represents a base deletion, such as “c.135_137delTTA”
levels of DNA, RNA, and protein, and changes in RNA and indicating a deletion of the coding DNA 135–137 base (TTA).
protein levels should be clearly demonstrated experimentally “ins” represents a base insertion. For example, “c.76_77insG”
or theoretically. When the mutation occurs in patients with indicates that the base G is inserted between 76 and 77 bases of
recessive genetic disease, it should also indicate whether the the coding DNA.
mutation is homozygous or heterozygous. ④ “dup” represents a repeat of the same base (repetitive
insertion is described as a repeat, but cannot be expressed
by an insertion variant, e.g., the sequence ACTTTGTGCC
6.2.2 Content of the Variant Description mutation to ACTTTGTGGCC cannot be described as
c.8_9insG, but should be described as c.8dupG).
Human Genome Variation Society (HGVS), is a nongovern- ⑤ “delins” or deletion/insertions (indels) represent inser-
mental academic organization whose website is available at tional deletions, e.g., p.Cys28_Lys29delinsTrp represents
http://www.hgvs.org/. The rule for the HGVS nomenclature 3 bases at codon 28 (encoding cysteine Cys) and codon 29
SNP method is tantamount to indicate the referenced nucleic (encoding lysine Lys). Deletion of the base results in the
acid sequence number (Reference Sequence, RefSeq) replacement of these two amino acids by tryptophan.
and the position of the SNP in the nucleic acid sequence. ⑥ “inv” stands for inversion, such as c.203_506inv, indi-
The description of the nucleic acid sequence variation cating that a total of 304 nucleotides have been inverted from
includes three parts, the cited nucleic acid sequence num- 203 to 506.
64 Y. Tong
⑦ “con” stands for conversion, e.g., g.123_678con Table 6.1 Common nucleotide representation information table
NG_012232.1:g.9456_10011, indicating that the 9546 Symbol Meaning Description
to 10011 nucleotide sequence in the reference sequence A A Adenine
NG_012232.1 in Genbank replaces the nucleotide sequence B C, G or T Not-A (B follows A in alphabet)
of 123 to 678 in the reference sequence. C C Cytosine
⑧ “fs” means frame shift; e.g., p.Arg456GlyfsTer17 (or D A, G or T Not-C (D follows C in alphabet)
G G Guanine
p.Arg456Glyfs*17).
H A, C or T Not-G (H follows G in alphabet)
⑨ “[]” represents an allele, e.g., c. [76A>C; 83G>C] indi-
K G or T Keto
cates that one gene in a chromosome has both c.76A>C and M A or C aMino
c.83G>C mutations. c. [76A>C]; [83G>C] indicates that N A, C, G or T aNy
one variation originates from the mother, and one variation R A or G purine
originates from the father, i.e., 76A>C mutation occurs in S G or C Strong interaction (3 H-bonds)
one chromosome, and 83G>C mutation occurs in the other T T Thymine
chromosome. The specific position of “()” for the occurrence V A, C or G Not-T/not-U (V follows U in alphabet)
of mutation is uncertain, and the possible range is indicated W A or T Weak interaction (2 H-bonds)
in parentheses. For example, c.(67_70)insG represents the Y C or T pYrimidine
Used in alignments only
insertion of a base G at a position from 67 to 70 bases.
X A, C, G or T Masked nucleotide
➉ “ext” indicates elongation, p.*110Glnext*17 (or – None Gap of indeterminate length
p.*110Qext*17), indicating that the stop codon variation of
110 is Gln, and the amino acid sequence is extended by 17
amino acids (including Gln). 6.3.1.2 The Representation of the Amino Acid Sequence
Follows the Following Rules
1. Genomic reference sequence: The nucleotide number of
6.2.5 Reference Sequence the genomic reference sequence is completely random,
and the first base of the reference sequence stored in the
The reference sequence code is authoritative and unique database is programmed as l, and then pushed backward,
included in the National Center for Biotechnology Information without “+”, “−” and other prefixes. The sequence should
(NCBI). Wherein the prefix “NM_” is denoted as an mRNA cover all nucleotides of the sequence of interest (gene),
sequence, “NP_” denotes a polypeptide sequence, and “NG_” starting with the 5′ promoter region of the gene (Fig. 6.2).
denotes a genomic sequence. The genomic reference sequence 2. Coding DNA reference sequence: The code starts at

should list the complete gene sequence, including the 5′ and 1, does not start at 0. The number 1 corresponds to the
3′ untranslated regions (UTRs). When a segment of a coding base A in the translation start codon ATG (T is 2, G is
DNA reference sequence is used to describe a mutation, the 3, and the translation sequence is pushed backward). The
appropriate transcript should be selected and the starting tran- translation start codon ATG upstream (5′ end) base num-
scription point of the transcript should be clear, such as selecting ber is −l, −2, and is pushed forward. The base number
the most common transcript, or the largest known transcript, or downstream of the translation stop codon (3′ end) is *l,
having tissue. Specific editing of transcripts. When a reference *2, which is pushed down in sequence. The intron num-
sequence has multiple transcriptions, the most comprehensive ber is numbered from the two sides to the middle of the
version of the NCBI database is selected. adjacent exon number plus (upstream) or minus (down-
stream) intron relative to the position of the exon. For
example, the l intron in Fig. 6.1 is located between the 1st
6.3 Naming Rules exon (base number 1–12) and the 2nd exon (base number
13–88), and the intron number is 12+1, 13-1, 12+2, 13-2,
Sequence variations can occur at the level of DNA, RNA, or push in the middle (Fig. 6.3).
protein, and we need to describe the specific rules of varia- 3. Base substitution: The replacement of a single nucleotide
tion from these three levels. is indicated by the symbol “>”. The presentation format
is “prefix” “position_substituted” “reference_nucleo-
tide” > “new_nucleotide”. “prefix” indicates the sequence
6.3.1 Specific Rules for DNA Levels type, such as g for the genomic sequence and c for the
coding DNA. “position_substituted” is the position where
6.3.1.1 the base substitution occurs. “reference_nucleotide” ref-
The numbering of nucleotides in DNA (Table 6.1) relates to erence base. “>” indicates replacement. “new_nucleo-
the precise localization of the mutated DNA and is critical in tide” is a newly mutated base. For example, c.85G>C
the description of the variation. describes the mutation of nucleotide 85 at the 85th posi-
Poly A-addition site

Splice acceptor site
Splice acceptor site

Transcription start
Transcription start
Transcription stop
Splice donor site
Splice donor site

(cap-site)
3’ gene flanking
First exon Intron 1 Exon 2 Intron 2/ .../intron 4 Last Exon
5’ UTR 3’ UTR
.catgcatgc agt.//.gaccATGGACG.//.CAG gtgagt.//.tttcag GCATT.//.TCG gtgagt...........//..........tttcag GCATG.//.CATGAcaatca.//.atgc atgcatgc..

1 271 301 313 413 489 1601 1631 1851 2000
genomic reference sequence

coding DNA reference sequence
–31 –30 –1 1 12 12+1.... 13–1 13 88 88+1 301–1 301 330 *1 *320 *321
–300 *470
protein reference sequence
5 30 42 101
1 110
coding sequence
Fig. 6.2 Schematic diagram of nucleotide naming in the genome reference sequence
Fig. 6.3 Schematic diagram Coding DNA reference

of nucleotide naming in the –93 1 351 *223
coding sequence of coding
DNA and non-coding DNA
–45 –44 –45 –44 *96 *97
gtgag..//..tttcag gtgag..//..ttctag gtgag..//..ctttag

–45+1 –44–1 187+1 188–1 *96+1 *97–1
–45+2 –44–2 187+2 188–2 *96+2 *97–2
–45+3 –44–3 187+3 188–3 *96+3 *97–3
Noncoding DNA reference

1 667
49 50 280 281 540 541
gtgag..//..tttcag gtgag..//..ttctag gtgag..//..ctttag

49+1 50–1 280+1 281–1 540+1 541–1
49+2 50–2 280+2 281–2 540+2 541–2
49+3 50–3 280+3 281–3 540+3 541–3
tion of the coding DNA to C; and c.-14A>C indicates the to C mutation occurs in the intron region of nucleotides
14th position before the 5′ end of the start codon ATG 88–89 of the coding DNA (variation point), two base
of the coding DNA. A>C variation occurs at the position positions upstream of the 89th nucleotide; c.*46T>A
of the nucleotide; likewise, c.89-2A>C indicates that A indicates that a substitution of T > A occurred at the
66 Y. Tong
base of the 3′ end of the translation stop codon. Two or or insertion of nucleotide 7 at the genomic reference
more consecutive base substitutions are indicated by the sequence. ACTTTGTGCC becomes ACTTTGTGTGCC,
symbol “delins”. For example, c.112_117delinsTG (or described as g.7_8dup (or g.7_8dupTG, instead of
c.112_117delAGGTCAinsTG) indicates that the nucle- g.5_6dup, or g.8_9insTG), indicating that TG repeats
otides 112–117 (AGGTCA) of the coding DNA were occur in the TG tandem sequence. ACTTTGTGCC
replaced by TG. becomes ACTTTGTGTGTGCC, described as g.7_8[4]
4. Nucleotide deletion: Adding the symbol “del” after
(or g.5_6[4], or g.5TG[4], instead of g.7_10dup),
the start and end of the deletion indicates a nucleotide indicating an additional second TG in the TG repeat
deletion in the format “prefix” “position(s)_deleted” sequence variation region. C.123+74TG[3_6] (or
“del”, “position(s)_deleted” indicates the occurrence of c.123+74_123+75[3_6]) indicates that the TG dinucle-
the deleted nucleotide Location or range. For example, otide repeat occurs at position 74 of the intron 74 after
c.7_8del (or c.7_8delTG) indicates the deletion of the encoding the DNA for 3 to 6 times.
nucleotide TG at the 7th and 8th nucleotides of the coding 7. Chromosomal translocation: The expression of trans-

DNA (sequence ACTTTGGCCC becomes ACTTTGCC); location is seen in cytogenetics, such as t(X; 4) (p21.2;
c.88_?_923+?del indicates the 88th nucleotide at the q35), i.e., the equilibrium translocation occurs between
coding DNA A deletion occurs from an unknown posi- x chromosome and chromosome 4, and the short crack
tion in the 5′ end intron to an unknown position in the points are Xp21.2 and 4q35. At the molecular level, a
3′ end of the nucleotide at position 923; it is worth not- specific sequence is added to make the breakpoint more
ing that the alignment of the nucleotide sequence is from precise. These translocation breakpoints should be sub-
an exon (or intron) the largest alignment between the mitted to the nucleic acid sequence database with their
two ends, the sequence is missing, e.g., ACTTTGTGCC serial number and version number. The database includes
becomes ACTTGCC, it must be described as c.5_7del the Genbank, European Molecular Biology Laboratory
(c.5_7delTGT, instead of c.4_6delTTG); the sequence (EMBL), and DNA Data Bank of Japan (DDJB). For
TCACTGTCTGCGGTAATC becomes TCACTG example, t(X; 4)(p21.2; q35) (c.857+101_857+102) indi-
CGGTAATC C.7_10del (c.7_10delTCTG) instead cates that p21.2 of the X chromosome and translocation
of c.4_7del (c.4_7delCTGT); AAAGAAGAGGAG of q35 of chromosome 4 occur. The cleavage site occurs
becomes AAAG GAG is described as c.5_9del (or between the 101 intron and the 102 intron after the 857
c.5_9delAAGAG) instead of c.3_7delAGAAG; intron nucleotide encoding the DNA.
sequence is deleted, e.g., ctttagGCATG It becomes cttag-
GCATG described as c.301-3delT instead of c.301-5delT.
5. Nucleotide insertion: The symbol “ins” may indi-
6.3.2 Detailed Rules for RNA Levels
cate the insertion of a nucleotide in the format “pre-
fix” “positions_flanking” “ins” “inserted_sequence”, The variation in RNA levels is the same as the variation
and “positions_flanking” indicates the position of two in protein levels, which is due to changes in DNA levels.
nucleotides flanking the insertion site. For example, Therefore, RNA level variation or protein level variation
c.56_57insG indicates insertion of G between nucleo- may be the result of experimental analysis or may be derived
tides 56 and 57 of the coding DNA; c.123+54_123+55 by deriving from DNA level variation. If the variation is
ins AB012345.2:g.76_420 indicates intron c.123+54 in derived from the experimental analysis, the description is
the coding DNA A 345 nucleotide sequence was inserted basically the same as the DNA variation description. Use
into 123+55, and the sequence was nucleotide sequence the symbol “r” and lowercase bases to represent the RNA
76–420 of the gDNA sequence in the reference sequence sequence (Table 6.2). The format is “prefix” “position_sub-
AB012345.2 in GenBank. stituted” “reference_nucleotide” > “new_nucleotide”, and
6. Nucleotide repeat: The symbol “dup” may indicate
“prefix” refers to the sequence type. “r” represents the RNA
repeated insertion of nucleotides in the format “prefix” sequence, and “position_substituted” is the position at which
“position(s)_duplicated” “dup”, and “position(s)_dupli- base substitution occurs. “reference_nucleotide” refers to a
cated” indicates the position of a repeating base or base reference base. “>” indicates replacement. “new_nucleotide”
cluster. For example, the sequence ACTCTGTGTCC refers to a newly mutated base.
becomes ACTCTTGTGTCC, described as g.5dupT (or For example, r.78u>a represents the 78th nucleotide U
g.5dup, but not g.5_6insT), indicating that a repeat or becomes A. However, DNA variation can affect the tran-
insertion occurs at nucleotide position 5 of the genomic scription process, resulting in two or more transcripts. In this
reference sequence. AGACTTTGTGCC becomes case, you can separate each transcript with “,” in “[]”. For
AGACTTTTGTGCC, described as g.7dupT (or g.7dup, example, r.[=, 73_88del], indicating that c.76A>C results in
instead of g.5dupT or g.7_8insT), indicating a repeat two RNA molecules, one is the normal transcript (r.=) and
Table 6.2 Table of nucleotide information in RNA 6.3.3.2 Silent Changes

Symbol Meaning Description Silent changes do not cause changes in protein levels. The
a A Adenosine expression p. (Leu54Leu) or p. (L54L) is wrong and should
b c, g or u Not-a (b follows a in alphabet) contain a representation of the level of DNA, as expressed
c C Cytidine as c.162C>G (p.(Leu54=), if expressed as p. (Leu54=), then
d a, g or u Not-c (d follows c in alphabet) indicates that there is no expected effect on protein levels.
g G Guanosine
h a, c or u Not-g (h follows g in alphabet)
k g or u Keto
6.3.3.3 Substitutions, Missense Changes
m a or c Amino Substitutions, missense changes, indicating that an amino
n a, c, g or u Any acid is replaced by another amino acid, represented by “pre-
r a or g Purine fix,” “amino_acid,” “position,” “new_amino_acid.” “prefix”
s g or c Strong interaction (3 H-bonds) refers to the sequence type. p represents a protein sequence.
u U Uridine Such as p.Arg54Ser. “amino_acid” is a reference amino
v a, c or g Not-u (v follows u in alphabet) acid. “position” refers to the location at which an amino acid
w a or u Weak interaction (2 H-bonds) substitution occurs. “new_amino_acid” refers to a newly
y c or u Pyrimidine mutated amino acid. As for p.Trp26Cys, the “>” symbol used
in DNA or RNA sequences is not used in the expression.
the other is the nucleotide deletion from 73 to 88, r.[76a>c,
73_88del], indicating that c.76A>C in the coding DNA 6.3.3.4 Amino Acid Deletion
sequence results in two RNA molecules, one carrying a Like the nucleotide deletion at the DNA level, it is rep-
76a>c variant and one being a nucleotide deletion from 73 to resented by the symbol “del” in the format of “prefix,”
88.,r.[=,88_89ins88+l_88+l0;88+2u>c], indicating that the “amino_acid(s) + position(s)_deleted” “del”. For example,
mutation at the coding DNA level c.88+2T>C results in two p.Lys2del indicates that the second amino acid Lys(K) of
RNA molecules, one being a normal transcript (r.=) and the the protein sequence MKMGHQ is deleted (the sequence
other is a nucleotide insertion from the intron 88+l to 88+10 becomes MMGHQ), while p.Cys28_Met30del indicates
with r.88+2t>c mutation. the deletion of the third amino acid at positions 28 to 30;
if the deletion is accompanied by the insertion, the sym-
bol “delins” is used. Indicates that the format is “prefix,”
6.3.3 Detailed Rules of Protein Levels “amino_acid(s) + position(s)_deleted,” “delins,” “inserted_
sequence.” For example, p.Cys28delinsTrpVal indicates that
Sequence variations in protein levels are described by the the 28th amino acid Cys is deleted and two amino acids of
symbol “p” and amino acids in a three-letter abbreviation TrpVal are inserted at this position. p.Cys28_Lys29delinsTrp
(initial capitalization) (Table 6.3), each amino acid encoded indicates that two amino acids Cys28 and Ly29 at positions
by three nucleotides (Table 6.4). Because sequence variation 28 to 29 are deleted and an amino acid Trp is inserted. p.
at the protein level may be the result of experimental analysis (Pro578_Lys579delinsLeuTer) indicates that the two amino
or may be derived from DNA level variation, this should be acids Pro578 and Lys579 at positions 578–579 are deleted
pointed out in the description at first. If it is an experimen- and two amino acid Leu and Ter deletions are inserted and
tally observed protein change, its protein sequence variation an amino acid Trp is inserted.
is directly described, rather than trying to mix it with DNA
level variation. 6.3.3.5 Frameshift Mutation
The frameshift mutation causes codon shift due to mutation
6.3.3.1 Amino Acid Coding at the DNA level, and is represented by the first amino acid
The protein sequence should be the original translation prod- after the addition of the symbol “fs” or “fsX#”, the latter
uct (including the signal peptide sequence) rather than the description being more specific, indicating the length of
posttranslationally modified mature protein sequence. The the stop codon, the format is “prefix” “amino_acid” posi-
translation initiation codon methionine (metheine) is coded tion “new_amino_acid” “fs” “Ter” “position_termination_
as l, written as Met l (or Ml), not as metl or Metl). The amino site.” For example, p.Arg97ProfsTer23 (also abbreviated
acids upstream of the translation initiation point, such as as p.Arg97fs, indicating that the amino acid Arg at position
nucleotides, are numbered −1, −2, −3⋯⋯, such as..., Gln-2, 97 as the first amino acid affected by the frameshift muta-
Thr-1; the amino acid downstream of the translation termination becomes GIy, generating a new reading frame until
tion point is *1,* 2, *3⋯⋯, such as Gln*1, Ser*2, ...; muta- the termination password is reached after 15 amino acids.
tion causes intron translation, numbered with nucleotides, Sub-(X16). It can be seen that the description does not spec-
such as Val4+1, Ser4+2, ..., Phe5-2, and Gln5-1. ify the specific amino acid change (deletion, insertion) from
68 Y. Tong
Table 6.3 Amino acids common information

One letter Three letter
code code Amino acid Possible codons Systemic name Formula
A Ala Alanine GCA, GCC, GCG, GCT 2-Aminopropanoic acid CH3-CH(NH2)-COOH
B Asx Aspartic acid AAC, AAT, GAC, GAT
or asparagine
C Cys Cysteine TGC, TGT 2-Amino-3- HS-CH2-CH(NH2)-COOH
mercaptopropanoic acid
D Asp Aspartic acid GAC, GAT 2-Aminobutanedioic acid HOOC-CH2-CH(NH2)-COOH
E Glu Glutamic acid GAA, GAG 2-Aminopentanedioic acid HOOC-[CH2]2-CH(NH2)-COOH
F Phe Phenylalanine TTC, TTT 2-Amino-3- C6H5-CH2-CH(NH2)-COOH
phenylpropanoic acid
G Gly Glycine GGA, GGC, GGG, Aminoethanoic acid CH2(NH2)-COOH
GGT
H His Histidine CAC, CAT 2-Amino-3-(1H-imidazol- CH2-CH(NH2)-COOH
4-yl)-propanoic acid
HN N
I Ile Isoleucine ATA, ATC, ATT 2-Amino-3- C2H5-CH(CH3)-CH(NH2)-COOH

methylpentanoic acid
K Lys Lysine AAA, AAG 2,6-Diaminohexanoic acid H2N-[CH2]4-CH(NH2)-COOH
L Leu Leucine CTA, CTC, CTG, CTT, 2-Amino-4- (CH3)2CH-CH2-CH(NH2)-COOH
TTA, TTG methylpentanoic acid
M Met Methionine ATG(translation 2-Amino-4-(methylthio) CH3-S-[CH2]2-CH(NH2)-COOH
initiation) butanoic acid
N Asn Asparagine AAC, AAT 2-Amino-3- H2N-CO-CH2-CH(NH2)-COOH
carbamoylpropanoic acid
P Pro Proline CCA, CCC, CCG, CCT Pyrrolidine-2-carboxylic
acid
COOH
N
H
Q Gln Glutamine CAA, CAG 2-Amino-4- H2N-CO-[CH2]2-CH(NH2)-COOH
carbamoylbutanoic acid
R Arg Arginine AGA, AGG, CGA, 2-Amino-5- H2N-C(=NH)-NH-[CH2]3-CH(NH2)-
CGC, CGG, CGT guanidinopentanoic acid COOH
S Ser Serine AGC, AGT, TCA, TCC, 2-Amino-3- HO-CH2-CH(NH2)-COOH
TCG, TCT hydroxypropanoic acid
T Thr Threonine ACA, ACC, ACG, ACT 2-Amino-3- CH3-CH(OH)-CH(NH2)-COOH
hydroxybutanoic acid
U Sec Selenocysteine TGA H2N-CH(COOH)--CH2-SeH
V Val Valine GTA, GTC, GTG, GTT 2-Amino-3-methylbutanoic (CH3)2CH-CH(NH2)-COOH
acid
W Trp Tryptophan TGG 2-Amino-3-(lH-indol-3- CH2-CH(NH2)-COOH
yl)-propanoic acid
N
H
X Xaa Unknown or NNN
other
Y Tyr Tyrosine TAC, TAT 2-Amino-3-(4-
hydroxyphenyl)-propanoic HO CH2-CH(NH2)-COOH
acid
Z Glx Glutamic acid

* *(Ter) Termination TAA, TAG, HGVS addition (V2.0)
TGA(translation
termination)
Used in alignments only
– – Gap of
indeterminate
length
Table 6.4 Amino acid codon Nucleotide position in codon

table
first second third
T C A G
TTT-Phe TCT-Ser TAT-Tyr TGT-Cys T
TTC-Phe TCC-Ser TAC-Tyr TGC-Cys C

T
TTA-Leu TCA-Ser TAA-Ter TGA-Ter A
TTG-Leu TCG-Ser TAG-Ter TGG-Trp G
CTT-Leu CCT-Pro CAT-His CGT-Arg T
CTC-Leu CCC-Pro CAC-His CGC-Arg C

C
CTA-Leu CCA-Pro CAA-Gln CGA-Arg A
CTG-Leu CCG-Pro CAG-Gln CGG-Arg G
ATT-Ile ACT-Thr AAT-Asn AGT-Ser T
ATC-Ile ACC-Thr AAC -Asn AGC-Ser C

A
ATA-Ile ACA-Thr AAA-Lys AGA-Arg A
ATG-Met ACG-Thr AAG-Lys AGG-Arg G
GTT-Val GCT-Ala GAT-Asp GGT-Gly T
GTC-Val GCC-Ala GAC-Asp GGC-Gly C

G
GTA-Val GCA-Ala GAA-Glu GGA-Gly A
GTG-Val GCG-Ala GAG-Glu GGG-Gly G
the frameshift mutation to the stop codon. For example, a acid(s)+position_repeat_unit” “[""copy_number""]”, where
sequence, from the 30th amino acid Leu to the 42th amino “amino_acid(s)+position_repeat_unit” represents the first
acid Cys, results in the frameshift mutation, described as repeated copy of the amino acid position or range, e.g.,
p.Leu30fs or p.Leu30SerfsX3, is not described as p.Leu30_ p.Arg65_Ser67 [12]. The format of the amino acid repeat
Cys42delinsSerfsX3. For the above mentioned, do not try and insertion description is p.Lys2_Met3insGlnSerLys,
to mix protein sequence variation and DNA level variation, p.Cys28delinsTrpVal or p.Gly4_Gln6dup.
for example, CTCAGAACGATATAG (Leu-Arg-Thr-Ile-X)
becomes CTAGAACGATATAG (Leu-Glu-Arg-X), and the
DNA level variation should be described as c.3delC. If the 6.3.4 G
ene Pharmacology Genotype
DNA level is combined, the protein level variation should Terminology
occur in the first amino acid. It is easy to misreport as
p.Leu1LeufsX4, but regardless of DNA level variation, in The most widely used nomenclature describes genetic
fact, the amino acid change is from the second amino acid, pharmacological genotypes that differ from other genetic
described as p.Arg2GlufsX3. tests, with the metabolic-related gene cytochrome p450
family as an example, the Human Cytochrome P450 (CYP)
6.3.3.6 Amino Acid Insertion, Repeat Allele Nomenclature Committee (http://www.Cypalleles.
The description rules for amino acid repeats and insertions ki.se) is recommended to be named with “*”, as shown in
are basically the same as those for DNA-level nucleotides. Fig. 6.4. In this system, the most common allele is desig-
The format of amino acid repeats is “prefix” “amino_ nated as “* 1”.
70 Y. Tong
1 2 3 4 5 genetic level and promote the new development of diagno-

Cytochrome P450 2 D 6 *4 sis and prevention technology. The analysis of endogenous
gene variation and the study of exogenous gene invasion has
1 led to the development of technical methods for diagnosing
Superfamily
diseases at the molecular level. Through continuous develop-
2 ment and improvement, this method has played an important
Family
role in the diagnosis of infectious diseases, genetic diseases,
and chronic diseases as well as tumors.
3
Subfamily Genetic diagnosis is the application of molecular biology
techniques to the diagnosis of human conditions and diseases at
4
Isoenzyme the DNA or RNA level by detecting the presence of a causative
gene (endogenous or exogenous), genetic defects, or abnormal
5 expression. The essence of its theoretical basis is to apply molec-
Allele variant
ular biology and molecular genetics theory and technology to
Fig. 6.4 Naming convention for cytochrome P450 CYP2D6 allele examine and analyze whether a certain or a certain group of
genes in a subject is normal or not, so as to diagnose the subject.
After decades of development and improvement, the gene
6.3.5 Other diagnosis technology based on the detection of nucleic acid
molecules has become a systematic molecular level detection
For more complex mutations, refer to the HGVS recom- technology platform more and more widely used in biomedi-
mended naming conventions to address the naming of other cal researches and clinical fields. Among them, high through-
complex mutations. put sequencing technology, also known as next- generation
sequencing (NGS), can obtain a large amount of genetic
variation information about disease populations and the gen-
6.4 pplication of Gene Mutation
A eral population, and explain the etiology and development of
in Disease Diagnosis human diseases from the overall genetic level. Exploring effec-
tive prevention and treatment measures is of great significance,
6.4.1 Genetic Variation and Genetic and it also brings clinical medicine to the era of individual
Diagnosis genome sequencing and personalized medicine. Genetic test-
ing has increasingly been used in clinical decision-making,
Genetic variation refers to the changes in the genetic material such as preventive mastectomy, cardiac defibrillator implan-
of an organism that can be passed on to the next generation. tation, oncology, and prenatal diagnosis. Misinterpretation of
It is this variation that causes organisms to embody genetic the meaning of sequence variation can have a serious impact
diversity at different levels. Genetic diversity is the material on patients. The rational and effective use of large genetic
basis for the survival and development of human society. The information will greatly promote the development of human
study of genetic diversity is important for both the conser- health. However, incorrect determination of the pathogenicity
vation of biodiversity and the sustainable use of biological of sequence variation can also have serious consequences for
resources, as well as the food supply of the future world [4]. patients, leading to misjudgment in treatment, prognosis, or
There are many ways of genetic variation in the genome, reproductive counseling. Given the potentially huge impact of
including microscopically visible chromosome inversions sequence variation detection techniques on medicine, it is nec-
to single nucleotide mutations. With the development of essary to distinguish true pathogenicity-related variability from
genomics research, genetic variation information has become numerous non-pathogenically relevant potential functional
more comprehensive, including single nucleotide polymor- variants from the variability of the human genome [1].
phism (SNP), insert and deletion of small fragments (Indel),
structural variation (SV), copy number variation (CNV), and
transposon variation. 6.4.2 Application of Genetic Variation
The development of molecular biology technology has Detection in Disease Diagnosis
laid a foundation for people to understand diseases from
the level of genetic material base nucleic acid molecules. 6.4.2.1 Molecular Diagnosis of Mendelian
The role of genetic factors in disease development has been Genetic Disease
paid more and more attention. With the rapid development Mendelian genetic disease is usually a relatively rare dis-
of molecular biology and molecular genetics, people gradu- ease with a strong genotype and phenotype correlation, and
ally understand the mechanism of disease occurrence at the a high rate of disability. Most Mendelian genetic diseases
are identified and diagnosed only by clinical features, and no wide association study (GWAS) is one of the important
relevant candidate genes and mutant genes have been found. components of human complex disease research, which is to
Linkage analysis and candidate gene screening are tradi- detect the genetic association between genome-wide genetic
tional methods for studying the pathogenesis of Mendelian variation and observed traits at the population level. The
genetic disease. They are usually time-consuming and labor- traditional GWAS has achieved a lot of results by relying
intensive, have low success rates, and are incapable of diag- on array technology and found some common variations of
nosis of incomplete penetrance, new mutations, sporadic, genetic susceptibility. However, the variation studied by this
and rare diseases. In addition, traditional methods have lim- method usually has a slight effect on the phenotype. The het-
ited research capacity for Mendelian genetic diseases with erogeneity of the results of different studies on some traits is
genetic heterogeneity (cause mutations occur in different weak, and the function of the significantly associated genetic
genes) and phenotypic heterogeneity (difficult clinical clas- variation site is difficult to explain, and clinical diagnosis
sification and phenotypic differentiation) [4]. and risk stratification are almost impossible. Those limits its
clinical application. NGS technology provides high through-
6.4.2.2 Prenatal Diagnosis and Prenatal put sequencing data, making it possible to study complex
and Postnatal Care diseases for rare mutations, low-frequency mutations, epi-
Prenatal diagnosis and screening can effectively reduce birth genetic abnormalities, etc.
defects [5]. Traditional prenatal diagnosis is mostly inva-
sive. Early detection of fetal chromosomal abnormalities 6.4.2.4 Diagnosis and Treatment of Tumors
and other genetic abnormalities by separation of maternal The tumor genome is significantly different from the nor-
blood nucleated cells. Recently, studies have found that not mal cell genome. The tumor genome is usually highly het-
only fetal nucleated cells, but also fetal free DNA (cffDNA) erogeneous and can contain multiple clones of the genomic
exist in peripheral blood of pregnant women, thus promoting type. Comparison of tissue and cancer tissue is critical
the emergence of noninvasive prenatal tests (NIPT) screen- to identifying somatic mutations. NGS technology has
ing technology characterized by cffDNA detection. In addi- achieved genome-wide, exome, and transcriptome sequenc-
tion, sequence variation detection techniques are also used ing of tumor samples, which greatly promoted the study of
in assisted reproductive medicine. The meiosis, chromo- acquired mutations in tumor cells. Tumor sequencing often
some recombination and aneuploidy status of human single has a large amount of data and a wide variety of mutations.
sperm cells were analyzed by multiple annealing and loop- A noninvasive method called liquid biopsy has emerged in
ing based amplification cycles (MALBAC)-based sequenc- recent years. The method uses blood circulating DNA instead
ing techniques. of tissue biopsy DNA for molecular detection of tumors.
For the techniques of routine prenatal diagnosis, the Studies have found that circulating system-free tumor DNA
method of cell culture and karyotyping of villus tissue has is present in serum/plasma of cancer patients and fluctuates
high requirements on specimens and high failure rate; the with changes in solid tumors and chemotherapy responses,
detection range of probes by fluorescence in situ hybridiza- suggesting the feasibility of monitoring cancer status by cir-
tion is limited. The NGS technology has the characteristics culating DNA. Detection of circulating tumor DNA can be
of high throughput, wide detection range, fast detection used to assist in imaging examinations, with low cost and
speed, noninvasiveness, etc. It is widely used in prenatal low side effects.
diagnosis. However, there are still many problems, such as Sequence variation detection can be used to find thera-
the difficulty in successfully applying twins, multiple tires, peutic targets, drug resistance genes, molecular markers, etc.
expensive testing costs, and difficult to interpret the results,However, there are still many problems in this method, for
which limits the application of high throughput sequencing. example, the amount of circulating tumor DNA will change
At present, the prenatal diagnosis of NGS is basically lim- in different disease stages, different types, different metasta-
ited to diseases with abnormal chromosome multiples, such ses and different treatments, and a small amount of circulat-
as 21-trisomy syndrome. Only a small part of the peripheral ing tumor DNA cannot guarantee the sensitivity of detection.
blood-free DNA of pregnant women comes from fetal DNA, The types of mutations detected by various types of sequence
and the content is very low. Therefore, high sensitivity and variation detection techniques are still limited. Sequence
specific detection are needed to obtain fetal whole genome variation detection also provides an effective means for the
data by increasing the depth of sequencing [6]. research and detection of drug targets, providing a possi-
bility for individualized medical treatment of patients. For
6.4.2.3 Molecular Genetic Testing of Complex example, drug-targeted therapy for patients with non-small
Diseases cell lung cancer, for a subset of NSCLC patients with epi-
The heritability of complex diseases is low, and is affected by dermal growth factor positive (about 15%), EGFR-targeted
genetic susceptibility and environmental factors. Genome- tyrosine kinase inhibitors (TKIs) can effectively improve
72 Y. Tong
EGFR progression-free survival in patients with mutations; 6.5.1 Content Covered by the Clinical Report
and crizotinib has anticancer effects in patients with positive
ALK gene rearrangement. Molecular testing of genetic variation is a laboratory test
based on genetic counseling. The laboratory test results
6.4.2.5 Detection of Pathogenic Microorganisms must also be reasonably explained by the clinical consul-
Infectious diseases are caused by the invasion of exogenous tant in combination with the patient’s clinical symptoms.
pathogens into the body, and pathogen diagnosis is generally Laboratory tests for genetic diseases should also be consis-
performed by pathogen culture or serological methods. These tent with existing or established laboratory test reports.
traditional methods have obvious deficiencies in the speed,
specificity, sensitivity, and correctness. The early diagnosis 6.5.1.1 The Test Report Should Include
of the detection and the methods of identification are limited the Following
by many factors. For example, it takes a certain period of 1. The name of the medical institution (and department), the
time for the pathogen to produce antibodies after the infec- name of the project, the unique number of the sample, the
tion. It is difficult to make a timely diagnosis by serological contact information of the hospital and department, and
methods, and serological tests can only determine whether or the inspection unit.
not the pathogen has been contacted, and it is not possible to 2. Identification information of the test sample includes the
determine whether there is an existing infection. In addition, patient’s name, gender, date of birth of the patient (prena-
the ability to identify mixed infections and unknown patho- tal diagnosis should also list current age and gestational
genic microorganisms is limited. In recent years, a large age), specimen collection time, sample reception time,
number of analytical work have been carried out on the gene experimental code, specimen type, doctor for examina-
sequences of various pathogens, and it has been possible tion, the time for the diagnosis of the disease, the report
to design specific probes for pathogen-specific nucleotide of the test result, etc.
sequences for molecular hybridization, or to amplify con- 3. Sample processing process, detection method, detec-

served sequences of pathogen genes by PCR, and to grasp tion process, test results, and related charts; if necessary,
pathogenic microorganisms as a whole. Genomic sequenc- include the normal range of detection, positive judgment
ing of clinical isolates allows early, clear pathogen diagnosis value (Cut-off).
of most infectious diseases, detection of carriers and poten- 4. Clearly describe and explain the detected results, clini-
tial infections, and classification and typing of pathogens. cal significance and recommendations, detection limita-
In addition, sequence compilation testing can also be used tions, references, and limitations of genetic testing for the
for antibiotic resistance monitoring and infection control, purpose of genetic testing (such as limitations of experi-
as well as for drug development and clinical trials. At pres- mental techniques and clinical effectiveness, and non-
ent, sequence mutation detection technology has also been parentality) should be clearly explained and described.
applied to the detection of viral diseases, bacterial diseases, 5. In the results report of the estimated risk rate, the infor-
and parasitic diseases, such as human immunodeficiency mation and data used to calculate the risk rate should be
virus (HIV), hepatitis C virus (HCV), and influenza virus. clearly described.
In addition, the new sequence mutation detection technology 6. The test results must be signed by the test personnel, the
is more efficient and convenient, and provides a more effec- result interpreter, and the report reviewer.
tive means for detecting virus mutation. In addition, genetic
diagnosis can be used for the detection of bacteria such as For different detection methods, corresponding test results
Neisseria gonorrhoeae, Helicobacter pylori, and Neisseria judgment and diagnostic criteria should be established in
meningitidis, and can also be used for the diagnosis of vari- the laboratory. In particular, the selection of test methods,
ous parasitic diseases such as spirochetes, malaria parasites, the selection of test markers (markers for linkage analysis),
and toxoplasma. detection systems, positive markers (such as the length of
PCR products), etc., should be evaluated for clinical validity,
sensitivity, and specificity.
6.5 hat Is Involved Before the Clinical
W The report of molecular genetic testing should include
Report the cause of the experimental/disease test, the test method
used, the target site of the test, the genotype of the individual,
The test results are issued in the form of test report sheets. the detected mutation site, the interpretation of the results
A paper version test report is required. The conditional test (clinical significance), and the need for detailed information
laboratory can issue reports in electronic form and establish on follow-up recommendations, genetic counseling recom-
a network query system. The check doctors can log into the mendations, etc. If the detection method is a linkage analysis
website to check the test results. method, the family report, and genotype information should
be included in the test report. It should be noted that any tion. For each genetic disease, the laboratory should first
genetic testing report should take care to protect the privacy use the corresponding database as a reference. If the
of patients and other family members. Therefore, it is rec- detected mutation is a new mutation, the nature and sig-
ommended that the testing laboratory provides experimen- nificance of the mutation may not be clear at the moment
tal tests for different versions and different information for and should be indicated in the report.
clinicians and probands. In the test report, the testing labo- 5. The clinical relevance of the test results should be noted.
ratory can provide a professional experimental report inter- Because information about the pathogenicity of the
pretation for the patient or clinician according to different mutation may change over time, it should also be noted
testing methods to help the clinician make a correct diagno- if the detected variation may have a potential clinical
sis. However, it should be noted that the interpretation of the effect.
test report is not a substitute for the clinician for diagnosis. 6. The technical limitations of the test method and the limi-
All test results should be interpreted independently by more tations of the interpretation of the test results should also
than two individuals, one of whom must be the laboratory be clearly stated. For example, when the test result is
director or laboratory manager or other qualified people. All negative, the reason for the possibility of the negative
results with inconsistencies or data inconsistencies must be result should be explained and described. For example,
judged by qualified personnel with additional supplemen- because sequencing is limited to the coding region of
tary experimental analysis. The test results can be analyzed the gene, the mutation may be in the uncovered intron
and explained by referring to the members of the family or promoter region, and the pathogenic mutation in the
using known genotypes as a control. Since the reliability other parts of the genome cannot be excluded, the sen-
of the quantitative analysis results is significantly less than sitivity of the detection is <100%, and the sequencing
the qualitative analysis results, an internal control must be method is applied. Large gene deletions and duplica-
applied to ensure the accuracy of the test. For PCR analysis, tions cannot be detected, and other genes may also cause
the possibility of different amplification products needs to be diseases.
considered. 7. All data analysis software packages, databases, etc. used
in the analysis of the results should indicate the name
6.5.1.2 DNA Sequencing Reports and version number. It is best to indicate the URL in the
and Explanations Should Include online database.
For clinical reports of sequencing results, the following 8. A summary of the genetic information associated with
information is important for understanding and interpret- the patient’s phenotype should be provided. If the
ing test results. When designing the final report template, it patient is recommended to continue testing other genes,
should include as much as possible: it is also recommended to include them in the report.
9. If it is speculated that the genes of other people need to
1. The genes and/or chromosomal regions analyzed by be further tested according to the results of the patient’s
sequencing should be clearly indicated by the HGNC test, it should also make recommendations in the report.
gene naming rules. Specific regions of the analyzed gene For example, family members of patients with genetic
and/or chromosome should be noted, such as coding diseases should follow the genetic type and the degree of
regions, exons, cleavage sites, and the like. emergence of genetic diseases. Make genetic screening
2. The reference sequence used (RefSeq access number) recommendations for family members.
should be noted. 10. If there is a correlation between the treatment of a cer-
3. For the sequence variations and exact sites found by tain mutation and the disease or the prognosis, recom-
sequencing, the HGVS nomenclature rules should be mendations should be given in the report.
specified. As indicated in the report, the position and
change of the reference sequence of the single nucleo- The results report requires a rigorous and effective pro-
tide in the Genebank, and the corresponding standard cess of issuance and review to ensure complete, effective,
position of the protein change. timely, correct, and private inspection information. First of
4. The relationship between the predicted base variation all, the test results report should be reviewed and analyzed,
and the known gene structure and other data, and the including the effectiveness of the audit process, the basic
influence of base variation on the gene. Changes in information of the subject, and the analysis of the result data.
amino acid coding due to nucleic acid variation, and The auditor shall be the staff member of the supervisor or
possible changes in protein function, should also be above, the person in charge of the professional laboratory,
based on HGVS rules and detailed references. The mis- the senior inspector, and the authorized person of the clinical
sense variation of the base needs to indicate whether it laboratory director. The auditor is responsible for the quality
represents a mutation, a polymorphism, or a rare muta- of the inspection report.
74 Y. Tong
6.5.1.3 Reports and Interpretations of Whole- insertional deletions, frameshift mutations, and splice
Exon or Whole-Genome Next-Generation sites AG/GT interfere with normal protein synthesis and
Sequencing Should also Include cellular transcriptional and translational regulation.
the Following 3. Sequence mutations have not been reported before, and
1. All detected genetic variations should be assessed and may or may not be related to the disease. Mismatched
classified according to international standards. changes, in-frame insertion deletions, and splice site
2. Assessing the genetic function or gene product caused by mutations may affect gene expression or intracellular
the mutation and the possibility of disease and the exist- processing and may be associated with disease. Further
ing evidence. examination of other tests is generally required to clarify
3. For the detection of a wide range of phenotypes, it should the clinical significance of these variations.
also be compared with the clinical manifestations of the 4. Sequence mutations have not been reported before, and
patients for comparative evaluation. it is likely that they will not cause disease. These varia-
4. If multiple clinically significant variants are detected, the tions generally do not alter the coding sequence, and base
relevance of each variant to clinical manifestations should changes do not affect known intracellular signal process-
be addressed. ing or regulatory pathways.
5. Sequence variation has been previously reported and is a
Accidental variability may or may not be of clinical signif- recognized neutral variation. There is evidence that this
icance, and testing laboratories should establish procedures sequence variation can be observed in normal population
and standards. The detected variations should be labeled and has no correlation with disease occurrence.
according to international standards (www.genenames. 6. Sequence variation is not the cause of the expected dis-
org). The method of labeling the variation should include ease, but has been reported to be related to the clinical
gene name, heterozygosity/homozygosity, cDNA naming, manifestations of another disease. In general, the inciden-
protein naming, and exon number. It should be clearly indi- tal findings found in the test that are not related to the
cated in the test report whether a mutation that can explain patient’s expected clinical phenotype may not be reported;
the patient’s disease has been detected. If the mutation does however, if the detected mutation may cause a clear dis-
not clearly explain the disease, explain the possible situa- ease to the patient or his or her family sex and hazard, it
tion. The corresponding supporting evidence should also be is recommended to report truthfully in the test results.
listed in the test report. When the next-generation sequenc- The laboratory should refer to existing literature to
ing method used does not cover all genes, the actual cover- develop reporting principles for sporadic mutations for
able genes and gene regions should be indicated in the test specific projects.
report. In the application of the next-generation sequencing
test report, the limitations of the test should be clearly indi- In general, reports of sequencing results should be based
cated, and the data processing methods should be described. on existing disease and/or genetically related literature
reports or disease diagnosis guidelines as much as possible,
and after thorough analysis, accurate interpretation, and
6.5.2 Clinical Interpretation of the Data reporting. For the literature that has not been reported yet,
and whether there is any clinically relevant variation, the
The sequence variation found needs to be described with ref- laboratory needs to formulate corresponding standards for
erence to the rules established by HGVS, and interpretation declaration and follow-up strategies.
should be given based on the results of existing literature to
indicate whether the detected variation changes the relation-
ship between protein coding and function, and the disease 6.5.3 C
linical Molecular Diagnostic Testing
phenotype. For the variations found in sequencing, the fol- Process and Precautions
lowing classifications are recommended:
In order to ensure that clinicians or other personnel obtain
1. Sequence variation has been previously reported and has accurate, clear, and unambiguous information from clinical
been recognized as being associated with disease. The molecular diagnostic test reports, clinical laboratories should
relationship between the clinical outcomes of sequence develop a rigorous clinical molecular diagnostic test proce-
variation is supported by more feasible literature reports. dure, and regularly follow the relevant disease diagnosis
2. Sequence variation has not been reported before, but is and treatment guidelines and clinical molecular diagnostics
expected to cause disease. In general, mutations that are related regulations to modify.
6.5.3.1 Clinical Molecular Diagnostic Testing query of the test results can usually be queried according
Process to the patient’s name, sample number, test item, and date
Sequence mutation detection in the clinical testing process, of inspection. After the test report is issued, the test report
the entire clinical molecular diagnostic test process, from the complaints need to be recorded and counted, and the rea-
patient’s medical treatment to the report issuance, is included sons are analyzed to avoid secondary errors.
in the supervision. 4. Posttest genetic counseling. Genetic counseling is the
provision of knowledge or information services related to
6.5.3.2 Precautions for Clinical Reports genetic diseases to patients or their families. The process
The preparation of clinical reports on sequence variation mainly includes: assessing the occurrence and recurrence
requires a rigorous and effective process to ensure that the of the disease through the interpretation of family his-
test information is complete, effective, timely, correct, and tory and the history of medical history and genetic laws;
protects the patient’s privacy. Test results are issued in the educating the counselor on the genetics, laboratory test-
form of test report sheets. A paper version test report is ing, treatment and prevention of the disease, and provid-
required. The conditional test laboratory can issue reports ing various treatments related to the disease, channels of
in electronic form and establish a network query system. help and awareness of the research direction; counseling
The check doctors can log in to the website to check the test consultants to make informed choices and knowledge
results. and acceptance of the disease and its recurrence risk. The
The laboratory should also establish a scientific and sys- scope of genetic counseling has expanded from simple
tematic interpretation of the test results and provide explana- fertility genetic counseling to counsel on genetically pre-
tions of the results. Ways and means of providing consulting disposed diseases including common tumors. For these
services on the report form, such as customer service line, diseases, the principle of non-instructive counseling is
configuration of professional consulting service personnel; no longer applicable, and information on the education
convenient clinicians and patients to feedback and advice at and research of the disease-related genetic content of the
any time. counselor is included in the scope of genetic counseling.
In addition, due to the extensive clinical application of
serial variation, it should be treated differently according to
the specific detection purpose in the review process of the 6.6 Reporting Model and Case Analysis
report, but the report of the sequence variation of the detec-
tion target should pay attention to the following problems: With the rapid development of clinical molecular diagnos-
tic techniques, the correlation between testing and clinical
1. Turnaround time. TAT is the time from the collection of a is getting closer. Continuous improvement of inspection
blood sample to the reporting of results. Appropriate quality depends on the support of clinical departments and
return time for results should be written in writing for the doctors, and is inseparable from the development of the lab-
actual testing process and clinical needs of each sequenc- oratory. Among them, the quality control after the clinical
ing project. test analysis refers to the process in which the test speci-
2. The confidentiality of the test reports. All test results are men is reported to the doctor after being analyzed by the test
confidential. The results can be used to guide clinical physician.
individualized care. If you report the results directly to Various inspection reports are the final result of the exper-
the individual, you need to have the appropriate guidance imental work and the most critical part of the quality control
to understand the results of the test and to understand the after analysis. The high-quality inspection report should be
deficiencies of the test methods. complete with the following contents: name of the hospital
3. Traceability of test records and traceability of patient laboratory, type of report, patient name, gender, age, (hos-
reports. The laboratory needs to determine the type and pital) department, bed number, inspection item, purpose of
timing of the various sequencing data in accordance with inspection, type of specimen, application Name of the doctor
the requirements of the local health administration. The to be examined, specimen collection time, laboratory receipt
general inspection report shall be kept for at least 2 years, of specimen time, report time, method of detection, test
and the test result data shall be kept for at least 2 years; the results, molecular diagnosis, recommendations, limitations
indoor quality control and the inter-room quality evalua- of detection methods, key parameters of this test (sequence
tion record and quality control information shall be kept coverage depth, etc.), interpretation of results information
for at least 2 years; the instrument status and maintenance such as literature references. Each inspection report should
records shall be retained for the life of the instrument. The be reviewed by two professionals before it can be issued. If
76 Y. Tong
the specimen is found to have hemolysis, lipemia, jaundice, in the United States (Fig. 6.5). In China, the incidence of lung
paraffin tissue specimen degradation, etc., which may affect cancer has increased year by year, and it has become the can-
the accuracy of the results, it should be indicated on the report cer with the highest mortality rate among malignant tumors.
form. The results of the question should be reviewed, and the The incidence of lung cancer is more than 40 years old, and
doctor should be contacted to ask for the situation, and if the peak incidence is between 60 and 79 years old. The aver-
necessary, re-collect the specimen for review. When the criti- age chance of men suffering from lung cancer for life is 1/13,
cal value is determined in the test results, the clinician should and for women it is 1/16. It is estimated that there are about
be notified immediately and the registration (test data, time, 1.8 million new cases of lung cancer every year in the world,
reporter, and notified person) should be completed. accounting for about 13% of the total cancer diagnosis. Lung
Once the test results are inconsistent with the clinical cancer is the leading cause of cancer diagnosis and death in
diagnosis, the inspector should communicate with the clini- men. Lung cancer is the leading cause of cancer death among
cian in time to find out the symptoms and the situation. With women in developed countries, while it is the second leading
the popularization of medical knowledge, many patients want cause of cancer death in developing countries. In males, the
to know their condition and cause. Therefore, the inspectors highest incidence of lung cancer is in Europe, eastern Asia,
will often explain the results of their tests. The inspectors northern Asia, and the United States, with the lowest inci-
should conduct targeted and comprehensive analyses of the dence in sub-Saharan Africa; the highest rates of lung cancer
condition according to the test results. in women are in the northern United States, northern and
western Europe, Australia/New Zealand, eastern Asia. The
rate of lung cancer among Chinese women (20.4 per 100,000
6.6.1 Tumor Molecular Diagnosis Report people) is higher than in some European countries, although
the prevalence of smoking is lower.
The molecular diagnosis of tumors is based on the diagno- Non-small cell lung cancer (NSCLC) includes several
sis information of disease target genes. Based on the results types of squamous cell carcinoma (squamous cell carci-
of evidence-based medicine research, it provides a scientific noma), adenocarcinoma, and large cell (undifferentiated)
basis for the clinical development of treatment programs, cancer. Among them, squamous cell carcinoma accounts for
and creates conditions for patients to obtain individualized 25–30% of NSCLC. This type of lung cancer is associated
medical treatments. It has become a trend of modern medi- with smoking and usually grows in the center of the lung
cal development. It has been widely used in clinical. By close to the bronchus. Lung adenocarcinoma accounts for
detecting gene mutations, gene SNP typing, gene and pro- approximately 40% of NSCLC and is usually found in the
tein expression status of biomarkers in biological samples of outer part of the lung. Large cell (undifferentiated) cancer
tumor patients, predicting drug efficacy and evaluating prog- accounts for about 10–15% of NSCLC and can occur any-
nosis, guiding clinical individualized treatment, improving where in the lungs. Non-small cell lung cancer accounts for
efficacy, reducing adverse reactions, and promoting medical 80–85% of the total number of lung cancer. Compared with
resources Rational use. In addition to the continuous devel- small cell lung cancer, non-small cell lung cancer cells have
opment of molecular diagnostic techniques, the produc- slower growth and division, and the diffusion and metastasis
tion of test reports and the timely and accurate issuance are relatively late. Although the surgery and chemotherapy
depend on the improvement of the quality assurance system techniques have been greatly improved, when people are
of molecular diagnostic laboratories, so that clinicians can diagnosed with NSCLC, 70% are advanced and it is dif-
understand the clinical purpose of the tested items, under- ficult to undergo radical treatment through surgery and
stand the clinical significance of the test results and for the radiotherapy.
role of treatment, the medical laboratory provides timely and Studies have shown that lung cancer occurs in a multifac-
accurate inspection reports for patients or clinical staff, and tor interaction (Fig. 6.6). After multiple steps and multiple
provides consulting services related to the report, in order to processes, cells undergo canceration through processes such
truly standardize and standardize the individualized detec- as proliferation, differentiation, dysplasia, transformation
tion of tumors. Take the report of clinical molecular diagno- and metamorphosis, and finally form tumors. It includes the
sis results of non-small cell lung cancer as an example. activation of the gene and the inactivation of the tumor sup-
pressor gene.
6.6.1.1 Introduction to Non-small Cell Lung There are different genetic variants in non-small cell
Cancer lung cancer. Common mutations include EGFR, EML4-
Lung cancer is one of the most common malignant tumors in ALK, KRAS, and ROS1 (Table 6.5). Among them, EGFR
the world and has become the leading cause of cancer death gene mutation is the most common in Asian non-small
Estimated cases Estimated deaths
Female Female
Male Male
Breast Breast
Lung, bronchus & trachea Lung, bronchus & trachea
1,676,600 521,900
1,241,600 1,098,700
Colon & rectum Lung, bronchus & trachea
Prostate Liver
614,300 491,200
1,111,700 521,000
Lung, bronchus & trachea Colon & rectum
Colon & rectum Stomach
583,100 320,300
746,300 469,000
Cervix uteri Cervix uteri
Stomach Colon & rectum
527,600 265,700
631,300 373,600
Stomach Stomach
Liver Prostate
320,300 254,100
554,400 Esophagus
Corpus uteri liver
Urinary bladder 281,200
319,600 224,500
330,400 Pancreas
Ovary Pancreas
Esophagus 151,300
238,700 156,600
323,000 Leukeima
Thyroid Ovary
Non-hodgkin lymphoma 200,700
229,900 151,900
217,600 Urinary bladder
Liver Esophagus
Kidney 123,100
228,100 119,000
213,900 Non-hodgkin lymphoma
Non-hodgkin lymphoma Leukemia
Leukemia 115,400
168,100 114,200
200,700 All sites
All sites All sites
All sites 4,653,400
6,633,000 3,548,200
7,427,100
Fig. 6.5 Global cancer incidence and mortality
Normal Precancer Early-stage cancer Advanced cancer
Smoking-related
Clonal patch
Tobacco smoke
Host susceptibility
Unknown factors
Not smoking-related
Fig. 6.6 Schematic diagram of lung cancer disease progression

78 Y. Tong
Table 6.5 Frequency of target gene mutations in NSCLC carcinoma, EGFR gene mutation detection and ALK gene
Gene Alteration Frequency in NSCLC detection may be considered, especially for patients with
AKT1 Mutation 1% no history of smoking or small biopsy specimens or mixed
ALK Rearrangement 3–7% histological types of puncture specimens, and ROS1 gene
BRAF Mutation 1–3% detection may also be considered. The guide also states that
DDR2 Mutation ~4% KRAS mutations are associated with EGFR TKI resistance,
EGFR Mutation 10–35%
so KRAS gene testing can be used as a candidate for EGFR
FGFR1 Amplification 20%
TKI treatment to further determine whether patients can
HER2 Mutation 2–4%
KRAS Mutation 15–25% benefit from treatment. KRAS oncogene mutation is also a
MEK1 Mutation 1% prognostic biomarker. Compared with KRAS mutant wild-
METa Amplification 2–4% type patients, KRAS mutations in non-small cell lung cancer
NRAS Mutation 1% patients have poor survival results.
PIK3CA Mutation 1–3% In addition, the guidelines also describe some genetic variants
PTEN Mutation 4–8% associated with new targeted drugs, such as BRAF V600E site
RET Rearrangement 1% variants, non-small cell lung cancer with non-V600E mutations
ROS1 Rearrangement 1% with variable enzymatic activity and for vemurafenib (vemu-
rafenib)), Dabrafenib alone, and Dabrafenib + Trametinib com-
cells. About 50% of lung cancer patients, about 10% of bined sensitivity; high level MET gene amplification or MET 14
non-Asian patients, EMN4-ALK fusion gene accounts exon skip mutation, using ketazole Crizotinib may benefit from
for 2–7%, KRAS gene mutation accounts for about 25%, it; RET gene rearrangement and HER2 gene mutation in non-
ROS1 gene mutation accounts for about 1%, and each gene small cell lung cancer patients can be used with Cabozinidinib
variant there is repulsion. Numerous studies have shown and trastuzumab/afatinib (afatinib) targeted drug administration
that there is a mutual exclusion between genetic variants was carried out (Table 6.6).
such as EGFR, EML4-ALK, and KRAS, and there are no
two or more such genetic variants at the same time, and Detection Method of NSCLC Target Molecule
these genetic variants are closely related to the treatment The selection of tumor target gene detection methods generally
and prognosis of NSCLC. needs to be selected according to the mutation pattern of the
target gene and its clinical significance, such as EGFR gene
6.6.1.2 Methods and Test Items for Clinical mutation. The clinical significance of the detection is to detect
Molecular Diagnosis of Non-small Cell whether EGFR mutation is clinically used for patients with non-
Lung Cancer small cell lung cancer. One of the indicators of EGFR-TKIs
With the development of molecular diagnosis and targeted drugs, its common mutation form and mutation frequency are
drug development for non-small cell lung cancer, the tar- also factors to be considered in the selection of detection meth-
geted therapy for non-small cell lung cancer has been ods (Fig. 6.7), followed by the detection platform and instru-
widely used clinically, especially in the treatment of patients ments and equipment in the clinical laboratory itself.
with advanced or metastatic non-small cell lung cancer. Epidermal growth factor receptor (EGFR) gene, anaplas-
Significant progress, more and more molecular diagnostic tic lymphoma kinase (ALK) gene, KRAS gene, and BRAF
projects are included in the NSCLC treatment guidelines, gene are important targets for targeted therapy of non-small
the detection of NSCLC-related target molecules, providing cell lung cancer. The detection method is shown in Table 6.7.
a solid basis for the individualized medical care of NSCLC. Among them, the most commonly used molecular
detection methods for detecting EGFR gene mutations in
Source of NSCLC Target Molecule Detection Project
In the National Comprehensive Cancer Network (NCCN)
clinical practice guidelines, NCCN experts believe that Table 6.6 NSCLC target molecule detection and individualized
medication
the application of molecular detection technology is a key
step in improving the treatment of patients with metastatic Genetic mutation Targeted drug
ROS1 rearrangement Crizotinib
non-small cell lung cancer, so patients with metastatic non-
PD-L1 expression Pembrolizumab
small cell lung cancer are strongly recommended. Perform
BRAF V600E site variation Verofenib
molecular testing. Among them, EGFR gene mutation detec- Non-small cell lung cancer with non-V600E Dabrafenib
tion, ALK gene detection, ROS1 gene detection, and PD-L1 mutation has variable enzyme activity
detection are recommended for patients with adenocarci- High level MET gene amplification Dabrafenib +
noma, large cell lung cancer, and non-specific non-small Trametiniib
cell lung cancer. In addition, for patients with squamous cell Or MET 14 exon skip mutation Crizotinib
Fig. 6.7 Schematic diagram of the relationship between EGFR gene mutations and TKIs in NSCLC
NSCLC include gene sequencing and amplification muta- reverse transcription-polymerase chain reaction is fast and
tion blockade, and immunohistochemistry can also detect simple, but only detects known fusion types.
partial EGFR mutations. Although the sequencing method
is the most direct, it can detect unknown mutations, but its For Example
sensitivity is relatively low, and the process is cumbersome. Sample Source
Therefore, the EGFR gene mutation is often used in the The NCCN guidelines state that for patients with advanced
clinic with simple and sensitive amplification and mutation lung cancer with local recurrence and distant metastasis,
methods. In addition to EGFR gene mutations, EGFR gene molecular testing should be considered. The source of the
amplification, and protein overexpression are also com- specimen can be:
mon in patients with non-small cell lung cancer, which is
thought to be associated with prognosis in patients with tar- 1. Paraffin tumor tissue: 10 paraffin-embedded tumor tissue
geted therapy. sections (4 wax packets ~10 embedded white tablets).
Fluorescence in situ hybridization (FISH), chromogenic 2. Fresh tumor tissue: The size of the tissue soybeans

in situ hybridization, and real-time fluorescent quantitative (2 cm*2 cm) determined by the pathologist is determined
PCR are commonly used to detect EGFR gene copy number, to be containing the tumor cells, washed 3–5 times with
and immunohistochemistry is mainly used for EGFR pro- normal saline, placed in a sterile container containing
tein expression. Both of these tests are included in the guide physiological saline, and stored at 4 °C for examination.
as a means of detecting the efficacy of a targeted therapy. 3. Pleural effusion: 50 mL was placed in a sterile container
The molecular detection methods of the EML4-ALK fusion and stored at 4 °C for inspection.
gene mainly include FISH, IHC, and quantitative reverse 4. Peripheral blood: Circulating tumor DNA (ctDNA),

transcription-polymerase chain reaction (qRT-PCR). Among 15 ml, EDTA anticoagulant tube (Streck vacuum blood
them, the FISH test method is the gold standard for ALK collection tube), stored at 4 °C for examination (sepa-
fusion gene detection, but the detection cost is high and the rate plasma within 6 h, extract free DNA, and store at
skill requirements of the tester are also high. Quantitative −80 °C).
80 Y. Tong
Table 6.7 Molecular diagnostic test methods for target molecules tumor cells or tumor cells in the sample. It affects the accu-
commonly used in NSCLC racy of the test results. The quality of the specimen extrac-
Detection Specimen tion and the positive control of the negative should be noted
Project method Source category
in the analysis. After the analysis, you should pay attention
EGFR gene Real-time National Paraffin
mutation PCR Comprehensive tumor tissue,
to regularly update the interpretation of the results to make
detection Cancer Network fresh tumor them consistent with the latest clinical guidelines or specifi-
NCCN tissue, cations to avoid misinterpretation.
pleural Data analysis was performed according to the kit instruc-
effusion,
peripheral
tions. Data analysis should pay attention to:
blood
EGFR protein First- National Paraffin 1. Determine the Ct value of the mutation group by manu-
expression generation Comprehensive tumor tissue, ally setting the threshold line by automatically setting the
sequencing Cancer Network fresh tumor baseline.
NCCN tissue
KRAS, BRAF High- National Paraffin
2. Negative and positive control analysis to determine

gene mutation throughput Comprehensive tumor tissue, whether the test results are under control.
sequencing Cancer Network fresh tumor
NCCN tissue, Results Report
pleural
effusion,
A molecular diagnostic report analysis process suitable for
peripheral clinical laboratories should be developed to ensure that each
blood examiner discards personal preferences and makes the report
EML4-ALK Real-time National Paraffin consistent in the analysis of the report. The content of the
gene fusion PCR Comprehensive tumor tissue, report should at least include the following:
Cancer Network fresh tumor
NCCN tissue
ROS1 Real-time National Paraffin 1. Results report basic information: Test name, specimen
rearrangement PCR Comprehensive tumor tissue, identification number, patient information (such as name,
Cancer Network fresh tumor gender, age, department, and hospital number), specimen
NCCN tissue,
pleural
type, clinical diagnosis, doctors sent, report content (test
effusion, method, test used Objectives, methods and principles, test
peripheral results, test results analysis, remarks), instructions, testers
blood and reporters for double-trial verification, report dates,
MET gene First- National Paraffin appendices, and references.
amplification generation Comprehensive tumor tissue,
sequencing Cancer Network fresh tumor 2. Gene information: Detailed description of the detected
NCCN tissue, gene information, including detection of gene name, gene
pleural ID, reference sequence, and mutation site.
effusion, 3. Description of test results: A detailed description of the
peripheral
blood detected results, for example, a mutation (p. L858R) was
HER2 gene High- National Paraffin detected in exon 21 of the region detected by the EGFR
mutation throughput Comprehensive tumor tissue, gene of the subject.
sequencing Cancer Network fresh tumor 4. Analysis of test results: Firstly, the quality control of
NCCN tissue,
pleural
this test should be described, whether it meets the test
effusion, standard; secondly, the molecular diagnosis of this test
peripheral is given, and a clear definition is given; finally, for the
blood purpose of this test, combined with the current authority
PD-L1 Fluorescence National Paraffin certification Clinical guidelines (e.g., NCCN and ASCO)
expression in situ Comprehensive tumor tissue,
hybridization Cancer Network fresh tumor provide clinically feasible clinical recommendations for
(FISH) NCCN tissue clinical and molecular evidence for individualized diag-
nosis and treatment.
5. Remarks: Briefly describe the purpose and significance
Tumor Tissue Testing Process of this test, the epidemiology and hazards of related dis-
Fluorescence quantitative PCR detection flow chart of tumor eases, the definition of the tested genes, and the clinical
tissue, should pay attention to clinical diagnosis and patho- application value.
logical diagnosis before analysis and quality control of spec- 6. Appendix: Supplement to the report content so that clini-
imens when collecting specimens, avoiding the absence of cians can better understand the test report.
6.6.2 M
olecular Diagnosis Report 6.6.2.2 Method and Test Item for Clinical
of Infectious Diseases Molecular Diagnosis of Hepatitis B Virus
Molecular biology is a new subject in the study of the Project Source

nature of life at the molecular level. It is based on the In the 2015 update of China’s chronic hepatitis B preven-
structure of biological macromolecules such as nucleic tion and treatment guidelines, the expert group believes
acids and proteins and its role in the transmission of that the molecular biological diagnosis of HBV mainly
genetic information and cellular information. It is the fast- includes HBV DNA quantitative detection, HBV genotyp-
est growing in current life sciences. And is in the field of ing, and drug-resistant mutant detection. HBV DNA quan-
cutting-edge research that is widely intersected and infil- titative detection is mainly used to determine the level of
trated with other disciplines. Infectious diseases are one of viral replication of chronic HBV infection, and can be used
the most common diseases in humans, especially in devel- for the selection of antiviral treatment indications and the
oping countries, causing a serious burden on humans. In judgment of curative effect. A real-time quantitative PCR
recent years, molecular diagnosis has made tremendous method with high sensitivity and accuracy is recommended.
contributions to human health, especially in the field of The main purpose of HBV genotyping is to determine the
diagnosis and individualized treatment of infectious dis- sensitivity and prognosis of HBV against viral drugs. The
eases. For example, the diagnosis and treatment of hepa- clinical outcomes of different genotypes are different. HBV
titis B virus, in clinical practice, found that the treatment has at least 9 genotypes (A ~ J), and China is mainly B and
of chronic hepatitis B is not only related to the viral load, C. The HBV genotype is associated with disease progres-
but also related to the virus genotype and gene mutation. sion and alpha-interferon (IFNα) response. Compared with
The detection of viral load, genotypes, and mutations is those infected with C genotype, those with genotype B are
inseparable from molecular diagnostic techniques. It is less likely to progress to chronic hepatitis and cirrhosis. The
precise because of the individualized treatment after the response rate of HBeAg-positive patients to IFNα treatment
introduction of molecular diagnostic techniques that it was higher in B genotype than in C genotype and higher
plays an important role in inhibiting the spread and spread in A genotype than in D genotype. Viral quasispecies may
of hepatitis B virus. Take the report of clinical molecular be important in HBeAg seroconversion, immune clearance,
diagnostic results of hepatitis B virus as an example. and antiviral therapy responses. At present, the therapeutic
drugs for hepatitis B are mainly nucleoside (t) ide analogues
6.6.2.1 Introduction to Hepatitis B (NAs). It is reported that genetic mutations at certain sites
HBV infection is worldwide, but the prevalence of HBV of HBV are associated with resistance to NAs. The detec-
infection varies widely among different regions. According tion of genetic mutations can help determine whether a virus
to WHO estimates, about 2 billion people worldwide have has developed resistance, whether the current treatment plan
been infected with HBV, of which 240 million are chronic needs adjustment and rational use of the drug.
HBV-infected, and about 650,000 people die each year from
chronic hepatitis B and its complications. HBV infection Detection Method
can be acute or chronic and may range from asymptomatic According to the purpose of detection, the selected detection
or mild lesions to severe or very few fulminant hepatitis. methods are different (Table 6.8). For example, quantitative
Worldwide, most people with hepatitis B are infected at birth detection of HBV DNA is generally performed by real-time
or early in childhood. The disease spectrum and natural his- PCR, and genotyping can be performed by real-time PCR,
tory of chronic hepatitis B are diverse. Some of the CHB is Sanger sequencing, or gene chip.
static and does not show symptoms of liver disease. However,
some patients may cause progressive liver fibrosis, leading to For Example
a significant increase in the risk of end-stage cirrhosis and Sample Source
hepatocellular carcinoma (HCC). In global cirrhosis and The Chinese chronic hepatitis B prevention and treatment
HCC patients, the proportions caused by HBV infection guidelines indicate that HBV DNA is mainly used to judge
were 30% and 45%, respectively. In Chinese patients with the level of viral replication of chronic HBV infection, and
cirrhosis and HCC, the proportions caused by HBV infec- can be used for the selection of antiviral treatment indica-
tion were 60% and 80%, respectively. Due to the widespread tions and the judgment of curative effect. The source of the
immunization of hepatitis B vaccine, the marked reduction in specimen can be: serum (Pulp), serum (pulp) isolated from
acute HBV infection, and the aging of HBV infected popula- whole blood is stored at 4 °C for 24 h, and −20 °C can be
tions, coupled with the widespread use of antiviral drugs, the stored for a long time. In addition, biopsy tissue, amniotic
proportion of HBeAg-negative CHB patients has increased fluid, milk, pleural effusion, ascites tissue wax block, etc.
in recent years. can also be used for fluorescent PCR detection of HBV
82 Y. Tong
Table 6.8 Detection methods and project sources non-invasive. At present, invasive prenatal diagnosis is the
Detection Specimen gold standard for diagnosing fetal chromosomal diseases,
Project method Source category mainly refers to the acquisition of fetal chromosomal infor-
HBV DNA Fluorescent Guidelines for the Plasma mation by collecting fetal cells or tissues by villus biopsy,
quantitative PCR prevention and
detection treatment of chronic
amniocentesis, and umbilical vein puncture. The accuracy of
hepatitis B in China invasive testing is 98–99%, but with a risk of miscarriage of
HBV genotype Fluorescent Guidelines for the Plasma 0.5–1%, and it is also associated with amniotic fluid leakage,
PCR prevention and intrauterine infection, and other risks, so invasive prenatal
First- treatment of chronic diagnosis is currently only used for children at high risk,
generation hepatitis B in China
sequencing in advanced pregnancy, or in families with genetic diseases
Gene chip (Table 6.9).
High- Traditional noninvasive prenatal examinations mainly
throughput include methods such as ultrasonography and maternal sero-
sequencing
Detection of Fluorescent Guidelines for the Plasma
logical testing. These methods can be used to avoid harm
HBV resistant PCR prevention and to the fetus and pregnant women, but their sensitivity and
mutants First treatment of chronic specificity are limited. For the past two decades, prenatal
generation hepatitis B in China screening based on ultrasound screening combined with
sequencing
Gene chip
serum screening of various proteins or hormones in preg-
High- nant women has developed rapidly. 21, 18-trisomy syndrome
throughput and neural tube defects are the target diseases of prenatal
sequencing serological screen. The detection rates in early pregnancy,
middle pregnancy, and early combined pregnancy are 83%,
81%, 94%, respectively. 96%, the false-positive rate is about
DNA, which is generally not commonly used due to the limi- 5%. The false-positive rate of these methods is high, which
tation of materials. brings great clinical pressure and laboratory pressure for
subsequent invasive prenatal diagnosis, and brings unneces-
Detection Procedure sary puncture.
The quality of the sample extraction and the efficiency of PCR In recent years, with the development of a new genera-
amplification should be noted in the analysis. After the analy- tion of high throughput sequencing technology, noninvasive
sis, you should pay attention to regularly update the interpre- prenatal testing (NIPT) technology based on next-genera-
tation of the results to make them consistent with the latest tion sequencing technology has emerged. Next-generation
clinical guidelines or specifications to avoid misinterpretation. sequencing of cell free fetal DNA (cffDNA) in pregnant
women’s plasma has great potential for improving the effi-
Data Analysis ciency of prenatal screening.
According to the description of the HBV DNA test kit, dur- The basic principle of NIPT is that the free DNA in the
ing the analysis, it should be noted that: peripheral plasma of pregnant women is mainly from the
pregnant women’s own tissue cells, and a small part of the
1. Determine the Ct value of the mutation group by manu- incomplete free DNA fragments from the placental tropho-
ally setting the threshold line by automatically setting the blast cells after apoptosis (Fig. 6.8), average 50–200 bases,
baseline. of which 80% of the fragments are <193 bp in length. In the
2. Negative and positive control analysis to determine
NGS instrument, the established 36 base-based large nucle-
whether the test results are under control. otide sequence database is identified by massively parallel
3. Whether there is an atypical fluorescence quantitative sequencing (MPS) method, and then divided into categories
amplification curve. and accumulated, and calculated. The number or frequency
of DNA fragments from different chromosomal locations
(unresolved DNA fragments from pregnant women or pla-
6.6.3 N
oninvasive Prenatal Screening Results centa), and then statistically statistical analysis of whether
Report the DNA fragment derived from a specific chromosome
exceeds the standard to predict the specific chromosome
6.6.3.1 Introduction to Noninvasive Prenatal number variation (Table 6.10). The Z-value obtained by the
Screening currently used analytical program exceeds 3; or normalized
According to the different methods of obtaining fetal speci- chromosome values (NCVs) exceeding 4 is considered to be
mens, prenatal examination is divided into traumatic and chromosomal aneuploidy (aneuploidy).
Table 6.9 Different prenatal screening and diagnosis methods

Invasiveness Test Comments Time
Non- Fetal cells in Based on enrichment of fetal cells that circulate in maternal blood. Since fetal cells First trimester
invasive maternal blood hold all the genetic information of the developing fetus, they can be used to perform
(FCMB) prenatal diagnosis.
Non- Cell-free fetal DNA Based on DNA of fetal origin circulating in the maternal blood. Testing can potentially First trimester
invasive in maternal blood identify fetal aneuploidy(available in the United States, beginning 2011) and gender of
a fetus as early as 6 weeks into a pregnancy. Fetal DNA ranges from about 2–10% of
the total DNA in maternal blood.
Cell-free fetal DNA also allows whole genome sequencing of the fetus, thus
determining the complete DNA sequence of every gene
Non- Preimplantation During in vitro fertilization (IVF) procedures, it is possible to sample cells from Before
invasive genetic human embryos before implantation. PGD is in itself non-invasive, but IVF usually implantation
diagnosis(PGD) involves invasive procedures such as transvaginal oocyte retrieval
Non- External examination Examination of the woman’s uterus from outside the body. First or second
invasive trimester
Non- Ultrasound detection Commonly dating scans (sometimes known as booking scans) from 7 weeks to First or second
invasive confirm pregnancy dates and look for twins. The specialized nuchal scan at trimester
11–13 weeks may be used to identify higher risks of Down’s syndrome. Later
morphology scans from 18 weeks may check for any abnormal development.
Non- Fetal heartbeat Listening to the fetal heartbeat First or second
invasive trimester
Non- Non-stress test Use of cardiotocography during the third trimester to monitor fetal wellbeing Third
invasive trimester
Less Transcervical Cervical mucus aspiration, cervical swabbing, and cervical or intrauterine lavage can First trimester
invasive retrieval of be used to retrieve trophoblast cells for diagnostic purposes, including prenatal genetic
trophoblast cells analysis. Success rates for retrieving fetal trophoblast cells vary from 40% to 90%. It
can be used for fetal sex determination and identify aneuploidies. Antibody markers
have proven useful to select trophoblast cells for genetic analysis and to demonstrate
that the abundance of recoverable trophoblast cells diminishes in abnormal gestations,
such as in ectopic pregnancy or anembryonic gestation
Less Maternal serum Including β-hCG, PAPP-A, alpha-fetoprotein, inhibin-A. First or second
invasive screening See separate section below trimester
More Chorionic villus Involves getting a sample of the chorionic villus and testing it. This can be done earlier After
invasive sampling than amniocentesis, but may have a higher risk of miscarriage, estimated at 1%. 10 weeks
Fig. 6.8 Free DNA Maternal Blood

composition in peripheral
blood of pregnant women
Placenta
Fetal DNA
Maternal DNA
An accurate NIPT test, the fetal ratio of cell-free DNA prenatal testing. In addition to the number of gestational
fragments in pregnant women’s plasma must be at least 4%, weeks affecting the fetal ratio of the free DNA fragments
i.e., most pregnant women can receive NIPT examination at in the plasma of pregnant women, there is a large inter-
12 weeks of pregnancy, compared to other prenatal screen- individual difference in the concentration of cffDNA in the
ing or prenatal Diagnostics, NIPT has the advantage of early peripheral blood of pregnant women of the same gestational
84 Y. Tong
Table 6.10 Detection sensitivity of NITP in specific chromosomal thousands to millions of DNA molecules at the same time. It
variation is possible to perform a detailed analysis of the transcriptome
Sensitivity and genome, which is also known as deep sequencing.
Condition (detection rate)
At present, the most mature clinical application of high-
Trisomy 21 (Down syndrome) >99%
throughput gene sequencing in genetic diseases is NIPT
Trisomy 18 (Edwards syndrome) 96.4%
Trisomy 13 (Patau syndrome) >99%
(prenatal diagnosis of noninvasive DNA), which can obtain
Monosomy X (Turner syndrome) 92.9% fetal genetic information from free DNA in maternal plasma
Triploidy/vanishing twin detection Detectable to detect whether the fetus has three major chromosomal
Male >99% diseases.
Female >99% Clinically, this test is mainly targeted at high-precision
22q11.2 deletion syndrome (DiGeorge 95.7% screening, deep screening, screening close to diagnosis,
syndrome) primary screening of high-risk groups, etc. Although the
1p36 deletion syndrome/Angelman syndrome/ 93.8 to >99%
names are different and there are certain limitations in
Cri-du-chat syndrome/Prader-Willi syndrome
clinical application, experts from various countries. The
level of NIPT’s high sensitivity and high specificity has
age, because the weight, race, serum markers, smoking, and been fully recognized, and it is hoped that the test will be
chromosomes of pregnant women Karyotype is also a factor more suitable for the actual clinical situation in the coun-
affecting the fetal ratio, and thus directly or indirectly affects try. Because NIPT is the target disease for precise prena-
the sensitivity and specificity of NIPT detection. The half- tal screening techniques, it is currently difficult to replace
life of the cell-free fetal DNA fragment is only 16.3 min, and existing prenatal screening diagnostic techniques. ACMG
no free fetal DNA fragments of this pregnancy can be found (American Society of Medical Genetics and Genomics)
2 h after the mother’s production. The detection of cff DNA updated its consensus on noninvasive prenatal screening
in the pregnancy is not affected by the last pregnancy. for fetal aneuploidy in July 2016 (Genet Med. 2016 Jul
In addition, because the primary source of fetal-free DNA 28. https://doi.org/10.1038/gim.2016.97.) Replace the ver-
fragments in the NIPT test is the placenta; in theory, this test sion released in 2013. ACMG’s technical positioning based
should be similar to the villus sampling in prenatal diagno- on fetal aneuploid noninvasive prenatal screening does not
sis, so karyotype false positives similar to villus sampling use the generic term NIPT (T is the testing abbreviation, ie
may also occur (False positive) report, or the result of a false detection), but directly uses NIPS (S means screening, ie
negative report. screening). For the first time, a professional association has
issued a statement recommending to all pregnant women
6.6.3.2 Noninvasive Prenatal Screening Method that the target is aneuploidy (Down’s syndrome, Edward’s
and Test Project for Clinical Molecular syndrome, Padua’s syndrome) for traditional screening
Diagnosis [21-trisomy, respectively), 18-trisomy, 13-trisomy, NIPS is
The development of clinical applications is inseparable from the most sensitive screening method.
the support of industry experts, including the American
College of Obstetricians and Gynecologists (ACOG), the For Example
National Association of Genetic Counselors (NSGC), the Sample
International Association for Prenatal Diagnosis (ISPD), The American College of Obstetricians and Gynecologists
and international maternity. Many professional organiza- (ACOG), the National Association of Genetic Counselors
tions, including the International Ultrasound Association (NSGC), the International Association for Prenatal Diagnosis
(ISUOG), provide guidance on the clinical application of (ISPD), the International Association of Obstetrics and
NIPT. The issuance of these statements and guidance reflects Gynecology Ultrasound (ISUOG), and our pre-diagnostic
the importance of the test in the international medical field expert group The agency issues guidance on the clinical
and the positive role and good results of NIPT in prenatal application of NIPT. These statements and guidance indicate
testing. The industry guide helps medical workers under- that the source of noninvasive DNA prenatal testing tech-
stand NIPT at a deeper level and know how to apply it in the nology is EDTA anticoagulation about 5–10 ml for pregnant
field of prenatal testing, making NIPT a healthier and more women aged 12–22+6 weeks. Hemolysis should be avoided
standardized direction. when collecting maternal blood, stored at 4 °C, separated
within 4 h. Plasma was stored at −20 °C. Storage at −20 °C
6.6.3.3 High Throughput Sequencing should not exceed 1 week, and −80 °C for long-term storage
Flux sequencing technology, also known as “next generation” (Table 6.11). At present, maternal blood fetal-free DNA has
sequencing technology, is a revolutionary change to traditional been used for the detection of fetal sex, Rh blood type, and
sequencing. It is epoch making for sequencing hundreds of fetal chromosome aneuploidy.
Table 6.11 The samples requirements for NIPT per chromosome is %chrN of the percentage of all autosomal
Sampling type Sampling requirements unique reads in the sample, i.e., the Reads ratio value. The Z
Pregnant 5–12 ml of peripheral blood of single-pregnant value of each chromosome is calculated again, and the detec-
woman pregnant women at 12–22+6 weeks of gestation, tion result of the sample is judged based on the Z value of
peripheral blood stored in EDTA-treated blood collection tubes.
Hemolysis should be avoided during maternal
each sample.
blood collection, stored at 4 °C, plasma By comparing the large sample negative pregnant plasma
separated within 4 h, and stored at −20 °C. samples with the positive pregnant plasma samples, the per-
centage of unique reads of large samples of negative samples
was analyzed by the Kolmogorov-Smirnov test and shapiro
Data Analysis test, and the results showed that chromosomes 21, 18, and
Abnormal fetal chromosome numbers cause a small change 13. The unique reads percentage satisfies the standard normal
in free DNA content in maternal plasma. Deep sequencing distribution (as shown in Fig. 6.9), so the Z test can be used
by a new generation of DNA sequencing technology com- to verify whether there is a significant difference between the
bined with bioinformatics analysis can detect this change, positive and negative samples.
which is the theoretical basis for noninvasive DNA prenatal
testing. Taking the most common Down syndrome (T21) in Result Report
birth defects as an example, the feasibility of cffDNA for The clinical application of NIPT should be targeted at “pre-
prenatal testing of fetal chromosomal diseases was analyzed natal diagnosis level” and “target disease pointing to precise”
by mathematical model. Normal 21 and normal mothers prenatal screening techniques. Although the technique has
have 2 chromosomes 21, and those with Down syndrome been successfully validated for 21-trisomy, 18-trisomy, and
have 3 chromosomes. It is assumed that the fetal free DNA 13-trisomy prenatal screening, it has been effectively vali-
content in the peripheral blood of the mother is 20%, and dated, but 21-trisomy, 18-trisomy, 13-three False-negative
the mother’s own free DNA accounts for 80%. For conve- cases and detection failures (2–5%) of body and sex chro-
nience of explanation, it is assumed that there are 10 copies mosome abnormalities still exist, and the coincidence rate
of DNA. The free DNA of chromosome 21 in the peripheral of noninvasive diagnosis of sex chromosome abnormalities
plasma of pregnant women with normal fetus is 10, 2 from is relatively low (about 25%). In addition, due to the limi-
the fetus and 8 from the mother. For pregnant women with tations of existing library construction and bioinformatics
Down’s syndrome, it is not difficult to calculate 11 copies analysis, approximately 5% of cases with chromosomal
of the free DNA of chromosome 21 in peripheral blood, 3 abnormalities in twin or multiple births, chimeras, and par-
from the fetus, and 8 from the mother. The proportion of free ents are not suitable for NIPT analysis. At this stage, the
DNA on chromosome 21 in maternal peri-week plasma with technique is abnormal for other chromosomes. There is still
Down syndrome and normal fetus is 11:10. Similarly, for a no effective screening indication for structural abnormalities
case where the fetal-free DNA content is 5%, the ratio of free and microdeletion syndrome. Therefore, only the detection
DNA of chromosome 21 in the maternal peri-week plasma of report of 21, 18, 13-trisomy syndrome is issued in the clinic,
Down’s syndrome fetus and normal fetus is 10.25:10. and other chromosome aneuploidy abnormalities will be
Therefore, there is no need to isolate fetal-derived free analyzed at the same time. The written report is not issued
DNA, and the total content of free DNA of chromosome at present. This method is not suitable for detecting abnor-
21 in the peripheral plasma of pregnant women with Down’s mal chromosome structure, and is not suitable for detecting
syndrome is generally increased slightly. In theory, by dis- twins. For the above multiple births, it is not possible to use
tinguishing this small difference, we can achieve prenatal this test result as the basis for whether or not to terminate the
detection of fetal chromosomal diseases using cffDNA. pregnancy. The test result is “high risk” pregnant women,
After the high throughput sequencing is completed, the and further prenatal diagnosis is recommended.
data is quality-evaluated and effectively filtered, and the
sequencing data is returned to a single sample according
to the label, and the sequencing result of each sample inde- 6.6.4 Genetic Disease Diagnosis Report
pendently generates a corresponding data file (.bin file), and
Align to known human genome sequences. Use the universal Each gene has a specific biological function that can be
function to compare and filter the unique matching Reads encoded into different proteins to maintain the structure and
number of each sample, that is, the unique reads number, and function in normal humans. Once a gene has a structural
perform multistep data correction on the data, such as GC change in base pair composition or sequence, we call it Gene
correction and RUN correction to achieve data normaliza- mutation. If the mutation of the gene causes the function of
tion, and calculate each sample. The number of unique reads the encoded protein to be abnormal, or the number of chro-
86 Y. Tong
Normal,
Bell-shaped curve
0.13% 2.14% 13.59% 34.13% 34.13% 13.59% 2.14% 0.13%

Percentage of
cases in 8 portions
of the curve
Standard Deviations –4α –3α –2α –1α 0 +1α +2α +3α +4α
Cumulative 0.1% 2.3% 15.9% 50% 84.1% 97.7% 99.9%
Percentages
Percentiles
Z scores –4.0 –3.0 –2.0 –1.0 0 +1.0 +2.0 +3.0 +4.0
T scores 20 30 40 50 60 70 80
Standard Nine 1 2 3 4 5 6 7 8 9
(stanines)
4% 7% 12% 17% 20% 17% 12% 7% 4%
Percentage
in stanine
Fig. 6.9 The percentage of unique reads of chromosomes 21, 18, and 13 meets the standard normal distribution
mosomes or structural variation will lead to disease in the screening for pathogenic gene carriers or high-risk individu-
body, called genetic disease, it is often characterized by ver- als in the patient’s family, effectively reducing the incidence
tical transmission and lifelongness. According to the changes of these diseases. Genetic counseling and prenatal guidance
of genetic material, genetic diseases can be divided into five for patients with fertility requirements and their families can
categories: chromosomal diseases, monogenic diseases, prenatal diagnosis of the fetus through amniotic fluid or cho-
polygenic diseases, mitochondrial diseases, and somatic rionic cells, which plays an important role in the early inter-
genetic diseases. Currently, there are more than 500 kinds of vention of genetic diseases.
chromosomal diseases, and there are many natural abortion Thus, genetic diseases are diseases caused by changes
fetuses. 20–50% are caused by chromosomal abnormalities, in genetic material, including chromosomal aberrations
and the incidence of chromosomal abnormalities in new life and genetic mutations that are invisible at the chromosomal
infants is also 0.5–1%. There are about 7000 single-gene level. At present, most genetic diseases do not have a good
genetic diseases confirmed in the world, and more than 3000 treatment method. Therefore, it is necessary to carry out tar-
kinds of therapeutic genes are included in the OMIM data- geted individualized medical tests based on the molecular
base, such as color blindness, hemophilia, and thalassemia, basis of the disease to achieve molecular diagnosis of the
all of which are monogenic diseases (Table 6.12). Cleft lip, disease. Below we will use the clinical molecular diagnosis
cleft palate, hypertension, diabetes, manic depression, rheu- of G6PD deficiency as an example to describe the interpreta-
matoid arthritis, and congenital heart disease all have mul- tion of the molecular diagnosis results of genetic diseases.
tiple genetic basis and each has its heritability. There are 117
somatic genetic diseases in the OMIM database. The most 6.6.4.1 Clinical Molecular Diagnosis of G6PD
common somatic genetic diseases are tumors, such as reti- Deficiency
noblastoma, hereditary breast cancer, and familial adenoma-
tous polyps. Introduction to G6PD Deficiency
At present, molecular diagnosis in developed countries G6PD deficiency is the most common X-linked incomplete
has been systematically and universally applied to the diag- dominant hereditary enzyme deficiency syndrome in the
nosis and genetic counseling of single-gene genetic diseases. world [7]. About 400 million people worldwide are affected,
By analyzing a specific gene (DNA) or its transcript (mRNA) and patients are eating broad beans. The cause of G6PD defi-
of a subject, it can not only patients can also be diagnosed by ciency is due to mutation of G6PD, which catalyzes the dehy-
Table 6.12 Common genetic diseases and their corresponding genes Table 6.12 (continued)
Number Single gene disease Chromosome Gene Number Single gene disease Chromosome Gene
1 Alpha-thalassemia chr16 HBA1,HBA2 34 Marfan syndrome chr15 FBN1
2 Beta-thalassemia chr11 HBB 35 ADA type severe chr20 ADA
3 Congenital adrenal chr6 CYP21A2 combined
hyperplasia immunodeficiency
4 Hemophilia A chrX F8 disease
5 Duchenne muscular chrX DMD 36 Limb-type muscular chr2 DYSF
dystrophy dystrophy (LGMD2B)
6 Adult autosomal chr16 PKD1 37 Rett syndrome chrX MECP2
dominant polycystic 38 Spherocytosis chr14 SPTB
kidney type I
7 Adult autosomal chr4 PKD2
dominant polycystic
kidney type II drogenation of glucose phosphate to maintain the reducibility
8 Phenylketonuria chr12 PAH of the antioxidant glutathione (GSH), eliminate the toxicity
9 Spinal atrophy chr5 SMN1 of intracellular peroxides, and protect hemoglobin and cell
10 Lieber’s congenital chr17 AIPL1 membrane thiol protein while maintaining the stability of red
cataract blood cell structure and function. The G6PD gene mutation
11 Congenital painless with chr1 NTRK1
leads to a decrease in its enzymatic activity, and the peroxide
no sweat
12 Congenital contracture chr5 FBN2 formed in the red blood cells is easily damaged, and the red
13 Congenital aniridia chr11 PAX6 blood cells cannot be destroyed by oxidative damage, caus-
14 HLA matching chr6 HLA-A, ing hemolytic anemia. The underlying cause is the insuffi-
HLA-B, cient production of NADPH and thus the low functional loss
HLA-DRA, of GSH and the lack of oxidative vulnerability of Cat and
HLA-DQB1
GSHPX antioxidant dysfunction (Fig. 6.10).
15 Wiskott-Aldrich chrX WAS
syndrome It is currently believed that G6PD deficiency is the result
16 Ornithine chrX OTC of natural selection of malaria, mainly distributed in the
carbamoyltransferase tropics, the Middle East, Southeast Asia, South America,
deficiency the Mediterranean coast, and southern China, with an inci-
17 Hepatolenticular chr13 ATP7B
dence rate of 5% to 25% (Table 6.13). China is one of the
degeneration
18 Hereditary non- chr7 SLC26A4
high-incidence areas of this disease, showing a distribution
syndromic hearing loss of high and low north, with a prevalence of 0.2–44.8%. It
19 X-linked ichthyosis chrX STS is mainly distributed in the provinces south of the Yangtze
20 Neurofibromatosis chr17 NF1 River, with high levels in Hainan, Guangdong, Guangxi,
21 Osteogenesis chr7 COL1A2 Yunnan, Guizhou, and Sichuan provinces. Since most of the
22 Less sweat or no chrX EDA disease is usually free of clinical symptoms, early diagnosis
sweat-type ectodermal
hypoplasia
and early intervention are very important for the prevention
23 Spinal epiphyseal chr12 COL2A1 and control of the disease. If necessary precautions are taken,
dysplasia the severity of the clinical manifestations of the deficiency
24 Viscous lipid storage chr12 GNPTAB can be greatly reduced or prevented.
disease G6PD deficiency is an X-linked incomplete dominant
25 Methylmalonic aciduria chr6 MUT inheritance. The manifestations of enzyme deficiency are
26 X-linked chronic chrX CYBB
different, so the clinical manifestations vary and vary greatly,
granuloma
27 Severe combined chrX IL2RG from asymptomatic to neonatal jaundice, drug-induced
immunodeficiency hemolysis, acute hemolysis caused by infection, etc. Severe
28 Huntington’s disease chr4 HTT jaundice in neonatal period causes death or permanent nerve
29 X-linked lymphocytic chrX XIAP damage. WHO classifies broad bean disease into five grades
proliferative disease type based on G6PD activity (Table 6.14).
II
Because most of the disease is usually without clinical
30 Ichthyosis vulgaris chr1 FLG
31 Cartilage hypoplasia chr4 FGFR3
symptoms, early diagnosis and early intervention are very
32 Citrullineemia type II chr7 SLC25A13 important for the prevention and control of the disease. If
33 Spinocerebellar ataxia chr14 ATXN3 necessary precautions are taken, the clinical manifestations
SCA3 of G6PD deficiency can be greatly reduced or prevented.
88 Y. Tong
Fig. 6.10 Schematic diagram Parasite plasma

of the molecular mechanism L-glutamate + L-cysteine membrane
of faba bean disease
γ - GCS
MRP
NADPH ne
io
a th te
g -L-glutamyl-L-cysteine ut ga
Gl nju
Co
glycine
T
GS
GS
Toxic nucleophile
GSH NADPH
Reduction of Pentose phosphat
hydrogen or pathway:G6PD and
GR
lipid peroxides 6-phosphoglucona
dehydrogenase
GSSG
NADP+
Table 6.13 Incidence of G6PD deficiency Table 6.14 G6PD variant WHO class and associated G6PD deficiency
phenotype
Region Total prevalence estimate Prevalence estimate for males
Africa 7.5% (7.1–7.9) 8.5% (7.9–9.1) Enzyme
Middle 6% (5.7–6.4) 7.2% (6.6–7.7) Class Enzyme activity activity ratio Disease
East Class Serious lack of enzyme <10% CNSHA
Asia 4.7% (4.4–4.9) 5.2% (4.7–5.6) I activity
Europe 3.9% (3.5–4.2) 3.8% (2.9–4.7) Class Serious lack of enzyme <10% AHA
Americas 3.4% (3.0–3.8) 5.2% (4.7–5.8) II activity
Pacific 2.9% (2.4–3.4) 3.4% (2.7–4.1) Class Moderate or slightly 10%–60% Intermittent
III reduced enzyme activity hemolysis
Class Normal enzyme activity 60%–100% Normal
IV
Methods and Test Items for Clinical Molecular
Class Increased enzyme 100%–150% Normal
Diagnosis of G6PD Deficiency V activity
The gene that causes faba bean disease is the G6PD gene, CNSHA chronic non-spherocytic hemolytic anemia; AHA acute hemo-
and the molecular diagnosis is to detect the G6PD gene lytic anemia
mutation. The detection of G6PD gene mutation can not only
provide direct evidence of the pathogenesis of the disease at that patients with G6PD deficiency must be tested for G6PD
the genetic level, but also make an etiological diagnosis for gene status.
the patient, and can make prenatal diagnosis for the fetus.
Different mutation types have different effects on G6PD Detection Method of G6PD Deficiency Target Molecules There
activity, so detecting G6PD gene mutations is also useful for are more than 180 types of G6PD gene mutations have been
determining prognosis and guiding treatment. reported so far. The genetic variant gene of G6PD deficiency is
located on the X chromosome in Xq28, and the patient’s gene
Source of G6PD Deficiency Molecular Testing Project consists of 13 exons and 12 introns. Most of the genetic muta-
The cause of G6PD deficiency has been described in detail in tions in patients with G6PD deficiency are single-clip replace-
the geneticist’s reference because of a decrease in the activity ment missense mutations (Fig. 6.11) [8].
or inactivation of its encoded protein due to mutations in the
G6PD gene. The Clinical Pharmacogenetics Implementation The distribution of mutation types has ethnic heteroge-
Consortium (CPIC) has published a relationship between neity. Up to now, there are more than 30 mutations found
genotypes of G6PD deficiency and drugs such as ralli- in the Chinese population, missense mutations including
zyme, and clearly indicates that G6PD gene status must be A95G, T196A, G202A, C274T, G392T, G442A, G487A,
detected in patients with G6PD deficiency. The World Health A493G, T517C, C519G, C519T, C563T, C592T, C703T,
Organization (WHO) also published a report on the relation- A835G, A835T, G871A, C1004A, C1004T, C1024T,
ship between G6PD deficiency and drugs, clearly indicating C1311T, G1360T, G1376T, G1381A, G1387T, G1388A,
A95G T1153C
A535T
G159C G593C T1141C G1154T
G40A T224C A527G C1155G
T1139C
G637T A1156G
C317G A1138G
T648G C1159T
C1089A
E1 C514T G1160A
A713G C1082T
G488A A1162G
E2 G1081A
G769C A1166G
E3 A376G G962A C1177G
G806A
G1178A
E4 G820A
G1180C
C833T
E5 C1187T
C241T G910T G1192A
E6 G1215A
G242A G921C
C131G G1228T
T323A E7
T143C C592T G1229M
G172A G337A E8
A634G G1246A G1462A
C185T A376G
E9 C1284A G1463T
G202A T383C G680A
G392T G1466T
T208C G871A
A835T E10 G1502T
A209G A404C G916A
C406T G844C A1376M E11
G929A
G466A G854A G1361A
Class I(<10%) G949A E12
G487A T968C C1360T
A493G T1358A
Class II(<10%) C977A E13
G497A G1003A G1347C
A542T C1004A A1342G G1388A
Class III(10% to 60%)
C563T G1339A C1387T
C1024T
Class IV(>60%) G1048c C1318T G1400C
G1052T G1316C
C1057T A1220C
A1193G
Fig. 6.11 Common mutation sites of G6PD gene and classification of WHO enzyme deficiency
A1414C, and the like. Among them, the most common 2. Blood spots: Blood spots are mainly used for newborn
G6PD gene mutation sites in the Chinese population: screening, generally collected from the newborn heel acu-
G1376T, G1388A, A95G, C1024T. Among them, G1376T, puncture. It can also be used for the collection of adult
G1388A, and A95G accounted for 60–72% of the total samples in case of difficult sample transportation and
mutation rate of G6PD gene in Guangdong, Guangxi, and storage. Blood samples are naturally dried after collec-
Hainan. In addition, G392T, C592T, G871A, and C1024T tion to avoid exposure to sunlight and ultraviolet light,
accounted for 10–20%. baking, and volatile chemicals. Filter paper dried blood
tablets should be placed in a sealed bag.
For Example 3. Saliva: Saliva is convenient and non-invasive, and the
Sample Source amount of DNA extracted is superior to the whole blood
Different testing purposes have different requirements on the sample; especially the salivary specimen is stable and
type and collection of samples. Each laboratory can formu- can be stored at room temperature for a long time. It is
late the required sample types, collection quantities, and spe- now used for population screening and genetic diagno-
cial requirements according to the testing items. sis. Sampling and mailing in remote areas. Currently, the
commercially available salivary sample collection tubes
1. Peripheral blood: Peripheral blood is the most com-
are provided with a saliva nucleic acid preservation solu-
monly used body fluid sample. The type of anticoagu- tion, which is very simple to use. The collected salivary
lant should be determined according to the experimental nucleic acid can be stably stored at room temperature for
requirements. When sampling, gently mix and mix the more than half a year.
blood collection tube several times to ensure sufficient 4. Oral swab: After the patient rinses the mouth, the medi-
anticoagulation, and the action should be gentle to avoid cal swab is repeatedly wiped several times on the cheek
hemolysis. mucosa on the inner side of the mouth, and the cotton
90 Y. Tong
swab is taken out and placed on the filter paper after ster- Results Report
ilization, and at least three are extracted from each sub- The molecular diagnostic report analysis process for Sanger
ject. The cotton swab is stored in a clean sealed plastic sequencing in clinical laboratories should be developed to
bag. ensure that each examiner discards personal preferences and
5. Prenatal diagnosis samples: Prenatal diagnosis samples makes the report content consistent when performing report
are mainly used for prenatal diagnosis of genetic dis- analysis.
eases, including amniotic fluid, villus, and cord blood.
Detection Process References

Care should be taken in clinical diagnosis and quality con-
trol of the specimens at the time of specimen collection 1. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the
interpretation of sequence variants: a joint consensus recommenda-
to ensure that there are sufficient cells for testing. At the tion of the American College of Medical Genetics and Genomics
same time, it is necessary to check whether the informed and the Association for Molecular Pathology. Genet Med.
consent is signed and whether the family history is clear. 2015;17:405–24.
The quality of the sample extraction and the efficiency of 2. Freed D, Stevens EL, Pevsner J. Somatic mosaicism in the human
genome. Genes (Basel). 2014;5:1064–94.
PCR amplification should be noted in the analysis. After 3. den Dunnen JT, Dalgleish R, Maglott DR, et al. HGVS recommen-
the analysis, you should pay attention to regularly update dations for the description of sequence variants: 2016 update. Hum
the interpretation of the results to make them consistent Mutat. 2016;37:564–9.
with the latest clinical guidelines or specifications to avoid 4. Biesecker LG, Harrison SM, ClinGen Sequence Variant

Interpretation Working Group. The ACMG/AMP reputable source
misinterpretation. criteria for the interpretation of sequence variants. Genet Med.
2018;20:1687–8.
Data Analysis 5. Armour CM, Dougan SD, Brock JA, et al. Practice guideline:

According to the analysis of the results of the sequencer joint CCMG-SOGC recommendations for the use of chromosomal
microarray analysis for prenatal diagnosis and assessment of fetal
analysis, the analysis should pay attention to: loss in Canada. J Med Genet. 2018;55:215–21.
6. Cherry AM, Akkari YM, Barr KM, et al. Diagnostic cytoge-

1. QC data analysis: GAPDH was used as a positive control, netic testing following positive noninvasive prenatal screening
and ultrapure water was used as a negative control to results: a clinical laboratory practice resource of the American
College of Medical Genetics and Genomics (ACMG). Genet Med.
monitor the PCR and sequencing reactions. 2017;19:845–50.
2. Results interpretation: The sequence was compared with 7. Luzzatto L, Nannelli C, Notaro R. Glucose-6-phosphate dehy-

the gene reference sequence in NCBI GenBank, and drogenase deficiency. Hematol Oncol Clin North Am. 2016;30:
the mutation was determined according to the results of 373–93.
8. Fiorelli G, Martinez di Montemuros F, Cappellini MD. Chronic
nucleic acid alignment. The mutation site was based on non-spherocytic haemolytic disorders associated with glucose-6-
the NCBI-ClinVar database and the gene mutation profes- phosphate dehydrogenase variants. Baillieres Best Pract Res Clin
sional database. Haematol. 2000;13:39–55.
Factors Associated with Variation
7
Xue Qin
Precision medicine is now undergoing rapid development tory results are not accurate enough, even if the instruments
and constitutes an inexhaustible driving force for innovation were well-functioned and the technologies were advanced.
and progress in laboratory medicine. The accuracy of labora- The accuracy of the laboratory results is responsible for the
tory reports requires not only the advanced laboratory instru- correct diagnosis and treatment in clinic; therefore it is only
ments with high precision but the systematic laboratory when the specimens are correctly sampled according to cor-
quality management and control [1]. In this course, the pre- responding methods and procedures, the laboratory results
analytical phase is a key process easy to be neglected for the are useful to the clinic.
majority of clinical staff, and is susceptible to numerous fac-
tors whose quality is difficult to control [2]. This section
focuses on pre-analytical quality control and introduces the 7.2.1 Specimen Collection
effect of sampling, patient’s physiological factors, and drugs
on laboratory reports. 7.2.1.1 Time of Collection
A fasting specimen is a specimen collected from a patient
after at least 8 h on an empty stomach. Clinical biochemical
7.1 The Concept tests usually use morning fasting specimens to reduce the
influence of diet, physiological factors, and exercise, thus
The pre-analytical phase refers to a process from the moment discovering true pathogenesis.
clinicians propose test orders to the time clinical laboratory A sporadic specimen is a specimen collected without time
receives the specimens, which includes test orders, patient restrictions. These specimens are generally used for compul-
preparation, and the collection and delivery of specimen to the sory inspection items of emergency patients, or for items
laboratory [3]. The completion of the pre-analytical phase with stable metabolism and low interference factors.
requires the cooperation of clinicians, collectors, patients, and A specially collect specimen refers to specimens collected
specimen deliverers; therefore the quality of such a process is within a certain period according to requirements of different
difficult to be controlled by laboratory alone, and laboratory inspection items. For example, bacterial blood culture speci-
error always emerges in reports. According to statistics, most mens should be collected during fever plateau before the use
of the reasons for the unsatisfactory test report according to of antibiotics, which has the highest positive rate. Cardiac
clinic feedback are mainly the unqualified specimens [4]. troponin T or I in myocardial infarction, however, is sug-
gested to be collected 4–6 h after onset. Urine HCG (human
chorionic gonadotropin) should be collected 35 days after
7.2 Sampling pregnancy, which also generates the highest positive rate.
The virally infected antibodies are recommended to be col-
The sampling, delivery, and preservation of specimens are lected from two sera at both the acute and convalescent
crucial in the pre-analytical phase of systematic laboratory phases of the disease.
quality management and control. If the way of sampling does
not meet the requirements of clinical laboratory, the labora- 7.2.1.2 Method of Collection
X. Qin (*) Body Position and Part in Blood Collection

Department of Clinical Laboratory, First Affiliated Hospital of The quantity of laboratory results varies with different body
Guangxi Medical University, Nanning, Guangxi, positions and different body parts for blood collection. If the

92 X. Qin
patient switches from supine to erect or sitting, the water and cases, the sampling volume may not be required, such as
small molecules in the body will be transferred from the people with difficulty in collecting blood (such as newborns),
blood vessels to the interstitial space, resulting in an increase cerebrospinal fluid specimens, and critical patients.
in the concentration of macromolecules that cannot filter the
blood vessels: albumin and immunoglobulin, for example, 7.2.1.4 Precautions for Sample Collection
the macromolecules substance of each will increase by
5–15%. Components in arterial blood, venous blood, and Collect Representative Specimens
capillary blood could also be different. Therefore, when col- Sputum bacterial culture should avoid the incorporation of
lecting blood specimen, the body parts of blood collection saliva; feces examination and culture should take the pussy
should be prudently selected according to different inspec- bloody stool; urine bacterial culture should take the middle
tion purposes, so as to ensure the laboratory results with bet- urine or catheter urine; puncture fluid collection should try to
ter accuracy. avoid mixing traumatic blood.
Cuff Proper Use of Anticoagulant

While collecting venous blood, the use of cuff can occlude According to different inspection items, different anticoagu-
venous vessels for too long, result in local anoxia which lants should be chosen: for example, sodium citrate antico-
increases anaerobic glycolysis, and lead to lactic acid agulant in coagulation test, EDTA-K2 anticoagulant in blood
increase and pH decrease, consequently affecting the accu- routine and peripheral blood cell examination, and heparin
racy of related laboratory tests. Therefore, the cuff should be anticoagulation in red blood cell osmotic fragility test.
released immediately on viewing the back blood in piercing
the needle into venous vessels, and this procedure is sug- Avoid Specimen Hemolysis and Container
gested to complete in 1 minute. If there is a need for blood Contamination
recollection, it is suggested to collect venous blood from the When collecting specimens, sterile containers should be
other arm. used to avoid contamination of bacteria or other impurities,
which may result in inaccurate test results. When collecting
Infusion venous blood, the needle should be prevented from penetrat-
If possible, blood collection should be avoided during infu- ing the blood vessel and causing hemolysis due to repeated
sion, because the blood can be diluted during infusion, and freezing and thawing.
the infused substance can interfere with the test results. In
general, for patients who infuse fat emulsions, the blood is Avoid Collecting Blood During Infusion
suggested to be collected 8 h after the infusion; for patients Infusion not only dilutes blood specimen but also mixes
who infuse electrolytes, proteins, or sugars, the blood is sug- additional components such as glucose and electrolytes,
gested to be collected 2 h after the infusion. which can adversely affect the accuracy of the laboratory test
results.
7.2.1.3 Volume of Specimen
Different inspection items require different sampling vol-
ume. The insufficient volume can lead to the following 7.2.2 Sample Delivery and Preservation
problems:
7.2.2.1 Sample Delivery
• Fail to meet the requirements of the inspection items.
• Fail to meet the requirements for reviewing the specimen Delivery Principle
and conducting the verifying experiment. Samples should be delivered immediately after collection
• Fail to meet the requirements of retaining traceable and should be protected against contamination, breakage,
specimens. and loss during transfer. It is recommended to separate the
• Reduce the positive rate of some inspection items. serum or plasma immediately after blood collection before
delivery to the clinical laboratory when the specimen trans-
Some tests require high sampling-volume accuracy. For port takes a long time. Emergency samples and bacterial cul-
example, the sampling volume of the coagulation should be tured specimens should be sent immediately to ensure the
strictly assigned in proportion to the anticoagulant according timeliness of the specimens and to avoid microbial contami-
to the scale of the anticoagulant tube; in semen analysis, the nations. Most specimens should be sent to relevant labora-
amount of semen is one of the important indicators for evalu- tory within 1 h after collection, because for some specimens,
ating its properties; 24-h proteinuria is required to record the their components may alter (for blood specimens, the water
patient’s accurate urine output. However, in some special can evaporate leading to hemoconcentration). Some special
7 Factors Associated with Variation 93
specimens should be sent in a specified way to the labora- 7.3.1.1 Neonatal

tory. For example, the specimens delivered in semen and tro- The mother supplies oxygen to the fetus via diffusing through
phozoite examination need thermostat conditions; the blood. The fetus, hence, is always in a physiological hypoxic
specimens for insulin and C peptide examination are sug- state. Its red blood cell count and hemoglobin are signifi-
gested to be put inside an ice box before delivery to cantly higher than those of adults; after birth, as the respira-
laboratory. tory system matures, the destruction of red blood cells
accelerates. The increased hemoglobin is converted to indi-
Specially Assigned People rect bilirubin by reticular endothelial. However, since the
Clear the regulations, and ensure the promptness of speci- neonatal liver is deficient in glucuronyltransferase, the indi-
men delivery after collection to relevant laboratory. The clin- rect bilirubin is blocked by the bilirubin pathway, resulting in
ical laboratory should provide training on the relevant elevated serum total bilirubin and indirect bilirubin levels in
content of the delivered specimens, including the require- neonatal clinical manifestation. The leukocyte count of neo-
ments for specimen delivery, biological hazards, and natal is higher than that of adults in circulation, and the neu-
precautions. trophils and monocytes are continuously elevated within
1–2 weeks after birth. Lymphocyte counts are higher, while
Receiving basophils only transiently increase on the day of birth.
Carefully check the specimen information, the inspection
items, and whether the specimen meets the receiving regula- 7.3.1.2 Children
tions. If the blood volume is low, empty tube, and sample Because children are in the growth stage, they are character-
collection errors occur, the specimen should be rejected, and ized by rapid bone growth and increased metabolism of
the clinician should be notified to re-collect specimens. osteoblasts. At this time, the alkaline phosphatase secreted is
about 3 times that of adults.
7.2.2.2 Specimen Preservation
Specimens that need to be reexamined or cannot be exam- 7.3.1.3 The Elderly
ined promptly should be appropriately treated and stored. As the age grows, the overall function of the body declines,
For example, specimens should be covered to prevent leak- including decreased renal function, decreased creatinine
age and evaporation; attention should be paid to avoiding clearance, increased vasopressin, IL-6 and thyroid stimulat-
light and air; blood specimens should be separated into ing hormone, and reduced dehydroepiandrosterone sulfate
serum and plasma according to inspection items. Storage and serum III iodine levels.
temperature varies with specimen type. Generally, the stor-
age temperature of routine samples such as serum, plasma,
and body fluids is 4 °C, while the specimens such as nucleic 7.3.2 Gender
acids and strains should be stored at −70 °C and repeated
freezing and thawing should be avoided. Due to endocrine, gender, and organ-specific differences, for
some of the inspection items, the results of males could be
higher than females, including creatinine, creatine kinase,
7.3 Biological Factors hemoglobin, red blood cell count, urea, uric acid, transami-
nase, acid phosphatase, alkaline phosphatase, cholesterol,
In the process of pre-analytical quality control, the biological glucose, total protein, etc. For copper, reticulocyte counts,
factors affecting the accuracy of the test results include age, and high-density lipoprotein-cholesterol tests, the results are
gender, season, altitude, biological cycle, pregnancy, and usually higher in females than males.
lifestyle.
7.3.3 Season
7.3.1 Age
The variation of season always has impacts on inspection
Different age groups yield difference in inspection results. In results, for example, 25-hydroxyvitamin D3 levels can be
the routine work of the clinical laboratory, the reference higher in summer than in winter, because of the long-time
range of the inspection results of the corresponding popula- exposure to sunshine, while triiodothyronine and total cho-
tion should be set according to the age group. lesterol levels are higher in winter than in summer.
94 X. Qin
7.3.4 Altitude Table 7.1 Changes in major blood indicators during pregnancy
Variety Mechanism
With the change of altitude, the levels of some compo- Fat mobilization Apo AI, A II, triglycerides, and total
nents in serum can vary to adapt the body to the environ- increases cholesterol (especially LDL-C, i.e., low
density lipoprotein-C) greatly increase
ment, for example, serum transferrin, plasma renin,
Plasma transport Alpha-fetoprotein, ceruloplasmin, thyroxine,
estriol, urinary creatinine, and creatinine clearance protein increases and lipid increase in plasma
decrease as altitude increases, while the levels of Plasma dilution Total protein and albumin content reduce
C-reactive protein, red blood cell count, and hemoglobin Weight and Glomerular filtration rate decreases and
increase in the same course. metabolism increase creatinine clearance increases
Coagulation system Activity of coagulation factor increases,
hyperfunction prothrombin time and activated partial
thrombin time shorten, fibrinogen content
7.3.5 Biological Cycle increases
Relative deficiency Iron, transferrin deficiency
Circadian rhythm is an important factor affecting the fluc- due to increased
tuation of various analyte levels. For example, white blood need
Protein increase Erythrocyte sedimentation rate elevates
cell count is higher in the afternoon than in the morning; during acute
bilirubin and serum iron reach the highest concentration in reaction
the early morning, while blood calcium concentration is
lowest at noon. Cortisol can promote glucose metabolism,
7.3.7 Lifestyle
and it varies in large scale between day and night, which
has significant impact on the glucose tolerance test. It is
Habits and dietary patterns can affect some inspection results.
because cortisol concentration is lower in the afternoon
For example, excessive intake of coffee increases the levels of
than in the morning; in the glucose tolerance test, the blood
catecholamines, blood glucose, and angiotensin; high-fat diets
glucose level is higher in the afternoon than in the
increase the level of triacylglycerol; for the protein dieters, uric
morning.
acid and urea levels will increase; with high fish oil diet, triac-
The levels of multiple hormones also vary during differ-
ylglycerol and very low-density lipoprotein levels (VLDL)
ent stages of the menstrual cycle.
decrease; for excessive smokers, the adrenaline, cortisol, and
aldosterone levels increase; for long-term drinkers, their serum
• During the menstrual period, the levels of progesterone
transaminase and glutamyl transpeptidase levels will increase.
and estrogen in the serum gradually increase.
• In the follicular phase, the estrogen levels in this period
gradually increase due to the secretion of more estrogen
from the follicle.
7.4 Drugs
• In the luteal phase, the corpus luteum is produced after
The interference of the drugs on inspection results is compli-
ovulation of mature follicles, and the progestogen secreted
cated. Drugs can affect the accuracy of inspection results by
by it acts on the endometrium.
their own properties or their metabolites, mainly including:
Therefore, the reference intervals for multiple hormone
• Interference with the results of certain items through their
levels associated with reproduction vary with the menstrual
physiological, biochemical, toxicological, and pharmaco-
cycle phase.
logical properties
• Through the toxic side reaction of metabolites
7.3.6 Pregnancy
During pregnancy, due to the maternal supply of fetal growth

7.4.1 I nterference of Drugs on Routine
and development, adaptive physiological changes occur with
Inspection in Clinic
the participation of some hormones, for example, increased
blood volume during pregnancy results in blood dilution and
7.4.1.1 The Impact on Routine Blood Tests
decreased concentration of trace elements; the main indica-
Results
Certain antibiotics affect platelet and white blood cell count.
tors of blood changes during pregnancy are shown in
Most anti-tumor drugs inhibit bone marrow hematopoietic
Table 7.1.
7 Factors Associated with Variation 95
Table 7.2 Effects of drugs on routine blood tests 7.4.2.2 Drugs Affecting Blood Sugar Assay
Test items Effect Drug Blood glucose assay is an indispensable test for diabetes
Red blood Increase Hormonal drugs such as diagnosis. Some drugs can affect blood glucose measure-
cell glucocorticoids, adrenaline ments. Glycosides, caffeine, and dextran can increase blood
Decrease Baotaisong, sulfa glucose, while vitamin C decreases blood glucose.
Leukocyte Increase Corticosteroids, adrenaline
Decrease Antitumor drugs, sulfonamides,
quinolones 7.4.2.3 Drugs Affecting Protein Assay
Platelet Increase Aminoglycosides, β-lactam Serum protein assay is susceptible to a variety of factors:
Decrease Sulfonamides, quinolones, clofibrate and phenol sulfonate can affect the detection of
cephalosporins, antitumor drugs light absorption and lead to false increase in serum total pro-
Hemoglobin Increase Iron agent tein; fat emulsion and dextran can affect serum turbidity and
Decrease Antitumor drugs, β-lactam lead to false increase in serum total protein; anti-epileptic
drugs cause a false decrease in total protein due to the toxic
function, thereby affecting some blood routine tests. The side effects.
effect of specific drugs on routine blood tests is shown in
Table 7.2. 7.4.2.4 Drugs Affecting Lipid Assay
Vitamin C, corticosteroids, and iodide can lead to elevated
7.4.1.2 The Impact on Routine Urine Test Results serum total cholesterol because of the interference with
Urine dry chemical analysis is widely used in clinical labora- assay. Chlorpromazine, sulfonamides, and androgens
tories because of its concise operation and timeliness, but increase serum total cholesterol due to cholestasis, while clo-
some drugs could interfere with the test strip module and fibrate esters inhibition of cholesterol synthesis leads to a
lead to false test results. For example, high concentration of decrease in serum total cholesterol. Oral estrogen and birth
vitamin C in urine may produce false negative results for control pills increase serum triglycerides, while vitamin C,
urine glucose, urinary bilirubin, and urinary nitrite. β-lactam metformin, and phenformin cause a decrease in serum
drugs can cause false positive results in urinary occult blood triglycerides.
and urinary tract. Penicillin could lead to false negative
results for urine protein. 7.4.2.5 Drugs Affecting Electrolyte Assay
Phenylbutazone and guanethidine lead to sodium retention,
7.4.1.3 The Impact on Routine Stool Test Results which can increase serum sodium and chloride ion levels;
In some clinical situations, medications can affect the results drugs acting on renal tubules such as spironolactone, methi-
of the stool routine examination and chemical tests. For cillin, and tetracycline are nephrotoxic and can increase
example, taking iron, tincture, activated carbon, or some serum potassium levels. Some drugs with diuretic effects,
Chinese medicine can darken or tarnish the stool, while tak- such as mannitol and furosemide, will reduce the sodium,
ing large amount of vitamin C can lead to false negative potassium, and chloride ion levels in the blood.
results in the fecal occult blood test.
References
7.4.2 I nterference of Drugs on Clinical
Biochemical Tests 1. Sciacovelli L, Aita A, Padoan A, et al. Performance criteria and
quality indicators for the post-analytical phase. Clin Chem Lab
Med. 2015;53:943–8.
7.4.2.1 Drugs Affecting Enzyme Assay 2. Plebani M, Sciacovelli L, Aita A, et al. Quality indicators to detect
Ethanol drugs, analgesics, and isoniazid can cause an pre-analytical errors in laboratory testing. Clin Biochem Rev.
increase in transaminase activity, and Schisandra can reduce 2014;432:44–8.
3. Lima-Oliveira G, Volanski W, Lippi G, et al. Pre-analytical phase
the activity of transaminase. Drugs such as corticosteroids, management: a review of the procedures from patient preparation to
furosemide, and dexamethasone cause increased serum amy- laboratory analysis. Scand J Clin Lab Investig. 2017;77:153–63.
lase and serum lipase activity due to pancreatitis; cholinergic 4. Cornes M, Van D-LE, Grankvist K, et al. Order of blood draw: opin-
drugs, morphine, and codeine cause Addis sphincter spasm ion paper by the European Federation for Clinical Chemistry and
Laboratory Medicine (EFLM) Working Group for the Preanalytical
and increase serum amylase and serum lipase activity. Phase (WG-PRE). Clin Chem Lab Med. 2017;55:27–31.
Quality Control and Quality Assurance
8
Gaowei Fan and Qingtao Wang
Molecular genetic tests are utilized widely. Nucleic acid The biggest problem concerning nucleic acid-based tests
amplification-based methods are sensitive and are prone to is contamination which may lead to false positive results. For
the contamination of amplification product. Besides, poor example, micro-droplets aerosolized by opening and closing
quality of the specimens, unskilled personnel, incorrect stan- amplifying tubes contain 10–100 copies of the amplified
dard operation procedures (SOPs), and unqualified instru- product. These micro-droplets then deposit on the surface of
ments may lead to false negative tests. Nucleic acid the laboratory such as bench tops, floor, instruments, exposed
amplification-based assays are generally complex and need skin, hair, etc., increasing the risk of contamination [1].
higher professional technical requirements. The interpreta- Besides, the capacity of standardization and harmoniza-
tion of the results is crucial and should be done by qualified tion across different detection methods also varies. Incorrect
personnel. Thus, proper quality system is necessary for the results may lead to delayed disease diagnosis, inappropriate
timely and correct report. In this chapter, we talk about treatment, or clinical decision. Some genetic tests may be
important components of quality system, highlighting the performed only once in a lifetime; incorrect results not only
importance of establishing and following quality control and have a major influence on the tested individuals but also on
quality assurance. This text is intended to serve as a good their relatives. There are some potential sources of errors
laboratory practice primarily in amplification-based assays. affecting nucleic acid-based assays (Table 8.1).
Given the importance of the molecular testing in clinical
diagnosis and treatment, the Organization for Economic
8.1 Overview Cooperation and Development (OECD) Guidelines for
Quality Assurance in Molecular Genetic Testing highlighted
Since the 1980s molecular diagnostic testing has grown the importance of quality and harmonization in genetic test-
steadily. A wide range of nucleic acid-based tests such as ing services by recommending that genetic testing laborato-
quantitative PCR, DNA sequencing analysis, and molecu- ries should be accredited [3].
lar amplification methods are emerging to detect bacterial Proper quality control (QC) and quality assurance (QAu)
and viral infections, identify heritable genetic disorders, are often used interchangeably. In fact, the two terms refer to
and confirm mutations, deletions, and duplications. These two different procedures. QC is defined as “part of quality
methods open new perspectives in diagnosing diseases, management focused on fulfilling quality requirements”
preventing the risk of a heritable genetic disorder, assisting (ISO 9000[3.2.10]) [4]. QC refers to those activities that
therapy decisions, predicting treatment response, and must be included to detect, reduce, and correct deficiencies
assessing prognosis. in a laboratory’s analytical process prior to the release of
Currently, there are thousands of molecular genetic tests patients’ results. In clinical laboratory, quality control mate-
for infectious diseases, cancers, heritable disorders, and rials are used to evaluate the reliability of a method. QAu is
reproduction. Some molecular tests are commercially avail- defined as “part of quality management focused on providing
able; some are developed in laboratories. New issues are confidence that quality requirement will be fulfilled” [4].
raising with the expended use of genetic testing. QAu involves a planned system of review procedures, which
monitor quality control procedures and verify that quality
objectives have been met. The successful implementation of
a QAu relies on an appropriate guideline or criteria.
G. Fan · Q. Wang (*) Accreditation, certification, and licensure are critical compo-
Department of Clinical Laboratory, Beijing Chaoyang Hospital,
Capital Medical University, Beijing, People’s Republic of China nents of QAu. The items accreditation, certification, and

98 G. Fan and Q. Wang
Table 8.1 Potential sources of errors that affect results of the test [2] cess by which a governmental authority grants permission to
Potential sources of errors an individual practitioner or health-care organization who
Pre-analytic Lacking appropriate informed consent meets minimum standards or criteria to carry out a specific
phase Incomplete requisition forms task [2, 6]. Licensure regulations are usually required by law
Patient identification error and regulation. For example, clinical amplification-based
Specimen misplacement genetic laboratories in China must follow the regulation doc-
Unnecessary or inappropriate test selection and ument “Medical Institution Clinical Laboratories Regulation
specimen submission
Incorrect specimen collection, processing,
for Amplification-Based Molecular Diagnostics” launched
handling, and storage by the National Health Commission of the People’s Republic
Contaminated specimens of China (http://www.nhc.gov.cn, last accessed February 26,
Incorrect criteria for acceptance/rejection of 2019). Another critical mechanism of QAu is External
primary samples Quality Assurance (EQA) and audit. Sometimes, EQA
Analytic phase Improper use of equipment schemes are not available; inter-laboratory comparisons are
Incorrect reagent preparation and storage
performed to assess the performance of the test.
Poor quality of nucleic acids
Several points should be kept in mind to produce a correct
Digestion of DNases/RNases
Inhibitors (e.g., heparin, ethanol, and genetic test. A rational setup of a molecular genetic labora-
hemoglobin) tory aids in decreasing the risk of aerosol contamination.
Inappropriate quantification of input nucleic Besides, skilled personnel and appropriate facilities are the
acids guarantee of good quality results. Practical and accurate
Non-adherence to standard operating procedures SOPs should be established and followed. All SOPs and pro-
(SOPs)
cedure documents should be reviewed regularly. Thus, docu-
Post-analytic Incorrect interpretation of laboratory test results
phase Deficiencies related to genetic test reports
ment control is needed to ensure all the documents are
Equipment Equipment is not validated, maintained, relevant, up-to-date, and written in a clear and accurate fash-
calibrated, or operated correctly ion. Before the clinical usage, all methods used should be
Personnel Inadequate personnel training validated properly. Appropriate quality control samples
Quality control Lacking internal quality control should be implanted to monitor the quality of the test.
Inadequate establishment or verification of Participating external quality control can be helpful to
method specifications
improve test and should be followed. All processes in the
Problems in external quality assurance (EQA)
laboratory should be monitored regularly through internal
Deficiencies related to inter-laboratory
comparison and external audits. When problems associated with quality
management occurs, the laboratory must take effective
actions to guarantee the test; thus, corrective actions should
licensure are also misunderstood and used inaccurately. be performed. There are many different techniques in molec-
Accreditation refers to a procedure by which a recognized ular tests, and some of them are under clinical development.
body, usually a non-governmental organization (NGO) such No general principles can be applied to all techniques.
as ISO 15189, gives formal recognition that a health-care
organization meets predefined standards. Many accreditation
programs including Laboratory Accreditation Program (LAP 8.2 Laboratory Setting
of the College of American Pathology, USA), PALC-Clinical and Contamination Control
Laboratories Accreditation (Brazilian Society of Clinical
Pathology/Laboratory Medicine, Brazil), and Standard ISO Nucleic acid-based amplification methods have the advan-
15189 (International Organization for Standardization) are tage of exquisite sensitivity. In theory, after about 30 cycles
specific for clinical laboratory [5]. Accreditation is often a of amplification, 10 million copies of molecules are pro-
voluntary process, in which an organization chooses to par- duced from several copies of template. Aerosolized amplifi-
ticipate to improve themselves continuously. Certification cation products will contaminate reagents, equipment, and
refers to a process by which an organization assesses whether ventilation systems. Potential sources of contamination
an individual or an organization meets pre-defined criteria. include cross contamination between specimens, amplifica-
Certification organization can be either a governmental or tion product contamination, work bench surfaces, and venti-
non-governmental organization. Accreditation and certifica- lation ducts.
tion are often used interchangeably. Both accreditation and Efficiently reducing contamination relies on laboratory
certification can apply to organizations. Certification can design, laboratory practices, and chemical and enzymatic
also be applied to individuals [6]. Licensure refers to a pro- controls.
8 Quality Control and Quality Assurance 99
8.2.1 Laboratory Setting DNA, it is necessary to have two separated rooms for tissue
DNA extraction and plasma DNA extraction.
To ensure the quality, safety, and efficacy of the work, the It should be noted that the air handling systems for each
laboratory should provide sufficient space and infrastructure independent room/area are completely independent. If not,
for its staff, health-care personnel, patients, and the commu- the reagent preparation room and nucleic acid room should
nity. The laboratory should be designed to have designated be located closed to the source of air in reference to nucleic
rooms for specific uses, for example, laboratory working acid amplification and detection rooms [1]. The air pressure
space, office, storage facilities, washrooms, and patient colin different work rooms could be different to prevent aerosol
lection area. Working spaces should be clean, safe, and dust- contamination. In reagent preparation room, in order to be
free. When designing the laboratory, one must account for free of temple or amplicon, the air pressure should be posi-
equipment placement, proper ventilation and lighting, tive, and blow out of the room. In the specimen preparation
reagent and specimen storage, and fire-resistant environment room, nucleic acid extraction is performed. There exist PCR
for archiving of data. The accommodation and environmen- temples in this room. To prevent air blowing from the speci-
tal conditions such as energy sources, lighting, ventilation, men preparation room to the reagent storage and preparation
water resources, and disposal of medical wasters should ful- room, the air pressure should be lower than that of the reagent
fill the routine work of the laboratory. Temperature and storage and preparation room. Thus, the air pressure in the
humidity should be monitored frequently. If the temperature specimen preparation room should be slightly positive. In the
and humidity exceed the tolerance range, corrective action nucleic acid amplification room where the amplification
should be taken. reaction occurs, the air pressure in this room should be
For a molecular genetic testing laboratory, rational labo- slightly negative, so the air can’t blow back to the pre-ampli-
ratory setting is critical for minimizing the risk of air con- fication areas. In the PCR product analysis room, PCR prod-
tamination and maximizing workflow. To reduce aerosol uct is analyzed by gel electrophoresis, sequencing, or
contamination, the different working areas should be com- hybridization, according to the method used. In this room,
pletely independent. The number of independent areas may aerosol particles from billions of amplicons fill the air, which
differ depending on the nature of the molecular assay and the become the primary source of contamination. In this room,
types of specimen. the air pressure should be negative to stop the air from blow-
In principle, molecular genetic testing laboratories per- ing out of the room (Fig. 8.1).
forming amplification-based assays should have separate Another feasible way to reduce aerosol contamination is
independent areas for reagent preparation, nucleic acid to construct an anteroom into each independent room. The
extraction, nucleic acid amplification, and PCR product anteroom can be either positively pressurized or negatively
detection [7]. Reagent preparation room and nucleic acid pressurized by having the fan draw air in or out of the ante-
preparation room are called pre-amplification rooms. Nucleic room. The positive/negative anteroom serves much the same
acid amplification and detection rooms are called post- purpose as preventing the air from blowing into or out of the
amplification rooms. Room setup can be different according laboratory (Fig. 8.1).
to the nature of assays. For example, it is unnecessary for a
laboratory who uses real-time PCR to have a separated detec-
tion room. Because the PCR product of a real-time PCR is 8.2.2 Facilities
kept in closed tubes, nucleic acid amplification and detection
are in a closed tube; thus work areas for PCR and PCR pro- Appropriate facilities are critical for the quality of the test.
duction detection can be performed in one room. For micro- To avoid carryover contamination, each room should have
fluidic PCR of which nucleic acid extraction and detection their own dedicated equipment, consumables, and cleaning
are performed in a microchip, only two separated rooms are tools such as swab or rag. No equipment such as pipettes or
needed; one is for reagent preparation room, and another one cleaning utensils is allowed to exchange between different
is for specimen preparation, amplification, and detection. For rooms. To assure the safety of the staffs, safeguard equip-
a clinical NGS laboratory, several additional separated rooms ment such as eye wash, shower, and medical aid box is
may be needed for both dry-bench and wet-bench experi- needed [8]. The laboratory should have appropriate rooms to
ment, for example, cancer sequencing using Illumina MiSeq host the machine.
platform, additional separate rooms for library preparation, For example, in a nucleic acid amplification-based clini-
library amplification, hybrid capture, cluster generation, cal laboratory, different facilities are needed for a routine
sequencing, and electrophoresis. Working rooms can differ run. In a reagent preparation room, benchtop, autoclavable
according to the types of specimen. For laboratories, who pipettes, UV light, vortex, centrifuge, and a freezer for
perform PCR using both tissue DNA and circulating tumor reagent storage are likely present. In a specimen preparation
Fig. 8.1 A scheme for an a

ideal nucleic-acid
amplification-based
laboratory. (a) Each working
room has different air
pressure. (b) Positive
anterooms are used to prevent
air from blowing out of
laboratory. (c) Negative
anterooms are served to
prevent air from blowing into
the laboratory
room, nucleic acid extraction is performed. Infectious cal safety cabinet should be available. In a nucleic acid
specimen should be performed under a safety hood to protect amplification room where the amplification reaction occurs,
health. Besides, centrifuge, automated extraction platform, a thermocycler is needed for this function. In a PCR product
UV light, freezer for sample storage, centrifuge, vortex, analysis room, PCR product is analyzed by gel electrophore-
heater, fluorometer, and eye wash are equipped for the func- sis, sequencing, or hybridization, according to the method
tion. If organic solvents are used in the laboratory, a chemi- used.
8.2.3 Laboratory Practices about 60–90 cm. It is noted that the penetrating power of UV
light is limited.
In routine practice, some simple practices can be utilized to
minimize the risk of contamination. The workflow must be
uni-directional only, from reagent preparation to specimen 8.3 Internal Quality Control
preparation, amplification, and amplification product detec-
tion and analysis (Fig. 8.1) [9]. It is prohibited to move back. Internal quality control (IQC) materials are used to monitor
Tubes that contain amplicons should never be opened in the day-to-day consistency and to verify whether the results are
pre-amplification rooms. If one must go against the uni- reliable enough to be reported [13]. IQC is the mainstay of
directional workflow, one should change to a clean lab coat, quality control. The laboratory shall establish proper internal
gloves, hairnet, and shoe covers. quality control systems to help detect immediate errors, thus
The workflow of housekeeping is equally important. The increasing the probability of error detection and decreasing
laboratory staff instead of the housekeeping staff is advised the probability of false rejection. A laboratory should docu-
to perform the housekeeping. Aerosol contamination risk can ment its quality control plan in detail including control mate-
also be minimized if the laboratory staff bags the trash from rials to be used, candidate IQC procedures such as conducting
each room and leaves it outside the laboratory for picking up IQC at appropriate intervals and using appropriate IQC rules,
by the housekeeping staff. the concentration of the materials, and the criteria of in con-
It is a good idea to soak used pipette tips or PCR product trol or out of control.
in containers containing decontamination liquids such as In a molecular genetic testing laboratory, the IQC mate-
hydrochloric acid equivalent or sodium hypochlorite solu- rial is used to monitor the complete analysis process, includ-
tion. It is recommended to wear separated laboratory coats ing the extraction and amplification phases. When key steps
and caps in different working areas. When the staff leaves in the process are not included in the IQC procedure, IQC
the working area, he should leave the laboratory coats and can fail to identify an out-of-control situation.
caps in this room. It should be noted that coats from differ- In order to independently assess system performance,
ent rooms can’t be washed together. The use of closed tube IQC materials shall be different from calibrator materials or
systems such as real-time PCR and full automation system those produced by the manufacturer of the test or analyzer
which coupled sample processing module to real-time PCR [14]. Sometimes, there is no IQC material from a third-party
can reduce aerosol contamination to a large extent [10]. source; controls from manufacture of the reagents or calibra-
0.9–1.8% hypochlorite is quite efficient to eliminate labora- tor may be used [15]. If calibrators are used as controls, the
tory contamination [11]. Cleaning bench surfaces with 1% lot number for calibration should be different from that used
bleach solution followed by 70% ethanol is still warranted for QC [16].
after finishing work. Powder-free gloves must not be used Quality controls can be either a commercial or an in-
since the powder on the powdered gloves inhibits the ampli- house control such as a previous patient’s sample. To mini-
fication. It’s recommended to change gloves frequently to mize matrix effects on the measurement, it is recommended
reduce cross-over contamination. No-template control to select IQC materials that are similar to or identified with
(NTC) can be used to check for the absence of contamina- patient sample matrix [15]. For example, human serum-
tion in the lab [12]. based controls are commended for method that assays serum
samples. The laboratory shall use IQC materials that cover
the analytical concentrations encountered. And the concen-
8.2.4 Chemical and Enzymatic Controls trations used should be at the clinically relevant levels.
IQC materials should be stable for a long period of time
Enzymatic contamination control systems (e.g., introduction to avoid unnecessary frequent reordering and verification.
of uracil-N-glycosylase), aerosol-resistant tips, and aliquoted Besides, the selected IQC materials should provide the
reagents are also helpful to minimize the risk of contamina- desired low vial-to-vial variability [17].
tion [10]. When in-house controls are used, the laboratory shall
Additional safeguard against contamination includes UV verify and record their utilization. In-house controls should
lights (254 nm wavelength) and 1% bleach solution. UV at least possess the characteristics of homogeneity and sta-
light exposure for at least 5 min is sufficient to deactivate bility [18]. When the laboratory prepares in-house controls
nuclease and destruct extraneous DNA on the surface. by nucleic-based amplification, it should be noted that high
Usually, the working bench surfaces are exposed by UV for copies of quality control materials may be substantial sources
30 min before and after work. To assure the efficient irradia- of contamination. As a result, special attention should be
tion of bench surfaces, the working distance of UV light is paid to monitor, detect, and prevent cross contamination.
IQC materials should be tested in the same manner as patient person reference interval, and a second one is with a target
specimens [19]. value that would be in a sick person. If three levels of control
QC material should be stored as a requirement by the are used, a control with target value in an abnormally low
manufacturer. Important information such as content, stor- patient range should be included.
age requirement, date opened, personnel who prepared the The laboratory must not use mean, standard deviation
QC material, and expiration date should be labeled to the QC (SD), and ranges supplied by manufacture directly. Instead,
materials. Outdated QC materials should be stopped to use they shall establish their own means and SD after running for
any longer. a period of time (e.g., 20 data points from 20 separated days)
The IQC procedure shall be performed at least once dur- [15]. When a new lot of QC material is used, the laboratory
ing each analytical run. A run refers that all tests are ana- should calculate the mean and SD values of the new lot.
lyzed under repeatability conditions. Due to the systematic Acceptable ranges of IQC values (e.g., whether 2SD, 3SD,
changes within a run, repeatability is an ideal that is never or other) should be optimized for clinical decision and patient
realized. If the time span from the first to the last analysis is management [15].
short enough, it is believed that there is negligible change in The laboratory should record every quality control value
the magnitude of errors. The length of the analytical run is by plotting the measurements on control charts to see if the
defined as the interval (period of time or group of patients’ control results fall within certain control limits. Quality con-
samples) for which a control measurement is made [20]. trol statistics should be calculated and reviewed at specified
Analytical run can be defined based on the six sigma or risk- intervals, for example, one month, to define analytic impreci-
based approach [20]. In order to yield a more valid estimate sion and to monitor trends over time.
of analytical imprecision, the placement of quality controls
is recommended to be random rather than fixing.
The results of IQC should be reviewed to be acceptable 8.3.2 Qualitative Tests
before the release of the report. When IQC exceeds defined
acceptability limits, the laboratory should find the root cause Ideally, template control (assay positive control), mutation
and take corrective actions. The corrective action for tests template control (mutation positive control), and non-
should be recorded. In this case, the test results since the last template control (assay negative control, or blank control)
acceptable run should be retested to make sure if a signifi- for each analyte are included in each run [21]. No template
cant clinical difference occurs. control is when there is no starting material in the PCR mix.
QC logs should be available to document control results. If a positive result appears, it indicates that the reagents or
QC records should at least contain personnel who performs consumables are contaminated with sample containing the
QC runs, date, information regarding QC materials (material desired target. The negative control is a sample that doesn’t
name, manufacturer, concentration, lot numbers, opened include the target gene. When there is a reaction, it indicates
dates, expiration dates), and results of controls (e.g., Levey- the presence of contamination, or the reagent is not specific
Jennings charts or control charts). QC records should be for the target. The positive control is a sample that contains
reviewed, signed, and dated regularly. QC records should be the target sequence. If the positive control doesn’t amplify,
retained for a specific period of time according to the require- the storage and the expiry dates of the reagents must be
ment of local regulatory bodies. checked. Besides, the laboratory also needs to investigate
Alternative control procedures can be used to monitor the whether or not there exists PCR inhibitor.
performance of the test when control materials are not avail- For tests with a large targeted panel for detection of vari-
able. The laboratory may use alternative procedures includ- ants, it is not feasible to include all positive controls in each
ing split sample testing with another method or with another run. One can rotate positive controls in a systematic fashion
laboratory, the test of previously tested patient samples, or at a defined frequency, and within a period, all positive con-
the repeat test of patient specimens in duplicate [15]. trols are included [16]. For tests that use a cut-off value to
distinguish positive from negative, the cut-off value shall be
verified when a lot of controls changes.
8.3.1 Quantitative Tests
For quantitative tests, the laboratory should first define qual- 8.4 Quality Assessments
ity requirements in the form of allowable total error (TEa)
[20]. Then, proper control rules and numbers of control mea- Quality assessments can help to identify and resolve noncon-
surements are selected. Commonly, at least two levels of formity that may affect testing performance. Complete qual-
control at relevant decision points are used in every run. For ity assessments include reviewing examination requests and
example, one control is with a target value in the healthy sampling requirements periodically; monitoring customer
feedback, staff suggestions, quality indicators, corrective national or regional level by organizations independent of the
actions undertaken; and participating in external quality participating laboratories [26]. They allow a laboratory to
assessment (EQA) and audits. compare its quantitative or qualitative analytical results with
other laboratories. The purpose of EQA schemes is meant to
improve harmonization of the test instead of punishing the
8.4.1 Quality Indicators participants. As a result, participating in EQA schemes
becomes a key tool for quality control of molecular genetic
Quality indicators (QIs) are objective measures enabling test [27]. Governments and regulatory and professional bodies
users to define and quantify errors in the test. Thus, reliable should encourage laboratories to participate in an EQA scheme
QIs in the total testing process are fundamental tools in for diseases they test, where such schemes are available.
reducing errors and improving the quality of test [22]. QIs EQA schemes help to reduce the percentage of laborato-
are observations, statistics, or data which can be used to pro- ries making errors through sending EQA samples to partici-
vide the evidence that the laboratory is meeting its quality pating laboratories by EQA organizations. The samples sent
goals. QI data are collected over time to evaluate critical care by EQA organization are with known and validated geno-
domains such as patient safety, effectiveness, equity, timeli- type. The participating laboratories are asked to genotype the
ness, and efficiency. The International Standard ISO samples and return the result within a defined period. EQA
15189:2012 for Accreditation of Medical Laboratory samples should be tested in the same manner as clinical
requires the establishment and implementation of QIs to specimens and by the person who routinely perform the clin-
measure, monitor, and improve laboratory performance [23]. ical testing. The reports returned are assessed for technical
Different QIs are developed and used worldwide to docu- accuracy. Sometimes, EQA schemes provide mock clinical
ment the quality of the service provided, to improve perfor- scenarios with the EQA samples to participating laboratories
mance and patient safety, to make comparison between and ask participants to interpret the results in the context of
laboratories or periods, to make judgments, and to set priori- the clinical scenario and report their findings. Thus, the abil-
ties [24]. Due to the difference in terminologies and data col- ity to interpret the result and produce clear and informative
lection, it is unlikely to generate QIs in a comparable manner, reports is assessed. EQA organizations score the reports and
which hampers the widespread use of QIs [22, 24]. It is return a report on performance with individual comments to
urgently important for laboratories to adopt harmonized QIs. participants. The laboratory performance in EQA schemes
The process of harmonization of QIs consists of identifying should not be made public by EQA schemes providers unless
common QIs and standardizing reporting system [24]. To the laboratory volunteers or so required by law. The partici-
promote the harmonized use of QIs and reduce errors in lab- pating laboratories who do not meet the standards expected
oratory testing, the Working Group on “Laboratory Errors should find the root cause of the mistake and take measures
and Patient Safety” (WG-LEPS) of the International to improve the test.
Federation of Clinical Chemistry and Laboratory Medicine EQA organizations should be competent to provide such
(IFCC) develop a Model of Quality Indicators (MQI) [25]. schemes, as established by accreditation to the ISO/
These MQIs include 53 measurements to monitor 27 QIs IEC17043 or equivalent recognition. EQA schemes should
related to pre-, intra-, and post-analytic phases. (http://www. be organized to assess all critical phases of the laboratory
ifcc.org/media/455725/Quality_Indicators_Key_Processes. process, including the pre-analytic, analytic, and post-
pdf, last accessed February 27, 2019). analytic phase. Some EQA samples are provided in the form
A laboratory is encouraged to select suitable QIs. The of DNA. It should be realized that such samples don’t moni-
selected QIs should monitor for all operations across pre-, tor all the analytic steps (e.g., nucleic acid extraction and
intra-, and post-analytic phases. QIs including number of preparation). They might be derived from tissue samples or
tests offered, the turn-around time, failure rates, number of cell lines with a known genotype, or synthesized by adding
referred analyses, percent of positive test results, and user analytes into a matrix such as plasma or urine.
satisfaction survey may be used in clinical molecular testing EQA results should be reviewed and dated by the labora-
laboratory. The laboratory should regularly monitor these tory directors. When discordant EQA results in clinical
indicators for their concordance with the defined objectives genetics occur, the laboratory should investigate the cause of
established in the laboratory. problem and take corrective actions. The following problems
including clerical problem, quality control materials prob-
lem, methodological problem, equipment problem, technical
8.4.2 External Quality Assessment problem, and equipment problem may need to be checked
(Table 8.2).
External quality assessment (EQA) schemes are also known There are several well-established EQA schemes all over
as proficiency testing schemes which are organized at a the world, e.g., the Clinical Molecular Genetics Society
Table 8.2 Common problems associated with incorrect EQA results

Problems associated Problems associated with Problems with control Problems associated Problems associated with
with the clerical methods materials/calibrators with equipment technique
Mislabeled PT Lack of written procedure Method affected by Equipment Samples are mixed up
samples temperature or humidity malfunction
Transcription error Incorrect procedure or Carryover contaminations Errors in equipment Improper handling of sample
method software
Incorrect units were Method not properly Inappropriate reference Detection system Dilution error
reported validated or verified prior to intervals error
clinical use
Select an incorrect Pipetting or dilution error
reporting
The lack of Failure to follow written
commutable EQA procedures or EQA instructions
materials
Improper translation Improper reconstitution of EQA
materials
Incorrect data entry by Improper storage of EQA
EQA provider materials
Inappropriate target Misinterpretation of test results
value Improper calibration
Calculation error
(CMGS) in the United Kingdom, College of American Table 8.3 Overview of the main EQA providers
Pathologists (CAP), Centers for Disease Control and Name Website
Prevention (CDC; Atlanta, Georgia), and American College The European Molecular Genetics Quality https://www.emqn.
of Medical Genetics (ACMG) in the United States [26], Network (EMQN) org
European Molecular Genetics Quality Network (EMQN) in Genomics Quality Assessment (GenQA) https://www.genqa.
org
Europe, and National Center for Clinical Laboratories
European Research Network for evaluation https://erndim.org/
(NCCL) in China [28] (Table 8.3). and improvement of screening, Diagnosis home/about.asp
EQA schemes for nucleic acid-based diagnosis of infec- and treatment of Inherited disorders of
tious diseases, like hepatitis B, hepatitis C, hepatitis G, her- Metabolism (ERNDIM)
pes simplex virus, enteroviruses, JC virus, cytomegalovirus, UK NEQAS https://ukneqas.org.
uk
HIV, toxoplasmas, and Mycobacterium tuberculosis, have
American Proficiency Institute (API) www.api-pt.com
been developed. Other EQA schemes such as Huntington
Bio-Rad Laboratories (EQAS) www.qcnet.com
disease, Y-chromosome microdeletions, factor V Leiden (The College of American Pathologists) www.cap.org
mutations, non-invasive prenatal testing, and cancer diagno- CAP
sis such as EGFR, KRAS, and ALK are also developed. Clinical Microbiology Proficiency Testing www.cmpt.ca
One of the important problems faced by EQA organiza- (CMPT)
tions is the lack of appropriate QC materials for many genetic Institute for Quality Management in www.iqmh.org
Healthcare (IQMH)
tests [29]. Thus, developing a sustainable process to improve
National Serology Reference Laboratory, www.nrlquality.org.
the availability of appropriate QC materials for genetic test- Australia (NRL) au
ing is of utmost urgency [29]. Multiple approaches are avail- Oneworld Accuracy Inc. (OWA) www.
able including establishing stably transformed cell lines by oneworldaccuracy.
using residual patient samples [29], genetically constructing com
mutation samples that resemble natural mutation-containing Quality Control for Molecular Diagnostics www.qcmd.org
(QCMD)
human cell lines [29], genotyping cell lines in multiple labo- National Center for Clinical Laboratory http://www.nccl.org.
ratories by using a variety of kits or platforms, and creating (NCCL) cn/
plasmid controls containing target sequence [30]. Swiss Quality Control Center (CSCQ) www.cscq.ch
For many rare diseases, there are no EQA schemes avail- QualiGene www.qualigene.co.il
able. Besides unstable analytes, the lack of commutable German Society for Clinical Chemistry and www.dgkl.de
control materials also results in the lack of formal EQA Laboratory Medicine—DGKL
Italian External Quality Assessment www.iss.it
programs. When not available, laboratories should use
(IEQA-ISS)
alternative methods to ensure the performance of the test.
Table 8.3 (continued) 8.4.3 Auditing Program

Name Website
European Research Network for evaluation www.erndim.unibas. Audit is referred by ISO 9001:2000 as “a systematic, inde-
and improvement of screening, Diagnosis ch pendent and documented process for obtaining evidence and
and treatment of Inherited disorders of
Metabolism—ERNDIM
evaluating objectively the extent to which audit criteria are
Labquality Ltd. www.labquality.fi fulfilled” [33]. Audits are part of the continuous quality
Instand e.V. www.instandev.de improvement process. The purpose of audit is to assess
whether actual practices within the laboratory are against the
laboratory’s policies and procedures or guidelines or stan-
Table 8.4 Alternative methods that can be used to assess the perfor-
mance of the test dards; to identify and correct procedures that are deficient; to
investigate user satisfaction and complaints; and to ensure
Splitting clinical samples with
different level of This method assesses laboratory safety. Auditing laboratories is an effective way to
concentrations or genotypes inter-laboratory ensure compliance with good laboratory practices (GCLP)
are sent outside laboratory for agreement and guidance and SOPs. ISO 19011 “Guidelines for auditing
comparison of results. The technical errors management systems” offers a guide to perform a good
method requires at best the use instead of accuracy.
of the same method by both When a reference audit.
Split sample laboratories. If not applicable, method is used, Audit can be categorized as internal audit and external
with another other methods with the same accuracy of the test audit. Internal audits are self-assessment performed by the
laboratory reference range can be used can be assessed laboratory personnel. ISO 15189 demands that the labora-
Internal split A single sample is split into This method
tory should carry out an internal auditing program annually.
sample multiple aliquots and tested assesses neither
method by different methods inter-laboratory Internal audit consists of horizontal and vertical audit. A
comparison nor horizontal audit is a detailed check of a particular aspect of a
accuracy quality management system or testing processes. A vertical
Audit A single specimen is divided Audit sample audit is a detailed check of all elements associated with a
sample into multiple aliquots, and assesses precision
method these aliquots are tested by of the method, but chosen test. For example, assessing all aspects of a test from
the same method at different does not evaluate request, sampling, to report is a vertical audit. Examining a
time points. Then, the results accuracy or number of requests is a horizontal audit. External audits
of aliquots are compared inter-laboratory known as assessments are performed by external inspection
over time. Make sure that the comparison
aliquots are stable and bodies. Participation in EQA is a form of external audits.
maintain integrity during this Audits consist of preparation, execution, reporting, and
process follow-up [3]. To perform a good audit, the laboratory man-
Testing The laboratory may use This method can be ager has the responsibility to schedule and coordinate audits.
product calibrator of a different lot or used to determine
Selecting or designating trained personnel to organize the
calibrator or other quality control accuracy
trueness materials provided by audit team is critically important. The auditors should be
control manufacturer that is independent of the work being audited. They should receive
material traceable to a reference audit training and have a good understanding of the proce-
material or procedure
dure and reference material related to auditing. They should
Clinical Compare the testing results This method can
correlation to the clinical findings only be applied to familiarize themselves with the audit procedures. When sev-
studies certain patient with eral auditors are selected, one of them can be designated to
specific disorder serve as the principal auditor. Personnel who participate in
diagnosis audits should be allocated sufficient time, as audit can be
time consuming. Usually, published guidelines and stan-
Such performance assessments include split sample from dards are choosing as criteria to which the performance will
another laboratory, an internal split sample, audit sample, be compared. If not available, the audit team need to develop
using product calibrator or trueness control material, and their own best practices before audit. Besides, it is necessary
clinical correlation studies [31] (Table 8.4). Alternative for the auditors to study Quality Manual, guidelines, proce-
performance assessment should be performed at least twice dures, and records of prior audits (if available) to develop
a year where applicable. However, one should keep in mind better checklists in advance. An audit checklist may contain
that deficiencies related to alternative performance assess- the following points: assessment criteria to assess whether
ment may exist [32]. actual practices/procedures or policies comply with require-
Table 8.5 Examples of audit in the laboratory [34, 35] may be defined as “control materials,” “controls,” or “refer-
Process of ence materials.” For example, reference material, according
analysis Contents of audit to the International Organization for Standardization
Pre-analytical Are the laboratory tests appropriately selected? [ISO]15195, is defined as “material or substance, one or
phase more of whose property values are sufficiently homogeneous
Whether or not procedures for specimen collection,
labeling, and processing are established and
and well established to be used for the calibration of a mea-
followed? suring system, the assessment of a measurement procedure,
Is proper collection container or device used? or for assigning values to materials” [37]. Reference materi-
Do the transport conditions ensure the quality of als are divided into “certified reference materials” (CRMs)
specimen? and “reference materials” (RMs) by ISO [38]. CRMs are
Is there a criteria for specimen acceptance or characterized for composition, accompanied by a certificate
rejection?
that provides the value with associated uncertainties. CRMs
Analytical Is the performance of the test established and
phase verified? can be used as primary reference materials to trace the SI
Are specimens handled according to the SOP? unit for secondary calibrants by the manufacturers to estab-
Do quality control practices meet the requirement? lish their own calibrants. There are three different kinds of
Post- Are the results correctly reported? traceability:
analytical
phase
1. Traceability to the international system of unit (SI). This
Does the retention of reports meet the requirement?
kind of traceability is universally valid, without depend-
ing one any validated method or artifact.
ments; noncompliant finding; recommended corrective 2. Traceability to a method. This option can only be valid
actions; verifying corrective actions; and detailed reason that when a specific measurement protocol is strictly
corrective actions not verified. Once finished, the checklist is followed.
then reviewed and approved by the Lead Auditor. The labo- 3. Traceability to an artifact. This option may be dependent
ratory manager should provide the necessary resources to or independent of a specific method. For most genetic
assure the audit. Appropriate findings are also necessary, and testing, the traceability is established to an artifact. When
most of the costs are related to the use of staff resources. the traceability to an artifact is independent of a method,
Before the audit, make sure that auditees and the supervisors the used method must be properly validated.
of the areas are informed in advance. During the audit, the
auditors record all findings. Make sure that each element of Standard reference materials (SRMs) are the version of
the quality management system including pre-analysis, anal- CRMs produced by the National Institute of Standards and
ysis, and post-analysis is examined regularly [34, 35] Technology (NIST, US Department of Commerce). European
(Table 8.5). The auditors complete a Deficiency Report. reference materials (ERMs) are, similarly, referring to CRMs
Corrective actions are taken and completed by the audited produced by the European Reference Materials consortium.
personnel within a defined date. Upon finishing the correc- For a genetic testing, the final goal is to identify variants
tive action for each of the deficient findings, the audit is or abnormalities in a nucleic acid sequence (e.g., mutation,
closed. Documents including checklist, Deficiency Report, translation, duplication, amplification, and deletion), or to
and corrective action response should be maintained. quantify the copy number of the target sequence, or to deter-
mine the number of nucleotide repeats or the length of
nucleic acid sequence. Therefore, potential RMs with a certi-
8.5 Quality Control Material fied DNA sequence should be stable, homogeneous, and
commutable. They should have the same matrix as patient
Control material is referred as “a device, solution, or lyophi- specimens, if possible. They should be generated continu-
lized preparation intended for use in the quality control pro- ously. They should be stable for an acceptable period, and
cess; NOTE 1: The expected reaction or concentration of should have minimal vial-to-vial variability. Some RMs are
analytes of interest is known within limits ascertained during derived from reference cell lines (e.g., NA12878, NA19240,
preparation and confirmed in use; NOTE 2: Control materi- and NA18507) that contain a range of mutations at varying
als should not be used for calibration in the same process in alley frequencies [39]. Some are synthetic DNA samples
which they are used as controls” [36]. Quality control mate- containing specific sequence variants at known positions
rials have a broad application in both quantitative and quali- [40]. Some are genetically characterized cell lines [41]. For
tative test to calibrate assays, validate or verify the analytical NGS, quality control materials for bioinformatic analysis are
performance of test, monitor precision of the test, and deter- also required. In silico manipulated sequence data represent-
mine accuracy of the test system. Quality control materials ing a diversity of variants may be used to assess the limits
and capabilities of the bioinformatic pipeline. In silico mance parameters to fulfill a clinical intended use. For FDA-
manipulated sequence can be generated from common data approved tests, clinical laboratory should verify the
sets (e.g., FASTQ or SAM/BAM format), and software tools performance of the test under actual testing conditions within
are available to produce human genomes containing known their own laboratory in a particular patient population. Since
genotypes [42]. Methods such as digital-PCR, real-time FDA-clear/approved tests have been validated by manufac-
PCR, and sequencing are used to develop genetic RMs. turer to establish performance characteristics, the clinical
Digital PCR, which is independent of calibration, provides a laboratory need fewer samples to verify the tests in their own
way to count single DNA molecules and is used to provide laboratory. Commonly, performance characteristics includ-
absolute quantification and traceability to SI unit—the mole ing accuracy, precision, reportable range, and reference
[43]. Recently, digital PCR has been used to establish value range must be verified for quantitative tests. For qualitative
of certified RMs such as CMV and BCR–ABL [37, 44]. tests, reportable range is often simply positive or negative,
There exist very few CRMs for molecular genetic testing. mutation or wild. So, the laboratory doesn’t need to specially
They are mainly produced by NIST (SRMs), National verify the reportable range. For modified FDA-clear/
Institute for Biological Standards and Control (NIBSC), approved test or laboratory-developed test (LDT), the labo-
IRMM, Coriell, ATCC, WHO, LGC, Maine Molecular ratory must validate both analytical and clinical characteris-
Quality Controls Inc., Asuragen, Sera Cara, BAM, HPACC tics prior to clinical use. Analytic characteristic focuses on
and NCCL, etc. Information regarding quality controls and potential sources of technical variation in the analysis of
reference material producers can be found on the website of clinical samples, while clinical characteristic focuses on
EuroGentest (http://www.eurogentest.org/index.php?id=726, potential sources of biological variation in the analysis of a
last accessed February 27, 2019). given sample [50]. Analytic validity includes accuracy, pre-
When there is a lack of commercial RMs, one has to precision, reportable range, reference range, sensitivity, speci-
pare a RM in house. There are several guidelines to generate ficity, and other important parameters (e.g., reagent stability,
reference materials [45–47]. The following points should be linearity, carryover) [16]. Clinical validity includes positive
obtained to develop a quality control at home: selecting predictive value, negative predictive value, clinical sensitiv-
appropriate materials (native specimen vs spikes), preparing ity, and clinical specificity [16]. They reflect the ability to
and packaging, homogeneity and stability (short-term and correctly classify individuals with respect to their disease
long-term) testing, accurate and traceable characterization status. It should be noted that predictive values are related to
(for CRMs), and commutability testing and application. the prevalence in the population being tested. Clinical char-
acteristics such as clinical sensitivity and specificity can also
be obtained from scientific literature if they can’t be estab-
8.6 Verification and Validation lished within a laboratory [16]. Clinical utility refers to the
outcome of the clinical use of the test result. When there is a
Verification and validation are two different procedures. change in clinical validity, the user of the test should be
Verification is defined broadly as “confirmation through the informed. Both clinical validity and clinical utility should be
provision of objective evidence, that specified requirements assessed for each genetic test. There should be an adequate
have been fulfilled” by both the Food and Drug Administration number, type, and variety of samples to establish perfor-
(FDA) and ISO [48]. Verification refers to confirmation that mance parameters and define limitations, so that the results
the manufacturer’s claims can be replicated when the labora- can be interpreted for specific populations. In certain situa-
tory perform a test according to the package insert by tions, the rare mutation or variants are hard to obtain from
CLIA. Validation is defined as “confirmation by examination naturally occurring samples; alternative methods such as
and provision of objective evidence that the particular control samples can be used.
requirements for a specific intended use can be consistently For NGS test system, the validation process should
fulfilled” by FDA and ISO [48]. The term validation refers to include wet-bench validation and bioinformatic validation.
“a quality assurance process of establishing evidence that Paradigms for wet-bench validation include accuracy, preci-
provides a high degree of assurance that a product, service, sion, analytic sensitivity and specificity, and reportable and
or system accomplishes its intended requirements” by the reference range [51]. Since NGS can detect both known and
manufacturing industry [49]. Validation and verification are novel sequence variants, it is not possible to validate all pos-
often mix-up. In fact, validation goes a step beyond verifica- sible variants that can occur. It is recommended to use a com-
tion. Performance parameters should be established by vali- bination of a “methods-based” and “analyte-specific”
dation by the manufacturer before they are replicated by validation approach to assess a test’s analytic performance
verification in the laboratory. [52]. Variant information may be obtained from Sanger
To assure the quality of the molecular genetic testing, sequencing or oligonucleotide microarray genotyping data
clinical laboratories should establish or verify the perfor- [52]. There is a variety of open-source and commercial bio-
informatics algorithms and software for NGS data analysis. There should be enough information to identify the source
Each has weakness and strength in data analysis. of the samples, the authorized person who requested the test,
Bioinformatic validation aims to establish the computational and clinical information including examination and treat-
software settings required to provide accurate sequence data ment, as well as time and date the sample is taken in the
and detection variations within the target genomic regions request form. Additional information such as pedigree and/or
[51]. When there is an upgrade of the informatics portion, racial/ethnicity is required for molecular genetic tests for
there should be a revalidation. Commonly, the laboratory use heritable diseases and conditions.
known patient samples and training data sets to test algo- Nucleic acid quality relies largely on appropriate speci-
rithms and software parameters. Then, a larger set of samples men handling. Inappropriate specimen handling may lead to
are used to determine analytic sensitivity and specificity for degradation of target sequence, which is true especially for
the types of variant assayed after establishing a working set RNA. Poor quality of nucleic acid may result in falsely nega-
of bioinformatics tools and parameters [52]. tive result or inaccurate quantitative result. Thus, specimen
handling, including transport and storage, should be criti-
cally monitored for fitness. The laboratory should follow
8.7 he Process of Detection and Quality
T manufacturer’s directions to establish procedures for sample
Control collection and transportation to prevent specimen loss, alter-
ation, or contamination. An audit trail for every specimen
The process of genetic analysis is subdivided into three from collection, transport, to storage should be maintained
phases: pre-, intra-, and post-analytic phase. Pre-analytic and documented.
phase includes providing information to users of laboratory Molecular genetic tests may be performed with DNA
services, informed consent, test selection, requisition, speci- extraction from any specimen (e.g., peripheral blood, fresh
men collection, processing, handling, and transporting to the or frozen tissues, paraffin-embedded tissues, prenatal speci-
testing site. The service users should be noted the character- mens). The laboratory should provide practical instruction
istics of the test, the limitation of the tests, the clinical utility for sample collection (the type and amount of specimens to
of the tests, and the charges of the tests. Sometimes, informed be collected, appropriate collection container or device, spe-
consent is needed prior to the test according to the local law. cial timing of specimen collection), handling, and transpor-
The majority of errors are found in pre-analytic phase [53]. tation (conditionals under which analytes or specimen can be
When there is a legal/ethical necessity, the persons who are kept stable and maintain integrity) to those who collect,
authorized to order the tests are responsible for obtaining and transport, and handle samples. All samples should be granted
documenting informed consent. The analytic phase includes a unique identifying number. During the transport and stor-
nucleic acid extraction, measurement, the performance of age, proper conditions (e.g., the correct temperature, the cor-
test procedures, monitoring the reliability of the result, and rect time frame, and the designated preservatives) may vary
documentation of the test. The post-analytical phase includes depending on the analyte and the type of the specimen. The
result analysis, recording, reporting, and posttest counseling. samples must not be exposed to conditions that may cause
Closely monitoring pre-, intra-, and post-analytic phase, is the degradation of the target sequence. Transport must be
critical to ensure quality results. Standard operational proce- safe for the carrier, general public, and the laboratory staffs.
dure (SOP) should be available and understood by the staff. When non-PCR-dependent detection and PCR-dependent
detection are ordered by the same patient, separated samples
for PCR-dependent detection are required in order to avoid
8.7.1 Pre-analytic Phase opening the tube for non-PCR-dependent detection to pre-
vent contamination. To minimize the chance of mix-ups, it is
The laboratory should provide patients and clinical physi- better to have a designated reception area. The laboratory
cians/requestors information before test selecting. The infor- should establish defined criteria for acceptance/rejection of
mation can either be obtained from information pamphlets or specimen. Rejection reasons in a clinical genetic testing lab-
brochures, instructions for specimen submission, test request, oratory include improper handling or transport of specimens,
or from websites. The service users should be properly insufficient volume or amount of sample, use of inappropri-
informed of the intended use of the test (including the pur- ate anticoagulants, poor quality of nucleic acids, etc. When a
pose of testing and the recommended population), indica- specimen is inadequate, or lacks critical information, the
tions for testing (including performance specifications and laboratory should notify the submitting physician/requester
the limitations of the test), and how to correctly collect, store, and record the notification.
handle, and transport the specimen. Thus, unnecessary or A molecular genetic testing laboratory faces the chal-
unwarranted testing can be avoided. lenge associated with handling multiple types of samples.
When handling formalin-fixed, paraffin-embedded (FFPE) ryover contamination occurred by using no-template control.
tumor specimens, some important issues should be kept in For ctDNA, it is recommended to use extraction kits with
mind to ensure the quality of the sample. The time interval high yield. Methods such as UV spectroscopy or fluores-
from specimen acquisition to fixation should be less than cence spectroscopy are used for quantification of DNA. But
24 h. Tissue should be stored at 2–8 °C prior to process. The these assays may be problematic for low concentration DNA
fixation duration should be optimal. It is recommended that such as cell-free DNA and circulating tumor DNA. It should
fixation duration for surgically resected specimen is no lon- be pointed out that spectrophotometric methods measure
ger than 24 h [54]. A decalcified tissue should be avoided. It total nucleic acids (including double-stranded DNA, single-
is necessary to have separated area for paraffin wax slice, stranded DNA, oligo, and free nucleotides) as well as impu-
H&E staining, and sample quality assessment. To avoid rities. Thus, spectrophotometric methods should be
cross contamination between samples, it is best practice to accompanied by caution. Double-stranded DNA-specific
change scalpel blades between cases, transfer sections to fluorometric quantitation methods can be helpful to obtain
glass slides by using disposable plastic ware, and clean accurate DNA quantitation. Quantitative PCR is often used
work surfaces with bleach frequently [39]. The purity of to quantify DNA with low concentration [59]. When assess-
neoplastic cells should at best be assessed and recorded by a ing RNA quality and quantity, the laboratory should ensure
pathologist before the extraction of DNA. If the tumor that the A260/A280 ratio is between 1.8 and 2.0. If a ratio is
purity exceeds the minimum requirement of tumor purity, below 1.8, it indicates protein contamination. Nucleic acids
the specimen can be used directly for DNA extraction. If the integrity can be assessed by using a denatured gel or the
tumor purity is below the minimum requirement of tumor Agilent Bioanalyzer system. Quality and concentration of
purity, the area of the section containing tumor cells on the nucleic acids should be documented. When the quality of
H&E slide should be marked by a pathologist for micro- extracted nucleic acid is inadequate or doesn’t meet require-
dissection or macro-dissection [55]. Areas with extensive ments of the laboratory, the laboratory should reject the spec-
necrosis should be avoided to assure the integrity of nucleic imen and request a new specimen. Sometimes, if it is not
acid [54]. FFPE DNA becomes fragmented during the stor- feasible for a fresh specimen, potential risks should be
age period of FFPE, which may lead to false negative results detailed in the report.
[56]. Besides, deamination reactions for cytosine to uracil in It’s necessary for the laboratory to determine the calibra-
DNA recovered from older FFPE blocks (e.g., >3 years) tion and control procedures. The following information
may lead to false positives [57]. Uracil N-glycolase may be should be recorded when performing molecular genetic test-
helpful to eliminate this effect, but this should be validated ing: operator, test to be processed, date for each step, lot
thoroughly before routine use. Less than two-year-old FFPE numbers of the reagents, important equipment used, list of
samples are recommended for molecular genetic tests. It is samples processed together, and information about controls
better to use plasma instead of serum to extract circulating used in the run (lot numbers, position, results of controls).
tumor DNA (ctDNA) because the genomic DNA released
by necrotic WBCs may dilute the ctDNA fraction. Blood
samples can be collected in EDTA anticoagulant tubes or 8.7.3 Post-analytical Phase
cell-free DNATM blood tubes. CfDNA in Streck Cell-Free
DNA™ BCT blood tubes can be stable for up to 14 days at The laboratory should establish procedures for test reports,
room temperature [58]. retention of records and reports, and specimen retention.
The test results should be reviewed and evaluated with the
clinical information by designated staff. It is recommended
8.7.2 Analytic Phase to report the results in International System of Units (SI).
Internationally accepted standard terminology and nomen-
When a sample is received, the laboratory begins to process clature should be adopted when reporting genetic testing
the sample. To ensure the quality of the test, the laboratory reports. The genetic test report should be concise, accurate,
should have written procedures for testing. The laboratory and comprehensive and communicate all essential informa-
should validate or verify the test performance properly to tion to enable effective decision-making by patients and the
make sure that intended uses are fulfilled. Reagents should clinicians. The laboratory should define alert or critical val-
be prepared with caution throughout the test, and appropriate ues in consultation with clinicians. The laboratory should
safety equipment is used to avoid contamination. The labora- notify in time the responsible clinic staff when the assay
tory should validate or verify extraction and quantification results fall within established critical ranges, and the notifi-
approaches before clinical use. If DNA is extracted by an cation should be recorded. When the reporting time exceeds
automated system, the laboratory should monitor that no car- the turn-around time (TAT) established by the laboratory, the
responsible clinicians or the patients should be notified of the 8.9 uality Control of Laboratory
Q
delayed testing. There should be availability of laboratory Equipment
consultations regarding results interpretation, and implica-
tions of the test. The laboratory should ensure confidentiality All instruments used for genetic test will affect the quality of
of patient information throughout all phases of the testing the results directly or indirectly. Proper space and other con-
process. The laboratory should establish the procedure for ditions (e.g., humidity, vibration, and electricity) should be
storage of samples for a specified time post-analysis to guaranteed and monitored to make sure that the instruments
enable re-examination if required. Copies or files of reported work under appropriate conditions. If out-of-range tempera-
results should be retained for a specific time according to the tures and other conditions occurred, the laboratory needs to
local laws. take corrective action. Besides, the laboratory should estab-
lish the policy and procedure to install, validate, maintain,
calibrate, and operate equipment [31]. A SOP on the use,
8.8 Personnel maintenance, and safety risks of the equipment should be
written in a language understood by the staff and is accessi-
Best-trained personnel is the key aspect in the development ble at the bench. Make sure that the laboratory staffs who use
of suitable QA. The laboratory personnel should meet the the instrument are trained properly according to manufac-
local law or recognized standards to have appropriate profes- turer’s instructions. When new equipment is installed, the
sional qualifications [31]. The laboratory should have written laboratory should perform and document validation prior to
personnel policies to address topics such as orientation, use for clinical service. Validation process is dependent on
training, and continuation education. Staffs in clinical labo- the type of equipment and its use. The laboratory should vali-
ratory should be notified that education, training, skills, and date reproducibility and accuracy in the testing environment
experience regarding molecular genetic testing can help to prior to clinical use. The laboratory should establish daily,
assure the quality of the test. Education and training in clinic weekly, and/or monthly maintenance plans for all equipment
genetics testing and consultation should be recognized by according to manufacturer’s instruction. Maintenance such
regulatory and professional bodies [31]. as cleaning and defrosting should be done regularly and
Professional/regulatory bodies should develop and hold recorded on the charts in Equipment Maintenance binder.
educational and training programs relevant to molecular Tolerance limits for acceptable performance and calibra-
genetic testing and consultation. All personnel involved in tion checks for each equipment in molecular genetic detect-
molecular genetic testing should participate continuing edu- ing laboratory should be defined and monitored regularly.
cation and training program. Laboratory employees should When performance or calibration checks fall outside the tol-
possess necessary knowledge to review test request for erance limits, the equipment should be repaired and re-
appropriateness, validate/verify and perform tests, under- verified prior to the clinical use. The laboratory should make
stand the clinical implications of the test result, communi- sure the instruments meet all the required criteria for its use.
cate with referrers such as patients or medical specialists, Each machine should be uniquely identified by serial num-
and identify and correct problems [2]. Laboratory proce- ber or unique number developed by the laboratory. A master
dures and protocols for test performance, quality control, file including information about name of the equipment,
results reporting, documentation, and problem correction brand manufacture, inventory number, serial number, model
should be followed by staffs. New staffs should be trained in year, location, cost, date of purchase, date of first use, main-
laboratory-specific biosafety, biohazard waste management, tenance, record of calibration, and record of repair should be
personal protective equipment, as well as procedures to use maintained for each piece of equipment. If the machine is not
all chemicals. They should be educated to handle waste dis- working correctly, the troubleshooting is required and
posal according to national regulations. They should be recorded. The laboratory should keep maintenance logs.
properly trained on the methodologies they are to perform. When multiple analyzers are used for the same test,
Upon completion of initial training, competency assessments equipment-between comparability should be performed to
must be carried out and recorded. The laboratory should ensure the results are consistent between instruments. In
establish policies and procedures to assess employee compe- order to minimize calibration difference, the laboratory must
tency regularly. Competency assessments need to be con- use the same lot reagents and the same calibrators to cali-
ducted regularly. The performance of testing personnel brate both analyzers [15].
should be evaluated and documented regularly. A laboratory Monitoring equipment temperature with thermometers
must have personnel files that contain documents concerning (e.g., incubators, oven, refrigerator, water baths, and freezer)
the staff qualifications (diplomas, training certificate, com- for which temperature is critical in molecular genetic testing,
petency assessments, etc.). at least once a day for every workday when used, with writ-
ten records kept. The thermometers should be calibrated reagents and consumables are stored under proper environ-
annually against a reference thermometer; if more than 1 °C mental conditions. Information including receipt date and
difference from the reference thermometer is observed, the the date it is opened should be recorded. It is recommended
thermometers should be discarded. to label reagents with expiry dates. Reagents, whether pur-
Thermocyclers are widely used in molecular genetic test- chased commercially or manufactured within the laboratory,
ing laboratories. Several common variations that cause fluc- should be verified or validated prior to routine application.
tuations in fluorescent signal may occur during the usage of When a new lot or shipment of reagents is used, the labora-
real-time PCR. For example, with age of real-time PCR, tory should compare new and old lots of reagent side by side
excitation strength and emission sensitivity across the wells to ensure reproducibility.
of the block will change. During cycling, temperature-related
phenomena (refluxing, small air bubbles, the pressure of the
steam) will lead to fluctuations in signal. Regular mainte- 8.11 Quality Improvement
nance and calibration should be performed by the staff
according to the manufacturer’s instruction. Besides, it is Quality improvement is one of the critical elements in a qual-
necessary to have a secure and surge-free electricity supply ity management system [3]. A laboratory is committed to
for a thermocycler to avoid any fluctuation in electricity that identifying, documenting, correcting, and preventing non-
could impact its performance. conforming events in respect of quality management system.
Pipettes are used during the process of molecular genetic Performing audits, using proper quality indicators, and par-
analysis. Critically maintenance of pipettes will reduce ticipating EQA schemes are effective approaches to evaluate
errors in absorption. The laboratory personnel should be and improve the quality system. Besides, a laboratory should
instructed how to use pipettes correctly. When using a consistently assess the process of the test, analytical test per-
pipette, the plunger is depressed with thumb to the first stop formance, and staff related to the test. When problems asso-
to empty air, and insert the plastic tip of the pipette below the ciated with quality management occur, the laboratory must
surface of the fluid. Then, release the thumb pressure on the take effective action to remove the causes. Several models
plunger slowly, allowing the fluid to rise up. During this pro- such as Six Sigma, plan-do-check-act, Lean, Lean Six
cess, the tip of pipette should remain beneath the surface of Sigma, and root cause analysis are helpful to identify poten-
the fluid. When expelling the fluid in the pipette, one should tial risks and prevent problems from occurring [61].
depress the plunger to the first stop slowly, to prevent aero- Suggestions from staff members may also improve the qual-
solizing the specimen. Depressing the plunger to the second ity and efficiency of the laboratory.
stop may create bubbles and droplets of liquid which may
lead to specimen carryover. For quantitative assays, reverse
pipetting is recommended. Pipette tips containing hydropho- 8.12 Document Control
bic filter should be used in a molecular genetic testing labo-
ratory. Used tips are suggested to be discarded into a The documents in a clinical laboratory can either be elec-
container containing sodium hypochlorite solution. The con- tronic, on paper, or a mix of both, covering SOPs, work-
tainer should be bagged before discard. sheets, log books, validation and training files, quality
When contamination is suspected, equipment such as manual, and personnel files [3]. The laboratory should have
thermocyclers and centrifuges should be cleaned with freshly a written document of control plan to ensure that SOPs are
made 5–10% bleach solution followed by 70% ethanol or accurate and relevant. Documents are suggested to be written
water to prevent corrosion of equipment surface [1]. in a standard format. In each page of the documents, a ver-
Certain types of “frost-free” freezers should not be used sion number, page number, total number of pages, a date of
for specimen storage since the temperature cycling that elim- issue, and the name of the authorizing person are suggested
inates the accumulation of ice can damage specimens [60]. to be contained, so that the document can be uniquely identi-
Powdered gloves should not be used throughout the process fied. It is recommended to have a master list of SOPs cur-
as the powder on the gloves inhibits the amplification. rently used in the laboratory. An audit trail should be existed
to make sure that all SOPs are reviewed, signed, and dated
frequently by the director or designee. If changes occur, the
8.10 R
eagents and Consumables Quality document should be updated and readily accessible to labo-
Control ratory staffs [62]. Retired or obsolete SOPs should be
removed from circulation. It is necessary to archive retired or
The laboratory should establish policy and procedure for the obsolete SOPs for a period time. Documents are written for
reception, storage, acceptance testing, and inventory man- use, but not for the auditors. Make sure that laboratory per-
agement of reagents and consumables. Make sure that sonnel review and understand all relevant policies and proce-
dures. Documents may be prepared by competent personnel, 6. Rooney AL, Van Ostenberg PR. Licensure, accreditation, and cer-
such as quality manager, the staff who knows the procedures tification: approaches to health services quality. Center for Human
Services, Quality Assurance Project; 1999.
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be established that SOPs are approved by laboratory director ment of molecular amplification methods in clinical diagnostics.
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for molecular based tests used in diagnostic laboratories. In Wide
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manager. the performance of Nucleic acid Amplification Technology (NAT):
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Precision Medicine
9
Yingping Cao and Xianjin Zhu
Precision medicine is a new medical model. As the next gen- As the next generation of medical model, precision medi-
eration of medical model, precision medicine in the diagno- cine in the diagnosis and treatment of disease has great tech-
sis and treatment of disease has great technical advantages nical advantages over traditional treatment model: First, it is
over traditional treatment model: First, it is accurate. Second, accurate. Mutation genes of cancer can be identified by gene
it is convenient. In this chapter, we mainly introduce the sequencing; thereby doctors can quickly identify symptom-
roles of companion diagnostics, pharmacogenomics, transla- atic drugs, save time for patients to try various treatment
tional medicine of molecular diagnostic tests, and evidence- methods, and improve the therapeutic effect. Second, it is
based laboratory medicine. It is foreseeable that precision convenient. Gene sequencing requires only the patient’s
medicine can provide patients with better personalized blood and even saliva. There is no need to get traditional
medicine. pathological sections from patients, which can reduce the
In 2011, the National Research Council of the United damage of the patient’s body in the process of diagnosis. It is
States firstly proposed the concept of “Precision Medicine” foreseeable that precision medicine can provide patients
in the article “Toward Precision Medicine: Building a with better personalized medicine.
Knowledge Network for Biomedical Research and a New
Taxonomy of Disease” [1]. In 2015, former US president
Barack Obama said that “doctors have always recognized 9.1 Evidence-Based Laboratory Medicine
that every patient is unique, and doctors have always tried to
tailor their treatments as best they can to individuals. You can Laboratory medicine is a branch of medicine and performs
match a blood transfusion to a blood type—that was an in vitro testing of blood, tissue, and other samples of body
important discovery. What if matching a cancer cure to our materials. The core of laboratory medicine is to provide
genetic code was just as easy, just as standard? What if figur- information for the diagnosis, prevention, treatment of
ing out the right dose of medicine was as simple as taking human diseases, or assessment of human health. Although
our temperature?” in his speech, and launched “Precision the results from laboratory provide a large amount of infor-
Medicine Initiative.” From this time on, the advanced con- mation, it is difficult to find systematic evidence that labora-
cept of precision medicine spread rapidly around the world. tory medicine contributes specifically to the whole medical
Precision medicine is a medical model that proposes the cus- process [3].
tomization of healthcare, with medical decisions, treatments, The shortcomings of laboratory medicine are the lack of
practices, or products being tailored to the individual patient. comprehensiveness in the research content, the lack of integ-
In this model, diagnostic testing is often employed for select- rity in the research direction, and the limitations in the
ing appropriate and optimal therapies based on the context of research methods [4]. For example, the pathophysiological
a patient’s genetic content, combined with proteomics, mechanism of disease in laboratory medicine is mainly from
metabolomics, and other internal environmental informa- animal experiments; however, the evaluation and selection of
tion, in order to achieve the maximum therapeutic effect and drugs are based on the improvement of clinical symptoms
minimize the adverse reaction [2]. and the changes of some clinical indicators. The application
of such individual diagnostic test results is often not appli-
cable to all or most of a disease. Astion and colleagues first
recognized that the errors in the testing process can reduce
Y. Cao (*) · X. Zhu the effectiveness of drugs and harm patients [5].
Department of Clinical Laboratory, Fujian Medical University
Union Hospital, Fuzhou, Fujian, People’s Republic of China

116 Y. Cao and X. Zhu
Evidence-based medicine comes into being in the process results into clinical applications. The emergence of new lab-
of exposing the disadvantages of experimental medicine, and oratory technologies and its integration with disease diagno-
the introduction and improvement of evidence-based medi- sis allow patients to receive more personalized, predictable,
cine promote the development of laboratory medicine. preventable, and participable medical services [7].
Evidence-based medicine continuously evaluates the meth- In 1992, Choi officially proposed the concept of “bench
ods and clinical values of tests through combing the vast lit- to bedside,” which emphasized the importance of transition
erature and clinical statistics to identify direct, effective, from “laboratory basic research results” to “clinical appli-
accurate, and reasonable marks for the diagnosis and prog- cations” [8]. Translational medicine is defined by the
nosis of disease, and finally obtains reliable test information European Society for Translational Medicine (EUSTM) as
for clinical decision-making [5]. Evidence-based medicine “an interdisciplinary branch of the biomedical field sup-
mainly uses randomized controlled clinical trial (RCT) and ported by three main pillars: benchside, bedside, and com-
pooled analysis to assess the efficacy and safety of drugs. munity” [9]. Since the concept of translational medicine
Based on the reliable conclusions of large-scale RCT, the cri- first appeared in The Lancet magazine in 1996, it has gradu-
teria of laboratory and clinical diagnostic are constantly ally attracted worldwide attention [10, 11]. In 2012, the
being developed or modified to guide clinical practice and Ministry of Health of China proposed to establish a direct
improve the efficiency of clinical tests [3]. link among basic medicine, clinical medicine, preventive
The application of evidence-based medicine in laboratory medicine, and drug research and development, to achieve
medicine is evidence-based laboratory medicine. In 1993, the interconnection of basic research results, clinical diag-
evidence-based laboratory medicine established the nosis, and treatment methods, so that the development of
International Organization of Evidence-Based Medicine. medical science and technology can benefit patients and the
With the development of evidence-based medicine research, public health.
evidence-based medicine puts forward higher requirements Molecular diagnosis is the detection of the structure and
for the diagnostic test of evidence-based laboratory medi- expression of genetic material, including DNA, RNA, and
cine. Evidence-based laboratory medicine is not only used to proteins, and molecular diagnostic techniques include poly-
diagnose disease by diagnostic techniques, but also to guide merase chain reaction (PCR) technology, nucleic acid
treatment decision-making and monitor prognosis. The labo- hybridization, and biochip [12]. The development of molec-
ratory should use evidence-based laboratory medicine to ular biology techniques improves the understanding of the
continuously evaluate the pros and cons of the experiment mechanisms underlying cancer; thus researchers can identify
and credibility, and apply the current reasonable testing tech- the association of genetic mutations with cancers. Molecular
nology and quality control system to evaluate the test results; diagnosis as one of the tools of precision medicine, the trans-
thus the laboratory can provide dynamic and effective labo- formation of its research results can bring great value to the
ratory evidence to clinicians and patients and provide the clinic. However, it is not easy to translate this scientific
most scientific and appropriate tests [3]. For example, stud- knowledge into the interests of patients, and there is a huge
ies have shown that BNP and NT-proBNP is an effective challenge in integrating new discoveries in basic science
method for diagnosis and prevention of heart failure and can with clinical practice. Vasen and his colleagues provide a
reduce initial hospitalization rate and total hospitalization clear example how transformational medicine affects clinical
costs [6]. practice, but this transformation from molecular genetics to
Evidence-based medicine promotes the development of clinical practice requires familiarity with the language of
laboratory medicine. We firmly believe that combined labo- molecular biology [10].
ratory medicine (using animal as its main research object) In addition, researches on biomarker, which is a central
with evidence-based medicine (using human body as its part in translational medicine, often use molecular diagnostic
main research object) will promote the development of techniques to screen biomarkers which are used for the diag-
medicine. nosis and classification, treatment response, and prognosis of
diseases. The development of molecular analysis, such as
proteomics, metabolomics, and genomics, has led to the
9.2 ranslational Medicine of Molecular
T standardized application of analytical methods in transla-
Diagnostic Tests tional medicine [10, 13]. The technologies, methods, and
related products in clinical practice are the products of clini-
Translational medicine refers to two-way transformation cal basic research to clinical transformation. Differentially
process between laboratory research and clinical application. expressed mass spectrometry peaks in serum of patients with
It is a bridge to link basic research with clinical research, ovarian cancer, and control group is screened to identify
makes the integration of basic medicine and clinical medi- potential new tumor markers using matrix-assisted laser
cine, and promotes the transformation of scientific research desorption/ionization time of flight mass spectrometry
9 Precision Medicine 117
(MALDI-TOF-MS) and weak cation exchange (WCX) mag- ciated with the severity of the disease, such as urokinase-
netic bead purification kit [14]. type plasminogen activator (uPA) and plasminogen activator
The new molecular and cellular technologies in labora- inhibitor 1 (PAI-1), carcinoembryonic antigen (CEA), and
tory are gradually transformed into clinical research to solve circulating tumor cells [22, 23]. The American Society of
the problem of diagnosis and treatment of disease. Basic Clinical Oncology (ASCO) Quality Tumor Practice Initiative
medical research and clinical diagnosis and treatment are recommends HER2 testing for all newly diagnosed invasive
forming more and more extensive cooperation. It is still the breast cancer women within 31 days of the first visit [24, 25].
goal for current laboratory researchers to find more accurate, When microscopic analysis and surgical staining methods
specific, and simple molecular detection methods and how to cannot provide a definitive diagnosis, a specially designed
successfully transform them into practice. Strengthening the molecular diagnostic test can identify the primary tissue of a
cooperation of multidisciplinary experts, increasing policy primary breast cancer subtype and an unknown primary met-
guidance, and implementing the concept of translational astatic carcinoma [26–30].
medicine will contribute to establishing a new medical However, one of the shortcomings of molecular diagnos-
research system and improving the medical quality of our tic tests is the lack of linkages between testing, therapy, and
country. treatment outcomes; thus many newly developed molecular
diagnostics are not widely accepted. Now, there is a lack of
unified evaluation criteria for whether the new molecular
9.3 Molecular Diagnostic Tests diagnosis can be applied in clinical practice [31]. Though
in Personalized Medicine there are still many uncertainties in the field of molecular
diagnosis, molecular diagnosis is far more than a trivial diag-
The “patient-centered” medical and health service opens the nosis, and it is important to identify individual risks of devel-
door to a new vision of precision medicine. The detection of oping disease, to develop safe and effective treatment, and to
biomarkers by molecular diagnostic technology and the use evaluate responses to treatment. In addition, the next-
of biomarkers to determine the susceptibility or severity of generation molecular diagnostic tests with higher accuracy,
diseases are conducive to the early detection, monitoring, higher throughput, shorter test times, and lower cost effec-
assessment of disease-related risks, as well as the guidance tiveness will promote its application in clinical.
of treatment decisions and the realization of personalized
medicine [15, 16].
Many early diseases diagnostic reagents developed by 9.4 The Application
pharmaceutical companies involve the use of specific bio- of Pharmacogenomics
markers. Molecular diagnostic tests (also known as molecu-
lar genetic testing or MDx) represent the fastest-growing With the completion of the Human Genome Project and the
diagnostic (Dx) market, driving personalized medicine to proposal of Precision Medicine Program in the United States,
measure trends in protein, nucleic acid, or metabolite the era of precision medicine has arrived. Precise administra-
changes. There are more than 26,000 diagnostic tests for tion, one of the reflecting forms of precision medicine, is a
more than 3600 genes in the United States alone, and person- personalized treatment. Pharmacogenomics, a significant
alized medicine has undoubtedly become one of the hottest part of precision medicine, analyzes the relationship between
areas in the global medical field. However, there is no direct genomic information and drug response, thus making precise
evidence of a patient’s prognosis; a diagnostic test is often administration possible [32]. Pharmacogenomics mainly
seen as a worthless service because it does not treat the explores the effects of genome or gene mutation on drug
patient as a treatment, but rather to improve the doctor’s absorption, metabolic efficacy, and adverse reactions by
decision on treatment intervention. In spite of this, it is esti- detecting individual gene polymorphisms or genome-wide
mated that the results of diagnostic tests have a significant sequencing analysis, so as to better predict the genetic sus-
impact on approximately 60–70% of clinical decisions, ceptibility of multi-pathogenic diseases and guide the
although they account for only 4–5% of medical costs [17]. research and development of new drugs [33–35].
Similarly, evidences shown that diagnostic tests have reduced Pharmacogenomics is often clinically used to guide the
hospital and outpatient direct charges by 30–50% by identi- safety and rational use of drugs. Because genetic polymor-
fying key changes in health status [18]. phism affects pharmacokinetics, pharmacogenomics
Molecular diagnostic tests have been widely used in vari- research can help clinicians to choose various treatments for
ous healthcare fields. For example, molecular diagnostic tar- different patients [36]. As we know, acute myeloid leukemia
geting to detect carcinogenic factors can improve survival in patients have large differences in response to chemotherapy
patients with lung cancer [19–21]. Studies have shown that and targeted therapy, which is the main factor for the recur-
other biomarkers detected in tumor tissue or serum are asso- rence and poor prognosis in some patients. Cytosine
d eaminase (CDA) is one of the main enzymes that mediate nostic is a medical device, often an in vitro device, which
the metabolic inactivation of cytarabine (Ara-C) and its provides information that is essential for the safe and effec-
active metabolite cytarabine (Ara-CTP). High expression of tive use of a corresponding drug or biological product.
CDA in acute myeloid leukemia patients is associated with Companion diagnostics is co-developed with drugs to deter-
Ara-C resistance and recurrence, while CDA expression is mine whether a particular therapeutic product’s benefits to
lower in Ara-C-sensitive acute myeloid leukemia cells [37– patients will outweigh any potential serious side effects or
39]. TP53 mutations cause the resistance to Ara-C combined risks [53–55]. The information of companion diagnostics
with anthracycline chemotherapy in acute myeloid leukemia plays significant roles in the safe and effective use of particu-
patients, while complete remission rates in acute myeloid lar drugs due to the importance of collecting targeted therapy
leukemia patients with TP53 mutations are only 20–30% response from patients. Precision results of companion diag-
[40]. Decitabine is often used alone in the treatment of nostics provide a powerful proof for the selection of targeted
elderly patients with acute myeloid leukemia, but its clinical drugs to help patients tailor the optimal treatment and to
efficacy is also affected by the mutation of tumor suppressor achieve maximize patient survival benefits [56].
gene TP53 [41]. As a metabolic enzyme of anti-leukemia In recent years, with the development of sequencing
drugs, thiopurine methyltransferase (TPMT) is affected by technology, high throughput sequencing (NGS) provides
genetic polymorphism and dose [42]. Besides, angiotensin- lots of helps for the research of tumor, the therapeutic target
converting enzyme gene polymorphisms, N-acetyltransferase identification, and the drug discovery. NGS-based compan-
gene differences, and apolipoprotein E mutations may affect ion diagnostic kit has made major breakthroughs in clinical
the clinical efficacy of some drugs [43–45]. applications [57]. In December 2016, the first NGS-based
The pharmacogenomics of cardiovascular drugs is also companion diagnostic kit Foundation Focus CDx BRCA
one of the active research fields, and the use of genetic infor- was approved by FDA [58]. As cancer is a dynamic devel-
mation to guide the use of drugs for cardiovascular diseases opmental disorder, analysis of single biomarker at some
has become increasingly widespread. Clopidogrel is a com- point in time provides limited information using the tradi-
monly used cardiovascular drug that prevents recurrent car- tional methods. NGS can be used for multivariate analysis
diovascular disease by inhibiting platelet function in patients which enables us to have a broader and more comprehen-
with acute coronary syndrome (ACS) or in patients undergo- sive understanding of the genes involved in the disease
ing percutaneous coronary intervention [40, 46]. However, [59]. At present, companion diagnostics is also widely used
the clopidogrel response has individual differences, and on the world. For example, a series of molecular targeted
studies have shown that this response is highly heritable precision therapy is used in lung cancer treatment, which
[47]. CYP2C19, which belongs to cytochrome P450 significantly prolongs the survival of patients [60–63]. It is
(CYP450), is a liver enzyme that metabolizes many drugs worth noting that companion diagnostics is not only used
[48]. The genotype of CYP2C19 affects treatment. Studies for predicting outcomes, but also for detecting treatment
have shown that clopidogrel in cytochrome P450 (CYP)-2C19 responses. For example, the DNA and RNA of the tumor
(CYP2C19) is a poor metabolite that leads to treatment fail- are released into the peripheral circulation due to apoptosis
ure, so clopidogrel is avoided in patients with known or necrosis of the cells; thus liquid biopsy, which can
CYP2C19 gene polymorphisms, and the doctor should con- replace tumor biopsy, can monitor the disease of therapeu-
sider alternative treatments [49]. tic response [64–68]. Brett et al. systematically analyzed
As a tool for precision medicine, pharmacogenomics the targeted drugs approved by Food and Drug
aims to determine the rational use of drugs based on the Administration from January 2000 to April 2014, and found
genome of patient and improves clinical outcomes. that targeted therapy with companion diagnostics has better
Pharmacogenomics helps to find and identify new prognos- safety and tolerability in general [69]. Although the value
tic biomarkers, design better targeted therapies, and fine new of companion diagnostics as a predictor is obvious, there
drugs. With the increasing application of pharmacogenom- are few approved targeted drugs developed using compan-
ics, people recognize its enormous potential impact and use ion diagnostics [70, 71].
it for drug development. With the change of drug development model of individu-
alized drugs, companion diagnostics play a central role in
guiding the treatment of patients; thus it is becoming increas-
9.5 Companion Diagnostics ingly important for the regulation of companion diagnostics.
The purpose of companion diagnostic is to make specific
An increasing amount of evidence showed that companion treatment decisions, but incorrect diagnosis may lead to
diagnostics plays important roles in precision medicine [50– inappropriate treatment [72]. The specificity and sensitivity
52]. The Food and Drug Administration (FDA) introduces of companion diagnostics are closely related to its clinical
the concept of companion diagnostics that companion diag- application [73, 74]. Jorgensen et al. suggest that researchers
9 Precision Medicine 119
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marker as strictly as therapeutic trials [75–77]. ing new diagnostic and therapeutic paradigms. Mol Diagn Ther.
2012;16:1–6.
FDA firstly issued a regulatory draft on in vitro compan- 20. Lococo F, Paci M, Rapicetta C, et al. Preliminary evidence on the
ion diagnostic equipment guidance in 2011, and the final ver- diagnostic and molecular role of circulating soluble EGFR in non-
sion was published in 2014 [78]. With the development of small cell lung cancer. Int J Mol Sci. 2015;16:19612–30.
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small cell lung cancer: optimizing the diagnostic journey. Transl
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research of companion diagnostic products should be strictly breast cancer: a clinical perspective. Breast (Edinburgh, Scotland).
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23. Dunn L, Demichele A. Genomic predictors of outcome and treat-
ment response in breast cancer. Mol Diagn Ther. 2009;13:73–90.
24. Wolff AC, Hammond MEH, Allison KH, et al. HER2 testing in
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Part II
Molecular Biomarkers and Signals
from Diseased Functional Organ
Molecules in Body Systems
10
Shiyang Pan and Xincen Duan
10.1 Overview spite of this, small organic molecules are much less abundant
than the organic macromolecules, accounting for only about
All biological systems are composed of the same types of one-tenth of the total mass of organic matter in a cell [1].
chemical molecules and employ similar principles of organi- Cells contain four major families of small organic mole-
zation at the cellular level [1]. cules: sugars (the building blocks of complex carbohydrates),
Water, inorganic ions, and a large array of relatively small fatty acids (the building blocks of biomembranes), amino
organic molecules (e.g., sugars, fatty acids, amino acids, and acids (the building blocks of proteins), and nucleotides (the
nucleotides) account for 75–80% of living matter by weight. building blocks of DNA and RNA) (Fig. 10.1). Some small
Most of the dry mass of a cell consists of macromolecules molecules function as precursors for the synthesis of macro-
that have been produced as linear polymers of amino acids molecules (Fig. 10.2). Sugar is the main source of cellular
(proteins) or nucleotides (DNA and RNA), covalently linked chemical energy and can be incorporated into polysaccha-
to each other in an exact order. Cells acquire and use these rides to store energy. Fatty acids are also important for
molecules of two sizes in fundamentally different ways. energy storage, but their key function is to form cell mem-
Cells also make and alter many small organic molecules by a branes. Polymers of amino acids make up a wide variety of
series of different chemical reactions. In contrast, cells can useful macromolecules called proteins. Nucleotides play a
obtain macromolecules only by making them. Their synthe- central part in energy transfer. They are also the subunits
sis entails linking together a specific set of small molecules from which the informational macromolecules, RNA, and
(monomers) to form polymers through repetition of a single DNA, are made [1].
type of chemical-linkage reaction.
10.2.1 Sugars Provide an Energy Source

10.2 F
our Major Families of Small Organic for Cells and Are the Subunits
Molecules of Polysaccharides
The small organic molecules of the cell are carbon-based The simplest sugars (the monosaccharides) are compounds
compounds that have molecular weights in the range 0.1 kDa with the general formula (CH2O)n, where n is an integer from
to 1 kDa and contain up to 30 carbon atoms. They are usually 3 through 7. Glucose, for example, has the formula C6H12O6.
found free in solution and have many different fates. Some Hexoses (n = 6) and pentoses (n = 5) are the most common
small organic molecules are used as monomer subunits to monosaccharides (Fig. 10.3).
construct the giant polymeric macromolecules of the cell. In Many biologically important sugars are hexoses, includ-
ing glucose, mannose, and galactose. Mannose is identical to
glucose except that the orientation of the groups bonded to
S. Pan (*) carbon 2 is reversed. Similarly, galactose, another hexose,
Department of Laboratory Medicine, The First Affiliated Hospital differs from glucose only in the orientation of the groups
of Nanjing Medical University, Nanjing, Jiangsu, attached to carbon 4. d-glucose (C6H12O6) is the principal
e-mail: sypan@njmu.edcu.cn
external source of energy for most cells in complex multicel-
lular organisms. In a series of reactions, it is broken down
X. Duan
Department of Laboratory Medicine, Zhongshan Hospital of Fudan
into smaller molecules, releasing energy that the cell can har-
University, Shanghai, People’s Republic of China ness to do useful work.

124 S. Pan and X. Duan
CH2OH
C O
H OH
H NH2
C C
OH H N
HO H N
C C O O O
H H H H H H H H H H H H H H
O
H OH –
O P O P O P O CH2 N
H C C C C C C C C C C C C C C C O N
A SUGAR O – O– O– O–
H H H H H H H H H H H H H H H
H3N+ C COO–
A FATTY ACID
CH3 OH OH
A NUCLEOTIDE
AN AMINO ACID
Fig. 10.1 The four main families of small organic molecules in cells
Fig. 10.2 Small molecules

function as precursors for
synthesis of macromolecules larger units of the cell
building blocks of the cell
SUGARS POLYSACCHARIDES
FATTY ACIDS FATS,LIPIDS,MEMBRANES
AMINO ACIDS PROTEINS
NUCLEOTIDES NUCLEIC ACIDS
Sugar molecules consist of a skeleton of carbon atoms, C– linkage between the two sugars (Fig. 10.4). Sugars can be
which are connected together in a linear array by single joined by a variety of different glycosidic bonds.
bonds. Each carbon atom in the main chain is attached to a A molecule consisting of only two sugar units is a disac-
hydroxyl group, and only one has a carbonyl group (CPO). A charide. Disaccharides are primarily used as readily avail-
sugar is a ketose if the carbonyl group is in an internal posi- able energy stores. Sucrose, or table sugar, is the main
tion. If the carbonyl group is at one end of the sugar, it forms component of plant juices, which carry chemical energy
an aldehyde group, which can be seen in molecules such as from one part of the plant to another. Lactose is found in the
aldose and glucose. Because of their large number of milk of most mammals and provides fuel for the early growth
hydroxyl groups, sugars tend to be highly water soluble. and development of newborn mammals. Lactose in the diet is
Monosaccharides can be joined together by dehydration hydrolyzed by lactase, which is present in the plasma mem-
reactions in which H2O is removed and the sugars are linked brane of intestinal cells. Following childhood in humans,
by a glycosidic bond between carbon atom C1 of one sugar loss of the lactase enzyme can cause digestive discomfort
and the hydroxyl group of another sugar, generating a –C–O– after consumption of dairy products.
10 Molecules in Body Systems 125
Fig. 10.3 Structure of simple

sugars ALDOSES KETOSES
3-carbon (TRIOSES)
OH OH OH O OH
O
H C C C H C C C H
H H H H H
5-carbon (PENTOSES) glyceraldehyde dihydroxyacetone
OH OH OH OH OH OH OH O OH
O
H C C C C C H C C C C C H
H H H H H H H H H
ribose ribulose
6-carbon (HEXOSES)
OH OH OH H OH OH OH OH H O OH
O
H C C C C C C H C C C C C C H
H H H OH H H H H H OH H
glucose fructose
Oligosaccharides are small chains of several sugars joined 10.2.2 Fatty Acids Are Precursors
together and are commonly found to covalently bind lipids for Phospholipids and Other Membrane
and proteins to convert them into glycolipids and glycopro- Components
teins respectively. Cells use simple polysaccharides com-
posed only of glucose units as long-term stores of energy. Fatty acids are an important energy source for many cells
The main functions of sugars are varied, which can include and are stored in the form of triacylglycerols within adipose
producing and storing energy, as well as making mechanical tissue. Fatty acids also are precursors for phospholipids and
supports. Oligosaccharides, which are smaller than polysac- many other lipids with a variety of functions. Fatty acids
charides, can be covalently linked to proteins to form glyco- consist of a hydrocarbon chain attached to a carboxyl group
proteins and to lipids to form glycolipids, which are found in (COOH) (Fig. 10.5). They differ in length, although the pre-
cell membranes. The sugar side chains of these polymers are dominant fatty acids in cells have an even number of carbon
often recognized selectively by other cells. Of note, differ- atoms, usually 14, 16, 18, or 20. Fatty acids often are desig-
ences between cell-surface sugars provide the molecular nated by the abbreviation Cx:y, where x is the number of
basis distinguishing the major human blood groups [1]. carbons in the chain and y is the number of double bonds.
HO HO collection of biological molecules with the common feature

that they are insoluble in water, while being soluble in fat and
organic solvents such as benzene. They typically contain
O O
either long hydrocarbon chains, characteristic of fatty acids
OH + OH and isoprenes, or multiple linked aromatic rings, such as in
OH
steroids [1].
O H
HO HO The most important function of fatty acids in cells is in
OH OH the construction of cell membranes (Fig. 10.6). Unlike pro-
teins, nucleic acids, and polysaccharides, membranes are
assembled by the noncovalent association of their compo-
H2O nent building blocks.
The main component of all biofilms is the phospholipid, a
small molecule that, like triacylglycerol, consists mainly of
HO HO fatty acids and glycerol. The difference is that in phospholip-
ids, glycerol is linked to two fatty acid chains, while in tri-
glycerides, glycerol is linked to three fatty acid chains. This
O O spermatogenic substance gives phospholipids different phys-
OH OH icochemical properties from triacylglycerols. While triacylg-
O lycerols are predominantly hydrophobic, molecules like
OH phospholipids, with both hydrophobic and hydrophilic
HO
OH
regions, are termed amphipathic [1].
OH
In addition to the basic functions mentioned before, fatty
acids have four major physiological roles.
1. Fatty acids are fuel molecules. They are stored as triacyl-

glycerols (also known as neutral fats or triglycerides),
a(1 4) glycosidic bond
which are uncharged esters of fatty acids and glycerol.
Triglycerides are stored in fat cells which compose adi-
Fig. 10.4 Formation of a glycosidic bond
pose tissue. Fatty acids mobilized from triglycerides are
oxidized to meet the energy needs of cells or organisms.
Fatty acids are our main source of energy during rest or
Fatty acids containing 12 or more carbon atoms are nearly moderate exercise.
insoluble in aqueous solutions because of their long hydro- 2. Fatty acids are components of phospholipids and

phobic hydrocarbon chains. Fatty acids without the carbon– glycolipids.
carbon double bond are called saturated fatty acids, and fatty 3. Many proteins are modified by the covalent attachment of
acids with at least one double bond are called unsaturated fatty acids, which target the proteins to membrane
fatty acids. Unsaturated fatty acids containing more than one locations.
carbon–carbon double bond are called polyunsaturated fatty 4. Fatty acid derivatives serve as hormones and intracellular
acids. Common fatty acids can be synthesized in mammals messengers.
except for the two “essential” polyunsaturated fatty acids lin-
oleic acid (C18:2) and linolenic acid (C18:3), which must be
added to the diet. 10.2.3 Amino Acids Are the Subunits
Fatty acids are a valuable food source because they can be of Proteins
broken down to produce twice as much energy as glucose.
Fatty acids are stored in the cytoplasm of many cells in The monomeric building blocks of proteins are 20 amino
the form of droplets of triglyceride molecules, which consist acids [2], all of which have a characteristic structure consist-
of three fatty acid chains, each joined to a glycerol molecule; ing of a central carbon atom bonded to four different chemi-
these molecules comprise animal fats. When required to pro- cal groups: an amino (NH2) group, a carboxyl (COOH) group,
vide energy, the fatty acid chains can be released from tri- a hydrogen (H) atom, and one variable group, called a side
glycerides and broken down into two-carbon units [1]. chain, or R group (Fig. 10.7). The specific chemical proper-
Fatty acids and their derivatives such as triacylglycerols ties of the different amino acid side chains determine the roles
are examples of lipids. Lipids comprise a loosely defined of each amino acid in protein structure and function [3].
Fig. 10.5 Structure of fatty o

acids
–o
Palmitate(C16)
–o
Stearate(C18)
HO
Oleate(C18)
Fig. 10.6 Phospholipid
monomers noncovalently
assemble into a bilayer Phospholipid
Phospholipid bilayer
structure
Polar group
Phosphate Hydrophilic
Glycerol head group
Hydrophobic
Fatty acid fatty acyl tails
Side chain Dietary proteins are key sources of essential amino acids
for mammals and humans, while nonessential amino acids
H H can be synthesized endogenously. Nine amino acids (histi-
dine, isoleucine, leucine, lysine, methionine, phenylalanine,
H N+
α
C C o– threonine, tryptophan, and valine) are considered “essential,”
which means humans are unable to synthesize them endog-
R
o enously but acquire them from the diet (Table 10.1). Most
H
nonessential amino acids such as alanine, arginine, aspara-
gine, aspartic acid, glutamate, glutamine, glycine, proline,
Amino group Carboxyl group and serine are synthesized from glucose. Tyrosine is metabo-
lized from phenylalanine, and cysteine is metabolized from
Fig. 10.7 Structure of amino acids
methionine. Cysteine, tryptophan, and methionine are rela- (N1). Cytosine (C), thymine (T), and uracil (U) are called
tively uncommon amino acids, accounting for only about 5% pyrimidine compounds because they are all simple deriva-
of the typical protein composition. Four amino acids (leu- tives of a six-membered pyrimidine ring; guanine (G) and
cine, serine, lysine, and glutamic acid) by contrast, are the adenine (A) are purine compounds, with a second five-
most abundant amino acids, constituting 32% of all the resi- membered ring fused to the six-membered ring (Fig. 10.9).
dues in a typical protein [2]. Each nucleotide is named by reference to the unique base
Proteins, three-dimensional structures, are folded by long that it contains.
chains, which are polymers of amino acids joined head-to- Adenine, guanine, and cytosine are found in both DNA
tail. The covalent linkage between two adjacent amino acids and RNA; thymine is found only in DNA, and uracil is found
in a protein chain is called a peptide bond (Fig. 10.8). The only in RNA. The linear sequence of nucleotides in DNA
polypeptide has a chain of amino acids, including an amino and RNA encodes the genetic information of the cell. The
(NH2) group at one end (its N-terminus) and a carboxyl ability of the bases in different nucleic acid molecules to rec-
(COOH) group at its other end (its C-terminus) [1]. ognize and pair with each other by hydrogen bonding (called
base pairing)—G with C, and A with either T or U—under-
lies all of heredity and evolution (Fig. 10.10) [2].
10.2.4 Nucleotides Are the Subunits of DNA Nucleotides can be used as short-term carriers of chemi-
and RNA cal energy. Most importantly, the ribonucleotide adenosine
triphosphate (ATP) is used to transfer energy in hundreds of
A nucleotide is a molecule consisting of a nitrogenous ring different cellular reactions. ATP is formed in a reaction
compound joined to a five-carbon sugar. This sugar can be a driven by energy released by the oxidative decomposition of
ribose or deoxyribose, which is lacking an oxygen atom in food. Its three phosphates are held in series by two phos-
comparison to ribose. Ribose nucleotides are called ribonu- phoric anhydride bonds, and their breaking releases useful
cleotides and deoxyribose nucleotides are called deoxyribo- energy. In particular, terminal phosphate groups are often
nucleotides. The nucleotide carries one or more phosphate hydrolyzed and separated, often transferring phosphoric acid
groups as well. In nucleotides, the 1′ carbon atom of the to other molecules, releasing energy that drives biosynthetic
sugar (ribose or deoxyribose) is attached to the nitrogen at reactions. Other nucleotide derivatives are used as carriers
position 9 of a purine (N9) or at position 1 of a pyrimidine for the transfer of other chemical groups. The most funda-
mental role of nucleotides in the cell, however, is in the stor-
age and retrieval of biological information. Nucleic acid
Table 10.1 Dietary requirements for amino acids in humans
chains are synthesized from energy-rich nucleoside triphos-
Essential Nonessential
phates by a condensation reaction that releases inorganic
Histidine Alanine
pyrophosphate during phosphodiester bond formation [1].
Isoleucine Arginine
Leucine Asparagine
Lysine Aspartate
Methionine Cysteine 10.3 T
he Chemistry of Cells Is Dominated
Phenylalanine Glutamate by Macromolecules with Remarkable
Threonine Glutamine Properties
Tryptophan Glycine
Valine Proline Macromolecules are the most interesting molecules in living
Serine systems. In a real sense, the evolution of life is the evolution
Tyrosine
of macromolecular structure.
H2O
R1 O R2
R1 H R2
O H3N+ C C N C COO–
H3 N+ C C + H N C COO–
O– H H H
H H H
Peptide bond
Fig. 10.8 Formation of a peptide bond

PURINES
Adenine (A) Guanine (G)
PYRIMIDINES
Uracil (U) Thymine(T) Cytosine (C)
Fig. 10.9 Purines and pyrimidines
Fig. 10.10 Complementary
pairing between nucleic acid
bases
By weight, macromolecules are the most abundant ing small organic molecules (called monomers) into long
carbon-containing molecules in a living cell (Fig. 10.11). chains [1]. They have remarkable properties that could not
They are the principal building blocks from which a cell is have been predicted from their simple constituents.
constructed, and also the components that confer the most Three major types of macromolecules are found in cells:
distinctive properties of living things. The macromolecules proteins, composed of amino acids linked by peptide
in cells are polymers that are constructed by covalently link- bonds; nucleic acids, composed of nucleotides linked by
growing chain, through the loss of one water molecule for

each added subunit. The gradual polymerization of mono-
mers into chains is an efficient way to make large and com-
plex molecules, because the subunits are added by the same
Protein(15%) reactions performed repeatedly by the same set of enzymes.
Apart from some of the polysaccharides, most macromole-
cules are made from a limited set of monomers that are
slightly different from one another—for example, the 20 dif-
ferent amino acids from which proteins are made. It is criti-
cal to life that the polymer chain is not assembled at random
from these subunits; instead the subunits are added in a pre-
Polysaccharide(2%) Small molecules(3%) cise order, or sequence [1].
Inorganic ions(1%)
DNA(1%) 10.3.1 Carbohydrates
Carbohydrates (commonly called polysaccharides) include

RNA(6%)
all the larger molecules that are composed of sugars. The
main function of carbohydrates is to store chemical energy
H2O(70%)
and to serve as durable building materials for biological con-
Phospholipid(2%) struction. Most sugars have the general formula (CH2O)n,
from which the name carbohydrate is derived (C = “carbo”
and H2O = “hydrate”). The sugars of importance in cellular
metabolism have values of n that range from 3 to 7.
Bacterial Cell
Sugars containing three carbons are known as trioses,
CELL VOLUME OF 2×10–12CM3 those with four carbons as tetroses, those with five carbons
as pentoses, those with six carbons as hexoses, and those
with seven carbons as heptoses. The six carbon (n = 6) sugar
Fig. 10.11 The distribution of molecules in cells
glucose (C6H12O6) is especially important in cells, since it
provides the principal source of cellular energy. Other mono-
phosphodiester bonds; and polysaccharides, composed of saccharides contain three to seven carbons, the most com-
monosaccharides (sugars) linked by glycosidic bonds. mon of which are 3–5. Sugars containing five or more
Phospholipids, the fourth major chemical building block, carbons can be cycled to form ring structures, which are the
assemble noncovalently into biomembranes [2]. main form of these molecules in cells. Carbohydrates are one
Proteins are the most abundant and functional macromol- of the four major groups of biomolecules, together with pro-
ecules in cells. Proteins are rich and amazingly versatile, per- teins, nucleic acids, and lipids. They are aldehydes or ketones
forming thousands of different functions in cells. Many with multiple hydroxyl groups. They make up most of the
proteins act as enzymes that catalyze many of the covalent organic matter on earth because they play a broad role in all
bond formation and bond-breaking reactions needed by forms of life.
cells. Enzymes catalyze all reactions in which cells extract First, carbohydrates are intermediates in energy storage,
energy from food molecules. For example, an enzyme called fuel, and metabolism. Second, ribose and deoxyribose form
ribulose bisphosphate carboxylase helps convert carbon part of the structural framework of RNA and DNA. Third,
dioxide from photosynthetic organisms into sugar, p roducing polysaccharides are structural elements in the cell walls of
most of the organic material needed for life on earth. Other bacteria and plants. For example, cellulose is the main com-
proteins are used to build structural components, such as ponent of plant cell walls and one of the most abundant
tubulin, a protein that self-assembles into cell tubules, which organic compounds in the biosphere. Fourth, carbohydrates
binds DNA in chromosomes. Other proteins, such as myosin are linked to many proteins and lipids, which play a key role
in muscles, act as molecular motors that generate force and in regulating cell–cell interactions and interactions with
movement. other elements in the cellular environment.
Although proteins, nucleic acids, and polysaccharides A key relevant property of carbohydrates as intermediar-
add subunits to their polymers differently, they share ies in cell interactions is their large structural diversity.
important characteristics. In condensation reactions, each Carbohydrates are made up of monosaccharides, and as
polymer is grown by adding a monomer to the end of the mentioned earlier, monosaccharides are small molecules,
usually containing 3–9 carbon atoms, that are different in and polysaccharides are important in a variety of cell sig-
size from the stereochemical configuration of one or more naling processes.
carbon centers. These monosaccharides can be joined
together to form a variety of oligosaccharide structures. In
the field of proteomics, the difficulties in unraveling these 10.3.2 Lipids
oligosaccharide structures, discovering their location at spe-
cific sites in proteins, and determining their function are for- Lipids are a group of different nonpolar biological molecules
midable challenges. Large polymeric oligosaccharides, whose common properties include their ability to dissolve in
formed by the linkage of multiple monosaccharides, are organic solvents (such as chloroform or benzene) and their
called polysaccharides (Fig. 10.12). Polysaccharides play inability to dissolve in water; properties that explain many of
vital roles in energy storage and in maintaining the structural their different biological functions. Important lipids in cel-
integrity of an organism. When a polymer is composed of all lular function include fats, steroids, and phospholipids.
of the same monosaccharides, these polymers are called Lipids have three major roles in cells. First, they provide
homopolymers. The most common homopolymer in animal an important form of energy storage. Second, lipids are the
cells is glycogen, the storage form of glucose. major components of cell membranes, which is of great
The carbohydrates include simple sugars as well as importance in cell biology (Table 10.2). Third, lipids actin
polysaccharides. These simple sugars, such as glucose, are cell signaling, both as steroid hormones (e.g., estrogen and
the major nutrients of cells. Finally, in addition to their testosterone) and as messenger molecules that convey sig-
roles in energy storage and cell structure, oligosaccharides nals from cell surface receptors to targets within the cell [4].
Fig. 10.12 The synthesis of

Glucose
polysaccharides
CH2OH
OH
Glycogen (n Glu)
OH
CH2OH CH2OH
OH HO
OH
OH OH
OH OH
NTP
H2O (energy from nucleoside triphosphate hydrolysis)
CH2OH CH2OH CH2OH
OH
OH OH OH
OH OH OH
Glycogen (n+1 Glu)

Table 10.2 Lipid compositions of some biological membranes

Lipid Human erythrocyte Human myelin Beef heart mitochondria E. coli
Phosphatidic acid 1.5 0.5 0 0
Phosphatidylcholine 19 10 39 0
Phosphatidylethanolamine 18 20 27 65
Phosphatidylglycerol 0 0 0 18
Phosphatidylserine 8.5 8.5 0.5 0
Cardiolipin 0 0 22.5 12
Sphingomyelin 17.5 8.5 0 0
Glycolipids 10 26 0 0
Cholesterol 25 26 3 0
The values given are weight percent of total lipid
Fig. 10.13 Lipid
components of the plasma Outside of cell
membrane Sphingomyelin
Phosphatidylcholine Glycolipid Cholesterol
Phosphatidylinositol Phosphatidylserine
Phosphatidylethanolamine Cytosol
Phospholipids are the main components of cell mem- 10.3.3 Nucleic Acids
branes and consist of two fatty acids attached to a polar head
group. Phospholipids are amphiphilic molecules, with some The nucleic acids—DNA and RNA—are the principal infor-
regions water soluble and others insoluble in water. mational molecules of the cell. Deoxyribonucleic acid
In addition to phospholipids, many cell membranes con- (DNA) has a unique role as genetic material located in the
tain glycolipids and cholesterol (Fig. 10.13). Glycolipids nucleus in eukaryotic cells. Different types of ribonucleic
consist of two hydrocarbon chains linked to polar head acid (RNA) participate in a number of cellular activities, act-
groups that contain carbohydrates. Cholesterol, in contrast, ing as an informational molecule for instance. Additionally,
consists of four hydrocarbon rings rather than linear hydro- RNA is capable of catalyzing a number of chemical reac-
carbon chains. The hydrocarbon rings are strongly hydro- tions, including those reactions involved in protein synthesis
phobic, but the hydroxyl (OH) group attached to one end of and RNA processing [6].
cholesterol is weakly hydrophilic, so cholesterol is also The three-dimensional structure of DNA, as mentioned
amphipathic [5]. before, consists of two long helical strands that are coiled
In addition to being part of cell membranes, lipids act as around a common axis to form a double helix (Fig. 10.14).
signaling molecules in and between cells. Steroid hormones DNA strands consist of monomers called nucleotides; these
such as estrogen and testosterone are derivatives of monomers are often called bases because they contain cyclic
cholesterol. organic bases. Four distinct nucleotides (abbreviated as A, T,
The information in DNA and RNA is conveyed by the

order of the bases in the polynucleotide chain. DNA is a
double-stranded molecule consisting of two polynucleotide
chains running in opposite directions [7]. The information
carried by DNA and RNA directs the synthesis of specific
proteins (Fig. 10.16), and thus controls most cellular
activities.
10.3.4 Proteins
A G T C
Proteins are the main functional and structural components

of all cells in the human body. The defining characteristic of
a protein or amino acid is its amino nitrogen group. This
nitrogen group distinguishes amino acids from, for example,
Parental strands
sugars. Proteins are large molecules composed of long chains
of amino acid subunits. In the protein molecule, the amino
acids are joined together by peptide bonds (Fig. 10.17). In
biological systems, the chains formed might be anything
from a few amino acids (di-, tri-, or oligopeptide) to thou-
sands of units (polypeptide). The sequence of amino acids in
the chain is known as the primary structure. A critical feature
of proteins is the complexity of their physical structures [8].
The polypeptide chain does not exist as a long chain, but
folds in a three-dimensional structure. Chains of amino acids
tend to curl into helices (secondary structures). Because of
the hydrophobic interactions between nonpolar side chains,
Nucleotide and disulfide bonds in some proteins, portions of the helix
(T) may fold into each other, so the entire molecule may be
spherical or rod shaped (tertiary structure). Their exact shape
depends on their function and, for some proteins, their inter-
actions with other molecules (quaternary structures).
Protein is the main executor of live cells, which is the
most abundant and versatile of the macromolecules. The cell
makes a linear chain from 20 different amino acids. The spe-
cial chemical properties of the side chains of different amino
acids determine the role of each amino acid in protein struc-
Daughter ture and function. During or just after its polymerization, a
strands linear chain of amino acids folds into a complex shape, con-
ferring a distinctive three-dimensional structure and function
Fig. 10.14 DNA consists of two complementary strands wound on the protein [2].
around each other to form a double helix Proteins are the most diverse of all macromolecules, and
each cell contains several thousand different proteins, which
C, and G) are joined together to form a DNA strand, with the perform a wide variety of functions (Fig. 10.18). The defin-
base portion protruding from the backbone of the strand. The ing characteristic of proteins is that they are polypeptides
two chains are joined by their bases and twisted to form a with specific amino acid sequences. Proteins are currently
double helix. Each DNA double helix has a simple construc- sequenced using automated methods, and the complete
tion: wherever one strand has an A, the other strand has a T, amino acid sequences of over 100,000 proteins are now
and each C is matched with a G [2]. The polymerization of known [9].
nucleotides to form nucleic acids involves the formation of The tertiary structure of proteins refers to the relative
phosphodiester bonds between the 5′phosphate of one nucle- spatial position of all amino acid residues in the whole pep-
otide and the 3′hydroxyl of another (Fig. 10.15). tide chain. Myoglobin, for instance, is a single peptide chain

nucleic acids
Fig. 10.16 The information

encoded in DNA is converted
into the amino acid sequences
of proteins by a multistep
process
protein composed of 153 amino acid residues with a heme The fourth level of protein structure, known as its qua-
cogroup. In the structure of myoglobin molecules, the α ternary structure, consists of the interactions between dif-
helix accounts for 75%, forming 8 helix regions. There is a ferent polypeptide chains in proteins. It should be noted
random curl between two helix regions, and proline is located that proteins with quaternary structures are distinctively
at the corner. Due to the interaction of R group of side chains, composed of more than one polypeptide. Hemoglobin, for
the polypeptide chain is wound to form a spherical molecule. example, is composed of four polypeptide chains held
The hydrophilic side chain is mainly on the surface, while together by the same types of interactions that maintain its
the hydrophobic side chain is located inside the molecule. tertiary structure [10].
The formation and stabilization of the tertiary structure Consequently, proteins constitute an extremely complex
mainly depend on secondary bonds such as hydrophobic and diverse group of macromolecules, suited to the wide
bonds, salt bonds, hydrogen bonds, and van der Waals forces. variety of tasks they perform in cell biology.

proteins
Fig. 10.18 Overview of

protein function
References 5. Kalisch B, Dormann P, Holzl G. DGDG and glycolipids in plants

and algae. Subcell Biochem. 2016;86:51–83.
6. Zhao Y, Chen F, Li Q, et al. Isothermal amplification of nucleic
1. Alberts B. Molecular biology of the cell (6th edition). Garland sci-
acids. Chem Rev. 2015;115:12491–545.
ence. New York, NY: Taylor and Francis Group, p. 1 volume (vari-
7. Traube FR, Carell T. The chemistries and consequences of
ous pagings); 2015.
DNA and RNA methylation and demethylation. RNA Biol.
2. Lodish HF. Molecular cell biology. 6th ed. New York:
2017;14:1099–107.
W.H. Freeman; 2008.
8. Chin JW. Expanding and reprogramming the genetic code. Nature.
3. Wu G. Amino acids: metabolism, functions, and nutrition. Amino
2017;550:53–60.
Acids. 2009;37:1–17.
9. Hashimoto SI. Discovery and history of amino acid fermentation.
4. Song MJ, Malhi H. The unfolded protein response and hepatic lipid
Adv Biochem Eng Biotechnol. 2017;159:15–34.
metabolism in non-alcoholic fatty liver disease. Pharmacol Ther.
10. Dill KA, MacCallum JL. The protein-folding problem, 50 years on.
2019;203:107401.
Science. 2012;338:1042–6.
Molecules in Signal Pathways
11
Shiyang Pan and Wei Zhang
Table 11.1 Some pathways and molecules in this section

11.1 Overview
Pathways Molecules
Apoptosis signal pathway BCL-2, Caspases, IAPs
Cell signal transduction is essential for human life. Signal PI3K/AKT/mTOR signal PI3K, AKT, mTOR
transduction pathways participate in all cell life activities, pathway
such as cell metabolism, cell division, cell differentiation, Wnt signal pathway Wnt, APC, β-catenin, c-Myc
cell functional activities, and cell death. During cell metabo- MAPK signal pathway ERK1/2, JNKs, p38
lism, cells ingest and metabolize nutrients that provide JAK/STAT signal pathway JAKs, STATs
energy. During cell division, DNA is replicated, and signal- Angiogenesis VEGF/VEGFR
ing pathways regulate the cell cycle, allowing for cell divi- Notch signal pathway Notch
Other molecules PD-1/PD-L1, HIF-1, p53, SP70
sion and proliferation. During cell differentiation, gene
products in cells are selectively expressed, thereby allowing
cells to differentiate into mature cells with specific functions.
During cell functional activities, cells release neurotransmit- tion pathways, and they may be biomarkers for some dis-
ters or biochemicals, leading to contraction or relaxation of eases. Here, we introduce some classical signal pathways as
muscle cells, cytoskeletal formation, or remodeling. Cell well as important molecules (Table 11.1).
death is the final event of the cell life cycle, which can occur
in a local and a certain number of groups in order to maintain
the overall benefit. 11.2 Functions
In a signal transduction pathway, when a signal transduc-
tion protein is mutated, a molecule is damaged, or a func- 11.2.1 The Change of Signal Transduction
tional change has occurred, disease may result. Various Molecules Is the Basis of Signal
extracellular molecules, which participate in receptor- Transduction
mediated cell signal transduction pathways, act on the
nuclear genome targeting gene expression, modify cell mac- 11.2.1.1 Signal Transduction Molecules
romolecules, regulate cell metabolism and organelle func- Constitute the Network of Signal
tion, and in some pathological conditions, can cause Transduction Pathways
pathological changes in cell signal transduction pathways. Membrane receptor-mediated signal transduction into cells
Many bioactive molecules convey their signals via diverse requires the participation of multiple signal transduction
signal transduction pathways, from small molecules, for molecules such as proteins or second messengers. They form
example, dopamine, acetylcholine, norepinephrine, sero- complex signal transduction pathways (signal transduction
tonin, and histamine, to glycoprotein hormones and large networks), which enable communication between different
peptides. Molecules play important roles in signal transduc- signal transduction pathways, so as to detect, amplify, and
integrate signals, and to act on the effector and target genes.
The basic action mode of signal transduction molecules
includes conformation and activity change, intracellular
S. Pan (*) · W. Zhang localization change, formation or depolymerization of signal
Department of Laboratory Medicine, The First Affiliated Hospital transduction molecular complexes, the content change of
of Nanjing Medical University, Nanjing, Jiangsu,
People’s Republic of China signal transduction molecule such as a second messenger
e-mail: sypan@njmu.edcu.cn small molecule.

140 S. Pan and W. Zhang
11.2.1.2 T he Change in the Conformation 11.2.2 Signal Transduction Molecules Are

and Activity of Signal Transduction Important Targets for Drug Action
Molecules Is the Basis of Signal
Transduction Increased understanding of cell signal transduction mecha-
Changes in the conformation and activity of signal transduc- nisms and the structure and function of signal transduction
tion molecules can cause three effects: first, to enhance or molecules in diseases provides opportunities for developing
inhibit the activity of the enzyme signal transduction mole- new diagnoses and treatment methods of disease, while also
cule; second, to expose the membrane or nuclear localization providing a target for the screening and development of new
domain after the conformational change, which can be trans- drugs. This leads to the concept of signal transduction drugs
formed into the cell or nuclear membrane; and third, to and targeted therapies. Agonists and inhibitors of signal
recruit new interacting protein molecules to dissociate the transduction molecules are research hotspots in drug devel-
original molecules. opment. Protein kinase inhibitors, which are widely used as
There are four main factors that cause changes in the anticancer drugs, are one such example.
conformation and activity of signal transduction protein
molecules: first, chemical modification changes the protein
conformation of signal transduction molecules, such as 11.3 Signal Pathways
phosphorylation and dephosphorylation, acetylation and
deacetylation, methylation and demethylation; second, the 11.3.1 Apoptosis
second messenger acts as an allosteric effector to cause
changes in the conformation of the signal transduction pro- Apoptosis, a synonym for programmed cell death (PCD),
tein molecule, such as the activation of cAMP after combi- eliminates damaged cells and maintains homeostatic bal-
nation with protein kinase A, or the activation of cGMP ances in multicellular organisms [1, 2]. Apoptosis signaling
after combination with protein kinase G; third, the protein pathways (shown in Fig. 11.1) include death receptor-
interaction can lead to protein conformational changes of mediated and mitochondrial-mediated pathways, which are
signal transduction molecules; and fourth, dimerization or also known as extrinsic and intrinsic pathways respectively
oligomerization of signal transduction protein molecules [3]. Apoptosis is regulated by different molecules. Apoptosis
can cause conformational changes and internal energy dysregulation can cause many different diseases, including
changes. degenerative disorders, autoimmunity, cancers, and ischemic
injury. In the processes of apoptosis, interactions between
11.2.1.3 C hanges of Intracellular Signal proteins play very important roles in apoptotic signal trans-
Transduction Molecule Content duction regulation. As apoptosis-related molecules are
and Translocation Are Important Ways attractive drug targets, compounds that modulate molecular
of Signal Transduction Regulation functions in apoptotic processes are currently undergoing
Cells can regulate the signal transduction molecule’s content pharmaceutical development [4, 5].
in a variety of ways. The content of small molecule second
messenger can be enzymatically upregulated or downregu- 11.3.1.1 The BCL-2 Family
lated. The protein signal transduction molecule’s content can BCL-2, was first identified through chromosomal mapping
vary depending on the level of gene expression, and also can in follicular lymphoma where constitutive BCL-2 expression
change due to the stability change of the protein molecule. is driven by the immunoglobulin heavy chain promotor by
Changes in the content of effector molecules can also affect the t [14;18] translocation [6–8]. BCL-2 facilitates tumori-
signal transduction and cellular responses. genesis by cell death resistance, whereas other known onco-
Cellular localization alteration (translocation) of signal proteins facilitate tumorigenesis by promoting cell growth
transduction molecules is also an important means of signal and proliferation [9]. At least 30 BCL-2 family members are
transduction regulation. For example, signal transduction currently known, and the BCL-2 family is a protein with
molecules can translocate molecules localized in the cyto- approximately 180 amino acid residues and has a function-
plasm to the cell membrane, nucleus, or other organelles, and ally related BH domain (BCL-2 homology region) that medi-
can also translocate molecules localized on the cell mem- ates interaction between members. BCL-2 family members
brane, the nucleus, or other organelles to the cell cytoplasm. can be divided into three categories based on their structural
Signal transduction molecule translocation to the corre- and functional characteristics: pro-apoptotic BH3-only pro-
sponding response site facilitates molecular interactions and teins (such as Bim, Bid, Bad, Puma, and Noxa), death effec-
produces downstream effects. tor proteins (such as Bak and Bax), and pro-survival BCL-2
11 Molecules in Signal Pathways 141
Fig. 11.1 Apoptosis signal

pathway
proteins (such as BCL-XL, BCL-w, BCL-2, A1, and MCL- phagocytosis and removal of cell debris. Of all the caspases,
1). Pro-survival BCL-2 proteins and death effector proteins the most frequently activated caspase is caspase-3 that cata-
contain multiple BCL-2 homology domains (BH1–BH4), lyzes the cleavage of major cellular proteins and leads to the
while BH3-only proteins only contain the conserved BH3 condensation of chromatin. Caspases also activate DNase
region. Interactions among BCL-2 family proteins determine enzymes that cause fragmentation of DNA followed by inter-
cell fate [10]. Pro-survival proteins promote cell survival, nucleosomal fragmentation.
whereas pro-apoptotic proteins mediate receptor-, mitochon-
dria-, or endoplasmic reticulum stress-dependent apoptosis. 11.3.1.3 Inhibitors of Apoptotic Proteins
High expression of BCL-2 promotes tumor development and Inhibitors of apoptotic proteins (IAPs) were originally dis-
can be a factor in drug resistance. covered in baculoviruses by Lois Miller and colleagues in a
genetic screen in order to identify viral factors that can sub-
11.3.1.2 Caspases stitute for the caspase inhibitor p35 encoded by baculovirus
Caspases, a family of cysteine proteases, are the central regu- [11]. Eight IAPs have been found, all with 1–3 baculovirus
lators of apoptosis. At least 14 caspases have been found in IAP repeat (BIR) domains. There is a conservative motif in
mammals, and they are generally synthesized in the form of the BIR domain, which is a zinc-finger structure. IAPs can
pro-caspase in cells. The caspases in the apoptosis signal inhibit tumor cell apoptosis in a caspase-dependent or a non-
pathway can be divided into two categories according to the caspase-dependent manner. XIAP is the only member that
original domain length and the caspase position in the path- inhibits caspase activity by directly binding to caspases,
way: initiators (caspase-2/8/9/10) and executors (caspase- while other IAPs indirectly inhibit caspase activity. XIAP is
3/6/7). Caspases exist in cells in an inactive form and require able to inhibit both the initiator (caspase-9) and the executor
proteolytic cleavage to an active form. Once caspases are (caspase-3 and caspase-7) caspases involved in the intrinsic
activated, the initiator caspases cleave, and these activated or BCL-2 blockable apoptotic pathway (Fig. 11.2). In addi-
caspases induce the activation of executor caspases. The tion, XIAP has been shown to bind to the caspases in the
active executor caspases lead to the cleavage of several pro- apoptosome (Apaf-1, caspase-9, and caspase-3) complex,
teins in the cell that brings about cell death and eventually which are activated by cytochrome c released from the mito-
as substrate. Type II PI3K is mainly on the cell membrane,

and types I and III are in the cytoplasm when not activated.
Types I and III moves to the cell membrane after activation.
The most studied is type I PI3K, which stimulates membrane-
bound phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)
P2;PIP2] conversion to phosphatidylinositol-(3,4,5)-trispho-
sphate [PtdIns(3,4,5)P3; PIP3] [19, 20]. PIP3 acts as a sec-
ondary messenger, facilitating the recruitment and activation
of kinases with pleckstrin homology domain, such as PI3K-
dependent kinase-1 (PDK1). The signal duration of PIP3 is
regulated by phosphatase and tensin homolog (PTEN),
which opposes PI3K activity.
11.3.2.2 Akt
The serine/threonine kinase Akt, also known as protein
kinase B (PKB), has a PH domain, and is recruited to the
plasma membrane together with PDK1. Akt consists of 480
amino acid residues, including the N-terminal regulatory
region, the middle serine/threonine kinase active region, and
the C-terminal region (HM region, which is a hydrophobic
region, rich in proline). PDK1 and mTORC2 phosphorylate
amino acid residues T308 and S473, respectively, which is
essential for complete Akt activation. Akt regulates cell sur-
vival by phosphorylating multiple downstream targets. After
Fig. 11.2 XIAP inhibits processed caspases-3 and -9 to block activa- activation, Akt can phosphorylate many target proteins, such
tion of the apoptosome
as glycogen synthase kinase3 (GSK3), caspase-9, PRAS40
(AKT1S1), and tuberous sclerosis2 (TSC2), which explains
chondria intermembrane space [12–14]. In several in vivo its extensive downstream roles in promoting cell differentia-
xenograft models, IAP antagonists (SM-164, BV6, and tion, proliferation, angiogenesis, apoptosis, and metabolism.
GDC-0152) have shown tumor suppressive activity [15–18], Akt activation can regulate cell growth, cell death, and cell
and several of them have entered phase I clinical trials. cycle progression, and control the rate of glucose uptake into
cells through the GLUT1 transporter [23]. In addition, Akt
activates FOXO3a to inhibit cell apoptosis and promote
11.3.2 PI3K/AKT/mTOR Signal Pathway mitochondrial biogenesis to sustain growing cells [24].
Phosphatidylinositide 3 kinases (PI3Ks), protein kinase B 11.3.2.3 Mammalian Target of Rapamycin

(Akt), and mammalian target of rapamycin (mTOR) make up Mammalian Target of Rapamycin (mTOR) is an evolution-
the core components of the PI3K/Akt/mTOR signal path- arily conserved serine/threonine protein kinase that belongs
way. This signal pathway (Fig. 11.3) facilitates a variety of to the PI3K-related kinase family. mTOR’s molecular weight
processes that are essential in many aspects of mediating cell is 289 kDa and consists of 2549 amino acid residues. mTOR
function, such as cell growth, cell survival, anabolic reac- is a translation regulator that phosphorylates the eukaryotic
tions, and nutrient uptake. PI3Ks, Akt, and mTOR have been initiation factor 4E-binding protein 1 (eIF4E-BP1), which is
shown to be overactivated in many cancers and have there- a suppressor of the 5′ CAP-dependent translation [25]. The
fore become a focus of research in this area [19–21]. accessibility of the mTOR active site is partially governed by
mTOR-related proteins that form two different complexes:
11.3.2.1 Phosphatidylinositide 3 Kinase mTOR complex 1 (mTORC1) and mTOR complex 2
PI3Ks were first discovered in the 1990s as part of research (mTORC2). Both can be activated by mitogens or growth
relating to the transformation ability of viral oncoproteins factors. The composition, activation pattern, and rapamycin
[22]. PI3Ks are a unique intracellular lipid kinase family that sensitivity of these complexes are different [26, 27]. mTOR
can phosphorylate the 3′-hydroxyl of the inositol ring of in the two complexes both interact with mLST8 and
phosphatidylinositides (PtdIns). PI3Ks include three types. DEPTOR, but other core components are different. mTORC1
Type I PI3K uses PI, PIP, and PIP2 as substrate, while type II has RAPTOR (RPTOR) and PRAS40, while mTORC2 con-
PI3K uses PI and PIP as substrate, and type III PI3K uses PI tains mSIN1 (mammalian stress-activated protein kinase
Fig. 11.3 PI3K/AKT/mTOR
signal pathway
interacting protein 1; MAPKAP1) and RICTOR (rapamycin- conserved Wnt family secreted glycolipoproteins, in a
insensitive companion of mTOR). β-catenin-dependent manner (canonical Wnt signal path-
way) or through β-catenin-independent mechanisms (nonca-
nonical Wnt signal pathway) [30–32].
11.3.3 Wnt Signal Pathway The Wnt pathway, especially under its canonical form
(Wnt/β-catenin pathway), is a signal pathway involved in the
The Wnt signal pathway is a central regulatory pathway that development of invertebrates and vertebrates (Fig. 11.4). It
controls key functions of normal and malignant epithelial plays an important role in embryogenesis and morphogene-
cells, and has become an important new target in cancer drug sis as well as stem cell proliferation and differentiation. This
development. The pathway consists of a set of signal trans- pathway is relatively complex, and all processes and physi-
duction elements that regulate calcium flux, cytoskeletal ological effects have not been fully interpreted. Some of the
changes, and gene transcription in epithelial cells. This path- proteins involved in the pathway are “Wingless,” “Frizzled,”
way is essential for cell cycle regulation, inflammation, and “Dishevelled.” They were initially designated as mutants
embryonic development, and tumorigenesis [28, 29]. Wnt of Drosophila development and proved the universality of
pathway’s main component is the Wnt protein family that this pathway in the animal kingdom.
activates cell membrane receptors in an autocrine and para-
crine manner. Wnt proteins secreted by cells can induce cel- 11.3.3.1 Wnt
lular mechanisms by activating Frizzled membrane proteins WNT, originally called Int-1, was identified in 1982 as a
and regulate gene expression through intracellular proteins gene that was activated by integrating a mouse mammary
and transcription factors. Wnt signal pathway is initiated by tumor virus’ proviral DNA into virus-induced breast tumors
Fig. 11.4 Mechanism of

action of canonical Wnt
pathway
[33]. To date, 19 different Wnt proteins have been found in mutations determine familial adenomatous polyposis coli,
humans. Wnt proteins are secreted proteins that typically which is one of the main syndromes of hereditary predisposi-
signal across the membrane by interacting with the trans- tion to colorectal cancer. Generally, these are nonsense muta-
membrane receptor Frizzled. They contain an N-terminal tions that lead to a truncated protein. It seems that the
signal peptide domain, receptor binding domain, signal pep- mutation in only one allele is sufficient to constitute this car-
tidase recognition domain, and a C-terminal-containing cys- cinogenic event by haploinsufficiency.
teine domain, but do not include a transmembrane domain.
Wnt can be divided into two groups, Wnt1 and Wnt5a. Wnt 11.3.3.3 β-Catenin
is the promoter of the wnt pathway, which activates the sig- β-Catenin is a multifunctional protein in the cytoplasm. It
naling pathway through autocrine and paracrine binding mediates cell adhesion in the plasma membrane and acti-
with plasma membrane receptors (Frizzled). Wnt proteins vates gene transcription in the nucleus. β-Catenin is a posi-
have certain specificity and can bind to different receptors, tive regulatory factor of Wnt signal pathway that can promote
with different effects. Proteoglycans on the cell surface can c-Myc and cyclin D1 activation, and promote cell prolifera-
promote afferent Wnt signaling. Lastly, Wnt secretion tion. The molecular weight of β-catenin is 94 kDa and con-
depends on the porcupine protein. tains about 800 amino acid residues. β-Catenin binds to the
actin cytoskeleton through an α-catenin molecule in the
11.3.3.2 Adenomatous Polyposis Coli cytoplasm, and binds to the junctional structures of
The adenomatous polyposis coli (APC) gene was indepen- E-cadherin (CDH1) in the extracellular space. Therefore,
dently discovered in a hereditary cancer syndrome called β-catenin plays a major role in epithelial cell adherens junc-
familial adenomatous polyposis [34, 35]. APC’s germline tions and in the maintenance of tissue structure.
11.3.3.4 c-Myc scriptional activation domain [43, 44]. Ten JNK isoforms
c-Myc, a proto-oncogene, is a well-known Wnt target gene have been identified, which are encoded by three genes:
both in colorectal cancer cells and in normal intestinal epi- JNK1, JNK2, and JNK3. JNK1 and JNK2 are widely
thelium and is often used as a readout of Wnt pathway expressed, while JNK3 is expressed in the heart, testis, and
activity [36, 37]. C-Myc has a molecular weight of 62 kDa brain. JNKs act downstream in the MAPK signaling cascade
and is mainly involved in transcriptional regulation. c-Myc and play a key role in regulating inflammatory/immunologi-
has been found to be upregulated in response to inactivation cal responses, tumor progression, neuron development,
of APC in murine intestinal epithelium, which suggests that apoptosis, and intercellular connection formation. JNK sig-
it is important in mediating abnormal Wnt activation [38]. naling mechanisms involve multiple stimuli, occur in many
cell types, and include many interactions. JNK promotes
stress tolerance and cell proliferation by activating transcrip-
11.3.4 Mitogen-Activated Protein Kinase tion factors Foxo and AP-1 [45, 46], and induces cell death
Signal Pathway by phosphorylation of cytoplasmic proteins (including Itch,
Bid, and Bim), which control cFlip stability [47]. JNK pro-
Mitogen-Activated Protein Kinase (MAPK) signal pathways motes autophagy by phosphorylation of BCL-2 and Beclin1
constitute one of the most important and evolutionarily con- release [48]. Excessive activation of JNK plays an important
served mechanisms for extracellular information perception role in tumor development.
in all eukaryotes. MAPK pathways are involved in the trans-
fer of information perceived from extracellular stimuli to the 11.3.4.3 p38
cell. The end result is different transcription factor activation The p38 MAPK family’s first member was independently
that regulates the expression of genes in response to the stim- identified as a 38 kDa protein (p38) by four groups. The pro-
uli. MAPK signal pathways play a key role in cell prolifera- tein was found to be the homologue of Saccharomyces cere-
tion, differentiation, and apoptosis. MAPK includes three visiae Hog1, which was an important regulator of the osmotic
types: extracellular signal-related kinases (ERKs), c-jun ter- response and is now called p38α (MAPK14). Other members
minal kinases (JNKs), and p38. All the MAPKs can translo- of p38 MAPK family, which are approximately 60% identi-
cate into the nucleus and activate transcription factors by cal in their amino acid sequence, were subsequently cloned
phosphorylation [39]. and named p38β (MAPK11), p38γ [SAPK3 (stress-activated
protein kinase), ERK6 (extracellular signal-regulated
11.3.4.1 E xtracellular Signal-Regulated Kinase kinase), or MAPK12] and p38δ (SAPK4 or MAPK13) [49–
1/2 54]. All the p38 MAPKs are encoded by different genes and
ERK1 (p44) and ERK2 (p42) are proteins encoded by two have diverse tissue expression patterns. Among them, p38α
splice variants of the same gene. ERK1/2 can be positively is universally expressed at a significant level in most cell
activated by MEK1/2 by phosphorylation of Thr and Tyr types, while others appear to be expressed in a tissue-specific
residues (Thr202 and Tyr204 of ERK1 and Thr173 and manner.
Tyr185 of ERK2 respectively). After phosphorylation by
MEK1/2, they translocate into the nucleus and activate vari-
ous transcription factors, such as NF-κB, ETS, ELK-1, 11.3.5 JAK/STAT Signal Pathway
c-Myc, and CREB [40]. ERK1 and ERK2 have hundreds of
substrates, many of which are involved in key physiological JAK/STAT signal pathway is one of the highly conserved
processes that control cell differentiation, survival, prolifera- metazoan pathways in a wide range of species [55], and
tion, and death [41]. Additionally, activation of the ERK sub- involves a variety of growth factors and cytokines [56].
strates will result in feedback loops, which in turn positively Compared to other complex major signaling pathways, the
or negatively regulate the ERK signal pathway depending on JAK/STAT pathway appears to be relatively simple. JAK/
the substrate [42]. Excessive activation of ERK1/2 leads to STAT signal pathways begin with JAK activation by binding
cell proliferation and tumorigenesis. ligands (including interleukins, interferons, or growth fac-
tors) to specific transmembrane receptors. JAK/STAT path-
11.3.4.2 c-Jun Terminal Kinases way activation is associated with multiple receptors,
JNKs were originally identified in the early 1990s as protein including cytokine receptors, G protein-coupled receptors
kinases that bind to a specific region of c-Jun and phosphory- (GPCRs), tyrosine kinases receptors, and homodimeric hor-
late c-Jun at serine 63 and serine 73 in the N-terminal tran- mone receptors [57] (Fig. 11.5).
Fig. 11.5 Schematic of JAK/STAT pathway
11.3.5.1 JAKs residues. They are a cytoplasmic protein family which can
JAKs, known as large tyrosine kinases, are a protein family bind to target gene promoters. The STAT family consists of 7
that belongs to intracellular non-receptor tyrosine kinases. In members: STAT1, STAT2, STAT3, STAT4, STAT5a,
mammals, the JAK family contains 4 members: JAK1, JAK2, STAT5b, and STAT6, which mainly function as transcription
JAK3, and TYK2. All members have similar structures and factors, and are the main substrate of JAKs [56, 64]. As the
are composed of a JH2 domain, an SH2-like domain, a name suggests, STATs act as both transcription factor and
C-terminal JH1 domain, and an N-terminal FERM (4.1R, signal transducer. However, two structural components make
ezrin, radixin, moesin) domain [58]. JAK1/2 and TYK2 are them unique among transcription factors: the highly con-
widely found in various tissues, and JAK3 is only found in served C-terminal tyrosine residue and the SH2 domain [65].
the lymphatic and immune systems. They are more than The tyrosine residue is phosphorylated by activated JAKs.
1000 amino acids in length and have molecular weights STATs have been shown to activate transcription of inactive
ranging from 110 to 140 kDa [59]. The JAK name originates genes within minutes [66]. STAT3 is by far the most studied
from the two-faced Roman God of doorways, Janus, because and well-known member of the STAT protein family. STAT3
JAK has two almost identical phosphate transferring activates the transcription of multiple genes that play an
domains: one domain demonstrates kinase activity, and the important role in proliferation, cell cycle regulation, and cell
other negatively regulates the kinase activity of the first survival, such as VEGF, Bcl-xL, c-Myc, and c-Fosandcyclin
domain [60, 61]. The JAK family’s first member was cloned D1.
and synthesized in 1990 [62]. The gene was named TYK2,
and attracted attention because of the “kinase-like” domain
next to the conserved and well-known protein tyrosine kinase 11.3.6 Angiogenesis
domain [63].
Angiogenesis, also termed neovascularization, occurs when
11.3.5.2 STATs new capillaries germinate from an existing blood vessel and
STATs are nuclear transcription factors with a molecular grow toward a chemoattractant in a directed manner.
weight of 85–115 kDa and containing 750–850 amino acid Angiogenesis is a complex process by which new vessels
develop from existing vessels. It is involved in various physi-

Hypoxia Tumor cells
ological processes such as tissue regeneration and wound
healing, and is also a pathological feature of many diseases
(inflammatory diseases, atherosclerosis, ischemia, and can- HIF-1 α
cer). It is speculated that angiogenesis is controlled by a bal-
ance between positive and negative modulators, and that
angiogenesis continues only when the activity of the angio-
VEGF
genesis stimulators exceeds the activity of the angiogenesis MMP
inhibitors. Therefore, the first stage of angiogenesis is to
express or overexpress angiogenic factors with or without
angiogenesis inhibitor downregulation, creating an angio- VEGFR
Endothelial Cells
genic environment. Cell growth, Migration,
ECM Proliferation
The research of angiogenesis was established 35 years degradation
ago, when Dr. Judah Folkman first described the angiogene-
sis process and determined that tumor growth depended on
neovascularization [67]. Dr. Judah Folkman was the first to
hypothesize that tumor growth and progression had to recruit
its own vascular network. Due to the limited distance of oxy-
gen diffusion in the tissues, tumors need to recruit their own
vascular network to reach a size greater than a few millime-
ters in diameter [68]. Angiogenesis
Since angiogenesis was first described in 1971, many
molecules have been identified that could positively regulate Fig. 11.6 VEGF/VEGFR induces angiogenesis
angiogenesis. However, the most prominent one is probably
vascular endothelial growth factor (VEGF). It is generally released by cancer cells and induces tumor angiogenesis
believed that VEGF is the most important angiogenic factor [74]. VEGF upregulation is induced by hypoxia-inducible
in terms of normal and pathophysiological angiogenesis. factor (HIF)-1a and platelet-derived growth factor (PDGF)
VEGF and VEGF receptors are considered to be part of one B. Additionally, extracellular matrix (ECM) can secrete
of the major signal pathways in angiogenesis [69] (Fig. 11.6). VEGF via MMP9 to activate the angiogenic switch, which
Many basic properties attributed to VEGF have a direct promotes tumor growth.
effect on promoting angiogenesis. First, it has a proliferation
effect on endothelial cells, which begin to grow under its
influence, thereby increasing cell survival and decreasing 11.3.7 Notch Signal Pathway
cell apoptosis. Second, it enhances vascular permeability,
which influences infiltration and migration of cells from cir- Notch signal pathway plays an important role in angiogen-
culation. In terms of its angiogenic effect, the induction of esis. Notch signal transduction is independent of a second
vasodilatation is another VEGF property. After VEGF binds messenger. Notch transmembrane proteins directly receive
to its receptor, there are different signaling pathways that can signals from neighboring cells, and the plasma membrane
promote endothelial cell proliferation, survival, and portion transfers to the nucleus, thereby initiating the
migration. expression of downstream transcription factor. Notch sig-
In mammals, VEGF ligands contain five members of naling conduction cannot amplify the signal, but precise
homodimeric disulfide-bound glycoproteins, namely control of differentiation is required during the initiation
VEGF-A (also known as VEGF), VEGF-B, VEGF-C, process. The Notch signal pathway is comprised of three
VEGF-D, and placenta growth factor (PlGF) [70–72]. parts: receptor, ligand, and DNA binding sequence
Particularly, VEGF is considered the most representative CSL. When a ligand binds to a Notch receptor between two
member of the VEGF family. Both VEGF and its receptor adjacent cells, the Notch signal pathway will be activated to
VEGFR-2 are major targets of the current anti-angiogenic determine cell fate and ultimately regulate organ formation
agents. VEGF is considered to be the strongest pro- and morphogenesis.
angiogenic stimulator. Even the loss of a single allele can Notch was discovered through a notch presence in the
lead to embryonic death because of vascular hypoplasia [73]. wings of fruit flies, which was later found to play an impor-
Endothelial cells are the main targets of VEGF. VEGF is tant role in embryonic development [75]. The Notch receptor
is a type I one-way transmembrane protein encoded by 2370 by subtractive hybridization. PD-1 was primarily involved in
amino acids [76]. In mammals, there are four isoforms of programmed cell death, and from that its name is derived
Notch receptors (Notch1/2/3/4 [77]) and five Notch ligands [86]. PD-L1 (B7-H1 or CD274) was the first ligand discov-
(Dll1/3/4, Jagged1/2 [78]). All Notch receptors have the ered for PD-1, belonging to the B7 family and located on
same overall structure: three Lin-12/Notch repeats (LNR) human chromosome 9 p24.2 [87]. The amino acid structure
and 36 homologous epidermal growth factor (EGF)-like tan- of PD-L1 is similar to PD-1.
dem repeats in the extracellular domain; a PEST sequence; PD-1 is a specific receptor for PD-L1. After binding to
seven ankyrin-like repeats (ANK); and a CSL-binding PD-L1, PD-1 can inhibit the activation of lymphocytes,
domain RAM in the intracellular domain [79]. Notch ligands reduce the secretion of lymphocyte cytokines, and thus pro-
are also type I transmembrane proteins, which contain extra- mote the apoptosis of lymphocytes. The PD-1 protein is a
cellular EGF-like repeats, a DSL (Delta/Serrate/Lag-2) motif type I transmembrane protein with a molecular weight of
that accounts for Notch interactions, and short and divergent 55–60 kDa, composed of a hydrophobic transmembrane
intracellular domains [80]. region, an intracellular region, and an extracellular IgV-like
Notch target genes vary in different tissue and cells, such domain. The C-terminal and N-terminal amino acid residues
as MMP-9, cyclin D1, Her2, c-Myc, HES and HEY families, of the PD-1 intracellular domain have two independent phos-
MMP-2, p21, and apoptosis-related genes. Notch may pre- phorylation sites: the immunoreceptors tyrosine-based
cisely regulate cell proliferation, differentiation, and tumori- switch motif (ITSM) and tyrosine-based inhibitory motif
genesis by modulating the expression of these genes. Notch (ITIM) [88]. ITSM is an important structural site for PD-1 to
plays a role in lymphatic development, arterial differentia- exert its biological function. When PD-1 binds to PD-L1, the
tion, sprouting angiogenesis, and lymphangiogenesis during ITSM region will be phosphorylated to activate a series of
development [81]. Notch receptors and ligands are widely intracellular signaling pathways and achieve immune
expressed in endothelial and perivascular cells such as peri- inhibition.
cytes, macrophages, and vascular smooth muscle cells. Evidence indicates that the PD-1/PD-L1 pathway activa-
Notch components are also expressed in tumor endothelial tion leads to the inhibition of antitumor adaptive responses
cells, and some studies have shown that the Notch signal through mechanisms involving induction of cytotoxic T-cell
pathway affects tumor angiogenesis. The Notch signal path- apoptosis, exhaustion, energy, and decreased cytokine pro-
way regulates the expression of VEGFR1, VEGFR2, and duction [89–91] (Fig. 11.7). Therefore, the interaction of
VEGFR3 in a complex pattern [82]. Dll4/Notch signaling PD-1 with PD-L1 leads to an increased resistance of the
controls the endothelial tip cell’s appearance, thereby con- tumor cell to pro-apoptotic signals and immune escape of
trolling the branching of vessels during sprouting angiogen- tumor cells, ultimately leading to cancer progression [92,
esis [83–85]. The VEGF signal pathway through VEGFR2 93]. Tumor-infiltrating lymphocytes (TILs) in many epithe-
upregulates the expression of Dll4 in tip cells, which in turn lial cancers express PD-1, indicating that the PD-1/PD-L1
triggers the activation of Notch in adjacent endothelial cells. pathway may influence antitumor immunity. Blockade of
Notch signal pathway activation downregulates the levels of immune checkpoints using monoclonal antibodies that target
VEGFR2 and VEGFR3, inhibiting the phenotype of the tip the PD-1/PD-L1 pathway have shown very promising results.
cell, causing these cells to be stalk cells that form the nascent Several PD-1 or PD-L1 targeted antibodies are currently
sprout proliferative core. being examined in clinical trials for a variety of malignancies
[94–102]. Along with immunotherapies, re-activation of the
tumor immune response remains a highly relevant research
11.4 Important Molecules topic. Because the PD-1/PD-L1 signal pathway is one of the
important pathways for tumor immune escape, regulating
A wide variety of signal molecules have been implicated in PD-L1 expression could manipulate the tumor microenvi-
multiple pathways, some of which we will emphasize below. ronment by inhibiting T cell activation, thereby eliminating
immune surveillance in the tumor microenvironment.
11.4.1 PD-1/PD-L1
11.4.2 HIF-1
PD-1, the cluster of differentiation 279 (CD279), was origi-
nally cloned in 1992 from hematopoietic progenitor cell The Warburg effect, first described by Otto Warburg in the
lines and drug-treated mouse hybridoma in an apoptotic state 1920s, has resurfaced as a controversial theory with both
a Tumor cell b Tumor cell

B cell T cell
1
-L
PD
M
PD
H
-L
Ag
C
1
Ag
BCR PD-1 PD-1 TCR
P P P
SHP1
Syk Igα/β SHP2 CD3 d
SHP2 P
ZAP70
PLCy2 ERK PI3K
PI3K PKCq RAS
Ca2+ mobilization Bcl-xl ERK1/2

Cell cycle progression
Antibody secretion
Cell survival
Cell proliferation
Cytokine production
Fig. 11.7 PD-1 signal pathways
supporting and opposing arguments. Although tumor sup- major regulator of numerous proteins and enzymes involved
pressor genes (TSGs), several oncogenes, and the tumor in the glycolytic pathway and glucose metabolism. HIF-1
microenvironment have been implicated in the Warburg pathway modulation represents an attractive target of
effect, hypoxia inducible factor-1 (HIF-1) has been identified therapy.
as a key regulator of cancer cell proliferation [103].
To date, three members of HIF family (HIF-1, HIF-2,
and HIF-3) have been identified in mammals, of which 11.4.3 p53
HIF-1 is the best characterized [104]. HIF-1 is a basic
helix-loop-helix transcription factor composed of discrete p53 was first discovered as a tumor suppressor in 1984, when
alpha and beta subunits (HIF-1α and HIF-1β, respectively) several independent research groups revealed that the protein
[105]. Although HIF-1β (also known as aryl hydrocarbon was essential for suppressing cancer [106]. p53 is a tran-
nuclear translocator, ARNT) is stably expressed, HIF-1α scription factor and cell regulator with tumor suppressor
subunits are continuously degraded by the 26S proteasome properties, and also is an important regulator of replicative
in physiologic conditions, thereby preventing HIF activity senescence and cell death in response to carcinogenic stress
in the normoxic state. In hypoxia, HIF-1α degradation is [107]. p53 crosses into the mitochondria and activates pro-
inhibited, the α and β subunits dimerize, translocate to the apoptotic genes’ expression, as well as inhibits anti-apoptotic
nucleus, and activate a family of genes’ expression that genes’ expression. These pro-apoptotic proteins are trans-
help cells adapt to hypoxia. HIF-1 has been shown to be a ported into the mitochondria together with the p53 protein,
where they induce increased mitochondrial membrane per- Table 11.2 Some DEGs with large fold changes
meability and cytochrome c release, which connects with the Gene Fold change Function
caspase-9 proenzyme and the Apoptotic protease activating HSPA8 13.3142 Autophagy
factor-1 (Apaf-1), to create the complex called the “apopto- DDIT3 5.6010 Apoptosis
some.” The apoptosome then causes the activation of cas- ARG2 5.1996 Metabolism
pase-9, which consequently promotes the caspase-3 GADD45A 2.4898 Apoptosis
proenzyme to the active protease stage, and then adheres to IFI16 2.1863 Apoptosis, proliferation, migration,
inflammation
effector caspases. These caspases are responsible for intra-
BRI3BP −7.8927 p53 inhibitor
cellular protein cleavage and the morphological changes of DIDO1 −6.9589 Invasion, migration, apoptosis
apoptosis [108]. Moreover, the p53 protein is involved in MALAT1 −5.9453 Invasion, migration, apoptosis
apoptosis pathway regulation, regulating gene coding recep- MCTS1 −5.787 Invasion, EMT
tor expression (such as DR5/KILLER, FAS/APO1), and the MTDH −5.1282 Apoptosis, invasion, drug resistance
development of the FAS receptor ligand (FasL). Furthermore, FAM83H −5.0505 Proliferation, Invasion
DR5 gene promotion is activated indirectly by the p53 pro- MUC1 −2.1177 Proliferation, apoptosis, invasion,
tein. The FAS receptor expression on the cell surface can be immunoregulation
PDPK1 −2.1063 Proliferation, apoptosis, invasion
enhanced by increasing the transport of FAS receptors from
NOTCH −2.3747 Proliferation, apoptosis, invasion,
the Golgi apparatus to the cell membrane [109]. p53 is angiogenesis
located in the nucleus, where it can receive signals from a
protein network that sense the health of cells and the cellular
DNA. When damage occurs, the p53 protein is activated and
helps cells make the decision between repair and induction sequently, miRNA and mRNA microarrays were conducted.
of apoptosis. However, if p53 is mutated, it will be unable to The miRNA results showed that there were 35 miRNAs with
effectively bind to DNA. This results in relatively unregu- the differential expression, among which 14 miRNAs were
lated cell division, allowing DNA damage accumulation upregulated and 21 miRNAs were downregulated. The
leading to potential tumorigenic cells. mRNA results demonstrated that there were 543 upregulated
genes and 818 downregulated genes after blocking SP70 of
SPC-A1 cells. The differentially expressed genes (DEGs)
11.4.4 Tumor Specific Protein 70 were closely associated with the occurrence and develop-
ment of tumors. Table 11.2 lists some DEGs with large fold
It was reported that NJ001 could react with non-small cell changes. These DEGs have been reported in multiple tumor
lung cancer (NSCLC) cells. NJ001. The corresponding anti- types, and play different roles.
gen of NJ001 is Tumor Specific Protein 70 (SP70), a protein Based on Gene Ontology (GO) enrichment and Kyoto
with a relative molecular weight of 70 kDa. SP70 has been Encyclopedia of Genes and Genomes (KEGG) pathway
demonstrated to be located in the cytoplasm of NSCLC cells, analysis, we found that SP70 could regulate many genes that
as well as on the cell membrane. NJ001 could induce apop- are involved in invasion, apoptosis, proliferation, and metas-
tosis of lung adenocarcinoma cell effectively [110]. SP70 is tasis, as well as pathways including MAPK, apoptosis, focal
highly expressed in NSCLC tissues, and the positive rate is adhesion, and cholesterol biosynthesis. SP70, as a new tumor
related to the clinical stage. Serum level of SP70 is a more biomarker, exerts a variety of roles by regulating multiple
sensitive indicator to predict timely response to chemother- downstream genes. Through bioinformatics analysis, we
apy and Progression-free survival (PFS) [111]. obtained some miRNA targeted genes involved in signaling
In order to assess the effect of SP70 on cancer, we treated pathways representing a spectrum of cell cycle and biochem-
lung adenocarcinoma cell line SPC-A1 with NJ001, and sub- ical activities (Fig. 11.8).
CIS SOCS4
a Apoptosis
STAT DNA Bcl-2 MCL1
STAT
STAT Bcl-xL PIMI
+p +p +p
Vav Rac PAK2 MEK1 ERK1/2 EIK1 XIAP
DNA
+p FA tumover
+p
+p MLCK MLC Actomyosin assembly
contraction
XIAP CASP3
Cytokine IFNAR1 JAK PDPK1
CASP9 Apoptosis
BCL2L.11
+p
PDGFA +p Bcl-xL
EGFR FAK/IRS P13K Akt/PKB Bad
EGF PIP3 Bcl-2 Cell cycle
+p Autophagy
FOXO
DNA Glycolysis/Gluconeogenesis
+p FasL
BCL2L11 Cell survival

+p
TSC1/2 Protein systhesis
Rheb mTOR
TBC1D7 Glycerolipid metabolism
Regulation of autophage
Rictor Regulation of actin cytokeleton
PDGFA PDGFRA
Differentiation
EGF EGFR GRB2 SOS Ras ERK Proliferation
DNA
FGF FGFR
TNF TNFR Differentiation

CASP PAK2 JNK Proliferation
IL1 IL1R DNA Inflammation
Apoptosis
FASL FAS Cell cycle
DAXX ASK1 p38
TGFB TGFBR DNA
c +p +p -p
GNA13 RhoA ROCK MLCP MLC Smooth muscle relaxation
Gs ADCY9 PKA ATP2A2 Cardiac muscle contraction

cAMP
Neurotransmitter Contraction
autacoid GPCR
Metabolism
GNA11 PLCβ Proliferation
IP3R Fertilization
Growth factor EGFR PLCγ IP3 Ca2+ Learning and memory
Other signaling pathways
Exocytosis Secretion
d
PROCR
Cell adhesion
uPA uPAR Migration
TFPI F10 F2 TM PC PAI Proliferation
tPA
NF2 PAR1 Platelet activation
Fig. 11.8 Some signal pathways of SP70 downstream genes

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Endocrine and Metabolism
12
Shichang Zhang and Xiaoting Chen
12.1 Overview to form a free AB chain, which is then inactivated by hydro-

lysis to an amino acid under the action of insulinase. The
Metabolic syndrome (Met S), a high incidence rate of dis- islet beta cells have a reserve of about 200 U insulin and
ease, is a combination of abnormal cardiometabolic risk fac- about 40 U daily secretions. The plasma insulin concentra-
tors characterized by dyslipidemia, impaired glucose tion is 5–15 μU/mL on an empty stomach, whereas it can be
tolerance, insulin resistance, inflammation, thyroid disease, increased by 5–10 times after a meal. The biosynthesis rate
obesity, and hypertension. of insulin is affected by plasma glucose concentration. When
These factors are associated with an increased risk of type the blood glucose concentration increases, the proinsulin
II diabetes mellitus and cardiovascular diseases including content in β cells increases, and insulin synthesis accelerates.
myocardial infarction in patients with Met S. Unfortunately, Insulin is secreted into the blood with an equivalent molecule
Met S is typically unrecognized with the heterogeneity of its of C-peptide. In patients with clinical insulin therapy, insulin
management, which can hamper clinical decision-making antibodies are present in the serum, which affects the deter-
and become an obstacle to achieve therapeutic goals of cere- mination of blood insulin levels by radioimmunoassay. In
bral vascular diseases (CVD) and diabetes prevention. The this case, the level of plasma C-peptide can be determined to
heterogeneity management urges the need for clinical bio- understand the endogenous insulin secretion status.
marker tests to improve and complement Met S diagnosis Exogenous insulin is mainly used for the treatment of diabe-
and treatment (Table 12.1). tes. Early use of insulin and super antioxidants in diabetic
patients can lead to longer-term stabilizers and less depen-
dence [1].
12.2 Insulin
12.2.1 Sources and Characteristic 12.2.2 Methods
Insulin is a type of protein hormone with 15 min half-life, The methods for detecting insulin by immunoassay include
secreted by pancreatic beta cells, stimulated by endogenous radioimmunoassay, ELISA, and immunoluminescence.
or exogenous substances such as glucose, lactose, ribose, Since the immunoluminescence method is automated and
arginine, and glucagon (Fig. 12.1). Insulin is the only hor- accurate, it is currently the most widely used in clinical
mone in the body that lowers blood sugar and promotes gly- practice.
cogen, fat, and protein synthesis. The molecular weight of
insulin is 5.70 kDa, which consists of two amino acid pep-
tide chains linked by two disulfide bonds. There are 11 kinds 12.2.3 Reference Interval for Healthy Persons
of 21 amino acids in the A chain, 15 kinds of 30 amino acids
in the B chain, and a total of 16 kinds of 51 amino acids. In Fasting: 2.6–24.9 mIU/L (17.8–173.0 pmol/L). Due to the
the liver, the disulfide bond in the insulin molecule is reduced different determination results of various methods and
reagents, it is recommended to establish respective labora-
tory reference intervals.
S. Zhang (*) · X. Chen
Department of Laboratory Medicine, The First Affiliated Hospital

156 S. Zhang and X. Chen
Table 12.1 Detection methods and reference ranges of molecular markers of metabolic syndrome
Reference interval for
Biomarkers Methods healthy persons Reference
Insulin Immunoluminescence 2.6–24.9 mIU/L Pascaline Aime et al. (2012)
C-Peptide Immunoluminescence 0.3–0.6 nmol/L Pascaline Aime et al. (2012)
HbA1c HPLC 4.2–6.5% American Diabetes Association (2010)
IGFBP-2 ELISA Negative V.C. Russo et al. (2014)
Fig. 12.1 The biological role lslet of

of the islet of the pancreas Human pancreas Langerhans Beta cell Muscle fibers
Glucose
Insulin Blood
vessel
12.2.4 Clinical Significance 12.2.4.2 T he Application in the Hypoglycemia

Syndrome
12.2.4.1 T he Application in the Diagnosis The occurrence of hypoglycemia syndrome can be caused by
and Treatment of Diabetes excessive secretion of insulin by exogenous or endogenous
Type I diabetes: The patients’ islet β cells are severely dam- (insulinoma).
aged, and the function of secreting insulin is significantly
lower. Whether it is fasting or after meals, serum insulin is 12.2.4.3 T he Application in the Diagnosis
often less than 5 mIU/L or cannot be detected. However, in of Insulin Beta Cell Tumor
patients who have been using insulin for a long time, the Insulinoma patients have autonomic and paroxysmal secre-
measured value is low due to the production of insulin anti- tion of INS, which is not regulated by blood glucose levels.
bodies. At this time, the function of β-cells can be under-
stood by measuring the concentration of C-peptide in serum. 12.2.4.4 T he Application of Other Diseases
Type II diabetes is abnormal insulin secretion, impaired in the Diagnosis and Treatment
insulin action, and/or insulin receptor deficiency. The insulin Insulin autoimmune syndrome: Insulin and C-peptide are
secretion is relatively insufficient, and the release reaction is elevated.
slow in type II diabetes patients. Abnormal insulin structure.
Secondary diabetes is when certain endocrine diseases,
drugs, pancreatic diseases, and hereditary diseases can cause • Insulin receptor abnormalities: The levels of blood sugar
diabetes secondary to insulin secretion, interference with and insulin increase.
peripheral effects of insulin, or defects in insulin receptors. • NIDDM with hypertension: the insulin and
Gestational diabetes usually happens 3 months after preg- insulin/C-peptide ratios of these patients are significantly
nancy. This is when the secretion of placental prolactin, cho- higher than those with normal NIDDM.
rionic gonadotropin, and other hormones increase, which • Pancreatitis: Due to impaired pancreatic function, insulin
have antagonistic effects on insulin. While placental insulin- levels are lower than normal. If blood glucose exceeds
ase can also accelerate insulin degradation, if β cells GDM 11.0 mmol/L within 24 h, it can cause permanent
can occur when the glucose response is deficient, insulin diabetes.
secretion is insufficient, and insulin resistance is difficult to • Insulin antibody-positive patients: Insulin levels in such
overcome. patients are very low or undetectable.
Obesity and obesity type diabetes: Obesity is often • About 1/3 of gout patients, starvation or malnutrition,
accompanied by hyperinsulinemia. islet alpha cell tumor, INS secretion of insulin decreased.
12 Endocrine and Metabolism 157
• Some patients with severe cirrhosis can reduce the inacti- inhibits gluconeogenesis, glycogenolysis, and ketogenesis
vation of INS by the liver, increase the concentration of and is degraded within 5–10 min. C-peptide, on the other
hormones (glucagon, growth hormone) against INS, hand, has limited degradation in the liver and is degraded by
decrease the number of receptors on the hepatocyte mem- the kidneys. Hence, the half-life of C-peptide is around
brane, decrease the activity of INS and increase the com- 30–35 min. Due to hepatic uptake, the half-life of insulin is
pensatory secretion. Obesity, hypertension, coronary much shorter than proinsulin and C-peptide. C-peptide (as a
heart disease, hyperlipidemia, and the like are dependent substitute for insulin) is considered to be a sensitive indicator
on the utilization of glucose in INS tissue, resulting in of islet β-cell function, which can be used to help distinguish
elevated serum INS. autoimmune diabetes from other diabetes subtypes [5].
Because C-peptide does not cross-react with insulin and is
not affected by exogenous insulin and insulin antibody, the
12.3 C-Peptide determination of C-peptide is an important means of under-
standing the function of cells, especially for diabetic patients
12.3.1 Source and Characteristics who have been treated with insulin. It is also great for diabe-
tes typing [6].
C-peptide, also known as a connective peptide, is a single
chain polypeptide containing 31 amino acids extracted from
the proinsulin secreted by cells with a molecular weight of 12.3.2 Methods
3.02 kDa [2]. C-peptide is removed in the Golgi apparatus
from proinsulin resulting in the formation of the mature insu- The methods for detecting C-peptide by immunoassay
lin molecule with both alpha and beta chains bound together include radioimmunoassay, ELISA, and immunolumines-
by disulfide bonds [3] (see in Fig. 12.2). It is negatively cence. Since the immunoluminescence method is more accu-
charged and plays an important role in the folding of insulin rate and efficient, it is currently the most widely used in
and the formation of disulfide bonds. C-peptides have not clinical practice.
been reported to show an orderly tertiary structure under
physiological conditions [4]. Both insulin and C-peptide are
stored in secretory vesicles and released in equimolar con- 12.3.3 Reference Interval for Healthy Persons
centrations upon stimulation of beta cells by glucose and
other secretagogues. Once secreted, both insulin and Fasting: 0.3–0.6 nmol/L.
C-peptide are routed through the liver. In the liver, insulin C-peptide release test: The peak time of C-peptide in nor-
binds to its receptors and initiates glucose uptake, which mal people after consuming sugar was basically the same as
Fig. 12.2 Molecular
structure of C-peptide
Foreshortened COOH
connecting peptide
A1
NH2 A20 A21
S
S Chain A
A6 S
A7
S
A11
S B30
S
B7
B19
Chain B
that of insulin, 0.5–1 h after the meal, which was four–six Pancreatectomy: C-peptide was not detected in the serum
times higher than the fasting value. of insulinoma patients after a total pancreatectomy. If the
Urinary C-peptide: 16.2–33.8 nmol/L. concentration of C-peptide increases, it indicates tumor
recurrence or functional metastasis.
Liver and Kidney Disease: The simultaneous measure-
12.3.4 Clinical Significance ment of C-peptide and insulin can help to understand the
changes in the liver and kidney. In patients with hepatitis or
Distinguish Type 1 and Type 2 Diabetes: C-peptide plays an liver cirrhosis, the level of insulin in the blood tends to
important clinical role in distinguishing Type 1 and Type 2 increase following less insulin was taken in the liver, while
diabetes. C-peptide levels can be used to understand the C-peptide is less affected, so the ratio of C-peptide to insulin
function of islet β cells in diabetic patients. Due to the large in the blood decreases. In kidney disease, C-peptide degrada-
number of islet β cells destroyed, the C-peptide level in Type tion slows down, so the ratio of C-peptide to insulin is sig-
1 diabetes is lower than normal but not in Type 2 diabetes. nificantly higher than normal. When kidney function is
Determining Prognosis: In patients with type 1 diabetes, severely impaired, the ratio is close to normal or reduced.
β-cell function measured using C-peptide is very limited,
which is also related to improving glycemic control, decreas-
ing hypoglycemia, and significantly reducing microvascular 12.4 Glycosylated Hemoglobin (HbA1c)
complications.
Diagnosis and Differential Diagnosis of Hypoglycemia: In 12.4.1 Sources and Characteristics
patients with hypoglycemia treated with exogenous insulin,
the c-peptide assay can identify whether the cause of hypo- Hemoglobin can be predominantly divided into three main
glycemia is due to insulin overdose or insufficient food intake. types: HbA (α2β2), HbA2 (α2δ2), and HbF (α2γ2). HbA1
Islet Transplantation: C-peptide assays can be used to accounts for about 6% of the total HbA, which can be further
determine graft survival rate and post-transplant secretory divided into four subtypes (HbA1a1, HbA1a2, HbA1b, and
function of β cells. C-peptide <0.1 nmol/l (fasting and/or HbA1c) according to the electrophoretic and chromato-
mixed-meal tolerance test) has been used as a criterion for graphic properties [7] (see in Fig. 12.3).
islet cell transplantation and determination of complete Glycosylated hemoglobin (HbA1c) is the product of a
transplantation failure. slow, non-enzymatic condensation of glucose with hemoglo-
Fig. 12.3 Hemoglobin A1c

HbA1a
from discovery to the
HbA1 HbA1b
diagnosis of diabetes
HbA1c
Adult hemoglobin
(HbA)
Non-glycated
HbA0
Glycated
HbF
Minor form in normal

Hemoglobin
adults
HbA2
HbS
HbC
Hemoglobin variants
HbD
HbE
bin in the blood [7]. The synthesis of HbA1c is slow and 12.4.3 Reference Interval for Healthy Persons
irreversible, and its synthesis rate is in positive correlation
with average blood glucose concentration, HbA1c can con- Fasting: 4.2–6.5%.
tinue to exist in the life cycle of RBC for 120 days, so its There are regional and ethnic differences in the setting
proportion in total hemoglobin can accurately reflect the pre- of the HbA1c reference interval, but what is common is
test 8–12 weeks of average blood glucose levels. HbA1c is that most laboratories do not consider the influence of age
convenient, stable to detect, and is not affected by the fluc- and gender when setting the HbA1c reference interval.
tuation of blood glucose. Thus, it has been listed as one of The new guidelines issued by ADA in 2010 have listed
the diagnostic criteria for diabetes by many foreign guide- HbA1c 6.5% as one of the diagnostic criteria for diabetes,
lines [8]. and HbA1c 5.7% as one of the screening criteria for
diabetes.
The laboratory uses many different methods to measure
12.4.2 Methods HbA1c (see in Table 12.2), but some of them may give
inaccurate results when patients have hemoglobin variants
Currently, there are more than 100 HbA1c detection methods such as sickle cell trait or elevated fetal hemoglobin (HbF)
on the market, all of which are based on one of the flowing levels [9].
two principles: separation based on charge differences or
separation based on structural differences, both of which will
occur when glucose binds to Hb. The former is used for ion- 12.4.4 Clinical Significance
exchange chromatography and electrophoresis-based analy-
sis, while the latter is used for immunoassays, boronate As an evaluation index of long-term blood glucose control in
affinity chromatography, and enzymology. patients with diabetes: HbA1c was determined to eliminate
One of the methods is based on charge differences: the effect of fluctuating blood glucose on disease control
charge-based differences depend on the extra negative charge observation. It can be used as a good indicator for the long-
that is generated when glucose is attached to the N-terminal term control of diabetes, which can reflect the measurement
valine of the HbA β-chain. These methods include electro- of the average blood glucose level in the first 8-10 weeks and
phoresis and high performance liquid chromatography monitor the degree of diabetes control. According to the
(HPLC). monitoring results, clinicians can adopt methods such as diet
Another one is based on structural differences. Such control or drugs to formulate correct treatment plans. It has
methods are mainly based on the structural characteristics of the advantages of small variability, less blood collection,
glycosylated groups on hemoglobin, including affinity chro- convenient detection, and no special preparation for patients.
matography, immunochemical method, and enzymatic However, the main disadvantage is the detection method is
method. not yet unified.
Table 12.2 The table below lists the methods most commonly used to measure HbA1c and whether this method is affected by HbC, HbS, HbE,
or HbD trait or elevated HbF
Interference from Interference from Interference from Interference from Interference from elevated
Method HbC HbS HbE HbD HbF
Bio-Rad D-10(A1c No No No No No <10% HbF
program)
Bio-Rad D-100(A1c No No No No –
program)
Bio-Rad Variant II NU No No No No No <10% HbF
Bio-Rad Variant II Turbo No No Yes↑ Yes↑ No <5% HbF
Bio-Rad Variant II Turbo No No No No No <25% HbF
2.0
Sebia Capillarys 2 Flex No No No No No <15% HbF
Piercing
Siemens DCA 2000/ No No No No No <10% HbF
Vantage
Tosoh G7 Yes↓ No Yes↓ No No ≤30% HbF
Tosoh G8 Yes↓ Yes↓ Yes↓ Yes↓ No ≤30% HbF
(No for ver. 5.24) (No for ver. 5.24) (No for ver. 5.24#) (No for ver. 5.24)
It is likely to be a useful diagnostic tool for confirming contains 4 exons, 496 nucleotides, and 3 introns [12]. The
gestational diabetes mellitus (GDM): evidence suggests that mRNA length of IGFBP-2 is about 1.6 kb, which plays a key
HbA1c is a significant marker for Chinese patients with part in the transcription and protein expression of the
GDM. With high specificity and sensitivity, the HbA1c test IGFBP-2 gene [13]. The molecular weight of human mature
plays an important role in confirming the diagnosis of GDM IGFBP-2 is about 31 kDa, containing 289 amino acids, and
in a Chinese population. its primary structure was first obtained by Binkert and col-
It is closely related to the degree of atherosclerotic steno- leagues Recent studies have found that IGFBP-2 functional
sis in cerebral infarction patients: Numerous studies have domains mainly include the integrin-binding domain (RGD),
shown that whether patients have diabetes or not, the increase heparin-binding domain (HBD) and nuclear localization sig-
of serum HbA1c level is an independent risk factor for acute nal (NLS) sequences, which may be related to the biological
ischemic stroke [10]. Therefore, as a new risk factor, HbA1c role of these structural regions [14].
is gradually receiving the attention of first-line neurologists.
In the comparative study of the anterior and posterior circu-
lation, the effect of increased HbA1c level in diabetes on ath- 12.5.2 Methods
erosclerosis of posterior circulation, namely vertebrobasilar
artery system, is more obvious than that of the anterior circu- Serum IGFBP-2 concentration was detected by ELISA. The
lation, which is an independent risk factor for posterior cir- main principle of ELISA is to use the specific reaction
culation cerebral infarction, especially cerebral stem between antigen and antibody. Firstly, antibodies or anti-
infarction [11]. gens are combined with solid phase carriers to maintain
the inherent immune activity. Secondly, antibodies or anti-
gens are connected with enzymes. During determination,
12.4.5 Conclusions and Prospects samples and enzyme-labeled antibodies or antigens react
with antigens that are bound to the solid phase carrier in
Unlike FPG, HbA1c reflects average levels of glucose over different grades. Antigen-antibody complexes are sepa-
the past 2–3 months, suggesting that HbA1c better reflects rated from the solid phase carrier by washing, and the
glucose fluctuation. Because the determination of HbA1c is amount of enzyme on the surface of the solid phase carrier
more repeatable than FPG, more convenient than 2-h PPG, is proportional to the target substance in the sample. These
and has less variation in individuals, it provides a method for enzymes then react with the substrate to produce colored
the comprehensive determination of glucose that is less sus- substances for quantitative determination. It can be an
ceptible to short-term effects. As a result, the application of antibody or an antigen. The method is highly sensitive.
HbA1c in the screening and diagnosis of diabetes has greatly ELISA is used to determine the level of IGFBP-2 in human
increased. samples. After sample treatment, anti-IGFBP-2 antibody
was added to biotin and then combined with streptavidin-
HRP to form an immune complex. Then, it is incubated by
12.5 IGFBP-2 using the chromogenic system. The chromogenic degree
was compared with the standard substance, and the spec-
12.5.1 Sources and Characteristics trophotometric value was measured to obtain the concen-
tration value.
IGFBP-2 is well known as an important family member of
insulin-like growth factors (IGFs), including insulin-like
growth factor ligand (IGFs), cell surface receptor (IGF-R), 12.5.3 Reference Interval for Healthy Persons
and insulin-like growth factor binding protein (IGFBPs).
IGFBPs mainly include 6 IGFBPs with high affinity to IGF IGFBP-2 is expressed in human serum, body fluids, and tis-
and 4 or more IGFBP-related protein members. There are a sues, and is highly expressed in embryo and fetus. After
lot of studies showing that IGFBP-2 has a high affinity with birth, the expression level gradually drops to a low stable
IGF-II and it mainly plays a role through binding IGFs path- level, but when the body suffers from severe stress response
way and non-dependent IGFs pathway. IGFBP-2 is impor- or disease, the expression level in various tissues will
tant for cell metabolism, cell proliferation, and cell migration. increase to varying degrees. The serum expression level of
IGFBP-2 was first discovered by Mottol et al. in the BRL-3A IGFBP-2 will be increased when the body experiences stress
cell line in 1986, and its gene structure and protein structure reaction, shock, trauma, benign and malignant tumors, and
were subsequently confirmed. The human IGFBP-2 gene acute or chronic inflammation [15].
12.5.4 Clinical Significance cation and could have an important significance in the devel-
opment of new prevention or treatment strategies.
IGFBP-2 and its role in diabetes: Clemens Wittenbecher
found IGFBP-2 concentrations were associated with the risk
of type 2 diabetes. The association remained significant even 12.6 Thyroid Markers
after controlling important molecular and other diabetes risk
factors [16]. The study organized by Kristina Hedbacker 12.6.1 The Function of Thyroid
demonstrated that IGFBP-2 excessively helps lower blood
glucose and strongly inhibits HGP, as well as genes related to Thyroid hormones are essential for normal growth and devel-
hepatic gluconeogenesis and fatty acid synthesis [17]. Under opment, particularly of the central nervous system, and they
non-experimental conditions, interpretation of the relation- play a key role in regulating metabolism. The synthesis of
ship between 245 circulating IGFBP-2 concentrations and thyroid hormones requires a normally developed thyroid
the risk of type 2 diabetes is complicated because IGFBP-2 gland, a functioning hypothalamic-thyroid axis, an adequate
expression is partly controlled by insulin and cross-sectional nutritional iodine intake, and a series of regulated biochemi-
associations of IGFBP-2 with insulin resistance were cal steps within thyroid follicular cells. The thyroid gland
reported [18]. One possible explanation for the relation secretes two hormones, thyroxine, and triiodothyronine,
between circulating IGFBP-2 and type 2 diabetes risk is an which short name are T4 and T3, respectively. In addition, the
involvement of IGFBP-2 in insulin-regulated pathways [19]. thyroid gland secretes small amounts of biologically inactive
In vitro experiments have shown that IGFBP-2 enhances rT3 and monoiodotyrosine (MIT) and diiodotyrosine (DIT),
GLUT4-mediated glucose uptake in adipocytes, suggesting a which are precursors of T3 and T4.
direct insulin-signaling pathway [20]. In several human Thyroid hormones have many important biological
whole-genome studies, IGFBP-2 and the relationship of dia- effects. A major function is their control of the basal meta-
betes show that it may play a role in regulating metabolism. bolic rate and calorigenesis through increased oxygen con-
IGFBP-2 levels associated with insulin resistance could be sumption in tissue via the effects of thyroid hormone on
used as a biomarker of insulin sensitivity and may play an membrane transport and enhanced mitochondrial metabo-
important role in the pathogenesis of diabetes. A recent large lism. Thyroid hormones are indispensable for neural devel-
prospective study in women have found that IGFBP-2 level opment, normal growth, and sexual maturation in mammals.
has a negative correlation to the risk of T2DM. Other actions include stimulation of adrenergic activity with
The relationship between IGFBP-2 and malignant tumor: increased heart rate and myocardial contractility, stimulation
IGFBP-2 performs a valuable function in regulating cell proof protein synthesis and carbohydrate metabolism, increased
liferation, differentiation, and the inhibition of cell apoptosis synthesis and degradation of cholesterol and triglycerides,
[21]. A large number of in vivo and in vitro experiments have increased requirement for vitamins, increased calcium and
identified the main pathway of IGFBP-2 malignant tumor phosphorus metabolism, and enhanced sensitivity of adren-
progression include the IGFs pathway and non-dependent ergic receptors to catecholamines. These effects are typically
IGFs pathway. Among them, the non-dependent IGFs path- magnified in patients with hyperthyroidism and subdued in
way mainly includes integrin receptor, extracellular mecha- patients with hypothyroidism.
nism components, cell membrane proteoglycan, and nuclear
EGFR-STAT3 signaling pathway, et al. [10]. The mechanism
of action is complex and there are interconnections among 12.6.2 Laboratory Evaluation of Thyroid
various pathways, which need to be further explored. Current
studies have shown that IGFBP-2 is closely related to the Thyroid function testing is the most used diagnostic evalua-
occurrence and development of glioma, ovarian cancer, and tion in endocrine practice. A profound understanding of the
prostate cancer, and has been proven to be related to the indications and limitations of thyroid testing is essential for
metastasis of cancer cells in patients with breast cancer, an adequate clinical diagnosis and the management of a wide
bladder cancer, and prostate cancer [22]. range of thyroid disorders [23]. Thyroid function tests are
used as a screening tool, to verify the clinical diagnosis of
hyper and hypothyroidism, to assess the adequacy of medical
12.5.5 Conclusions and Prospects treatment, and in the follow-up of differentiated thyroid
cancer.
In conclusion, Plasma IGFBP-2 has strong inverse associa- These tests cover the assessment of the hypothalamic-
tions with the risk of type 2 diabetes both in men and women. pituitary-thyroid axis, radioiodine uptake tests, determina-
IGFBP-2 may play an important role in diabetes risk stratifi- tion of urinary iodide and serum thyroglobulin, as well as
Table 12.3 Classification of the most important thyroid function tests hormones. Biologically active free thyroid hormone frac-
Test classification Test item tions (FT4 and FT3) can enter cells by specific membrane
Tests that assess the hypothalamic- Serum total thyroxine (TT4) transport mechanisms.
pituitary-thyroid axis Serum total In the pituitary, the negative feedback of thyroid hormone
triiodothyronine (TT3) on TSH secretion is mediated primarily by T3 produced
Serum free thyroxine (FT4) locally from the deiodination of T4. Most thyroid function
Serum free triiodothyronine
tests are performed on serum and are based on automated
(FT3)
Serum thyrotropin (TSH) immunoassays. Liquid chromatography-tandem mass spec-
TRH test trometry (LC-MS/MS) has recently been introduced in this
Indicators of iodine intake and Urinary iodine field, in order to measure total and free thyroid hormones or
utilization by the thyroid Radioactive iodine uptake urinary iodine [25]. Technically, it is easier to develop meth-
test (RAIU) ods to measure total thyroid hormone (TT4 and TT3) concen-
Other tests related to thyroid Serum thyroglobulin (Tg) trations, as compared to tests that estimate free concentrations.
function Perchlorate discharge test
The former are measured at nanomolar levels, whereas the
Tests for the evaluation of thyroid Thyroid peroxidase
autoimmunity antibody (TPO-Ab)
latter are measured in the picomolar range and require assays
Thyroglobulin antibody that have to be free from interference by the much higher
(TG-Ab) total hormone concentration [26].
TSH receptor antibody
(TR-Ab) 12.6.2.2 T ests for Evaluation of Thyroid
Autoimmunity
investigation of variables suggestive of thyroid autoimmu- Thyroid autoantibodies are directed predominantly against
nity (see in Table 12.3). thyroglobulin, thyroid peroxidase and the TSH receptor,
although other components of the thyroid cell may also be
12.6.2.1 T
ests That Asses the Hypothalamic- autoantigenic targets in a small proportion of patients. In
Pituitary-Thyroid Axis terms of pathogenesis, any role for thyroglobulin or thyroid
peroxidase antibodies is confined to the secondary phase of
T3 and T4 autoimmune destruction. In contrast, stimulating antibodies
Triiodothyronine (T3), the biologically active hormone, and against the TSH receptor cause Graves’ disease, and in a
Thyroxine (T4) are the major secretory products of the thy- small proportion of patients with autoimmune hypothyroid-
roid; more precisely, T4 is synthesized exclusively in the thy- ism, blocking antibodies against the TSH receptor is respon-
roid gland, whereas approximately 80% of T3 is produced by sible for thyroid dysfunction.
peripheral conversion of T4 using monodeiodination, medi-
ated by 50-monodeiodinase operating at different tissue TG-Ab and TPO-Ab
levels. TG was the very first autoantigen to be described; the now
classic experiments undertaken by Rose and Witebsky in
TSH and TRH 1956 showed that immunization of rabbits with thyroid
Thyroid hormone synthesis and secretion are regulated by extract led to the production of TG-Ab and a lymphocytic
intrathyroidal and extrathyroidal control, the most impor- thyroiditis (around three-quarters of thyroid protein is TG)
tant factor being pituitary thyrotropin (TSH). In turn, TSH [27]. TPO was initially described as the thyroidal micro-
production and secretion are under the regulation of a feed- somal antigen by Belyavin and Trotter more than half a
back inhibition via thyroid hormones, and stimulated by the century ago [28]. Twenty-six years later, two groups simul-
hypothalamic tri-peptide thyrotropin-releasing hormone taneously characterized this autoantigen as TPO [29]. TPO
(TRH). T4 and T3, in the circulation, are mostly bound to a belongs to the family of mammalian peroxidases and has a
set of plasma proteins that differ widely in their concentra- high sequence homology with myeloperoxidase [30]. In
tion and affinity for these hormones. The three major trans- man, TPO is a membrane-associated protein of 933 amino
port proteins are T4-binding globulin (TBG), transthyretin acids with five potential glycosylation sites and is encoded
(TTR or T4-binding pre-albumin, TBPA) and albumin by a single gene on chromosome 2, spanning 17 exons.
(ALB) [24]. Human TPO-Ab and monoclonal TPO antibodies recog-
nize multiple conformational epitopes on TPO which map
FT4 and FT3 to two major, closely associated domains, A and B, within
Protein-bound thyroid hormones do not cross plasma mem- an immunodominant conformational region on the protein
branes and are therefore considered to be biologically inac- located in an area that shares homology with
tive and functioning as a reservoir for circulating thyroid myeloperoxidase.
TR-Ab central medulla. The adrenal cortex accounts for about 90%
TSHR, composed of 764 amino acids, is the key autoantigen of the gland. Its structure is divided into three layers: the
in GD and plays a role in some patients with autoimmune globular zone, the fascicular zone, and the reticular zone.
hypothyroidism [31]. The TSHR belongs to the G protein- The cells in the three zones can secrete steroids (The globu-
coupled receptor family and is composed of three extracel- lar zone mainly secretes aldosterone, the fascicular zone
lular loops containing the amino terminus, seven mainly secretes glucocorticoid, and the reticular zone mainly
transmembrane segments, and three intracellular loops end- secretes androgen). The adrenal medulla, about 10% of the
ing with a carboxyl terminus. The gene encoding TSHR, volume of the adrenal gland, located in the center of the adre-
located on chromosome 13, is composed of ten exons, the nal gland and mainly composed of medullary cells, which
first nine of which encode the extracellular domain (ECD). can be divided into adrenaline cells and noradrenaline cells.
The ECD has multiple TSH binding sites as well as TSHR
antibody (TR-Ab) binding sites. The receptor undergoes
important modifications during synthesis including glyco- 12.7.2 Function of Adrenal Gland
sylation, which is essential for the proper folding of the
receptor and for its antigenic properties. As an endocrine organ, the adrenal gland regulates the body’s
function mainly by secreting hormones. Below we introduce
the hormones secreted by the adrenal gland.
12.6.3 Conclusions and Prospects
(i) Aldosterone secreted by zona glomerulosa cells belongs
Nowadays, the assessment of thyroid function allows the to saline corticosteroids, which can promote the reab-
diagnosis of subclinical alterations with high precision. sorption of Na+ and K+ in distal renal tubules and collect
However, even the most sensitive free thyroid hormone and ducts. At the same time, it can stimulate gastric mucosa,
TSH assays may be prone to interference and artifactual salivary gland, and sweat gland to absorb Na+, increase
measurement due to the presence in the serum sample of blood Na+, and decrease blood K+, in order to maintain
interfering factors. For this reason, the lucid analysis of inex- blood volume.
plicable discrepancies in thyroid function tests should lead to (ii) Glucocorticoid is produced by fascicular zone cells,
the reanalysis of such cases employing alternative platforms mainly cortisol and corticosterone. There are glucocor-
to determine whether the abnormality can be verified. When ticoid receptors in the cytoplasm of many kinds of cells
considering the presence of possible interfering factors, it is in the human body, so glucocorticoid has many biologi-
helpful to examine the linearity of recovery in serial dilutions cal functions. Its main function is to promote the
of the patient’s serum with appropriate diluents. decomposition of protein and fat into sugar (gluconeo-
Since the early 1990s, the diagnosis criteria for many thy- genesis). It also has anti-inflammatory and immunosup-
roid lesions have been refined, new immunohistochemical pressive effects.
markers have emerged, and a variety of novel genetic altera- (iii) Both adrenaline and norepinephrine secreted by medul-
tions responsible for the familial and sporadic forms of thy- lary cells are catecholamines. Adrenaline can accelerate
roid cancer have been discovered. Most importantly, the heart rate and dilate the blood vessels of the heart
molecular genetics has penetrated all fields of medicine and and skeletal muscle. Norepinephrine can cause wide-
has increased impact on the diagnosis of nontoxic goiter, spread contraction of blood vessels in various organs of
thyroid nodule, thyroiditis, hypothyroidism, hyperthyroid- the body and accelerate blood flow in the heart, brain,
ism and thyrotoxicosis, toxic adenoma and multinodular and skeletal muscle.
toxic goiter, thyroid carcinoma.
12.7.3 Laboratory Examination of Adrenal

12.7 Adrenal Gland Markers Gland
12.7.1 Structure of Adrenal Gland As an important endocrine organ, a variety of clinical dis-
eases can cause hyperactivity or insufficiency of adrenal
The adrenal glands are located above the kidneys on both function, and the examination of adrenaline and its metabo-
sides and are wrapped in the renal fascia together with the lites can be combined with other types of examinations to
kidneys, but the adrenal glands have independent fibrous complete the diagnosis of the diseases. Therefore, we should
sacs and fat sacs. The left adrenal gland is half-moon-shaped, have a comprehensive understanding of these commonly
and the right adrenal gland is triangular or elliptic. The adre- used examinations, so as to better complete the diagnosis of
nal parenchyma consists of the surrounding cortex and the the diseases (Table 12.4).
Table 12.4 Laboratory examination of the adrenal gland mance liquid chromatography (HPLC), radioimmunoassay,
Test classification Test item etc. Chemiluminescence immunoassay (CLIA) is the most
Tests that assess the level of Plasma norepinephrine (NE) commonly used method in clinical laboratories because of
adrenal medullary hormone Plasma adrenaline (E) its rapidity, simplicity, and sensitivity.
Plasma dopamine (DA) There is a circadian rhythm of cortisol secretion in normal
Urinenorepinephrine
Urine adrenaline people, which disappears in patients with hypercortisolism,
Urine dopamine it’s one of the bases for the diagnosis of hypercortisolism.
Urineanillylmandelic acid (VMA) Increased serum cortisol concentration is mainly seen in
Urine homovanillic acid (HVA) adrenal hyperfunction, adrenal tumors, oral contraceptives,
Tests that assess the level of Serum (plasm) cortisol
adrenocortical hormone Free cortisol in urine and saliva
etc. The decrease is mainly seen in hypoadrenocorticism and
Urine 17-hydroxycorticoid hypopituitarism.
(17-OHCS) 17-OHCS mainly reflects the secretory function of the
Urine 17-ketocorticoid (17-KS) adrenal cortex. 17-OHCS increases in hyperfunction of the
Plasma adreno-cortico-tropic-
hormone (ACTH)
adrenal cortex, such as Cushing’s syndrome and adrenocorti-
Plasma N-terminal peptide of cal tumors, and decreased 17-OHCS is found in hypofunc-
proopiomelanocortin (N-POMC) tion of the adrenal cortex, hypophysis, and hypothyroidism
ACTH stimulation test after adrenalectomy.
Desamethasone suppression test
Urine 17-KS test mainly reflects testicular function and
adrenal cortex secretion function. The increase is mainly
seen in adrenocortical hyperfunction, pituitary hyperfunc-
12.7.3.1 T ests That Assess the Level of Adrenal tion, and Leydig cell tumors of the testis. The decrease of
Medullary Hormone urinary 17-KS is found in the hypofunction of the adrenal
The hormones secreted by the adrenal medulla include E, cortex, pituitary gland, testis, and thyroid.
NE, and DA, collectively known as catecholamines hor-
mones. The main end product of epinephrine and norepi- Examination of ATCH and N-POMC in Plasma
nephrine is VMA. The main end product of dopamine is ACTH exists at the level of pg/mL in peripheral blood.
HVA. Urinary catecholamine test is sensitive and reliable, Immunoassay is often used in the test of ACTH in the clinic.
but it needs higher level of technology. In recent years, sensi- Monoclonal antibodies targeting the C-terminal and
tive and specific radioenzyme analysis technology or high N-segment of the ACTH peptide chain are often used in the
performance liquid chromatography (HPLC) analysis tech- examination. The double antibody sandwich method has
nology is used in the tests of norepinephrine, adrenaline, and high sensitivity and specificity.
dopamine in the blood. Although the experimental condi- The increase or decrease of plasma ACTH and the disap-
tions are demanding and the price is expensive, they’re the pearance of circadian rhythm suggest the existence of adre-
most sensitive and accurate method of diagnosis of pheo- nocortical dysfunction. However, plasma ACTH examination
chromocytoma, especially useful for the diagnosis of pheo- is generally not the preferred screening item. It is often used
chromocytoma with normal blood pressure. Urine with cortisol examination to diagnose the types of adreno-
catecholamine and its metabolites VMA and HVA can be cortical dysfunction and lesions. Both ACTH and cortisol
evaluated by spectrophotometry or fluorescence increase, suggesting adrenocortical hyperfunction caused by
spectrophotometry. hypothalamic, pituitary lesions, or heterogenous ACTH
syndrome.
12.7.3.2 T
ests That Asses the Level
of Adrenocortical Hormone Dynamic Functional Test
ACTH Excitation Test: This test is used to diagnose primary
Examinations of Glucocorticoids and Their or secondary cortical dysfunction. Because ACTH can
Metabolites in Blood, Urine, and Saliva promptly stimulate the synthesis and release of cortisol from
Clinical biochemical indicators include glucocorticoids the adrenal cortex, so the excitability of the adrenal cortex
and their metabolites in blood, urine, and saliva, free corti- can be evaluated by intravenous injection of ACTH.
sol in urine and saliva, 17-hydroxycorticosteroids in urine
(17-OHCS), 17-ketocorticosteroids (17-KS). Methods to Dexamethasone Inhibition Test
measure these indicators include fluorescence spectropho- This experiment is suitable for the diagnosis and differential
tometry, chemiluminescence immunoassay, high perfor- diagnosis of Cushing’s syndrome. Dexamethasone is a pow-
erful synthetic glucocorticoid (GC) drug, which can produce simultaneously they will be helpful to judge the location and
strong cortisol-like negative feedback inhibition on CRH and nature of the lesion. In recent years, genetic testing is widely
ACTH secretion, and then affect the function of the adrenal carried out, which is a more effective and accurate method to
cortex in the secretion of GC. diagnosis adrenopathy.
12.7.3.3 Genetic Testing

At present, with the intensive study of the etiology of adrenal 12.8 The Relevant Progress
diseases, many genes related to the occurrence and develop-
ment of adrenal diseases have been reported. Such as Metabolic syndrome associated with other diseases like
CYP21A2, CYP111B1, CYP17A1, SD3B2, STAR, and Alzheimer’s disease, cardiovascular diseases, and/or cancer
POR. At present, some diagnostic companies and hospitals will arise more often. Additionally, other associations with
begin to develop related gene testing, so as to achieve the unforeseen diseases may emerge (see in Table 12.5). In such
purpose of predicting the occurrence of diseases and diag- cases, a comprehensive personalized biomarker model is
nosing early. essential for assessing an individual’s health, as the com-
bined impact of multiple diseases may be different from the
impact each individual disease has on a person’s health. In
12.7.4 Conclusion such cases, adequate monitoring of an individual’s health
will be a major challenge. Recent discoveries highlight the
The adrenal gland consists of two independent endocrine combined detection of multiple markers as an attractive tar-
glands, the cortex, and medulla. The best choice for the diag- get for the detection, prevention, and treatment of obesity
nosis of adrenocortical dysfunction is the examination of and diabetes-related diseases. We expect that instead of using
total cortisol in plasma, saliva, and urine. If ACTH in plasma a single biomarker, there will be a shift to biomarker panels/
and the necessary dynamic function test can be carried out models to more accurately describe an individual’s health.
Table 12.5 Related diseases with biomarkers and its intended uses
Biomarkers Related diseases Intended use References
Insulin Type 2 diabetes IR itself has been shown to significantly increase the incidence and prevalence of the Natalie
mellitus cardiovascular disease in individuals with T2DM. J. Haywood et al.
(2018)
Alzheimer’s Insulin-sensitizing drugs are performed on multiple aspects of AD pathology in Hilaree
Disease human patients. Dissection of the actions of insulin in neuronal and glial cells is likely N. Frazier et al.
to foster knowledge on the roles of insulin and related signaling pathways in CNS (2018)
physiology.
C-Peptide Diabetes It suggests that the physiological C peptide concentration in type 2 DM patients may Arturo Pujia
delay the development of DN. et al. (2017)
Hypoglycemia C-peptide assay helps to identify the causes of hypoglycemia. Suzy V. Hope
et al. (2018)
HbA1c Diabetes As an evaluation index of long-term blood glucose control in patients with diabetes Chewng BY
It is likely to be a useful diagnostic tool for confirming gestational diabetes et al. (2012)
mellitus(GDM) Whiting D R
It is closely related to the degree of atherosclerotic stenosis in cerebral infarction et al. (2011)
patients
IGFBP-2 Diabetes IGFBP-2 over-expression protects against diabetes via its modulation of insulin Natalie
sensitivity. J. Haywood et al.
(2018)
Obesity IGFBP-2 over-expression protects against obesity via its mediated inhibition of Natalie
adipogenesis. J. Haywood et al.
(2018)
Glioma IGFBP-2 is increasingly recognized as a glioma oncogene, promoting glioma Faping Shen
development and progression. et al. (2018)
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cancer cells and is found elevated in the serum of patients with prostate carcinoma, (2014)
where its levels positively correlate with tumor stage/grade.
Breast cancer Expression levels of IGFBP-2 strongly correlate with the grade of malignancy, with V.C. Russo et al.
increased cytoplasm levels and cell surface association of IGFBP-2 as a distinctive (2014)
feature of the carcinoma in situ and infiltrating/invasive breast carcinomas.
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Immune System
13
Xiuru Guan
13.1 Overview tem, the damaged tissues will produce related protein
degradation products, such as inflammatory factors.
Immune system is a large and complex network regulatory Basing on the indirect immunofluorescence (IIF) and
system composed of multiple organs, tissues, cells, and radioimmunoassay (RIA), some high-throughput, high-
immune molecules in the body. It coordinates with other sys- precision methods, like protein chip technology and che-
tems and jointly maintains the homeostasis of the internal miluminescence immunoassay, will be more widely used
environment through recognizing and getting rid of antigenic to detect the biological markers of autoimmune diseases.
foreign bodies. Molecular markers can not only be patho- 2. Nucleic acid markers: The evolution of molecular biology
genic molecules that cause immune diseases, but also serve has led to more precise and reliable molecular markers
as indicators of the immune reaction process. Therefore, that have been widely studied. Among them, DNA and
there are countless links between the immune system and MicroRNA have gradually become research hotspots for
molecular biomarkers, and molecular biomarkers have the prevention and diagnosis of autoimmune diseases.
important clinical significance for the prevention, diagnosis, 3. Other types of markers: In addition to the above two types
and treatment of immune system diseases. of markers, cell metabolites, specific molecular signals,
and some genetic genes related to autoimmune diseases
are also potential markers.
13.1.1 Autoimmune Diseases
Molecular biomarkers, a new direction for the precise
Autoimmune diseases, AID, refer to the diseases caused by development of immunomedicine, are widely used in all
the breakdown of the body’s self-tolerance state and the sorts of illnesses. However, the application of molecular
immune system’s immune response to autoantigens, which markers in immune system diseases is still in its infancy
react with its own cells, tissues, and/or organs, resulting in owing to the large composition of the immune system, the
pathological damage or functional disorders of organs or large number and variety of molecules involved in the
systems. immune response, and the complex mechanism of immune
Autoantibody is the first substance to be discovered and regulation. This section will start from the two parts of
used as a marker in the diagnosis of autoimmune diseases. At organ-specific autoimmune disease markers and systemic
present, the accuracy and speed of detection of autoantibod- autoimmune disease markers to describe the corresponding
ies have been constantly improved, and the research on diag- relatively common landmark autoantibodies.
nostic biomarkers of autoimmune diseases has also been
greatly promoted.
There are several categories of molecular biomarkers that 13.1.2 Hypersensitive Diseases
are now widely studied for autoimmune diseases:
Hypersensitivity reactions can be divided into four catego-
1. Specific protein markers: In autoimmune diseases, fol- ries. Type I hypersensitivity reactions, also called allergies,
lowing local tissues are attacked by the autoimmune sys- are immediate-type allergic reactions triggered by 1gE anti-
bodies. After exposure to allergens, individuals first produce
specific IgE antibodies. Subsequent exposure to the same
X. Guan (*) allergen can induce the activation of basophils and mast cells
The First Affiliated Hospital of Harbin Medical University,
causing special effects of such allergic reaction. Other dis-
Harbin, Heilongjiang, People’s Republic of China

168 X. Guan
eases, for instance, serum sickness, allergic contact dermati- Table 13.1 Criteria for judging delayed-onset skin test results
tis are classified into type II, III, or Th1- or CD8 T-cell type Result of
IV hypersensitivity reactions. reaction Intradermal Patch
No response No response or less No response or less
(–) than control than control
13.1.2.1 M arkers of Type I Hypersensitivity
Weak positive With only redness Mild redness and
Diseases (+) pruritus
Type I hypersensitivity, also known as allergic reaction or Positive (++) Redness with Redness and sometimes
immediate hypersensitivity, is the most common hypersensi- induration erythema
tivity reaction in the clinic. It is mainly mediated by specific (0.5–1 cm) marked
Strong positive Sclerosis, blister, Erythema with bean
IgE antibodies and hypertrophied after the second exposure
(+++) erythema rash, blister
to the same antigen. The release of biologically active medi- Strong positive Bullae or (and) ulcers Swelling, Blisters,
ators by effector cells such as mast cells and basophils lead (+++) Ulcers
to clinical symptoms. Its main features are rapid occurrence,
rapid regression; usually causing physiological dysfunction,
stimulating with the same antigen, so it gets the name delayed
less serious tissue cell damage; mediated by specific IgE
hypersensitivity. Table 13.1 shows the criteria for judging
type antibody, no complement participation; with obvious
delayed-onset skin test results.
individual differences and genetic background.
13.1.2.2 M arkers of Type II Hypersensitivity 13.2 Tissue-specific Autoantibodies

Diseases
Type II hypersensitivity reaction is also known as cytolytic 13.2.1 Anti-intrinsic Factor Antibody (AIFA)
type or cytotoxic type hypersensitivity reactions. It refers to
the combination of antibodies to specific cells or tissues sur- Anti-endogenous factor antibody is an autoantibody target-
face antigen forming cell–antibody complex. Then, the acti- ing a glycoprotein came from gastric mucosal parietal cells.
vation of the complement system or other mechanisms leads It is combined with vitamin B12, which is an accomplice
to the damage to the target cells. Type II hypersensitivity factor for intestinal absorption. Enzyme-linked immunosor-
reaction is rarely associated with exogenous antigens usually bent assay (ELISA) has been widely used for its detection.
because of the production of autoantibodies to their own The reference interval of ELISA is negative.
components or microbial antigens that cross-reacted with Intrinsic factor antibody is the basis of autoimmune gas-
autoantigens. tric atrophy [1]. Enclosed type AIFA is a specific marker
antibody of pernicious anemia (PA), and its positive can help
13.1.2.3 M arkers of Type III Hypersensitivity to differentiate gastric atrophy with PA from simple gastric
Diseases acid deficiency.
Type III hypersensitivity is also known as immune complex
type hypersensitivity refers to the deposition of antigen–anti-
body complexes in tissues, which leads to tissue damage 13.2.2 Anti-parietal Cell Antibody
caused by activation of complement. Complexes with appro-
priate size can be localized and activate the complement sys- Anti-parietal Cell Antibody (APCA) targets the α subunit
tem mainly in the blood vessels, kidneys, lung skin and joints. of H+/K+ATPase and binds to the β subunit. This character-
istic affects the transport of H+ and thus regulates the secre-
13.1.2.4 M arkers of Type IV Hypersensitivity tion of gastric parietal cells. A variety of laboratory methods
Diseases can be used to detect circulating serum APCA: ELISA,
Type IV hypersensitivity is a cellular immune response which is the most frequently used method, fluorescence
induced by an antigen. After sensitized T cells bind to the immunoassay (FIA), and IIF. The reference interval of IIF
corresponding antigen, hypersensitivity inflammation is and ELISA for Healthy Persons is weakly positive and neg-
characterized by mononuclear cell infiltration and tissue ative, respectively.
damage. Independent of antibodies and complement, it is APCA is a biomarker for autoimmune gastritis, which is
primarily immune damage mediated by effector T cells, elevated in autoimmune atrophic gastritis (AAG) and PA [2].
phagocytic cells, and their resulting cytokines or cytotoxic The positive ACPA is also found in other autoimmune dis-
mediators. This type of hypersensitivity reaction occurs eases, such as thyroid diseases and familial malignant
slowly, usually inflammatory reaction occurs 48–72 h after anemia.
13 Immune System 169
13.2.3 Anti-smooth Muscle Antibody 1. Thyroglobulin (TG), a target antigen of TGA, is a soluble
iodine glycoprotein synthesized and secreted by thyroid
In 1965, Johnson and colleagues found autoantibodies epithelial cells. TGA can activate NK cells through inter-
against smooth muscle tissue as target antigen in patients action between Fc receptor and binding antibody, and
with chronic hepatitis: anti-smooth muscle antibodies attack target cells, destructing thyroid cells, thus causing
(ASMA). Enzyme immunoassay can be used to detect anti- various thyroid diseases.
smooth muscle antibodies, but the most commonly used 2
. Thyroperoxidase (TPO) is a key agent during the thyroid
method is IIF using smooth muscle as an antigen sheet. The hormone synthesis. TMA can directly combine with TPO
reference interval of ELISA and IIF for healthy people is to inhibit the synthesis of thyroid hormones.
negative.
ASMA mainly appeared in the type I autoimmune hepati- In addition to detecting TMA and TPO by IIF and ELISA,
tis (AIH), the most common type is IgG, characterized by both can be detected by radioimmunoprecipitation (RIP)
high titer, specific target antigen for F-actin. ASMA is usu- involving I. Current sensitive luminescence method for the
125
ally characterized by low titer in other liver diseases and determination of anti-thyroid antibody. The reference inter-
non-liver diseases, most of which are IgM type, and the spe- vals for healthy persons of TPO, TGA, and TMA are
cific target antigen is non-actin. 0–34 IU/mL (luminescence method), <115 IU/mL (lumines-
cence method), and associativity <15% (RIA) or negative
(ELISA), respectively.
13.2.4 Anti-liver/Kidney Microsome Antibody TPOs are commonly performed in the work-up of
Hashimoto thyroiditis and Graves’ disease [3]. Chronic lym-
Anti-liver/Kidney Microsome (ALKM) can react with renal phocytic thyroiditis (CLT) and HT are positive for TPO and/
proximal tubule and liver cell. It can be divided into ALKM- or TGA. It can be seen that TGA and TPO have a higher rate
1, ALKM-2, and ALKM-3. Its target antigens are LKM-1 of detection in diffuse hyperthyroidism. What is more, the
(cytochrome P450IID6), LKM-2, LKM-3, and hepatic levels of TGA and TMA in SLE, RA, thyroid cancer, AIH,
microsomes. The common methods for ALKM detection are and other patients were increased to varying degrees.
IIF, ELISA, and western blotting. The reference interval for
healthy persons is negative.
Anti-liver/kidney microsome antibody is an autoanti- 13.2.7 Anti-glutamic Acid Decarboxylase
body, which is a serological indicator for the diagnosis of Antibody
type II AIH.
The target antigen of Anti-glutamic Acid Decarboxylase
Antibody (GADA) is the key point to induce type 1 diabetes
13.2.5 Anti-pituitary Antibody mellitus (T1DM), glutamate decarboxylase, which can
inhibit the biosynthesis of neurotransmitter γ-aminobutyric
Anti-pituitary Antibody (APA) was first discovered in people acid. ELISA and RIA are used to detect GADA in a clinic.
who had autoimmune thyroiditis several years ago. ELISA The reference interval of ELISA for healthy people is nega-
and IIF are in common used to detect APA. The reference tive. Previous studies have indicated that GADA is a good
interval for healthy people is negative. immune antibody for T1DM [4].
APA detection is an auxiliary diagnostic index of endo-
crine system autoimmune diseases, and its positive results
are found in idiopathic hypophyseal hypofunction syndrome 13.2.8 Anti-islet Cell Antibody
or autoimmune polyendocrine gland syndrome.
Anti-islet cell antibodies (ICAs) were first discovered in
T1DM by indirect immunofluorescence. IIF is widely used
13.2.6 Anti-thyroid Antibody as a basic method for ICA detection because of its high posi-
tive detection rate. The reference interval of IIF is negative.
Anti-thyroid antibodies include anti-thyroid globulin anti- ICA is one of the important serological biomarkers of
body (TGA), and anti-thyroid microsomal antibody (TMA). T1DM, which helps to distinguish T1DM from T2DM. It is
These antibodies are organ-specific and species-specific. often detected in the early stage of T1DM, and the detection
Autoimmune thyroid diseases are a group of utterly common rate is up to 60–70%. In addition, ICA has potential thera-
thyroid diseases, containing Hashimoto’s thyroiditis, Graves’ peutic effects in predicting T1DM and judging the prognosis
disease, and so on. of the disease.
170 X. Guan
13.2.9 Anti-insulin Antibody sue components. The presence of autoantibodies does not
always indicate disease. For example, about 5% of the nor-
Anti-insulin Antibody (IAA) can bind insulin to form a com- mal population is rheumatoid factor positive. The elderly
plex that inactivates insulin, which is one of the main reasons population may be more likely to be rheumatoid factor posi-
for insulin resistance in diabetic patients [5]. Anti-insulin tive due to long-term stimulation of a variety of foreign anti-
antibodies have two major clinical implications. (1) gens than the general population. Autoantibodies can be
Treatment of insulin-dependent diabetes mellitus (IDDM): organ-specific that people have mentioned before, such as
Detection of anti-insulin antibodies can guide the dosage of TGA, APCA, and IAA. They can also be systemic, which
insulin. (2) Judging the prognosis of IDDM: The high titer of means that they are not organ-specific and are related to a
insulin antibody indicates that the patient is not islet failure. variety of systemic autoimmune diseases.
On the contrary, it shows insulin failure and poor prognosis.
13.3.1 Anti-mitochondrial Antibody

13.2.10 Anti-adrenocortical Antibody
Anti-mitochondrial Antibody (AMA) targeting mitochon-
Anti-adrenocortical Antibody (AACA) targeting glycolipids drial inner membrane is divided into nine mitochondrial anti-
in adrenocortical cell microsomes can be detected by com- gen/antibody (M1–M9). M2, M4, M7, M8, and M9 are
plement binding assay, indirect hemagglutination assay, and associated with PBC. ELISA, Immunoblotting (IBT), and
IIF. Among them, complement binding assay (C.F.T) and IIF have been extensively applied to the detection of
indirect hemagglutination assay were applied early, and IIF AMA. The reference interval of ELISA, IBT, or IIF is
is the most important test of the current application. negative.
Anti-adrenocortical antibodies are mainly used for the AMA is a specific biomarker for the diagnosis of PBC
diagnosis and differential diagnosis of idiopathic Addison’s that is a chronic cholestatic liver disease (CLD). The eleva-
disease. AACA in patients with this disease (60–70%) is tion of AMA in chronic graft-versus-host disease (GVHD) is
positive. instructive.
13.2.11 Anti-glomerular Basement 13.3.2 Human Anti-globulin Antibody

Membrane Antibody
The serum or erythrocyte membrane of patients with auto-
Various factors stimulate the glomerular basement mem- immune hemolytic anemia (AIHA) often contains incom-
brane (GBM) and induce the autoantibody—Anti-glomerular plete antibodies that attach to the erythrocyte membrane and
Basement Membrane Antibody (AGBMA) produced by the sensitize red blood cells. When human anti-globulin anti-
body. The latter binds with the glomerular basement mem- bodies bind to these antibodies, the sensitized red blood
brane to form antigen–antibody complex, which damages cells agglutinate. It was found that anti-globulin test was
the glomerulus. The most commonly used method for detect- divided into direct against human globulin test (DAGT,
ing GM antibody is IIF with kidney tissue as antigen. Its Coombs test) and indirect anti-human globulin test (IAGT).
fluorescence features are typical petal-like, spot-like, and The former is commonly used for detecting IgG type incom-
granular staining on the GBM. ELISA using crude GM prod- plete antibodies on the erythrocyte membrane; while the lat-
ucts digested by collagenase as antigen and RIA could also ter detecting whether or not the serum contains incomplete
be used. The reference interval of IIF or indirect hemaggluti- antibodies. The reference intervals of DAGT and IAGT are
nation is negative (or serum titer < 4). negative.
As a powerful basis for the diagnosis of anti-GBM nephri- Antiglobulin antibodies play an iconic role in diagnosing
tis (especially Goodpastures syndrome), AGBMA also exists AIHA, and a positive Coombs test is also found in other
in severe glomerular diseases and acute glomerulonephritis. types of hemolytic diseases and autoimmune diseases.
On the other hand, AGBMA can also play an auxiliary role
in kidney transplantation.
13.3.3 Anti-cold Agglutinin Antibody
13.3 Systemic Autoantibodies Anti-cold agglutinin antibodies, cold-reactive autoantibod-

ies, are most likely to bind to erythrocyte membrane antigens
Normal immune regulation is difficult to maintain under the at 0–4 °C, which can agglutinate red blood cells in vivo,
stimulation of various factors. The body produces antibodies leading to cold agglutinin syndrome (CAS), multiple
named autoantibodies that react with normal or changed tis- myeloma (MM), mycoplasma pneumonia, and other dis-
eases. D–L antibody caused a cold-antibody type autoim- 5. Centromere pattern: The main target antigen is centro-
mune anemia—Paroxysmal cold hemoglobinuria (PCH), mere B antigen, and the related autoantibody is anti-
and cold–hot hemolysis test of it is positive. centromere antibody (ACA).
Generally speaking, the titer of ANA antibody in rheuma-

13.3.4 Anti-nuclear Antibody tism involving SLE, RA, MCTD, scleroderma, and SS is
relatively high. Furthermore, the older the normal person is,
Anti-nuclear antibodies (ANA), also known as anti-nucleic the higher the positive rate is (that of people who are over 60
acid antigen antibodies, are a crowd of autoantibodies pro- years old is 20–25%), but the titer is low. The significance of
duced by nucleic acid, protein, or molecular complexes of ANA positive needs to be comprehensively analyzed in com-
these substances. According to the different properties of bination with clinical data. ANA positive cannot establish a
each molecule in its nucleus, ANA can be distinguished as certain clinical diagnosis, on the contrary, ANA negative
anti-DNA antibody, anti-histone antibody, anti-non-histone cannot completely exclude autoimmune venereal diseases. If
antibody, and anti-nucleolar antibody. Now antinuclear anti- the ANA titer (> 1:1000) is considered as connective tissue
body test is a screening test for autoimmune diseases. It can disease or autoimmune disease, the specific disease should
observe the response of disease treatment, assist in monitor- be considered in combination with medical history, symp-
ing disease activity, and judging prognosis. toms, signs and other laboratory results.
ANA is mostly IgG, but also IgM and IgA, and even IgD
and IgE. ANA can react with nuclei from different sources
without organ-specificity or species-specificity. ANA is most 13.3.5 Anti-extractable Nuclear Antibody
common in serum.
The various tests include IIF, ELISA, nephelometry, and ENA is a general term for extractable nuclear antigens,
immunoblotting. Bead array and chip array are being which can be extracted from the nucleus by saline or phos-
expected to improve automation degree. The reference inter- phate buffer. The antigens of ENA mainly include Sm, RNP,
val of IIF is < 1:10. SSA, SSB, Jo-1, and Scl-70. Anti-ENA Antibodies (Anti-
There are five fluorescence karyotypes of ANA: homoge- ENAs) have high clinical diagnostic value for connective tis-
neous pattern, speckled pattern, membrane pattern, nucleolar sue diseases, such as SLE, MCTD, SS, polymyositis (PM),
pattern, and centromere pattern. and dermatomyositis (DM).
At present, anti-ENA detection is still widely carried out
1. Homogeneous pattern: Homogeneity is mostly caused by in the market with techniques such as immunoblotting (IBT)
anti-DNP antibodies (or Lupus factor) or anti-double- and luminescence immunoassay (LIA) in the 1970s. Now,
stranded DNA (anti-dsDNA) antibodies. High titers were DELISA, an improved ELASA, has better sensitivity and
mainly found in patients with SLE at the active stage, specificity than ELISA. More and more immunological
while low titers were found in patients with RA, chronic detection techniques and methods have been gradually
liver disease, infectious mononucleosis, or drug-induced applied to clinical detection of anti-ENA. For example,
lupus. Multiple Flow Immunoassay (MFI) can realize multiple
2. Speckled pattern: Autoantibodies associated with the simultaneous detections of anti-ENA at one time, but also

speckled pattern are anti-extractable nuclear antibodies actualizes automation and high throughput. Next, we will
(anti-ENA). High titer is common in mixed connective introduce several representative antibodies with clinical
tissue disease (MCTD), but also in SLE, scleroderma, significance.
and SS.
3. Membrane pattern/Peripheral pattern: It is mainly caused 13.3.5.1 Anti-Smith Antibody
by antibodies against dsDNA. High titers are almost The antibody was first found in the blood of a patient named
exclusively found in SLE, especially during the active Smith and named after him. Anti-Smith Antibody (Anti-Sm
stage of SLE, while it is rare in other autoimmune antibody) is one of the specific markers of SLE, but the posi-
diseases. tive rate is low, about 30–40%. Simultaneous detection of
4. Nucleolar pattern: Nucleolar-type-related antibodies are anti-Sm antibodies and other related antibodies can improve
anti-nucleolar specific low molecular weight RNA, such the diagnostic rate of SLE.
as anti-nucleolar antibodies, anti-RNA polymerase-1
antibodies, and anti-U3RNP antibodies. High titers are 13.3.5.2 Anti-SSA/Ro Antibody and Anti-SSB/La
most specific for scleroderma, but they are also found in Antibody
the Reynolds phenomenon and occasionally in systemic Anti-SSA autoantibodies (Anti-Sjögren’s syndrome-related
lupus erythematosus (SLE). antigen A, also called anti-Ro) targeting ribonucleoproteins
172 X. Guan
are linked to SLE, SS/SLE overlap syndrome, subacute cuta- responding autoantibodies. The target antigen of anti-dsDNA
neous lupus erythematosus (SCLE), neonatal lupus, and pri- antibody is DNA double helix structure in nucleus; the target
mary biliary cirrhosis [6]. These two proteins are markers of antigen of anti-ssDNA antibody is ribose and deoxyribose;
SS, but the specificity of anti-SSB antibody is higher than and the target antigen of anti-Z-DNA antibody is left double
that of anti-SSA antibody. helix DNA.
The main methods for laboratory detection of anti-DNA
13.3.5.3 Anti-RNP Antibody antibodies are RIA, IFF, dot immunogold filtration assay
Anti-RNP antibody, autoantibodies against ribonucleopro- (DIGFA), IBT, and ELISA. ELISA combined with quantita-
tein is also called anti-U1-RNP antibody. The presence of a tive detection of anti-dsDNA antibody and anti-ssDNA anti-
significant level of it serves as a possible indicator of mixed body screening is currently recommended. For suspected
connective tissue disease (MCTD) when detected in con- patients, IFF can be used to assist confirmation. The positive
junction with several other factors, which is also detected in rate of anti-dsDNA could be judged by the combined rate of
nearly half of patients of SLE patients. DNA with RIA > 20%, or the titer of IIF > 1:5. Next, we will
introduce the representative antibodies with clinical
13.3.5.4 A nti-topoisomerase Antibody (ATA)/ significance.
Anti-Scl-70 Antibody
Anti-Scl-70 antibody is one of the anti-extractable nuclear 13.3.7.1 Anti-dsDNA Antibody
antigen (ENA) antibody profiles. Its name comes from the High concentrations of anti-dsDNA antibodies are almost
molecular weight of the corresponding antigen is 70 kDa. It exclusively found in SLE with a positive rate of 70–90%,
is considered as a marker antibody for systemic scleroderma and are closely associated with disease activity, especially
(SSc). Natural Scl-70 antigen is a degradation product of with active lupus nephritis. Other autoimmune diseases such
DNA topoisomerase I with a molecular weight of 100 kDa. as RA and MCTD, can also be positive for anti-dsDNA, but
It is not only found in diffuse systemic scleroderma, but also the positive rate is small and most of them are overlapping
in the more limited form of systemic scleroderma named syndromes combined with SLE.
CREST syndrome.
13.3.7.2 Anti-ssDNA Antibody
Although it has the same pathogenicity as the anti-dsDNA
13.3.6 Anti-cardiolipin Antibody antibody, the specificity is poor, the diagnostic value of SLE
is small. It can also be seen in inflammatory diseases, drug-
Anti-cardiolipin Antibody (ACA) is a molecule targeting induced lupus, and chronic active hepatitis.
negatively charged cardiolipin on platelets and endothelial
cell membranes. ACA is one of the components of antiphos-
pholipid antibodies (APA). ELISA is the common method 13.3.8 Anti-histone Antibody
for the quantitative determination of ACA. The reference
interval of ELISA is negative. Histones are an important part of the nucleosome that is the
Many factors are closely related to the production of basic structure of chromatin. Anti-histone antibodies (AHAs)
ACA. are autoantibodies that belong to a part of the antinuclear
antibody family and are specifically targeted at histone pro-
1. Autoimmune diseases such as SLE and scleroderma of tein subunits or histone complexes. ELISA and dot enzyme-
RA. linked immunosorbent assay (DELISA) were used to
2. Viral infection such as adenovirus, rubella virus, varicella determine whether AHA existed. Homogeneous fluores-
virus, and mumps virus. cence in IIF can also indicate the presence of AHAs, chroma-
3. Oral administration of certain drugs, such as chlorproma- tin, or some dsDNA. The reference interval of ELISA or
zine, and phenothiazine. DELISA is negative.
4. A small number of normal people without obvious
AHAs are mostly found in SLE and drug-induced lupus
organic diseases, especially the elderly. (DIL) patients. In patients with lupus induced by penicilla-
mine, isoniazid, or methyldopa, almost 100% of the AHAs
were positive. AHAs can also be detected in patients with
13.3.7 Anti-DNA Antibody RA. In 70% of patients with Sjogren’s Syndrome (SS) and
systemic sclerosis, AHAs are present, and about 76% of
Anti-DNA antibodies contain double-stranded DNA patients with primary biliary cirrhosis (PBC) are positive for
(dsDNA), single-stranded DNA (ssDNA), and Z-DNA cor- AHAs in non-rheumatic autoimmune diseases.
13.3.9 Anti-centromere Antibody • Tuberculosis (10–20)

• Infective endocarditis (15–20)
The centromeric antigen consists of three kinds of centro- • Healthy individuals (5 increasing to 20 over the age of 65
meric proteins (Cen P), including Cen P-A (17 kDa), Cen years)
P-B (80 kDa), and Cen P-C (140 kDa), among which Cen
P-B is the main component of the target antigen of anti-
centromeric antibody. Anti-centromeric antibodies are the 13.3.12 Anti-citrullinated Peptide Antibody
marker antibodies of the limited type of systemic sclerosis. (Anti-CCP Antibody)
Anti-CCP antibody spontaneously came from IgG-type B

13.3.10 A
nti-p62 Antibody (AP62A)/ lymphocyte in RA, mainly IgG type. In the following years,
Anti-sp100 Antibody/Anti- researchers developed several methods for detecting Anti-
glycoprotein-210 Antibody (AGPA, CCP antibody, employing mutated citrulline protein vimen-
Anti-gp210, Anti-nup210, tin, serine-derived peptides, and viral citrulline peptides.
Anti-np210)
The autoantigen of anti-p62 antibody closely related to pri- 13.3.13 Anti-glucose-6-Phosphate Isomerase
mary biliary cirrhosis (PBC) is the nucleoporin 62 kDa pro- (Anti-G6PI)
tein. Anti-glycoprotein-210 antibodies are directed at gp210
and are found within PBC patients in high frequency. G6PI is a multifunctional cytoplasmic enzyme, which mainly
exists in articular cavity. It not only plays a necessary role in
glycolysis and gluconeogenesis, but also activates cells and
13.3.11 Rheumatoid Factor growth factors. G6PI in serum and joint fluid is usually
detected by sandwich ELISA. It had been shown that anti-
Rheumatoid Factor (RF) is an autoantibody targeting dena- G6PI antibodies were usually positive in serum of inflamma-
tured IgG Fc fragment. Rose and colleagues initially found it tory arthritis patients, including RA, crystal-induced arthritis,
in the serum of patients with RA in 1984. RF is mainly IgM seronegative spondyloarthropathies, traumatic arthritis, and
class autoantibodies, but there are IgG class, IgA class, IgD other inflammatory arthritis.
class, and IgE class. There are many methods to detect RF,
such as latex agglutination test and ELISA. The latex method
mainly detects IgM RF, while the ELISA method can be 13.3.14 Antineutrophil Cytoplasmic
used to determine different Ig RF. The reference interval of Antibody
ELISA is <20.0 U/mL (quantitative analysis).
Detection of RF is of great significance in the diagnosis, Antineutrophil cytoplasmic antibodies (ANCAs) targeting
classification, and observation of curative effect of rheuma- the components of neutrophil plasma antigens are classified
toid arthritis. The detection rate of RF in patients with rheu- into two types: cytoplasmic ANCA (c-ANCA) and perikary-
matoid arthritis is very high. The titer of RF is positively otic ANCA (p-ANCA). The target antigen of c-ANCA is
correlated with clinical manifestations, that is, the titer of RF protease-3 (PR3), and the corresponding antigen of p-ANCA
increases with the aggravation of symptoms. In addition, is mainly myeloperoxidase (MPO). ANCA was initially
50% of patients with SLE are RF positive, with varying detected by IIF for screening test, and now it is also com-
degrees of positivity in other desmosis such as Sjogren’s syn- monly detected by immunochemical analysis such as ELISA
drome (SS), scleroderma, chronic active hepatitis, and in the for confirmation experiment, using purified specific neutro-
elderly. Disease associations of rheumatoid factor are shown phil cytoplasm as antigen. The reference interval for ELISA
(Prevalence) [7]: or IIF is negative.
ANCA has the strongest correlation with immune vascu-
• RA (60–70) litis with granuloma and polyvasculitis (GPA) and micro-
• SS (75–85) scopic polyvasculitis (MPA) [8]. There is a special correlation
• Systemic sclerosis (20–30) between c-ANCA and Wegener granulomatosis (WG)
• SLE (15–30) characterized by granulomatous necrosis of arterioles,

• Infectious mononucleosis (5–10) venules and capillaries, and the positive detection rate is as
• Hepatitis (15–40) high as 90%. The positive rate of ANCA increases in SLE,
• Juvenile idiopathic arthritis (10–12) polyarteritis nodosa, autoimmune liver disease, chronic
• Leprosy (10–50) infection, or acute progressive glomerulonephritis.
174 X. Guan
13.3.15 Rh Antibody should be detected regularly before transplantation to deter-

mine the level of antibodies and the characteristics of anti-
Landsteiner and Wiener examined human red blood cells bodies. Its main clinical significance is: Recipients with
with antibodies produced by rabbits immunized with rhesus positive HLA antibodies must be cross matched before trans-
monkeys’ red blood cells and Rh blood group in 1940, the plantation. In HLA antibody positive (>10%) recipients,
latter is the most complex and polymorphic erythrocyte graft survival was significantly lower than antibody-negative
blood group system. Rh-negative people are stimulated by (<10%) recipients. In patients with antibodies >50%, the sur-
Rh-positive red blood cells (with Rh antigen) to produce vival rate was significantly lower than that of the antibody
antibodies. Anti-D immunoglobulin can be extracted from level of 11–50%. More than 80% of positive recipients are
their serum to produce Rh antibodies. Saline agglutination generally considered to be contraindications for transplanta-
method and commonly used enzyme medium method. tion. According to the level and nature of HLA antibodies in
Rh-negative individuals can receive D antibodies by the recipient, the transplanted organ can be selected and the
receiving D antigen stimulation through organ transplanta- timing of the transplant operation can be determined.
tion, blood transfusion, pregnancy, and other factors. A
hemolysis reaction can occur when this individual receives D
antigen-positive blood again. Therefore, in the blood transfu- 13.3.18 Prospects for Autoantibodies
sion of patients with anemia whose ABO blood group is
identical, if the anemia phenomenon has not been alleviated Based on the above elaboration and enumeration, autoanti-
or there are no signs of hemolysis, hemolysis after transfu- bodies may be considered as molecular biomarkers of auto-
sion and aggravation of hemolysis on the basis of the original immune diseases from a macro perspective. Especially when
hemolysis should be detected for Rh antibody in patients’ ANAs are mentioned, we cannot help associating them with
serum. If Rh antibody is positive, Rh-negative blood transfu- systemic autoimmune diseases such as SLE, SS, and
sion with ABO blood group is needed. MCTD. So, there is no doubt that the detection of ANAs in
healthy individuals is of clinical value. Table 13.2 summa-
rizes the relationship between ANAs and clinical manifesta-
13.3.16 Anti-erythrocyte Antibody tions of SLE.
In recent years, with the development of molecular bio-
In 1945, British immunologist Coombs and colleagues technology and gene technology, many novel biomarkers
established the Coombs test, which can detect erythrocyte related to organ-specific autoimmune disease diagnosis have
surface antibodies. And it is usually used to detect incom- emerged. Molecular markers, the new direction of the pre-
plete anti-erythrocyte antibodies. Most of the incomplete cise development of immunomedicine, will exert irreplace-
antibodies are IgG monoclonal antibodies of 7S. Although able functions in the future development of autoimmune
they are small and can bind firmly to the corresponding anti- diseases.
gens, they do not show visible reactions under general condi-
tions. The common methods for anti-erythrocyte antibody
detection are direct coombs test and indirect coombs test. 13.4 Immunoglobulin E
There is no incomplete antibody IgG in healthy subjects.
Direct Coombs test can check whether the subject’s red Immunoglobulin E (IgE) is a secretory immunoglobulin with
blood cells have been sensitized by incomplete antibodies, a molecular weight greater than IgG and monomeric IgA,
positive can be seen in neonatal hemolysis, hemolytic trans- which is 196,000. Among the five immunoglobulins, IgE has
fusion reaction, and AIHA. Indirect Coombs test can identify the shortest half-life and the highest decomposition rate and
the presence or absence of incomplete antibodies in Rh blood the lowest synthesis rate. So, the serum content is the lowest,
group and serum. Positive can be found in blood transfusion, only 0.002% of the total serum Ig.
blood products, pregnancy, organ-transplanted immune
blood group antibodies, and autoimmune blood group anti-
Table 13.2 ANAs related to clinical manifestations of SLE
bodies. What is more, indirect Coombs test can also be used
Clinical manifestations ANAs
for crossmatching.
Skin lesion, Vasculitis, and Anti-SSA/Ro antibody and
purpura anti-SSB/La antibody
Raynaud phenomenon Anti-ssDNA antibody and anti-RNP
13.3.17 Anti-HLA Antibody antibody
Lupus nephritis Anti-Nucleosome antibody (ANuA)
In organ transplantation, such as heart, liver, and kidney Thrombocytopenia Anti-SSA/Ro antibody
transplantation, the presence of HLA antibodies in serum Nervous system injury Anti-RNP antibody
For total IgE measurement, ELISA, together with immu- 13.5 Relevant Activated Cell Markers
noturbidimetry and chemiluminescence immunoassay are
commonly used clinically. The reference interval of total IgE 13.5.1 Mast Cells
is Male: (631 ± 128) U/mL, (31–535) ng/mL, Female:
(337 ± 128) U/mL, (31–2000) ng/mL. Mast cells are multi-functional cells which is widely distrib-
The detection of specific IgE in serum is determined by uted in tissues and secretes multiple mediators. MC is a pri-
radioallergen adsorption test (RAST), immunoblot assay, mary effector cell of immediate allergic reactions, and its
and fluorescence immunoassay which are used clinically, activation directly affects the recruitment and activation of
as well as intradermal test; picking test and inhalation eosinophils and neutrophils. After the activation of MC, the
allergen screening test (Phadiatop). Intradermal test with secreted media mainly include histamine, tryptase, chymase,
negative results means no change in the skin, no swelling denatured protein degrading enzyme, bradykinin, platelet-
around the skin, or red halo around the skin, diameter activating factor and prostaglandin D2 [10].
<1 cm, patients without conscious symptoms; positive: Histamine acts on the nasopharynx, which causes nasal
local skin hill uplift, and red halo diameter more than congestion, runny nose, and bronchial asthma. If the release
1 cm, or around pseudopodia, local itching. Skin prick test dose of histamine is large which affecting systemic tissues
with negative results means no wind mass reaction; (+): and capillaries, blood pressure may decrease due to a reduc-
wind mass reaction is 1/3 of the positive control; (++): tion in effective blood volume. Thus, anaphylactic shock
wind mass reaction is 2/3 of the positive control; (++): may occur.
wind mass reaction is the same as the positive control;
(+++): wind mass reaction is greater than the positive con-
trol. ELISA method with positive means leukocyte injury 13.5.2 Eosinophil Activation Marker
index 0.2–0.9 and that with negative results means leuko-
cyte injury index: below 0.01. Eosinophils (Eos) are important secondary effector cells in
Increased total IgE in serum is common in allergic asthma, allergic diseases. The released cellular components mainly
allergic rhinitis, idiopathic rash, eczema, drug-induced inter- contain eosinophil cationic protein (ECP), eosinophil pri-
stitial pneumonia, bronchopulmonary aspergillosis, parasitic mary basic protein (MBP), eosinophil-derived neurotoxin
infection, thymic dysplasia, high IgE syndrome, soft tissue (EDN), and eosinophil peroxidase (EPO). They were ele-
Eosinophilic granuloma, acute and chronic hepatitis, and IgE vated in sputum, bronchoalveolar lavage fluid, serum, and
multiple myeloma [9]. urine in asthma patients.
Anaphylactic shock caused by drugs such as penicillin, In the serum of patients with allergic dermatitis, anaphy-
Allergic reactions to penicillin include immediate and lactic shock, allergic purpura, food allergies and other aller-
delayed allergic reactions. The former manifests as anaphy- gic diseases, ECP, MBP, EDN, EPO can be detected, and the
lactic shock, urticaria, angioedema, etc. degree of elevation is related to the severity of the disease.
The use of animal immune serum such as tetanus toxin Evaluation is beneficial to control the symptoms of allergic
and diphtheria antitoxin caused serum anaphylactic shock. diseases and to make reasonable treatment.
Because animal immune sera are heterologous proteins in
the human body, a small number of people with allergies can
produce specific IgE antibodies. When a serum product of 13.5.3 The Activation Markers of Basophil
the same source is injected again, a type I hypersensitivity and Neutrophil
reaction can be induced, and symptoms similar to those of
the drug anaphylactic shock can occur. The level of leukotrienes increased significantly in both the
In allergic rhinitis, allergic asthma, allergic gastroenteritis fast-acting and the late-onset phases in allergen-induced
and other diseases, atopic individuals produced inflamma- nasal allergic reactions.
tory mediators after exposure to allergens, which are mainly 2D7 is an IgG-type monoclonal antibody and its ligand is
mediated by IgG and have various noninfectious inflamma- a component 2D7 antigen of human basophil secreting gran-
tory diseases involving immune active cells and cytokines. ules. 2D7 is currently considered to be a specific marker for
Skin allergic reactions are caused by food. Food-specific IgE identifying basophil activation in tissues.
testing in children with specific dermatitis found that nearly BB-1 is a monoclonal antibody of IgG2a. Basophils
one-third of children had IgE-mediated food allergic reac- release the BB-1 antigen under the stimulation of anti-IgE
tions. Allergic airway inflammation is a harmful immune and calcium ionophore.
response mediated by IgE, and serum IgE levels are closely In patients with asthma, basophils in the blood express
related to the severity of asthma. high levels of CD69, which is expressed more in bronchoal-
176 X. Guan
veolar lavage fluid. CD69 is primarily produced by IL-3 50–100 U/mL; complement C3: (1.12 ± 0.55) mg/L; and
stimulation and acts as a marker for IL-3 activation of complement C4: (0.553 ± 0.109) mg/L.
basophils. The clinical observation of changes in complement values
In IgE-mediated food allergy, human basophils express is of great significance for the diagnosis, etiology, and prog-
LTD4 and CD63 after activation. MPO is a member of the nosis of this type of disease. Multiple sclerosis (MS) and
mammalian peroxidase family and the MPO gene of human neuromyelitis optica spectrum disorder (NMOSD) are auto-
is located on chromosome 17. immune diseases of the central nervous system. Four com-
plement proteins (C1inh, C1s, C5, and FH) were higher in
NMOSD compared to MS or controls.
13.5.4 T Lymphocyte Immunophenotype
T lymphocyte is a heterogeneous cell population, which can 13.7 Circulation Immune Complex
be categorized into different categories and subgroups owing
to its dissimilar cellular or molecular biological characteris- When the ratio of specific antigen to specific antibody is
tics. For example, mature T cells can be split into CD4+ or appropriate, Circulation Immune Complex (CIC) with larger
CD+ cells. Type IV hypersensitivity is mainly a type of cel- molecular weight is produced. The formation of circulating
lular immune response mediated by CD4+ Th1 cells and immune complexes belongs to the normal immune response
CD8+ effecting CTL cells. CD4+ Th1 cells bind to corre- of the organism, which is conducive to the elimination of
sponding antigens and release a variety of cytokines. CTL harmful antigens. However, when the amount of antigen is
cells mediate cytotoxicity and directly slay target cells. slightly more than that of antibody, a medium-sized (8.8S–
Hence, the recognition of CD3+CD4+ labeled helper T cells 19S) soluble immune complex can be formed, which is not
(Th) and CD3+CD8+ labeled cytotoxic T cells (CTL) can lay easy to be swallowed and cleared by phagocytes, nor can it
the clinical immunological foundation for the diagnosis of be filtered out by glomeruli and circulated in blood and other
type IV hypersensitivity and disease development. body fluids for a long time. When the permeability of blood
CD3+CD4+ Th and CD3+CD8+ CTL are involved in vessel wall increases, this kind of IC is easily deposited on
delayed-type hypersensitivity, transplant rejection, and path- the capillary wall or embedded on the glomerular basement
ological processes of certain autoimmune diseases. The phe- membrane. It can activate complements, infiltrate neutro-
notypic detection of Th and CTL by flow cytometry showed phils, and activate platelets, thus causing immune damage
higher than normal value which has certain auxiliary diag- and leading to the occurrence of immune complex diseases
nostic significance for the occurrence of type IV hypersensi- [11].
tivity, acute GVHD, and transplant rejection. Antigen-specific immune complex such as hepatitis virus
HBsAg-CIC can be detected by ELISA and trypsin dissocia-
tion; thyroglobulin TG-CIC can be detected by ELISA; and
13.6 Complement System renal syndrome Ag/IgE and Ag/IgD-CIC can be detected by
capture ELISA. Non-antigen-specific immune complex can
The complement system consists of 22 plasma proteins and be measured by polyethylene glycol (PEG) method, C1q
some membrane proteins closely related to plasma proteins. solid phase method, anti-C3-CIC-ELISA, raji cell test, and
These membrane proteins are either receptors of plasma sys- monoclonal rheumatoid factor (mRF) gel diffusion test.
tem activating peptides or participating in the regulation of Systemic lupus erythematosus, rheumatoid arthritis, par-
complement activation. The electrophoretic mobility of most tial glomerulonephritis, and vasculitis are immune complex
complement components is beta globulin, while a few are disease (ICD). The effect has a certain meaning. When tested
alpha globulin and gamma globulin with molecular weight by C1q binding method, the positive rate of CIC in systemic
ranging from 25 to 390 kDa. lupus erythematosus was 75–80%, rheumatoid arthritis was
Detection of single complement components: Indicators 80–85%, and vasculitis was 73–78%.
commonly used for detection of single complement compo- The human body also has a small amount of CIC under
nents include C3, C4, C1q, factor B, and C1 esterase inhibi- healthy conditions. The detection rate of CIC will also
tors. The determination methods include immunohemolysis, increase when the body has malignant tumors but there are
immunochemistry, and automated immunoscattering turbi- immunological damage symptoms of type III hypersensitiv-
dimetry. Serum total complement hemolytic activity (CH50): ity reaction that which is called clinical occult ICD.
when sheep red blood cells are combined with correspond- Evidence for the diagnosis of immune complexes is as
ing antibodies, sensitized sheep red blood cells are formed. follows: (1) Localized IC deposition; (2) CIC levels are sig-
The reference intervals for healthy persons are CH50: nificantly elevated; (3) Clear antigenic properties in IC. The
third criterion is sometimes difficult to detect, but at least the human bone marrow-associated protein complex (MRP8\14)
first two are required. In addition, circulating immune com- concentration is significantly increased, and CRP is also ele-
plexes also increase in serum diseases. vated [12].
13.8 Other Markers 13.9 The Relevant Progress
13.8.1 Cytokines Based on the rapid development of immunology and molec-

ular biology technology, detection of specific nucleic acids,
When foreign antigens enter the body, effector Th1 cells rec- antigens, and antibodies in patients with immune system dis-
ognize and activate, releasing a series of cytokines, IL-2, eases has profound significance in early diagnosis of dis-
TNF, and IFN-γ, for example. These factors activate mono- eases, and these specific molecules often have the potential
cytes and macrophages, phagocytize and remove antigens, to become molecular markers related to certain immune sys-
release lysosomal enzymes and other inflammatory media- tem diseases. The molecular markers introduced in this sec-
tors, and mediate tissue damage. They can also induce adhe- tion are closely related to the accurate and rapid diagnosis of
sion and the aggregation of monocytes–macrophages in autoimmune diseases, hypersensitivity and immunodefi-
blood to the antigen site, and directly produce cytotoxic ciency diseases, the evaluation of therapeutic effects, and the
effects on target cells and surrounding tissue cells, causing monitoring of treatment, which can be used as new targets
tissue damage. At present, the detection types of for disease treatment.
transplantation-related cytokines mainly include IL-26,
IL-22, and its receptors, TNF-2α, MUC-2, MUC-4, IFN-2γ,
FGF-21, and FGFR et al. And the detection methods include References
immunospot assay, ELISA, RIA, and IBT.
When transplant rejection occurs after liver transplanta- 1. Toh BH. Pathophysiology and laboratory diagnosis of pernicious
anemia. Immunol Res. 2017;65:326–30.
tion, the expression of TNF-α will be increased in trans- 2. Chobot A, Krzywicka A, Rusak E, et al. Anti-parietal cell antibod-
planted liver mononuclear macrophages. The expression of ies—diagnostic significance. Adv Med Sci. 2016;61:175–9.
mucin MUC-2, MUC-4, interferon (IFN-γ), and TNF-2α 3. Loh TP, Tee JC, Tee NW, et al. Association between thyroid func-
was increased when early rejection occurred after intestinal tion tests and anti-thyroid peroxidase (TPO) antibodies in preg-
nancy. Endocrine. 2016;53:865–7.
transplantation. Increased transplantation of renal fiber 4. Achenbach P, Warncke K, Reiter J, et al. Stratification of type 1
growth factor (FGF-21) and fibroblast growth factor receptor diabetes risk on the basis of islet autoantibody characteristics.
(FGFR) suggests chronic rejection. In acute rejection of kid- Diabetes. 2004;53:384–92.
ney transplant patients, transplanted kidneys showed 5. Singla R, Homko C, Schey R, et al. Diabetes-related autoantibodies
in diabetic gastroparesis. Dig Dis Sci. 2015;60:1733–7.
increased expression of IL-22 receptors. 6. Franceschini F, Cavazzana I. Anti-Ro/SSA and La/SSB antibodies.
Autoimmunity. 2005;38:55–63.
7. Aggarwal A. Role of autoantibody testing. Best Pract Res
13.8.2 Proteins and Enzymes Rheumatol. 2014;28:907–20.
8. Weiner M, Segelmark M. The clinical presentation and therapy of
diseases related to Anti-neutrophil cytoplasmic antibodies (ANCA).
There are many proteins and enzymes for detecting organ Autoimmun Rev. 2016;15:978–82.
transplant rejection, but C-reactive protein and heat shock 9. Rath N, Raje N, Rosenwasser L. Immunoglobulin E as a biomarker
protein are widely used. C-reactive protein (CRP) was disin asthma. Immunol Allergy Clin North Am. 2018;38:587–97.
10. Gonzalez D, Alvarez Twose I. Mast cells as key players in allergy and
covered by Tillet and Francis in 1930. CRP is a part of non- inflammation. J Investig Allergol Clin Immunol. 2018;28:365–78.
specific immune mechanism, which combines 11. Wang T, Zhang M, Cheng J, et al. Establishment and evaluation of a
C-polysaccharide and phosphocholine on the cell membrane general dissociation technique for antibodies in circulating immune
when Ca2+ exists. The concentration of CRP in serum of complexes. Clin Exp Med. 2018;19:65–75.
12. Kollu V, Mott SL, Khan R, et al. C-reactive protein monitoring pre-
healthy people was very low (<5 mg/L). Serum CRP is usu- dicts neutropenic fever following autologous hematopoietic stem
ally measured by immunoturbidimetry. When rejection cell transplantation for multiple myeloma. Cureus. 2018;10:e2945.
occurs in small intestine or liver transplant patients, serum
Lipoproteins
14
14.1 Overview mally transported in the aqueous phase of plasma. The cores
of CM and VLDL are dominated by TG, while the cores of
14.1.1 Basic Concepts LDL and HDL are dominated by CE.
Using electrophoresis, the plasma lipoprotein can be
Blood lipids, also known as plasma lipids, only account for a divided into chylomicron (CM), β-lipoprotein, pre-β-
portion of the organic constituents of plasma; however, they lipoprotein, and α-lipoprotein. Using ultracentrifugation, the
are very metabolically active. These include cholesterol, tri- plasma lipoproteins can be divided into very low-density
glycerides, phospholipids, and free fatty acids. Because lip- lipoprotein (VLDL), intermediate-density lipoprotein (IDL),
ids are poorly soluble in water, they all bind apolipoproteins low-density lipoprotein (LDL), and high-density lipoprotein
that have high solubility to form compounds to be trans- (HDL).
ported in the circulation. The total volume of blood lipids is
4.0–7.0 g/L. Degradation and synthesis maintain a dynamic
equilibrium. The change in content of each blood lipid con- 14.1.2 Functions and Metabolic Pathways
stituent is also stable within a certain range. Blood lipid of Various Lipoproteins
determination may reflect the lipid metabolism of organisms.
Lipids play a significant role in maintaining normal psy- 14.1.2.1 Chylomicrons Transport Exogenous
chological activities, notably energy metabolism and mem- Triglycerides and Cholesterol
brane formation. Blood lipid abnormalities are among the The CM metabolic pathway is also known as the exogenous
independent risk factors responsible for ischemic cardiovas- transport pathway or the exogenous lipid metabolic pathway.
cular diseases. Blood lipid detection provides a quantitative After dietary fat is digested, mucous membrane cells of the
basis for diagnosing blood lipid abnormalities, which play a small intestines synthesize triglycerides with ingested
vital role in tertiary prevention of metabolic diseases. medium and long-chain fatty acids. Triglycerides, synthe-
Total cholesterol (TC), triglycerides (TG), phospholipids sized and assimilated phospholipids, and cholesterol,
(PL), free fatty acids (FFA) are collectively known as blood ApoB48, ApoAI, ApoAII, and ApoAVI are assembled into
lipids. Lipids have poor water solubility and are difficult to new CM, that enters the blood via lymphatic channels and
transport. Therefore, lipids in peripheral circulation exist in obtain ApoC and ApoE. Some ApoAI, ApoAII, and ApoAIV
the form of compounds. are transferred to HDL, thereby forming new CM. ApoCII
Either exogenous or endogenous lipids bind with proteins activates surface lipoprotein lipase (LPL) of capillary endo-
to form compounds with high water solubility, known as thelial cells of tissues such as skeletal muscle, myocardium,
lipoproteins (LP). Various lipoproteins have similar basic and fat, causing TG phospholipid in CM to hydrolyze gradu-
structures. Lipoproteins are generally spherical compounds ally, thereby producing glycerol, fatty acid, and lysolecithin.
covered by apolipoproteins, phospholipids, and free choles- A large amount of fatty acids is released, ingested, and uti-
terol monomolecular layers with TG and CE as the internal lized by the myocardium, skeletal muscle, fatty tissue, and
core, thereby guaranteeing that insoluble lipids can be nor- hepatic tissue while the TG core of CM is being hydrolyzed.
The CM particles increasingly become smaller and excess
ApoAI, ApoAII, ApoAIV, ApoC, phospholipid, and choles-
C. Wang (*) · Z. Li terol on the surface leave the CM particles to form new
Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur HDL. Finally, CM transforms into CM remnants rich in CE,
Autonomous Region, Urumqi, Xinjiang,
People’s Republic of China ApoB48, and ApoE, identified and bound by proteins related

180 C. Wang and Z. Li
to the cytomembrane LDL receptor and radically degraded 14.1.3 Lipoprotein Metabolic Disturbances
after being ingested by hepatocytes. The half-life of CM of a
normal individual in plasma is 5–15 min. Therefore, the Because most blood lipid constituents exist in blood in the
plasma of a normal person contains no CM when he or she form of lipoproteins, lipoprotein metabolic disturbances are
fasts for 12–14 h. the leading cause of abnormal levels of various types of lipo-
proteins and abnormal levels of blood lipids. Depending on
14.1.2.2 V ery Low-Density Lipoproteins the expression levels of lipoproteins in plasma, lipoprotein
Transport Endogenous Triglycerides metabolic disturbances may present as hyperlipoproteinemia
VLDL is an important means of transporting endogenous or hypolipoproteinemia. The former is more common and
TG. The plasma metabolite LDL is an important means for also known as hyperlipidemia (HLP). This means that one or
transporting endogenous cholesterol. VLDL and LDL meta- several lipoproteins in plasma has an excessively high con-
bolic pathways are also known as endogenous lipid trans- centration, commonly found in hypercholesteremia and
porting pathways or endogenous lipid metabolic pathways. hypertriglyceridemia. Hyperlipidemia can be divided into
Hepatocytes synthesize TG with intermediate products of two categories, primary and secondary. Hyperlipidemia aris-
glucose catabolism or synthesize TG using food-derived ing from genetic defects is primary, e.g., familial hypercho-
fatty acids as well as fatty acids in fatty acid depots. TG, lesteremia. Hyperlipoproteinemia arising from diabetes and/
ApoB100, ApoE, phospholipid, and cholesterol are assem- or kidney diseases is secondary.
bled into VLDL. Furthermore, mucosal cells of small intes-
tines may also synthesize small amounts of VLDL. After 14.1.3.1 Hyperlipoproteinemia
VLDL enters the bloodstream, ApoC is obtained from Hyperlipoproteinemia is a disease characterized by elevated
HDL. ApoCII activates LPL on the surface of capillary levels of cholesterol and/or triglycerides in plasma arising
endothelial cells of extrahepatic tissue. Under the action of from various factors, notably, elevated levels of one or sev-
LPL, TG in VLDL is hydrolyzed to release fatty acids and eral lipoproteins in plasma. It can be divided into primary
glycerol to be used by extrahepatic tissue. Meanwhile, ApoC and secondary hyperlipoproteinemia. Primary hyperlipopro-
phospholipids and cholesterol on the VLDL surface transfers teinemia can be subdivided into familial and sporadic. The
to HDL and the HDL cholesteryl ester transfers to onset of the former may be influenced by genetic factors and
VLDL. This process repeats continuously. The half-life of the latter is subject to neither genetic nor secondary factors.
VLDL in blood is 6–12 h.
Primary Hyperlipoproteinemia
14.1.2.3 Low-Density Lipoprotein Transports Primary hyperlipoproteinemia is a genetic plasma lipid met-
Endogenous Cholesterol abolic disturbance chiefly characterized by elevated levels of
The liver is the principal organ for ingesting and degrading plasma lipids. The Fredrickson typing of primary hyperlipo-
LDL. About 50% of LDL is degraded in the liver. Tissues proteinemia was introduced in 1967. The major basis of
such as the adrenal cortex, ovary, and testis also have strong determination is lipoprotein electrophoresis. The typing
capacities to ingest and degrade LDL. Plasma LDL degrada- method ignores molecular defects, an important cause of
tion can either be achieved via the LDL receptor or the abnormal lipid metabolism and does not consider
mononuclear–phagocyte system. Its half-life is 2–4 days. HDL. However, it is well known because the typing method
is simple. It was amended and supplemented by the WHO in
14.1.2.4 High-Density Lipoprotein Transports 1970. Based on the appearance of fasting serum, biochemi-
Cholesterol in Reverse Fashion cal detection indexes TG and TC, and the lipoprotein electro-
HDL is primarily synthesized in the liver and some HDL phoresis diagram, the primary hyperlipoproteinemia is
may be synthesized in the small intestine. During CM and divided into six types. At present, the existing known causes
VLDL metabolism, surface ApoAI, ApoAII, ApoAIV, ApoC, of primary hyperlipoproteinemia include three classes, lipo-
phospholipid, and cholesterol may also form. HDL can be protein metabolism-related enzymes, apolipoprotein, and
divided into HDL1, HDL2, and HDL3 by density. HDL only lipoprotein receptor genetic mutations or defects. LPL is an
exists in the plasma of a person who ingests food with high important enzyme in the hydrolysis of triglycerides during
cholesterol. The plasma of a normal individual primarily the lipoprotein metabolic process. Its genetic mutation or
contains HDL2 and HDL3. The metabolic process of HDL is defects make LPL synthesis impossible or cause declines in
actually the reverse transport process of cholesterol. During activity, thereby causing levels of triglyceride in serum to
the process, cholesterol in extrahepatic tissue is transported increase significantly, resulting in Type I hyperlipoprotein-
to the liver via the circulation and is discharged after being emia. ApoCII plays an important role in activating
transformed into bile acid. Some cholesterol may also be dis- LPL. Deficient ApoCII leads to inactivation of LPL and Type
charged into the intestinal lumen together with bile. I hyperlipoproteinemia. The decline in LPL activity is related
14 Lipoproteins 181
to Type IV hyperlipoproteinemia. The increased expression Diagnostic Criteria for Hyperlipoproteinemia

of ApoCIII and the increase in LPL activity resulting from Epidemic research results have shown that blood lipids are
ApoCII deficiency are among the important causes of Type V associated with populations, races, regions, ages, genders,
hyperlipoproteinemia. ApoE deficiency is usually closely living styles, habits, labor intensity, educational levels, and
associated with Type III hyperlipoproteinemia. Deficiency of genetic factors. Normal and abnormal blood lipid levels are
apolipoprotein E and B receptors is associated with Type II artificially defined. Currently, there is no definite and uni-
hyperlipoproteinemia. form method for defining the diagnostic criteria for hyper-
lipidemia. To prevent and control atherosis and coronary
Hyperlipoproteinemia heart diseases, an appropriate level of plasma cholesterol
Hyperlipoproteinemia is characterized by excessively high should be kept below 5.17 mmol/L (200 mg/dL). Most
HDL content in serum. Plasma HDL levels over 2.6 mmol/L scholars in China believe that plasma TC > 6.21 mmol/L
are defined as hyperlipoproteinemia. It is generally accepted (240 mg/L) can be defined as hypercholesteremia. Plasma
that HDL plays a role in resisting atherosis; an excessively TG > 2.3 mmol/L (200 mg/dL) can be defined as hypertri-
high concentration of blood indicates a pathological state. glyceridemia. The diagnostic criteria for hyperlipidemia
Hyperlipoproteinemia is also divided into primary and sec- developed in various regions vary due to differences in pop-
ondary types. Primary hyperlipoproteinemia may be caused ulations and detection methods. The conventional classifica-
by cholesteryl ester transfer protein defects or declines in tion method divides the lipoproteinemia into six types
hepatic lipase activity. The causes of secondary hyperlipo- (Table 14.1).
proteinemia include ataxia, excessive alcohol intake, pri-
mary liver cirrhosis, and use of medications for treating 14.1.3.2 Hypolipoproteinemia
hyperlipidemia. Both primary and secondary are closely Compared to hyperlipoproteinemia, hypolipoproteinemia
associated with cholesteryl ester transfer protein and hepatic is less commonly seen in clinical practice. There are four
lipase activity. common types: hypolipoproteinemia with autosomal domi-
nant inheritance; hypolipoproteinemia with the mode of
Secondary Hyperlipoproteinemia inheritance unknown; abetalipoproteinemia with autosomal
Some primary diseases may cause lipid metabolic distur- recessive inheritance; and Tangier disease (hypoalphalipo-
bances during onset, further leading to hyperlipoprotein- proteinemia) with autosomal recessive inheritance.
emia. Hyperlipoproteinemia disappears gradually while the
primary disease is being treated. Therefore, it is called sec- 14.1.3.3 L ipoprotein Metabolic Disturbance
ondary hyperlipoproteinemia. Systemic diseases resulting in and Atherosis
raised levels of blood lipids include diabetes, nephrotic syn- Atherosclerosis (AS) is a chronic progressive disease charac-
drome, and hypothyroidism. Furthermore, such factors as terized by deposition of endarterium lipid and blood constit-
drugs (diuretics), high-cholesterol foods, smoking, and drink- uents, proliferation of smooth muscle cells and collagenous
ing may cause secondary hyperlipoproteinemia. Secondary fibers, necrosis, and calcification. The lipoprotein metabolic
hyperlipoproteinemia is common in clinical practice. Accurate disturbance is dominated by hyperlipoproteinemia that may
identification of the cause is the key to clinical diagnosis. promote formation of atherosis by accelerating deposition of
lipids in the endarterium and development of atheromatous
Clinical Manifestations of Hyperlipoproteinemia plaques.
The clinical manifestations of hyperlipoproteinemia include
two aspects: giant cell tumors of the tendon sheath caused
Table 14.1 Typing of lipoproteinemia
by deposition of lipid within the dermis and atherosis caused
Typing Changes in plasma lipoprotein Changes in blood lipid
by deposition of lipid within the blood vessel endothelium.
I Elevated levels of chylomicrons Triglyceride↑↑↑
Giant cell tumors of the tendon sheath are abnormal soft, Cholesterol↑
yellow, orange, or nodular, plaque-shaped, or brownish red IIa Elevated levels of low-density Cholesterol ↑↑
papular localized raised skin lesions. They are the result of lipoprotein
accumulation of macrophages (foam cells) absorbing lipids IIb Elevated levels of low-density and Cholesterol↑↑
in the corium. Familial hypercholesteremia may present as very low-density lipoproteins Triglyceride↑↑
arcus corneae, also known as arcus senilis. The deposition III Elevated levels of intermediate- Cholesterol↑↑
density lipoprotein (a wide β band Triglyceride↑↑
of large-grained lipoprotein rich in triglycerides in retinal present during electrophoresis)
arterioles may lead to astigmatism. Significant hypertriglyc- IV Elevated levels of very low-density Triglyceride↑↑
eridemia may lead to acute pancreatitis. Severe hypercholes- lipoprotein
teremia, particularly the homozygous type, may present as V Elevated levels of very low-density Triglyceride↑↑↑
migrating arthritis, less commonly seen in clinical practice. lipoprotein and chylomicrons
Factors Leading to Atherosis determinants and finally form OxLDL. OxLDL, following
The gradual deposition of lipids in the endarterium is among oxidization and modification, may promote secretion of
the important factors leading to atherosis. Therefore, lipo- inflammatory factors and adhesion molecules, accelerate the
proteins that can help transport and deposit cholesterol are rate at which the macrophages ingest LDL, cause buildup of
among the factors resulting in atherosis, including lipopro- cholesterol in the macrophages and transform them into
tein remnants, degenerated LDL, Type B LDL, LP(a), and foam cells. Many foam cells derived from macrophages
OxHDL. accumulate below the arterial endothelium and cause athero-
sis. OxLDL may also directly stimulate proliferation of
Lipoprotein Remnants smooth muscle cells by toxic damage to the endothelial cells
Triglyceride rich-lipoprotein (TRL) remnants are generated of the vascular wall or may act on vascular endothelial cells,
by hydrolysis of CM and VLDL via LPL. The products of macrophages, and platelets, thereby leading to release of bio-
further catabolism are remnant-like particles (RLP). RLP is active factors and proliferation of vascular smooth muscle
rich in ApoE. As a ligand, ApoE ingests RLP via macro- cells. For example, endothelial cells produce basic fibroblast
phages (Mø), platelets, vascular endothelial cells, and other cell growth factors, epidermis growth factors, and endothe-
cells that express LDL receptor or remnant receptor, playing lin-1 that accelerate formation of AS. The saccharification
an important role in the removal of plasma CH and and modification of LDL begin with the covalent binding
TG. Nevertheless, the functions of these cells change signifi- between the reducing sugar and the protein amino group.
cantly after they ingest RLP rich in ApoE. RLP promotes the The resulting Schiff base finally produces irreversible
formation of AS primarily by damaging arterial endothelial advanced glycation end-products (AGEs) following a series
cells and degrading their barrier functions. RLP may damage of sequential intramolecular structural rearrangements,
endothelium-dependent vasodilatation responses by sub- dehydration, and redox reactions. Some studies reported that
stances such as acetylcholine and may also damage erythro- AGEs exist in ApoB and lipid of LDL. GlyLDL may be
cyte membranes. Such damage may be caused by the ingested by macrophages via a unique high-energy receptor
independent action of the receptor via the apoprotein and with low affinity and may then transform macrophages into
may be associated with reduced nitric oxide (NO) biological foam cells. The aggregation rate of GlyLDL in macrophages
activity in coronary arteries. RLP contains a large amount of is significantly higher than that of non-glycosylated
lecithin peroxide. These lipid constituents also lead to oxida- LDL. Some scholars have found that the glycosylated LDL
tive damage to the cytomembrane. Such damage can be is more likely to be further oxidized and modified.
inhibited by antioxidants. RLP may strengthen the aggrega-
tion activity of platelets under the action of ADP and colla- Type B LDL
gen induction in whole blood, causing leakage of ADP from LDL is generally divided into Types A and B. Type B is
red blood cells, mediating aggregation of platelets, and small and dense LDL and is one of the high-risk factors of
solely inducing aggregation of platelets on the surface of red occurrence of AS. The mechanism of action of its role in
blood cells. Such a role of RLP may be the result of RLP- triggering AS includes susceptibility to oxidization, low rate
dependent interactions. This may be common in patients of plasma removal, susceptibility to adhesion to the vascular
with hyperlipidemia. wall, and susceptibility to entry into the arterial wall, thereby
leading to an increase in the synthesized thromboxane
Modified LDL (TXA2) in vascular endothelial cells. Endothelial cells and
Modified LDL, also known as degenerated LDL, is the prod- platelets synthesize prostacyclin (PGI2), disrupting PGI2/
uct of chemical modification of the protein components of TXA2 balance, causing aggregation of platelets, and pro-
LDL. LDL experiences changes in both normal morphology moting formation of AS. In vitro experiments have shown
and biological activity after being modified. The existing that large and light LDLs significantly elevate the concentra-
known degenerated LDL versions include AcLDL, OxLDL, tion of calcium ions within vascular smooth muscle cells. As
and GlyLDL. AcLDL is a modified LDL after the the second most important messenger within the cells, cal-
ApoB100lysine residue in LDL is acetylated. AcLDL acti- cium ions participate in several processes involved in AS
vates macrophages and causes macrophages to extract formation.
AcLDL and transform into foam cells under the mediation of
the scavenger receptor, thereby promoting the formation of LP(a)
AS. The natural low-density lipoprotein (LDL) contains a It is generally accepted that high Lipoprotein (LP)(a) is an
large amount of unsaturated fatty acid that is oxidized to trig- independent risk factor for cardiovascular and cerebrovascu-
ger a series of chain reactions of free radicals and generate lar AS diseases. LP(a) is a unique independent plasma lipo-
several types of active aldehyde. These active aldehydes bind protein with high molecular mass, formed by covalent
apolipoprotein ApoB in LDL to generate new antigenic binding between apolipoprotein A and low-density lipopro-
14 Lipoproteins 183
tein components via the disulfide bond. In 1963, Berg as bile acids. Esterified cholesterol is found in blood and
reported that LP(a) is more likely to deposit in the vascular binds with protein to form lipoprotein. CH is a basic compo-
wall, promoting growth of smooth muscle cells, inhibiting nent of human semantic tissue cells. All human tissues,
dissolution of fibrous proteins, and promoting atherosis and except for brain tissue, synthesize CH. Therefore, humans
thrombosis. generally have no lack of CH except in some special cases
Oxidized HDL (OxHDL) is a recently described modified such as congenital β-lipoprotein deficiency disease.
protein capable of leading to atherosis that is a product of Generally, the decline in serum CH has no significant clinical
oxidized and modified natural HDL. Natural HDL is thought manifestations; however, long-term low levels of cholesterol
to be a protective lipoprotein that negatively correlates with influence memory and reaction capacity. Excessive ingestion
cardiovascular disease. However, it loses the ability to resist of cholesterol-rich food, smoking, drinking, and some patho-
AS and gains the ability to cause AS after being oxidized and logical states such as hepatic and renal diseases may cause
modified. The possible pathogenic mechanisms include the high levels of cholesterol and further hypercholesteremia.
following: HDL promotes damage to endothelial cells by This is a lipid metabolic disease seen in clinical practice that
inhibiting the synthesis of nitric oxide within endothelial is considered one of the most important causes and risk fac-
cells and the activity of the nitric oxide synthase. HDL weak- tors for atherosis and coronary heart disease.
ens the process of transfer of cholesterol from vascular
smooth muscle cells. HDL promotes movement of mononu-
clear cells into the space below human blood vessel endothe- 14.2.2 Detection Methods and Reference
lium. They are activated and differentiate into macrophages Values of Healthy People
that ingest lipid to form foam cells. HDL inhibits inverse
transport of cholesterol. HDL loses the ability to inhibit LDL 14.2.2.1 Detection Methods
oxidization and modification. HDL influences blood clotting There are more than 200 methods for determining total
and fibrinolytic activity. cholesterol. They can be divided into five major categories:
Factors Resisting Atherosis HDL is the most common colorimetric method, enzyme analysis method, fluores-
anti-AS lipoprotein that exerts its anti-AS function by par- cent method, gaseous phase, and high-performance liquid
ticipating in reverse transporting of cholesterol, inhibiting chromatography. As the gaseous phase and high-perfor-
LDL oxidization, neutralizing the activity of the ligand of the mance liquid chromatography methods have strict require-
modified LDL and inhibiting the expression of adhesion ments, the most common methods in clinical laboratories
molecules in endothelial cells. are the chemical reagent method and the enzyme method.
The reference method for detecting total cholesterol is the
improved Lieberman-Burchard method. The conventional
14.2 Total Cholesterol method is the holoenzyme method. The recommended
methods include the o-phthaldialdehyde method and the
14.2.1 Sources and Characteristics ferric acid color development method. The isotope dilution
liquid chromatography-tandem mass spectrum method is a
TC refers to the total cholesterol contained in all lipoproteins new method that is now recommended as the confirmation
in the blood. Cholesterols are divided into esterified choles- method.
terol (CE, accounting for 60–70%) and free cholesterol (FC, Reaction principle of Lieberman-Burchard: Serum is
accounting for 30–40%). The ratio of the two types in healthy treated with cholesterol esterase and dissolved in glacial ace-
individuals is constant. Cholesterol, with its scientific name tic acid. Acetic anhydride-concentrated sulfuric acid is added
of 5-Cholesten-3beta-ol, is an unsaturated sterol derived (20:1). The test result may be judged as positive if the reac-
from perhydrocyclopentanophenanthrene. A hydroxy group tion solution experiences a series of changes in color, pale
is bound to C-3 of the steroid nucleus while an alicyclic side purple/pink-blue-green-blackish green.
chain with eight or more carbon atoms is bound to the C-17 Principle of the holoenzyme method: Cholesteryl ester is
site. It is insoluble in water but is freely soluble in solvents decomposed into free cholesterol and fatty acid by choles-
such as ethanol and chloroform. About 1/3 of the cholesterol terol esterase. Using cholesterol oxygenase, free cholesterol
is obtained by the exogenous route (absorption of food via consumes oxygen and then is oxidized, thereby causing H2O2
the small intestine). About 2/3 of cholesterol is obtained by levels to increase. This reaction may be detected by a com-
somatic cell synthesis. Cholesterol exists in the serum in two plete set of procedures, including peroxidase-positive color
forms: about 1/4 free, and 1/2 esterified. CH in most tissue developing agent or the spectrophotometric method;
cells is free cholesterol, also known as nonesterified choles- catalase-positivity; NADH+NAD peroxidase spectrophotom-
terol. It is an essential constituent of all cytomembranes as etry; or the fluorescent method. Among these methods, the
well as a precursor of the steroid hormone vitamin D as well most common one is the Trinder reaction. It is a red quinone
compound of benzoquinone imine formed by phenol and proteinemia; (2) secondary hypercholesterolemia, including
4-amino antipyrine under the catalysis of peroxidase. The atherosis, nephrotic syndrome, hypothyroidism, diabetes,
absorption peak occurs at 550 nm. The optical density value pregnancy, and intestinal obstruction; (3) longstanding high
is directly proportional to the cholesterol concentration. The cholesterol, high saturated fatty acid, high-calorie diet, and
enzyme method is currently the routine application method. excessive alcohol intake.
It is fast and accurate with high specificity, small sample size Decreased serum TC may be found in (1) primary hypocho-
requirement, no need for extraction, and it is convenient for lesterolemias such as familial no P or low P-lipoproteinemia;
automatic analysis and batch determination. It is a routine (2) secondary hypocholesterolemias such as hyperthyroid-
method for determining cholesterol recommended by the ism, severe hepatic failure, hemolytic anemia, infections,
Examination Science Branch of the Chinese Medical and malnutrition.
Association.
14.2.2.4 Related Progress
14.2.2.2 Reference Values of Healthy Cholesterol (hypercholesteremia) is closely associated to
Individuals atherosis, demonstrated by the following research: (1) ani-
Blood lipid levels of populations depend on life factors, par- mal experiments [1]; (2) histopathological and chemical
ticularly diet and nutrition. Therefore, the reference values research on human arterial atheromatous plaques [2]; (3)
obtained by investigation in various regions may vary. Each clinical examinations of blood lipids in patients with athero-
region has its own division criteria for high TC. At present, sis [3]; (4) premature coronary heart disease induced by
the TC level that significantly increases the risk of coronary genetic high blood lipid disease [4]; (5) epidemiological
heart disease (medical decision level) acts as the division research [5]; and (6) intervention with preventive treatment
criterion. Based on the methodological standardization, a tests [6]. Therefore, cholesterol is believed to be one of the
common division criterion is used to help avoid confusion. important risk factors of atherosis.
The standard proposed by the 2007 Guide for Prevention Elevated cholesterol levels often lead to cardiovascular
and Control of Abnormal Blood Lipid in Adults in China and cerebrovascular diseases, including coronary heart dis-
is: ideal range: <5.18 mmol/L (<200 mg/dL); marginal ease, myocardial infarction, and stroke. However, it may
elevation: 5.18–6.1 mmol/L (200–239 mg/dL); elevation: only be used as a risk factor for assessing atherosis instead of
≥6.22 mmol/L (≥240 mg/dL). The medical decision level a diagnostic indicator because of its insufficient specificity
proposed by National Cholesterol Education Program, Adult and sensitivity [7]. In academic circles, it is believed that
Treatment Panel is: ideal range: <5.1 mmol/L (< 200 mg/ high cholesterol is an important risk factor of coronary heart
dL); marginal elevation: 5.2–6.2 mmol/L (200 mmol/L, disease and is one of the major substances contributing to
239 mg/dL); elevation: ≥6.21 mmol/L (≥240 mg/dL). atherosis. In recent years, some scholars have suggested that
the TC/HDL ratio may predict coronary heart disease in a
14.2.2.3 Clinical Significance more accurate manner. When TC/HDL > 5, the incidence of
TC is used to diagnose primary and secondary lipid meta- coronary heart disease rises sharply [8].
bolic anomalies, to predict the risk of atherosis disease, to
diagnose severe hepatic diseases, and to evaluate nutrition.
TC concentration may serve as a baseline value, indicating 14.3 Triglycerides
whether further testing is necessary to determine other
experimental indexes of lipoprotein. TC predicts the proba- 14.3.1 Sources and Characteristics
bility of cardiovascular and cerebrovascular diseases, includ-
ing AS, coronary heart disease, and myocardial infarction. Triglycerides are among the major components of blood lip-
Generally, the risk of cardiovascular and cerebrovascular ids. They are the most abundant lipids in humans. Also
diseases is moderate when TC < 4 mmol/L (160 mg/dL). known as triacylglycerol, they are molecules formed by tri-
Meanwhile, 5.2 mmol/L (200 mg/dL) serves as the threshold molecular long-chain fatty acids and monomolecular glyc-
value; the risk of coronary heart disease increases when TC erol. Humans store fat in the form of monocarboxylic acid
is greater than this value; the risk increases significantly (with or without double bonds) composed of 18 or 16 carbon
when it is larger than 5.4 mmol/L (250 mg/dL). A decrease atoms, particularly oleic acid, and palmitic acid. Therefore,
in cholesterol suggests protein-energy malnutrition. The triglyceride is a compound composed of various types of
probability of infection and neoplastic diseases increases. triacylglycerol with average molecular weight of about
Elevated serum TC may be found in (1) primary hyper- 875 kDa. Triglyceride is insoluble in water and can only be
cholesterolemias such as familial hypercholesteremia (low- transported when it binds lipoprotein; it is located in the core
density lipoprotein receptor defect), familial ApoB defects, of the lipoprotein. TG in plasma has two sources: the first is
multifocal high diacylglycerol (TC), and mixed hyperlipo- exogenous TG derived from food. It is emulsified via cholate
14 Lipoproteins 185
in the small intestine, hydrolyzed into glycerol, fatty acid, Finally, a colored dye is used (often quinone-imine) or an
and a small amount of semi-hydrolyzed products, diacylg- ultraviolet absorbing substance forms. The appropriate TG
lycerol and monoglyceride by pancreatic lipase. The hydro- concentration is computed using the spectrophotometric
lyzed products are esterified into TG under the action of method. The lipoprotein lipase-glycerol phosphoric acid
transaldolase in the smooth endoplasmic reticulum of muco- oxidase-4-amino antipyrine phenol (GPO-PAP method)
sal cells of the small intestines. TG, several apolipoproteins, method is the most common enzyme method. This method is
a small amount of phospholipid, and cholesterol combine simple, fast, and convenient with high precision and high
into CM containing large amounts of TG in the rough endo- specificity. It is easy to reach the end point and has a wide
plasmic reticulum that enters the blood via thoracic duct linear range. The one-step enzyme method determines total
lymph vessels. TG in CM is hydrolyzed into glycerol and glycerides in serum (defined as the sum of TG, FG, and a
fatty acid by lipotropin, thereby supplying energy. Most tis- small amount of diacylglycerol and monoglyceride, collec-
sues supply energy via diacylglycerol decomposition. Excess tively referred to as TG). To eliminate interference on the
glycerol and fatty acid may transform into TG again. part of FG, the examination branch of the Chinese Medical
Therefore, in addition to TG, a small amount of diacylglyc- Association recommended the GPO-PAP two-step enzyme
erol, monoglyceride (the sum of the two accounting for less method as the serum conventional determination method.
than 3% of TG), and free glycerol (FG) may be present in This method requires no increase in reagent cost and work-
peripheral blood. Under normal conditions, the content of load and is suitable for automated analyses. Because the
free glycerol in blood is about 1.1 mmol/L (50 mg/dL). The reagent is added in two portions, the requirements for accu-
second source is endogenous TG, primarily synthesized in rately setting the analytical determination parameters are
liver and adipose tissues. Liver can only synthesize TG and strict. Some have raised doubts regarding whether this
is unable to store it. By contrast, adipose tissue not only syn- method can completely remove free glycerol. In response to
thesizes TG but also stores surplus TG. Adipose tissue sup- this situation, the examination branch of the Chinese Medical
plies energy for cellular metabolism by means of fat Association recently suggested that eligible laboratories
mobilization when the energy supply to cells is insufficient. (such as Grade A Class 3 hospitals) should consider conduct-
ing FG determination. Accurate content of serum TG should
be the difference between the concentration of total glycerol
14.3.2 Detection Methods and Reference and the free glycerol.
Values of Healthy Individuals Principle of the GPO-PAP method
LPL
14.3.2.1 Detection Methods triglyceride + H 2 O glycerol + fatty acid
TG detection methods are divided into three major catego-
ries: chemical methods, enzyme methods, and chromatogra- glycerol ATP GK phosphoric acid-glycerol ADP
phy. The enzyme method is recommended.
Chemical method: TG in the sample is extracted by an GPO
organic solvent. After interfering substances such as phos- triglyceride + H 2 O glycerol + fatty acid
pholipid in the extraction solution are removed, TG is sapon-
ified by alkaline hydrolysate. Glycerol is oxidized with POD
3-phosphoric acid-glycerol + 4-AAP quinone-imine
periodic acid to generate formaldehyde. Then, the color
development reaction is used to measure the content of form- + H2 O
aldehyde. The dichloromethane-silicic acid-chromotropic
acid method (Van Handel-Carlson) is accurate. This method Chromatography is a TG direct detection method, including
achieves complete extraction and removes phospholipid and gaseous phase chromatography and liquid chromatography.
glycerol with high sensitivity of color development of chro- The nuclide dilution/gaseous phase chromatography/mass
motropic acid and stable color development. This method spectrum technique (ID/GC/MS), is used to establish a quali-
remains the reference method used by the US Centers for tative method, prepare reference substances and set v alues in
Disease Control. However, it is not suitable for routine use the reference system. This method is difficult to recommend
because of the various operating steps and strict technical as it is costly and involves complex procedures for sample
requirements. treatment.
Enzyme method: This method includes three basic steps: FG determination may use the enzyme method in TG deter-
Appropriate LpL is used to hydrolyze TG to generate glyc- mination except that no hydrolysis step is required. Reagent
erol and FFA. Then, they are subjected to enzymatic conver- kits that have recently become commercially available use
sion (for example, glycerol kinase is used to phosphorylate the chemiluminescence reaction of isoluminol- peroxidase
glycerol). Otherwise, intermediate products are generated. for serum glycerol analysis. These kits have higher sensi-
tivity, wider linear ranges, lower costs, and higher accuracy Increased TG levels are found in those with coronary
compared to other enzyme methods. These kits can be recom- heart disease, obesity, diabetes, pancreatitis, hypothyroid-
mended easily and are applicable for manual detection, semi- ism, glycogen storage, nephrotic syndrome, primary hyperli-
automatic, and fully automatic detection. Chromatography poproteinemia Types I, IIb, III, IV, and V and people who
may also be used for detection of glycerol, but only limited to fast for long periods, those fed a high-fat diet, and those who
experimental research. It is difficult to use in clinical practice. drink excessively.
Decreased levels of TG are found in those with hyperthy-
14.3.2.2 Reference Values of Healthy roidism, hepatic failure, and adrenal dysfunction.
Individuals Elevations in both LDL and TG increase the risk of car-
Levels of TG in normal individuals are affected by living con- diovascular diseases. Decreased levels of TG and HDL and
ditions. Average levels of TG in China are lower than those of other risk factors of coronary heart disease, including a fam-
Europe and America. Levels of TG rise with age in adulthood. ily history of coronary heart disease, drinking, and smoking
Individual differences in TG levels are greater than those of are particularly significant for assessing the risks of cardio-
TC; the data on population investigation are dispersed and vascular and cerebrovascular diseases, including coronary
exhibit a significant positively skewed distribution. heart disease and myocardial infarction.
The judgement criterion proposed by China in its
Suggestion for Prevention and Control of Blood Lipid 14.3.2.4 Related Progress
Anomaly is as follows: It is not yet certain been determined whether hypertriglyceri-
demia is a risk factor for coronary heart disease [9]. However,
• Ideal range: <1.7 mmol/L (<150 mg/dL) based on epidemic data and on the progress of pathological
• Elevated: > 1.7 mmol/L (>150 mg/dL) physiological research, triglycerides are not only closely
associated with the onset and progression of coronary heart
The medical decision level proposed in the second report disease, but they also play an important role in assessing the
(ATPIII) for the NCEP adult treatment group is as follows: risks of cardiovascular diseases. With respect to the depth of
knowledge, TG may be considered a conditional risk factor
• Ideal range: <1.7 mmol/L (<150 mg/dL) for coronary heart disease. Therefore, deepening the under-
• Marginally increased: 1.7–2.25 mmol/L (<150–199 mg/ standing of hypertriglyceridemia will help better control the
dL) risk factors of cardiovascular diseases and lower the risk of
• Increased: 2.26–5.64 mmol/L (<200–499 mg/dL) onset of coronary heart disease to a greater extent.
• Very high: ≥5.65 mmol/L (≥500 mg/dL)
14.3.2.3 Clinical Significance 14.4 Free Fatty Acids

The levels of TG in healthy populations are influenced by
living habits, dietary conditions, and age. Levels of TG fluc- 14.4.1 Sources and Characteristics
tuate significantly between individuals. The levels of TG in
China are lower than those of western countries and are Fatty acids are found in several serum lipids. Free fatty acid,
closer to those in Japan, because the amount of fat in the also known as nonesterified fatty acid (NEFA), is composed
Chinese diet is smaller than that of the western diet. To of oleic acid, palmitic acid, and linoleic acid. It is the most
exclude the influence of diet, samples should be drawn while active metabolic lipid transported in serum after binding
the subjects are fasting. Individuals fasting for long periods with protein. NEFA is released from the decomposition of
may have elevated levels of endogenous TG due to fat mobi- neutral fat and may be utilized as energy in peripheral tissue.
lization. Generally, levels of TG in children are lower than Under normal conditions, the content of NEFA in serum is
those of adults. TG levels rise with age, particularly after age very low. Its main components include oleic acid (C18:1) 54%,
30 years. Levels of TG in male adults are somewhat higher palmitic acid (Cl6:1) 34%, stearic acid (C18:1) 6%, a small
than those of adult females. TG levels may decline after age amount of lauric acid (C22:1), myristic acid (C14:1), and arachi-
60 years. The levels of TG in postmenopausal female adults donic acid (C20:1). NEFA is susceptible to various physiologi-
are higher than those of male adults. TG in serum is primar- cal and pathological changes, notably lipid metabolism,
ily found in VLDL and CM. Hypertriglyceridemia is among glucose metabolism, and endocrine function. Starvation,
the risk factors of cardiovascular diseases. In clinical exami- exercise, rage, diabetes, and changes in some endocrine fac-
nations, the concentration of TG is used in definitions of tors may cause FFA levels to rise; FFA levels decline after a
hyperlipidemia, pancreatitis, hepatic and renal diseases, ath- meal or use of glucose.
erosis, and in nutritional evaluations.
14 Lipoproteins 187
14.4.2 Detection Methods and Reference tumor, pituitary hypofunction, and overdoses of antidiabetics
Values of Healthy Individuals and insulin.
14.4.2.1 Detection Methods

The detection methods for FFA include nonenzymatic and 14.5 Phospholipids
enzymatic determination. Nonenzymatic determination
methods include the titration method, colorimetric method, 14.5.1 Sources and Characteristics
atomic absorption spectrophotometric method, high-
performance liquid chromatography, liquid chromatography Lipids containing fatty acids are called phospholipids, pri-
fluorescent determination, and mass spectrum identification marily comprising glycerol or sphingosine, fatty acid, phos-
method. The first three methods have low accuracy and the pholipid, and nitrogen-containing compounds. Phospholipids
last two methods require costly instruments, making batch may be divided into two major classes according to glycerol
operation impossible. The enzyme method determination is components: One is phospholipid including glycerol, called
accurate, reliable, fast, and suitable for clinical detection. phosphoglyceride; the other is phospholipid including sphin-
Principle of the FFA colorimetric method: Free fatty acid gosine, called sphingolipid. The phospholipids in serum
in serum is extracted with a high-density copper reagent. include lecithin (60%), sphingolipid (20%), lysolecithin
Copper soap forms in the upper solvent phase. (2–10%), and phosphatidylethanolamine (2%).
Diphenylcarbazide is used for color development and
quantification.
Principle of the enzyme method determination: Free fatty 14.5.2 Detection Methods and Reference
acid in serum generates a purple compound with its color Values of Healthy People
shade directly proportional to the FFA content under the
action of the enzyme reagent. 14.5.2.1 Detection Methods
Methods include the inorganic phosphorus method and the
FFA ATP CoA acetyl CoA synthetase acetyl CoA
enzyme method. The enzyme method is widely used in clini-
AMP PPi cal detection.
acetyl CoA O2 acetyl CoA synthetase 2, 3-enol acylCoA
14.5.2.2 Reference Values of Healthy
H 2O2
Individuals
Adults: 0.56–1.70 mmol/L.
2H 2 O2 N-acetyl-N- 2-hydroxyl-3-thiopropionamide
3-toluidineCoA TOOS 4-amidogenantipyrine 14.5.2.3 Clinical Significance
peroxidase claret-colored compound 4H 2 O Serum phospholipid detection is associated with obstructive
jaundice, lipoprotein anomaly, Tangier disease, and lack of
LCAT.
14.4.2.2 Reference Values of Healthy
Individuals
Adults: 0.4–0.9 mmol/L; slightly higher for children and 14.6 Lipoprotein
obese adults. It is recommended that laboratory reference
intervals should be established. Lipoprotein is a compound containing protein, cholesterol,
and phospholipid. There is no ideal method for quantifica-
14.4.2.3 Clinical Significance tion. Currently, the methods for determining plasma lipopro-
Physiological elevations occur in cases of starvation, exer- teins include the ultracentrifugation separation and
cise, rage, and drinking excessive amounts of caffeinated purification methods, the electrophoresis separation method,
beverages. A physiological decline is often found after a the plasma standing test, and the plasma lipoprotein choles-
meal. Pathological elevations occur in diabetes, glycogen terol determination method. This is because the content of
storage diseases, hyperthyroidism, brown cytoma, acromeg- cholesterol in lipoprotein is stable. Currently, the method for
aly, gigantism, Cushing’s syndrome, severe hepatic impair- determining total cholesterol in lipoprotein is used as the
ment, myocardial infarction, the latter half of gestation, quantification basis for lipoprotein, i.e., determination of
obstructive jaundice, hepatitis, liver cirrhosis, acute pancre- cholesterol in HDL, LDL, or VLDL. For Lp(a), in addition to
atitis, and hemochromatosis. Pathological declines occur in the immunology method, the electrophoresis method may be
hypothyroidism, Addison’s disease, pancreatic islet cell used to determine the cholesterol in plasma Lp(a) (Lp(a)-C).
14.6.1 High-Density Lipoprotein HDL components are separated, by means of agarose gel or
gradient electrophoresis. The former has simple require-
14.6.1.1 Sources and Characteristics ments for apparatus and reagents and the operation is rela-
Particle sizes, components, and functions of human plasma tively simple. The latter has very strict requirements for gel
HDL are very uneven. The density gradient ultracentrifuga- quality and laboratory conditions. Specially customized gels
tion method may be used to separate HDL particles into par- are required. Furthermore, the changes in laboratory condi-
ticles of three densities: HDL1, HDL2, and HDL3. HDL in tions should be closely monitored to guarantee accuracy.
plasma is dominated by HDL2 (1/3) and HDL3 (2/3). HDL is Chemical deposition method: with the dextran sulfate and
the esterified cholesterol in high-density lipoprotein. HDL2 polyethylene glycol as the precipitants, solution density, and
and HDL3 are the cholesterols in these two subtypes of pH values are adjusted. HDL2 and HDL3 are separated. The
HDL. HDL is a generally accepted anti-AS factor. Therefore, content of cholesterol in the solution is measured for quanti-
HDL is called “good cholesterol.” Based on the existing lit- fication. This method has a simple operation process and
erature, HDL2 is theoretically more protective than is HDL3. requires no special equipment; however, it is subject to the
levels of triacylglycerol. Subsequently, the complex solution
14.6.1.2 D
etection Methods and Reference selective deposition method was invented to separate and
Values of Healthy Individuals determine the HDL subcomponents. This method is cost-
effective and fast. However, this method has insufficient
Detection Methods experimental stability and reproducibility and is difficult to
For HDL detection, a method is generally used to deposit be standardized because different results may be obtained by
VLDL and LDL. Then, the method related to detection of different laboratories for the same sample.
total cholesterol (the most widely used enzyme method) is The TC/HDL ratio is computed with the TC and HDL
employed to detect cholesterol content in HDL. Currently, values.
there is no reference method. The available deposition meth-
ods include phosphotungstic acid /MgCl2, heparin/MnCl2, 14.6.1.3 Reference Values of Healthy
glucan sulfate/MgCl2, polyethylene glycol 6000, and quanti- Individuals
tative lipoprotein electrophoresis. Glucan sulfate/MgCl2 is The levels of HDL-C in male adults in China range from
recommended as the preferred method for clinical chemical 1.16 to 1.42 mmol/L (45–50 mg/dL). Levels in female adults
laboratories. The Centers for Disease Control recommends are higher, ranging from 1.29 to 1.55 mmol/L (50–60 mg/
the d = l.006 g/mL ultracentrifugation method to separate dL). HDL-C in normal individuals accounts for about
CM and VLDL. Then, the heparin//MnCl2 is used to deposit 25–30% of TC.
LDL. The phosphotungstic acid/MgCl2 deposition method is The judgement criterion proposed by China in its
widely used in Europe. Currently, some scientific research Suggestion for Prevention and Control of Blood Lipid
institutions also use the ELISA method to detect HDL. The Anomaly is as follows:
double antibody sandwich ABC-ELISA method is most
common. Some research institutions use high-performance • Ideal range: >1.04 mmol/L (>40 mg/dL)
liquid chromatography to separate and measure HDL. • Decline: <0.91 mmol/L (<35 mg/dL)
The density gradient ultracentrifugation method: HDL is
subjected to centrifugal deposition or precipitation balance Medical decision level proposed by NCEP, ATPIII:
in a gradient medium. Under the action of centrifugal forces,
HDL subcomponent particles are distributed to various sites • <1.03 mmol/L (40 mg/dL) indicates a decline and an
in the gradient solution, thereby forming zones and achiev- increased risk of CHD
ing mutual separation of various subcomponents of • > 1.55 mmol/L (60 mg/dL) indicates a negative risk
HDL. The advantages of this method are high resolution, factor
high accuracy, excellent separation effects, ability to obtain
pure particles at one time, no compression and deformation ATPIII elevates the original level of HDL-C from <35 mg/
of particles, ability to maintain the activity of particles, and dL (0.9 mmol/L) to <40 mg/dL (1.03 mmol/L) to persuade
ability to prevent the resulting zones from mixing due to con- more individuals to undergo preventive treatment. A total of
vection. The disadvantages include expensive equipment, 40% of males (originally 5%) and 15% of females (origi-
long centrifugation time, necessity of preparing a gradient nally 5%) are classified as high-risk populations.
solution, strict operation requirements, and difficulties with
mastery of the method. 14.6.1.4 Clinical Significance
Electrophoresis: according to the levels of electric charges • HDL is a protective factor for AS. HDL may be used to
of HDL particles and characteristics of the components, assess the onset risks of AS and coronary heart disease
14 Lipoproteins 189
and is one of the important indicators for monitoring 14.6.2.2 D

etection Methods and Reference
hyperlipidemia, diet adjustment, exercise effects, and Values of Healthy Individuals
medication efficacy.
• Declines in HDL levels may be found in smoking, high- Detection Methods
fat diets, obesity, less exercise, hypoalphalipoprotein- The classical methods for LDL detection include the homo-
emia, hypertriglyceridemia, increased steroid hormones, geneous phase direct determination method and the compu-
diabetes, uremia, and use of some drugs (diuretics, tational method. Immune transmission turbidimetry is a new
β-receptor retardant, probucol, neomycin). method for fully automatic analyzers established in recent
• Increased levels of HDL are found in moderate exercise, years. It is suitable for batch clinical detection. The commer-
diet, cholesteryl ester transfer protein (CETP) deficiency, cially available ELISA detection kits of LDL similar to HDL
chronic obstructive pulmonary disease (COPD), primary are generally applied in scientific research.
biliary liver cirrhosis, alcohol consumption, and long- Homogeneous phase direction determination method:
term physical labor. Similar to HDL, to measure LDL, VLDL, or HDL should be
• HDL2 is one of the major components for protecting first deposited. The deposition reagents include macromolecule
coronary heart disease from occurring in HDL. HDL3 glucose sulfate, polyanions, polyethylene sulfate, and PH5.12
has little or no protective effects. Therefore, detecting heparin. Generally, the difference between total cholesterol
the content of HDL2 is more significant than detecting and the amount of cholesterol in the supernatants is obtained
HDL. by precipitation of VLDL and HDL. In the case of polyanion
• The TC/HDL ratio plays a role in predicting the occur- precipitation, the electrophoresis method may also be used to
rence of coronary heart disease. When TC/HDL ≤4.0, precipitate LDL; however, the accuracy cannot be guaranteed.
there is a very low risk of coronary artery disease. By con- Friedewald computation method: LDL may also be com-
trast, there is a very high risk of heart attack when TC/ puted using the mathematical method via the Friedewald for-
HDL ≥8.0. mula based on the concentrations of cholesterol, triglyceride,
and HDL.
14.6.1.5 Related Progress
A low level of high-density lipoprotein cholesterol (HDL-C) LDL = TC-TG/5-HDL (mg/dL)
is associated with an increased risk of cardiovascular disease LDL = TC-TG/2.2-HDL (mmol/L)
(CVD). High levels of HDL-C are associated with decreased
risk of CVD; however, the causal relationship has not been These two formulas are only applicable to fasting serum with
confirmed [10]. Currently, there is no unambiguous evidence CM and TG concentrations lower than 400 mg/dL (4.7 mmol/L).
indicating that drug treatment for low HDL-C is beneficial It is clinically recommended that a combination of the
[11]. Nevertheless, changes in lifestyle, including exercise, ultracentrifugation method and the polyanion precipitation
weight loss (for obese people), and smoking cessation can method should be employed as the reference methods for
raise levels of HDL-C [12]. Therefore, this method is recom- LDL detection; however, they are not suitable for routine
mended for patients with low levels of HDL-C. Some studies clinical applications.
reported that increase in the HDL-ApoC III/VLDL-ApoC III Principle of transmission turbidimetry: A special surface-
ratio is a good predictive index of patients with coronary active agent is used to modify LDL and a dispersing agent is
heart disease and is even superior to conventional lipid mark- used to control LDL to prevent precipitation of large parti-
ers [13]. cles and to maintain the reactants in a reactant state. The
absorbency is directly proportional to the LDL content at
wavelengths of 600–630 nm.
14.6.2 Low-Density Lipoprotein
Reference Values of Healthy Individuals
14.6.2.1 Sources and Characteristics Levels of LDL-C increase with age. Levels in middle-aged
LDL is a particle rich in cholesterol and lipoprotein. LDL and elderly people range from 2.7 to 3.1 mmol/L on average
is called “bad cholesterol” because it contributes to forma- (105–120 mg/dL).
tion of AS. LDL is one of the major carriers of cholesterol The judgement criterion proposed by China in its
in plasma. About 60–80% of LDL enters the cells via the Suggestion for Prevention and Control of Blood Lipid
low-density lipoprotein receptor on the cell surface and is Anomaly is as follows:
degraded to release free cholesterol. When LDL is exces-
sive, cholesterol carried by LDL accumulates in arterial • Ideal range: <3.12 mmol/L (<120 mg/dL)
walls, often leading to arteriosclerosis and coronary heart • Marginal elevation: 3.15–3.61 mmol/L (121–139 mg/dL)
disease. • Elevation: >3.64 mmol/L (>140 mg/dL)
Medical decision level proposed by NCEP, ATPIII: B with density close to 1.06 g/mL, also known as small dense
low-density lipoprotein (SD-LDL). SD-LDL is a subset of
• Ideal level: <2.58 mmol/L (<100 mg/dL) LDL with a small percentage of cholesterol and a large per-
• Close to the ideal level: 2.58–3.33 mmol/L (100–129 mg/ centage of protein. SD-LDL particles contain less choles-
dL) teryl ester and the CHOL/ApoB ratio is lower.
• Marginal elevation: 3.64–4.11 mmol/L (130–159 mg/dL)
• Elevation: 4.13–4.88 mmol/L (160–189 mg/dL) 14.6.3.2 D
etection Methods and Reference
• Very elevated: >4.91 mmol/L (>190 mg/dL) Values of Healthy Individuals
14.6.2.3 Clinical Significance Detection Methods

A raised level of LDL is a risk factor for AS and one of the There are various methods for SD-LDL detection. The density
major factors that trigger accumulation of plaques in athero- gradient ultracentrifugation method is the gold standard for
sis. The content of LDL and the morbidity and severity of determining LDL subtypes. Gradient gel electrophoresis is the
cardiovascular and cerebrovascular diseases exhibit a signifi- most common method. The heparin-magnesium precipitation
cantly positive correlation. Therefore, LDL concentrations method is one of the hot areas of research in terms of SD-LDL
are measured to assess lipid metabolism disorders and to pre- detection methods. We obtain supernatants of SD-LDL and
dict the risks of atherosis. An elevated level of LDL may be HDL with densities greater than 1.044 g/L by means of sepa-
found in smoking, high fat diet, obesity, less exercise, ration using this method. In this method, heparin magnesium
hypoalphalipoproteinemia, high triglyceride, diabetes, ure- ions selectively precipitate the lipoprotein with a density
mia, familial hypercholesterolemia, type IIa hyperlipopro- smaller than 1.044 g/L. An automatic biochemical analyzer is
teinemia, and use of some drugs (diuretics, β-receptor used to selectively determine the content of SD-LDL and
retardants, probucol, neomycin). Decreased levels of LDL SD-LDL Apo B in supernatants, thereby quantifying SD-LDL.
may be found in moderate exercise, people on diets, hyper-
thyroidism, acute myocardial infarction, myeloma, trauma, Reference Values of Healthy Individuals
severe hepatic diseases, and Reye syndrome. Males aged 20–44 years, 93–538 mg/L
Females aged 20–54 years, 94–428 mg/L
14.6.2.4 Related Progress Males above 45 years and females above 55 years,
Elevated levels of LDL are among the major lipid risk factors 102–526 mg/L
for incidence and progression of atherosis. In recent years,
research has found that the oxidative modification of LDL is It is recommended that each laboratory should establish
one of the critical promoters of atherosis [14]. Relevant its own reference range.
research has demonstrated that oxidized low-density lipo-
protein (OX-LDL) may serve as a risk factor for coronary 14.6.3.3 Clinical Significance
heart disease in an early stage instead of advance stage [15]. As SD-LDL and high TG are closely associated in terms of
The cytotoxicity of OX-LDL may lead to changes in struc- metabolism and high TG is accompanied by low HDL, the
ture and function of vascular endothelial cells, accelerating concurrence of high TG, low HDL, and increased SD-LDL is
the formation of fatty streaks, causing accumulation of a often collectively referred to as atherosis lipoprotein pheno-
large amount of cholesterol within cells, formation of foam type or lipid triad syndrome in clinical practice. The level of
cells and promotion of atherosis plaques [16]. SD-LDL is an effective indicator of metabolic syndrome of
patients with coronary heart disease.
14.6.3 Small and Dense Low-Density 14.6.3.4 Related Progress

Lipoprotein SD-LDL may promote the incidence and progression of AS
and is one of the independent risk factors of cardiovascular
14.6.3.1 Sources and Characteristics and cerebrovascular disease [17]. SD-LDL has a stronger
Non-denaturing gradient gel scanning is performed to deter- ability to cause AS than does LDL [18]. Measuring levels of
mine the peak particle diameter (PPD) of LDL. LDL can be various LDL subtypes have higher clinical value than does
divided into two subtypes: When PPD >25.5 nm, LDL is measurement of LDL levels alone. Furthermore, it is more
classified as Type A, i.e., large LDl, with its density close to important to quantitatively measure levels of SD-LDL in
1.02 g/mL. When PPD <25.5 nm, LDL is classified as Type high-risk patients.
14 Lipoproteins 191
14.6.4 Lipoprotein (a) thrombosis. Lp(a) also interacts with LDL to form polymers,
thereby extending its retention in the intima and contributing
14.6.4.1 Sources and Characteristics to the formation of foam cells. Lp(a) can activate the trans-
LP (a) is a special lipoprotein in humans. It is the result of forming growth factor β (TGF-β), stimulating proliferation
connecting apolipoprotein (a) and ApoB via a disulfide bond. of smooth muscle cells, and strengthening their activity.
It has a structure similar to that of LDL and contains a spe- Apo100 in Lp(a) often binds with extracellular matrices
cific Apo(a) with a structure similar to that of fibrous protein (protein mucopolysaccharide, fibronectin) in the intima. Free
zymogen in addition to ApoB. They are connected by a Apo(a) entraps more particles rich in cholesterol and allows
disulfide bond (Fig. 14.1). Apo(a) and fibrous protein zymo- macrophages to ingest receptor-mediated LDL and Apo(a)
gen are very similar in structure and both contain a kringle- on a larger scale, suggesting that Lp(a) is closely associated
like structure composed of three pairs of monosulfide bonds with AS. The physiological functions of Lp(a) remain
(K, relative molecular weight 12,700). Apo (a) includes a K4 unknown at present; however, a great deal of clinical epi-
copy, one K5, and a protein hydrolase area. K4 is divided demic data have demonstrated that Lp(a) is an important risk
into 10 types, from K4-1 to K4-10. K4-2 has a variable num- factor of coronary heart disease, closely associated with
ber of replicates. Three to 40 copies form polymorphisms heredity.
with Apo(a) molecular weights ranging from 187,000 to
662,000. The level of plasma Lp(a) is controlled by genes 14.6.4.2 Detection Methods and Reference
and is associated with polymorphisms of Apo(a). Apo(a) is Values of Healthy Individuals
positively correlated with AS, coronary heart disease, myo-
cardial infarction, and stroke. It participates in AS lipid accu- Detection Methods
mulation and formation of foam cells, promoting AS. Lp(a) The detection methods for Lp(a) are of two types: immuno-
may also be oxidized and modified. Oxidized and modified logical and non-immunological detection. The non-
Lp(a) produces excessive amounts of plasminogen activation immunological detection method primarily measures the
inhibitors, thereby inhibiting fibrinolysis and leading to content of cholesterol in Lp(a).
Fig. 14.1 Structure of

lipoprotein (a)
At present, among the common Lp(a) immunochemical Decrease: Lp(a) is synthesized primarily in the liver. Any
quantifications methods are immune transmission turbidim- hepatic disease may influence normal biological synthesis.
etry, latex-enhanced immune transmission turbidimetry, Decreased levels of Lp(a) may be found in patients with
electrophoresis immune diffusion, radio-immune diffusion, hyperthyroidism and patients who are receiving estrogen,
immune diffusion, and ELISA. The double antibody sand- niacin, and neomycin.
wich method is the reference method for detection. The cap-
turing antibody is a-6 (specific to Site K4-2) or a-40 (specific 14.6.4.4 Related Progress
to Site K4-9). The detecting antibody is a-40. As the Apo(a) Lp(a) is not recommended as one of the CHD risk indexes in
molecule only contains one copy of K4-9, this method is ordinary populations; however, it is suitable for patients with
insensitive to polymorphisms of Apo(a). an increased risk of familial CHD. In recent years, research
Determining the content of cholesterol in Lp(a) may elim- has found that high levels of Lp(a) are closely associated
inate the polymorphism of Apo(a). Ultracentrifugation or with acute coronary artery syndrome, coronary artery vascu-
lectin affinity chromatography separation of Lp(a), both lar remodeling, degree of coronary artery stenosis, calcifica-
enzyme methods for determining the content of cholesterol, tion, and spasm [19]. Lp(a) is a major risk factor of
have complex operation procedures and are inconvenient for myocardial infarction (AMI) independent of LDL and may
clinical applications. Recently, a method combining the elec- become a basis for risk stratification for AMI patients [20].
trophoresis technique and in situ enzyme color development Elevated levels of Lp(a) may serve as a predictive factor of
or optical density scanning has been utilized to determine the CHD disease severity and it is expected to become an effec-
content of cholesterol in Lp(a). However, the clinical value tive marker of high incidence of disease in patients with
of determining the Lp(a) cholesterol is expected to be stud- coronary artery spasm [21]. A meta-analysis of studies of
ied in the future. patients receiving statin treatment found that an increased
baseline and statins Lp(a) and the risk of cardiovascular dis-
Reference Values of Healthy Individuals eases exhibit an independent approximate linear relationship
The Lp(a) data of normal individuals exhibit significantly [22].
skewed distributions. Levels of Lp(a) of individuals can be
up to more than 1000 mg/L. However, 80% of the normal
people have Lp(a) levels below 200 mg/L. The average levels References
of Lp(a) in the literature range from 120 to 180 mg/L. The
medians of Lp(a) levels range from 81 to 117 mg/L. Usually, 1. Stamler J, Wentworth D, Neaton JD. Is relationship between serum
cholesterol and risk of premature death from coronary heart disease
300 mg/L acts as the demarcation line. People with Lp(a) continuous and graded? Findings in 356,222 primary screenees
levels higher than 300 mg/L have a significantly higher risk of the Multiple Risk Factor Intervention Trial (MRFIT). JAMA.
of coronary heart disease. About 14% of the people have 1986;256:2823–8.
Lp(a) levels higher than this. In some studies, 450 or 2. Fallaize R, Carvalho-Wells AL, Tierney AC, et al. APOE genotype
influences insulin resistance, apolipoprotein CII and CIII according
480 mg/L serve as the upper limits of normal. There are no to plasma fatty acid profile in the metabolic syndrome. Sci Rep.
significant differences in Lp(a) levels between people of dif- 2017;7
ferent genders and ages. 3. Wang HH, Garruti G, Liu M, et al. Cholesterol and lipoprotein
metabolism and atherosclerosis: recent advances in reverse choles-
terol transport. Ann Hepatol. 2017;16:S27–42.
14.6.4.3 Clinical Significance 4. Genest JJ Jr, Martin-Munley SS, McNamara JR, et al. Familial lipo-
Independent risk factors of cardiovascular disease protein disorders in patients with premature coronary artery dis-
<200 mg/L, low risk; 200–300 mg/L, moderate risk; 310– ease. Circulation. 1992;85:2025–33.
500 mg/L, high risk; and > 500 mg/L, very high risk. 5. McGill HC Jr, McMahan CA, Zieske AW, et al. Association
of coronary heart disease risk factors with microscopic quali-
Pathological increase: Significantly elevated levels of ties of coronary atherosclerosis in youth. Circulation. 2000;102:
Lp(a) in patients with ischemic cardiovascular and cerebro- 374–9.
vascular diseases, hyperlipidemia, AS, coronary heart dis- 6. Arroyo-Olivares R, Alonso R, Quintana-Navarro G, et al.
ease, and cerebral infarction are not associated with Adults with familial hypercholesterolaemia have healthier
dietary and lifestyle habits compared with their non-affected
hypertension, smoking, diet, or other blood lipids. Some relatives: the SAFEHEART study. Public Health Nutr. 2019;22:
have argued that high levels of Lp(a) only indicate risks 1433–43.
when accompanied by high levels of LDL. Lp(a) has acute 7. Wang HH, Garruti G, Liu M, et al. Cholesterol and lipoprotein
time phase protein reactivity and increases during acute metabolism and atherosclerosis: recent advances in reverse choles-
terol transport. Ann Hepatol. 2017;16:s27–42.
responses such as myocardial infarction, surgery, acute 8. Aryal M, Poudel A, Satyal B, et al. Evaluation of non-HDL-c and
trauma, and acute inflammation. Others include nephrotic total cholesterol: HDL-c ratio as cumulative marker of cardiovas-
syndrome, uncontrolled diabetes, thyroid dysfunction, ure- cular risk in diabetes mellitus. Kathmandu Univ Med J (KUMJ).
mia, and malignant tumors (except for hepatic carcinoma). 2010;8:398–404.
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9. Reiner Z. Hypertriglyceridaemia and risk of coronary artery dis- 17. Packard CJ. Small dense low-density lipoprotein and its role as an
ease. Nat Rev Cardiol. 2017;14:401–11. independent predictor of cardiovascular disease. Curr Opin Lipidol.
10. Vitali C, Khetarpal SA, Rader DJ. HDL cholesterol metabolism and 2006;17:412–7.
the risk of CHD: new insights from human genetics. Curr Cardiol 18. Sumino H, Nakajima K, Murakami M. [Possibility of new cir-
Rep. 2017;19:132. culating atherosclerosis-related lipid markers measurement in
11. Briel M, Ferreira-Gonzalez I, You JJ, et al. Association between medical and complete medical checkups: small dense low-density
change in high density lipoprotein cholesterol and cardiovascu- lipoprotein cholesterol and lipoprotein lipase]. Rinsho Byori.
lar disease morbidity and mortality: systematic review and meta- 2016;64:298–307.
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12. Escola-Gil JC, Julve J, Griffin BA, et al. HDL and lifestyle inter- nosis, controversies, and emerging therapies. J Am Coll Cardiol.
ventions. Handb Exp Pharmacol. 2015;224:569–92. 2017;69:692–711.
13. Koch M, Furtado JD, Jiang GZ, et al. Associations of anthropom- 20. Andreassen AK, Berg K, Torsvik H. Changes in Lp(a) lipoprotein
etry and lifestyle factors with HDL subspecies according to apoli- and other plasma proteins during acute myocardial infarction. Clin
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14. Gao S, Liu J. Association between circulating oxidized low-density 21. Mellwig KP, Horstkotte D, van Buuren F. Lipoprotein (a) and coro-
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Radic Res. 2017;51:439–47.
Transport and Carrier Proteins
15
15.1 Overview weight of 29 kDa, primarily found in LDL and HDL. It is an

endogenous lipid transporting protein that inhibits the cho-
Apoprotein (Apo) is the major protein component of lipopro- lesteryl ester transport protein from mediating the transport
tein that determines its properties. Apolipoproteins are gener- of CE and TG. In vitro testing demonstrates that ApoF selec-
ally divided into 5–7 types. Most of their amino acid sequences tively inhibits the transport of lipids toward LDL, increases
have been determined. The types of apolipoproteins are named the rate of the transport of CE and TG from HDL to VLDL,
according to the classification system suggested by Alaupovic and plays an important role in the reverse transport of choles-
in 1972. The types are encoded with the English alphabet. terol. Similarly, ApoL is closely related to reverse transport
Each type is further subdivided into subtypes. These include of cholesterol; however, the specific mechanism is unclear.
ApoAI, ApoAII, ApoAIV, ApoB100, ApoB48, ApoCI, ApoAV accounts for a low percentage of apolipoprotein in
ApoCII, ApoCIII, ApoD, ApoE, ApoH, ApoJ, and Apo(a) humans. Recent research has found that ApoAV activates
(Table 15.1). The primary location of apolipoprotein synthesis lipoprotein lipase (LPL) and inhibits ApoCIII to accelerate
is the liver. A small amount of apolipoprotein is synthesized in the catabolism of VLDL, thereby decreasing TG levels. In
the small intestine. Recently, it was found that visceral organs rats and mice, ApoAV binds HDL in a manner similar to that
(excluding the liver) including brain, kidney, adrenal gland, of other lipoproteins. ApoAV activates the acyl transferase of
and spleen, and even the macrophages, can synthesize apoli- lecithin cholesterol (plecithin: cholesterol acyl transferase,
poproteins. Most apolipoproteins have an amphipathic α-helix LCAT). ApoAV is predicted to control the outflow of choles-
structure. On the surface of lipoproteins, the nonpolar domain terol in lipoprotein and promotes lipid exchange between
is connected with triglycerides (TG) and cholesterol ester lipoproteins after binding with HDL. ApoM is primarily
(CE) via hydrophobic amino acid residues. Therefore, the found in HDL. Its content in HDL is only second to that of
lipoprotein is a compound with its surface covered by apolipo- ApoAI. It is closely associated with the role of HDL in cho-
proteins and phospholipids, with TG and CE at the core. lesterol outflow; however, the specific mechanism of action
Types, contents, and functions of the apolipoproteins remains unknown.
vary. They have important physiological functions: activat-
ing enzymes with lipoprotein as their metabolic routes,
maintaining the structural integrity of the lipoprotein, identi- 15.2 Apolipoprotein AI (AI, ApoAI)
fying the lipoprotein receptor on the cell surface, and mediat-
ing lipoprotein entry into cells. Lipoproteins also weakly 15.2.1 Sources and Characteristics
bind large amounts of plasma protein. However, this mecha-
nism has not yet been explained. Apolipoprotein AI is the major structural protein of HDL,
In recent years, newly identified apolipoproteins include synthesized in the liver and small intestine, and also found in
ApoF, ApoL, ApoAV, ApoM, and ApoO. chylomicrons (CM) in the small intestine. It plays an impor-
Research regarding metabolic routes and mechanisms of tant role in the reverse transport of cholesterol, inhibits oxi-
action is insufficient. ApoF is a sialoprotein with molecular dation and modification of LDL, stabilizes prostacyclin,
protects vascular endothelial cells, and stabilizes the cyto-
membrane structure. ApoAI accounts for about 75% of
C. Wang (*) · Z. Li ApoA. ApoAI converts cholesterol into cholesteryl ester and
Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur
transports excessive esterified cholesterol to the liver for
Autonomous Region, Urumqi, Xinjiang,
People’s Republic of China treatment. Its concentration positively correlates with levels

Table 15.1 Structural characteristics and functions of common apolipoproteins

Amino acid Plasma
Molecular number of Lipoprotein concentration
Apolipoprotein weight (kDa) residues carrier Function Synthesis site (g/L)
AI 28.3 243 HDL, CM Stabilizing the HDL structure and LCAT Liver and 1.00–1.60
cofactors and identify the HDL receptor intestine
AII 17.5 77×2 HDL Activating HTGL, inhibiting LCAT, and Liver and 0.30–0.40
participating in identifying the HDL receptor intestine
AIV 46 371 CM, HDL Participating in fat absorption, inversely Intestine 0.10–0.18
transporting the cholesteryl ester, and
activating LCAT
B100 513 4536 VLDL, IDL, Transporting TG and TC and identifying the Liver 0.60–1.12
LDL LDL receptor
B48 264 2152 CM Promoting formation of CM in the intestine Intestine
and transporting exogenous TG
CI 6.5 57 CM, VLDL, Activating LCAT Liver 0.03–0.07
HDL
CII 8.8 79 CM, VLDL, LPL co-factors Liver 0.03–0.05
HDL
CIII 8.9 79 CM, VLDL, Inhibiting Apo CII and activating LPL Liver 0.08–0.12
HDL
D 22 169 HDL Transporting cholesteryl ester Liver 0.02–0. 04
E 34 299 CM, VLDL, Promoting ingestion of CM remnants and IDL Liver 0.03–0.06
HDL
H 36 326 CM, VLDL, Activating LPL and inhibiting the endogenous Unknown Unknown
IDL, HDL blood coagulation bypass
J 70 427 HDL, VHDL Dissolving and transporting the lipid Liver Unknown
(a) 187–662 4529 Lp(a) Inhibiting the activity of fibrous protein Liver 0–0.3
lysozyme
of HDL-C. Apo AI synthesized by hepatocytes enters the cir- immunoassay, immunological turbidimetry, ELISA, and
culation together with the new dish-shaped HDL and radioimmunoassay. Immunological turbidimetry is most
exchanges with free ApoAI in the plasma, Apo AI in other widely used in clinical practice. This method combines the
lipoproteins, or other apolipoproteins. In this manner, ApoAI antigen–antibody and the dynamic determination methods,
plays a role in removing lipids and resisting atherosclerosis. and is subdivided into immunological transmission turbi-
ApoA includes AI, AII, and AIV but the content of ApoAI is dimetry, immunological scattering turbidimetry, and immune
the highest. ApoAI is frequently evaluated in clinical prac- latex turbidimetry.
tice. ApoAI is primarily distributed in plasma CM, HDL2, In immunological transmission turbidimetry, antigen
and HDL3. It consists of 243 amino acids. The relative and antibody are combined to form immune complexes.
molecular mass is 28.30 kDa. The regulatory processing of Within a certain period of time, the complexes aggregate
ApoAI occurs following transcription, as opposed to occur- to cause turbidity. When light passes through the solution,
ring at the transcription level. During the progression of ath- it is absorbed by the immune complex. A larger amount of
erosclerosis, enhanced expression of the ApoAI gene reduces immune complex leads to larger amounts of light absorbed.
the formation and accumulation of foam cells within the The amount of light absorbed is directly proportional to
arterial wall. High-concentration ApoAI in plasma and HDL the amount of the immune complex within a certain range.
inhibit the formation of atherosclerosis. Low-concentration A turbidimeter is used to determine the optical density
ApoAI in plasma and low-concentration HDL-C alone are value. The content of the complexes is directly propor-
not sufficient to induce atherosclerosis. tional to the optical density. Similarly, when the amount of
antibody is fixed, the optical density value is also directly
proportional to the content of antigens. This method is
15.2.2 Detection Methods and Reference more sensitive, faster, and more convenient than general
Values of Healthy Individuals immunochemical quantification methods such as one-way
agar diffusion and rocket electrophoresis. However, the
15.2.2.1 Detection Methods quantity and molecular weight of immune complexes are
The measurement of ApoAI largely uses immunological required to reach a certain level, otherwise, it is difficult to
methods, including radial immunodiffusion (RID), enzyme determine the values.
15 Transport and Carrier Proteins 197
In immunological scattering turbidimetry, light at particu- ApoAI may be found in patients with acute and chronic hep-
lar wavelengths shines along the horizontal axis, passes atitis, liver cirrhosis, extrahepatic biliary obstruction, isch-
through the solution, and is refracted by the particles and emic diseases, artificial dialysis, coronary artery disease,
deflects when meeting antigen–antibody complexes. The diabetes, nephrotic syndrome, and malnutrition.
angle of light deflection is closely associated with the wave- An increase in ApoAI is less common. Increasing levels
length of the emitted light and the size and quantity of the of ApoAI can be found in patients with alcoholic hepatitis or
antigen–antibody complex particles. The intensity of the scat- hypoalphalipoproteinemia.
tered light is directly proportional to the content of the com- Levels of ApoAI in the serum of patients with ApoAI
plexes. In other words, a larger number of antigens to be deficiency (for example, Tangier disease, a rare hereditary
tested would result in more complexes and more intensely disease) and fish eye disease are significantly decreased.
scattered light. The intensity of the scattered light is also ApoAI is determined and analyzed together with serum
closely associated with various physical factors, including the cholesterol, triglycerides, and apolipoprotein B, and can help
time when the antigen or antibody is added, intensity of light stratify risk of cardiovascular disease.
source and wavelength, and the measuring angle. Scattering
turbidimetry is further subdivided into velocity scattering tur-
bidimetry and end-point scattering turbidimetry. 15.2.4 Related Progression
In immunological latex turbidimetry, antibody corre-
sponding to the substance to be tested is enveloped on latex In recent years, research has found that ApoAI is closely
particles with diameters ranging from 15 to 60 nm, thereby associated with the occurrence of tumors. Low levels of
increasing the volume of the antigen–antibody compound. ApoAI expression have been reported in gastrointestinal and
After light passes through the substance, changes in intensity gynecological tumors [1, 2]. Research suggests that ApoAI
of the transmitted light and the scattered light are more sig- is able to promote tumor growth. Some scholars believe that
nificant, thereby improving the sensitivity of the test. joint measurement of ApoAI and CRP is an independent
prognostic of carcinoma [3]. Li and colleagues indicated that
15.2.2.2 Reference Values of Healthy ApoAI is a potential molecular marker for measurement and
Individuals diagnosis of carcinoma of the urinary bladder [4]. ApoAI has
The reference values of healthy are grouped by age immunoregulatory function. Several types of immunity-
(Table 15.2). related receptors are anchored on the lipid raft of the cyto-
membrane surface. ApoAI and HDL may play roles in
reducing inflammation by transmitting signals to cells when
15.2.3 Clinical Significance receiving cholesterol from the lipid raft. Understanding the
mechanism of action of ApoAI in tumors may facilitate new
The level of ApoAI in blood is influenced by drugs and hor- pathways for prevention, diagnosis, and treatment of tumors.
mones, including contraceptives, estrogen, menopause, and
alcohol consumption. ApoAI levels fluctuate somewhat with
age. Levels of ApoAI in females are somewhat higher than 15.3 Apolipoprotein B
those of males. Levels of ApoAI in both males and females
decline after they reach the age of 80 years. Levels of ApoAI 15.3.1 Sources and Characteristics
in Chinese are close to those of Americans.
Decreased levels of ApoAI are among the risk factors of Apolipoprotein B is the largest apolipoprotein in humans,
cardiovascular and cerebrovascular diseases. Decreases in found in LDL and chylomicrons (CM). It is the structural
protein of the two. ApoB has two subtypes, ApoB48 and
ApoB100. The relative molecular weight of ApoB48 is
Table 15.2 Reference values of healthy individuals
240.00 kDa. Its sequence is consistent with the sequence of
Age (years) Male (g/L) Female (g/L) most amino acids of ApoB100. It is primarily found in CM
4–5 1.09–1.72 1.04–1.63
and participates in digestion, assimilation, and transport of
6–11 1.11–1.77 1.10–1.66
exogenous lipids.
12–19 0.99–1.65 1.05–1.80
20–29 1.05–1.73 1.11–2.09 Derived from the small intestine, ApoB100 is only found
30–39 1.05–1.73 1.10–1.89 in intestinal lipoproteins such as CM. Mature ApoB100 con-
40–49 1.03–1.78 1.15–1.95 sists of 4536 amino acids, the molecular weight is 55.00 kDa.
50–59 1.07–1.73 1.17–2.11 It participates in the transport of exogenous cholesterol and
60–69 1.11–1.84 1.20–2.05 does not bind the LDL receptor. Under normal conditions, it
>69 1.09–1.80 1.18–1.99 is difficult to measure mature ApoB100. ApoB100 is the
major component of ApoB, constituting over 95% of the Chinese are lower than those of Europeans. Under normal
LDL protein components. Fasting ApoB measured in normal conditions, levels of ApoB fluctuate with levels of TG and
individuals is ApoB100. After being synthesized in the liver, LDL-C.
ApoB100 transports endogenous cholesterol and binds the Levels of ApoB in plasma negatively correlate with those
LDL receptor on the surface of peripheral tissue cells. It is of ApoAI. Even if levels of TG and LDL are within normal
closely associated with the deposition of the intracellular range, increases in levels of ApoB may directly indicate the
cholesterol. ApoB100 is further degraded into such products risk of cardiovascular disease. Increased levels of ApoB are
as ApoB48, 75, 41, 36. Epidemiology and clinical experi- found in atherosclerosis, obesity, U-type hyperlipidemia,
ence have shown that ApoB100 is closely associated with cholestasis, kidney diseases, and thyroid dysfunction.
coronary artery disease. Some studies have indicated that Decreased levels of ApoB in plasma are found in hepatic
patients with increased ApoB100 are linked with increased disease and hyperthyroidism.
LDL-C. The mechanisms of ApoB100-induced atherosclero- ApoB deficiency leads to abetalipoproteinemia or hypo-
sis include binding with cell receptors below the intima, betalipoproteinemia, including fat absorption disturbance
thereby leading directly to deposition of LDL-C below the (fatty diarrhea), deformation of red blood cells (acantho-
endarterium; directly stimulating proliferation of smooth cytes), and ataxia.
muscle cells and entering the lower layer of intima; causing
necrosis of foam cells in atherosclerotic plaques; and forma-
tion of lipid stripes by activating protein kinase-p38 partici- 15.3.4 Related Progress
pation in signal transduction within endothelial cells.
Research has indicated that ApoB is closely associated with
cardiovascular diseases, kidney diseases, and tumors. ApoB
15.3.2 Detection Methods and Reference is among the major factors responsible for atherosclerosis.
Values of Healthy Individuals ApoB is closely associated with LDL and cardiac surgery
scores (SS) [5]. In many kidney diseases, the distribution of
15.3.2.1 Detection Methods ApoB and ApoE in the kidney accelerates the progression of
The methods for ApoB measurement are primarily immuno- glomerular hardening. Preventing the buildup of ApoB in the
logical. In clinical practice, the two most commonly used kidney by inhibiting TGF-β improves survival rate and kid-
methods are velocity scattering immunological turbidimetry ney prognosis [6]. During tumorigenesis, ApoB mRNA edit-
and immunological transmission turbidimetry. ing catalytic polypeptide-like family (APOBEC) enzyme
shows deaminase activity, converting cytosine into uracil
15.3.2.2 Reference Values of Healthy while RNA is editing and reversely transcribing. The geno-
Individuals type and the highly expressed subtype may lead to drug
The reference values of healthy are grouped by age resistance, associated with clinical results [7].
(Table 15.3).
15.4 A
polipoprotein B/Apolipoprotein AI
15.3.3 Clinical Significance Ratio (ApoB/ApoAI Ratio)
Levels of ApoB in plasma increase with age both in males 15.4.1 Sources and Characteristics
and in females. Levels of ApoB in males are higher than
those of females throughout the lifespan. Levels of ApoB in Apolipoprotein B (ApoB) is the major apolipoprotein of
LDL, while apolipoprotein AI (ApoAI) is an important part
of HDL. Determining the levels of the two in plasma directly
Table 15.3 Reference values of healthy individuals
reflects plasma levels of LDL and HDL. The content of cho-
Age (years) Male (g/L) Female (g/L) lesterol in lipoprotein changes under some pathological con-
4–5 0.58–1.03 0.58–1.04
ditions. Therefore, LDL-C and HDL-C are not able to replace
6–11 0.56–1.05 0.57–1.13
the measurement of ApoB and ApoAI. It is generally believed
12–19 0.55–1.10 0.53–1.19
20–29 0.59–1.30 0.59–1.32 that ApoAI decreases and ApoB increases in cases of
30–39 0.63–1.43 0.70–1.32 atherosclerosis and coronary artery disease. Particularly in
40–49 0.71–1.52 0.75–1.36 coronary artery disease, increases in ApoB are more signifi-
50–59 0.75–1.60 0.75–1.68 cant than increases in TG and LDL-C. Some data have shown
60–69 0.81–1.56 0.75–1.73 that individuals with elevated plasma levels of ApoB have a
>69 0.73–1.52 0.79–1.68 90% higher risk of coronary artery disease compared with
those who have normal levels of ApoB. In the case of cere- 15.5 Apolipoprotein CII and CIII
brovascular disease, levels of ApoAI and HDL-C decrease
more significantly while levels of ApoB are usually normal. 15.5.1 Sources and Characteristics
In the case of cerebral hemorrhage, levels of ApoB may be
lower. Some studies have used multiple stepwise regression Apolipoprotein C is the main apolipoprotein of VLDL, also
analysis, and found that the ApoB/ApoAI ratio was signifi- found in HDL and LDL, a low-content structural protein in
cantly positively correlated with HDL and significantly neg- CM, VLDL, and HDL. Apolipoprotein C has three types:
atively correlated with TG and LDL, suggesting that ApoB/ ApoCI, ApoCII, and ApoCIII. Apo CI is the apolipoprotein
ApoAI is a reliable indicator predicting AS. The ApoB/ with the smallest relative molecular weight and strongest
ApoAI ratio is more significant than the single indicators of alkalinity. It is a single-stranded polypeptide composed of 57
ApoB and ApoAI. Therefore, some scholars have argued that amino acid residues with a relative molecular weight of
the ApoB/ApoAI ratio can replace the LDL-C/HDL-C ratio 6.60 kDa. In the secondary structure, 55% of the α-helical
as an onset risk indicator of AS. structure easily binds phospholipid. Currently, the physio-
logical functions of Apo C have not been completely eluci-
dated. A great deal of data has shown that it has effects on
15.4.2 Detection Methods and Reference many enzymes and receptors participating in lipoprotein
Values of Healthy Individuals metabolism, including lipoprotein lipase (LPL) and HL
activity, inhibiting the ingestion of the TG-rich lipoprotein
15.4.2.1 Detection Methods by the hepatic lipoprotein receptor, and participating in regu-
ApoB and ApoAI are detected by immunological methods, lating the activity of LCAT and CETP.
including immunodiffusion, enzyme immunoassay, immu- ApoCII is primarily expressed in the liver and small intes-
nological turbidimetry, ELISA, and radioimmunoassay. tine, and is the structural protein of CM, VLDL, and
Immunological transmission turbidimetry is most widely HDL. ApoCII is a single-stranded polypeptide composed of
used in clinical applications. 79 amino acid residues with a relative molecular weight of
8.80 kDa. LPL is an intermediate enzyme of triglyceride-
15.4.2.2 Reference Values of Healthy rich lipoprotein (TRL) steatolysis. ApoCII is an essential
Individuals activator of LPL; however, it may inhibit the activity of LPL
0.35–0.60 in high concentrations. Nevertheless, its specific mechanism
of action remains unknown. ApoCII participates in the TRL
exogenous metabolic pathway (Fig. 15.1) [9]. A great deal of
15.4.3 Clinical Significance experimental research has demonstrated that ApoCII may
only activate LPL after binding with lipid. Therefore, it is
Evaluating the important indicators of coronary artery dis- clear that the lipid binding region of ApoCII is essential for
ease (CAD) is more significant than evaluating cholesterol its function of activating the lipid enzyme. ApoCII also
and the LDL/HDL ratio. An ApoB/ApoAI ratio of 0.7 indi- inhibits the binding of ApoE-mediated β-VLDL and LDL
cates that the risk is moderate, a ratio of 0.9 indicates that the receptors and the LDL receptor-related protein (LRP). It also
risk is twice higher, and a ratio of 1.0 indicates that the risk inhibits the binding between CM and VLDL and the
is three times higher. steatolysis-stimulated receptor, and the activity of LCAT and
A significant pathological decrease in the ApoB/ApoAI CETP. The amount of ApoCII in humans is insufficient to
ratio is found in atherosclerosis, CAD, diabetes, hyperlipid- predict hypertriglyceridemia. ApoCII deficiency is charac-
emia, and obesity. terized by chylomicrons, giant cell tumors of the tendon
sheath, and periodic pancreatitis.
The exogenous pathway. The intestine absorbs fat in food
15.4.4 Related Progress to form CM, and CM combines with apoCIII and apoE
derived from mesenteric lymphatic vessels to enter the circu-
The ApoB/ApoAI ratio has value for the prognosis of some lation. CM is hydrolyzed by LPL to free fatty acids (FFA)
tumors, including gastric cancer and colorectal cancer and monoglycerides (MG), and converted to CM remnants.
(ApoB/ApoAI-32/38). Some research has shown that a large CM remnants are degraded by the liver in the end.
ApoB/ApoAI ratio in metastatic renal cancer patients before ApoCIII has a signal peptide with a length of 20 amino
surgery is closely associated with progression-free survival acid residues when initially synthesized within hepatocytes.
and overall survival [8]. Before entering the circulation, the signal peptide is cut and
Fig. 15.1 TRL exogenous metabolic pathways
distributed in HDL, VLDL, and CM. Mature ApoCIII is an 15.5.2 Detection Methods and Reference
acid glycoprotein containing 79 amino acid residues with a Values of Healthy Individuals
relative molecular weight of 8.7 kDa. It is a very strong
inhibitor of LPL. ApoCIII has the highest concentration in 15.5.2.1 Detection Method
the C family, primarily regulating the decomposition and Immunological transmission turbidimetry
metabolism of the triacylglycerol lipoprotein (TRLs) in
hypertriglyceridemia. Serum LPL and purified ApoCIII can 15.5.2.2 Reference Values of Healthy
be utilized to conduct in vivo dynamic experiments. Individuals
Experiments have revealed that it noncompetitively inhibits • ApoCII: 25.3–48.1 mg/L
the effects of LPL on Apo CIII and oleic acid glycerol, sug- • ApoCIII: 53.6–101.0 mg/L
gesting that ApoCIII may directly inhibit LPL. The N2 ter-
minal region of ApoCIII appears to be essential for inhibiting
the activity of lipoprotein lipase. In addition, ApoCIII may 15.5.3 Clinical Significance
influence the removal of plasma VLDL through the apolipo-
protein CIII receptor. The ApoCIII receptor is a newly found ApoCII is an essential activator of lipoprotein lipase; how-
special receptor on the hepatic cytomembrane. Unlike the ever, high concentrations of ApoCII may inhibit the activity
lipoprotein receptor, it is independent of Ca2+, is not subject of lipoprotein lipase. Elevated levels of ApoCII may be
to EDTA restriction, and is sensitive to trypsin. When the found in patients with Types I, IIb, III, IV, and V hyperlipid-
activity of the Apo CIII receptor is high, the ability of the emia and obstructive jaundice; decreased levels of ApoCII
liver to bind VLDL decreases. It is inferred that the func- may be found in patients with liver cirrhosis. The lack of
tions of the receptor on the hepatic cytomembrane to remove ApoCII is one of the causes of hyperchylomicronemia
the VLDL change when ApoCIII in VLDL binds its receptor syndrome.
on the hepatic cytomembrane. This also leads to the decline In recent years, it has been found that ApoCIII is the
of effects of binding with VLDL and reduced internalization molecular connection of blood lipid disorders, metabolic
and removal of plasma VLDL. Similar to ApoCII, ApoCIII syndrome, and the characteristics of CVD. The concentra-
also inhibits the biological functions of LCAT and tion of ApoCIII (particularly in lipoproteins containing
CETP. The increase in the concentration of ApoCIII in ApoB) in plasma may be more appropriate as a predictive
plasma is usually accompanied by early-stage and medium- factor for progression of CVD than the concentration of tria-
stage hypertriglyceridemia. cylglycerol in plasma. An increase in the concentration of
Table 15.4 Common causes of anomalies of ApoAII, ApoCII, ApoCIII, and ApoE
Type Apo A II ApoC II Apo C III Apo E
Type I hyperlipoproteinemia Decrease Significant increase Significant increase Significant increase
Type IIa hyperlipoproteinemia Decrease Normal Normal Normal or increase
Type IIb hyperlipoproteinemia Normal Increase Increase Normal or increase
Type III hyperlipoproteinemia Normal Significant increase Significant increase Significant increase
Type IV hyperlipoproteinemia Normal Significant increase Significant increase Increase
Type V hyperlipoproteinemia Normal Significant increase Significant increase Significant increase
acute hepatitis Decrease Normal Decrease Significant increase
liver cirrhosis Decrease Decrease Decrease Significant increase
obstructive jaundice Significant decrease Increase Increase Significant increase
ApoCIII is a common characteristic of patients with insulin phatidylinositol (GPI) binds with the antibody of protein 1
resistance and obesity. Regulating ApoCIII metabolism may (GPIHBP1), which may lead to hypertriglyceridemia and
be one of the important targets for controlling the blood lipid severe non-familial hyperchylomicronemia syndrome [10].
anomalies in patients with metabolic syndrome and CVD. A case report from Japan found that the anti-ApoCII anti-
ApoCIII promotes various types of inflammation and ath- body was identified in patients with severe hypertriglyceri-
erosclerosis by activating vascular endothelial cells and demia uncorrelated with myeloma. An in vitro analysis
mononuclear cells. The expression of adhesion molecules on demonstrated that the antibody damages the interactions
endothelial cells is increased to activate this function and the between ApoCII and lipid substrate, suggesting an etiologi-
nuclear transcription factor NF-kB. NF-kB is one of the key cal role of the anti-ApoCII antibody in severe hypertriglyc-
regulatory factors in the inflammatory reaction of eridemia [11].
atherosclerosis. Overexpressed ApoCIII inhibits the hydrolysis of
The distribution of ApoCIII in various types of lipopro- VLDL-TG and leads to hypertriglyceridemia in human
teins regulates lipoprotein metabolism and further influences ApoCII transgenic mice. This suggests that overexpression
the occurrence of atherosclerosis (Table 15.4). In clinical of ApoCIII leads to hypertriglyceridemia. Some studies have
studies, it has been observed that the percent decrease in revealed that decreased ApoCIII levels in the circulation are
ApoCIII in the plasma ApoB lipoprotein in patients with good for CVD. Research demonstrated that ApoCIII
myocardial infarction was significantly higher than that of decreases triglycerides primarily by inhibiting TRL particles
the control group. Elevated levels of ApoCIII lead to anoma- and the removal of LDLR and LRP1. Volanesorsen can
lies of protein structure in HDL and metabolic disorders of effectively reduce triglyceride levels in plasma [12]. A study
HDL, further promoting atherosclerosis. showed that the existence of ApoCIII in HDL influences the
capacity of cholesterol excretion, suggesting a substitution
effect of ApoCIII in development of atherosclerosis [13].
15.5.4 Related Progress In recent years, a new type of ApoCIV has been found;
however, human plasma does not contain ApoCIV. The
All subtypes of ApoC participate in regulation of lipoprotein ApoCIV gene is expressed in a low level in the liver, suggest-
metabolism and notably play an important role in catabolism ing that ApoCIV may play a minor role in lipoprotein metab-
of TG-rich lipoprotein in plasma. Animal experiments have olism. ApoCIV may also influence the removal of VLDL TG
shown that overexpression of ApoCI in transgenic mice may by inhibiting the hydrolysis of VLDL. This is similar to the
lead to hyperlipidemia, suggesting that overexpression of effects of ApoCII and ApoCIII on VLDL.
ApoCI may significantly inhibit the ingestion of VLDL by
the liver. Endogenous ApoCI deficiency has the same func-
tion; however, the latter has a weaker inhibitory function. 15.6 Apolipoprotein E
ApoCI may degrade its capacity to ingest VLDL by directly
acting on the hepatic lipoprotein receptor or by indirectly 15.6.1 Sources and Characteristics
inhibiting the ingestion of VLDL in the liver by replacing
ApoE in VLDL. Apolipoprotein E is an alkaline protein rich in arginine. It is
ApoCII is associated with VLDL regulation. ApoCII is a primarily found in the VLDL, CM, CM residues, and some
physiological activator of lipoprotein lipase; however, exces- high-density lipoprotein subtypes. It is a ligand of the LDL
sive ApoCII in VLDL may inhibit the activity of lipoprotein receptor, VLDL receptor, and LDL receptor-related protein.
lipase, thereby inhibiting VLDL degradation. High-density Mature ApoE contains 299 amino acids with a relative
lipoprotein anchored by LPL, ApoCII, and glycosyl phos- molecular weight of 34.20 kDa. ApoE is primarily synthe-
sized in the liver and brain. ApoE is produced by astrocytes nostic criteria include elevated levels of total cholesterol and
in the central nervous system and is synthesized by macro- triglycerides.
phages in the peripheral nervous system. ApoE may influ- ApoE plays an important role in lipid transport and its
ence plasma lipoprotein metabolism by regulating the polymorphism is one of the decisive factors for several types
storage and redistribution of cholesterol and its lipid sub- of human diseases. Apo E is closely associated with levels of
stances. Meanwhile, ApoE participates in tissue repair, inhi- individual blood lipids as well as coronary artery disease and
bition of platelet aggregation, immunoregulation, inhibition cerebral vascular sclerosis. Various subtypes have signifi-
of cell proliferation, pathological, and physiological pro- cantly varying physiological functions.
cesses of senile dementia, and plays a role in the process of ApoE participates in cephalin metabolism, and maintains
normal growth of the nervous system and the process of redistribution of intracellular lipids and the stability of the
repair after its injury. ApoE participates in lipid metabolism environment within cholesterol. It is also involved in neural
in the liver, binds with several receptors (VLDL, LDL recep- development, activities of lipids during repair and regenera-
tor, sulfuric acid acetyl heparin proteoglycan, and VLDL tion of injured nerve cells, immunoregulation of lipids, and
receptor-related protein), and influences the role of TRL in cellular regulation.
hydrolysis of the residue lipoprotein constituting atheroscle- ApoE regulates bone metabolism as an important link to
rotic plaques. Meanwhile, VLDL and the chyle particle resi- lipid metabolism. Its genetic diversity influences the patho-
due synthesized in the intestine after meals further provide genesis and medication efficacy of post-menopause meta-
endogenous lipids for the body with ApoE as the carrier bolic disturbances. It induces bidirectional differentiation of
point. marrow mesenchymal stem cells via the Wnt/β-catenin sig-
The ApoE gene has polymorphisms. The polymorphism nal pathway, the antagonizing effect of PPARγ, and influ-
is found in the exchange between the cysteine (Cys) at site ences the levels of serum osteocalcin, thereby regulating
112 of the amino acid sequence and the arginine (Arg) at site bone quality.
158. ApoE2 has Cys in the two positions and ApoE4 has Arg Evidence has shown that the patients carrying the ApoE
in the two positions. ApoE3 has Cys and Arg, respectively. ε allele have a higher risk of Alzheimer’s disease (AD) and
The ApoE isomer is controlled by three alleles at one genetic their ages of onset tend to be younger. The risk of AD
point. The three alleles are ε2, ε3, and ε4. The polymorphism depends on the level to which ApoE ε is expressed. The age
leads to six genetic phenotypes in the population, i.e., three of AD onset decreases with the increase in the expression
heterozygotes (E2/3, E3/4, and E2/4) and three homozygotes of ε4.
(E2/2, E3/3, and E4/4). ε3, with ApoE3/3 being the most
common allele and genetic phenotype.
15.6.4 Related Progress
15.6.2 Detection Methods and Reference Research has indicated that the ApoE genotype is among the
Values of Healthy Individuals decisive factors for concentrations of blood lipids. Common
ApoE polymorphisms are responsible for 7% of the mutation
15.6.2.1 Detection Methods of serum cholesterol and low-density lipoprotein cholesterol
Quantitative measurement of ApoE in serum requires immu- in humans, increasing the risk of death in patients with coro-
nological transmission turbidimetry. nary artery disease [6]. Substituting saturated fatty acid for
ApoE genotype measurement requires PCR-RFLP. chemical compounds with a low blood glucose index in indi-
ApoE phenotype measurement uses immunoblotting after viduals carrying the ApoE4 gene polymorphism demon-
isoelectric focusing. strates benefits in reducing low-density lipoprotein
cholesterol [14]. ApoE4 levels correlate with insulin resis-
15.6.2.2 Reference Values of Healthy tance and metabolic syndrome. Scholars have identified that
Individuals ApoE4 influences insulin resistance and levels of ApoCII
• Serum ApoE: 27.7–55.9 mg/L. and ApoCIII via the profile of plasma fatty acid. Therefore,
patients with metabolic syndrome may benefit from person-
alized diet intervention based on the ApoE genotype [15].
15.6.3 Clinical Significance One study showed that the ApoE4 allele was closely asso-
ciated with the occurrence of Alzheimer’s disease. A total of
ApoE may be used to assess the risk of occurrence of cardio- 30–50% of the patients with Alzheimer’s disease were
vascular diseases. The ε2/ε2 genotype is a high-risk type that ApoE4 gene carriers while the percentage was 14–16% in
particularly leads to Type III hyperlipoproteinemia. Its diag- normal populations. In terms of pathology, individuals
c arrying the ApoE4 allele have more amyloid protein depos- 15.7.3 Clinical Significance
iting in the brain and a higher risk of disease onset [16].
ApoE has an immunoregulation function. ApoE immuno- Apolipoprotein H is closely associated with risk factors for
regulation receptors are found on the surface of lymphocytes. stroke, including antiphospholipid antibody syndrome,
Lipoprotein containing ApoE is able to antagonize factors hypertension, blood lipid metabolism disturbances, and
stimulating the division of lymphocytes. This is likely abnormal coagulation mechanisms. The ApoH gene has
because the binding between lipoprotein and its receptor hereditary polymorphisms and its phenotype influences lipid
inhibits the early-stage transformation required for activation metabolism, hypertension, and the generation of antiphos-
of lymphocytes. pholipid antibodies.
Serum ApoH is used to diagnose cardiovascular diseases
and assess risk. Genetic polymorphism analyses are used to
15.7 Apolipoprotein H predict disease risk. For example, the high morbidity of cere-
bral hemorrhage is associated with the polymorphism of No.
15.7.1 Sources and Characteristics 3 exon G341A (Ser88Asn) of the ApoH Gene 3. The poly-
morphism of the ApoH gene G341A (Ser88Asn) may act as
Apolipoprotein H, also known as β2 microglobulin, was first one of the indicators for hereditary susceptibility and can be
discovered by Schultze and colleagues. ApoH is a glycopro- used for predicting individuals susceptible to cerebral hem-
tein, and the molecular weight is 50.00 kDa. It is a single orrhage and preventing cerebral hemorrhage in an early
polypeptide chain composed of 326 amino acid residues. stage.
Among the constituents of amino acid, proline (pro) has the
highest content, followed by cysteine (cys). Of the total apo-
lipoprotein H in serum, about 16% is found in chylomicrons 15.7.4 Research Progress
and VLDL, 2% in LDL, 17% in HDL, and the remaining
65% in the underlying lipoprotein components. ApoH in The physiological functions of ApoH remain unclear. Some
plasma participates in the metabolism of such lipids as tri- studies demonstrated that ApoH correlates with cell signal-
glycerides and cholesterol and is associated with AS and ing and hypercoagulability in patients with certain diseases.
many cerebral vascular diseases. ApoH also functions in The anti-ApoH antibody has several types of biological
coagulation. activity after it binds to ApoH. In the presence of the anti-
Human ApoH has polymorphisms. It is encoded by four ApoH antibody, the structural domain of the I-IV zone of
alleles at a single genetic point. The ApoH gene is located on ApoH demonstrates an open conformation, gradually
chromosome 17. The three most common alleles have six strengthening the binding between zone V and the receptors
phenotypes: three homozygote phenotypes, H1/l, H2/2, and on the cell surface, activating the intracellular downstream
H3/3, and three heterozygote phenotypes, H2/1, H3/1, and signal of the cell surface receptors [17]. Studies have shown
H3/2. ApoH2 is the most common allele. ApoH4 is only that ApoH has a physical relationship with many types of
present in black individuals at a frequency of 0.11%. plasmid membrane receptors, including TLR4, TLR2, and
Currently, it is considered to be a unique marker of black possibly the apolipoprotein E receptor 2. Under the stimula-
individuals. tion of the ApoH/antibody complex, membrane phospholip-
ids may be an essential substance for TLR signal transduction
and cell activation [18, 19]. A clinical study found a potential
15.7.2 Detection Methods risk of thrombosis in ApoH IgG titers and is associated with
thrombosis in lupus and pregnancy [20].
15.7.2.1 Detection Methods
Quantitative measurement of ApoH in serum requires immu-
nological transmission turbidimetry. 15.8 Apolipoprotein M
ApoH genotype measurement usually involves
pCR-RFLp. 15.8.1 Sources and Characteristics
ApoH phenotype measurement may use the post-
electrofocusing immunoblotting method. Apolipoprotein M (ApoM) was recently described. It is a
hydrophobic molecule binding protein. ApoM is found in all
15.7.2.2 Reference Value of Healthy Individuals mammals with an extremely high degree of homology.
• Serum ApoH: 185–327 mg/L. ApoM is synthesized and secreted by parenchymal cells such
as hepatocytes and epithelial cells of kidney tubules. It can The negative ion protein may be the receptor of
be glycosylated and has a relative molecular weight of about ApoM. ApoM enters proximal tubule epithelial cells by
24.00 kDa. The comparative analysis of amino acid binding to its surface proteins. Platelet-activating factor
sequences of the apolipoprotein D (ApoD) and ApoM indi- (PAF) is a pro-inflammatory active phospholipid medium
cates that ApoM and ApoD both belong to the hydrophobic that promotes aggregation of platelets and release of neutro-
molecular binding protein family and can bind with and phil granulocytes, thereby producing a large amount of
transport hydrophobic bioactive factors in plasma. ApoM is inflammatory reaction media. ApoM may participate in
primarily found in lipoproteins. High-density lipoprotein inflammatory reactions via PAF. The expression of ApoM in
(HDL) has the highest content of ApoM, followed by the diabetic patients tends to be low, suggesting that high levels
triacylglycerol apolipoprotein (TGRLP). Low-density lipo- of blood glucose, insulin resistance, or insulin deficiency
protein (LDL) has the lowest content of ApoM. Furthermore, may influence the expression of ApoM.
levels of ApoM in plasma are lower than those of other apo-
lipoproteins. The human ApoM gene is located on the short
arm of chromosome 6 (6p21.3) with a length of about 750 15.8.4 Research Progress
base pairs, comprising six exons and five introns, encoding
188 amino acid residues. Studies of the promoter genes and In recent years, it has been found that ApoM/S1P (ApoM
the genetic structure indicate that expression of Apo M is tis- ligand, 1-phosphoric acid sphingosine) regulates particle
sue specific. ApoM is only highly expressed in human hepa- size and functions of HDL. ApoM gene mutations are
tocytes and epithelial cells of kidney tubules and is rarely associated with elevated risks of coronary artery disease,
expressed in other tissues and organs. The synthetic route of rheumatoid arthritis, and diabetes. The bioactive lipid s1p
ApoM in the liver remains unclear. The expression of ApoM influences angiogenesis, transport of lymphocytes, migra-
in the liver indicates that ApoM is associated with the trans- tion of endothelial cells, and inflammation. A drug for the
port and metabolism of blood lipids and may correlate with s1p system (fingolimod) is currently used to treat multiple
the generation of HDL and VLDL. The physiologic signifi- sclerosis. It improves the function of the blood–brain bar-
cance of ApoM expressed in kidney tubules remains unclear. rier and inhibits migration of lymphocytes into the brain.
The ApoM/S1P axis plays an important role in diabetes
and the metabolism of insulin. Studies have shown that
15.8.2 Detection Methods and Reference overexpression of ApoM promotes formation of large HDL
Values of Healthy Individuals particles rich in ApoM/S1P by promoting formation of
large new HDL particles and stimulating the synthesis of
15.8.2.1 Detection Methods sphingolipid and secretion of ApoM/S1P. These unique
The ApoM plasma measurement uses the ELISA method. HDL particles may transport S1P to extrahepatic tissue
offering protection from atherosclerosis, among other
15.8.2.2 Reference Values of Healthy functions [21].
Individuals
Serum ApoM:
References
• 0.58–1.18 μmol/L (For women 18–49 years)
1. Dieplinger H, Ankerst DP, Burges A, et al. Afamin and apolipo-
• 0.61–1.30 μmol/L (For men and women 50+ years) protein A-IV: novel protein markers for ovarian cancer. Cancer
Epidemiol Biomarkers Prev. 2009;18:1127–33.
2. Ehmann M, Felix K, Hartmann D, et al. Identification of potential
15.8.3 Clinical Significance markers for the detection of pancreatic cancer through comparative
serum protein expression profiling. Pancreas. 2007;34:205–14.
3. Mao M, Wang X, Sheng H, et al. A novel score based on serum apo-
ApoM plays a special role in lipid metabolism and may par- lipoprotein A-1 and C-reactive protein is a prognostic biomarker in
ticipate in transport of cholesterol or other hydrophobic mol- hepatocellular carcinoma patients. BMC Cancer. 2018;18:1178.
ecules with bioactivity. 4. Li C, Li H, Zhang T, et al. Discovery of Apo-A1 as a potential blad-
der cancer biomarker by urine proteomics and analysis. Biochem
The reason why HDL has an anti-atherosclerotic role is that Biophys Res Commun. 2014;446:1047–52.
it promotes outflow of cholesterol from cells. In the reverse 5. Lin T, Wang L, Guo J, et al. Association between serum LDL-C and
transport of cholesteryl ester, HDL transports cholesterol from ApoB and SYNTAX score in patients with stable coronary artery
peripheral cells to the liver; finally, cholesterol is transformed disease. Angiology. 2018;69:724–9.
6. Wilson PW, Schaefer EJ, Larson MG, et al. Apolipoprotein E
into bile acid and is discharged from the biliary tract, thereby alleles and risk of coronary disease. A meta-analysis. Arterioscler
maintaining normal level of cholesterol in blood. Thromb Vasc Biol. 1996;16:1250–5.
7. Gao J, Choudhry H, Cao W. Apolipoprotein B mRNA edit- wild type (E3/E3), after replacement of dietary saturated fats with
ing enzyme catalytic polypeptide-like family genes acti- low glycaemic index carbohydrates. Nutrients. 2018;10:1524.
vation and regulation during tumorigenesis. Cancer Sci. 15. Fallaize R, Carvalho-Wells AL, Tierney AC, et al. APOE genotype
2018;109:2375–82. influences insulin resistance, apolipoprotein CII and CIII according
8. Zhang F, Xie Y, Ma X, et al. Preoperative apolipoprotein B/A1 ratio to plasma fatty acid profile in the metabolic syndrome. Sci Rep.
is an independent prognostic factor in metastatic renal cell carci- 2017;7
noma. Urol Oncol Semin Orig Invest. 2019;37:184.e189–17. 16. Rebeck GW. The role of APOE on lipid homeostasis and inflamma-
9. Wolska A, Dunbar RL, Freeman LA, et al. Apolipoprotein C-II: tion in normal brains. J Lipid Res. 2017;58:1493–9.
New findings related to genetics, biochemistry, and role in triglyc- 17. de Groot PG, Meijers JC. beta(2)-Glycoprotein I: evolution, struc-
eride metabolism. Atherosclerosis. 2017;267:49–60. ture and function. J Thromb Haemost. 2011;9:1275–84.
10. Reichlin M, Fesmire J, Quintero-Del-Rio AI, et al. Autoantibodies 18. Chaturvedi S, McCrae KR. Recent advances in the antiphospho-
to lipoprotein lipase and dyslipidemia in systemic lupus erythema- lipid antibody syndrome. Curr Opin Hematol. 2014;21:371–9.
tosus. Arthritis Rheum. 2002;46:2957–63. 19. Meroni PL, Borghi MO, Raschi E, et al. Pathogenesis of antiphos-
11. Yamamoto H, Tanaka M, Yoshiga S, et al. Autoimmune severe pholipid syndrome: understanding the antibodies. Nat Rev
hypertriglyceridemia induced by anti-apolipoprotein C-II antibody. Rheumatol. 2011;7:330–9.
J Clin Endocrinol Metab. 2014;99:1525–30. 20. Banzato A, Pozzi N, Frasson R, et al. Antibodies to domain I of
12. Ramms B, Gordts PLSM. Apolipoprotein C-III in triglyceride-rich beta(2)Glycoprotein I are in close relation to patients risk cat-
lipoprotein metabolism. Curr Opin Lipidol. 2018;29:171–9. egories in Antiphospholipid Syndrome (APS). Thromb Res.
13. Luo M, Liu A, Wang S, et al. ApoCIII enrichment in HDL impairs 2011;128:583–6.
HDL-mediated cholesterol efflux capacity, Sci Rep. 2017;7 21. Borup A, Christensen PM, Nielsen LB, et al. Apolipoprotein M in
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benefit in lowering plasma cholesterol and apolipoprotein B than 2015;26:48–55.
Coagulation and Fibrinolysis
16
Hong Wang and Yun Ling
16.1 Overview body constantly in a liquid state and maintaining smooth

flow within the pipe. The fibrinolytic system includes fibri-
In the normal person, the hemostatic system is in a corre- nolytic enzymes, activators, and inhibitors of plasmin. The
spondingly balanced state, because the vascular endothelial basic process of fibrinolysis can be divided into two phases:
cells and the blood (including the inactivated clotting factors) activation of plasminogen and degradation of fibrin. Several
function to maintain fluidity. When patients encounter infec- commonly used coagulation and fibrinolysis biomarkers are
tion, inflammation, malignant carcinoma, vascular injury, shown in Table 16.1.
trauma, or surgery, this balanced state will be destroyed,
which may trigger platelet activation, coagulation cascades,
anticoagulation, and fibrinolysis. 16.2 Fibrinogen
Blood coagulation, referred to as coagulation, is a pro-
cess in which blood changes from a flowing state to a gel 16.2.1 Sources and Characteristics
state, and it is indispensable to the hemostatic system. Series
of proteins with coagulation function (zymogen, coenzyme) Fibrinogen, also clotting factor I, is a glycoprotein composed
and calcium ions, exist in blood, constituting the coagulation by the liver and has a molecular weight of about 340.00 kDa.
system. The coagulation process refers to a cascade reaction Fibrinogen includes three pairs of polypeptide chains
in which a coagulation factor is activated under certain con- (Fig. 16.2): two Aα, two Bβ, and two γ. They are connected
ditions (the zymogen converts to an enzyme with proteolytic together by 29 disulfide bonds. The fibrinogen structure is
activity), and activation of various factors of the coagulation composed of E-domain (N-terminal regions) and D-domain
system, called tissue factor pathway, contact factor pathway, (C-terminal regions).
and common pathway, occur sequentially to produce throm- The concentration of fibrinogen in plasma is about
bin. As a result, soluble fibrinogen transforms into insolu- 2.0–4.0 g/L, which is one of the most abundant proteins in
ble fibrin, which is the last step of coagulation cascades plasma. Under the activation of thrombin, the amino ter-
(Fig. 16.1). The anticoagulant system refers to the process by minus of the fibrinogen alpha chain and beta chain release
which humoral factors in plasma inactivate clotting factors fibrin peptide A (FPA) and fibrin peptide B (FPB) frag-
having been activated by inhibiting or hydrolyzing. The fibri- ments, which promote the formation of fibrin monomers.
nolytic system is actually part of the anticoagulation system. Due to the decrease in electronegativity, the fibrin mono-
The process that fibrin formed during blood coagulation is mers aggregate spontaneously, and its structural changes
decomposed and liquefied, called fibrin dissolution. Related make it possible to coagulate with factor XIIIa, platelet
chemical substances involved in the fibrinolysis process con- glycoprotein GpIIa/IIIb, Ca2+, plasmin, tissue plasminogen
stitute the fibrinolytic system. Fibrinolysis is an important activator (t-PA), and so on, thus participating in the process
anticoagulant process in the body. Like the blood coagula- of coagulation and hemostasis in the body. Studies have
tion process, it is also a protective physiological response found that when the amino acid residues at the combining
of the body. It plays a major role in keeping blood in the site of fibrinogen are mutated, the function of fibrinogen
could be affected, which may be clinically characterized
by hemorrhage, thrombosis, or asymptomatic state. So far,
H. Wang (*) · Y. Ling more than 300 natural variants of fibrinogen have been
Department of Laboratory Medicine, The First Affiliated Hospital found, of which about 55% are asymptomatic, 25% have
bleeding, and 20% are thrombotic.

208 H. Wang and Y. Ling
Blood vessel
Injury
Intrinsic pathway Extrinsic pathway
Factor XII Exposed

Prekallikrein Circulation Tissue factor
HMWK Factor VIIa Anionic membrane
“ Surface” Ca2+
Factor x
AT FXIa Intrinsic tenase Extrinsic tenase Factor IX
TFPI
Factor IXa AT Factor VIIa
Factor VIIIa Tissue factor
Factor IX FIXa Anionic membrane FIXa Anionic membrane Factor X
Ca2+ Ca2+
FXa FXa
FXIa AT Prothrombinase
AT
TFPI
Factor Xa AT Fibrinogen
Factor II Factor Va
Anionic membrane
Ca2+ Factor XIII
Activated platelets
Factor XI
AT FIIa FIIa
FIIa
(FXIIa) FXIIIa
Thrombin
FVa
Thrombomodulin Crosslinked
FVIIIa APC TAFIa
Anionic membrane fibrin
Ca2+
protein Case
Protein C TAFI
Fig. 16.1 Overview of hemostasis
16.2.2 D
etection Method and Reference tissue disease, acute nephritis and uremia, post-radiation
Range therapy, burns, myeloma, shock, after major surgery, late
pregnancy and pregnancy-induced hypertension, mild hepa-
• Clauss method: 2.0–4.0 g/L. titis, sepsis, acute infections, and malignant tumors.
Reduced fibrinogen (less than 1.5 g/L) can occur in: dis-
seminated intravascular coagulation and primary fibrinolysis,
16.2.3 Clinical Significance severe hepatitis and cirrhosis, dysfibrinogenemia, neonatal
and preterm infants, certain obstetric accidents, malignancy
Increased fibrinogen (more than 4.0 g/L) can occur in: dia- tumors, the treatment of defibrillation drugs (such as anti-
betes and diabetic acidosis, arterial thromboembolism (acute thrombotic enzymes, defibrase), and thrombolytic therapy
myocardial infarction), acute infectious diseases, connective (UK, t-PA).
16 Coagulation and Fibrinolysis 209
Table 16.1 Several commonly used coagulation and fibrinolysis biomarkers

Reference
Biomarkers Clinical significance Detection method range References
Fibrinogen Increase: Diabetes and diabetic acidosis, arterial thromboembolism Clauss method 2.0–4.0 g/L [1, 2]
(acute myocardial infarction), acute infectious diseases, connective
tissue disease, acute nephritis, and uremia
Decrease: Disseminated intravascular coagulation (DIC), primary
fibrinolysis, severe hepatitis and cirrhosis, dysfibrinogenemia, neonatal
and preterm infants, certain obstetric accidents, malignancy tumors, the
treatment of defibrillation drugs, and thrombolytic therapy
D-dimer Increase: hypercoagulable state, DIC, kidney diseases, organ transplant LAT <0.5 mg/L [3]
rejection, various thrombotic and thrombolytic disorders, thrombolytic
therapy
FDP Increase: Primary fibrinolysis, secondary fibrinolysis, hypercoagulable LAT <5.0 mg/L [4, 5]
state, DIC, renal disease, organ transplant rejection, and thrombolytic
therapy
PC Increase: Prethrombotic states and thrombotic diseases such as deep Chromogenic 70–140% [6]
vein thrombosis, DIC, and acute myocardial infarction method
Decrease: Congenital protein C deficiency and acquired protein C
reduction, such as liver disease, uremia and major surgery
Antithrombin Increase: Certain hemorrhagic conditions, such as hemophilia, aplastic Chromogenic 80–120% [7, 8]
anemia, heart valve disease (heart failure with liver enlargement) method
Decrease: DIC, renal diseases, cirrhosis, septicemia, thrombotic
diseases
α2-Plasmin Increase: Arterial and venous thrombosis, postpartum, malignant Chromogenic 80–120% [9, 10]
inhibitor tumors method
Decrease: Hyperfibrinolysis, live diseases, DIC, and congenital α2-PI
deficiency
von Willebrand Increase: myocardial infarction, glomerulopathy, DM, pregnancy- Latex VWF:Ag: [11]
factor induced hypertension, and post-major surgery agglutination- 50–200%
Decrease: VWD scattering
turbidimetry
Ristocetin Cofactor vWF:RCo: [12]
assay 50–150%
Thrombomodulin Increase: SLE, diabetes, rheumatoid arthritis, leukemia, multiple RIA method Ag: [13]
myeloma, DIC 232–352 ng/
mL
Chromogenic Activity: [14]
method 87–113%
TFPI Increase: Elderly, pregnancy, fatal sepsis, and chronic renal failure ELISA 44.3– [15]
Decrease: Prethrombotic state and thrombotic diseases, DIC, major 151.0 μg/L
surgery, and sepsis Chromogenic 78–154%
method
Fig. 16.2 Structure of Domain D Domain E Domain D

fibrinogen
C–terminus C–terminus
Aα
Bβ
γ
Fibrinopeptide A
Fibrinopeptide B
16.3 D-Dimer 16.3.3 Healthy Person Reference Range
16.3.1 Sources and Characteristics Importantly, the reference range varies with the tests used,
and there is no international D-dimer standard. Moreover,
Fibrin degraded by plasmin releases fragments of vari- every laboratory should build its own cut-off value to
ous sizes (Fig. 16.3), of which the smallest is the D-dimer exclude VTE.
(MW = 180.00 kDa) and the largest fragment is XXD, X=D−
E−D, with a mass of 595.00 kDa. D-dimer is a specific maker
of fibrinolytic process. The determination of plasma D-dimer 16.3.4 Clinical Significance
is an experiment to understand the function of secondary
fibrinolysis. In 1983, Elms and colleagues first reported Elevated D-dimer level can be found in secondary fibrino-
the preparation of monoclonal antibodies against D-dimers lysis, such as disseminated intravascular coagulation (DIC),
using D-dimer antigens. At present, the most popular anti-D- hypercoagulable states, deep venous thrombosis, pulmonary
dimer monoclonal antibodies in the world are D-DlC3/108, embolism, various thrombotic organ transplant rejection
D-D3B6/22, D-D13, and MA-15C5. These antibodies not and thrombolytic disorders, thrombolytic therapy, cerebral
only specifically bind to D-dimer (thrombolytic degradation infarction, myocardial infarction, tumor, surgery, infection,
product) in blood, but also to cross-linked fibrin (containing and tissue necrosis.
D-dimer) in thrombus. The successful preparation and appli-
cation of D-dimer monoclonal antibodies have opened up a
novel frontier for the diagnosis and therapy of thrombotic 16.4 Fibrinogen/Fibrin Degradation
diseases. Product
16.4.1 Sources and Characteristics

16.3.2 Detection Method
Fibrinogen/Fibrin Degradation Product (FDP) is com-
There are more than 30 different methods for D-dimer posed of all degradation products of fibrinogen and/or
detection, and the antibodies are for different epitopes of fibrin. Plasminogen is activated into plasmin, which cleaves
D-dimer. fibrin clot and/or fibrinogen to generate a series of soluble
D E D D E D D E D
E D D E D D E D D E
D E D D E D D E D
E D D E D D E D D E
D E D D E D D E D
E D D E D D E D D E
Plasmin
D D
D dimers
E D D
D D
D D D D
Fig. 16.3 Degradation of cross-linked fibrin

f ragments of different molecular weights. All these products 16.5.2 Detection Method
are called FDPs.
Chromogenic assay is the generally used method. Patient’s
platelet poor plasma is incubated with the PC activator at
16.4.2 Detection Method 37 °C for 5 min. Then a chromogenic substrate for APC is
added. The optical density changes are captured and trans-
• Latex agglutination test (LAT). ferred into corresponding PC level.
16.4.3 Healthy Person Reference Range 16.5.3 Healthy Person Reference Range
• LAT method: <5.0 mg/L. • Chromogenic method: 70–140%.
16.4.4 Clinical Significance 16.5.4 Clinical Significance
FDP is increased in primary fibrinolysis, secondary fibrino- Elevation of PC is more common in prethrombotic states and
lysis, renal disease, hypercoagulable state, organ transplant thrombotic diseases such as deep vein thrombosis, DIC, and
rejection, thrombolytic therapy, disseminated intravascular acute myocardial infarction.
coagulation, vascular embolism (pulmonary embolism, myo- Reduction of PC is more common in congenital PC defi-
cardial infarction, occlusive cerebrovascular disease, deep ciency and acquired PC reduction, such as liver disease, ure-
venous thrombosis), post-leukemia chemotherapy induction mia, and major surgery.
period, hemorrhagic thrombocytosis, uremia, liver diseases,
or various tumors.
16.6 Protein S
16.5 Protein C 16.6.1 Sources and Characteristics
16.5.1 Sources and Characteristics Protein S (PS), a vitamin K-dependent glycoprotein, is

also a member of the natural anticoagulant PC system. In
Protein C (PC) is a vitamin K-dependent serine protease, and the inactivation of factors Va and VIIIa, PS is the cofactor
is a core component of the natural anticoagulant PC system. of APC. PS also has the APC-independent anticoagulant
It is transferred by thrombin to its active form, activated pro- activity by direct binding to factor Va, factor Xa, and factor
tein C (APC). APC, with its cofactor protein S, can degrade VIIIa.
factor VIIIa and factor Va. PS has a similar structure with PC (Fig. 16.5). PS consists
PC includes 9–13 Gla residues (NH2-terminal Gla of two forms, bound and free. About 65% is the bound form
domain) and an epidermal growth factor (EGF)-like domain without activity. Only the free form is with anticoagulant
(Fig. 16.4). activity.
Fig. 16.4 Schematic S S
representation of PC structure
NH2 Gla EGF1 EGF2 AP Serine protease COOH
R169–L170
Fig. 16.5 Schematic
representation of PS structure
SHGB
NH2 Gla TSR EGF1 EGF2 EGF3 EGF4 COOH
domain
16.6.2 Detection Method 16.7.2 Detection Method
Both functional and immunological assays can be used for Chromogenic method: AT in the sample and thrombin in
PS detection. Functional assays measure only free PS. the reagent form thrombin–antithrombin complex. Then the
residual thrombin binds with the substrate in the reagent. The
• Total PS antigen: ELISA and radio-immunoassays meth- absorbance change is detected at the wavelength of 405 nm.
ods are usually used. The concentration of AT in the sample can be calculated
• Free PS: Most laboratories adopt methods for free PS according to the standard curve.
activity detection. The principle includes a process of
C4bBP- bound PS removement and the detection of
remaining unbound PS. 16.7.3 Healthy Person Reference Range
• AT activity: 80–120%.
16.6.3 Healthy Person Reference Range
• Free PS: 80–120%. 16.7.4 Clinical Significance
Increased AT activity: Certain hemorrhagic conditions, such

16.6.4 Clinical Significance as hemophilia, aplastic anemia, and heart valve disease.
After taking an anticoagulant, the activity of AT increases
Elevation of PS is more common in prethrombotic states and and returns to normal after stopping.
thrombotic diseases such as deep vein thrombosis, DIC, and Reduced AT activity: Disseminated intravascular coagula-
acute myocardial infarction. tion, renal diseases, cirrhosis, septicemia, thrombotic diseases
Reduction of PS is more common in congenital PS defi- (myocardial infarction, venous thrombosis, and so on), con-
ciency and acquired PS reduction, such as liver disease, ure- genital contraceptive deficiency, oral administration of AT.
mia, and major surgery.
16.7 Antithrombin 16.8 Plasminogen
16.7.1 Sources and Characteristics 16.8.1 Sources and Characteristics
The anticoagulant system is a counterbalance to the coag- Plasminogen (PLG) is synthesized in the liver and has
ulation system in the human body. Antithrombin (AT), an two forms: Lys-Plasminogen and Glu-Plasminogen. Glu-
inhibitor of the serine proteases, is the most important Plasminogen contains a glutamic acid residue at the N-terminal
anticoagulant component. Most of its target proteases are region. Glu-Plasminogen is activated by cleavage at Arg561
the activated clotting factors involved in the coagulation and Val562 to produce Glu-Plasmin, which is composed of
cascade, such as factor Xa, factor IXa, factor VIIa, fac- two chains. The heavy chain has the lysine binding sites, and
tor XIa, factor XIIa, kallikrein, and HMWK. When AT is the light chain has the serine protease activity.
combined with heparin (Fig. 16.6), the rate of inhibition
of these clotting factors is accelerated by up to 10,000-
fold. Determining the activity of AT in plasma can be used 16.8.2 Detection Method
for the diagnosis of diseases with increased or reduced
synthesis of AT and monitoring the effect of replacement Plasminogen activity can be determined according to its abil-
therapy. ity to turn into plasmin (activated by either streptokinase or
Fig. 16.6 Schematic S S
representation of AT structure Serine protease
S S target site
NH2 CHO domain COOH
S S
Heparin
binding
urokinase). And then the functional activity of plasmin can be 16.9.4 Clinical Significance
measured by using a variety of substrates.
Increased α2-PI activity is seen in arterial and venous throm-
bosis, postpartum, malignant tumors. Decreased α2-PI activ-
16.8.3 Healthy Person Reference Range ity is found in hyperfibrinolysis, liver disease, DIC, and
congenital α2-PI deficiency.
• PLG activity: 75–150%.
16.10 Von Willebrand Factor

16.8.4 Clinical Significance
Increased PLG is seen in arterial and venous thrombosis.
Plasminogen is an acute phase protein and the increased lev- Von Willebrand factor (VWF) is a glycoprotein synthe-
els occur in infection, trauma, inflammation, and malignancy. sized and secreted by vascular endothelial cells and mega-
Decreased PLG: Can be seen in hyperfibrinolysis, heredi- karyocytes. VWF is a large multimer and can be degraded
tary deficiencies, DIC, liver diseases, during and after throm- to various molecular weight products by the metalloprote-
bolytic therapy, and the newborn infant. ase ADAMTS13 (A Disintegrin-like and Metalloprotease
domain with Thrombospondin type I motifs). Therefore,
the lack of ADAMTS13 leads to the accumulation of high
16.9 α2-Plasmin Inhibitor molecular weight VWF multimers, which is the reason for
thrombotic thrombocytopenic purpura (TTP). VWF exists
16.9.1 Sources and Characteristics in plasma, endothelial cell surface, and platelet α granules.
VWF can interact with collagen and platelet membrane
α2-plasmin inhibitor (α2-PI), a primarily physiological glycoprotein GPIb or GPIIb/IIIa, which plays an important
inhibitor of plasmin, is the important regulation molecular role in platelet adhesion and aggregation. VWF is the car-
of fibrinolysis. α2-PI is synthesized by the liver and is also rier protein of factor VIII and participates in coagulation
secreted by activated platelets. α2-PI can be used as a diag- and hemostasis (Fig. 16.7). VWF gene is located in the
notic marker of arterial and venous thrombosis, malignant short arm of chromosome 12. The deficiency of VWF can
tumors, and α2-PI deficiency. cause von Willebrand disease (VWD).
The plasma α2-PI has two forms: (1) 30% of the α2-PI is
Met-α2-PI, which is a 464-residue protein with methionine at
the N-terminal region, (2) 70% of the α2-PI is Asn-α2-PI, an 16.10.2 Detection Method
N-terminally shortened form in which the N-terminal amino
acid is an asparagine residue. Asn-α2-PI seems to have more VWF antigen (VWF:Ag) can be detected by immuno-
physiological activity than Met-α2-PI. electrophoresis, ELISA or latex agglutination-scattering tur-
bidimetry methods.
The Von Willebrand Ristocetin Cofactor (VWF:RCo)
16.9.2 Detection Method assay gives the information of the ability of platelet aggre-
gation in the presence of the antibiotic Ristocetin, which is
α2-PI can be detected by either immunological or func- related to the plasma VWF concentration and activity.
tional method. α2-PI activity commonly detected by chro-
mogenic substrate method: an excess of plasmin is added to
the plasma to form a complex between α2-PI and plasmin. 16.10.3 Healthy Person Reference Range
The remaining plasmin acts on the chromogenic substrate
(HD-Nva-CHA-Lys-PHA) to release PNA, and the color • VWF:Ag: 50–200%.
intensity is positively correlated with the remaining plasmin • VWF:RCo: 50–150%.
in the plasma. There is a negative correlation between plas-
min and α2-PI in plasma. In CLSI H-51A (2002), it was suggested that each labora-
tory should determine its own VWF reference range according
to different ABO blood types. However, the National Institutes
16.9.3 Healthy Person Reference Range of Health made recommendations (2009) to establish a single
VWF reference range, regardless of ABO blood types.
• α2-PI activity (chromogenic method): 80–120%.
Fig. 16.7 The role of vWF in

hemostasis
Intact Vessel wall
Platelet plug
formation
Platelet Non–activated αIIbβ3 Activated αIIbβ3 Gplbα
VMF collagen
16.10.4 Clinical Significance anti-coagulation function. TM–thrombin complex is also

an activator of PC, which can accelerate the activation of
VWF reduction is mainly used in the diagnosis and classi- thrombin-dependent PC and inactivation of FVa and FVIIa.
fication of VWD. VWF is an acute phase reaction protein, TM is a specific and sensitive molecular marker for endo-
which is frequently seen in thrombotic diseases such as myo- thelial cell damage. TM antigen (TM-Ag) and TM activity
cardial infarction, glomerulopathy, DM, pregnancy-induced are often used in the laboratory to reflect the status of TM
hypertension (PIH), and post-major surgery. in vivo.
16.11 Thrombomodulin 16.11.2 D

etection Method and Healthy
Person Reference Range
• TM-Ag (RIA method): 232–352 ng/mL
Thrombomodulin (TM) is an I-type transmembrane glyco- • TM activity (Chromogenic substrate method): 87–113%
protein that is present on the vascular endothelial cell mem-
brane, which is composed of 554 amino acids and does not
have intrinsic enzymatic activity. In 1982, Naomi L. Esmon 16.11.3 Clinical Significance
and colleagues, for the first time, successfully isolated and
purified TM in the lung tissue homogenate using affinity TM increases in diseases involving vascular endothelial
chromatography. TM–thrombin complex inhibits the inter- damage, such as SLE, diabetes, rheumatoid arthritis, leuke-
action of thrombin with the fibrinogen and the protease- mia, multiple myeloma, and DIC. The decrease of TM activ-
activated receptor-1 (PAR-1), and can execute a direct ity is found in TM deficiency.
16.12 P
lasminogen Activator Inhibitor by GATA-2 elements. The expression of transcription factor
Type 1 GATA-2 is significantly correlated with TFPI. TFPI contains
three Kunitz domains: K1, K2, and K3. TFPI also includes
16.12.1 Sources and Characteristics two connecting domains, an N terminal rich in acidic amino
acids and a C terminal rich in basic amino acids. Domains
Plasminogen Activator Inhibitor Type 1 (PAI-1) is a serine K1 and K2 are the key sites for TFPI to exert anticoagulant
protease inhibitor, which is mainly secreted by endothelial activity. K3 is not necessary for this role, but its existence can
and adipose cells. PAI-1 is the main inhibitor of t-PA (tis- make TFPI better to inhibit blood coagulation. In recent years,
sue plasminogen activator) and urokinase. PAI-1 also plays a many studies have shown that K3 and C terminals can bind to
role in angiogenesis and thus is related to cancer prognosis. heparin, glycoproteins, and polysaccharides on the membrane
PAI-1 may also be a risk factor for cardiovascular disease surface, and this region is also related to the anti-inflammatory
and postoperative thrombosis. effect of TFPI. Glycosylation modification is almost inde-
pendent of the biological activity of TFPI. Its activity can be
inhibited by thrombin. Under physiological conditions, TFPI
16.12.2 Detection Method is mainly synthesized by microvascular endothelial cells, most
of which are anchored to endothelial cells, and a few of which
• ELISA Assays: These provide the quantification result of exist in free form, or enter the blood circulation after bind-
total PAI-1 antigen. The sample PAI-1 is combined with ing with lipoproteins and platelets. The constant expression
immobilized t-PA and then quantified with a peroxidase- of TFPI is very important for the anticoagulation function of
labeled monoclonal anti-PAI-1 antibody. endothelial cells and the maintenance of normal blood flow.
• Functional assays: The sample PAI-1 binds to the immo-
bilized t-PA. The remaining activity of t-PA indirectly
reflects the PAI-1 in the test sample. 16.13.2 Detection Method
TFPI antigen is commonly detected by ELISA. TFPI activ-

16.12.3 Healthy Person Reference Range ity is usually assayed by chromogenic method: the effects of
plasma on excessive TF/FVIIa and FX were measured. The
• PAI-1 Ag (ELISA): 4–43 ng/mL remaining TF/FVIIa hydrolyzes chromogenic substrates and
• PAI-1 activity (chromogenic assay): 0.1–1.0 AU/mL releases p-nitroaniline. The color depth is negatively corre-
lated with the concentration.

16.13.3 Healthy Person Reference Range
• Elevated levels: Obesity, physical inactivity, high levels of
blood lipids, and metabolic syndrome (insulin resistance). • TFPI Ag: 44.3–51.0 μg/L
• Decreased levels: Primary or secondary fibrinolysis. • TFPI activity: 78–154%
16.13 Tissue Factor Pathway Inhibitor 16.13.4 Clinical Significance
16.13.1 Sources and Characteristics TFPI elevation is common in the elderly, pregnancy, fatal
sepsis, and chronic renal failure. TFPI reduction is seen in
Tissue Factor Pathway Inhibitor (TFPI), a heat-resistant prethrombotic states and thrombotic diseases, DIC, major
single-chain glycoprotein composed of 276 amino acid resi- surgery, and sepsis.
dues, is synthesized mainly in endothelial cells and is a nat-
ural anticoagulant protein in vivo that controls the start-up
stage of coagulation. TFPI has a specific inhibitory effect on 16.14 L
aboratory Tests for Clotting Factor
tissue factor pathway (extrinsic pathway). Deficiency Screening
Because most of TFPI in plasma exists in lipoprotein, it was
called lipoprotein-associated coagulation inhibitor in the early 16.14.1 Prothrombin Time
stage. The total length of TFPI gene is about 86 kb, which is
located in 2q31–2q32.1 and consists of nine exons and eight 16.14.1.1 Sources and Characteristics
introns. The upstream regulatory region of the gene lacks Extrinsic pathway means that not all participating clotting
TATA box and GATA box. Its transcription is mainly regulated factors come from blood. The process is initiated by tissue
factors released into the blood. Tissue factor is a specific from factor XII activation to factor X activation. The activated
transmembrane protein that is present in a variety of cyto- partial thromboplastin time (APTT) is often used clinically to
plasmic membranes. When the tissue is damaged, tissue fac- measure the activity of the intrinsic pathways of coagulation.
tor is released, and with the participation of calcium ions, it When the vessel wall is damaged, the subendothelial tissue is
forms a 1:1 complex with Factor VII. It is generally believed exposed, and the negatively charged subendothelial collagen
that either Factor VII alone or tissue factor has no procoagu- fibers are in contact with the clotting factor, and Factor XII is
lant activity. However, Factor VII binds to tissue factor and is bound to it, and is activated to XIIa with the participation of
rapidly activated by activated factor X to VIIa, thereby form- HK and PK. Factor XIIa activates factor XI without relying
ing a VIIa tissue factor complex that is 16,000 times more on calcium ions. Activated XIa activates Factor IX in the pres-
potent than VIIa alone. Prothrombin Time (PT) measures the ence of calcium ions. IXa binds to VIIIa to form a 1:1 complex
activity of the extrinsic and common pathways of coagula- that activates factor X to form fibrin [18].
tion. PT is an index reflecting the activity of blood coagula-
tion factors I, II, V, VII, and X, or a monitoring index for 16.14.2.2 Detection Method
clinical anticoagulant therapy. PT is commonly used in sec- Platelet poor plasma is incubated at 37 °C, then phospho-
onds and international normalized ratio (INR), where INR lipid and a contact activator are added, and finally calcium is
is the preferred indicator for monitoring the amount of oral added. The time from calcium addition to fibrin clot forma-
anticoagulant (Warfarin), with an INR value between 2.0 and tion is APTT.
3.0 as the suitable range for the therapeutic dose [16].
16.14.2.3 Healthy Person Reference Range
16.14.1.2 Detection Method The reference range for APTT is between 27 and 35 s.
PT is the time required to form a fibrin clot by adding tissue However, the APTT reference range varies between labo-
factors, phospholipids, and calcium to platelet poor plasma. ratories because of different detection principles, phospho-
lipid, activator, and the incubation time used in the test.
16.14.1.3 Healthy Person Reference Range
The reference range of PT is affected by many factors includ- 16.14.2.4 Clinical Significance
ing source of TF (the type of thromboplastin), the exact APTT is widely used to screen for deficiency of coagulation
technique used, and method of end-point determination. factors in intrinsic pathways, such as factor VIII, factor IX,
Each laboratory should establish its own reference range. factor XI, and factor XII. APTT can also be used for primary
The International Sensitivity Index (ISI) was calculated by screening diagnosis of hemorrhagic diseases and laboratory
calibrating individual thromboplastin with an international monitoring of heparin anticoagulation therapy.
WHO reference thromboplastin. APTT prolongation can be seen in hemophilia A, hemo-
The International Normalized Ratio (INR) is now com- philia B, liver disease, intestinal sterilization syndrome,
monly used to correct the effect of thromboplastin reagent oral anticoagulant, DIC, factor XI deficiency, factor XII
differences on PT. INR is calculated according to the for- deficiency, and increased blood anticoagulant substances
mula: INR= (patient PT/ normal PT) ISI . The INR reference (coagulation factor inhibitors, lupus anticoagulants, warfa-
value is 0.8–1.2. rin, or heparin) [19, 20]. APTT shortening is seen in DIC,
prethrombotic state, and thrombotic disease.
16.14.1.4 Clinical Significance
PT prolongation occurs mainly in: Warfarin usage, congeni-
tal factor II, factor V, factor VII, factor X, and fibrinogen defi- 16.14.3 Thrombin Time
ciency; acquired coagulation factor deficiency such as DIC
[17], primary fibrinolysis, obstructive jaundice, and vitamin 16.14.3.1 Sources and Characteristics
K deficiency. PT shortening is seen in hypercoagulable states Activation of factor X to fibrin formation is the common
and thrombotic diseases such as deep vein thrombosis and coagulation pathway. In the common coagulation pathway,
myocardial infarction. the produced thrombin converts fibrinogen to fibrin, the time
of which can be reflected by thrombin time (TT).
16.14.2 Activated Partial Thromboplastin 16.14.3.2 Detection Method

Time Add human thrombin (or bovine thrombin) to platelet poor
plasma at 37 °C, and record the time required to form fibrin clots.
16.14.2.1 Sources and Characteristics
The intrinsic pathway means that all participating coagula- 16.14.3.3 Healthy Person Reference Range
tion factors are derived from blood and refers to the process The reference range for TT is 16–18 s.
16.14.3.4 Clinical Significance 16.14.4.4 Clinical Significance

TT prolongation is more common in DIC, congenital fibrin- Coagulation factor assay can provide information on coagu-
ogenemia [21], primary fibrinolysis, and liver disease, as lation factor deficiencies such as hemophilia A, hemophilia
well as heparin or heparin-like substances and increased B, combined factor V and factor VIII deficiency, combined
FDP. TT shortening is mainly seen in abnormal proteinemia deficiency of vitamin K-dependent factors, and so on.
or macroglobulinemia.
16.15 M
olecular Diagnosis of Inherited
16.14.4 Coagulation Factor Assay Bleeding Disorders
16.14.4.1 Sources and Characteristics In the late 1970s and early 1980s, molecular medicine helped
Fourteen clotting factors participate in the classic coagula- people to better understand the genetic basis of benign hema-
tion cascades, including factor I–V, factor VII–XII, prekalli- topathy, including the defects and/or dysfunction of clot-
krein (PK), and high molecular weight kininogen (HMWK). ting factors. A number of molecular abnormalities that cause
Factor IV is calcium ion (Ca2+), except which other clotting hereditary hemorrhagic diseases have been identified, includ-
factors are proteins. ing mutations in the genes that encode not only the clotting
Factor III, also called tissue factor (TF), usually lies factors themselves but also their regulatory regions. Molecular
in tissue and vascular endothelial cells. TF is a trans- diagnosis is focused on causative mutation identification in
membrane glycoprotein. Its N-terminal is the receptor of genes encoding corresponding coagulation factors. However,
factor VII, they bind together and initiate the extrinsic combined FV and FVIII deficiency and vitamin K-dependent
coagulation pathway. The C-terminal insert into the cyto- clotting factor deficiency (VKCFD) are exceptions [22].
plasm to provide a catalytic surface for the coagulation
reaction.
Factors II, V, VII, and X are also called vitamin 16.15.1 M
olecular Diagnosis of Hemophilia
K-dependent factors. Their molecular structure characteris- A and Hemophilia B
tic is that the N-terminal contains different amounts of car-
boxyglutamic acid (γ-Gla), and the biological synthesis of Hemophilia is the hemorrhagic disorder caused by factor
γ-Gla in hepatocytes depends on the mediation of vitamin VIII and factor IX gene defects, gene mutations, gene dele-
K. Factor II is usually called prothrombin. tions, and gene insertions. Hemophilia A and hemophilia B
Factors XI, XII, PK, and HMWK are called contact fac- are characterized by the deficiency of factor VIII and fac-
tors because they can start intrinsic coagulation pathway tor IX, respectively. Because factor VIII and factor IX genes
through contact reactions. Moreover, these factors also are located on the X chromosome, hemophilia A, and hemo-
participate in the activation of fibrinolytic and complement philia B typically follow an X-linked inheritance pattern. As
systems. a result, males are affected most often.
Factors I, V, VIII, and XIII are thrombin-sensitive factors. More than 90% of hemophilia A cases are the result of
They can be directly activated by thrombin. Factor I is com- factor VIII inversion mutation, such as those sequences in
monly called fibrinogen. intron 22 or intron 1. Gene sequencing can be employed
The principle depends upon the ability of a sample con- to find other mutations, especially for mild and moderate
taining the factor under investigation to correct or shorten hemophilia A. Approximately 80% of mutations in hemo-
the delayed clotting of plasma completely deficient in that philia B are point mutations. The reminder abnormalities of
factor. Such deficient plasmas must contain less than 1% of hemophilia B are frameshift, splice site, and rearrangements.
the clotting factor under investigation and normal levels of Amniocentesis and chorion villus sampling are performed
all other relevant clotting factors. for the prenatal diagnosis of hemophilia at 3- and 4-month
gestation, respectively.
16.14.4.2 Detection Method
One-stage clotting assay is widely employed for the
detection of clotting factor activity. The principle 16.15.2 M
olecular Diagnosis of Von
depends upon the ability of a sample to correct or shorten Willebrand Disease
the prolonged clotting time of a completely factor-defi-
cient plasma. VWD is caused by the deficiency of VWF and is the most
frequent autosomal inherited bleeding disorder. Most sub-
16.14.4.3 Healthy Person Reference Range types of VWD have an identifiable mutation in the VWF
For one-stage factor assays, the reference range is between gene, although about half of type 1 VWD patients do not.
50 and 150% for each clotting factor. In type 1 VWD, missense mutations, frameshifts, nonsense
mutations, or deletions have been reported. Missense muta- 1 in 500,000 to 1 in 2,000,000 (Table 16.2). Inheritance pat-
tions in VWF A2 domain and A1 domain have been found tern is autosomal recessive or dominant for all RBDs. The
in type 2A and 2B VWD, respectively. A few heterogeneous International Society on Thrombosis and Hemostasis (ISTH)
mutations are identified in type 2M VWD. Missense muta- mutation database provides some information on RBD iden-
tions in FVIII-binding domain of VWF are the reason for tified mutations. Missense mutations represent 50–80% of
type 2N VWD. In type 3 VWD, partial or total gene deletions identified mutations. As for fibrinogen, factor V, and fac-
have been reported [23, 24]. tor XIII genes, insertion or deletion mutations account for
20–30%. In other clotting factor gene abnormalities, less
than 15% are insertion or deletion mutations. Splicing and
16.15.3 M
olecular Diagnosis of Rare nonsense mutations are found in 5–15% of identified muta-
Bleeding Disorders tions. Variants located in the 3′ and 5′ untranslated regions
are rare, and have been reported in fibrinogen, factor VII,
The inherited clotting factor deficiency disease hemophilia factor XI, and factor XIII. Moreover, there are 5–10% of
and VWD have high prevalence, 133 in 1,000,000 males patients who have negative gene mutation results.
and 4–10 in 100,000, respectively. However, other clotting
factors deficiency are rare and the diseases are called Rare
Bleeding Disorders (RBDs). Two large surveys (WFH and 16.16 Related Progress
EN-RBD) have evaluated the worldwide RBD distribution
(Fig. 16.8) [25, 26]. The prevalence of RBDs ranges from The human blood system itself has a complex and perfect
mechanism of hemostasis and coagulation. Normally, this
mechanism is in a dynamic balance due to the mutual con-
FV+FVIII 3.0%
FV 9.0% straints of coagulation, anticoagulation, and fibrinolysis
systems. In pathological conditions, abnormalities in the
FII 1.5%
function of any system can affect this dynamic balance, lead-
FX 8.0% FVII 37.5% ing to bleeding or thrombosis. In recent years, with more
research, new detection items and parameters have emerged,
such as soluble vascular endothelial cell protein C receptor
(sEPCR), as a new molecular marker for clinical detection,
FXIII 6.5%
and the elevated plasma level reflects vascular endothelial
injury. Due to the improvement of science and technology,
the specificity and sensitivity of laboratory testing have
also improved, and more attention has been paid to intel-
ligence. In today’s medical examination, automated analyz-
FXI 26.5%
ers have been used instead of manual screening tests, which
Fibrinogen 8.0%
greatly reduce the inaccuracy caused by manual operation
errors and improve the accuracy of diagnosis. Meanwhile,
Fig. 16.8 Worldwide distribution of RBDs provided by the WFH and some molecular diagnostic techniques are gradually applied
EN-RBD in clinic, such as denaturing gradient gel electrophoresis
Table 16.2 Molecular diagnosis in rare bleeding disorders

Plasma level Hemostatic level
Deficiency Plasma half-life (μg/mL) (%) Prevalence Gene (chromosome)
Fibrinogen 2–4 days 1500–4000 50 (mg/dL) 1:1,000,000 FGA, FGB, FGG (4q28)
FII 3–4 days 100 20–30 1:2,000,000 F2 (11p11–q12)
FV 36 h 10 15–20 1:1,000,000 F5 (1q24.2)
FVII 3–4 h 0.13–1.0 15–20 1:500,000 F7 (13q34)
FX 40–60 h 10 15–20 1:1,000,000 F10 (13q34)
FXI 40–70 h 3–6 15–20 1:1,000,000 F11 (4q35.2)
FXIII 11–14 days 10–20 2–5 1:2,000,000 F13A1 (6p24–p25)
F13B (1q31–q32.1)
FV + FVIII 36 h (FV) As for each factor 15–20 1:1,000,000 LMAN1 (18q21.3–q22)
10–12 h (FVIII) MCFD2 (2p21–p16.3)
VKCFD As for each factor As for each factor 15–20 <50 families GGCX (2p12)
VKORC1 (16p11.2)
(PCR-DGGE). After screening for point mutations in the 11. Giurdanella G, Montalbano G, Gennuso F, et al. Isolation, cul-
tivation, and characterization of primary bovine cochlear peri-
hemophilia A gene, gene chip technology can now provide a cytes: A new in vitro model of stria vascularis. J Cell Physiol.
“panoramic” picture of human platelet molecules, which not 2019;234:1978–86.
only helps people to understand the function of platelets, but 12. Loomans JI, Stokhuijzen E, Peters M, et al. Administration of
also provides a rapid detection method for some hereditary DDAVP did not improve the pharmacokinetics of FVIII concentrate
in a clinically significant manner. J Clin Transl Res. 2018;3:351–7.
hemorrhagic diseases. With the development of gene cloning 13. Tateishi K, Imaoka M, Matsushita M. Dual modulating functions
technology, all of the clotting factors and genes that play an of thrombomodulin in the alternative complement pathway. Biosci
important role in the coagulation process have been cloned, Trends. 2016;10:231–4.
which provide a new direction for the diagnosis and treat- 14. Moore SF, Hunter RW, Hers I. Protein kinase C and P2Y12 take
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2014;12:748–60.
15. Basavaraj MG, Sovershaev MA, Egorina EM, et al. Circulating
monocytes mirror the imbalance in TF and TFPI expression in
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Cardiovascular System
17
Haitao Ding and Juan He
17.1 Overview to the actin subunit and then cause the myocardium to shrink.
cTnT is similar to cTnI and has myocardial tissue specificity,
Hypertension, atherosclerosis, and coronary heart disease are while cTnC is not extremely specific for myocardial damage.
the most common cardiovascular diseases. With the change Therefore, cTnT and cTnI are currently used for clinical lab-
of lifestyle, especially unhealthy diet, the morbidity and mor- oratory diagnosis. Currently, commonly used detection
tality of cardiovascular diseases have risen. In the past, inap- methods for cTnT and cTnI include bedside detection (Point
propriate risk stratification of cardiovascular diseases was due of Care Testing, POCT) and fully automated chemilumines-
to the low sensitivity of related clinical manifestation and cence or electrochemiluminescence.
electrocardiogram (ECG) indicators. So far, cardiovascular In October 2007, the ESC (the European Society of
biomarkers have received more and more attention in the Cardiology), together with the American Heart Association
diagnosis and treatment in recent years. The examination of (AHA) and the World Heart Federation (WHF), issued a uni-
cardiovascular disease is the “bottleneck” science in the fied definition of global myocardial infarction. The first diag-
whole cardiovascular field. Only when the disease is correctly nostic basis is the serum myocardial marker (mainly
initially diagnosed can the risk for sudden death be effectively troponin) increase (at least over 99% of the upper reference
reduced. Ideal markers should be high sensitivity, high speci- limit). In 2008, China also recommended the use of cardiac
ficity, high accuracy, good repeatability, short detection time, troponin as the preferred marker for the global definition of
and reduced effects by individual factors. In order to provide myocardial infarction. Since cTnC has no myocardial spe-
a reliable basis for diagnosis and prognosis, this chapter will cific phenotype, cTnT and cTnI are currently used for labo-
introduce markers of the cardiovascular system in the follow- ratory diagnosis of acute infarction. cTn usually increases in
ing aspects. See Fig. 17.1 for details. peripheral blood 3–6 h after the injury of myocardial organ,
peaks at 12–24 h, and increases continuously for almost
10–14 days, with high sensitivity and specificity [1]. cTn is
17.2 Myocardial Damage Markers also valuable in judging minor myocardial damage. Patients
with unstable angina often have minor myocardial damage.
17.2.1 Cardiac Troponin (cTn) For this tiny myocardial injury, the positive rate of cTn is
much higher than that of CK-MB, which is very helpful for
Cardiac troponin is composed of three different subunits, the initial diagnosis of the disease. The detection of hs-cTn
namely, cTnT, cTnI, and cTnC. Cardiac troponin complex is can detect a small amount of cTn in many healthy people.
a regulatory protein of myocardial contraction. At rest, the Therefore, troponin levels detected with hs-cTn need to be
binding site of myosin head and actin on the thin filament is referenced to the normal range and are defined as “elevated”
covered by tropomyosin. Immediately after the ion concen- when they are above 99% of the reference population [2].
tration increases, calcium ions bind to TnC, and TnI moves
away from the inhibition position, causing the tropomyosin
to be open. The myosin head on the thin filaments can bind 17.2.2 Myoglobin (Mb)
Myoglobin (Mb) is a small molecule protein containing 154

H. Ding (*) · J. He amino acids, which is composed of globin and heme. It
Inner Mongolia People’s Hospital,
Huhhot, Inner Mongolia Autonomous Region, reversibly binds to oxygen and acts as a transporter and
People’s Republic of China stores oxygen in muscle cells [3]. Myoglobin is widely

222 H. Ding and J. He
Fig. 17.1 Classification of

specific content in this chapter
p resent in myocardium and skeletal muscle cells. Under nor- 100 Myoglobin and CK isoforms
mal circumstances, the peripheral blood Mb content is very Troponin (large MI)
low. Only when the heart or skeletal muscle is damaged, it Troponin (small MI)
releases and enters the peripheral blood. Because of its rela- CK–MB
Multiples of the ULN 20 10% CV/99th percentile
tively small molecular weight and abundant cytoplasmic
content, Mb is one of the earliest elevated markers of acute
10
myocardial infarction and can be elevated within 2 h after
developing chest pain. The method of measuring the Mb 5
content includes the RIA method, the ELISA method, and
the high performance liquid chromatography. 2
Myoglobin can be used as a cardiac marker to diagnose or 1
exclude cardiac events with cardiac troponin. Serum or plasma 0
0 1 2 3 4 5 6 7 8
Mb rises 1–3 h after myocardial necrosis, usually reaches the
highest point at 6–9 h, and decreases to ordinary levels after Days after AMI
24 h [1]. Mb is elevated earlier than troponin, but it is not spe-

Fig. 17.2 Days after onset of AMI
cific to the heart and is present in the blood for a shorter time
than troponin. Although negative Mb detection excludes car-
diac events, a positive detection does not represent myocardial adding up to 81–82 kDa and consists of an M subunit and a B
damage because Mb also exists in skeletal muscle. Therefore, subunit. There are many detection methods for CK-MB. The
Mb in the blood of patients with trauma and surgery can also early methods of chromatography and electrophoresis meth-
be elevated. To diagnose myocardial damage, you must com- ods are complicated and time consuming. The luminescence
bine CK-MB, troponin T or I, electrocardiogram, and clinical method is used to determine CK-MB mass recently. The clin-
symptoms. Although Mb has low specificity for myocardial ical application of CK-MB has several aspects. First, CK-MB
injury, it still has a certain clinical value for early exclusion of detection was adopted for AMI detection. The plasma con-
acute myocardial infarction (AMI). centration of CK-MB increases in 4–6 h and reaches the
maximum level within 24 h after AMI onset. The CK-MB
level will go down within 48–72 h. In comparison with serum
17.2.3 Creatine Kinase Isoenzyme MB (CK-MB) CK and CK-MB activity, serum CK-MB mass is more helpful
for early diagnosis of AMI [4]. At the same time, combined
Creatine kinase (CK) refers to a type of enzyme that which with myocardial specific markers such as troponin T, it can
is directly connected to the transportation of energy within improve the accuracy of clinical diagnosis. The peak time of
cells and the muscle contraction as well as the reproduction various cardiac biomarkers is shown in Fig. 17.2.
of ATP. It is shown that CK has four types of enzyme: MM, In addition, CK-MB can be used for the prognosis of
BB, MB, and MiMi, respectively. MM mainly exists in all unstable angina, the risk stratification of ischemic myocar-
types of muscle cells and BB mainly exists in the brain cells dial injury, and the evaluation of myocardial reperfusion
and MB mainly exists in the cells of cardiomyocytes. CK-MB effect after thrombolysis. The serum level of CK-MB is one
refers to a dimeric enzyme, and it has the molecular weight of the important indicators for clinical diagnosis of AMI.
17 Cardiovascular System 223
17.2.4 Heart Type-Fatty Acid Binding Protein whether there is myocardial ischemia in patients with chest
(H-FABP) pain with normal ECG, but it cannot identify myocardial
infarction and myocardial ischemia. Therefore, it is proposed
Heart Type-Fatty Acid Binding Protein (H-FABP) consists to test both IMA and cardiac troponin T for the patients diag-
of 132 amino acid molecules and has a molecular weight nosed by the serious coronary syndrome, because the combi-
adding up to 15 kDa. It is highly cardiac-specific, but also nation of the two can improve the sensitivity of myocardial
has low concentrations in other tissues such as the brain, kid- infarction and reduce the time to diagnosis.
neys, and skeletal muscle. The main detection methods are Similar to IMA, CD40 ligand and pregnancy-related
enzyme-linked immunosorbent assay, rapid immunoturbidi- plasma protein A also has shown good value in evaluating
metric assay, and POCT. H-FABP is released into the blood myocardial ischemia and risk classification of ACS, but its
during myocardial injury. This feature is similar to myoglo- clinical specificity needs more research for confirmation.
bin, which is significantly increased within 0.5–2 h after
myocardial ischemia or injury, peaks at 6–8 h, and returns to
normal levels at 24–36 h. Because of this feature, it will be 17.4 Heart Function Markers
adopted to become an efficient way for diagnosing AMI [4].
If a patient with chest pain is tested again for H-FABP after 17.4.1 Brain Natriuretic Peptide (BNP) or
1 h, the sensitivity can reach 100%. Negative results can NT-proBNP
quickly diagnose and exclude AMI.
Brain natriuretic peptide (BNP) is another member of the
natriuretic peptide family. The gene of human BNP is located
17.2.5 Copeptin in the chromosome 1 and encodes pro-BNP (a kind of pro-
hormone). Cardiomyocytes are the main source of BNP
The Copeptin is composed of 30 amino acid residues which related peptides. Human myocardial cells are first synthe-
constitute the glycoprotein at the C-terminus of arginine sized with pro-B-type natriuretic peptide (pro-BNP) contain-
vasopressin (AVP). The relative molecular weight is 5 kDa. ing 108 amino acids, and then cleaved into 76 amino acids by
Copeptin and hypothalamic AVP are synthesized together, endonuclease N-terminal pro-B-type natriuretic peptide
and together with AVP enter the blood circulation from the (NT-proBNP) and 32 types of amino acid C-terminal poly-
neurohypophysis in an equal amount. Compared with AVP, peptide BNP. NT-proBNP has no biological activity [8]. At
Copeptin has better stability at room temperature and mea- present, commonly used detection methods include immu-
surement is relatively easy. Research has found that the level noassay and POCT.
of Copeptin is significantly elevated in various diseases such BNP or NT-proBNP can be adopted for the diagnosis, lev-
as hypertension, myocardial infarction, and mental exhaus- eling, and prognosis of the heart problems. Elevated levels of
tion. It is expected to be a potential biomarker for heart dis- them often indicate severe impairment of cardiac function.
eases such as heart failure [5]. Detection of Copeptin in BNP or NT-proBNP levels will be definitely measured once
patients with normal cTn but suspected ACS can be used as the heart problem is doubted. The increase of BNP or
a rapid and early exclusion marker for AMI [6]. NT-proBNP has a certain lag compared with myocardial
injury, so during the early stages of the disease, the levels can
be normal or slightly increased, prompting the need for
17.3 Myocardial Ischemia Markers repeated measurement within a short period.
17.3.1 Ischemic Modified Albumin (IMA)

17.4.2 Soluble Suppression
Ischemic modified albumin (IMA), an albumin with a of Tumorigenicity-2 (sST2)
reduced cobalt-binding affinity, is clearly increased in
patients with ischemic symptoms and is also regarded as a As a matter of fact, the ST2 protein is in the category of the
biomarker for the first-time detection of ischemia with myo- interleukin group and is a product of the IL-1 receptor-like
cardial symptoms, which is before the onset of the irrevers- 1 gene. It is present in two important subtypes of the blood:
ible cardiac injury. In the case of ischemia/regeneration, the one is the membrane receptor (ST2L) and the other is trun-
ability of albumin to bind to cobalt metal is reduced due to cated soluble form (sST2). ST2, as a kind of orphan recep-
the destruction of amino acid sequences of serum proteins by tor, is only associated with inflammatory and immune
free radicals, which should be approved by the US Food and diseases. Subsequent studies have found that IL-33 is a
Drug Administration (FDA) for the albumin cobalt binding ligand for ST2, which contributes to the understanding of
(ACB) test indirect measurement [7]. IMA can determine ST2’s function. IL-33 is produced after cell damage as an
endogenous risk signal or alarm that warns of tissue damage 17.5 Inflammatory Markers
in the immune system. The interactive function between
IL-33 and ST2L seems to have the role of protecting human 17.5.1 C-Reactive Protein (CRP)
hearts, which can also help them reduce the heart fibrosis
and the death of myocardium, and can also avoid hypertro- C-reactive protein (CRP) has the capability to bind pneumo-
phy. It can be used to improve the functions of heart muscles coccal C polysaccharide in the presence of calcium ions. It is
and can make different reactions to different stimulation. mainly synthesized by liver and epithelial cells under the
However, the sST2 produced by cells can increase, which stimulation of inflammatory lymphokines. The half-life of
will hinder the beneficial roles of IL-33, because the recy- CRP within the blood system is usually about 19 h in total.
cled sST2 will combine with IL-33 to reduce the cell signal, The concentration depends on the amount liver produced. It
which can result in fibrosis. Under this condition, ST2 is the begins to rise within a few hours after the injury of the body
cell intermediary and the refraining inhibitor of IL-33. In tissue, peaks in 24–48 h, and gradually returns to normal
recent years, sST2 has become the biological symbol of the after tissue repair [15]. Therefore, the level of CRP in serum
acute heart disease. Multiple studies have shown that con- is related to the degree of inflammation of the disease, and is
tinuous monitoring of sST2 is useful for the assessment of one of the most sensitive acute stage reactants in humans.
prognosis and myocardial remodelling after heart diseases The main functions of CRP are the following: (1) identify
onset [9, 10]. Clinical studies have found sST2 level to be foreign substances through the classical pathway and acti-
elevated in the mature acute myocardial infarction and HF vate the complement system [16]; (2) enhance the lympho-
patients, as well as connection to the severity of the injury cyte conditioning and phagocytosis of phagocytic cells; (3)
and inaction. In addition, the combination between sST2 combine with platelet activating factor to reduce inflamma-
and NT-proBNP can be added to stratify the patients’ risk of tion; and (4) combine with chromosomes to eliminate cellu-
death [11]. lar DNA in necrotic tissue. Since CRP levels in healthy
people are usually <3 mg/L, screening should be performed
in a highly sensitive manner, i.e., hypersensitive C-reactive
17.4.3 Adrenomedullin (ADM) protein (hs-CRP). At present, commonly used detection
methods include ELISA, scatter nephelometry, and nephe-
In 1993, a new cardiovascular active peptide, Adrenomedul- lometry. Multiple detections of hs-CRP > 3 mg/L are a per-
lin (ADM), was isolated from human pheochromocytoma sistent signal of inflammation, suggesting the risk of
tissue, which is also known as antihypertensive peptide due atherosclerosis. So hs-CRP is used as an indicator of cardio-
to its powerful antihypertensive effect [12]. ADM is mainly vascular disease risk assessment. Research has found that hs-
produced by endothelial cells and vascular smooth muscle CRP is a more innovative and efficient unique disease
cells. The main functions of ADM are as follows [13]: (1) predictor than LDL-C [17], and hs-CRP plays an increas-
dilate blood vessels and lower blood pressure; (2) dilate ingly significant role in the diagnosis and prediction of CHD
bronchial smooth muscle and improve bronchial micro- and stroke.
circulation; (3) promote gastric acid secretion; (4) dilate
renal artery, enter the small arteries, increase renal blood
flowing, and improve the rate of glomerular filtration; and 17.5.2 Interleukin-6 (IL-6)
(5) inhibit the secretion of aldosterone (ALD), insulin,
catecholamines, and ACTH, thereby participating in the Interleukin-6 (IL-6) is a core member of cytokines and can
regulation of blood pressure, glucose metabolism, and salt be produced by fibroblasts, mononuclear macrophages, T
metabolism. Commonly used detection methods for plasma lymphocytes, B lymphocytes, epithelial cells, glial cells, and
ADM are radioimmunoassay RIA and ELISA. Plasma various tumor cells. IL-1, TNF-a, viral infection, double-
ADM level is related to the progression of cardiovascular stranded RNA, and cAMP can induce IL-6 production in
disease and can be used as a judgment indicator of disease normal cells. IL-6 can regulate a variety of cellular functions
severity and treatment effect. In the initial stage of acute [18]: (1) chemotaxis to neutrophils and monocytes; (2) pro-
myocardial infarction, plasma ADM concentration is posi- mote the expression of vascular endothelial cell adhesion
tively correlated with the degree of heart failure [14]. When molecules, inflammatory mediators, and coagulation factors;
heart failure treatment is implemented, ADM levels can be (3) promote B cells become antibody-secreting cells and
reduced to normal. Plasma ADM levels are proportional promote T cell growth and IL-2 production; (4) induce
with the severity of hypertension, and can suggest that hepatocytes to synthesize fibrinogen, CRP, and other acute
elevated plasma ADM levels in patients with hypertension phase proteins; and (5) enhance the growth of bone marrow
may be the body’s compensatory response under pathologi- stem cells and increase the active platelet number. At present,
cal conditions. IL-6 is commonly used in clinical ELISA. In 1994, Seino
found IL-6 expression in human atherosclerotic plaques, and sclerotic plaques. Lp-PLA2 enhances the penetration of
studies have shown that IL-6 is involved in coronary athero- inflammatory cells and the formation of atherosclerotic
sclerosis as an autocrine transmitter. It is related to the stabil- plaques [24].
ity of the plaque [19]. The level of IL-6 positively correlated Plasma Lp-PLA2 can bind to a variety of lipoproteins,
with increased risk of coronary heart disease. The level of of which about 30% bind to the extremely low density lipo-
IL-6 in patients with detection can reflect the changes of the protein and high level density lipoprotein, and the remaining
disease to some extent [20]. In addition, IL-6 can also reflect 70% bind to low density lipoprotein, which combines to form
the development of autoimmune diseases, hematological lipoprotein. The binding complex is shown in the circulation
tumors, cervical cancer, and other diseases, and IL-6 will be of the human body and is dealt with inflammatory media-
significantly increased in postoperative burns, acute infec- tors to participate in the formation of atherosclerotic plaques.
tions, and organ transplant rejection. As for the pathogenesis There is a gender difference in Lp-PLA2 levels. In general,
of coronary heart, immune mechanism is one of its initiating premenopausal women have lower Lp-PLA2 levels than
factors. Inhibiting the expression of IL-6 in myocardium is a men, and the levels gradually increase with age. Elevation
potential therapeutic target for the treatment of different car- of Lp-PLA2 is common in coronary heart disease, ischemic
diovascular diseases [21]. In the future, anti-inflammatory stroke, carotid atherosclerosis, insulin resistance, and other
and immunomodulatory drugs will have broad therapeutic diseases. Recent researches have shown that Lp-PLA2 is
prospects. closely related to inflammatory response of arterial endothe-
lial cells in human blood circulation [25]. Therefore, plasma
Lp-PLA2 is used as an inflammatory marker for assessing
17.6 M
arkers That Predict Heart-Risk the risk assessment of cardiovascular events, and it has been
Events gradually proved that it can be used to evaluate the stability
of plaque and the seriousness of coronary artery disease in
17.6.1 Homocysteine (Hcy) terms of the coronary heart problems [26].
In 1931, Vincent du Vigneaud first isolated homocysteine

(Hcy) from bladder stones. It is a sulfur-containing amino 17.6.3 Myeloperoxidase (MPO)
acid that is an important intermediary in the metabolism of
methionine. Methionine is converted to S-adenosylmethionine Myeloperoxidase (MPO), also known as peroxidase, is an
by ATP, which is converted to S-adenosine-Hcy by methyl- essential content of lysosome, mainly found in neutrophils
transferase. Hcy is finally formed after de-adenosine. At and monocytes. As an inflammatory marker, it participates in
present, Hcy is detected by enzymatic circulation method in many pathophysiological processes in the body [27]. MPO
clinical practice. Recent research has indicated that it can refers to a kind of functional thing and stimulus thing, and its
cause atherosclerosis (AS), which turns out to be a unique level and activity changes as well as some heart and blood
risk factor for cardiovascular and cerebrovascular diseases, disease are closely related to each other. MPO promotes
and is closely linked to their happening and progress [22]. plaque formation and instability by generating free radicals
Although the pathogenesis of Hcy is not totally compre- and a variety of reactive substances, causing the progression
hended, epidemiological data have shown a close relation- of atherosclerosis as well as a variety of complications [28].
ship between plasma Hcy levels and vascular risk, where Studies have shown that MPO has a good application pros-
every 5 μmol/L increase in Hcy causes a 32% increased risk pect in understanding the risk classification of early myocar-
of heart disease. When Hcy decreases by 3 μmol/L, the risk dial ischemia and acute coronary syndrome, especially
of developing heart disease is reduced by 16% [23]. Increased reflecting the instability of atherosclerotic plaque, and con-
Hcy levels can be seen in atherosclerosis, myocardial infarc- sidered to be one of the unique risk factors for ACS [29]. The
tion, stroke, dementia, diabetic complications, and other current research has demonstrated that MPO is closely con-
diseases. nected to the severity of cardiovascular disease.
17.6.2 Lipoprotein-Associated Phospholipase 17.7 Other Markers

A2 (Lp-PLA2)
17.7.1 MicroRNAs
The elevated lipoprotein-associated phospholipase A2
(Lp-PLA2) level activates the body’s oxidative reactions, MicroRNAs are non-coding small RNA and are involved in
accelerates the destruction of endothelial cell structure, the performance of gene transcription and expression.
and thus exacerbates the inflammatory response in athero- Therefore, the occurrence and development of many life
activities and diseases in human body are inseparable from change of SCN5A gene has been linked to various heart
the changes of miRNA. Because the changes of miRNAs problems, including Brugada syndrome, LQT3, sick sinus
occur earlier than the changes of genes and proteins, and the syndrome, familial atrial fibrillation, familial dilated cardio-
occurrence of disease symptoms, the detection of dynamic myopathy, left ventricular noncompaction, and other disor-
changes of miRNAs may provide clues for the occurrence ders. Syncope, ventricular tachycardia, and cardiac arrest
and development of diseases, and then guide early clinical may occur in Brugada syndrome. It has a high threat of sud-
interventions to effectively control disease development. den cardiac death (SCD). SCN5A is the most common
Recent studies have shown that microRNAs as biomarkers of pathogenic gene of Brugada syndrome, so genetic testing for
cardiovascular disease may have important research value this disease has a strong predictive value.
and practical application prospects [30]. At present, the
detection of microRNA mostly uses PCR technology, which
is quick and easy. Research has shown that the blood nucleic 17.7.4 RYR2
acids, including microRNAs (miRs), mRNA, and DNA, are
significant for the treatment and supervision of the heart fail- RYR2 gene is located in chromosome 1q43 and encodes a
ure [31]. In particular, miR-155, miR-22, and miR-133 protein named ryanodine receptor 2 [33]. In heart myocytes,
appear to be promising for the diagnosis, prognosis, and there are two proteins with the ryanodine receptor. These
management of HF patients [32]. In addition, microRNAs channels can be included into an outside membrane of a cell
can also serve as targets for the treatment of cardiovascular structure. The sarcoplasmic reticulum is the storing center of
diseases and guide the molecular treatment of cardiovascular the calcium ions, and the calcium ions exit through RYR2. In
diseases. order to have effective and constant heart beats, the myocar-
dium should coordinate the balance of tension and relax-
ation. Under this condition, the calcium ions can accurately
17.7.2 KCNQ1 control the muscles, which can play an important role in the
tense and relaxed cycles. It is obvious that RYR2gene dys-
The KCNQ1 gene is located in chromosome 11p15.5 and is function will damage its control for the flowing of the cal-
approximately 404 kb in length. Its mRNA is 2031 bp long cium ion, which can result in an abnormal heart beating with
and includes 17 exons [33]. The KCNQ1 gene encodes an catecholaminergic polymorphic ventricular tachycardia
alpha subunit that slowly activates a voltage-gated potassium (CPVT) in human. In recent years, it is known that almost 70
channel. The protein produced by the KCNQ1 gene is located mutations of RYR2 gene can result in CPVT [35].
in the third-stage repolarization of myocardial action poten-
tial. The mutation of KCNQ1 gene leads to the disorder of
cellular ion channel in terms of composition, regulatory 17.7.5 KCNE1
functions, and electrophysiological functions. Eventually it
caused a variety of serious arrhythmias and even sudden car- KCNE1 gene is located in human chromosome 21q22.1–
diac death. Changes in the KCNQ1 gene have been linked to 22.2 and encodes the beta subunit of delayed rectifying
the different situations linked to heart problems, including potassium channel protein (Iks), which is slowly activated by
long-QT syndrome (LQTS), Jervell-Lange-Nielsen syn- human myocardium [33]. This protein plays a significant
drome (JLNS1), short-QT syndrome2 (SQT2), lone atrial role in the repolarization stage of myocardial action poten-
fibrillation, and familial atrial fibrillation3 (ATFB3). tials. Research has confirmed that mutations of the KCNE1
Long-QT 1 (LQT1) caused by the KCNQ1 mutation accounts gene can result in Jervell-Lange-Nielsen syndrome (JLNS),
for 40–55% of the entire long-QT syndrome. There are more familial long-QT syndrome 5 (LQT5), familial atrial fibrilla-
than 200 KCNQ1 mutation sites that have been discovered tion, and malignant arrhythmia.
so far [34].
17.7.6 KCNH2
17.7.3 SCN5A
KCNH2 gene is identified with the human ether-a-Go-Go-
SCN5A gene is located in chromosome 3p22.2 and encodes related gene (hERG). This gene encodes the alpha subunit of
an alpha subunit of the cardiac sodium channel, which has the Kv11.1 channel, which is associated with the rapidly
four homodomains (DI–DIV) [33]. Each domain is com- activated delayed rectifier potassium current (IKr) in the
posed of 6 transmembrane segments (S1–6). The SCN5A heart [33]. The IKr outward potassium current is the major
gene mutation induces arrhythmia by affecting protein repolarization process in the fast repolarization section of the
expression and gating function of the sodium channel. The human cardiomyocyte action potential. Pathogenic muta-
tions on hERG reduce or disable the function of the Kv11.1 15. Strang F, Schunkert H. C-reactive protein and coronary heart dis-
ease: all said—is not it? Mediat Inflamm. 2014;2014:757123.
channel, thereby prolonging the QT interval and leading to 16. Danesh J, Wheeler JG, Hirschfield GM, et al. C-reactive protein
LQT2. Approximately 30–35% of the hereditary LQTS is and other circulating markers of inflammation in the prediction of
derived from KCNH2 pathogenic mutations. 29% of LQTS2 coronary heart disease. N Engl J Med. 2004;350:1387–97.
patients experience syncope during rest or sleep, and only 17. Li H, Sun K, Zhao R, et al. Inflammatory biomarkers of coronary
heart disease. Front Biosci (Schol Ed). 2018;10:185–96.
13% of syncopal events are reported to occur during exer- 18. Malaguarnera M, Trovato BA, Laurino A, et al. Interleukin-6 in
cise. In addition, KCNH2 mutation is also associated with hepatitis C cirrhosis. Panminerva Med. 1996;38:207–10.
SQT1 [34]. 19. Schrader LI, Kinzenbaw DA, Johnson AW, et al. IL-6 deficiency
protects against angiotensin II induced endothelial dysfunction and
hypertrophy. Arterioscler Thromb Vasc Biol. 2007;27:2576–81.
20. Kanda T, Takahashi T. Interleukin-6 and cardiovascular diseases.
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Pregnancy
18
Xianzhang Huang and Enyu Liang
18.1 Overview 18.2 H

uman Chorionic Gonadotropin
(hCG)
Pregnancy is a complex, irreversible dynamic process in
which there are dramatic changes in the mother for accom- 18.2.1 Resource and Characteristics
modating embryo implantation and nourishing its growth
[1]. During pregnancy, both the mother and the fetus are at Human chorionic gonadotropin (hCG) is produced primarily
some uncertain risk. Many complications related to gestation by differentiated syncytiotrophoblasts and is among the most
include preeclampsia, ectopic pregnancy, and placental commonly used indicators of early pregnancy [4]. hCG is
abruption, leading to the mortality and morbidity of the comprised of an α-subunit (93-amino acid, 14.5 kDa) and
mother and the fetus [2]. Moreover, the vast majority of birth β-subunit (145-amino acid, 22.2 kDa) with non-covalent
defects or genetic disorders are largely incurable and can be interaction which shows 85, 46, and 36% homogeneity to its
a heavy burden for the family and the society. For this rea- glycoprotein hormone family members including luteinizing
son, regardless of risk, all pregnant women should undergo hormone (LH), thyroid-stimulating hormone (TSH), and
prenatal genetic testing to predict and reduce pregnancy- follicle-stimulating hormone (FSH), respectively [8, 9]. Due
related complications and birth defects. to its high structural homogeneity to LH, hCG stimulates
Most prenatal testing is intended for screening with the corpus luteal cells to produce sufficient progesterone to
goal to identify women with high risk of complications, maintain pregnancy by binding to luteinizing hormone/cho-
chromosomal abnormalities, or birth defects [3]. Prenatal rionic gonadotropin receptor (LHCGR) during the first
testing includes serum screening, carrier screening, and 3–4 weeks of pregnancy [10, 11]. This is the physiological
ultrasound. Maternal serum screening is an effective, eas- reason why it can be the early indicator of pregnancy and the
ily accessible, noninvasive method for evaluating pregnant therapeutic drugs for threatened abortion.
risk. Biochemicals produced by the placenta are present in In addition to regular hCG, there are other 15 variants and
the maternal circulation which perform as convenient, just three of which (regular hCG, hyperglycosylated hCG,
effective, and trimester-dependent markers for predicting hyperglycosylated hCG free β) perform as independent hor-
pregnancy outcomes and identifying risk in the fetus. mones with different functions [12]. Hyperglycosylated hCG
These multiple markers include human chorionic gonado- (hCG-H) is produced in states characterized by embryo
tropin (hCG) [4], alpha fetoprotein (AFP) [5], pregnancy- implantation or extravillous invasion cytotrophoblast cell
associated protein (PAPP-A) [6], estradiol (uE3) [7], and invasion [13, 14]. Therefore, it may be the earliest hCG pro-
inhibin A (DIA) [3]. duced in the first week of pregnancy following implantation
or development and advancement of invasive hydatidiform
mole and choriocarcinoma. Hyperglycosylated hCG free β
also produces in early pregnancy and maintains very low
proportions in the whole pregnant process [12]. Each of
those three hormones dominates in different disorders like
X. Huang (*) · E. Liang pregnancy and pregnancy abnormalities, trophoblastic
Department of Laboratory Medicine, The Second Affiliated
Hospital of Guangzhou University of Chinese Medicine,
malignancies and non-trophoblastic malignancies, etc.,
Guangzhou, Guangdong, People’s Republic of China which we will discuss below [12].

230 X. Huang and E. Liang
The biologically active hCG in serum and urine rises of serum hCG varies greatly depending on the detection
exponentially in the first trimester, doubling every 48 h, and methods [19, 21]. Usually, hCG exceeds the upper limit of
reaches a peak at 10 weeks of gestation. From thereon after, the reference value in about 10–14 days after conception
it decreases to the 16th week and maintains approximately which doubles every 2–3 days for the first 4 weeks of preg-
one-fifth of peak concentrations until labor [15]. Serum hCG nancy. It reaches a peak at 10 weeks and drops to lower
is metabolized primarily by the liver with approximately steady-state levels [19, 22].
20% excreted by the kidneys [16]. Therefore, serum or urine
hCG levels provide a variety of important information for
multiple clinical situations, such as diagnosis and monitor- 18.2.4 Clinical Application
ing of pregnancy, prenatal screening, pregnancy-related dis-
orders, and gynecological cancers. 18.2.4.1 D iagnosis and Monitor Normal/
Classically, serum samples are preferred for quantitative Abnormal Pregnancy
hCG, whereas urine samples are used for pregnancy tests and Both diagnosing and monitoring pregnancy can be achieved
to identify false-positive results in serum samples [17, 18]. by detecting either hCG alone or together with hCG. In non-
pregnant women, serum hCG is present at a very low level
[20]. After embryo implantation, the syncytiotrophoblasts
18.2.2 Detection Methods begin to synthesize hCG which will increase rapidly until the
8th week of gestation [23]. Serum hCG levels which are
The early method to detect hCG was radioimmunoassay lower than the lower limit or decrease gradually may be
which gradually was replaced by various nonradioactive associated with early pregnancy loss [4, 24].
immunoassays including chemiluminescent immunoassay, It is proposed that the hCG-H is a better biomarker than
electrochemical immunoassay, fluorescence immunoassay, regular hCG to indicate an early viable embryo implantation
chromatographic immunoassays, resonance scattering spec- because of less hyperglycosylated hCG produced by a failing
trometry, atomic emission spectrometry, MS, and others [19]. pregnancy [25, 26]. The hCG in urine of multiple pregnan-
Currently, the most common quantitative method for detect- cies is often higher than that in singleton pregnancy.
ing serum hCG is the chemiluminescent immunoassay which A single quantitative detection of serum hCG cannot reli-
characterizes with high efficiency, high specificity, high sen- ably distinguish a viable intrauterine pregnancy from a spon-
sitivity, and accuracy. Colloidal gold immunochromatogra- taneous abortion or an ectopic pregnancy because there is a
phy, also known as a pregnancy test strip, is usually used to significant overlap of hCG values among these three clinical
detect hCG in the urine specimens with obvious advantages situations [27, 28]. However, serial hCG measurements, espe-
of simple operation, high efficiency, and low cost. cially two measurements over 48 h, can be helpful for deter-
mining the diagnosis [28]. The possible differential diagnoses
for these clinical situations are summarized as Table 18.1.
18.2.3 Reference Range
18.2.4.2 Diagnosis of Gestational Trophoblastic
Qualitative analysis for hCG in urine: generally, the preg- Disease (GTD)
nancy test strip shows a positive reaction when hCG in urine hCG is the most sensitive tumor marker to diagnose tropho-
is more than 25 mIU/mL. blastic tumors. In addition, molar hCG β concentrations > 5%
Quantitative analysis for hCG in serum: in men and non- are strongly associated with aggressive gestational tropho-
pregnant women, hCG is produced by the pituitary and pres- blastic disease (GTD) [9]. Usually, a combination of hCG
ent at low serum levels. The hCG level is less than 3 mIU/mL and hCGβ are used to diagnosis and monitor GTD or to eval-
in cycling women, which may increase to 6 mIU/mL at uate benign and malignant trophoblastic diseases [30, 31]. A
menopause [20]. The absolute value of serum hCG changes high level or sustained increase of hCG in postoperative
greatly in different weeks of pregnancy. The reference range patients with GTD indicates residual and recurrence [30].
Table 18.1 The possible differential diagnoses of viable intrauterine pregnancy, spontaneous abortion, miscarriages, or ectopic pregnancy [29]
Clinical situations Initial hCG Sonographic evidence Incidence rate (%) The change of hCG over 48 h
Viable intrauterine pregnancy Above 1500–2000 mIU/mL Intrauterine pregnancy 99 Increase ≥ 53%
Ectopic pregnancy Above 1500–2000 mIU/mL No intrauterine pregnancy 21 Increase ≥ 53%
10 Decrease ≥ 30%
Miscarriages 2000 mIU/mL Intrauterine pregnancy 90 Decrease ≥ 30%
Spontaneous abortion 1000 mIU/mL Intrauterine pregnancy 95 Decrease ≥ 28%
18 Pregnancy 231
Fig. 18.1 All variants are Early Pregnancy (3-5 weeks)

present in both serum and General Pregnancy (6 weeks-term)
urine samples. The length of
the column chart in different Down’s syndrome pregnancy
colors are represented the * Abnormal pregnancy
relative proportion of regular
Regular hCG
hCG, hyperglycosylated hCG Pituitary Hyperglycosylated hCG
and hyperglycosylated hCG
free β. *Abnormal pregnancy Hyperglycosylated hCG free β
contains biochemical Quiescent trophoblastic disease
pregnancy, spontaneous Hydatidiform mole
abortion, ectopic pregnancy Invasive mole
Placental site trophoblastic tumor
Choriocarcinoma/Testicular germ cell tumor
Non-gestational malignancies
18.2.4.3 M arker for Down’s Syndrome albumin-like protein family which contains vitamin d-
Screening binding protein, l-albumin, and human serum albumin
hCG and hCGβ concentrations can be used in different tri- (HSA) [36]. Fetal serum AFP can be detected as early as
mesters as a screening test to evaluate for Down’s syn- 29 days after implantation and increases throughout the first
drome. It is recommended to combine serum hCGβ and trimester of gestation reaching a peak level at 10–13 weeks
pregnancy- associated plasma protein-A (PAPP-A) with (3–5 mg/mL) followed by a decline [37]. At around the 35th
maternal age and fetal nuchal translucency (NT) thickness week of pregnancy, the levels of AFP in fetal serum have
at 11th–13th weeks to screen for Down’s syndrome [32, decreased to 200–300 μg/mL and further reduced to 50 μg/
33]. However, during the 15th–22th weeks, hCG, inhibin A, mL in cord blood at birth [38]. AFP is passed from fetal
α-fetoprotein (AFP), and unconjugated estriol are more serum to amniotic fluid (AF) through the kidneys and then
suitable makers [4]. Furthermore, several hCG such as swallowed by the fetus and recirculates through its body
hCG, hCGβcf (hCG β-core fragment, a degradation product [39]. In the end, it is eventually degraded by the fetal liver
of hCG), and hCG-H are present in the maternal urine with with a half-life of 12–24 h [40]. AFP transfers across the pla-
a fetus with Down’s syndrome [33]. hCGβcf is the major centa into maternal circulation and rises from about the 14th
metabolic product of hCG in maternal urine with second- week of pregnancy up until about the 32nd week which is
trimester levels increasing in pregnancies complicated by about 10–150 ng/mL in 15th–20th week [35]. Therefore,
Down’s syndrome [34]. The different hCG variants used in maternal blood AFP level is often part of the screening test
different clinical situations are summarized in Fig. 18.1. for birth defects and pregnancy outcomes.
The changes of hCG have been discussed above, but the
proportion of hyperglycosylated hCG and hyperglycosyl-
ated hCG free β in total hCG gradually decreases along 18.3.2 Detection Methods
with the weeks of gestation.
Diagnosis and monitoring of pregnancy, prenatal aneu- There are various methods to measure the concentration of
ploidy screening, and detection of gynecological cancers are serum AFP, including Radioimmunoassay (RIA), Enzyme-
three important functions of hCG. Additionally, although not Linked ImmunoSorbent Assay (ELISA), Chemiluminescence
discussed here, hCG can be used for early detection of Immunoassay (CLA), Time-Resolved Fluoroimmunoassay
pregnancy-related disorders and treating infertility. (TRFIA), Electrochemiluminescence Immunoassay (ECLI),
etc. [41–43].
18.3 Alpha-Fetoprotein (AFP)

18.3.3 Reference Range
18.3.1 Resource and Characteristics
According to the instruction manual of Roche Electroche-
Alpha-fetoprotein (AFP), a major protein of the embryonic miluminescence Kit for AFP, the level in 95% healthy indi-
plasma, is primarily produced by the embryonic yolk sac viduals < 5.8 IU/mL or 7.0 ng/mL. The absolute values of
which is replaced by fetal liver at around 12 weeks of gesta- serum AFP in different weeks of gestation are summarized
tion [35]. AFP, a 69 kDa single-polypeptide chain, belongs to in Table 18.2.
Table 18.2 Serum AFP in different weeks of gestation Table 18.3 The normal or disease states with abnormal levels of AFP
in different pregnancy stages [5, 36]
Weeks 14 15 16 17 18 19
N 382 1782 2386 975 353 148 First and second trimester of
IU/mL 23.2 25.6 30.0 33.5 40.1 45.5 pregnancy Third trimester of pregnancy
ng/mL 27.9 30.9 36.1 40.4 48.3 54.8 I. High AFP levels
1. Multiple gestation 1. Preeclampsia
Conversion factor: IU/mL × 1.21 = ng/mL
ng/mL × 0.83 = ng/mL 2. Incorrect gestational age 2. Placental previa
levels
3. Placental obstructions 3. Premature labor
In a more recent research, the reference range of men and 4. Fetal growth restriction 4. Placental abruption
non-pregnant women is 0–40 ng/mL and of pregnant women 5. Hypertension 5. Perinatal loss
is 10–150 ng/mL in the 15th–20th week of gestation. AFP 6. Maternal tumor (liver cancer, 5. Fetal urinary tract
gastrointestinal cancer, germ malformations (obstruction,
levels greater than 200 ng/mL in patients with liver cirrhosis cell tumor, etc.) congenital nephrosis, etc.)
strongly indicate hepatocellular carcinoma [35]. 7. Open neural tube defect 6. Fetal digestive tract
malformations (duodenal
atresia, esophageal atresia, etc.)
18.3.4 Clinical Applications 8. Gastrointestinal defects 7. Prematurity
9. Renal agenesis 8. Fetal tumor
10. Cystic hygroma
AFP has long been employed as a gestational age-depen-
11. Tyrosinemia
dent fetal defect marker for aneuploidy chromosome
II. Low AFP levels
abnormalities, neural tube defects, and related spinal dis- 1. Trisomy-21 1. Fetal demise
orders [36]. Moreover, AFP is not only an embryo-specific 2. Trisomy-18 2. Intrauterine growth
protein but also a tumor-associated protein [36]. Therefore, retardation
it has been used as a tumor marker for diagnosis and moni- 3. Spontaneous abortion
toring of hepatocellular carcinoma and other germ cell 4. Non-pregnancy
tumors [35]. 5. Overestimated gestational age
18.3.4.1 Prenatal Diagnosis

The neural tube defects and brain/spinal cord malformations lar cancer, metastatic liver cancer, liver cirrhosis, and
were the first developmental abnormalities found to be asso- hepatitis [47, 48]. AFP usually increases moderately with
ciated with the aberrant AFP levels followed by chromo- less than 100 ng/mL in acute or chronic liver disorders
somal disorders (trisomy) and various anatomical such as liver hepatitis and liver cirrhosis which could be
abnormalities [44, 45]. In the routine clinical diagnosis, increased to greater than 1000 ng/mL [47]. Further, AFP-
serum AFP levels not only assist to diagnose placental abnor- L1 fraction is a major fraction of total AFP in the non-
malities, fetal death, growth restriction/retardation, and pre- malignant liver diseases like chronic hepatitis and liver
term labor, etc., but also perform as an indicator for perinatal cirrhosis, and AFP-L3 fraction appears to be synthesized
distress conditions like preeclampsia, fetomaternal transfu- only by cancer cells which make them a sensitive indica-
sion, intrauterine growth retardation (IUGR), fetal demise, tor for tumor diagnosis [47, 49]. However, it is noted that
etc. [5, 46]. Also, AFP levels in different biologic compart- there are about 30–40% of hepatocellular carcinomas are
ments are also meaningful for prenatal diagnosis [5]. The AFP negative which serve as a reminder to use a combi-
diagnostic functions of AFP levels are summarized in nation of multiple indicators to perform diagnosis of liver
Table 18.3 [5, 36]. cancer [50].
An elevated AFP can also assist in diagnosing yolk sac
18.3.4.2 Diagnosis and Monitoring tumor, germ cell tumors, etc. In adult embryonal carcinomas,
of Hepatocellular Carcinoma the AFP concentration of about 50% of patients is less than
and Other Germ Cell Tumors 50 ng/mL, 40% is 500–1000 ng/mL, and just 1–2% is 1000–
AFP is thought to be the most excellent oncobiomarker 10,000 ng/mL, while 70% of patients with hepatocellular
which is used for diagnosing and monitoring hepatocel- carcinoma is 500–1000 ng/mL and 85% of non-malignant
lular carcinoma for its development, metastasis, progno- liver diseases is less than 50 ng/mL [51]. The serum AFP
sis, and antitumor therapeutic measures [47]. The elevated in almost all the patients with yolk sac tumor, seminoma,
total AFP or its fractions, especially AFPL1 and AFP-L3, dysgerminoma, teratoma, and chorion epithelioma is under
can be used for the differential diagnosis of hepatocellu- 50 ng/mL [51].
18 Pregnancy 233
18.4 Pregnancy-Associated Plasma Table 18.4 The factors associated with the level of serum PAPP-A
Protein-A (PAPP-A) during pregnancy [6, 61, 62]
Maternal and pregnancy-related factors Level of PAPP-A
18.4.1 Resource and Characteristics 1. Singleton pregnancy 1.03–1.07 MoM
2. Twin pregnancy 1.86–2.12 MoM
3. Maternal weight
Pregnancy-associated plasma protein-A (PAPP-A) is a gly-
Low body weight (<45 kg) 1.55 MoM
coprotein derived from the syncytiotrophoblast which is
High body weight (>115 kg) 0.42 MoM
classified as a metzincin metalloproteinase with the charac- 4. Ethnicity (vs. Caucasian)
teristic of the metalloproteinase modules and hence is a East Asian 1.09–1.20 MoM
member of the large family of matrix metalloproteinases [6, South Asian 1.03–1.08 MoM
52]. PAPP-A regulates the formation of the placenta and the Afro-Caribbean 1.55–1.57 MoM
growth of the fetus by insulin-like growth factor system [6]. Adverse pregnancy outcome
Soon after implantation, PAPP-A in the maternal blood 1. Preeclampsia 0.74–0.86 MoM
becomes detectable and increases throughout the whole 2. Stillbirth/intrauterine growth restriction 0.79–0.89 MoM
Chromosomal abnormalities
pregnancy, especially rapidly during the first trimester with a
1. Trisomy-21 0.34–0.62 MoM
doubling time of 3–4 days and maximum levels at term
2. Trisomy 13 0.25 MoM
which make it very dependent on gestational age [53–55]. In 3. Trisomy 18 0.15–0.22 MoM
addition to gestational age, numerous pregnancy-associated 4. Triploidy
factors have an effect on maternal PAPP-A concentration Maternal origin 0.06 MoM
like the factors related to the mass of trophoblastic tissues Paternal origin 0.75 MoM
[6]. In the circulation of pregnant women, almost all PAPP-A MoM multiples of the median, PAPP-A pregnancy-associated plasma
is covalently bound to proMBP (proform of eosinophil major protein-A
basic protein, expressed in extravillous cytotrophoblasts) to
form a hetero-tetrameric complex (PAPPA/proMBP) with standardized by gestational age, maternal weight, method of
two PAPP-A and two proMBP subunits [56, 57]. It should be conception, smoking status, ethnicity, etc.
noted that approximately 1% of the total PAPP-A at term is In non-pregnant healthy individuals, the concentration is
uncomplexed active form which is reported to be a marker of only 3–5 mIU/L [6]. The factors associated with the level of
plaque rupture and increases in patients with acute coronary serum PAPP-A during pregnancy are listed in Table 18.4.
syndrome (ACS) [58]. This will not be discussed here.
Maternal PAPP-A levels reflect the placental volume
which are associated with the amount of trophoblastic tissue 18.4.4 Clinical Applications
and hence are used to screen fetal biometrics and pregnancy
outcomes such as spontaneous abortion, preeclampsia, and PAPP-A is a highly efficient serum marker for chromosomal
preterm delivery [6]. ProMBP was reported to be a marker abnormalities in the first trimester. Additionally, it is closely
for trisomy 21 in the first and second trimester [59]. However, related to adverse pregnancy outcomes like preeclampsia,
the clinical significance of proMBP is less clear. Therefore, intrauterine growth retardation, preterm delivery, and still-
we will not discuss here. birth [63].
PAPP-A is an important index in Down’s syndrome
screening programs because there is a strong correlation
18.4.2 Detection Methods between low maternal serum PAPP-A and trisomy 21, 18,
and 13 [62, 64]. Generally, PAPP-A is 0.34–0.62 MoM for
There are a variety of methods to detect PAPP-A including trisomy 21 which is 0.34–0.44 MoM in or before 11th week
enzyme immunoassay (EIA), immunofluorometric assay gestation, and 0.5–0.62 MoM after 11th week [6]. Compared
(IFMA), electrochemiluminescence immunoassay, and with the PAPP-A in trisomy 21, it is much lower for trisomy
immunochromatographic assay [58, 60]. 18 and trisomy 13, just 0.15–0.22 MoM and 0.25 MoM,
respectively [6, 59, 60]. For triploidy, there is a significant
difference between paternal origin (diandric) and maternal
18.4.3 Reference Range origin (digynic) with the PAPP-A values of 0.75 MoM and
0.06 MoM. For sex chromosome aneuploidies, the PAPP-A
The primary unit of measurement for serum PAPP-A is is 0.49 MoM in Turner syndrome (45, X) and 0.88 MoM in
mIU/L, whereas the multiple of median (MoM) is more other sex aneuploidies (such as 47, XXX; 47, XXY; 47,
commonly used in the risk assessment of pregnancy which is XYY) [6].
Although PAPP-A is closely related to chromosomal 18.5.2 Detection Methods

abnormalities, the combination of other serum makers,
pregnant characteristics, or ultrasonographic features can
The original method of inhibin was radioimmunoassay
promote the detection rate. The detection rate of the combi- which was gradually replaced by enzyme-linked immuno-
nation screen test which includes double serum markers sorbent assay (ELISA).
(PAPP-A and β-hCG), maternal age, and nuchal translucency
thickness is 85–94% and 86–100% for Down’s syndrome
and Edward’s/Patau syndromes, respectively [62, 64, 65]. 18.5.3 Reference Range
Low serum PAPP-A level has been reported to be an inde-
pendent marker of numerous adverse pregnancy outcomes or The median level of inhibin A in maternal serum from sin-
pregnancy complications, such as pregnancy-induced hyper- gleton pregnancy is listed in Table 18.5.
tension, pre-eclampsia, preterm delivery, spontaneous abor- There are various covariates affecting inhibin A concentra-
tion, and others. From a retrospective study with 961 tions including maternal body weight (heavier women have
pregnant women in the first trimester whose PAPP-A levels lower inhibin A), twin pregnancy (twice as high as a single-
were below 0.3 MoM (Reference PAPP-A level 0.9–1.1 ton pregnancy), race (Black women < Caucasian < Southeast
MoM), the odds ratio (OR) of chromosomal anomalies, Asian), and maternal smoking (increasing by almost 50%)
spontaneous abortion, pre-term delivery, and small for gesta- [80–83].
tional age was 116.0, 7.7, 2.3, and 4.9, respectively [63].
18.5.4 Clinical Application

18.5 Inhibin
18.5.4.1 A Marker of Trophoblast Viability
18.5.1 Resource and Characteristics and Placental Dysfunction
Maternal serum inhibin A is mainly derived from the pla-
Inhibin is a hypophysiotropic hormone which regulates pitu- centa and fetal membranes for which it is an appropriate
itary hormone secretion by acting on the pituitary cells [66]. marker for monitoring embryonic viability in early preg-
It is a glycoprotein with two subunits, an α-subunit and a nancy. A rapid decline of inhibin A implies nonviable clini-
β-subunit which form two mature inhibins, inhibin A (αβA) cal pregnancies with embryonic failure [74, 84]. The
and inhibin B (αβB) with short half-lives of about 3–6 min cut-offs 0.553 MoM is the best value to diagnose failing
and 3 min, respectively [67–69]. Within the pituitary-gonadal pregnancy with a sensitivity of 90.6% and a specificity of
axis, inhibin regulates the synthesis of FSH released from the 99.5% [85]. Moreover, inhibin A is also used to monitor
anterior pituitary as an endocrine negative regulatory hor- abortion miscarriage, similar to hCG. Inhibin A and hCG
mone [70]. In general, serum inhibin inversely correlates are lower in complete than in incomplete miscarriage [86].
with the FSH level in adults [71]. Therefore, inhibin has been Therefore, inhibin A is an effective index to predict early
used to assess fertility, pregnancy-related conditions, and pregnancy outcome.
reproductive function.
The ovarian granulosa cells, ovarian theca cells, pituitary 18.5.4.2 P renatal Serum Screening for Down’s
gonadotropes, testis, placenta, fetal membranes, and fetopla- Syndrome
cental unit have all been reported as the candidate source of Maternal serum inhibin A is the predominant variant of
inhibin in which it performed as a paracrine regulator [72– inhibins in the second trimester which make it the fourth
74]. In pregnancy, inhibin A is the predominant form of member in the quadruple screening test (maternal age plus
inhibin in maternal serum which is derived from the placenta
and fetal membranes. Maternal serum inhibin A keeps
Table 18.5 The median level of inhibin A in maternal serum from
increasing throughout the course of pregnancy, but maintains
singleton pregnancy [78, 79]
a relatively low level in the first and second trimesters, and
Gestation weeks Median (pg/mL) Gestation weeks Median (pg/mL)
then rises dramatically during the third trimester which
10 177.5 17 111.9
would significantly decrease within the first hour after the 11 164.3 18 156.0
removal of placenta [66, 75]. However, the placenta pro- 12 159.1 19 146.2
duced excessive inhibin when encountered adverse environ- 13 133.9 20 180.3
mental conditions such as hypertension, infection, and 14 157.4 23–24 352
hypoxia, as part of an adaptive response which made it a 15 142.5 37–42 872
marker for several gestational diseases [76, 77]. Therefore, 16 119.2
inhibin A is commonly used for prenatal screens. The detection method of the above results is ELISA
18 Pregnancy 235
AFP, uE3, hCG, and inhibin A) performed at 15th–18th 18.6 Unconjugated Estriol
weeks of pregnancy for Down’s syndrome, bringing a detec-
tion rate of about 80% at a 5% false-positive rate [82, 87]. 18.6.1 Resource and Characteristics
The maternal serum levels of inhibin A in the second trimes-
ter are twofold higher in Down’s syndrome than in normal Estrogen is the primary female sex hormone which regulates
pregnancies [82]. In Down’s syndrome pregnancy, the serum the development of female secondary sex characteristics and
markers derived from the fetus (AFP) and fetoplacental unit reproductive system. There are three major endogenous
(uE3) are decreased, while the markers from the placenta active estrogens in females including estrone (E1), estradiol
(hCG and inhibin A) tend to be increased [88]. According to (E2), and estriol (E3), among which estradiol is the potent
a report, the maternal serum inhibin A in normal pregnancy and important estrogen during reproductive years. However,
was 1.03 MoM, while in Down’s syndrome was significantly during pregnancy, estriol is the predominant circulating
elevated to 2.06 MoM [89]. estrogen with the most important functions for early preg-
nancy maintenance and fetal development [95].
18.5.4.3 P renatal Serum Screening for Other Estriol (E3) is originated from dehydroepiandrosterone
Chromosome Abnormalities sulfate (DHEA-S) which is released from the fetal adrenal
Maternal serum inhibin A in pregnant women who were glands at the 8th gestational week under the control of hCG
affected with Turner syndrome and hydrops had significantly [96, 97]. From the 10th week, under the stimulation of adreno-
increased with a median level of 3.91 MoM, while in women corticotropic hormone (ACTH), DHEA-S is converted to E3
who were affected with Turner syndrome but without after hydroxylation in the fetal liver and desulfation in the pla-
hydrops, inhibin A decreased markedly at about 0.64 MoM centa before secreting into maternal and fetal circulation [96,
[90]. However, the hydrops didn’t have an effect on inhibin 98]. Therefore, E3 levels are detectable from the 8th week and
A in Down’s syndrome [91]. Also, the median inhibin A increases markedly after 10th week which may achieve more
level in a sex chromosome abnormality (47, XXY) was 1.7 than 1000-fold [99]. Estriol is metabolized via glucuronida-
MoM [92]. tion and sulfation and is excreted through urination [100].
Triploidy, resulting from maternal (digynic) or paternal Although 90–95% of E3 is conjugated with sulfate or
(diandric) errors in meiosis, has variable phenotypes fol- glucuronate, the unconjugated E3 (uE3) showed more clini-
lowed by different maternal marker patterns. The phenotypes cal utility diagnosing adverse fetal development and predict-
caused by maternal errors lead to fetal growth restriction and ing pregnancy complications such as fetal chromosomal
a small placenta, whereas the phenotypes originated from abnormalities, intrauterine growth retardation, and gesta-
paternal errors have a hyperplastic placenta [93]. Since tional diabetes [101–103].
inhibin A and hCG were closely associated with placental
growth, they were simultaneously decreased in maternal
triploidy and were simultaneously increased in maternal trip- 18.6.2 Detection Method
loidy with a media level of 0.3 MoM and 9.0 MoM, respec-
tively. This date was from a study with a small sample size There are various methods for the detection of uE3 including
(n = 5 and 1) [93]. radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), time-resolved fluorescence immunoassay
18.5.4.4 A Marker to Evaluate the Development (TRFIA), and chemiluminescent immunoassay (CLIA).
of Hypertensive Disorders Chemiluminescent immunoassay (CLIA) has been com-
of Pregnancy monly used for its hypersensitivity and high specificity [104,
Multiple studies have shown that preeclamptic tropho- 105]. In addition to these immunoassays, several mass-
blasts expressed excessive inhibin subunits mRNA and spectrometric methods have been developed for accurate
protein leading to an increased level of inhibin A in women quantification and standardization of serum uE3 [106].
with preeclampsia [77, 94]. As is reported, inhibin A is
increased several weeks before the onset of clinical signs
of preeclampsia. The pregnant women whose inhibin A in 18.6.3 Reference Range
15–24 gestational weeks exceeds 1.8 MoM are more likely
to develop preeclampsia with the sensitivity range from 18 • Maternal E3 level [107]
to 48.6% and the specificity of 92% [77]. However, the • 10th week: 5.0–18.5 ng/mL
addition of hCG would not increase the sensitivity for • 29th week: 31.4–301 ng/mL
preeclampsia. • Delivery: 35.4–707 ng/mL
Table 18.6 The detection rates for different tests for Down’s syn- week increasing from 9th to 20th weeks and 0.6%/week
drome during the second trimester [36, 80, 108] from 21st week onwards [112, 113]. Placental cfDNA is
Screening test Detection rates (%) cleared rapidly after birth, so the cfDNA in the maternal
Maternal age alone 30–32 serum reflects the current conceptus only [114].
Maternal age, plus Since most circulating cfDNA in maternal serum is
AFP 36
derived from the mother, it makes its isolation and measure-
hCG 49
ment more difficult and complicated. Currently, the DNA
uE3 48
AFP + hCG 63 size is the most frequently used parameter to differentiate
AFP + hCG + uE3 69–71 fetal cfDNA from maternal cfDNA with a length of 143 bp
AFP + hCG + uE3 + inhibin A 79 and 166 bp, respectively [115]. This characteristic is
employed to improve the accuracy of circulating cfDNA pre-
natal screening.
18.6.4 Clinical Application
18.6.4.1 P renatal Serum Screening for Down’s 18.7.2 Detection Method

Syndrome
uE3 is a part of the triple test (AFP, hCG, and uE3) for There are three commercial cfDNA-based noninvasive
Down’s syndrome which improves the detection rate from screening (NIPS) methods. The first one is massively parallel
58 to 69% at a 5% false-positive rate in the second trimester shotgun sequencing (MPSS) based on a universal amplifica-
when it increases rapidly [108]. The detection rates for dif- tion and sequencing of all cfDNA with the advantage of need-
ferent tests for Down’s syndrome during the second trimester less separation and enrichment of fetal DNA and with the
are summarized in Table 18.6 [36, 80, 108]. disadvantage of high price and sophisticated algorithms. The
As it is reported, pregnancies with very low or undetect- second one is targeted massively parallel sequencing (t-MPS)
able levels of uE3 are associated with fetal abnormalities just with sequencing and counting the selected regions on the
such as trisomy 18, open neural tube defects, and Smith- chromosomes of interest which reduce the work and costs
Lemli- Opitz syndrome (SLOS) [109]. Additionally, the significantly. The third one is single nucleotide polymor-
undetectable uE3 during the second trimester should take phism (SNP)-based approach with specific amplification and
steroid sulfatase deficiency (STSD) into consideration which sequencing of SNPs which could return accurate copy num-
will interfere with placental estriol production leading to ber calls across chromosomes. This method is not suitable for
undetectable uE3 [109]. high genetics homology test, pregnancy from egg donation,
and bone marrow transplantation [113].
18.6.4.2 M onitor Placental Function and Fetal
Development
The precursor for placental E3 is dehydroepiandrosterone 18.7.3 Clinical Application
sulfate (DHEA-S) which is released from the fetal adrenal
glands. Although the fetal liver is a site for the hydroxylation The use of fetal cell-free DNA (cfDNA) to screen for chro-
of DHEA-S, it has limited capacity to supply 16a-hydroxy- mosome abnormalities was introduced in 2011 [116].
DHEA-S for which maternal E3 reflects the steroidogenic Although cfDNA is well known for its high sensitivity and
activity of the fetal adrenal glands and the integrity of specificity to screen trisomy 21 (Down’s syndrome), along
maternal-placental-fetal unit [110]. It is also commonly used with the development of detection technology, its clinical
to monitor the placental function and fetal development. applications are constantly expanding from screening of tri-
somy 13, trisomy 18, sex chromosome aneuploidies to
microdeletions and microduplications, and even single gene
18.7 Cell-Free DNA (cfDNA) mutations [117, 118]. For this reason, the extension and
application of fetal cfDNA reduce the invasive tests for fol-
18.7.1 Resource and Characteristics low-up validation.
Fetal cell-free DNA (cfDNA) is detectable in maternal blood 18.7.3.1 Trisomy 21 (Down’s Syndrome)
from early pregnancy. The placenta, precisely the outer cyto- The fetal cfDNA in pregnant women affected by trisomy 21
trophoblastic layer, is the primary source of circulating fetal is 17% higher than euploid pregnancies with the median fetal
DNA which is just a small portion of circulating total cell- fraction 1.045 MoM [119]. As it is reported, the detection
free DNA in maternal plasma [111]. The fetal cfDNA is rate of fetal cfDNA for trisomy 21 is more than 99.0% at a
about 8–10% in the 9th gestational week and maintain 0.1%/ false-positive rate lower as 0.1%, whereas the detection rate
18 Pregnancy 237
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Urine
19
Bingfeng Zhang and Qing Li
19.1 Overview character and can be collected noninvasively. Urinalysis

can be used to detect a variety of urinary system diseases,
The kidney is a vital organ of the human body with the such as chronic kidney disease, kidney stones, urinary tract
basic function of generating urine (Fig. 19.1). Renal blood infection, and bladder infection. Urine is produced by the
flow accounts for about 1/4–1/5 of systemic blood flow kidneys, and its properties can be affected by the condition
which generates approximately 1500 mL of urine. Certain of the kidneys. In order to improve the accuracy of diag-
wastes and toxins, such as creatinine, urea, uric acid, and nosis and prognosis of AKI and CKD, biomarkers in the
other metabolites, are partially or wholly removed from the urine need to be detected. Common biomarkers in urine
body through glomerular filtration and renal tubular secre- are shown in Table 19.1.
tion. At the same time, renal tubules reabsorb water and
other useful substances, such as glucose, proteins, amino
acids, sodium ions, potassium ions, and sodium bicarbon- 19.2 Urea
ate, to regulate water and electrolyte balance and maintain
acid-base balance. Furthermore, the kidney also has an 19.2.1 Sources and Characteristics
endocrine function, which produces renin, erythropoietin,
vitamin D3, prostaglandins, and kinins, which participate Urea is the main nitrogen-containing metabolite of human
in the regulation of vasodilation and contraction of intra- protein catabolism, which was discovered in 1773 by Hilaire
renal and extrarenal vessels in the kidney, and is the site Rouelle, accounting for more than 75% of the excreted non-
of endocrine hormone degradation and the target organ of protein nitrogen. The ammonia biosynthesis of urea derived
extrarenal hormones. These functions of the kidney ensure from amino nitrogen is performed only by the liver enzymes
the stability of the internal environment of the body and of the urea cycle. In protein catabolism, amino acid nitrogen
maintain normal metabolism. is converted into urea in the liver by the action of the urea
When the kidney develops a lesion, it will affect its nor- cycle enzymes [1]. The molecular structure of urea is shown
mal function, causing a series of clinical symptoms related in Fig. 19.2.
to the course of the disease, such as high blood pressure, More than 90% of urea is excreted through the kidneys,
proteinuria, and edema. At the same time, some body fluid and the loss through the gastrointestinal tract and skin
components change with the development of kidney dis- accounts for a small part of the rest (Fig. 19.3). Therefore,
ease, revealing to some extent the location of the lesion kidney disease is related to the accumulation of urea in the
and the severity of the disease. At present, serum creati- blood. Increased plasma urea concentration is character-
nine (SCR) and other functional biomarkers have been istic of a uremic state. Urea is neither actively reabsorbed
used to diagnose acute kidney injury (AKI) and chronic nor secreted by the renal tubules but is freely filtered by the
kidney disease (CKD). However, urine is more suitable glomeruli. In a normal kidney, 40–70% of the highly diffus-
for clinical diagnostics than blood because it is stable in ible urea is passively transferred from the renal tubule to the
interstitial space and eventually into plasma. The reabsorp-
tion of urea also depends on the urine flow rate, with less
B. Zhang (*) · Q. Li urine entering the interstitium during high flow conditions
Department of Laboratory Medicine, The First Affiliated Hospital (e.g., pregnancy) and vice versa. Therefore, urea clearance is
of Nanjing Medical University, Nanjing, Jiangsu, usually underestimated.

242 B. Zhang and Q. Li
Fig. 19.1 Structural relationship between the cortex, medulla, and nephron segments
19.2.2 Detection Methods 19.2.4 Clinical Significance
Enzyme Coupling Rate Method Urease hydrolyzes urea An elevated serum urea level can be found in renal insuf-
to produce ammonia and carbon dioxide. In the presence of ficiency caused by various diseases, including acute
α-ketoglutarate and reduced coenzyme I (NADH), glutamic nephritis, chronic nephritis, toxic nephritis, severe pyelo-
acid dehydrogenase (GLDH) catalyzes ammonia to form nephritis, renal tuberculosis, renal vascular sclerosis,
glutamic acid. At the same time, NADH is oxidized to oxi- congenital polycystic kidney disease, and renal malig-
dized coenzyme I (NAD), and the decreased rate of NADH nancies. Especially for the diagnosis of uremia, the
at 340 nm is proportional to the urea concentration in the increase of its degree is directly proportional to the sever-
sample. ity of the disease. However, due to the lack of sensitivity,
it cannot be used as an indicator of early renal damage. In
addition, some prerenal diseases (congestive heart fail-
19.2.3 Normal Reference Interval ure, severe vomiting, diarrhea, renal blood flow reduc-
tion, shock, gastrointestinal bleeding, severe infection,
Enzyme coupling rate method: serum, 3.2–7.1 mmol/L. diabetic acidosis, adrenal insufficiency.) and postrenal
19 Urine 243
Table 19.1 Common biomarkers

Biomarkers Clinical significance Detection technology Reference range
Urea Increase: Enzymatic methods 3.2 ~ 7.1 mmol/L (serum) [1]
1. Acute and chronic nephritis
2. Toxic nephritis
3. Diabetic acidosis, etc.
Creatinine Increase: The Jaffe reaction Male: 80 ~ 115 μmol/L (serum)
1. Renal drainage dysfunction, Female: 53 ~ 97 μmol/L (serum)
such as renal failure [2]
2. Uremia
3. Hyperthyroidism, etc.
Urinary microalbumin Increase: Immunoturbidimetry 0 ~ 20 mg/L (urine) [3]
More common in diabetes and
hypertension
Cystatin C Increase: Immunoturbidimetry Male: 0.63 ~ 1.25 mg/L (serum)
1. Renal damage in diabetic Female: 0.54 ~ 1.15 mg/L
patients (serum)
2. Rejection after renal
transplantation
3. Cardiovascular disease, etc.
Transferrin Increase: Glomerular injury Immunoturbidimetry <2.0 mg/L (serum) [4]
Uric acid Increase: Enzymatic method Male: 0.208 ~ 0.428 mmol/L
1. Primary hyperuricemia and (serum)
gout Female: 0.155 ~ 0.357 mmol/L
2. Leukemia
3. Other malignant tumors, etc.
α1-microglobulin Increase: Radioimmunoassay 10 ~ 30 mg/L (serum)
1. Primary glomerulonephritis 0.48 ~ 4.24 mg/L (urine) [5]
2. Interstitial nephritis
3. Diabetic nephropathy
4. lupus nephropathy, etc.
Decrease: Immunoturbidimetry 10 ~ 30 mg/L (serum),
1. IgA myeloma <12.5 mg/L (urine)
2. Severe liver function damage,
3. Hepatocellular carcinoma, etc.
β2-microglobulin Increase: Scattering nephelometry 1 ~ 3 mg/L (serum),
1. Acute and chronic 0.1 ~ 0.3 mg/L (urine) [6]
pyelonephritis
2. Rejection after renal
transplantation
3. Diabetic nephropathy, etc.
Neutrophil gelatinase-associated Increase: ELISA 34 ~ 106 ng/mL (serum),
lipocalin Acute renal lesion biomarker 0.7 ~ 9.6 ng/mL (urine) [7]
N-Acetyl-β-d Glucosamine Increase: Chemical method 7.5 ~ 28.7 U/L (serum),
Glycosaminase 1. Early renal injury and 0 ~ 11.5 U/L (urine)
microvascular disease
2. Renal vascular hypertension
3. Monitoring of renal transplant
patients, etc.
Retinol binding protein Increase: Immunotransmission 25 ~ 70 mg/L (serum),
1. Chronic kidney disease turbidimetric method 0 ~ 0.7 mg/L (urine)
2. Nutritional excess fatty liver
3. Coronary artery disease, etc.
Decrease:
1. Hepatobiliary diseases
2. Hyperparathyroidism
3. Malabsorption syndrome, etc.
diseases (prostate enlargement, urinary calculi, urethral

stricture, bladder tumor) can also lead to increased serum
urea level.
19.3 Creatinine
Creatine is synthesized in the kidney, liver, and pancreas with

two enzyme-mediated reactions. Firstly guanidinoacetic acid
is formed by transamidation of arginine and glycine, and
then S-adenosylmethionine, as the methyl donor, contributes
to methylation of guanidinoacetic acid. Finally, creatine is
transported in blood to other organs, such as the muscle and
brain, with the process of phosphorylation to phosphocre-
atine (a high-energy compound) [2]. The main routes of Cr
metabolism in the mammalian body are shown in Fig. 19.4.
Endogenous creatinine clearance rate refers to how much
volume of plasma endogenous creatinine is removed by the
kidney per unit time. It is a sensitive index to evaluate glo-
Fig. 19.2 Molecular structure of urea merular filtration rate.
Fig. 19.3 Urea cycle

19 Urine 245
pounds could produce a Jaffe-like chromogen, including

protein, glucose, ascorbic acid, ketone bodies, pyruvate, gua-
nidine, blood-substitute products, and cephalosporins.
Enzymatic Method Due to the lack of specificity of chemi-

cal methods, the enzymatic method with creatininase c oupled
sarcosine oxidase is suitable for routine work in clinical
laboratories.
19.3.3 Normal Reference Interval
Jaffe methods:
• Serum: male, 80–115 μmol/L; female, 53–97 μmol/L.

• Urine: male, 124–230 μmol/kg/day; female, 97–177 μmol/
kg/day.
• Urinary creatinine excretion decreases with age; typi-
cally, for a 70 kg man, creatinine excretion will decline
from approximately 14.5 to 9.1 mmol/day from age 30 to
80 years. Measurement of that can be a useful marker for
the completeness of a timed urine collection.
Fig. 19.4 Main routes of Cr metabolism in the mammalian body 19.3.4 Clinical Significance
Interconversion among phosphocreatine and creatine Serum creatinine can be seen in renal failure [1]; severe con-
is a remarkable characteristic for the metabolic processes gestive heart failure, hyperthyroidism, salicylic acid treatment,
of muscle contraction. A proportion of the free creatine in and eating food containing a large amount of creatinine will
muscle (thought to be between 1 and 2% day) spontaneously also lead to a significant increase in blood creatinine. To avoid
and irreversibly converts to creatinine, making the amount the influence of exogenous creatinine, serum creatinine, or
of creatinine produced each day fairly constant and related endogenous creatinine clearance rate, it is recommended to
to the muscle mass (and body weight). In healthy people, take in a no creatinine diet 2–3 days before testing.
the blood creatinine concentration is also fairly constant,
although diet may influence the value, dependent on the indi-
vidual’s meat intake. Creatinine is present in all body fluids 19.4 Urinary Microalbumin
and secretion, which is freely filtered by the glomerulus. For
the existence of non-reabsorption by the renal tubules to any 19.4.1 Sources and Characteristics
great extent, there is a small but significant tubular secre-
tion [1]. Creatinine production also decreases when there is When blood is filtered from the glomerulus, the macromo-
an increasing circulating level of creatinine; several related lecular protein cannot pass through the basement membrane,
mechanisms have been proposed, including feedback inhibi- and only a very small amount of albumin leaks out and is
tion of the production of creatine, reconversion of creatinine reabsorbed by the renal tubule. Under physiological condi-
to creatine, and conversion to other metabolites. tions, only a small amount of albumin appears in the urine.
When the glomerulus is damaged, the permeability of the
basement membrane increases, leading to a large amount
19.3.2 Detection Methods of protein entering the filtrate and the urine. The amount of
protein and the molecular weight of the protein is highly cor-
The Jaffe Reaction Measurement Most chemical methods related with the degree of lesion of the glomerulus. When
are primarily based on the reaction with alkaline picrate, the glomerulus is slightly damaged, a small amount of albu-
while creatinine reacts with picrate ion in an alkaline min appears in the urine, which is considered to be the most
medium, yielding an orange-red complex. The Jaffe reaction reliable diagnostic indicator for early kidney damage [1]
is not specific for creatinine. It is reported that many com- (Fig. 19.5).
Fig. 19.5 Filtration of kidney
19.4.2 Detection Methods Due to the influence of drinking water and dietary factors,
it is recommended to measure urine creatinine at the same
Immunoturbidimetry Albumin and an antibody rapidly time. The albumin/creatinine ratio report can better reflect
form an antigen-antibody complex in a special buffer, and the the degree of glomerular damage: 0–20 mg/g.
reaction solution exhibits a change in turbidity. When the anti-
body is kept in excess in the reaction solution, the formed com-
plex increases as the amount of albumin increases, and the 19.4.4 Clinical Significance
turbidity of the reaction solution also increases. The results are
compared with the standard to calculate the albumin content. Increased urinary microalbumin is more common in diabe-
tes and hypertension [8]. The results of urine microalbumin
determination, combined with the onset of symptoms, and
19.4.3 Normal Reference Interval medical history, can be used to diagnose the disease more
accurately, which is particularly important for judging the
Immunoturbidimetry: urine, 0–20 mg/L. course of disease and prognosis.
19 Urine 247
19.5 Cystatin C 19.5.4 Clinical Significance
19.5.1 Sources and Characteristics Serum cystatin-C is considered to be an endogenous indi-

cator that reflects glomerular filtration rate simply, quickly,
Cystatin C (CysC) is also named cysteine protease inhibi- and accurately. The linear relationship is significantly bet-
tor. In 1983, high-purity cysteine protease inhibitor was ter than the inulin clearance rate and phenol red excretion
first isolated and purified from egg white by Anastasi. CysC test. There is a high degree of correlation, which avoids the
gene with a length of 4.3 kb exists on the short arm of chro- source of error in urine retention compared with endogenous
mosome 20. All nucleated cells in the body can produce creatinine clearance and has obvious advantages. It is com-
CysC gene constantly. It is widely distributed in nucle- monly used in clinical evaluation of creatinine clearance
ated cells and body fluids of various tissues. Its molecular in children, renal damage in diabetic patients and rejection
weight is 13.3 kDa and it is composed of 122 amino acids. after renal transplantation, and prediction of nephrotoxicity
The residue consists of an alkaline non-saccharified low in the treatment of cancer patients. Recent studies have also
molecular protein having an isoelectric point of 9.3. CysC found that serum concentrations in liver fibrosis, liver cancer
in the circulation is completely filtered by the glomerulus lesions, and cardiovascular disease are slightly elevated.
and is metabolized and decomposed after being re-absorbed
in the proximal convoluted tubules. Renal tubular epithelial
cells also do not secrete cystatin-C into the lumen. It is an 19.6 Transferrin
endogenous marker that reflects changes in glomerular fil-
tration rate [9]. 19.6.1 Sources and Characteristics
CysC varies with age, and there are also large differences
in different tissues of the human body. The number at birth is Transferrin (Tf) is a glycoprotein consisting of 679 amino
higher, ranging from 1.64 to 2.59 mg/L, and decreases rap- acids with a molecular weight of 76.5 kDa and a half-life
idly within 4–5 months. The concentration is constant over period of 7–10 days. It is mainly synthesized in the liver and
1 year, close to the level in adults. Women under the age is the main protein that transports Fe3+ [10]. Tf has a molecu-
of 60 years have slightly lower levels of CysC than men of lar weight close to Alb and a diameter similar to that of Alb
the same age, although it has been shown that female hor- (tf 3.91 nm, alb 3.60 nm). Under physiological conditions, Tf
mone levels and drinking do not affect the concentration of is difficult to pass through the glomerular filter [11]. When
CysC, and there was no significant difference between the the glomerular charge barrier is damaged early, Tf leaks out
levels found in males and females over 60 years old. CysC of the urine, which is a sensitive indication of glomerular
is found in various body fluids of the human body, with the filter damage.
highest concentration in semen (40–60 times higher than in
serum), followed by cerebrospinal fluid (5 times higher than
in serum), while the concentration in urine is only about 2% 19.6.2 Detection Methods
of serum [5].
Scattering Turbidity Method Urine transferrin detection
methods include radioimmunoassay, enzyme-linked immu-
19.5.2 Detection Methods nosorbent assay, particle-enhanced scatter immunoturbidi-
metric assay on a special protein analyzer, and transmissive
Immunoturbidimetry The CysC in the sample causes spe- immunoturbidimetric assay on biochemical analyzers. At
cific anti-antibody reaction of the anti-human CysC poly- present, the latter two methods are used more in favor [12].
clonal antibody latex particles in the buffer to produce
turbidity. Since the degree of turbidity is positively corre-
lated with the density of CysC in the sample, the decrease in 19.6.3 Normal Reference Interval
absorbance can be monitored at 540 nm wavelength, and the
density of CysC in the sample is calculated in comparison Scattering turbidity method: serum, <2.0 mg/L.
with the standard.

The increase of urine Tf is earlier than mAlb in the occur-
Immunoturbidimetry: serum—male, 0.63–1.25 mg/L; female, rence of glomerular injury. Some people in the study of urine
0.54–1.15 mg/L. Tf concentration in diabetic patients with different urine pro-
tein concentrations found that uTf is a more sensitive sign of and 3,5-dichloro-2-hydroxybenzoic acid (DHBs) react to form
early detection of changes in diabetic nephropathy [13]. It blue substance. The absorbance value at the wavelength of
mainly reflects that the charge selective barrier of glomeru- 660 nm is proportional to the concentration of uric acid in the
lar filtration membrane is damaged. The diagnostic value of sample. This method has been used in many kinds of biochem-
urine transferrin for primary glomerulonephritis may be the ical automatic analyzers.
same as that of cystatin C, and it also has a certain refer-
ence value for disease monitoring [14]. Combined detection
of urinary microglobulin, urinary microalbumin, and urinary 19.7.3 Normal Reference Interval
transferrin is helpful to understand the early renal injury in
patients with diabetes, and is more conducive to the early Enzymatic method: serum—male, 0.208–0.428 mmol/L;
diagnosis of diabetic nephropathy [15]. female, 0.155–0.357 mmol/L.
The concentration of serum uric acid increases gradu-
ally with age, and it is increased in postmenopausal women.
19.7 Uric Acid During pregnancy, serum uric acid concentrations decrease
in the first 3 months of pregnancy and begin to rise around
19.7.1 Sources and Characteristics 24 weeks, and finally surpass the concentrations seen in a
non-pregnant state.
Uric acid (2, 6, 8-trihydroxypurine), with the molecular for-
mula C5H4N4O3 (Fig. 19.6), was discovered by Karl William
Scheler in 1776. Its molecular weight is 168.11Dalton, and 19.7.4 Clinical Significance
its alcohol formula is weak acid. In the human body, purines
derived from the catabolism of food nucleic acids are converted Increased uric acid in blood can be seen in gout. However,
directly into uric acid. The daily synthesis rate of uric acid is normal blood uric acid levels can be seen in a small number
about 400 mg. Of this, about 300 mg comes from food. All uric of patients during an acute gout flare. When cell prolifera-
acid in the blood is filtered from the glomeruli, secreted and tion cycle is fast and the nucleic acid catabolism is increased,
reabsorbed by the proximal convoluted tubules, and eventu- serum uric acid value is often increased in leukemia and other
ally about 6–10% uric acid is excreted from the urine. Various malignant tumors, multiple myeloma, and erythremia. The uric
factors can result in overproduction or under-excretion of uric acid in the blood increases significantly after chemotherapy.
acid, leading to increased uric acid concentration in the blood. Declining kidney function is often accompanied by elevated
As a result of this, urolithiasis or gout can occur. uric acid. This can be seen in acute and chronic nephritis,
renal tuberculosis, pyelonephritis, hydronephrosis, and so on.
Chloroform poisoning, carbon tetrachloride poisoning, lead
19.7.2 Detection Methods poisoning, carbuncle, pregnancy reaction, and eating food rich
in nucleic acids can increase uric acid in the blood as well.
Enzymatic Method The uric acid detection procedure is Hypouricemia can be seen in severe hepatocellular dis-
based on the oxidation of uric acid by uricase to allantoin, ease in which purine synthesis or xanthine oxidase activity
carbon dioxide, and hydrogen peroxide. Under the catalysis of is reduced, or if there is a reabsorption defect of uric acid in
peroxidase, hydrogen peroxide, 4-aminoantipyrine (4-AAP), renal tubule such as Fanconi’s syndrome [16]. Overtreatment
with allopurinol or uricosuric drugs and cancer chemother-
apy may also cause hypouricemia.
19.8 α1-Microglobulin
In 1975, Ekstrom isolated α1-microglobulin from the urine

of patients with chronic cadmium poisoning. It is a kind of
glycoprotein synthesized by human liver and lymph cells
with a molecular weight of 27.0 kD [16]. It is composed of
and widely exists on the surface of various body fluids and
lymphocyte membranes. α1-MG in the blood exists in two
Fig. 19.6 The structure of uric acid forms: free α1-MG and IgA-binding α1-MG (α1-MG-1gA).
19 Urine 249
Normally, α1-MG-1gA accounts for about 40–70% of the are transported to the outer membrane after assembly in the
total α1-MG in the blood, and the level of immunoglobulin endoplasmic reticulum. In order to enhance the antigen pre-
in blood has an effect on the ratio between the free form and sentation of MHC-1, β2-MG is upregulated in early stages of
the binding types. The free α1-MG in the blood can be freely infection [19]. In addition, β2-MG can increase susceptibility
absorbed and metabolized through glomerular filtration to age-related chronic neurodegenerative diseases by weak-
membranes [17]. 95–99% of the free α1-MG is reabsorbed ening hippocampal dependent cognitive and regenerative
and metabolized in proximal convoluted tubules of the kid- capabilities in recent studies [20]. β2-MG is a polypeptide
ney, and only trace amounts are excreted from the final urine. that has a relative molecular weight of 11.8 kD and consists
However, conjugated alpha-1-MG cannot pass through of 99 amino acids. It is the β-chain (light chain) portion of the
glomeruli [18]. It is generally believed that the determination cell surface human lymphocyte antigen (HLA) with a pair of
of alpha-1-MG in serum and urine can be used as a sensitive disulfide bonds in the molecule and contains no sugar; it is
index to reflect impaired renal tubules. similar in structure to the immunoglobulin stable region. It
is widely found in plasma, urine, cerebrospinal fluid, saliva,
and colostrum. All nucleated cells in the human body can
19.8.2 Detection Methods synthesize β2-MG, while lymphocytes contribute mainly.
After glomerular filtration, renal tubular epithelial cells take
Radioimmunoassay ELISA and Immunoturbidime- up 99% of them through the form of pinocytosis. The lyso-
try With the development of technology, detection methods somes decompose them into amino acids, which are reab-
have changed. Radioimmunoassay and ELISA have gradu- sorbed by renal tubules. The β2-MG no longer flows back
ally been replaced by simple, rapid, and stable immunoturbi- into the blood circulation. Under normal circumstances, the
dimetry. The results of different methods were different. The concentration of β2-MG in the urine is very low. An increase
serum concentration of males is slightly higher than that of means that the renal tubular function is impaired. The syn-
females, and there is no significant difference between males thesis and release of β2-MG is very constant, regardless of
and females in urine concentration. gender, age, and time. When the glomerular filtration rate
decreases, or when the renal blood flow decreases, the blood
concentration of β2-MG increases.
Radioimmunoassay: serum, 10–30 mg/L; urine, 0.48–4.24 mg/L. 19.9.2 Detection Methods

Immunoturbidimetry: serum, 10–30 mg/L; urine, <
12.5 mg/L. Scattering Nephelometry Anti-human β2-microglobulin
antibody and β2-microglobulin in the sample combine to
form an immune complex, suspended in a buffer to change
19.8.4 Clinical Significance the scattered light signal, and calculate β2-microglobulin
concentration by measuring the change of the signal.
The main reasons for the increase of α1-MG in serum and
urine are the decrease of renal tubular reabsorption and
metabolism of α1-MG, impaired glomerular filtration func- 19.9.3 Normal Reference Interval
tion, excessive synthesis in vivo, and destruction and release
of lymphocytes. Clinically, it is common in the early stages Scattering nephelometry: serum, 1–3 mg/L; urine, 0.1–0.3 mg/L.
of glomerular injury, primary glomerulonephritis, interstitial
nephritis, diabetic nephropathy, lupus nephropathy, acute, and
chronic renal failure [8]. The decrease is rare, and is related to 19.9.4 Clinical Significance
insufficient synthesis. It is found in IgA myeloma, severe liver
function damage, and hepatocellular carcinoma [18]. An increase of serum β2-MG may reflect the glomerular fil-
tration function or the increase of filtration load. An increase
of β2-MG excretion in urine indicates that there is tubular
19.9 β2-Microglobulin damage or increased filtration load. In acute and chronic
pyelonephritis, urinary β2-MG is elevated due to kidney
19.9.1 Sources and Characteristics damage. β2-MG gradually increases in diabetic patients with
renal dysfunction. It is considered to be a simple, accurate,
α-microglobulin and β2-microglobulin (β2-MG) are two and sensitive method for measuring renal dysfunction and
polypeptide chains that make up the MHC-1 molecule and efficacy.
Renal transplantation patients with significantly 19.10.2 Detection Methods

increased blood and urine β2-MG indicate that the body
is undergoing rejection as evidenced by elevated blood ELISA Lipocalin-2 (NGAL) is typically assessed for clini-
β2-MG from accelerated synthesis of β2-MG. Generally, cal or research purposes using ELISA or immunoturbidimet-
blood β2-MG rises to a peak 2–3 days after transplanta- ric assays. The meta-analysis showed that the detection of
tion, and then gradually decreases. Continuous measure- NGAL by the immunoassay analyzer (Architecture and
ment of blood and urine β2-MG after renal transplantation Triage) was better than that of the ELISA method. The area
can be used as a sensitive indicator of glomerular and under the ROC curve was 0.732 and 0.83, but the detection
tubular lesions. Decreased blood β2-MG following kid- limit (1.5 and 50 ng/mL) was inferior to ELISA (0.11 ng/
ney transplantation suggest good prognosis. The increase mL). In January 2011, the NGAL testing program began in
of blood β2-MG prior to rejection, and the determination major hospitals in Europe.
of β2-MG, is helpful in diagnosing subclinical kidney
rejection. The assay is also used to monitor the function
of proximal tubules. In the case of acute tubular injury 19.10.3 Normal Reference Interval
or necrosis, chronic interstitial nephritis, or chronic renal
failure, urinary β2-MG can be significantly increased. ELISA: serum, 34–106 ng/mL; urine, 0.7–9.6 ng/mL.
Renal transplantation patients with blood and urine β2-MG
increased significantly suggest that the body is undergoing
rejection; continuous measurement of β2-MG after kidney 19.10.4 Clinical Significance
transplantation can be used as a sensitive indicator to eval-
uate glomerular and tubular function. In the early stages It is considered that traditional markers of kidney injury,
of diabetic nephropathy, there is a change in renal tubular such as creatinine and blood urea nitrogen, are neither sensi-
function, and urinary β2-MG is also elevated. Cases such tive nor specific for the diagnosis of AKI. Increases in these
as systemic lupus erythematosus, hematopoietic malig- traditional markers are significant only after important kid-
nancies, and chronic lymphocytic leukemia also can have ney injury and a substantial time delay. They are therefore
elevated urine β2-MG. not optimal to achieve early diagnosis of AKI [25].
β2-MG is an important aging factor which can affect Because NGAL is released quickly after renal tubu-
nerve and cognitive functions [21]. Higher β2-MG levels lar injury, it is often used as a biomarker for renal injury
also increase the risk of cardiovascular disease (CVD) and [7]. The level of NGAL in the urine and plasma increases
stroke among those with and without chronic kidney dis- within 2 h of kidney injury. In AKI, the level of NGAL in
ease [22]. blood increases to 300 times (0.1–30 g/mL) and 1000 times
Urine β2-MG determination also helps identify upper and (0.04–40 mg/mL) in urine. Studies found that NGAL can be
lower urinary tract infections. Upper urinary tract infections detected in both plasma and urine, and it can be used for
easily affect renal tubular reabsorption of molecular pro- early prediction of AKI and its severity, and a high level of
teins, leading to elevated urinary β2-MG. However, urinary serum NGAL is associated with an increased risk of death. In
β2-MG does not increase in patients with lower urinary tract addition, urine and serum NGAL concentrations have been
infection. shown to be sensitive, specific, and highly predictive markers
of AKI after cardiac surgery.
19.10 Neutrophil Gelatinase-Associated

Lipocalin 19.11 N
-Acetyl-β-d Glucosamine
Glycosaminase
Neutrophil gelatinase-associated lipocalin (NGAL) is
a type of lipocalin, originally a small molecular weight N-acetyl-beta-d glucosamine glycosaminase (NAG), widely
secreted protein found in activated neutrophils. NGAL has existing in various tissues and organs, body fluids, and
since been described in many other cell types. NGAL is cells in the lysosome, is a type of high molecular weight of
expressed in renal, endothelial, liver, and smooth muscle lysosomal enzyme (a kind of acid hydrolysis enzyme). It is
cells (SMCs), as well as cardiomyocytes, neurons, and vari- mainly from the renal proximal convoluted tubule epithelial
ous populations of immune cells, such as macrophages and cells and is one of the most sensitive indicators of renal tubu-
dendritic cells [23, 24]. lar function.
19 Urine 251
19.11.2 Detection Methods 86.7% of them all showed increased urine NAG, while for
the patients with primary hypertension, increased urine NAG
Chemical Method NAG catalyzes the hydrolysis of pnp- only accounted for 16.7%.
nag to produce p-nitrophenol (PNP). After a certain period of In addition, NAG detection plays an important role in
time, the reaction is terminated, and PNP develops color in the early rejections after renal transplantation, urinary tract
an alkaline solution. The absorbance value at the wavelength infection localization, and monitoring of nephrotoxicity.
of 410 nm is proportional to the activity of NAG in the
sample.
19.12 Retinol Binding Protein
19.11.3 Normal Reference Interval 19.12.1 Sources and Characteristics
Chemical method: serum, 7.5–28.7 U/L; urine, 0–11.5 U/L. In 1961, Berggard discovered retinol binding protein (RBP)
via immunoelectrophoresis, where a long sedimentation
line was formed in the 2-globulin region, which was once
19.11.4 Clinical Significance called long 2-globulin. In the following decades, the molec-
ular structure and biological characteristics of this protein
Both urine microalbumin and NAG can be used to detect were comprehensively studied, and it was named as retinol-
early renal injury in diabetic patients, but the increase of binding protein (RBP). Retinol binding protein is a low
the two levels does not show a completely balanced rela- molecular weight protein with a molecular weight of about
tionship, which may reflect the renal injury from different 21 kDa, which is mainly synthesized by the liver and widely
aspects. Clinical data shows that in diabetes mellitus patients distributed in blood, cerebrospinal fluid, urine, and other
without uremic albumin elevation, about 42–72% of cases body fluids. The main function is to transport retinol from
have elevated urinary NAG, and the time of elevation is ear- liver cells to surrounding tissues.
lier than uremic albumin. In diabetic patients complicated RBP is excreted from glomeruli under normal conditions,
with microvascular diseases, most of them have increased of which 99.9% is absorbed and degraded by the epithelial
urinary NAG, which can appear within 1–2 years after the cells of the proximal convoluted tubules, with low urinary
onset of diabetes, prior to the increase of urinary microal- excretion. When the glomerular filtration rate decreases,
bumin. Studies have shown that the level of urine NAG is serum RBP increases. When the proximal convoluted tubules
related to the severity of microvascular disease, and its activ- are damaged, the RBP excretion increases, which is a sensi-
ity decreases after treatment, but remains higher than that of tive index to evaluate the renal proximal convoluted tubules
healthy people. function. The change of RBP content can also sensitively
As a monitoring index during pregnancy, serum NAG of reflect the degree of proximal liver function damage and is a
normal pregnant women show a progressive increase after the sensitive indicator of the development and outcome of kid-
sixth week of pregnancy. After every 3 months of pregnancy, ney, liver, and nutritional diseases.
serum NAG approximately doubles and rapidly increases
in the ninth month. The activity of serum NAG in the tenth
month is about 9 times more than that of normal non-preg- 19.12.2 Detection Methods
nant women. After normal delivery and caesarean section,
the serum NAG decreases sharply and drops to the reference At present, the methods for the determination of RBP in
range of non-pregnant women after 3 days. After termina- hematuria include radioimmunoassay, immunoelectropho-
tion of pregnancy, serum NAG activity also decreases rapidly resis, enzyme-linked immunosorbent assay, and immuno-
to the level of non-pregnant women. During pregnancy with turbidimetric assay, among which radioimmunoassay and
diabetes mellitus, serum NAG activity is significantly higher enzyme-linked immunosorbent assay are highly sensitive
than the reference level of corresponding gestational months and practical, while immunoturbidimetric assay is widely
in normal pregnant women, and is earlier than the change used in clinical practice, as it is simple, rapid, sensitive, and
of HbA1C. Therefore, to observe the dynamic changes of accurate.
serum NAG activity during pregnancy is a valuable indicator
to monitor the pregnancy development process of pregnant
women and gestational diabetes mellitus. 19.12.3 Normal Reference Interval
It is helpful for the diagnosis of renal vascular hyper-
tension (RVH). For the suspicious RVH patients who were Immunotransmission turbidimetric method: serum, 25–70 mg/L;
diagnosed as renal artery stenosis by renal arteriography, urine, 0–0.7 mg/L.
19.12.4 Clinical Significance 10. Mizutani K, Toyoda M, Mikami B. X-ray structures of transfer-
rins and related proteins. Biochim Biophys Acta. 2012;1820:
203–11.
When kidney disease occurs, RBP in serum and urine is sig- 11. Brummett LM, Kanost MR. The immune properties of Manduca
nificantly increased due to decreased glomerular filtration sexta transferrin. Insect Biochem Mol Biol. 2017;81:1–9.
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ferrin using a microfluidic compact disc and MALDI-MS. Anal
athy, hypertensive nephropathy, etc. cause a decrease in Bioanal Chem. 2016;408:4765–76.
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7. Di Grande A, Giuffrida C, Carpinteri G, et al. Neutrophil gelatinase- 24. Latouche C, El Moghrabi S, Messaoudi S, et al. Neutrophil
associated lipocalin: a novel biomarker for the early diagnosis of gelatinase- associated lipocalin is a novel mineralocorti-
acute kidney injury in the emergency department. Eur Rev Med coid target in the cardiovascular system. Hypertension.
Pharmacol Sci. 2009;13:197–200. 2012;59:966–72.
8. Lee SY, Choi ME. Urinary biomarkers for early diabetic nephropa- 25. Bagshaw SM. Novel biomarkers for early diagnosis of acute kidney
thy: beyond albuminuria. Pediatr Nephrol. 2015;30:1063–75. injury. Expert Opin Med Diagn. 2008;2:1041–54.
9. Herget-Rosenthal S, Marggraf G, Husing J, et al. Early detec-

tion of acute renal failure by serum cystatin C. Kidney Int.
2004;66:1115–22.
Bone
20
Lieying Fan
Metabolic osteopathy generally includes osteoporosis, endo- is slow, and osteoclasting proceeds as usual, causing senile
crine bone diseases, renal osteodystrophy, osteoarthritis, and osteoporosis. Normal bone development, growth, and min-
hereditary bone diseases (Fig. 20.1). Among them, osteo- eral metabolism are primarily ensured by proper nutrition
porosis is the most common metabolic bone disease. From and normal endocrine gland regulation. Undernutrition, vita-
birth to old age, osteogenesis and bone destruction as well as min deficiency, and endocrine gland dysfunction can cause
bone mineral deposits and removals are carried out regularly. systemic bone changes. Different performances occur in dif-
During the period from birth to adulthood, bone development ferent developmental and growth stages. The levels of bone
and growth predominate. The formation and absorption of biochemical markers in normal human blood circulation and
bone in adulthood are balanced. In the elderly, osteogenesis urine vary with age. A variety of metabolic bone diseases can
affect bone metabolism and lead to changes and abnormali-
ties in bone function.
20.1 Overview
Bone metabolism markers are generally classified into bone

formation markers, bone resorption markers, and other mark-
ers related to bone metabolism. Bone formation markers are
the metabolites during bone formation, which include bone
alkaline phosphatase (BAP), osteocalcin (BGP), N-MID
Osteocalcin, N-terminal propeptide of type I procollagen
(PINP), C-terminal propeptide of type I procollagen (PICP),
total procollagen type 1 amino-terminal propeptide (tPINP),
etc. Bone resorption markers are the metabolites during bone
resorption, which mainly include type I collagen carboxy
terminal peptide (CTx also known as β-Crosslaps), type I
collagen amino terminal peptide (NTX), urinary type I colla-
gen nitrogen terminal peptide (uNTX), tartrate-resistant acid
phosphatase (TRACP), urinary hydroxyproline, deoxypyr-
idinoline, pyridinoline, etc. Other markers related to bone
metabolism include parathyroid hormone (PTH), calcito-
Healthy Bone Metabolic Disorders of Bone
nin (CT), 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D
[1,25-(OH)2D3], serum calcium (Ca), and phosphorus (P)
Fig. 20.1 Metabolic osteopathy (Table 20.1).
L. Fan (*)
Department of Clinical Laboratory, Shanghai East Hospital,
School of Medicine, Tongji University, Shanghai, People’s
Republic of China

254 L. Fan
Table 20.1 Detection methods and reference ranges of molecular markers of bone diseases
Biomarkers Methods Reference interval for healthy persons References
BAP ELISA Healthy children: 31.89–89.47 ng/mL [1]
Adults: 8–14.5 ng/mL
N-MID Osteocalcin ECLIA Men: 6.00–24.66 ng/mL [2]
Women (premenopausal): 4.11–21.87 ng/mL
Women (postmenopausal): 8.87–29.05 ng/mL
tPINP ECLIA Men: 9.06–76.24 ng/mL [3]
Women (premenopausal): 8.53–64.32 ng/mL
Women (postmenopausal): 21.32–112.8 ng/mL
PINP ECLIA 20–100 μg/L [3]
PICP ECLIA Men: 79–213 μg/L [4]
Women: 90–181 μg/L
β-Crosslaps, CTx ECLIA Healthy men: 43–783 ng/L [5]
Women (premenopausal): 68–680 ng/L
Women (postmenopausal): 131–900 ng/L
NTX ELISA / /
TRACP ELISA 3.1–5.4 U/L [1]
Urinary hydroxyproline Colorimetry Hydroxyproline (free) [6]
Adults ≤ 2.7 mg/24 h
Hydroxyproline (total)
Men: 9–73 mg/24 h
Women: 7–49 mg/24 h
Children
Premature infants
3-week-old 6–35 mg/24 h
Full-term infants
3 day 8–20 mg/24 h
1 month 32–63 mg/24 h
1–10 years 15–150 mg/24 h
11–14 years 68–169 mg/24 h
Parathyroid hormone ECLIA 15–65 pg/mL (1.6–6.9 pmol/L) [7]
Calcitonin ECLIA Women: 0–6.4 pg/mL [8]
Men: 0–9.52 pg/mL
25-Hydroxyvitamin D3 ECLIA Women: 6.23–49.9 ng/mL (15.6–125 nmol/L) [9]
Men: 4.92–42.7 ng/mL (12.3–107 nmol/L)
HLA B27 Flow cytometry / [10]
HLA-B*5801 (allopurinol) PCR-SSOP / [11]
PCR-SSP
ECLIA electrochemiluminescence immunoassay, SSOP sequence-specific oligonucleotide probes, SSP sequence-specific primers
20.2 BAP core part of skeletal system metabolism, the proliferation, dif-
ferentiation, and maturation of osteoblasts are closely associ-
20.2.1 Sources and Characteristics ated with normal skeletal development and growth. Since a
large amount of BAP is produced by osteoblasts during bone
In humans, alkaline phosphatase (ALP) exists in all tissues formation, BAP is a good marker of bone formation and bone
of the whole body and is especially widely distributed in the turnover and can be used in the evaluation of skeletal status
liver, bone, intestine, kidney, and placenta. Bone alkaline [13]. Low serum BAP activity is sometimes seen in hypo-
phosphatase (BAP) and liver alkaline phosphatase are the thyroidism. Serum BAP may be elevated in increased bone
two main types of ALP in human serum. BAP, an extracellu- metabolism, such as metastatic carcinoma of bone rickets and
lar enzyme in osteoblasts, can hydrolyze phosphatase to pro- osteomalacia. A considerable rise in the BAP activity is fre-
vide phosphoric acid for the deposition of hydroxyapatite and quently seen in children and during healing of a fracture [14].
hydrolyze pyrophosphate to relieve the inhibition of bone salt It is caused by increased osteoblast activity following acceler-
formation, which is conducive to osteogenesis [12]. As the ated bone growth.
20 Bone 255
20.2.2 Methods Most of osteocalcin is deposited to bone matrix, while the

rest is released into circulation and finally catabolized and
The commonly used methods for detecting BAP include degraded in renal glomeruli. Osteocalcin has a high affinity
heat inactivation, chemical inhibition, gel electrophoresis, with hydroxyapatite and plays an important role in inhibiting
ELISA, dry chemistry, immune concentrated technique, abnormal chondrocyte mineralization.
chemiluminescence immunoassay (CLIA), and colorimetric Serum osteocalcin is considered as a bone-specific marker
assay. of osteoblastic activity and used to measure the rate of bone
formation. Osteocalcin concentration in serum exhibits a
diurnal variation and is related with age, alcohol intake, and
20.2.3 Reference Interval for Healthy Persons glucocorticoids [2].
Accordingly, the serum osteocalcin concentration is
• Method: ELISA applied to various disorders of bone metabolism, e.g., osteo-
• Healthy children: 31.89–89.47 ng/mL porosis in particular, but also in hyperparathyroidism and
• Adults: 8–14.5 ng/mL Paget’s disease. In primary osteoporosis, there is a significant
increase of serum osteocalcin in postmenopausal osteoporo-
sis compared with a slightly increase in senile osteoporosis.
20.2.4 Clinical Significance The intact osteocalcin in serum is degraded rapidly when
samples are left at room temperature for a few hours due to
BAP is an effective blood index for monitoring normal bone proteolysis between amino acids 43 and 44. The N-mid
metabolism and skeletal status. Serum BAP may be increased fragment in the range of amino acids 1–43, which is the
physiologically during the normal skeletal development of largest product from cleavage, is considerably more stable
children and the healing of a fracture. Many bone metabolic [16]. In clinical use, conventional immunoassays of intact
diseases will elevate blood levels of BAP, for example, pri- osteocalcin are gradually replaced by osteocalcin assay that
mary and secondary hyperparathyroidism, Paget’s disease, recognizes both the intact molecular and the N-mid frag-
and osteomalacia. Other diseases, like hypothyroidism and ment using two monoclonal antibodies specifically directed
pernicious anemia, usually show lower BAP level in the against epitopes on the N-MID fragment and the N-terminal
blood. Additionally, BAP can monitor the therapeutic effi- fragment.
cacy of osteoporosis and rickets.
20.3.2 Methods
The methods for detecting intact osteocalcin by immu-
Here we introduce that BAP plays an irreplaceable role in noassay include ELISA and RIA while for detecting
bone metabolic system. Generally, serum BAP will be signif- N-MID Osteocalcin by immunoassay include ELISA and
icantly increased in adolescence, which contribute to growth ECLIA. Since ECLIA is automated and this assay could
and development of bone. For clinical application, BAP is detect more epitopes that make the values more accurate, it
a conventional marker for detecting bone metabolism, often is currently the most widely used in clinical practice.
providing clinical diagnosis for various bone diseases, and
monitors the therapeutic effect of bone diseases.
20.3.3 Reference Interval for Healthy Persons
20.3 N-MID Osteocalcin • Method: ECLIA

• Men: 6.00–24.66 ng/mL
20.3.1 Sources and Characteristics • Women (premenopausal): 4.11–21.87 ng/mL
• Women (postmenopausal): 8.87–29.05 ng/mL
Osteocalcin (OCN), also known as bone gamma-carbox-
yglutamic acid-containing protein (BGP), has a molecu-
lar weight of about 5800 Daltons and is the most abundant 20.3.4 Clinical Significance
noncollagenous protein in bone matrix and regulates bone
remodeling and metabolism diseases. Osteocalcin is released Osteocalcin is used as a biochemical marker to monitor bone
from osteoblasts, while that process is dependent on vitamin formation process. In patients with osteoporosis, osteocal-
K and promoted by vitamin D3 [15]. cin is applied for distinguishing the subtype of diseases.
256 L. Fan
Moreover, osteocalcin is used as a biomarker on the effec- 20.4.2 Methods

tiveness of drugs on bone formation.
Osteocalcin concentration is elevated in patients with pri- The methods for detecting total PINP by immunoassay include
mary or secondary hyperparathyroidism, hip fracture, Paget’s ELISA and ECLIA (electrochemiluminescence immunoas-
disease, and bone metastases. say). Since the ECLIA method is automated and accurate, it
With further research, studies demonstrate there is a is currently the most widely used in clinical practice.
crosstalk of osteocalcin between bone diseases and metabo-
lism. Osteocalcin stimulates beta cell proliferation and insu-
lin secretion in the pancreas and promotes increased 20.4.3 Reference Interval for Healthy Persons
adipocyte sensitivity to insulin. Therefore, osteocalcin plays
a key role in glucose metabolism. A low concentration of • Method: ECLIA
osteocalcin is regarded as a risk factor for type 2 diabetes. • Men: 9.06–76.24 ng/mL
• Women (premenopausal): 8.53–64.32 ng/mL
• Women (postmenopausal): 21.32–12.8 ng/mL
Mounting evidence shows that osteocalcin is not only cor- 20.4.4 Clinical Significance
related with bone formation but also involved in metabo-
lism processes. Osteocalcin acts as a bone-derived hormone The concentration of PINP is much higher in infants and
that regulates adipose, pancreas, muscle, and the brain. children than in adults. Concentrations of total PINP in
However, the mechanisms of osteocalcin regulation in serum decreased from age 30 to 35 years and then remained
these organs remain unclear. Additionally, osteocalcin can stable up to 50 years in healthy premenopausal women. In
also be detected in urine [17]. All of the above suggest that postmenopausal women with osteoporosis, serum total PINP
osteocalcin has more breadth and depth prospect in clinical concentrations were much higher than in premenopausal
application. Meanwhile, there is still a lot of research on women. Moreover, the joint Working Group on Bone Marker
the function of osteocalcin, which needs to be addressed Standards (WG-BMS) of the International Federation of
in the future. Clinical Chemistry and Laboratory Medicine (IFCC) and the
International Osteoporosis Foundation (IOF) recommend the
clinical use of PINP as bone synthesis reference marker in
20.4 Total PINP observational and intervention studies [19].
Besides bone diseases, PINP has been found to be associ-
20.4.1 Sources and Characteristics ated with breast cancer with metastases and renal failure [20].
Type I collagen comprises more than 90% of the bone

matrix. This collagen is synthesized from type I procollagen, 20.4.5 Conclusions and Prospects
which is produced from fibroblasts and osteoblasts. During
the extracellular maturation of collagen, there is cleavage of The baseline of PINP is very important, even though it is
N-and C-terminal extensions from type I procollagen, which just the by-product during bone matrix formation. Different
is referred to as the amino terminal [N-terminal propeptide pathogenic mechanisms of skeletal and non-skeletal tissues
of type I collagen (PINP)] and carboxy terminal propeptide contribute to serum concentration of PINP. In clinical use,
(PICP), respectively. Both of the propeptides, produced in total PINP assay detects both forms of PINP antigen and is
equimolar amounts with each other and with the collagen, more applicable to monitor the process of bone formation
are set free into the blood stream [18]. than PINP assays.
PINP consists of three subunits, which include two pro-
α1 chains and one-pro-α2 chain that contribute to the hetero-
trimeric or homotrimeric isoform of PINP. However, the 20.5 PINP
trimeric form of PINP is rapidly degraded to a monomeric
form by thermal effect. Both the trimeric and the mono forms 20.5.1 Sources and Characteristics
in serum are called total PINP. Total PINP can be considered
as a bone turnover marker to assess bone formation and is As described in Sect. 20.4 total PINP, PINP has been con-
valid for instructing clinical diagnosis and prognosis of firmed to be sensitive for monitoring changes of bone for-
patients with osteoporosis. mation in osteoporosis and other metabolic bone diseases.
20 Bone 257
However, PINP detection has some limitations. Compared the maturation of type I collagen, PICP breaks off and is
to total PINP, PINP detection only recognizes the intact tri- released to blood at a ratio of 1:1 with type I procollagen.
meric form of PINP. However, the trimeric form of PINP Therefore, PICP can be used as a marker of type I collagen
is easily transited to the monomeric form at physiological synthesis, reflecting the formation of non-mineralized bone-
temperature in vitro, while the assays that detect only the like substance, and it is an osteogenic index. In the process of
trimeric form of PINP may underestimate the rate of type bone formation, type I collagen appears in the proliferation
I collagen biosynthesis. Moreover, the smaller antigens of phase of osteoblast precursor cells, prior to the expression of
PINP increase in some medical conditions, such as renal alkaline phosphatase, forming a collagen framework which
insufficiency and metastatic breast cancer. lays a foundation for matrix mineralization.
The serum PICP reflects the ability of collagen synthesis
of osteoblasts, which is a specific index for monitoring the
20.5.2 Methods viability of osteoblasts and bone formation. The level of
PICP is influenced by age and menopausal status [21].
The methods for detecting PINP by immunoassay include It is also reported that serum PICP may increase when
chemiluminescence and RIA. bone metastasis occurs in cancer. Type I collagen is ubiqui-
tously distributed and exists both in soft tissue and in bone,
where it forms the subsequent mineralization [22]. Thus,
20.5.3 Reference Interval for Healthy Persons PICP reflects a combination of bone and soft tissue growth.
• Method: ECLIA
• Healthy adults: 20–100 μg/L 20.6.2 Methods
PICP in serum can be detected by methods including ELISA,

20.5.4 Clinical Significance RIA, and ECLIA, while ECLIA is the main method used
presently.
In healthy adults, there is little difference between PINP and
total PINP assays. Both of them could be used as a marker
of bone turnover. However, PINP assay could not reflect the 20.6.3 Reference Interval for Healthy Persons
glomerular function in patients with renal insufficiency. The
level of PINP is not affected in breast cancer patients with • Method: ECLIA
metastases. • Healthy men: 79–213 μg/L
• Healthy women: 90–181 μg/L

In summary, as described in total PINP, the level of PINP is
crucial even though it is not the major constituent of bone 1. There are diurnal differences in serum PICP, which is about
matrix. However, its instability and the antigen recognition 20% higher in the first half of the night than in the morning.
limit its clinical use. It is not as convenient as total PINP The level of PICP increases in children during develop-
assay to be used as standard of care. ment, 3 months after pregnancy, bone tumors, deformity
osteitis, alcoholic hepatitis, pulmonary fibrosis, etc.
2. PICP may not be sensitive to acute events of chronic heart
20.6 PICP failure, but it is a very beneficial biomarker with high sen-
sitivity and specificity in diagnosis of low-LVEF heart
20.6.1 Sources and Characteristics failure. Serum PICP as well as coronary PICP are posi-
tively correlated with myocardial collagen content.
Bone matrix consists of 90% type I collagen. The syn- However, it is not correlated with patients with HCM and
thesis of collagen undergoes the processes from amino DCM compared to controls.
acid-
precollagen-
procollagen-collagen. Type I collagen is 3. PICP is a very early promoting marker in the acquisition
synthesized by osteoblasts, and its extensions are amino ter- by the tumors of prometastatic phenotype. It has an
minal propeptide of type I procollagen (PINP) and carboxyl important role in the productive interactions between
terminal propeptide of type I procollagen (PICP). During stroma and tumor cells.
258 L. Fan
20.6.5 Conclusions and Prospects therapy in osteoporosis or other bone diseases. Changes

induced by the therapy can be demonstrated after just a few
The carboxyl terminal extensions of type I pro-collagen months [26].
(PICP) is a stromal component cleaved from the proteolytic
fragments of pro-collagen I. During the modeling of bone,
type I collagen is synthesized by osteoblasts during their 20.7.2 Methods
early proliferative phase, releasing PICP into the circulation
[23]. Therefore it reflects the growth of bone and soft tissue. The CTx can be detected in both serum and urine. The meth-
PICP is abundant in tissues with high expression of col- ods include ELISA, RIA, and ECLIA, which use polyclonal
lagen I such as bone and the stroma of cancer. It can predis- antibody to a specific amino acid sequence of C-terminal
pose tumor cells to respond to the proliferation stimuli and peptide crosslinked by type I collagen. ECLIA is the main
may activate signaling by engagement of CXCR4 by cyto- method used currently.
kines and promoting their pervasion, due to the induction of
vascular development [24].
20.7.3 Reference Interval for Healthy Persons
20.7 β-Crosslaps • Method: ECLIA

• Healthy men: 43–783 ng/L
20.7.1 Sources and Characteristics • Women (premenopausal): 68–680 ng/L
• Women (postmenopausal): 131–900 ng/L
Type I collagen carboxyl terminal peptide (β-Crosslaps,
CTx) is an important component of the bone matrix, existing
in mature bone collagen. The components of 90% bone col- 20.7.4 Clinical Significance
lagen are type I collagen. As the osteoclast activity increases,
the release of type I collagen by the dissolution of bone colla- 1. Correlated to high conversion type increase of primary
gen will increase, which then degrades into CTx. CTx is the osteoporosis; the level in premenopausal women is higher
product of extracellular collagen fibers and can be detected in than postmenopausal women; elderly women with frac-
both serum and urine. During normal bone metabolism, bone ture express more than women without fracture.
matrix follows orderly synthesis and degradation, while type 2. Dramatically raised in metabolic osteopathy, primary

I collagen is synthesized in bone and broken into fragments hyperparathyroidism, hyperthyroidism, and Paget’s
and then released into blood and excreted from the kidney. disease.
CTx is an indicator of bone resorption and metabolism [19]. 3. Available for measurement in monitoring the efficacy
The degradation of type I collagen increases under physi- of anti-resorptive therapy for osteoporosis and other
ological and pathological enhancement of bone resorption osteopathy.
(e.g., in old age or as a result of osteoporosis), and there is a
commensurate rise in the level of degradation fragments in
blood. The most important fragments are the β-isomerized C 20.8 Conclusions and Prospects
(carboxy)-terminal cross-linking telopeptides (β-CTx), pro-
duced by osteoclastic hydrolysis of type I collagen [25]. This guide has made a clear recommendation for the applica-
CTx, as a specific product of type I collagen, is an inter- tion of biochemical markers of bone metabolism in the diag-
nationally recognized marker of bone resorption. Serum CTx nosis and treatment of metabolic bone diseases (Table 20.2).
reflects osteoclast activity and is significantly increased in However, there are large individual variations in each marker
metabolic osteopathy with elevated activity of osteoclasts. of patients. Thus, the clinical application of this guide should
The rise of CTx level represents the enhancement of bone highly consider individual differences and clinical manifesta-
resorption and bone loss, which commonly occurs in osteo- tion in patients. In addition, researchers need to further study
porosis and Paget’s disease. Determination of CTx in serum the biochemical markers of bone metabolism and provide
is recommended for monitoring the efficacy of anti-resorptive better evidence for the revision of this guide in the future.
20 Bone 259
Table 20.2 Change of bone metabolic biochemical markers

Bone formation Bone resorption 25-Hydroxyvitamin Serum Serum
Metabolic bone pain markers markers PTH D calcium phosphate
Hyperparathyroidism ↑ ↑ ↑↑ N or ↓ ↑ ↓
High turnover uremic ↑ ↑ ↑ N or ↓ ↓ ↑
osteodystrophy
Low turnover uremic N or ↓ N or ↓ N N or ↓ ↓ ↑
osteodystrophy or ↓
Bone tumors or Tumor bone ↑ ↑ ↓ N or ↑ N or ↑ N or ↓ or ↑
metastasis
Osteomalacia or Rickets ↑↑ ↑ ↑ ↑ or ↓ N or ↓ N or ↓
Vitamin D deficiency N or ↑ N or ↑ ↑ ↓ N or ↓ N or ↓
1 4. Kaplan MM. Alkaline phosphatase. N Engl J Med. 1972;286:200–2.

15. Hauschka PV, Lian JB, Cole DE, et al. Osteocalcin and matrix
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Cancer
21
Wenling Zhang, Yumei Huang, and Jian Xu
The discovery of tumor biomarkers with high specificity and 21.1 Overview
sensitivity has become a major focus of cancer research.
Here we describe those classical tumor markers (TM) based Malignant tumor, the leading cause of non-communicable
on their biochemical properties, including oncofetal pro- diseases (NCD) deaths together with cardiovascular diseases
teins, proteins, enzymes, sugar esters or glycoproteins, and and chronic respiratory diseases, seriously endangers human
hormones. We also depict tumor biomarkers in oncogenesis life and health [1]. The number of new cases of tumor world-
and development of tumors for early diagnosis, invasion and wide in 2018 has already increased to 18 million [2]. With
metastasis, and prognosis. Due to the prosperous develop- the aging of the population, smoking, infection, environmen-
ment of “Precision Medicine,” tumor biomarkers that are tal pollution, and changes in diet, tumor diagnosis is facing
critical and relevant to Precision Medicine have been embod- huge challenge. Early detection and intervention are likely to
ied in this chapter as well. In addition, with the promotion of be the most effective approaches to lessen morbidity and
technologies, emerging molecular biomarkers of tumor have mortality of human cancers. TM is of great practical value in
also been investigated, including CTC, ctDNA, DNA meth- tumor screening, diagnoses, prognoses, evaluation of thera-
ylation in blood, circulating miRNAs, lncRNA, circRNA, peutic effect, and follow-up observation of high-risk popula-
endosomes, and exosomes. The study of tumor molecular tion. Therefore, the discovery of TM with high specificity
markers is still a long process, which needs to go through and sensitivity has become a major focus of cancer research.
several important stages, including clinical trials, confirma- Tumor markers (TMs) are referred to as any products that
tion period, retrospective study period, prospective screening produced directly by tumor or non-tumor cells responding to
period, and tumor control period, and to evaluate the signifi- the presence of tumors. These substances exist in blood,
cance of these molecular markers. body fluids, or tissues. They are required to be “measurable”
by chemical, immunological, or molecular methods in order
to discern tumors from the normal tissues or to predict the
existence of tumors.
The exploration of TMs has been going on for more than
170 years. The first TM, a protein that precipitates when
W. Zhang (*) heating a urine specimen, was discovered in 1846 by Henry
Department of Medical Laboratory Science, The Third Xiangya
Hospital, Central South University, Changsha, Hunan,
Bence-Jones in the urine of multiple myeloma patients and
People’s Republic of China thus was named as Bence-Jones protein (BJP) [3]. The dis-
Department of Medical Laboratory Science, Xiangya School of
covery of this protein opened up the research of tumor mark-
Medicine, Central South University, Changsha, Hunan, ers and represented the first stage of this field. It was the
People’s Republic of China second stage that hormones, enzymes, and their isozymes
Y. Huang were discovered in the early twentieth century, such as ecto-
Department of Laboratory Medicine, Hunan Cancer Hospital and pic hormones, gonadotropins, alkaline phosphatase (ALP),
Affiliated Cancer Hospital of Xiangya School of Medicine, lactate dehydrogenase (LDH), and their isozymes. In the
Central South University, Changsha, Hunan,
1960s, the discovery of some classical and significant embry-
onic protein markers, such as α-fetoprotein (AFP) and carci-
J. Xu
noembryonic antigen (CEA), was considered the third stage.
of Nanjing Medical University, Nanjing, Jiangsu, An epoch-making discovery of monoclonal antibody (McAb)
People’s Republic of China in 1975 hastened the birth of many TMs that can be used in

262 W. Zhang et al.
clinical diagnosis, such as the cancer antigen series identified the complicated source and nature of these TMs, there is no
by monoclonal antibodies: CA15–3, CA19–9, CA125, et al. uniform classification method for TMs so far. Here we will
From then on, TM garnered extensive attention and was for- describe the classification of TMs in the following aspects.
mally nominated as “tumor marker” by Herberman in 1978.
It was confirmed and quoted publicly since then. 21.1.2.1 C lassification by Biochemical
As tumor molecular biology and immunology advance, Properties
dozens of biomarkers have been discovered and gradually According to their own biological properties, TMs can be
applied to clinical diagnosis and treatment. Oncogenes, tumor divided into a number of categories, such as protein markers,
suppressor genes, and their expression products, such as nucleic acid markers, and others. This classification contrib-
BRCA1, BRCA2, Ras, and p53, are always abnormally utes to choosing proper test method.
expressed in most tumor tissues, and thus can be reasonable,
broad-spectrum TMs. In addition, increased DNA levels in 21.1.2.2 Classification by Sources
blood are found mainly from the release of tumors. Brouckaert Although there are many types of molecular markers for
and colleagues clarified the release mechanism of cell-free clinical use, they can generally be divided into two catego-
DNA (cfDNA) that DNA fragments released by malignant ries as far as nucleic acids are concerned:
tumor cells during phagocytosis by macrophages were het-
erogeneous vs. homogeneous by normal apoptotic cells [4]. 1. Exogenous nucleic acids, emerging when infected by

These studies indicate that circulating DNA (cDNA) can be a pathogenic microorganisms, such as EBV for nasopha-
novel TM for early diagnosis and monitoring of tumors. ryngeal carcinoma (NPC), HBV for hepatocellular carci-
noma (HCC), HPV for cervical cancer, Helicobacter
pylori (Hp) for gastric cancer, and so on.
21.1.1 The Ideal Biomarker for Cancer 2. Endogenous nucleic acids from human cells, which are
peculiar for certain tumor. For example, BRCA1/2 gene
Ideal TM should be specific to tumor cells, but non-existent mutation can predict breast cancer risk.
in healthy people or benign tumors. They can be detected in
body fluids and can be used for early screening of asymp- 21.1.2.3 C lassification by Oncogenesis
tomatic malignant tumors. The ideal TM should own the fol- and Development of Tumors
lowing characteristics: Multiple molecules participate in each stage of tumorigene-
sis and progression, where tumor cells themselves release
1. High sensitivity, so that the tumor can be found and diag- certain tumor-related molecules or metabolites as well.
nosed early. These molecules can be used to monitor the occurrence and
2. High specificity, that is, it is positive in patients with development of tumors, the severity of patients, the efficacy
tumors, but negative in other patients. of clinical treatment, and to evaluate the recurrence and
3. Capacity to orientate tumor, that is to say, it has organ prognosis of patients, as well as to provide evidence for clin-
specificity. ical targeted therapy.
4. Property in association with the severity of tumor, tumor
size, or stage, that is, the larger or more advanced the
tumor, the higher the concentration of tumor markers. 21.2 T
umor Markers with Different
5. Ability to monitor the therapeutic effect of tumor, that is, Biochemical Properties
the fluctuation of concentration is closely related to the
therapeutic effect. 21.2.1 Oncofetal Protein
6. Capability to monitor the recurrence of tumor, that is to
say, the concentration will decline after tumor treatment, Oncofetal protein, just as its name implies, is a set of proteins
and ascend significantly when tumor recurs. which typically exists only during embryonic development.
7. Prediction of the prognosis of tumor, that is, the higher the But these proteins can appear in the blood of adults with cer-
concentration, the worse the prognosis, and vice versa. tain kinds of cancer and thus are used as tumor markers for
both diagnoses and following treatments of the tumors.
But so far, none of the tumor markers could fully meet the Representatives of oncofetal antigens are AFP and CEA.
above criteria.
21.2.1.1 AFP
AFP is a main plasma protein normally generated in early
21.1.2 Classification of Tumor Biomarkers life by the yolk sac and the fetal liver during embryonic
development. AFP levels reach 15.7–146.5 μg/mL at birth,
With the lucubrating of tumorigenesis and development of but decrease rapidly to a normal range, 10–20 ng/mL, over
biotechnology, more and more TMs were identified. Due to the first year of life [5]. Elevated expression in serum is
21 Cancer 263
a ssociated with some tumors, such as HCC, hepatoblastoma, below 2.5 ng/mL in nonsmokers and below 5 ng/mL in
metastatic diseases that affect the liver, and nonseminoma- smokers [13]. However, CEA rises in some types of cancer,
tous germ cell tumors. AFP concentration elevated greater such as breast, gastric, pancreatic, and ovarian. Consequently,
than 400 ng/mL in patients with cirrhosis highly supports it can be used as a TM although it is not specific for malig-
diagnosis of HCC [6]. The patients with low AFP elevation nant tumors. 45–80% patients with primary colorectal cancer
should be observed dynamically and compared with the (CRC) suffer increase of CEA, and 50–70% positive for
changes of liver function, which is helpful for diagnosis. patients with pancreatic, gastric, esophageal, lung cancer,
About 30% of HCC patients were estimated to have normal breast, and urinary system tumors [14]. CEA may also
levels of AFP. Because of low sensitivity and specificity, AFP increase in nonmalignant diseases such as cirrhosis, gastritis,
is not a routine screening marker for HCC any more in west- diverticulitis, pancreatitis, and inflammatory bowel disease,
ern countries. However, AFP is often related to tumor size but the concentration is much lower than in malignant
and volume in AFP-secreting tumors. Resection usually tumors. Serum CEA level > 20 ng/mL frequently indicates
results in a decline in concentration. AFP may thus be used existence of a digestive system tumor. Therefore, CEA is
to monitor the treatment efficacy or tumor recurrence of recommended for differential diagnosis between benign and
treatment [7]. AFP is an important prognostic factor as well. malignant tumors rather than for early screening and diagno-
Prolonged decrease of AFP levels is in correlation with lon- sis of cancer alone.
ger survival of HCC patients when compared to slowly CEA is also useful for monitoring treatment efficacy and
increasing levels of AFP [8]. When serum AFP levels evaluation prognosis, especially when combined with other
≤1000 ng/mL, the incidence of vascular invasion in HCC tumor markers, such as CA242 and CYFRA 21–1 [15]. CRC
patients was 32%, but the incidence was 61% when serum patients with low preoperative serum CEA levels have better
AFP levels >1000 ng/mL [9]. prognoses than those with CEA concentrations exceeding
10 ng/mL [16]. In addition, CEA can also be used to evaluate
21.2.1.2 AFP-L3 the prognosis of lung cancer, especially lung adenocarci-
AFP-L3 is the Lens culinaris agglutinin (LCA)-reactive noma. High levels of serum CEA are generally regarded risk
type among three isoforms of AFP. AFP-L3 is widely modi- for brain metastasis and always along with poor prognoses.
fied at posttranslational levels, and its changes are specific In lung adenocarcinoma patients with plasma CEA > 40 ng/
to malignant tumors. The production of AFP along with mL, the brain metastasis rates in 1 year and 2 years were
regeneration of hepatocytes may affect the diagnostic value 27% and 32%, respectively [17].
of AFP for tumors to a certain extent. But AFP-L3 is a spe- Moreover, serum CEA levels have been shown clearly
cific substance in HCC cells; hence, the percentage of AFP- correlated with TNM stages of CRC. The more advanced the
L3 in total AFP (AFP-L3%) and AFP-L3 levels have already disease, the higher the CEA concentration. Nevertheless,
been considered as a tumor marker to distinguish HCC from there are contrary statements [18].
benign liver diseases and then for early diagnosing HCC in
the clinic.
However, the application of AFP-L3 alone in the diagnosis 21.2.2 Protein
of HCC still has some limitations, and the diagnostic value
cannot be satisfied. In recent years, the combination of triple 21.2.2.1 TPA and TPS
biomarkers AFP, AFP-L3, and PIVKA-II has been widely Tissue polypeptide antigen (TPA) is a single-chain polypep-
used to diagnose HCC. AFP-L3 in combination with tide existing in the cell membrane and cytoplasm of placenta
PIVKA-II can improve the detection rate of HCC [10]. In and most tumor tissues. Serum TPA can be detected in almost
Japan, this combination has been routinely used for screening 70% of patients with malignant tumors. However, its increase
for HCC and achieved increased early HCC detection rate. has no correlation with the locations and types of tumors.
This combination was thus covered by Japan’s national health Clinically, it is used to assist the diagnosis of malignant
insurance and recommended be tested at intervals of 3 to tumors with rapid proliferation and to monitor the therapeu-
4 months in the very-high-risk group and at 6-month intervals tic effects. Serum TPA in patients with malignant tumors is
in the high-risk group [11]. The combination was described to significantly higher than normal, but it will decrease after
significantly improve discovery rate of A stage HCC, with a treatment. An increase of TPA later indicates a recurrence of
specificity of 93.3% and sensitivity of 85.6% [12]. tumors. Besides, TPA is well known to differentiate cholan-
giocarcinoma (TPA elevated) from HCC (TPA not elevated)
21.2.1.3 CEA [19]. The simultaneous use of TPA and CEA is helpful for
Carcinoembryonic antigen (CEA) normally exists in gastro- the differential diagnosis of malignant and non-malignant
intestinal tissue during embryonic development, but it almost breast diseases. In addition, TPA may rise in patients with
disappears after birth. The normal serum level of CEA is lung cancer, acute hepatitis, pancreatitis, or pneumonia.
264 W. Zhang et al.
Tissue polypeptide-specific antigen (TPS) was expressed to 84% compared with AFP or PIVKA-II alone, respectively
in a small amount in some active somatic cells, such as hepa- [24]. Moreover, it has been recommended to discriminate
tocytes and urogenital tract cells, while it was highly HCC from non-malignant liver diseases, such as chronic
expressed in epithelial malignant tumors and metastatic can- liver diseases and benign liver tumors. In addition, PIVKA-II
cers. TPS is synthesized and released into blood or other is useful to monitor therapeutic effect as well as recurrence
body fluids at S and G phases of the cell cycle. Serum con- of tumors. An important characteristic of PIVKA-II is that its
centration of TPS is a more specific indicator to evaluate the half-life is 2.5 days, so it falls back quickly after resection.
degree of tumor cell division and proliferation activity. The PIVKA-II level may reach to a significantly high level,
Different from traditional tumor markers such as CEA, but AFP stays at normal level when tumor recurs, which indi-
CA15–3, and CA125, TPS is “tumor activity dependent.” Its cates an application of dynamically detected PIVKA-II and
serum level is related to the number of cells under dividing AFP to predict early recurrence of HCC. PIVKA-II was
and proliferating, that is, to the activity of tumors. In con- regarded as the best prognostic predictor in patients with
trast, others are “tumor volume dependent,” which are related HCC after radiofrequency ablation (RFA) therapy [25].
to the tumor load, that is, the counts of cancer cells. Therefore,
in the early stage of cancer, before recurrence or metastasis,
the serum levels of those markers reflecting tumor volume 21.2.3 Enzymes
are often very low because of the small amount of tumor
cells. Meanwhile, TPS can be very high due to active divi- 21.2.3.1 PSA
sion and proliferation of tumor cells, which is conducive to Prostate-specific antigen (PSA) is an organ-specific glyco-
the early diagnosis, treatment, prediction, and prognosis of protein enzyme. Only a small amount of PSA exists in the
tumors [20]. serum of men with healthy prostates. The amount will
increase when the prostates get inflammation, hyperplasia,
21.2.2.2 CYFRA21-1 or even malignant transformation. The normal value is
Cyfra21–1 is also known as KRT19. It is a soluble fragment ≤4 ng/mL.
of cytokeratin 19. CYFRA21-1 is expressed in the cytoplasm Total PSA (tPSA) has a low diagnostic sensitivity of pros-
of epithelial tumor cells and thus widely used as a TM for tate cancer (merely 21%) when the cutoff is set on 4 ng/mL
non-small cell lung cancer (NSCLC). It is useful in deter- [26]. Nevertheless, the percent of free PSA (%fPSA), the
mining tumor stage, predicting prognosis and the efficacy of small amount of total PSA unbound to serum proteins,
treatment, and reflecting tumor burdens [21]. Patients of increases the diagnostic specificity of prostate cancer.
esophageal squamous cell carcinoma (ESCC) with low pre- %fPSA is in negative correlation to the probability of pros-
operative serum Cyfra21–1 had longer OS (91.9 months) tate cancer. Moreover, several calculated parameters such as
than those with high level (46.6 months) [22]. Serum PSA-density (PSAD), PSA-velocity (PSAV), and PSA-
Cyfra21–1 in patients with early oral/oropharyngeal squa- doubling time (PSADT) have been developed for prostate
mous cell carcinoma (stage I + II) was more concentrated cancer [27, 28].
than that with benign tumor and healthy control group. The Despite routinely determination, using PSA to screen
diagnostic cutoff level, sensitivity, and specificity of prostate cancer is still controversial. But PSA is a valuable
Cyfra21–1 were 2.17 ng/mL, 60.36%, 81.03%, respectively tool to evaluate the prognosis and the therapeutic effect. PSA
[23]. Cyfra21–1 is an independent prognostic factor for concentration will rise proportionally to the clinical stage
NSCLC, especially squamous cell carcinoma. Serum level of and the tumor volume of prostatic cancer in untreated patient.
Cyfra21–1 is not affected by smoking and blood collection PSA exceeding 20 ng/mL highly indicates the existence of
skills, so the false positive rate is low. bone metastases. An upward tendency of PSA is more mean-
ingful than an individual detection in patients treated with
21.2.2.3 PIVKA-II radiotherapy or androgen-deprivation therapy. Imaging
PIVKA-II is an abnormal form of the coagulation protein, examination after determination of raised PSA to evaluate
prothrombin. PIVKA-II displays high specificity and stabil- the recurrence site is a recognized strategy for identifying
ity. It is only detectable in 91% of HCC patients but not metastatic site.
found in other liver diseases. In addition, PIVKA-II level
will not alter with the administration of vitamin K. It has 21.2.3.2 NSE
been written into the guidelines by the Asia-Pacific Society Neuron-specific enolase (NSE): serum NSE is an acid prote-
of Hepatology and by Chinese Liver Cancer Diagnosis and ase specific to the neurons and neuroendocrine cells. It is a
Treatment Guidelines in China. Combined detection with specific TM for small cell lung cancer (SCLC) and neuroen-
AFP can improve the diagnostic efficiency of HCC. The sen- docrine tumors including neuroblastoma and medullary car-
sitivity of combined detection is improved from 73% or 76% cinoma of the thyroid [29].
21 Cancer 265
NSE is a preferred marker for the detection of SCLC, cancer in asymptomatic patients because of low sensitivity in
while CYFRA 21-1 is superior to NSE in the detection of early stage cancers and its lack of specificity, especially in
NSCLC. NSE in patients with SCLC is significantly higher premenopausal women [32]. CA125 is also useful to reflect
than with NSCLC, which, hence, can be used for differential the response to treatment and to predict prognosis after treat-
diagnosis. It is also useful for monitoring the therapeutic ment. Also, an increase in CA125 levels after debulking sur-
effect of SCLC after radiotherapy and chemotherapy. When gery and chemotherapy strongly predicts the recurrence with
the treatment was effective, the concentration of NSE gradu- 95% accuracy.
ally decreased to the normal level, and the serum NSE level
increased when the patients relapsed. 21.2.4.2 CA15–3
NSE is also helpful to distinguish neuroblastoma from Carcinoma antigen 15–3 (CA15–3) is encoded by MUC1
nephroblastoma. NSE significantly rises in neuroblastoma, gene. Generally, the higher the level elevated, the more
but the change in nephroblastoma is not obvious. It also has malignant the tumor presented. Serum CA15–3 has no role
high clinical value in the early diagnosis, monitoring the in screening asymptomatic populations and early diagnosis
state of illness, and prediction recurrence of neuroblastoma. of patients with breast cancer. CA15–3 was always used in
Serum NSE levels in patients with neuroendocrine cell combination with other biomarkers. Elevated CA15–3 is
tumors, such as pheochromocytoma, islet cell tumor, thyroid related to more risk of early recurrence in breast cancer along
myeloid carcinoma, melanoma, retinoblastoma, and so on, with ALP [33].
are also elevated.
21.2.4.3 CA19–9
21.2.3.3 LDH Carbohydrate antigen 19–9 (CA19–9) is the most frequently
Lactate dehydrogenase (LDH or LD) is a cellular enzyme adopted TM in the management of pancreatic cancer. The
existed in almost all living cells (animals, plants, and pro- normal value is <37 U/mL. But setting the cutoff value on
karyotes). It is considered a TM due to its participation in 37 U/mL, the sensitivity and specificity in diagnosis of pan-
tumor initiation and metabolism, although not sensitive and creatic cancer is 79% and 85%, respectively [34].
specific. LDH is propitious to indicate the prognosis of can- CA19–9 is more useful in monitoring the recurrence of
cers because of its strong correlation with tumor burden [30]. pancreatic cancer rather than screening for cancer, due to its
LDH should normalize after curative surgery; otherwise low positive predictive value. A significant or sustained
residual disease may exist. In addition, it can be used to mon- increase in postoperative serum level of CA19–9 suggests
itor response to treatment and to find recurrence and metas- recurrent or progressive disease. Besides, it is helpful to dis-
tasis of tumors, even to indicate immune suppression in tinguish benign from malignant pancreatic pathologies. In
cancer. Moreover, LDH has been regarded as a promising addition, CA19–9 can be used as a predictor to assess
therapeutic target through inhibiting its activity and thus pre- response to systemic therapy as well.
venting carcinogenic cells from proliferating [31].
21.2.5 Hormone
21.2.4 Sugar Esters or Glycoproteins
21.2.5.1 HCG
The variation of glycosylation process in cancer cells results Human chorionic gonadotropin (HCG) is a hormone secreted
in the change of glycosylation sequence in glycoproteins or by cytotrophoblasts of the placenta. HCG is composed of
glycosyl esters on cell secretion or cell membranes, forming two different subunits, α and β subunit. The α subunit is same
a special antigen different from normal glycoproteins. to that of luteinizing hormone (LH), follicle-stimulating hor-
Monoclonal antibody technology can be used to detect gly- mone (FSH), and thyroid-stimulating hormone (TSH), while
coprotein antigens. The commonly used glycoprotein tumor the β subunit is unique to HCG. Thus, β-HCG is applied as
markers include CA125, CA15–3, CA549, CA27–29, an excellent TM to monitor germ cell tumors in conjunction
CA19–9, CA19–5, CA50, CA72–4, CA242, and so on. with AFP and LDH.
The normal range for males or nonpregnant females is
21.2.4.1 CA125 between 0 and 5 mIU/mL. The increase of β-HCG may occur
CA125, also known as mucin 16 or MUC16, is a glycopro- in 60–80% of patients with nonseminomatous germ cell
tein. It is the most commonly used TM for ovarian cancer. tumors (NSGCTs), 10–20% with stage I seminoma, and
Around 90% of women with stage III or IV ovarian cancer 30–50% with disseminated seminoma. Usually, β-HCG con-
have elevated serum levels of CA125, which can be used to centration < 500 mIU/mL presented in patients with semi-
detect ovarian cancer patients with symptoms. But CA125 noma while >1000 mIU/mL is more often seen in NSGCT
has not yet been extensively employed to screen for ovarian [35]. Interestingly, some studies reported that β-HCG may
266 W. Zhang et al.
also be elevated in select non-trophoblastic solid tumors development. In different stages of tumor initiation and devel-
such as tumors of the small and large bowel, hepatomas, opment, at least two or more different cancer-related genes are
lung, and stomach cancers, HCC, renal cell carcinoma required for abnormal activation or inactivation to cause tumor
(RCC), and bladder carcinoma. The β-HCG is overexpressed transformation. This is mainly because the proliferation and
in the CRC patients, indicating poorer outcome of early- differentiation of cells are controlled by many factors, and the
stage CRC [36]. In addition, as β-HCG is not able to cross synergistic effect of multiple cancer-related genes is needed to
the blood-brain barrier, presence of β-HCG in the cerebro- get rid of these controls. The discovery of oncogenes and
spinal fluid and the ratio being more than 1:60 strongly indi- tumor suppressor genes marks the beginning of the era of
cate the brain metastasis of the malignant tumor. molecular oncology. The development of NPC is a multi-step
process. In the process, it experienced from normal nasopha-
21.2.5.2 CA ryngeal epithelium to epithelial atypia, carcinoma in situ, tiny
Catecholamine (CA) is a general term for a class of sub- infiltrating carcinoma, and obvious infiltrating carcinoma such
stances containing catechols in their structures, including a cancerous process. In every stage of cancer, there are multi-
epinephrine (adrenaline), norepinephrine (noradrenaline), ple genes to regulate the development of NPC. Different sus-
and dopamine. Detection of epinephrine in urine can indi- ceptibility genes at different stages could control the incidence
rectly understand the secretion of catecholamine in vivo. of NPC. The activation of these oncogenes and the inactiva-
Extremely high levels of catecholamine can be seen in tion of tumor suppressor genes as well as the imbalance of
pheochromocytoma. their interaction are the molecular basis for the occurrence and
development of NPC (Fig. 21.1) [37].
21.3 Multistage Biomarkers

of Tumorigenesis and Development 21.3.1 Molecular Biomarkers for Early
Diagnosis
Tumor is a genetic disease with multiple genes. Its occurrence
and development is an extremely complex biological phenom- In the early stage, the first change occurs in molecular level,
enon with multiple factors and multiple genes acting synergis- including chromosome, DNA, or RNA, and protein expres-
tically and finally forming after many stages. Tumor initiation sion eventually leads to tumorigenesis. The prognosis of
mainly includes initiation, promotion, transformation, and tumors partially depends on whether they can be diagnosed
The changes of SNP fom BRD7, EBER-1 expression, loss of SPLUNC1, The class forecast model consisting of up-regulated RBI, STMN1,
NGX6, NORI and UBAP1 RASSFIA,CDH3, pl6, p27 DSP and down-regulating SERPINB6, AGTRL1, SYTL2,
normal NPE dysplastic NPE early NPC early invasive NPC
Loss of LTF, NGX6, Ezrin, THYI, nm23, TIMP, E-cadherin, activation of

vitonectin, MMPI6, Tiam-1, OPN
Cyclin DI, Survivin and HPA, a group of biomarkers

related to the prognosis of NPC;
Bcl-2. EGFR and Ki67 proteins, a group of perfect
candidate biomarkers for forecasting radiation
sensitivity of NPC;
SAA and cox-2, the candidate biomarkers monitoring
NPC recurrence;
constructing a biomarkers system of different clinical
stages of NPC composed of I39 genes.
invasive NPC metastasis NPC
Fig. 21.1 Model of biomarkers involved in the multi-step process of NPC, and metastasis NPC. There were many biomarkers involved in
NPC. The development of NPC included multi-step process, from nor- each step of NPC progress
mal NPE to dysplastic NPE, early NPC, early invasive NPC, invasive
21 Cancer 267
accurately and treated reasonably in the early stage. cancers, indicating the tumor suppressor function of this
Therefore, early molecular diagnosis for tumors is particu- gene. The hypermethylation of CpG-island promoter region
larly important. is closely related to the silence of RASSF1A and is consid-
ered the early and more significant, compared with gene
21.3.1.1 SPLUNC1 mutation or deletion, event involved in the occurrence of
Short palate lung and nasal epithelium clone 1 (SPLUNC1) NPC [41]. Other than early diagnosis, this gene is useful for
is specifically expressed in the respiratory tract where it detecting invasion, metastasis, and treatment of NPC as well.
plays a pivotal natural defense role. It functions to eliminate
foreign pathogens, especially Gram-negative bacteria, and is 21.3.1.4 EBERs
able to bind with nanobacteria, to regulate immune response EB virus-encoded RNAs (EBERs), a general term for
and inflammation. SPLUNC1 get connection with many EBER1 and EBER2, is expressed in all populations with
human respiratory diseases during participation in these bio- known latent infections of EB virus [42]. In situ hybridiza-
logical processes. It plays an intrinsic immune protective tion with EBER1 showed that EBER1 was only occasionally
role in the very early stage of NPC and can be considered as positive in normal nasopharyngeal and atypical hyperplasia
a very promising TM for early diagnosis, efficacy of treat- epithelial tissue, while EBER1 was always positive in the
ment, and prognosis of NPC and NSCLC. Studies have majority of keratinized squamous cell carcinoma and non-
shown that in the very early stage of NPC, the expression of keratinous carcinoma cells (Fig. 21.3) [43]. This result indi-
secretory protein SPLUNC1 is down-regulated in atypical cates that latent infection of EB virus existed in normal
hyperplasia and in nasopharyngeal secretions of patients nasopharyngeal epithelium, representing an early event of
with NPC, making it an ideal molecular target for early NPC.
molecular diagnosis and risk screening of NPC [38]
(Fig. 21.2). 21.3.1.5 DAPK
Death-associated protein kinase (DAPK) is a serine/threo-
21.3.1.2 APC nine protein kinase regulated by calcium/calmodulin, which
Adenomatous polyposis coli (APC), a tumor suppressor can regulate cell survival and apoptosis and can inhibit can-
gene, was first cloned from colon cancer and officially nomi- cer as well. DAPK, as a positive regulator of apoptosis, can
nated as DP2, 5 or APC. It is in close correlation with the be activated by many factors such as INF-γ, Fas, TNF-β,
occurrence of malignant tumors such as CRC [39]. APC ceramide, ERK, C-myc, and E2F and be induced to apopto-
gene mutation is earlier than K-ras and p53 gene mutation in sis through p19ARF/p53 and other pathways. Methylation of
the tumorigenesis of colon cancer, which is called gate CpG islands in the promoter region results in silence of
keeper in colon cancer as a result. APC gene inactivation DAPK gene and thus inhibits apoptosis, which may lead to
happens in the early stage and stabilizes in the whole process the formation of tumors. This methylation is an early event
of occurrence and development of colon cancer. The deletion of tumorigenesis, which can possibly be detected before cell
of APC gene is closely related to the genetic susceptibility of canceration. DAPK promoter methylation was found in
colon cancer. Loss of heterozygosity (LOH) of APC gene many cancers, such as NSCLC, HCC, and cervical cancer, as
has been reported in sporadic CRC in China [40]. Germline well as some benign conditions, such as in bronchial epithe-
mutation of APC gene exists in typical familial adenomatous lial cells of smokers [44]. Therefore, DAPK provides a new
polyposis syndrome. Around one third of the sense muta- biomarker for early diagnosis of tumors.
tions concentrate between codons 1061 and 1309, while the
rest are scattered from codons 200 to 1600. Mild familial 21.3.1.6 NES1
adenomatous polyposis syndrome usually resulted from The normal epithelial cell-specific-1 (NES1) gene belongs to
mutations at 5′ or 3′ terminal of APC gene. Moreover, APC the kallikrein (KLK) family and encodes a serine protease
mutation has been reported deeply involved in the transfor- designated as human kallikrein 10 (hK10) with high homol-
mation of gastric adenoma to adenocarcinoma. The research ogy to the glandular KLK family. It is revealed as a novel
on LOH of APC gene gastric cancer suggests that the loss tumor suppressor implicated in carcinogenesis and as a
rate was higher in the early stage. This discovery indicates a potential biomarker for tumors. The expression of NES1
possibility that APC gene mutation may be an early event gene is absent in breast cancer, thus it is regarded as an early
and it thus possibly plays a key part in the early stage of gas- TM of breast cancer [45]. The expression of NES1 is signifi-
tric cancer. cantly decreased in patients with acute lymphoblastic leuke-
mia (ALL) and prostate cancer [46], and is in correlation
21.3.1.3 RASSF1A with remission, disease-free survival (DFS), and overall sur-
RASSF1A is a tumor suppressor gene located on the 3p21.3 vival (OS) of tumors. Inactivation of NES1 gene is an early
chromosome and belongs to the RAS region-related family event in the occurrence of ALL, and methylation of exon 3 is
gene. Inactivation of this gene is very common in various an indicator of poor prognosis. NES1 can assist CA125 in
268 W. Zhang et al.
Fig. 21.2 The expression of secretory protein SPLUNC1 was down- glands. VII: SPLUNC1 was weak positive in dysplasia NPE. VII:
regulated in NPC tissue microarray using IHC. I: NPC tissue microar- SPLUNC1 was negative in NPC
ray. II-VI: SPLUNC1 was positive in normal NPE, nasopharyngeal
21 Cancer 269
a b
c d
Fig. 21.3 The expression of EBER-1 was up-regulated in NPC tissue positive in keratinized squamous cell carcinoma. (d) EBER1 was posi-
microarray using ISH. (a) EBER1 was negative in normal NPE. (b) tive in keratinized squamous cell carcinoma
EBER-1 was negative in undifferentiated NPC. (c) EBER1 was weak
early diagnosis of ovarian cancer. NES1 is a promising TM DCC is related to the degree of differentiation and Dukes
that the expression and methylation of NES1 can provide stage of tumors. The lower the degree of differentiation and
valuable information for early diagnosis and prognosis of the later the Dukes stage, the more obvious the loss of DCC
various tumors. protein expression is. Therefore, the detection of DCC
expression in colon cancer has certain reference value for
21.3.1.7 DCC early diagnosis, evaluation of treatment, and prognosis of
Deleted in Colorectal Carcinoma (DCC) is an important colon cancer.
tumor suppressor gene located in 18q21.3 [47]. The protein
product of DCC is a cell adhesion factor which shares the
same interchangeable name DCC. It is closely correlated 21.3.2 Molecular Biomarkers for Invasion
with the occurrence, development, metastasis, and prognosis and Metastasis
of CRC. Its normal expression can lead to strong adhesion
between cells, limiting the transfer of cells. But reduced Invasion and metastasis, as important features of malignant
expression due to a point mutation, deletion, or insertion in tumors, undergo abating cell adhesion, penetrating through
sequence leading to weakness of the interaction between basement membrane into blood, escaping from immune sur-
cells will probably result in metastasis of tumor cells. LOH veillance and growing at distant sites. Although significant
of DCC is one of the most frequent genetic abnormalities progress has been made in early diagnosis and treatment of
that occur in advanced CRC. The expression of DCC in cancer, a great majority of patients that suffered tumor still
colon cancer tissues has been reported down-regulated com- die from metastasis. In order to prevent cancer metastasis
pared with normal colon tissues. The absent expression of and to reduce the mortality of cancer patients, it is urgent to
270 W. Zhang et al.
seek for molecular markers related to cancer metastasis, and have 24 different combinations of heterodimers, made up by
to explore the mechanism of their involvement in the inva- 1/18 αand 1/8 β subunit. The expression pattern varies
sion and metastasis of cancer, and to utilize them to develop between two different subunits and among different types of
new methods and targets against metastasis. cells. The biological function of different heterodimer varies
as well. Integrin mainly mediates the adhesion between cells
21.3.2.1 Adhesion Molecules and ECM, which enables cells attached to form integration,
Adhesion molecules (AM) are a subset of cell adhesion pro- hence its name. And this adhesion and attachment are closely
teins located on the cell surface or distributed in extracellular related to tumor invasion and metastasis. Vitronectin (VTN
matrix (ECM) that assist cells in sticking to each other and to or VN) belongs to the hemopexin family. It is widely secreted
ECM in cell adhesion process. Most of them are glycopro- in serum, the extracellular matrix, and bone. It contains an
teins and a few are glycolipids, acting in the form of ligand- RGD (Arg-Gly-Asp) sequence, which is a binding site for
receptor binding. These molecules take integral parts in cell membrane-bound integrins, and initiate integrin signaling
adhesion, recognition, activation, proliferation and differen- and activate related pathways such as STAT3, Akt, and
tiation, and signal transduction, and cell extension and move- Erk1/2 pathway, leading to cell adhesion as well as invasion
ment, forming the molecular basis of a series of important and metastasis [49].
physiological and pathological processes such as immune
response, inflammation, coagulation, wound healing, and CD44
tumor metastasis. Based on its structure and function, adhe- CD44 is a cell-surface glycoprotein participated in cell–cell
sion molecules are subclassified into five categories: interactions, cell adhesion, and migration. CD44 plays a vital
E-cadherins, integrins, immunoglobulin superfamily, selec- role in lymphocytes homing, migration, and binding to
tins, and CD44. E-cadherins, integrins, and CD44 are mainly hypervascular endothelial cells. CD44 splice variants com-
involved in the metastasis of tumors. prising variable exons are named CD44v which is secreted
by metastatic tumor cells. CD44 is associated with the patho-
E-Cadherins logic activities of cancer cells, such as proliferation, inva-
Cadherins are AMs reliant on calcium ions (Ca2+) to func- sion, metastasis, and prognosis [50]. It has been widely
tion, hence their name. Mammals mainly have E, N, P, and H studied as a potential therapeutic target.
types of cadherin, among which E-cadherin (E-CD) is cor-
related to invasion and metastasis. Epithelial-mesenchymal 21.3.2.2 Proteolytic Enzymes and Their
transition (EMT) is an important biological process for Inhibitors
epithelial-derived malignant tumor cells to acquire ability of Tumor cells must degrade ECM and penetrate basement
migration and invasion, which is characterized by decreased membrane in order to separate from the primary tumor and
expression of AMs such as E-CD, transformation of cyto- form new metastatic foci. Studies have shown that tumor
skeleton from cytokeratin to vimentin, and acquisition of cells can produce substantial proteolytic enzymes to degrade
morphological features of mesenchymal cells. Through the matrix and basement membrane, leading to the dissolu-
EMT, epithelial cells lose cell polarity and junction with tion of local tissues and the formation of channels for tumor
basement membrane, but obtain some mesenchymal pheno- cell metastasis. Proteolytic enzymes include matrix metallo-
types, such as migration, invasion, and ability against apop- proteinase (MMP), plasminogen activator, and cathepsin.
tosis and degradation of ECM. E-CD is located on the
extracellular surface of epithelial cells, which consists of MMP
extracellular domain (making cells adhere to each other) and MMP, also known as matrixin, is a calcium-dependent zinc-
intracellular domain. Intracellular regions form complexes containing endopeptidase, which is named for its need for
by connecting the cytoskeleton microfilaments with their Ca2+, Zn2+, and other metal ions as cofactors. MMPs are cor-
associated protein α-cadherin. E-CD has also been shown to related with the cleavage of cell surface receptors, the release
be critical in cell adhesion and aggregation, except partici- of apoptotic ligands (such as the FAS ligand), and chemo-
pating in information transmission among cells, maintaining kine/cytokine inactivation. MMPs are also capable of
normal tissue formation and epithelial cell differentiation. It destroying local tissue structure, promoting tumor prolifera-
was found that the expression of E-CD was down-regulated tion; ruining basement membrane barrier, facilitating tumor
in many tumors and negatively related to the grade and metastasis; and remodeling ECM, accelerating tumor angio-
metastasis of tumors [48]. genesis. Among MMPs, MMP-2 and MMP-9 are considered
to be important in metastasis.
Integrins The activity of MMPs is prohibited by specific endoge-
Integrins, one of the major classes of transmembrane recep- nous tissue inhibitor of metalloproteinases (TIMPs), which
tors within the ECM, mediate cell-ECM interactions. They consist of a family of four protease inhibitors: TIMP1–4.
21 Cancer 271
They are widely distributed in vivo and are able to inhibit 21.3.2.3 Tumor Microangiogenesis-Related
MMPs through binding to the activated MMPs at a ratio of Molecules
1:1. The precise regulatory mechanism between MMPs and Angiogenesis has a vital role to play in the progression and
TIMPs, as well as many other regulatory factors, ensures cell metastasis of tumors. Inhibiting this process will obviously
migration and extracellular matrix remodeling in the physi- prevent the spread and metastasis of tumors. Thereby,
ological state of the organism. The imbalance between restraint of angiogenesis of tumors as a therapeutic target has
MMPs and TIMPs may lead to invasion and metastasis of been aroused more and more attention.
tumor cells [51].
Promoting Microangiogenesis Factors
uPA
Urokinase-type plasminogen activator (uPA), also known as FGF
urokinase, is a serine protease present in humans and other Fibroblast growth factor (FGF) functions as pleiotropic fac-
animals. The synthesis and secretion of uPA are influenced tor including angiogenesis on tumor and stromal cells. The
by many factors, such as proto-oncogene V-Src and Ras, and over-expression of FGF in the peripheral and invasive mar-
cytokines including TNF, INF, IL-1, TGF-β, IGF-1, FGF, ginal cells of the tumor tissue, while the low level of FGF in
and MMP. Elevated expression level of uPA is found to be tumor tissue with early stage, indicates the heterogeneity of
correlated with tumor malignancy. uPA stimulates cancer the tumor cell genes, that is, there are differences in invasion
cells to produce plasminogen which is further catalyzed by and metastasis related expression in the same tumors, and
uPA to activated plasmin. Plasmin is able to degrade a large cells with high expression of FGF gene will invade and
amount of ECM proteins and then facilitates tissue invasion metastasize. Inhibitors of the FGF/FGFR system have been
and, thus, contributes to metastasis. This makes uPA a prom- shown to act as “two compartment” targeting drugs impli-
ising drug target, and, hence, inhibitors have been sought to cated for the therapy of FGF/FGFR-driven tumors [54].
be used as anticancer agents [52].
VEGF
Cathepsin Vascular endothelial growth factor (VEGF) is the most direct
Cathepsins, a major member of the cysteine protease family, angiogenic active protein and is a vascular endothelial cell-
are proteases found in cells, especially lysosomes, of all ani- specific cytokinin and angiogenic factor which can bind to
mals as well as other organisms. More than 20 members have endothelial cell tyrosine kinase receptor and KDR factor.
been found and 11 members were in human body. Cathepsins VEGF is involved in several biological processes, such as
are closely related to many major diseases such as tumor, survival, metastasis, or further differentiation. Hence, VEGF is
osteoporosis, arthritis, and so on, attracting much attention a promising therapeutic target in cancer. A monoclonal anti-
as a target protease in recent years. body named bevacizumab is a VEGF targeted inhibitor which
Cathepsin B (CB) has been found to be related to the inva- can inhibit the biological activity of human VEGF, as a result
sion and metastasis of tumors. CB can directly or indirectly promoting endothelial cell mitogenic activity and increasing
activate and dissolve enzymes of ECM such as collagen, vascular permeability and angiogenesis activity, so as to
laminin, and basement membrane in the process of metasta- achieve the anti-tumor effect. Bevacizumab was the first anti-
sis of cancer cells to promote the infiltration of cancer cells VEGF drug approved in 2004 and was usually combined with
into deep tissues, thereby opening channels for expanding of standard chemotherapy regimen in clinic. Around 10–15% of
cancer cells. High expression of CB has been found in a vari- patients profit by bevacizumab therapy. However, biomarkers
ety of human tumors, such as gastric, lung, intestinal, breast, for bevacizumab efficacy still underwent observation [55].
ovarian, prostate, kidney cancer, and other cancer cells.
Development of potent and selective cathepsin B and L MVD
inhibitors as therapeutic agents for treating diseases has Microvessel density (MVD) refers to the counts of microves-
aroused much attention in recent years [53]. sels per unit volume in tumors. Elevated counts of MVD are
Cathepsin D (CD) is a recently discovered mitogen in all closely correlated with histological grade, clinical stage,
kinds of cells and is able to degrade various substrates such lymph node metastasis, recurrence, and prognosis of tumors.
as fibronectin and laminin. This ability indirectly destroys Highly increased MVD found in the bladder, colorectal, gas-
the basement membranes of blood vessels and activates CB, tric, and NSCLC is prone to metastasis.
triggers chain reaction, and induces activation of The infiltration and metastasis of tumors are related to the
uPA. Different from other cathepsins, CD has some protease entry of tumor cells into lymph and blood circulation. With
activity at neutral pH. High levels of CD in tumor cells the increase of blood vessels in tumors, the possibility of
appear to be correlated with greater invasiveness and tumor cells entering blood circulation is also increased. MVD
metastasis. is considered to be a reliable predictor of metastatic potential
272 W. Zhang et al.
in many tumors. It is suggested that application of MVD dur- 21.3.3 Molecular Biomarkers for Prognosis
ing pathological examination would provide an important
evidence for clinical evaluation of prognosis and treatment. In Prognosis refers to predicting the possible course and out-
addition, tumor MVD can be considered as a potential predic- come of disease. The prognosis is not only a simple cure or
tive marker for targeted angiogenesis therapy [56]. death but also includes a variety of outcomes related to cure
and death, such as recurrence, remission, deterioration, com-
Inhibitory Microangiogenesis Factors plications, and so on. The most important prognostic indica-
tors include OS, DFS, and quality of life. The factors
TSP-1 influencing the prognosis of tumors include their own char-
Thrombospodin-1 (TSP-1), also known as thrombin-sensitive acteristics, TNM stages, genetic factors, early diagnosis,
protein, is first isolated from the platelet membrane after the treatment, psychological status, nutrition, social and family
thrombin stimulation. TSP-1 is widely distributed in blood and factors, and so on. Biomarkers that are closely related to the
tissues such as heart, cartilage, lung, and brain. It is expressed prognosis of tumor can be used to provide important evi-
in many cell lines, such as lung fibroblasts, epithelial cells, vas- dence for the design of therapeutic regimen and the accurate
cular endothelial cells, and monocytes as well. TSP-1 is capa- evaluation of prognosis.
ble of resisting angiogenesis, inducing the apoptosis of vascular
endothelial cells. It can reduce the vascular density by acting on 21.3.3.1 Survivin
the CD36 receptor on the vascular endothelial cells, resulting in Survivin is a member of the inhibitor of apoptosis (IAP) fam-
necrosis of the tumor cells [57]. The TSP-1 can also regulate ily. The survivin protein abundantly exists in fetal tissue and
the proliferation of the tumor cells and induce the apoptosis of a great majority of tumors. However, it is absolutely absent
the cells through the TGF-1-dependent mechanism but inhibits in terminally differentiated human cells. It functions to pre-
angiogenesis through TGF-1-independent way. It has been vent cells from suicide (i.e., apoptosis or programmed cell
found that exogenous gene TSP-1 could prevent the metastasis death) by inhibiting the activation of caspase. Survivin is
of dormant microtumor after radiotherapy, and it could also enriched in various cancers, such as lung cancer, colon can-
inhibit the metastasis of tumor by inducing apoptosis of tumor- cer, pancreatic cancer, prostate cancer, and breast cancer.
associated microvascular endothelial cells. Survivin level is proportional to progress of disease and his-
tological stage. These data suggest survivin might act as a
Angiostatin and Endostatin biomarker for bad prognosis of cancer [60].
Angiostatin (AS) and endostatin are anti-angiogenic com-
pounds which are proteolytic fragments of plasminogen and 21.3.3.2 Cyclin D1
collagen XVIII, respectively. They are reported to serve as Cyclin D1 is one of the cell cycle regulators, which posi-
anti-angiogenic agents both in vitro and in vivo. Prior results tively regulates the cell cycle. Dysfunction of cell prolifera-
indicated that they are able to obviously inhibit the formation tion cycle resulted from uncontrollable expression of cyclin
of microangium and hamper the proliferation and metastasis D1 will lead to the occurrence of malignant tumors.
formation of tumor in several animal tumor models. Overexpression of cyclin D1 has been found in many malig-
Therefore, it is very appealing to use these naturally occur- nant tumors such as NPC, laryngeal cancer, breast cancer,
ring anti-angiogenic molecules in clinical practice [58]. esophageal cancer, lung cancer, and so on. Significantly high
expression of cyclin D1 protein is able to independently pre-
21.3.2.4 Autocrine Motility Factor dict the prognosis of head and neck squamous cell carcinoma
(HNSCC) after curative resection. Tumors in NPC patients
AMF that do not express cyclin D1 are prone to recur locally than
Autocrine motility factor (AMF) is a family of thermo-soluble those with high expression. In addition, the 13-year survival
proteins with molecular weight (MW) of 55–66 kDa. It is rate of cyclin D1-negative NPC patients is lower than those
secreted by cancer cells and binds to AMF receptor (gp78) on positive cases, which suggests that cyclin D1 is a promising
the same cell and stimulates cell growth and motility by auto- prognostic molecule in NPC patients (Fig. 21.4).
crine. MAPK/ERK or PI3K/AKT pathways are thought to be
involved in cancer metastasis mediated by AMF [59]. 21.3.3.3 P53
P53, the most widely studied human gene, is a tumor suppres-
Gp78 sor gene. It encodes a nuclear phosphoprotein with MW about
AMFR is a glycoprotein with MW of 78 kDa. The expres- 53 kDa, hence its name p53. Normally, the protein exists in the
sion of gp78 in esophageal cancer and colon cancer with nucleus of all types of cells and functions as a transcription
lymph node metastasis is noticeably increased, and the factor. Wild type p53 gene is a kind of anti-oncogene whose
5-year survival rate of gp78 negative patients is markedly inactivation plays a pivotal role in tumor formation. P53 pro-
higher than that of positive ones. tein that leads to tumor formation or cell transformation is the
21 Cancer 273
a b
1.0 1.0
P=0.010 P=0.019
0.8 0.8
Disease Free Survival
Overall Survival
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
0 50 100 150 200 0 50 100 150 200

Time (months) Time (months)
Fig. 21.4 Kaplan-Meier survival analysis for NPC patients based on (green) and negative expression (blue). (a) DFS: P = 0.010, log-rank
differential expression of cyclin D1. Cyclin D1 expression levels dif- test; (b) OS: P = 0.019, log-rank test [61]
ferentiate DFS and OS in the 2 patient groups: positive expression
product of mutated p53 gene. It is a tumor promoting factor mobility. The lack of TSLC1 expression is a vital factor in
which can eliminate the effects of normal p53. The death of tumor proliferation and invasion [63].
severely damaged DNA cells is beneficial to the body due to
prevention of persistent replication of harmful mutant cells. 21.3.3.5 BAG-1
P53 expression levels are correlated with the high proliferation Bcl-2-binding anti-apoptosis gene (BAG-1) does not belong
of cells and the degree of differentiation of tumor cells. Tumors to Bcl-2 protein family. It is a new, independent apoptosis-
with lower differentiation and higher degree have higher levels related protein. BAG-1 closely links with the occurrence and
of p53 genes. Moreover, higher p53 level is in correlation with development of tumor. It displays low or absent expression
recurrence in breast cancer patients with positive estrogen in normal tissue, but significantly higher expression in
receptor (ER) [62]. tumors, such as endometrial carcinoma, breast cancer, and
esophageal carcinoma. Besides, its expression is correlated
21.3.3.4 TSLC1 with pathological grade, clinical stage, distant metastasis,
Tumor suppressor in lung cancer 1 (TSLC1) is a member of and prognosis of the tumor. Therefore, BAG-1 is believed to
the immunoglobulin superfamily and thus also known as become a valuable biomarker for early diagnosis and prog-
immunoglobulin superfamily 4 (IGSF4). Tumor formation is nosis of tumor [64].
inhibited when NSCLC cell line A549 transfected with
TSLC1 were injected subcutaneously into nude mice, indi- 21.3.3.6 Bmi-1
cating an anti-tumor effect of TSLC1. It was found nega- B-cell-specific Moloney murine leukemia virus insertion site
tively correlated with the stage of cancer progression, 1 (Bmi-1) has been reported as an oncogene which directly
invasion and metastasis to lymph node, and vascular infiltra- regulates cell growth and proliferation. Bmi-1 is required for
tion in lung cancer, making it a potential biomarker for prog- self-renewal of adult hematopoietic stem cells (HSC) as well
nosis. It has been reported that the prognosis of patients with as leukemia stem cells with high efficiency. Aberrant expres-
negative expression of TSLC1 in lung adenomas is poorer, sion of Bmi1 appears to be involved in a variety of cancers,
which is worse in males than in females. TSLC1 deletion, such as breast, ovarian, bladder, prostate cancer, as well as
more common in patients with clinically advanced meningi- hematological malignancies. It abundantly amplifies and sig-
omas, leads to a remarkable decrease in survival rate. Around nificantly overexpresses in myelodysplastic syndrome (MDS)
50% of patients with esophageal cancer have decreased and is positively proportional to the score of international
expression of TSLC1 which has close relationship with prognostic scoring system (IPSS) [65]. This makes Bmi-1 a
grade of lesions, tumor invasion and metastasis, and cell potential biomarker for progression and prognosis of tumor.
274 W. Zhang et al.
21.4 M
olecular Biomarkers for Precision 21.4.1.2 ER and PR
Medicine Estrogen receptors (ERs) are a group of proteins that are
bound to and activated by the hormone estrogen inside cells.
Precision medicine (PM) is a novel medical concept that High expression of ERs has been shown in about 70%
develops grounded on personalized medicine along with the patients with breast cancer, defined as “ER-positive.” It is
advances in omics and the cross-application of bioinformat- strongly associated with the histological and clinical stage,
ics and big data science. In this model, omics such as genom- and proliferation activity of breast cancer. The positive
ics and proteomics and other medical cutting-edge techniques expression rate of ER in patients with stage I and II is signifi-
were employed to analyze large populations and to identify, cantly higher than that in patients with stage III and IV. The
verify, and apply results to specific disease in essence in decreased expression of ER in advanced breast cancer is
order to accurately seek for the etiology and optimal thera- mainly due to the loss of normal structure caused by cell
pies of the disease. depolarization and proliferation, which is also in correlation
Molecule-targeted therapy based on aberrant molecules with the occurrence of estrogen-independent breast cancer.
during occurrence and development of cancer is a significant The progesterone receptor (PR) is the product of estrogenic
progress for cancer therapy in the twentieth century. Different action. The expression of PR is based on the expression of
from traditional radiotherapy and chemotherapy which kill ER and is capable of promoting and synergizing the effect of
both cancer cells and normal cells with active proliferation, estrogen on ER. It has been proved that breast cancer patients
this method only targets tumor cells. It is based on the prin- with simultaneously positive ER and PR have a better prog-
ciple that targeted agents which are designed to aim at the nosis. Endocrine therapy for breast cancer such as tamoxifen
known carcinogenic site which is either a protein molecule and anastrozole is recommended when these two receptors
or a gene fragment in the tumor cells will specifically bind to are detected in breast cancer tissues [67].
corresponding carcinogenic site and cause specific death of
cancer cells without influence on normal cells around the 21.4.1.3 BRCA1/BRCA2
tumor. Generally, BRCA1 and BRCA2 exist in normal breast cells
The anti-CD20 monoclonal antibody clinically approved and other tissues. They usually function to assist in repairing
in 1997 is the first drug in this field. And a large amount of damaged DNA, but they will destroy cells if they failed.
targeted agents have emerged in the past 20 years. Molecule- Mutations in BRCA1 or BRCA2 lead to improperly repair of
targeted therapy has gradually been recognized as an increas- damaged DNA, which increases the risk for breast cancer
ingly important anti-cancer therapy. Precision medicine, as and ovarian cancer. Poly (ADP-ribose) polymerase (PARP)
proposed by President Obama in his White House speech in is an important enzyme involved in DNA repair and a useful
2015, is a further extension of molecule-targeted therapy. indicator for apoptosis. Its inhibitor can hence promote
apoptosis of tumor cells and enhance the therapeutic effect
of radiotherapy or chemotherapy such as alkylating agent
21.4.1 Breast Cancer and platinum drug. Olaparib, a PARP inhibitor, has been
found to not only prolong the PFS but also improve the
21.4.1.1 HER2 objective remission rate (ORR) of metastatic breast cancer
HER2, originally known as neu, is an oncogene. It encodes a patients with HER2-negative but germline BRCA mutation
protein which is a member of human epidermal growth fac- compared with standard chemotherapy [68]. Olaparib was
tor receptor (HER/EGFR/ERBB) family. HER2 is normally thus approved by FDA as the first PARP inhibitor for breast
inactivated (proto-oncogene), and dimer must be formed to cancer.
produce activation signals. After activation, the transcription
of early nuclear response genes such as c-fos and c-jun is
increased, which promotes cell proliferation, differentiation, 21.4.2 Lung Cancer
migration, and tumorigenesis. HER2 is overexpressed in
about 15–30% of breast cancers. It is closely related to 21.4.2.1 EGFR
increased recurrence and a poor prognosis of breast cancer EGFR (ErbB-1; HER1 in humans) is a member of the ErbB
[66]. Trastuzumab (marketed as Herceptin) is a monoclonal family of receptors. This family consists of four closely
antibody drug targeted HER2. It has been found that tumors related receptor tyrosine kinases: EGFR (ErbB-1), HER2/
with HER2+ acquired accelerated apoptosis and decreased neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4).
tumor volume after administration of trastuzumab in breast Mutation or amplification of EGFR could result in cancer.
cancer patients, which provides direct evidence that HER2 is PI3K-AKT and RAS-MEK-ERK signaling activated by
the driving gene of breast cancer. EGFR mutation promote the growth, proliferation, and
21 Cancer 275
migration of cancer cells. The most common mutations are ing in aneuploidy, chromosome genome amplification, and
deletions in exon 19 and a missense mutation in codon 858 high-frequency of LOH. CIN is common in solid and hema-
(causing arginine to be replaced by leucine, L858R). tological cancers, especially CRC. The most frequent single
NSCLC with these mutations, especially lung adenocarci- gene mutations are APC and K-Ras genes, involving Wnt
noma, is highly sensitive to tyrosine kinase inhibitor (TKI). signaling pathway, K-Ras signaling pathway, p53, and its
The targeted EGFR drugs include gefitinib, erlotinib, downstream signaling pathway.
osimertinib, and afatinib [69].
21.4.3.2 MSI
21.4.2.2 ALK Microsatellites are repetitive short nucleotide sequences
Anaplastic lymphoma kinase (ALK) belongs to the insulin scattered throughout the genome. MSI represents a condition
receptor subfamily of the tyrosine kinase receptor (RTK) that DNA is prone to mutate during duplication. Therefore,
family. Mutation, acquisition of additional gene copies, the presence of MSI strongly indicates that DNA mismatch
and formation of a fusion gene with any of several other repair (MMR) is not functioning normally. MSI may exhibit
genes of ALK gene can all lead to tumors. The EML4- one of following three states: MSI-High (MSI-H), MSI-Low
ALK fusion gene is a key driven gene which is responsible (MSI-L), or Microsatellite Stable (MSS) in colon cancers.
for around 3–5% of NSCLC. Therefore, it has been adopted CRC with MSI-H was reported to have a better prognosis,
as a new therapeutic target of NSCLC. Clinical trials have but a less sensitivity to the 5-FU-based chemotherapy com-
confirmed that ALK heterotopic lung cancer patients are pared to patients with MSI-L or MSS [72].
highly sensitive to the ALK inhibitors, such as crizotinib Pembrolizumab is an important immunotherapy drug
and brigatinib [70]. (PD-1 inhibitor) for the treatment of solid tumor patients
with MSI-H or dMMR. This is the first anti-tumor therapy
21.4.2.3 ROS-1 that is independent of tissue type and tumor location or
ROS1 is also a RTK which shares the similar structure with differentiation stage but merely according to whether it is
ALK. Usually, ROS1 rearrangements are mutually exclusive MSI-H, which is a real milestone.
of ALK rearrangement and EGFR mutation. ROS1 gene
rearrangement has been shown an incidence of approxi- 21.4.3.3 CIMP
mately 1% in patients with NSCLC, making it a unique CpG Island Methylator Phenotype (CIMP): DNA methyla-
molecular subset of NSCLC with a therapeutic target. tion usually occurs in 5’-CG-3′ (CpG) dinucleotides. Many
Rizotinib, used in ALK rearrangement, was also approved genes involved in CRC are silenced by methylation, includ-
for the treatment of metastatic NSCLC patients with ROS1- ing APC, MCC, MLH1, MGMT, and so on. CIMP refers to
positive. Attentions must be paid that there is drug resistance the coexistence of methylation in multiple genes that meth-
in ROS1+ lung cancer due to possible mechanisms including ylation in at least three markers can be defined as CIMP posi-
kinase domain mutations in ROS1 and bypass signaling via tive. At present, five molecules can be used as CIMP markers,
RAS and EGFR [71]. including CACNA1G, IFG2, NEUROG1, RUNX3, and
SOCS1. CRC with high CIMP accounts for 15% to 20% of
sporadic CRC.
21.4.3 Colorectal Cancer Targeted therapy for CRC is mainly focused on EGFR
and angiogenesis of tumor tissues. Pembrolizumab and
Mutations in many genes are involved in the occurrence of nivolumab are suitable for patients with MSI-H or non-
CRC. These mutations consist of three conditions: chromo- resectable or metastatic cancer with dMMR. Panitumumab is
somal instability (CIN), microsatellite instability (MSI), and recommended for metastatic CRC with wild type RAS
CpG island methylator phenotype (CIMP). These mecha- (including KRAS and NRAS).
nisms are not independent of each other in the development
of sporadic and hereditary colon cancer, but complementary
to each other to promote tumorigenesis. 21.5 Novel Molecular Biomarkers of Tumor
21.4.3.1 CIN With the development of molecular biology technology,

CIN refers to a type of genomic instability in which the scientists have discovered many biomacromolecules that
integrity of whole chromosomes or parts of chromosomes is are closely correlated with the occurrence and development
destructed during tumorigenesis. CIN is mostly resulted of tumors through the study of various tumors and normal
from chromosome segregation disorders, telomere dysfunc- controls. We collectively call these newly discovered mol-
tion, and defective DNA damage repair mechanism, result- ecules as potential new molecular markers of tumors. The
276 W. Zhang et al.
discovery of these novel molecular markers is of great sig-

nificance for the diagnosis and treatment of tumors. They
can be independent types of molecules, such as epitopes of
antigens or antibodies, specific molecular fragments of
nucleic acid molecules, DNA and DNA methylation, or
RNA (mRNA and miRNA). It can also be a large particu-
late matter, such as the circulating tumor cells (CTC) in the
blood or nanoscale endosome (endosome), as well as the
virus particles, etc.
21.5.1 Tumor Specific Protein 70 (SP70)
21.5.1.1 Sources and Characteristics

SP70 is a novel tumor marker, which is over-expressed in the
tissue and serum of some malignant diseases, including
NSCLC, liver cancer, CRC, pancreatic cancer, ovarian can-
cer, breast cancer, and thyroid carcinoma.
SP70 was discovered with monoclonal antibody library
technique and was isolated with affinity chromatography by
Fig. 21.6 Laser confocal micrograph of SP70 expression in SPC-A1
Prof. Shiyang Pan in 2012 [73]. The MW of SP70 is about cells
70 kDa (Fig. 21.5). Immunofluorescence results showed that
SP70 protein was mainly located in the cell membrane and
cytoplasm (Fig. 21.6). Immunoelectron microscopy indi-
cated that SP70 protein was also expressed at a low level in
the nucleus (Fig. 21.7).
Fig. 21.7 Immunoelectron micrograph of SP70 expression in SPC-A1

cells
Purified SP70 protein was identified with mass spectrum.

The sequence was compared with NCBI database. The result
proved that SP70 was a new protein containing 648 amino
acids and had some homology with Rho guanine nucleotide
Fig. 21.5 Purified SP70 protein electropherogram exchange factor (RhoGEF).
21 Cancer 277
Fig. 21.8 NJ001 mAb inhibits tumor cell invasion (Transwell experiment)
Table 21.1 Differential gene expression analysis after NJ001

treatment
Fold change Increased genes Decreased genes
≥6.0 3 7
5.0–5.9 10 8
4.0–4.9 28 36
3.0–3.9 75 119
2.0–2.9 435 652
Total 551 823
Encyclopedia of Genes (KEGG) pathway analysis, we found

that SP70 could affect many genes and participate in prolif-
eration, apoptosis, invasion, and metastasis of tumor cells.
The involved pathways include MAPK, cell apoptosis, focal
adhesion, and cholesterol biosynthesis. SP70 exerts a variety
of roles by regulating multiple downstream genes which are
involved in diverse signal pathways (refer to Fig. 11.8).
21.5.1.2 Clinical Detection Methods

Several assays were developed for SP70 detection.
Immunohistochemistry was used to detect SP70 protein in tis-
Fig. 21.9 NJ001 acting on tumor cells to induce apoptosis (electron sues. ELISA was used to detect SP70 protein released from
micrograph)
cells to peripheral blood. Flow cytometry assay and immuno-
magnetic beads capture based liquid biopsy were used to detect
Functional studies showed that SP70 could promote cell the positive expression of SP70 protein in cell membrane.
proliferation and metastasis and resist cell apoptosis (Fig. 21.8
and Fig. 21.9). The expression of 1374 genes changed more 1. Immunohistochemistry: Based on the specific-binding

than two times when lung adenocarcinoma cells were treated principle of antigen and antibody, the enzyme chromo-
with anti-SP70 monoclonal antibody NJ001 (Table 21.1). genic reagent of labeled antibody can be used to localize
Data have been uploaded to the NCBI database (GSE59655). and verify the antigen in tissue and cell by redox chemical
Based on Gene Ontology (GO) enrichment and Kyoto reaction.
278 W. Zhang et al.
2. ELISA: SP70 in serum was detected by double antibody when they intrude into the bloodstream. In the process of
sandwich ELISA. transfer, CTCs leave the support of microenvironment and
3. Specific circulating lung cancer cell (SCLCC) assay:
meet the challenge of survival including immune attacks,
SP70 antigen positive cells in body fluid were counted by the shear stress, and apoptosis. Most cells cannot survive in
flow cytometry assay using CF-488A fluorescence dye the blood, eventually undergoing apoptosis. The probability
labeled monoclonal antibody NJ001. of successfully penetrating through the vessel wall into
4. Immunomagnetic beads capture based liquid biopsy: The interstitial tissue is extremely low, only less than 0.1% of
immunomagnetic beads coated with monoclonal anti- the CTC to survive. Sometimes, CTCs aggregate in blood
body NJ001 are incubated with pleural effusion sample. vessels to form microemboli CTM, which can improve the
SP70 positive tumor cells are captured by immunomag- viability of tumor cells during metastasis and thus increase
netic beads, thereby achieving to separate the tumor cells the probability of tumor metastasis. At the same time, syner-
from pleural effusion. Further after Pap staining, the path- gistic effects with mesenchymal cells provide binding sites
ological properties of pleural effusion exfoliated cells are for CTC with high activity and strong mobility, and are
determined by microscopic examination. more conducive to the growth and reproduction of meta-
static tumor cells. Metastasis is the major reason of death of
21.5.1.3 Clinical Application tumor patients. Tumor cells invade into the surrounding tis-
SP70 can be used for auxiliary diagnosis, therapy efficacy sues of primary tumor, enter the blood and lymphatic sys-
monitoring, and prognosis prediction in NSCLC, thyroid tem, form CTC, which are transferred to the remote tissues,
carcinoma, pancreatic cancer, ovarian cancer, breast cancer, exudate, adapt to the new microenvironment, and finally
etc. settle down to form metastasis. Therefore, the detection of
CTC plays an important guiding role in the early diagnosis,
prognosis judgment, recurrence risk assessment, efficacy
21.5.2 CTC evaluation, and individualized treatment of patients [75]. At
present, the application of CTC in tumor screening still
In 1869, Australian doctor Ashworth first proposed the con- faces technical obstacles such as enrichment, screening,
cept of CTC. In 1976, Nowell defined CTC as tumor cells detection, and cut-off value determination of tumor cells in
derived from primary or metastatic tumors that acquire the peripheral blood circulation, with low sensitivity and speci-
ability to break away from the basement membrane and ficity, so its application in tumor screening still needs to be
invade blood vessels through the tissue matrix [74]. At pres- further updated and matured by detection technology.
ent, CTC is a general term for all types of tumor cells exist-
ing in peripheral blood. A large number of studies have
shown that the CTC exist in different forms in the peripheral 21.5.3 ctDNA
blood. CTC entering the blood circulation can exist alone,
also can form tiny bolts through the migration, adhesion, Circulating tumor DNA (ctDNA) is released into the circula-
and mutual interaction, called circulating tumor emboli or tion of the blood system by the tumor cells [76]. When tumor
circulating tumor microemboli (CTM). Under certain con- cells die and are cleaved, they release intracellular sub-
ditions, they can enter into interstitial tissues and thus stances, including ctDNA, the fragments of tumor genomes
develop metastases. Tumor cells in peripheral blood circula- that constantly flow through the body’s bloodstream. CtDNA
tion will happen in the process of EMT, so there are differ- can be released from macrophages by apoptosis, necrosis, or
ent types of CTC, including epithelial phenotype, interstitial absorption. In tumor patients, ctDNA is derived from normal
cell phenotype, and mixed epithelial and stromal cell pheno- cells and tumor cells, but in patients with very advanced can-
type. CTM and mesenchymal phenotype CTC have stronger cer, ctDNA is mainly derived from tumor cells. In general,
metastatic potential. EMT is a key step in the process of the amount of circulating tumor DNA in the blood is very
helping CTC entering the peripheral blood circulation. small, accounting for only 1% or even 0.01% of the total
When the tumor cells enter the blood, they will migrate in circulating DNA, but the amount of variation is very large.
the form of a single cell or CTM. When the flow reaches the CtDNA has a very short half-life of only 16 minutes, indicat-
appropriate location, the tumor cells will metastasize ing that ctDNA contains rather complex cellular and non-
through the blood vessel wall. Then they metastasize from cellular components, which can better reflect the latest
the blood to other tissues, proliferate, and form new lesions. dynamic information of the tumor (generally reflecting the
The EMT process can also promote the degradation of tumor situation for nearly a week), and facilitate the real-
extracellular matrix by MMP, induce the EMT transforma- time monitoring of the disease of tumor patients by clini-
tion of primary tumor cells, and increase the chances of end- cians. Compared with serum, the proportion of cfDNA
osmosis and exosmosis of tumor cells. CTCs are not safe released by normal tissues in plasma is lower, which can bet-
21 Cancer 279
ter reduce the background noise of ctDNA detection and ylation occurred in p16, RASSF1A, E-cadherin, H-cadherin,
improve the detection rate of ctDNA. Therefore, plasma MGMT, DAPK, DCC, COL1A2, TAC1, SST, and GALR1.
samples are more suitable for ctDNA detection than serum In 65% of head and neck cancers, more than two tumor sup-
samples. pressor genes were methylated, and patients with multiple
Studies have found that ctDNA can be detected in over gene methylation had a worse prognosis than those without
75% of patients with advanced pancreatic cancer, ovarian or with less than two genes methylation [77]. This indicates
cancer, CRC, bladder cancer, breast cancer, and melanoma that DNA hypermethylation is closely correlated with the
and liver cancer, but the detection rate in patients with early occurrence, development, and prognosis of tumor tissues.
brain cancer, kidney cancer, prostate cancer, and thyroid can- Specific gene hypomethylation can cause oncogene activa-
cer is less than 50%. The relative detection rates of patients tion and promote tumor growth and metastasis. Common
with local tumors, such as CRC, gastric cancer, pancreatic DNA methylation markers are (1) cyclin-dependent kinase
cancer, and breast cancer, were 73%, 57%, 48%, and 50%, inhibitors (p16INK4a); (2) O6-methyl guanine-DNA meth-
respectively, indicating that ctDNA has certain limitations in yltransferase (MGMT); (3) glutathione s-transferase1
the detection of early and local tumors and is mainly used for (GSTP1); (4) MLH1 genes; (5) breast cancer I susceptibility
the detection of advanced tumors. Clinical trials have shown gene (BRCA1); and (6) SEPT9 genes.
that (1) the ctDNA concentration is proportional to the tumor
load size, but the tumor stage, location, and size cannot be
determined. (2) ctDNA testing, especially the detection of 21.5.5 Circulating miRNAs
ctDNA mutations, can monitor tumor progression and prog-
nosis. Tests have shown that the level of ctDNA can be used MiRNAs are a class of non-coding small molecule RNAs
as an independent indicator to judge the prognosis of some with a length of 20–24 nucleotides. Mature miRNAs may
tumors. (3) It can detect specific mutations in tumor patients’ have incomplete pairing with the bases in the 3’-UTR region
tissues and plasma, accurately classify tumors, and guide cli- of the target gene, leading to degradation or translation inhi-
nicians to target treatment. (4) ctDNA test can reflect whether bition of the target gene. MiRNA takes a key part in a variety
the tumor has recurrence and metastasis and whether there is of physiological and pathological processes, such as partici-
minimal residual disease. (5) ctDNA can reflect the informa- pating in regulating ontogenesis, apoptosis, proliferation,
tion of whether anti-cancer treatment is effective and whether differentiation, and other life activities, which are closely
drug resistance occurs, so that clinicians can timely adjust correlated with the occurrence, metastasis, drug resistance,
the treatment plan, reduce expensive ineffective treatment, and other pathological processes of tumors. Most of the
and achieve individualized medication and treatment. miRNAs that promote tumorigenesis are located in the
amplification region of tumor genes, while most of the miR-
NAs that inhibit tumor are located in the region of gene dele-
21.5.4 DNA Methylation in Peripheral Blood tion, indicating that miRNAs play multiple roles in tumor
formation and development. Studies have found that differ-
DNA methylation refers to the process in which organisms ent types of miRNA are up-regulated or down-regulated in
combine a methyl group on the cytosine 5′-carbon site cova- the serum of patients with certain diseases or are specifically
lent bond of cytosine and guanine dinucleotide to form expressed in tissues, such as mir-211 is highly specifically
5-methylcytosine under the catalysis of DNA methyltrans- expressed in the lung, mir-122 is highly specifically
ferase (DNMT) and SAM as the methyl donor. expressed in the liver, and mir-124 abundantly exists in the
Hypermethylation of tumor suppressor genes and hypometh- brain. The miRNA expression profiles of different types of
ylation of specific oncogenes are the main forms of DNA tumors in serum/plasma were also different. As early as
methylation abnormalities, and the main locus of occurrence 2008, domestic and foreign scholars have found that miRNA
is in CpG islands. Because DNA methylation is closely with good stability exists in plasma/serum, which is the main
related to human development and tumor, the overall DNA component of small and medium molecular RNA in plasma/
methylation level of tumor cells is lower than that of normal serum, and the expression of circulating miRNA in normal
cells, but some specific genes, such as tumor suppressor gene people and tumor patients is significantly different. Compared
CpG islands, are in a state of hypermethylation. In tumor with traditional protein biomarkers, miRNA has the follow-
cells, the hypermethylation of tumor suppressor gene pro- ing advantages: low complexity; no post-processing modifi-
moter hinders the expression of tumor suppressor gene, and cation; high affinity “capture” reagents can be synthesized;
the tumor suppressor effect cannot be exerted, leading to the tissue restricted expression profile; and signal amplification
occurrence of tumor. Misawa K and colleagues studied the can be performed. Changes in circulating miRNA expression
methylation status of 11 tumor-related genes in 133 head and profiles have been found in various cancers, and their expres-
neck cancer tissues, and the results showed that gene meth- sion patterns appear to be tissue-specific. Due to their size,
280 W. Zhang et al.
concentration, tissue specificity, and relative stability in cir-

cells. Down-regulated ROR can effectively prohibit the
culation, plasma circulating miRNAs show great potential as growth and lung metastasis of breast cancer cells. Down-
new biomarkers for noninvasive tumors [78]. regulation of lncRNA LET expression is closely correlated
with the metastasis of a variety of tumors in the low-oxygen
environment. LncRNA LET can significantly reduce the
21.5.6 LncRNA invasion and metastasis ability of liver cancer cells in vivo
and in vitro experiments. These abnormal lncRNAs function
LncRNA is a class of non-coding RNA molecules with nonin the occurrence, development, metastasis, and apoptosis of
coding proteins and transcripts longer than 200 nt. Its coding cancer. In a variety of tumor cells, the expression level of
genes are widely distributed in the genome, located in the certain lncRNA will be changed, which may be regarded as
introns or exons of the genes encoding messenger RNA a TM for cancer diagnosis and prognosis.
(mRNA), or between the genes encoding mRNA. To date,
more than 8000 lncRNAs have been identified. LncRNAs
were originally thought to be the “dark matter” in gene tran- 21.5.7 CircRNA
scription, but recent studies have shown their significant role
in regulating cell growth and metabolism. Most of the non- In 1976, the Sanger research team discovered a single-
coding RNAs in the human body are lncRNAs. LncRNA stranded circular closed RNA virus, which did not have 5′ or
expression is time-specific and space-specific, which further 3′ ends, but covalently joined to form a closed circular struc-
confirms that lncRNA expression has a strict regulatory ture, which was called circular RNA [80]. This was the first
mechanism. The aberrant expression of lncRNA is in close time that the concept of circular RNA was proposed. Circular
correlation with the proliferation, apoptosis, invasion, migra- RNA is widely present in eukaryotic cells, as well as in
tion, and drug resistance of tumor cells. LncRNA is a kind of nucleated red blood cells, platelets, and human plasma exo-
new potential biomarkers and targets for cancer treatment. somes. Circular RNA plays a role in regulating miRNA
The expression of lncRNA is abnormal and dysfunction in sponge, gene transcription, RNA binding protein, and pro-
many malignant tumors such as lung cancer, breast cancer, tein translation. CircRNA can regulate gene expression and
liver cancer, and prostate cancer [79]. MALAT-1 is a non- even directly encode proteins at the transcriptional and post-
coding RNA which regulates related gene expression and transcriptional levels, thus affecting the development of
cell movement from the transcription level. The study found tumors [81]. CircRNA can regulate gene expression by spe-
that MALAT-1 will not change the splicing, but regulating a cifically binding miRNA through competitive miRNA ele-
set of transfer related gene expression and promoting lung ments. Compared with ceRNA network composed of
cancer formation. MALAT-1 expression level is involved in lncRNA and mRNA, circRNA network is more complex and
the prognosis of patients. MALAT-1 is expected as a thera- more stable and complete. According to genomic origin and
peutic target for lung cancer. H19 imprinted gene is the first sequence composition, circRNAs can be categorized into the
discovered lncRNA, which is directly induced by the lack of following three groups: exon circRNAs, intron circRNAs
c-myc and p53 genes. It is a powerful oncogene and is stably (ciRNAs), and exon-intron intron circRNAs (EIciRNAs).
expressed in the maternal allele. Abnormal expression of Exons circRNAs are the most abundant circRNAs in vivo,
H19 can be found in various solid tumors. It is associated most of which are produced by coding genes, but they do not
with ER/PR in women and regulated by hif-1, p53, E2F1, encode proteins. Like other long non-coding RNA transcripts,
and other factors, thus affecting the proliferation, invasion, circRNA competitively binds miRNA and down-regulates
and metastasis of breast cancer cells. There exists obvious the expression of EGFR, insulin receptor substrate-1 (irs-1),
difference of HOTAIR transcription in primary tumors and irs-2, and p21-activated kinase-1 (p21-activated kinase-1,
metastases of breast cancer. The abnormal expression of Pak1), P53 and oncogene Raf1, and other important carcino-
HOTAIR 5′ end functional domains binding with PRC2 genic factors. These genes are of great importance in the
induced genome relocation of PRC2 and caused JAM2, occurrence and development of prostate cancer, CRC, mela-
PCDH, EPHA1 gene abnormal expression. These will cause noma, malignant glioma, liver cancer, breast cancer, and
cell adhesion dissociation, promote breast cancer microvas- other cancers. Cirs-7/CDR1as is a widely studied circRNA
cular formation and the invasion and metastasis of breast with more than 70 mir-7 binding sites. Cirs-7/CDR1as pro-
cancer cells, and accelerate the progress of breast cancer. hibits the activity of mir-7 by binding to mir-7 and further
ROR is a lncRNA which was first discovered in induced plu- regulates the expression of mir-7 target genes. Mir-7 is
ripotent stem cells and abnormally highly expressed in breast involved in various biological pathways and can directly act
cancer tissues. ROR inhibits the expression of downstream on the relevant target genes of tumorigenesis to regulate the
miRNA-205 target gene ZEB2 and promotes EMT and expression of tumor factors. CircRNAs play different roles in
increases the migration and invasion ability of breast cancer different cancers. Some circRNAs may be carcinogenic
21 Cancer 281
while others may act in tumor inhibition [82]. In addition, amounts of exosomes secretion. Exosomes secreted by can-
the same circRNAs may have multiple effects. CircRNA is cer cells are closely involved in the occurrence and progres-
rich and conserved, is stable in saliva, blood, and exosomes, sion of cancer. They can promote the proliferation, spread,
and has the specificity of tissue development stage, which is metastasis, and deterioration of cancer cells and help cancer
a promising new biomarker and therapeutic target for cancer cells escape from the clearance mechanisms in the body or
treatment. in vitro. For example, exosomes produced by glioma cells
can accelerate their proliferation. Gu and colleagues found
that mesenchymal stem cells could release exosomes to
21.5.8 Exosome advance the growth of gastric cancer cells [85]. These results
suggest that exosomes can promote carcinogenesis and
Exosome is utricle bubble secreted by cells, a diameter of development. It almost exists in all body fluids in the human
30–200 nm double lipid package structure, with maternal body and is an important part of the liquid biopsy. It has the
cells biological information characteristic molecules, such as following three main advantages: (1) its level is higher than
protein, lipid, DNA, mRNA, microRNAs, and non-coding the CTC in body fluids and more sensitive than the
RNA biological activity [83]. They can be absorbed by CTC. There’re more than 10 exosomes in per milliliter blood;
receptor cells and implement the material transport and (2) it has a lipid bilayer structure, which can protect the inter-
information transmission between cells. Exosomes secreted nal nucleic acid molecules from degradation; (3) due to its
by different cells have common biological molecules, such small size and strong permeability, it is easy to enter body
as TSG101, CD9, CD63, FASN, and VCP proteins. These fluids and thus be detected. Fully recognizing and utilizing
common protein molecules can be used as biomarkers for the the characteristics and advantages of exosomes, screening
evaluation of exosome quality and purity. Exosomes cannot and finding new markers is expected to overcome the prob-
be simply understood as vesicle structures secreted by cells lems of tumor heterogeneity, which will have important clin-
directly into the external environment. Their formation pro- ical significance in early diagnosis, treatment monitoring,
cess has complex regulation, which can be divided into three and prognosis evaluation of tumors. In addition, exosomes
stages: cell membrane invasion to form intracellular bodies, are expected to be used as drug carriers and may have great
intracellular polyvesication, and fusion of polyvesicles with potential in tumor therapy.
plasma membrane to release exosomes. Before, it was
believed that the role of exosomes was only to transport met-
abolic wastes from inside cells to outside cells. Until 1996, 21.5.9 Endosome
Raposo and colleagues [84] found that exosomes could
deliver antigens to stimulate T cell proliferation. Gradually, Endosome refers to a membrane-bound organelle in eukary-
it is found that exosomes also have certain physiological otic cells, belonging to a vesicular structure. As a compart-
functions and are involved in immune regulation. B lympho- ment of the transport pathway in endocytosis, the endosome
cytes release antigen-presenting protein-carrying exosomes is transferred from the cytoplasmic membrane to the lyso-
to induce T lymphocyte immune response, leading to the some for degradation or recycling back to the cytoplasmic
occurrence and development of some diseases, such as neu- membrane [86]. As a hub for the transportation of vesicles,
rodegenerative disease Alzheimer’s disease. Exosomes pro- endosomes engage in a variety of vital activities including
mote virus infection and transmission. For example, HIV cell metabolism, the release of neurotransmitters, hormone
virus can find the optimal cells through exosomes for infec- secretion, natural immunity, and the sorting of sperm egg
tion and transmission. Exosomes are also significant in can- combination. They are known as sorting centers or sorting
cer. Cancer cells tend to release exosomes higher than normal machines. Transportation disorders after endosomal sorting
cells. EpCAM positive exosomes in the blood of ovarian can lead to defects of various organelles, which are corre-
cancer patients are considerably higher than that of normal lated with the occurrence and development of diabetes,
people and patients with benign ovarian diseases. Compared infection and immune deficiency, neurodegenerative dis-
with the low metastatic cell lines, the let-7 miRNA family eases, schizophrenia, infertility, cancer, and other diseases.
was much more abundant in the exosomes secreted by the Endosomes can be divided into early endosome, late endo-
ovarian cancer high metastatic cell lines, while the members some, and recycling endosome according to the different
of the mir-200 family were only present in the exosomes time stages of endocytosis, and they can be distinguished by
secreted by the low metastatic cells. Cancer cells secreting protein markers such as GTP binding protein rabs, and the
large amounts of exosomes may be caused by mutations in three endosomes are also different in morphology. Once ves-
some proto-oncogenes or tumor suppressor genes which lead icles in endocytosis are released, they first fuse with the pri-
to increased exosome production or secretion, and low pH mary endosomes, then grow into the secondary endosomes,
microenvironment around cancer cells can also induce large and fuse with the lysosomes. Many viruses enter the cell
282 W. Zhang et al.
through this way. Take HIV virus as an example; the virus

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Translation Research of Novel
Biomarker 22
Shiyang Pan and Yuexinzi Jin
22.1 Overview 22.2 Discovery
A biomarker has been defined as “an indicator that can be 22.2.1 Characteristics of Molecular Biomarkers
objectively measured and evaluated of a physiological as well
as a pathological processes or pharmacologic responses to a Large amounts of molecular substances related to diseases can
therapeutic intervention.” Millions of biological molecules are appear in the process of disease development. The most critical
related to diseases, but there are only thousands of molecules feature of a good biomarker is that it can reflect the state of a
that can be used as biomarkers in clinical practice. With the disease or the process of a disease, and it needs to contain rich
increasing demand of clinical medicine development and the and comprehensive disease state information of a certain dis-
rapid development of modern molecular detection and analysis ease [1]. A biomarker with high quality needs to meet the fol-
technology, various novel molecular targets of diseases have lowing criteria: (1) specimen is easy to obtain and minimally
emerged. There is a certain rule from the discovery of novel invasive such as blood or urine; (2) with high sensitivity and
biomarkers to the clinical application. At the same time, it is specificity to realize early diagnosis and differential diagnosis;
necessary to follow certain principles to establish new experi- (3) reflect the progress of the disease or the effect of treatment
mental diagnostic technology by using novel biomarkers, and and intervention promptly; (4) contribute to deeper understand-
carry out demonstration procedures in terms of relevant molec- ing of the molecular mechanism of the occurrence and devel-
ular targets, detection and analysis technology, and ethics in opment of disease; and (5) provide certain value for risk factor
accordance with standard and legal approaches. A novel bio- stratification and prognosis evaluation of the disease.
marker usually needs to go through the following processes of
translation research procedures from scientific research to
clinical application, including discovery, qualification, verifica- 22.2.2 Biomarker Classification and Samples
tion, assay optimization, and clinical validation.
In this chapter, we first propose the concept of using active From the relationship between molecular markers and dis-
antigen groups to prepare monoclonal antibody library to eases, biomarkers could be divided into the following cate-
screen and isolate active antigens, so as to further screen and gories. One is the pathogenic molecule of diseases, which
identify novel biomarkers, which has unique advantages in plays a crucial role in the occurrence and development of
the process of novel biomarker discovery and development. diseases. Such markers are not only diagnostic markers but
also can provide targets for clinical treatment, which is the
most ideal type of markers. Secondly, molecules produced
due to the concomitant effects of diseases, which have no
decisive effect on the diseases themselves, but only have cer-
tain correlation with the process of diseases, and can hardly
be used as molecular targets for diagnosis.
During the occurrence and development of diseases,
abnormal molecules can be produced at the gene level, tran-
S. Pan (*) · Y. Jin scription level, and protein level. The molecular mechanism
Department of Laboratory Medicine, The First Affiliated Hospital of diseases mainly includes changes in gene structure, gene
of Nanjing Medical University, Nanjing, Jiangsu, expression caused by cell regulatory factors or other factors,
e-mail: sypan@njmu.edu.cn
foreign pathogenic genes, post-translation processing, and

286 S. Pan and Y. Jin
degradation of proteins. Since proteins are the main mole- disease. Given that most biological processes are controlled
cules performing life functions, errors in the transcription, by proteins, which could be quantified efficiently with high
translation, degradation, and interaction of various molecules sensitivity, there is no doubt that proteins have developed to
related to protein synthesis can lead to the occurrence of dis- be the most potential biomarkers.
eases. Briefly, biomarkers can be basically divided into gene,
protein, carbohydrate, and lipid (Table 22.1).
The National Cancer Institute (NCI) defines a biological 22.2.3 Existing Detection Techniques
specimen as blood, urine, or other biological sample that is for Clinical Application
available for diagnosis and analysis. Samples include every-
thing from subcellular structure (DNA, RNA, protein) to a cell, Biomarker detection techniques for clinical application are
tissue (muscle, bone, skin), organs (liver, lung, kidney, bladder), shown in Table 22.2.
blood, gametes (sperm and eggs), embryonic and fetal tissue,
urine, feces, and others (sweat, hair, nails, loss of epithelial cells,
the placenta). Standards for biological sample database of China
Table 22.2 Detection techniques for clinical application
medical biotechnology association (trial) define a sample as
Types Technology Application
“any biological substance containing biological information of Gene Northern blotting, NB RNA qualitative analysis
the human body, including human tissues, blood, secretions, Southern blotting, SB DNA qualitative analysis
excretions and their derivatives.” In situ hybridization, DNA, RNA qualitative analysis
Biomarkers are indicators that can objectively measure ISH
and evaluate biological process, pathological process, or Electron microscopy DNA, RNA qualitative analysis
response to drug intervention and play an important role in hybridization in situ,
EM-ISH
disease diagnosis, treatment, and efficacy monitoring [2]. In
Polymerase chain DNA quantitative analysis
addition to the classic markers of proteins and nucleic acids reaction, PCR
in the blood, biomarkers are increasingly found in a variety Protein Radioimmunoassay, Hormone, tumor marker,
of body fluids (such as saliva, sweat, urine, etc.). RIA receptor quantitative analysis
Molecules such as DNA, RNA, proteins, peptides, and Radioimmunoimage, Tumor localization diagnosis
RII
metabolites can all become biomarkers. However, due to the
Immunofluorescence Localization of antigens and
complex regulatory mechanism of gene expression, the final assay, IFA antibodies in tissues or cells
product of the same gene may be different, the phenotype of Immunohistochemistry, Antigen localization,
an organism may be the same, and similarly, different pheno- IHC qualitative and quantitative
types may have the same gene sequence. Therefore, markers study in tissue
at the gene level may not accurately reflect the progression of Enzyme linked Detect antigens, antibodies
immunosorbent assay, quantitative analysis
ELISA
Western blotting, WB Analyze protein qualitatively
Table 22.1 Classification of biomarkers
and semi-quantitatively
Classification Samples Application Flow cytometry, FCM Single cell quantitative analysis
Gene DNA Nuclear cells, BRCA1 and sorting
pathological mutations Chemiluminescence Antigens, haptens, antibodies,
DNA cells, body APC immunoassay, CLIA hormones, enzymes, fatty
methylation fluids (blood, methylation acids, vitamins, and drugs
RNA saliva) miR-638 quantitative analysis
mRNA miR-9 Electroche Antigens, haptens, antibodies,
Non-coding LncRNA miluminescence hormones, enzymes, fatty
RNA RHPN1-AS1 immunoassay, ECLIA acids, vitamins, and drugs
Protein Plasma proteins Body fluids AFP, PSA quantitative analysis
Tissue proteins (urine, blood, Microparticle enzyme Antigens, haptens, antibodies
saliva), tissues immunoassay, MEIA quantitative analysis
Carbohydrate Glycosylation Body fluids CA-125, Time-resolved Hormones, viral hepatitis
(urine, blood, CA-153 immunofluorescence marker, tumor antigen,
saliva), tissues assay, TRFIA and drugs
Lipid Fats, lipids Body fluids LDL, HDL Qualitative analysis
(urine, blood, Immunoelectron Protein localization
saliva), tissues microscopy, IEM
22 Translation Research of Novel Biomarker 287
22.2.4 Biomarker Discovery Approaches High-throughput and large-scale microarray techniques

in transcriptome identify differentially expressed genes
22.2.4.1 Strategy of Biomarker Discovery between diseased populations and healthy controls through
studies of RNA transcripts. Nevertheless, RNA sequencing
Research Subjects (RNA-seq) has gradually replaced microarray technology
During the discovery phase, potential candidate biomarkers due to its higher resolution and reproducibility and the abil-
could be screened identified by testing and analyzing sam- ity to discover alternative splicing events, novel genes and
ples which derive from cell lines, animal models, patients transcripts, and fusion transcripts. RNA-seq workflow rou-
undergoing clinical trials, or an established biobank [3]. The tinely consists five steps as shown in Fig. 22.1 [6].
samples are preferred from large prospective case-control The purpose of proteomics is to make a list of all proteins,
studies involving a group of patients or individuals with or understand the structure and function of these proteins and
without a specific disease, and a comprehensive collection of the interactions within the protein population, and compare
clinical diagnosis, treatment, and outcome information the changes in their expression levels in disease and health
should be included. In order to obtain reliable and repeatable conditions. Proteomics research includes protein level (“top
screening results for candidate markers, the collection, stor- down”) and polypeptide level (“bottom up”); the former is an
age, and processing of samples need to follow standardized ideal proteomics research model, but it is difficult to achieve
protocols and analytical methods [4]. high throughput due to technical difficulties. The latter is
relatively mature and is the mainstream of proteomics
Omics Technologies research [7]. The basic procedures of proteomics research at
In the past few decades, high-throughput technologies such the protein level are protein extraction, protein separation,
as omics have been developing rapidly. Genomics deals with and mass spectrometry identification (Fig. 22.2). The classi-
the whole or part of the genome. Transcriptomics is applied cal analysis methods based on 2D-PAGE include (1) sample
to study transcription and regulation of all genes in cells at a preparation and quantification (extraction of proteins from
holistic level. Proteomics focuses on the full set or most of the control group and various experimental groups); (2) pro-
proteins in cells, tissues, or organisms. Epigenomics investi- tein separation (protein staining after 2D-PAGE separation);
gates covalent modifications that change activity of genetic and (3) qualitative and quantitative analysis of proteins
materials without altering DNA sequences, which include (compared with the control group, the difference points in
DNA methylation and histone modification. Metabolomics the experimental group are analyzed, and the difference
analyzes the complete set of amino acids, lipids, sugars, and point proteins are identified using mass spectrometry, and
other low molecular weight metabolites. Glycomics is a the changes in their expression levels are analyzed by
comprehensive approach to explore the structural and func- software).
tional relationships of complex carbohydrates and lipomics, Metabolomics have a large range of applications
which is a new subject for systematic analysis of whole lip- because metabolites are closely related to phenotypes. The
ids by comparing the changes of lipid metabolism network experimental steps of metabolomics can be divided into
under different physiological conditions. The representative the following four stages including sample collection and
techniques for each omics are listed in Table 22.3. preparation, metabolites separation and detection, data
Data from omics technologies are analyzed to generate mining and extraction, and data analysis and interpretation
particular biomarkers which are related to the occurrence (Fig. 22.3) [8].
and development of the disease or provide information on
prognosis or therapeutic effects. Genomics have been applied 22.2.4.2 Biomarker Function Identification
to study the profile of genetic risk factors and mutations
associated with a disease. On the one hand, genome-wide Role Transformation of P53
association studies (GWAS) identify genetic risk factors by The question is that: can differentially expressed molecules
comparing the distribution of common mutations in the screened by various techniques be considered as markers of
genome of a large amount of patients and controls. On the diseases? The answer is no. Take the tumor suppressor gene
other hand, next generation sequencing (NGS) could provide p53 as an example, which is of the highest correlation with
mutation profiles which are known to be involved in a par- human tumor so far. However, it had been widely believed
ticular clinical context [5]. that p53 was a biomarker of cancer since mid-1980s,
Table 22.3 Detection techniques for biomarker research

Types Technology Application
Genomics GeXP Quantitative analysis of gene expression profile
SNP stream Single nucleotide polymorphism analysis
Sequenom mass array Single nucleotide polymorphism analysis
Biological barcode detection technology DNA
High-throughput sequencing DNA
Proteomics Two-dimensional gel electrophoresis Protein identification, protein isolation
Isoelectric focusing gel electrophoresis Protein identification, protein isolation
Mass spectrometry Protein identification
Affinity chromatography Protein isolation, pure protein
Chromatin immunoprecipitation Regulation of gene expression at chromatin level
Genome-wide mapping technique, GMAT Distribution of histone modifications throughout the genome
Nucleosome phase analysis Changes in nucleosome localization
Detection of chromatin DNA enzyme sensitive sites Transcriptional genes and trans-acting factor action sites
Bisulfite genomic DNA sequencing DNA methylation
Methylation specific PCR DNA methylation
Restriction landmark genome scanning capture, RLGS DNA methylation
Chromosome conformation capture, 3C Determine the frequency of contact between any two genomic
loci
Electrophoretic mobility shift assay, EMSA Analyze DNA binding proteins in vitro
Methylation sensitive specific restriction footprint, Detect the methylation status of the target fragment and genome
MSRF
Metabonomics Nuclear magnetic resonance, NMR Identification and analysis of metabolites
Gas chromatography mass Spectrum, GC-MS Identification and analysis of metabolites
Liquid chromatography-mass spectrometry, HPLC-MS Identification and analysis of metabolites
Capillary electrophoresis mass spectrometry, CE-MS Identification and analysis of metabolites
Glycomics Sugar capture technique Analyze sugar structure
Frontier affinity chromatography Analyze sugar structure
Two-phase gel electrophoresis combines fluorescence Analyze the sugar conformation
staining and mass spectrometry
Capillary electrophoresis mass spectrometry, CE-MS Analyze the sugar conformation
Nuclear magnetic resonance, NMR Point the exact sites of interaction with proteins in the sugar
chain, confirm the epitope, and detect the conformation of the
sugar chain
Microarray technology Identify the interactions between sugars and proteins
Lipidomics Electrospray ionization mass spectrometry, ESI-MS Target lipid omics analysis for a class of lipid compounds
Gas chromatography mass Spectrum, GC-MS Separation and analysis of lipid molecules
Liquid chromatography-mass spectrometry, HPLC-MS Separation and analysis of lipid molecules
Multidimensional mass spectrometry based shotgun Target lipid omics analysis for a class of lipid compounds
lipidomics, MDMS-SL
Mass spectrometry imaging, MSI Spatial distribution of lipid molecules
Nuclear magnetic resonance, NMR Identification and quantitative analysis of lipid molecules
Raman spectroscopy Identification and quantitative analysis of lipid molecules
because it was found that many tumor cells expressed p53 in clones could show cell transformation activity in experi-
large quantities, but not in normal tissue cells. It took sev- ments. However, overexpression of wild-type p53 gene in
eral years for a new understanding of p53 to emerge, not as cells not only fails to transform cells, but can effectively
an oncogene but as an oncosuppressor [9]. Important evi- inhibit the transformation effect of oncogenes such as MYC
dence was that the researchers compared p53 gene sequences gene and HRAS gene on cells. So the wild type p53 gene is
obtained from mouse tumor cell lines with wild-type Trp53 tumor suppressive. The function of p53 is still under further
gene sequences obtained from normal mouse tissues, and exploration. A latest study comparing missense and com-
found that p53 gene clones obtained from mouse tumor cell plete deletion mutations in p53 with the normal p53 gene
lines were usually mutated clones, and only those mutated found that missense mutations, similar to complete muta-
Fig. 22.1 RNA-seq data analysis workflow
tions, could inhibit cell apoptosis due to the loss of DNA Current Novel Tumor Biomarkers
binding and transcriptional catalytic ability of the mutated
protein. It further indicates that p53 mutation itself has no Human Epididymis Protein 4 (HE4)
pro-cancer activity, but can interfere with the anti-cancer The discovery and validation of novel biomarkers for dis-
activity of normal p53 and promote the development of eases is currently under intense research. In the field of can-
tumor [10]. Thus, the differences in proteomic patterns cer, the reality is that only a few novel biomarkers have been
observed between healthy controls and cancer patients may applied in clinic in the last decade [11]. As a putative serum
be high-abundance molecules which are not expressed or tumor marker for ovarian cancer, human epididymis protein
secreted by the tumor cells but rather represent epiphenom- 4 (HE4) was previously identified to be overexpressed in
ena of tumor presence. ovarian carcinomas by gene-expression profiling study in
Protein Bottom up Top down

extraction Extract cellular proteins
Fractionate
2D-gel Shotgun digest
Protein
isolation
Separate
MS/MS
Digest −100%
MS/MS X X
Mass spectrum X
identification Fragment ions
X X
5-90%
X X
N C
Fig. 22.2 The basic procedures of proteomics research
Data preprocessing
V1 V2 V3 V4
alansf 0.009 0.008 0.343 0.280
bsnsj 0.233 V1 V2 V3 V4
Data acquisition
cnfkd 0.343 alansf 0.009 0.008 0.343 0.280
Sample
preparation dafbfj 0.427 bsnsj 0.233 0.875 0.043 0.948
cnfkd 0.343 0.932 0.282 0.945
Sample dafbfj 0.427 0.821 0.936 0.924

collection NMR spectroscopy
Peak alignment; data normalization; signal extracting;
MS-based techniques
baseline correction; integration
Multivariate statistical
analyses
Pathway analysis
8 Control group
Biomarkers biological identification of
Possible biomarkers 7
interpretation discriminative variables 6
5
4
3
diseased
2
Biomarker validation 1
group
1000 2000 3000 4000 5000 6000 7000 8000
Clinical application
Fig. 22.3 The flow chart of metabolite biomarker discovery based on metabolomics
1999. HE4 has been proved to have high sensitivity and new frameworks for deep neural networks (DNNs) imple-
specificity in the diagnosis of ovarian cancer. For I/II stage of mentation. The application of AI in biomarker development
ovarian cancer, its diagnostic sensitivity and specificity was was first published in 2016. The researchers screened the
62.4% and 96%; for III stage of ovarian cancer, the sensitiv- blood samples and identified top 5 important biomarkers for
ity and specificity was 74.6% and 96% [12]. Despite its diag- human chronological age prediction with multimodal data
nostic potential, HE4 has been only FDA-approved to integration, which may lead to uncomplicated, minimally
monitor the progression or recurrence of ovarian cancer and invasive, and economical approaches to track integrated bio-
should be used in combination with other clinical approaches. markers of aging in humans and perform cross-species fea-
The reasons for the limitation of its application may involve ture importance analysis [14]. This research opens the way
that it is still unclear whether HE4 is a decisive factor for for the application of AI in biomarker development field, but
ovarian cancer; there is no definitive cut-off value available; is limited to screening existing biomarkers. In the future, one
as well as tissue type specificity, patient age, premenopausal, of the most promising branches for AI to be applied in bio-
and other issues, especially early diagnosis, still need to medicine field could be novel biomarker discovery. With the
solve the application range of these indicators through a join of AI, the process of research and development of novel
large number of clinical validation data [13]. Since individu- biomarkers may be greatly shortened, and the efficiency
als with non-malignant diseases may also have elevated HE4 could be significantly improved.
levels, the level of HE4 concentration cannot be used as cru- Since the beginning of the whole human genome project,
cial evidence for the determination of malignancy and is not the use of big data to search for biomarkers in diseases has
suitable for cancer screening. been rapidly developed and popularized. Biological data
mainly include genomics data, transcriptome data, proteomics
Pro-Gastrin-Releasing Peptide (ProGRP) data, epigenomics data, and metabolomics data. These data
Pro-gastrin-releasing peptide (ProGRP) is precursor of gastrin- bring great convenience to the research. They are mainly
releasing peptide (GRP), which is a 27-amino-acid peptide stored in the database such as the Cancer Genome Atlas
homologous to the C-terminal of bombesin isolated from por- (TCGA) and Gene Expression Omnibus (GEO) related to
cine stomach and may act as an autocrine growth factor of human tumors. As an interdisciplinary research, medical big
small cell lung cancer (SCLC) cells and promote rapid tumor data still has some defects. For example, (1) medical big data
growth. The diagnostic performance of ProGRP in SCLC is research is difficult to be carried out in general research insti-
superior to NSE, and it has become one of the most reliable tutions due to the problems of medical data acquisition and
markers of SCLC. The sensitivity is 60–70% in the limited information silos caused by the data control system of medical
period diagnosis of SCLC and 75–90% in the extensive period. system; (2) rationalization of data storage structure (with the
Therefore, the molecules playing key roles in the disease, increasing diversification of medical big data sources, the
rather than the concomitant effects of the disease, have the existing storage architecture can no longer meet the require-
potential to become the best molecular biomarkers. ment of big data application); (3) precision and intelligence of
data retrieval (intelligent integrated hardware equipment based
Protein Induced by Vitamin K Absence/Antagonist-II on the data platform await to be widely applied); (4) database
(PIVKA-II) security (how to desensitize and store critical data such as
Apart from HE4 and ProGRP, PIVKA-II (also named Des- name, position, work unit, phone, and other sensitive informa-
gamma-carboxy prothrombin, DCP) are the most promising tion securely); and (5) lack of policy (effective management of
novel tumor markers in recent years, which could be helpful medical big data needs system guarantee).
in early diagnosis and prognostic monitoring of hepatocel-
lular carcinoma (HCC). However, the diagnostic perfor-
mance of PIVKA-II for HCC varies greatly among different 22.2.6 Monoclonal Antibody Library
detection platforms, and there is no authoritative cut-off Technique
value at present. As a result, it is still unable to replace classic
liver cancer markers such as AFP. With the development of highly powerful omics technolo-
gies, a series of tumor antigen screening technologies has
been established, including recombinant cDNA expression
22.2.5 Artificial Intelligence and Big Data libraries (SEREX) technology, serological proteome analy-
sis (SERPA), as well as protein microarray technology [15].
The concepts of artificial intelligence (AI) and deep learning However, the most inevitable drawback of the strategy of
have attracted extensive attention in various fields. The establishing protein expression library through molecular
unprecedented progress in this area benefits from the accel- cloning and surface display technology of these technologies
erated development of high-performance computing and is that protein molecules expressed by surface display tech-
Fig. 22.4 Schematic diagram of using active antigen group to develop monoclonal antibody library for screening novel tumor biomarkers
nology have structures and functions that are not naturally to obtain a set of monoclonal antibody library which are suc-
present in the human body [15, 16], leading to low screening cessively labeled so as to identify and isolate these active anti-
positive rate and a large number of identified antigens have gens they recognize, which could be further investigated
no general significance for clinical diagnosis. about their characteristics and functions to discover novel
disease biomarkers. Compared with high-throughput tech-
22.2.6.1 Usage of Monoclonal Antibody Library niques such as mass spectrometry, this technique has a sig-
nificant advantage in using monoclonal antibodies to
Research on Diagnostic Tumor Biomarker recognize and isolate antigens and maintain their original
Kohler and Milstein created the mouse hybridoma technique activity in physiological state. Take screening for tumor asso-
that produced monoclonal antibodies in 1975, opening up a ciated antigens as an example (Fig. 22.4). Firstly, tumor tis-
new era in clinical immunology. In the 1980s, Prof. Robert sues or tumor cells containing a group of active antigens are
C. Bast Jr. cultured the cancer tissues of a patient with ovarian used to immunize animals, and monoclonal antibodies against
serous papillary cystadenocarcinoma in vitro and obtained this group of active antigens are generated in the animal.
166 monoclonal antibodies with hybridoma technique, which Subsequently, techniques such as hybridoma are used to
were successively numbered OC1-OC166. Screening establish monoclonal antibody library, and these antibodies
revealed that antibody No. 125 (OC125, Ovarian Cancer 125) are successively labeled. Each monoclonal antibody corre-
had a high sensitivity and specificity to ovarian cancer cells, sponds to a specific antigen epitope. These monoclonal anti-
making it an ideal monoclonal antibody to detect ovarian can- bodies contain all antigen epitopes, which are further screened
cer. Subsequently, Bast named the cell-surface material iden- and classified into three main categories, including antibodies
tified by OC125 as oncoantigen 125 (CA125) [17]. Up till that only recognize tumor cells, antibodies that only recog-
now, CA125 is still of high diagnostic value for gynecological nize normal cells, as well as that recognize both these compo-
diseases such as epithelial ovarian cancer, cervical adenocar- nents. The identified antigens are isolated with monoclonal
cinoma, and stromal cell cancer. This classic case indicates antibodies to conduct further studies on the characteristics
the important role of monoclonal antibody library technology and functions of the antigens to discover novel biomarkers.
in the discovery of novel biomarkers. Translational medical research using large amount of samples
The main concept of monoclonal antibody library tech- from patients and healthy controls should be carried out to
nique is to immunize animals with groups of active antigens validate the diagnostic performance of the novel biomarker.
Antigen
Cell-culture (Tumor cells)
myeloma line
myeloma line
Tissue microarry
Spleen cells
fuse in
polyethylene glycol
select & grow select cells making
hybrid cells desired antibody
NJ001
propagate Affinity chromatography
clones SP70
ELISA
McAbs library Fast screen platform Protein purification system
Fig. 22.5 Discovery of SP70
Following the above process, a home-made tumor mono- tumor antigens. Monoclonal antibody library provides a
clonal antibody library was established by Prof. Shiyang valid method for the identification of malignant cells and
Pan, from which a monoclonal antibody, named NJ001, was their corresponding normal tissue components, and in this
found and could react to non-small cell lung cancer (NSCLC) way, molecular biomarkers with high tumor specificity could
cells. The recognized antigen of NJ001 is a protein with the be found, and their monoclonal antibodies with high affinity
relative molecular mass (Mr) of 70 kDa designated tumor and specificity make it possible for clinical diagnosis and
specific protein 70 (SP70) (Fig. 22.5). SP70 is currently immunotherapy.
being validated for its sensitivity and specificity in patients
with NSCLC and has been proved to have clinical signifi- Advantages of the Monoclonal Antibody Library
cance in cancer auxiliary diagnosis, therapy efficacy moni- Monoclonal antibody library is also big data. Labeling all the
toring, and prognostic prediction [18]. antibodies in the monoclonal antibody library is equivalent
Hybridoma technique is traditional to establish monoclo- to labeling all the disease-induced antigens they recognize.
nal antibody library. Modern technology can directly obtain B By screening antibodies and finding corresponding antigens
lymphocytes in the spleen of immunized animals to establish for subsequent verification studies and multi-center clinical
monoclonal antibody gene pool, and it oriented monoclonal trials, the efficiency of screening new markers is greatly
antibody library to develop novel biomarkers. The advantage improved. Although most of the current studies screen out
of this technology lies in the screening of antibody genes by various new markers through various omics technologies,
high-throughput technology and in vitro recombination, the problem of data island exists in most of the studies for a
which can directly humanize the monoclonal antibody and variety of factors, for instance, limited sample size involved
overcome the deficiency of immunogenicity of heterogenic and differences in sample processing methods. At the same
antibodies obtained by hybridoma technology, so that it can time, the antigens in the natural state of the body cannot be
be used not only for detection but also for treatment. obtained by mass spectrometry and other technologies, so
that a large number of antibodies prepared against the
Cancer Target Therapy screened differentially expressed proteins cannot detect the
Human monoclonal antibodies against various special types original antigens, resulting in the denial of these novel bio-
of human tumor cell membrane phenotypic antigens, such as markers in the verification stage. Monoclonal antibody
PD-1/PD-L1, have opened up a new way for clinical immu- library technology, on the other hand, combines the advan-
nodiagnosis and immunotherapy [19]. Tumor immunologists tages of big data characteristics, high accuracy and strong
have freed themselves from the limitations of conventional authenticity, playing a fatal role in the development of novel
serums and taken a big step toward revealing the nature of biomarkers.
22.2.7 Transformation Determinants 22.3.1 Samples from Clinical Cohort
It usually takes more than 20 years for a novel molecular A few representative cases and corresponding controls in a
biomarker from discovery to clinical practice. Several fac- cohort are applied in the discovery stage, and the screened
tors determine the clinical practicability of novel biomark- biomarkers will be detected in the same samples in the quali-
ers. Firstly, it depends on the degree of disease information fication phase. At the verification stage, an independent,
contained in the novel biomarker and its association with larger number of similar samples which come from multiple
the disease, that is, whether the marker is the cause of the clinical research centers should be involved. The case defini-
disease or an accompanying result. If a novel biomarker tion criteria adopted at this stage should be reproducible and
can be used both as a diagnostic target and as a treatment observer-independent.
target, this kind of marker usually has close relationship In addition, in the process of uniform sample collection and
with the onset of disease and is an important factor for dis- storage, great attention should be paid to details. It is increas-
ease occurrence. This biomarker is of great significance, ingly recognized that various schemes in sample collection,
not only for the study of the pathogenesis of the disease, storage, and processing could have a significant impact on the
but also for the clinical diagnosis and intervention of the different biomarker development phases. To overcome this
disease. problem, multicenter clinical studies should be conducted, and
Secondly, it depends on the molecular stability of bio- standard operating procedures (SOPs) for sample collection/
markers which includes molecular mass and half-life, and archiving should be developed and strictly followed. Despite of
the concentration of the molecule in biological samples. A the technical limitations of quality assessment and control of
biomarker with shorter half-life may be less reliable than proteomic samples, the quality of samples is recommended to
those with longer half-life. The concentration of the mole- be evaluated before qualification and verification analysis.
cule in the sample should be within the detection range of the The included sample size should be sufficiently powerful
existing detection technology platform. to evaluate the specificity and sensitivity of the potential bio-
Thirdly, it depends on the availability of appropriate tech- markers at verification stage. Several factors determine the
niques for clinical detection. Whether the novel biomarker sample size, which includes the biological individual differ-
can be applied in clinic not only depends on its diagnostic ence of patients, analytical variation of the assays, concen-
value to the disease, but also the clinical practicability of the tration variation of the molecule in different samples, as well
research approach. Some research techniques have limita- as the different abundance of the biomarkers between the
tions in their clinical application due to their high cost and case and control group. These factors need to be taken into
low cost performance ratio. Currently, although large consideration when statistically designing for sample size in
amounts of novel biomarkers have been discovered by using verification studies [20].
chip and mass spectrometry, the identification and clinical
application research and promotion of these markers will
take longer and have a lower success rate. Therefore, the 22.3.2 Platforms for Qualification/Verification
simpler the detection technology is, the easier to be applied
in clinical practice. 22.3.2.1 E nzyme-Linked Immunosorbent Assay
(ELISA)
ELISA has extraordinary sensitivity (low pg/mL) [21],
which has been extensively applied in verification of bio-
22.3 Qualification and Verification markers. With high sample throughput, this technique can
analyze hundreds of samples with high precision. However,
To isolate real biomarkers from a mass of proteins filtrated only a few potential biomarkers have immunoassay-grade
from the discovery phase has become the most challenging antibody available. ELISA assay is characterized as time-
bottleneck. The suitability or appropriateness of biomarkers and cost consuming and has high failure rate of clinical
should be discussed in the following qualification phase. For transformation. Because of the possibility of cross-reactions
example, if a specific nucleic acid (or protein) marker found between antibodies, it is hard for ELISA to quantify numer-
in diseased cells can be detected in a patient’s blood or body ous protein targets [22].
fluid by established methods, it can be determined whether it
is a suitable molecular marker for clinical testing. Only when 22.3.2.2 Mass Spectrometry
this point is satisfied, the molecular marker may be used for In recent years, many targeted mass spectrometry (MS)
further verification. methods have been developed, for example, accurate
Fig. 22.6 Comparison of advantages and disadvantages of target MS approaches used in biomarker verification
inclusion mass screening (AIMS), selected reaction moni- observed changes of candidate biomarkers derived from tri-
toring (SRM), parallel reaction monitoring (PRM), and age step. Besides, this step will also evaluate whether candi-
data-independent acquisition (DIA-MS/MS), coupled with date proteins in the clinical samples could be quantified
a quantitative, digitalized proteomic recording (SWATH directly, or additional sample fractionation or enrichment is
maps). They have great potential for the specificity, repeat- required.
ability, and quantitative measurement of protein changes. In the second step of quantification, based on the concen-
Among these approaches, SRM has been most widespread tration of candidate protein biomarkers in clinical samples,
used in qualification and verification of biomarkers. The they can be divided into several groups: extremely low abun-
advantages and disadvantages of these technical platforms dance proteins, medium-low proteins, and classic plasma
applied in biomarker verification are shown in Fig. 22.6 [23]. proteins. For proteins with extremely low abundance includ-
ing cytokine, ELISA is the preferred assay because of its
22.3.2.3 Selection of Assays extraordinary sensitivity, while SID-SRM is the priority for
Qualification and verification of biomarkers could be com- quantification of medium-low proteins as well as classic
posed by three steps: triage, quantification, and verification plasma proteins (Fig. 22.7). SRM could directly extract clas-
based on the number of candidate biomarkers, the abundance sical plasma proteins from the samples without prior frac-
of biomarker in the samples, the feasibility of implementing tionation. For medium and low abundance proteins, sample
novel assays, as well as the analytical effectiveness and fractionation or enrichment is required first to quantify can-
development cost of the assays [24]. didate proteins.
In the triage step, the relative abundance changes of target The objectives of verification step are threefold. Firstly, it
protein were semi-quantitatively estimated. Additional should be confirmed that the few biomarkers screened out by
quantification with SIDSRM-MS is required to confirm the the triage procedure can objectively reflect the occurrence,
Fig. 22.7 Comparison of the lower limit of quantification (LLOQ) of different quantification approaches for plasma protein biomarkers
severity, or outcome of the disease. Secondly, the specificity required to develop suitable corresponding antibodies to
and sensitivity of biomarkers should be established to deter- quantify each candidate biomarker.
mine their intended use. Thirdly, if the biomarker is appli-
cable, the appropriate sample fraction or enrichment methods
should be implemented [23]. 22.4.1 Development of Clinical Diagnostic
Monoclonal Antibodies
22.4 Assay Optimization Immunological techniques such as ELISA and radioimmu-

noassay (RIA) are readily available in clinical laboratory,
A list of screened candidate biomarkers in verification phase which are more sensitive than those advanced non-
has been confirmed to be differentially expressed between immunological technologies including LC-MS/MS [25].
disease and normal states. In the following stage of valida- Sandwich reactions are formed by identifying different epit-
tion and assay development and optimization, the single- opes of proteins with two antibodies, one for capture and the
digit coefficient of variation (CVs) requires to be measured other for detection. Both monoclonal and polyclonal anti-
in thousands of samples from patients. Since MS is not yet bodies could be used and could be produced using the puri-
capable of achieving an integration of high throughput and fied, synthesized, or recombinant protein. Besides, the
high detection precision and accuracy, it has not been exten- specificity of the antibodies needs to be determined by west-
sively used, nor has it been routinely accepted by FDA, so ern blot and immunostaining or other appropriate techniques
changes to the platform are required again. Therefore, it is prior to the usage in an immunoassay.
To date, monoclonal antibodies are usually produced in assessment data of the protein assay can determine whether
animals. The advantage of this method is simple and eco- protein concentration could be influenced by certain drug
nomical. Antibodies in ascites with high concentration and intake, other ancillary pathological conditions, or lifestyle
large yield could be sustainably extracted and have no need factors such as alcohol intake, smoking, exercise, diet, and
for culture medium and other costs, no need to repeat aseptic obesity.
operation. However, ascites is often mixed with various
mouse proteins and nonspecific antibodies (including hetero-
phil antibody, HA), so they can only be used after purifica- 22.4.3 Analytical Evaluation
tion, and there is the risk of contamination with animal
viruses, so it is best to use SPF mice. The analytical performance of newly developed assay must
Alternatively, monoclonal antibodies can be produced by be carefully examined before evaluating their clinical appli-
in vitro culture techniques such as hybridoma cell fermen- cation. For an established assay, multiple factors contribute
tation tank. The advantage of this approach is monoclonal to the performance characteristic limits, which include clini-
antibody concentration, and purification is more convenient. cal requirement, goals set by regulatory agencies, published
With the development of hybridoma cell fermentation tank professional recommendations, proficiency testing findings,
technique, the amount of monoclonal antibody prepared by and outcome research, while for a novel assay, such perfor-
in vitro culture method is very large, which is undoubtedly mance specification may be unavailable. However, a similar
suitable for large-scale production of monoclonal antibody. analytical performance to that of an established assay system
Mass and high density culture of hybridoma cells is neces- should be expected. The following are the main aspects of
sary to prepare monoclonal antibodies in vitro. The higher analytical evaluation.
the number of cells per unit volume, the longer the cell lives,
the higher the concentration of monoclonal antibodies and 22.4.3.1 Indicators of Accuracy
the higher the yield. With the deepening of research and the Trueness and accuracy are employed to assess analytical per-
improvement of technology, they will accelerate develop- formance. They are similar but not identical. Trueness is the
ment of industrialization of monoclonal antibody production. consistency between the average measured value of a bio-
marker from different samples and their true concentration,
which represents systematic error. Accuracy is the consis-
22.4.2 Preanalytical Variation tency between the measured value and the true concentration
of a biomarker in that sample, which reflects uncertainty,
Preanalytical variation also needs to be characterized and including systematic errors and random errors. Standard and
controlled in order to measure the analytes correctly. control are used to evaluate the accuracy of assay. A standard
Physiologic and non-physiologic components are both is a substance of definite content in a certain matrix and can
involved in preanalytical variability. To minimize this vari- be divided into three grades: first, second, and third.
ability, sample collection procedures should be standardized, Standards are usually classified as natural and synthetic. The
and the limitations of the assay under a variety of physiologi- control is the substance whose content is known and whose
cal and pathological conditions should be recognized. characteristics are clear in the same matrix as the actual
specimen.
22.4.2.1 Sample Collection and Processing
To determine whether the samples are collected based on 22.4.3.2 Indicators of Precision
expected clinical use of biomarkers is of great significance. Precision refers to the degree of similarity between the
The time of sample collection is closely related to factors results obtained from the same uniform test sample after
such as diet, exercise, biorhythmicity and half-life of the bio-
multiple sampling under specified conditions. Precision is
marker. Samples collected should be processed in a timely generally expressed as deviation, standard deviation, or rela-
manner. If not, appropriate storage ways should be deter- tive standard deviation. Precision could be indicated by
mined according to the stability of biomarkers. reproducibility and repeatability, which are applied to quan-
titatively express the consistency of different measured val-
22.4.2.2 Physiological Factors ues under certain conditions [26]. Repeatability and
Reference range study will help to determine whether the reproducibility refer to measurements conducted under the
protein biomarker concentration could be affected signifi- same and different conditions, respectively. Standard devia-
cantly by factors which include age, gender, or ethnicity. tion (s.d.) or (CV%), a statistical measure that reflects ran-
These data are essential to make interpretive criteria and to dom error, could be used to indicate imprecision. Precision
determine whether it is necessary to establish specific cut- indicates the degree of accidental error in the measurement
points for the different sub-groups. In addition, clinical results. High precision means that in multiple measurements,
the dispersion of the data is small and the accidental error is studied by statistical method of parameter analysis and pre-
small. Accuracy represents the degree of systematic error in sented in the form of mean ± 2 s.d. However, if it conforms
the measurement results. High accuracy means that the devi- to skewed distribution, nonparametric analysis should be
ation of the average value of multiple measurement data adopted to establish reference intervals, which could be
from the true value is small and the system error is small. shown as the lower 2.5th and the upper 97.5th percentiles.
Precision is related to the choice of methodology, and differ- Logarithmic transformation of the data to approximate the
ent methods are selected according to different precision normal distribution is an alternative approach.
requirements. Because the “normal” reference interval of different age
groups and different groups is different, the cut-off value of
22.4.3.3 Analytical Measurement Range (AMR) different groups needs to be determined. Kit manufacturers
AMR refers to the range of analyte values obtained by direct should indicate how the recommended cut-off value is deter-
measurement of samples, which do not require any pretreat- mined and in what group of people. Ideally, the lab would
ment such as dilution or concentration. Within this range, the determine interval and cut-off values in its own population.
measured values of analytes of different samples are linearly However, care should be taken when deviating from the stan-
proportional to their actual concentrations. In order to ensure dard of the product description.
the accuracy and precision of the measurements to be accept-
able, the limits and linearity of detection and quantification
must be evaluated. The limit of detection (LOD) refers to the 22.4.5 Cut-off Value
minimum concentration of the analyte to be measured that
the sample could be detected by an analytical method with a Cut-off value is the quantity value of the analyte to be tested,
given degree of reliability. In simple terms, the calculation which is used to determine whether the result is higher or
method of LOD is the mean value of repeated measurements lower than the clinical or analytical decision point. The set-
of blank sample plus 3 times the standard deviation, which is ting of cut-off value gives a brief combination of sensitivity
practical and has been extensively used in clinical laborato- and specificity. For most immunoassays, there is an overlap
ries. Linearity refers to a certain range of measured values, of test results between samples from diseased and healthy
and there is a constant relationship between the expected and populations, suggesting that an experiment is generally
the observed values in this range, which also determines the unlikely to have complete (100%) sensitivity, specificity, or
maximum detectable value under specified conditions. It is predictive value. Sensitivity, specificity, or predictive value
generally evaluated by continuous dilution of a high-volume should be considered when selecting cut-off values and
sample using a diluent similar to a neat sample matrix. To reporting test results. If the biomarker is intended to be
assess the linearity, a continuous dilution of a sample con- applied in screening tests, which are often used to detect spe-
taining a high concentration of analyte using a diluent simi- cific indicators in a population (or a specific population), in
lar to the matrix of blank sample is required. Visual method general, screening qualitative tests should be highly sensitive
or statistical fitting method can be used to evaluate whether to ensure true positive results. This can lead to false posi-
the actual measured values decrease in proportion to the tives, but can be compensated for by better specific confir-
dilution factor. mations than false negatives that result in transmission or
delay in treatment. If the biomarker is intended to be used in
diagnostic tests, which are usually used to clinically suspect
22.4.4 Reference Intervals the presence or absence of a specific disease, the diagnostic
test should have good sensitivity and specificity to meet the
To establish reference intervals that can be compared with requirement of timely treatment. If the diagnostic test is fol-
the results of the patients, the biomarker frequency distribu- lowed by a confirmation test, the diagnostic test specificity
tion should be detected in healthy individuals. More than 120 requirements can be slightly reduced. Confirmatory test is
values should be involved to establish the reference interval, used to verify the results of a screening test or diagnostic
and the population must be similar to that for which the test test. If the test confirms previous results, a diagnosis can be
is applicable. If statistically significant differences are shown made. Biomarkers of confirmation tests should generally
in the test values between subgroups of different gender, age, have a high specificity (even at the expense of sensitivity)
ethnicity, or physiological status, more samples should be and a high positive predictive value. Therefore, the cut-off
included to stratify the reference intervals into appropriate value of biomarkers is not fixed, but adjusted according to
subgroups. When the concentration of the biomarker con- different clinical needs and the expected clinical use of the
forms to normal distribution, the reference interval should be biomarkers.
22.5 Clinical Evaluation • Phase 2: The patient groups involved in this challenge
phase are usually similar to those in phase 1. This phase
Biomarkers can be used for disease diagnosis, staging, or requires determining whether the results of the test should
evaluating the safety and efficacy of new drugs or therapies use different cut-off values for specificity and sensitivity
in target populations. The first step in the discovery of bio- to predict whether the disease present or not. After the
markers is to screen candidate molecules and then validate diagnostic accuracy in this subset of diagnosed patients
their diagnostic accuracy and predictability of a disease. has been established, the performance of the biomarker
Statistical indicators and analytical methods such as sen- test in different populations should be evaluated through
sitivity, specificity, likelihood ratio, and receiver operating the next phase.
characteristic (ROC) curve are often employed to evaluate • Phase 3: Independent multicenter research should be con-
diagnostic accuracy of biomarkers for target diseases [27]. ducted in this advanced clinical phase to validate the diag-
Sensitivity represents the percentage of the diseased persons nostic tests and establish the diagnostic and predictive
who are correctly identified by the examined test, while values in populations from different regions. Phase 4:
specificity is the proportion of non-diseased persons who are This outcome phase requires evaluation of whether the
correctly excluded by the examined test. The likelihood ratio ultimate outcome of patients who underwent the test and
is mainly used in epidemiology, especially in exploration of the subsequent diagnosis or intervention has been posi-
risk factors of a disease or prediction of occurrence probabil- tively influenced compared with those who did not accept
ity of a disease according to the risk factors. ROC curve the test. This step could be conducted after the biomarker
enables to compare the performance of two or more different detection assay has been commercially available and
diagnostic experiments and to determine appropriate cut-off applied in clinic [28].
values based on the expected clinical application of the test.
Positive and negative predictive values indicate the ability To conclude, it is urgent to develop novel better biomark-
of a test to detect the presence or absence of a disease, ers to improve auxiliary diagnosis, and provide treatment
respectively, which are representative indicators of diagnos- target, as well as monitor therapeutic efficacy and prognosis
tic predictability. The predictive values vary with the preva- of a disease. Biomarker development is a multiple step pro-
lence of the disease within the population examined. If a cess which includes novel biomarker identification in rele-
disease is not common among the subjects, even with a vant samples, candidate biomarker qualification, candidate
highly specific diagnostic test, the majority of positive test verification in different group of samples, optimization of
results will be false positive and with a low positive predic- pre-clinical assay, clinical validation, as well as the final
tive value. Methodological validation is the evaluation of the assay approval by the FDA or other health administrations
determination method, the research content and basis of (Fig. 22.8) [29]. A better understanding of the strategies and
establishing the new method, and the validation type: full challenges in the various stages of biomarker development is
validation, partial validation, and cross validation. Validation essential to optimize experimental study design, which could
needs to be as comprehensive as possible: precision of the in turn improve the efficiency of novel biomarker develop-
method, linear analysis range, comparison with the reference ment and facilitate their clinical application.
method, inaccuracy of the results, detection limit, and anti-
interference capability. Clinical validation is more concerned
with the assessment of sensitivity and specificity. 22.6 Progress
Ideally, a series of clinical evaluation studies including
phase 1–4 trials should be carried out to establish the utility In recent years, the intensified studies have been conducted
of a novel biomarker: on the development of novel biomarkers due to the fact that
biomarkers applied in clinic currently suffer from apparent
• Phase 1: First, whether the novel biomarker is differently limitations. Despite the completed human genome sequenc-
expressed in patients with a certain disease, or the indi- ing project and promising advanced technologies, only a few
viduals without this disease should be examined in this of novel biomarkers have been approved by the FDA in the
exploratory phase. The development process needs to be past decade. The development of biomarkers involves mul-
immediately terminated if the area under the ROC curve tiple successive phases and faces many potential challenges.
(AUCROC) is ≤0.5, which means futility of the bio- The history of available clinical biomarkers suggests that it
marker or the test. However, if there is a significant dis- will take at least a decade to transition from the experimental
crimination between the two subject groups, the next to the clinical phase. Different novel biomarker discovery
phase of study is possibly continued to verify diagnostic and screening technologies have their own advantages and
accuracy. disadvantages, but what we need to consider is how to organ-
Gene, Assay
transcript or Candidate Assay Clinical approval
Verification
protein qualification optimization validation by health
identification agencies
Samples: <10 100s >1,000
Analytes: <1,000 100s 10-100 <10 1-5
Approach: • Whole genome • Selection of candidates • LC-SRM or ELISA • Protein expression • ELISA
sequencing based on quantitative or • Low sample and purification • No fractionation
• RNA sequencing semi-quantitative data fractionation • Development of • High throughput
• DNA or protein • Statistical analysis • Moderate throughput monoclonal
arrays • Selection of secreted, antibodies
• LC-MS/MS membrane or tissue- • Immunoassay
• Thorough sample specific proteins validation
fractionation • Availability of assays for
• Low throughput verification
Fig. 22.8 Novel biomarker development process
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ligence, high throughput sequencing, monoclonal antibody clinic: why, and what can be done to address the problem? BMC
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Clin Chem Clin Biochem. 1997;35:141–73.
Part III
Advanced Molecular Diagnostic Techniques
Next-Generation Sequencing (NGS)
23
Min Wang
23.1 Overview 23.2 Basic Principle
Next-generation sequencing (NGS) is an established method 23.2.1 Library Construction Principle

of simultaneously sequencing millions of fragments of DNA and Characteristics
(or complementary DNA). In recent years, NGS has been
rapidly evolved and adopted in the clinical laboratory. NGS In order to distinguish different fragments of the target to be
was originally developed from pyrophosphate sequencing tested and better anchor them on the sequencing chip and
principle. At present, more mature application platforms simultaneously amplify a sufficient number of sequencing
are various types of Ion Torrent, Illumina, and Complete templates in large quantities, it is necessary to connect the
Genomics (CG). Since its first appearance more than 10 years DNA molecules to be tested before performing the sequenc-
ago, NGS technology had become increasingly mature. One ing on the machine to specific detection linker. That is,
of the characteristics of high-throughput sequencing technol- sequencing library establishment is performed. A sequencing
ogy is the fact that it has many steps and complicated pro- library refers to a series of DNA fragments linked to a cor-
cedures; any problem in any part will affect the accuracy of responding linker, the length and the linker sequence being
the test results and then affect the clinical decision-making. suitable for processing by a sequencer. Different sequencing
That means it plays an important role in precision medicine. types require different and specific libraries for subsequent
Because high-throughput sequencing can detect numerous sequencing. Studies have demonstrated that the selection of
target genes and their mutation sites at one time, it has high appropriate library construction methods can produce on-
sensitivity and specificity and has both qualitative and quan- machine samples with as little bias as possible, thus mini-
titative detection. Moreover, the cost of detection is lower mizing sequencing errors [1].
than that of the same number of genes and loci detection. With the development of sequencing technology, nucleic
Therefore, it has broad clinical and scientific application acid preparation methods for sequencing and construction
prospects in many biomedical scientific fields, such as non- of NGS libraries are also improving. Generally speaking,
invasive prenatal screening (NIPS), mutation of tumor genes, library construction mainly includes the following steps:
genetic diseases, preimplantation genetic screening (PGS), sample extraction, fragmentation, anchoring, and amplifi-
preimplantation genetic diagnosis (PGD), pathogenic micro- cation (Fig. 23.1). According to the different sample types
organisms, and metagenomics. As with any new technology, of sequencing, the constructed sequencing libraries can be
NGS used in the clinical laboratory is constantly evolving divided into DNA libraries and RNA libraries [2].
at the same time. This chapter is a comprehensive overview
of NGS technology, including its basic principle, technology 23.2.1.1 Construction of DNA Library
development, as well as its clinical application. High-throughput sequencing technology can test DNA
samples mainly from genomic DNA of tissues, cells, and
various microorganisms, as well as small DNAs of various
body fluids (such as circulating free tumor DNA in plasma,
fetal-free DNA in maternal plasma, etc.). The preparation
M. Wang (*) steps of these DNA samples are basically the same. First,
Department of Laboratory Medicine, The Second Xiangya the sample DNA is extracted and then fragmented, and the
Hospital of Central South University, Changsha, Hunan,
People’s Republic of China appropriate size fragments are selected by gel electropho-
e-mail: wangmin0000@csu.edu.cn resis or magnetic beads. The DNA is then end-repaired and

306 M. Wang
ity is acceptable, the laboratory can use magnetic beads for

fragment size selection. However, when the sample quality is
not high, such as formalin-fixed paraffin-embedded (FFPE),
samples should be selected by gel electrophoresis for frag-
ment recovery.
Once DNA fragmentation begins, the ends of the frag-
ments are passivated and 5′ phosphorylated using a mixture
of enzymes: Klenow and T4 DNA polymerase. And Klenow
can be replaced by Taq DNA polymerase. The purpose of the
adapter is to anchor the fragment to be tested to the sequenc-
ing chip/semiconductor bead. At the same time, other prim-
ers next to the adaptor can amplify a sufficient number of
sequencing fragment templates to increase the detection rate.
The primer sequences and barcode sequences in the linker
sequences also differ according to the detection platform and
sequencing principles. Currently, there are two ways to add
a link sequence: one is a TA clone connection; the other is a
PCR mode connection. Both methods have good connection
efficiency and have different applications in different library
construction.
23.2.1.2 Construction of RNA Library

DNA is the main carrier of biological genetic information,
but if it is necessary to transform genetic information into
Fig. 23.1 Basic process for high-throughput sequencing library
a phenotype, RNA as a bridge has an indispensable impor-
construction tance. The molecular mass of RNA is relatively small and
diverse compared to DNA molecules. In addition to trans-
port RNA (tRNA), ribosomal RNA (rRNA), and messenger
then phosphorylated at the 5′, and the appropriate linker is RNA (mRNA), the main RNA involved in protein synthe-
added at its 3′ to amplify and quantify the final library [3, 4]. sis, small molecule nuclear RNA (snRNA), small molecule
At present, the methods available for DNA extraction are cytoplasmic RNA (scRNA), small molecule nucleolus RNA
mainly organic solvent extraction, spin column extraction, (snoRNA), chromatin RNA, antisense RNA, and various
and magnetic bead adsorption extraction. Organic solvent viral RNAs, also play an irreplaceable role in expression
extraction method is time-consuming and laborious, cannot and regulation of genetic information. Because of the wide
be extracted in large quantities, and is difficult to automate variety of RNA molecules, the primary objective of RNA
and thus has limited application in high-throughput sequenc- sequencing experiment is important for deciding on the best
ing. Spin column extraction method applies to large-scale library protocol. If the objective is to find complex and global
and high-throughput processing. Magnetic bead adsorption transcriptional events, the entire transcriptome including
extraction eliminates the influence of sample blocking adsorp- noncoding, coding, antisense, and intergenic RNAs should
tion membranes and is easy to operate and easy to automate. be captured in the library as comprehensive as possible. If
Therefore, the current use ratio of the method in high-through- the study is only for mRNA transcripts, the interference of
put sequencing detection is greatly improved [2]. rRNA needs to be removed during extraction, and the library
After DNA extraction of the sample, the extracted DNA is only constructed to screen for enriched RNA with poly-A
sample is fragmented to match the read length of each tail. If the research aims to focus on small RNAs such as
sequencing platform. An important parameter for NGS library miRNA, snoRNA, Piwi-interacting RNA (piRNA), snRNA,
construction is the size of the target DNA fragments. At etc., it is necessary to enrich the small RNA by fragment
present, DNA fragmentation is mainly obtained by physical selection before constructing the library. In the case of circu-
methods (such as ultrasonic disruption, atomization, etc.) and lar RNA sequencing, it is necessary to first degrade the linear
enzymatic digestion (nonspecific endonuclease digestion). RNA molecule using RNase R and then construct the library.
The fragmented DNA sample can be subjected to gel elec- In addition, depending on the purpose of the research, there
trophoresis or magnetic bead adsorption to screen a DNA are different applications such as RIP-seq and CLIP-seq. The
fragment of a suitable size for further library preparation. specific library construction method needs to be optimized
When the sample concentration is sufficient and the qual- for different application scenarios.
23 Next-Generation Sequencing (NGS) 307
Taking transcriptome sequencing as an example, the first The DNA strand to be tested was fragmented by ultrasonic
step in the general procedure for preparing a RNA-seq library
wave, and the fragment length was 200–500 bp, and specific
is to perform total RNA or mRNA extraction on the sample. linkers such as P5 and P7 were added to both ends of the frag-
At present, total RNA/mRNA extraction methods include ment. This method of database construction is cumbersome
Trizol extraction, spin column extraction, and magnetic beadand time-consuming. At present, transposon Tn5 is another
adsorption extraction [5]. Since the direct sequencing of option for high-throughput sequencing. The in vitro trans-
the sample total RNA will generate a lot of useless rRNA posable element of Tn5 for high-throughput sequencing con-
redundancy information, it is necessary to remove the inter-struction includes the end sequence of the transposon, target
ference from the rRNA. There are two main ways to remove DNA, transposase, and Mg2+ (activator); the P5 and P7 termi-
rRNA interference: one is poly-A purification [6]; the othernal linker sequences (Adapter1/2) were added to the transpo-
is rRNA direct removal [7]. son end sequence to synthesize donor DNA. The transposase
After the rRNA is cleared, the obtained mRNA is con- recognized the transposon end to form a Tn5 transposome
structed by the library. There are usually two ideas: one iscomplex with a P5 and P7 terminal linker. The complex rec-
to perform oligo(dT) binding reverse transcription on the ognizes the target sequence of the acceptor DNA, cleaves the
mRNA and to fragment the cDNA; the other is to perform acceptor DNA, and inserts the carried donor DNA to form a
mRNA first, then fragmentation, followed by random prim- DNA having a P5 partial linker (linker 1) at one end and a P7
ers for reverse transcription. At present, the research shows
moiety linker (linker 2) at the other end. The DNA sequence
that the library that first interrupts the mRNA and then per-
is then amplified by PCR plus a tag sequence (barcode, also
forms reverse transcription construction finally obtains theknown as index) and the rest of the linker to form a complete
sequence reads mainly for the gene ontology, while the linker containing the P5 and P7 ends.
reverse transcription is first performed, and then the sequenc- Different oligonucleotide sequences (P5’ and P7’) are
ing reads are obtained, for the transcript 3′ end has a strong
randomly distributed in the flow cell and can be complemen-
preference [8]. Therefore, a library construction method in tary to the linkers P5 and P7 on the DNA to be tested, respec-
which mRNA is first disrupted and reverse transcribed is rec-
tively. Flow cell is a glass plate with 2 or 8 lanes. Each lane
ommended in RNA-seq. can detect one or more samples according to different needs.
At present, the methods of RNA fragmentation mainly The DNA sequence to be tested is complementary to
include alkali treatment, divalent cation solution treatmentthe sequence of the flow cell. By the linker sequence, the
(Mg2+, Zn2+), and enzyme treatment (RNase III). The first complementary DNA strand is extended with the DNA
two treatments need to be performed at high temperatures sequence to be tested, and next the template strand is cleaved
(70 °C) to reduce RNA structural changes [7]. Subsequent and washed away; then the complementary strand is hybrid-
end-repair, 5′-end phosphorylation, and addition, ligation, ized with the linker sequence of the flow cell. Finally , the
complementary strand synthesize s a new strand. This pro-
and quantification of the final library are similar to the DNA
library construction process after the cDNA reverse tran- cess is bridge PCR. The next synthesized double strand is
scription process is completed. After completion of library then melted, hybridized with the junction on the flow cell,
extended, and repeated for multiple cycles, and finally each
quantification and standardization, the RNA library is finally
constructed. DNA fragment is concentrated into bundles at their respec-
tive positions, each bundle containing multiple DNA tem-
plate fragments. Each lane in the flow cell has two columns,
23.2.2 High-Throughput Sequencing each column has 60 titles, and different DNA clusters can be
Principles and Features generated in each title.
SBS determines the sequence of DNA by capturing the
High-throughput sequencing, also known as NGS, is epoch- markers of the newly synthesized ends. Reversible block-
making compared to traditional Sanger sequencing. Since ing technology is used. The reaction system consists of
the birth of the second-generation sequencing technology DNA polymerase, linker primers, and four kinds of NTPs
represented by Illumina (Solexa), Roche (454), and ABI with specific fluorescent labels. These dNTPs are chemi-
(SOLiD), it has quickly triggered fierce competition in the cally protected and only one dNTP can be added at a time.
innovation and development of sequencing technology. This Subsequently, the reaction is eluted, the reagent required for
part focuses on the principles and features of the commonly excitation of fluorescence is added and excited by a laser, the
used high-throughput sequencing technologies. base is identified by fluorescence signal analysis, the fluo-
rescent signal is quenched by chemical reagent to remove
23.2.2.1 Illumina Sequencing the protection of the dNTP 3′ end, and the next reaction is
Illumina’s core principle is sequencing by synthesis (SBS), carried out (Fig. 23.2). Illumina’s sequencing principle of
which contains the following four steps [9]. adding only one dNTP at a time makes it possible to solve
308 M. Wang
Fig. 23.2 The principle of Illumina sequencing
the problem of inaccurate sequencing due to the same base semiconductor chip. Subsequently, ACGT was sequentially
polymerization (such as the repetitive sequence of AAAAAA incorporated into the micropores of the sequencing chip.
in the DNA strand, most sequencing platforms are prone to With the incorporation of each base, if a binding of dNTP
occur). The current Illumina sequencing error rate can be as occurs, it will release H+. As hydrogen ions pass through
low as 0.1% (such as the HiSeq series); the main source of the bottom of each microwell, a change in potential can be
error is base substitution [10]. induced and can be detected. Real-time base interpretation
is finally achieved by detecting hydrogen ions and convert-
23.2.2.2 Ion Torrent Semiconductor ing them into electrical signals (Fig. 23.3). Currently, Life
Sequencing Technologies (acquired by Thermo Fisher Science in 2014)
The core technology of Ion Torrent sequencing is to use sequencers mainly include PGM platform, Ion Proton sys-
semiconductor technology to combine chemical signals and tem platform, Ion S5, and Ion S5 XL.
digital information to establish a direct connection between Ion Torrent sequencing also uses the SBS strategy, which
the two, so as to read and transform the sequencing informa- is different from Illumina sequencing. The hydrogen ions
tion. After the library to be tested is put on the machine, the released by the synthesis of DNA induce pH changes to
DNA strand in the library is fixed in the microwell of the obtain base information for sequencing.
a b
c d
Fig. 23.3 The principle of Ion Torrent semiconductor sequencing. (a) microwell of the semiconductor chip; (c) ACGT was sequentially incor-
Break the DNA to be tested into small fragments, and add different adapt- porated into the micropores of the sequencing chip; (d) With the incorpo-
ers to the fragments; (b) T he DNA strand in the library is fixed in the ration of each base, if a binding of dNTP occurs, it will release H+
23.2.2.3 C omplete Genomics Sequencing MDA-PE sequencing has the advantages of fast synthe-
Platform sis and high accuracy. The sequencing steps mainly include
The CG sequencing platform (taking BGISEQ-500 as an random six-base primer anneal to the template DNA at mul-
example) contains five key technologies: patterned array of tiple sites and then simultaneously start at multiple sites in
DNA nanoball (DNB), combinatorial probe-anchored liga- the DNA by a DNA polymerase (such as Phi29 DNA poly-
tion (cPAL), multiple permutation expansion, multiple dis- merase) with higher continuous synthesis ability and strand
placement amplification-pair end (MDA-PE) sequencing displacement ability. In the initial replication, synthesis of
method, and sCMOS technology. DNA along the DNA template, while replacing the comple-
DNB-forming genomic DNA is first fragmented, coupled mentary strand of the template, the replaced complementary
with a linker sequence, and cyclized to form single-stranded strand becomes a new template for subsequent amplification,
circular DNA, followed by amplification of single-stranded thereby obtaining a large amount of high molecular mass
circular DNA using rolling circle amplification (RCA) tech- DNA. After the first strand sequencing is completed, a sec-
nology. The resulting amplification product is called DNB. ond strand is formed by the action of the enzyme, and the
The DNB loading DNB is fixed on the arrayed silicon chip second strand is sequenced by the DNA molecular anchor.
by the DNB loading technique. The regular array of sequenc- DNB enhances the signal by linear amplification and reduces
ing chips adopts advanced semiconductor precision processing the error rate of a single copy. In addition, the size of the
technology to form an array of DNB binding sites on the surface DNB matches the size of the active site on the chip, and each
of the silicon wafer, thereby realizing the regular arrangement site combines with a DNB to improve the utilization effi-
and adsorption of DNB. The silicon wafer was finally cut into ciency of the sequencing chip. Finally, imaging inspection is
25 mm × 75 mm pieces to form the base of the sequencing chip. performed by high-performance sCMOS technology.
First, the DNA molecular error and the fluorescent probe are
polymerized on the DNB, and then the high-resolution imaging 23.2.2.4 Single-Molecule Sequencing
system collects the optical signal, and the optical signal is digi- High-throughput sequencing is a class of technologies
tally processed to obtain the sequence to be tested. MDA-PE based on the principle of non-Sanger sequencing, which
was sequenced. has been gradually applied to clinical services in individu-
310 M. Wang
alized medical and genetic diagnosis. In 2008, another type 23.3 Technology Development
of DNA sequencing technology based on the non-Sanger
principle using single-molecule signal detection was also Since the analysis of DNA double helix structure, people
introduced. This kind of DNA sequencing technology is have been devoted to exploring the complexity and diver-
called single-molecule sequencing (SMS) or third-gener- sity of genomes. Sequencing technology has undergone
ation sequencing (TGS). These new technologies include many technological innovations and large-scale growth in
HeliScope single-molecule sequencing, PacBio’s single- recent decades. It has experienced the development of the
molecule real-time (SMRT) sequencing, and Oxford’s first generation of sequencing, large-scale parallel sequenc-
nanopore sequencing. SMRT technology uses four-colored ing (also called high-throughput sequencing), and the third
fluorescently labeled dNTPs and nanostructures called generation of sequencing technology (TGS) represented by
zero-mode waveguides (ZMW) based on the SBS method Sanger sequencing. So far, high-throughput sequencing tech-
to achieve real-time sequencing of individual DNA mol- nology has become more and more mature and is entering
ecules. The long read length is a major advantage of SMRT the diagnosis and treatment of clinical diseases. In the appli-
sequencing technology. The sequencing of the technology cation stage, Illumina, Ion Torrent, and CG are the three plat-
is fast, with 10 dNTPs per second. At present, SMRT tech- forms. This part introduces the development history, main
nology is applied more and more widely and can be applied principles, application fields, and development prospects of
to serial repeat sequencing, highly polymorphic region sequencing technology.
sequencing, pseudogene recognition, tumor gene detection,
and reproductive omics research. ZMW is a kind of pore-
shaped nano-optical structure with a diameter of 50–100 m 23.3.1 Summary of Technology Development
and a depth of 100 mm. It is micro-machined to form a
microarray on the thin metal layer of silica matrix. The Nucleic acid is the main carrier of genetic information. DNA
light will exponentially decay after entering ZMW. Only and RNA sequences are known as genetic codes, which are
the portion of the hole that is close to the substrate can be the basis of analyzing gene structure, function, and their
illuminated. After immobilizing DNA polymerase at the relationship. They are also the most accurate criteria for
bottom of the ZMW, the templates, primers, and four-col- molecular diagnosis of clinical diseases. Gene sequencing
ored fluorescently labeled dNTPs were added. When the technology is the basic means to interpret these life codes.
DNA synthesis is performed, the dNTP on the connection In 1977, dideoxy chain termination method proposed
stays longer at the bottom of the ZMW (about 200 ms), by Sanger and chemical degradation method proposed by
and the wavelength of the detection laser is longer than the Gilbert marked the birth of the first-generation sequencing
outer diameter of the ZMW. After the laser is lifted from technology. Owing to significant contributions to nucleic
the bottom, it cannot penetrate the small hole and enter the acid sequencing technology, Sanger, Gilbert, and Berg, who
upper solution area. At this time, the energy is limited to a discovered DNA recombination technology, shared the 1980
small range (volume about 20 × 10−21 L). This range just Nobel Prize in Chemistry. From then on, more sequenc-
covers the part that needs to be detected, thus distinguishing technologies have emerged. From 2005 to 2007, it was
ing the fluorescent signal from the noise floor. Fluorophore marked by Roche’s 454 technology and Illumina’s Solexa
is attached to the phosphate of the dNTP, and when the next technology based on sequencing while synthesis prin-
base is added for extension, the fluorophore attached to the ciple and Life Technologies’ SOLiD technology based on
phosphate of the previous dNTP is cleaved to ensure the sequencing while connection principle. With the birth of
continuity of detection and the detection speed. In the SBS the second generation of high-throughput sequencing tech-
process, the DNA polymerase and the template are com- nology, a breakthrough has been achieved in large-scale
bined. In the base pairing stage, the addition of different sequencing. Although high-throughput sequencing technol-
bases emits different lights, and the wavelength and peak ogy has largely improved sequencing outcome and cut down
of light can be used as a basis for judging the type of input the cost, the short number of bases generated in the reaction
base [11], so that each continuous detection can be per- is always a technical bottleneck that is difficult to overcome.
formed in real time. The fluorescent signal of the nanopore Since 2008, the TGS technology marked by single-molecule
acquires the nucleic acid sequence to be tested [12, 13]. If sequencing and long reading length has emerged as the times
the base is changed, such as methylation modification, the require. The TGS technology can read up to hundreds of kb,
speed will be slowed down by the polymerase, and the dis- and real-time detection of DNA molecules can be realized
tance between adjacent peaks will increase, thereby detect- without PCR amplification. Development of nucleic acid
ing methylation information. testing technology is shown in Fig. 23.4.
2008
1977 2005 the birth of third-generation sequencing
the birth of first-generation sequencing the birth of second-generation sequencing
2009
SMRT single molecular
1975 2006 sequencing system
addition-subtraction 1995 Illumina was introduced
1983 the first single-channel launched
sequencing method
PCR was invented capillary electrophoresis Solexa
was reported
sequencer was launched sequencing 2010
system Life Technologies launched Ion Torrent
sequencing system
1996 2007 2010

1950 1960 1970 1980 1990 pyrophosphate 2000
Life-
2014
sequencing was technologies
Huada Gene launched BGIS-EQ-1000/500
established launched SoLiD
sequencing system
1952
DNA has been 1977
Sanger sequencing 1986
proved to be 2005 2008
method and the the first commercial
genetic material Roche launched 454 Heliscope single molecular sequencing
chemical degradation automatic sequencer
sequencing system system and Nanopore single molecular
sequencing method ABI Prsim370A was
launched sequencing system were introduced
were established
Fig. 23.4 Development of nucleic acid testing technology
23.3.2 First-Generation Sequencing methods are quite different, they all produce independent
Technology groups of radiolabeled oligonucleotide mixtures, which
have a common starting point and terminate randomly at
The germination period of nucleic acid sequencing tech- one or more specific bases. The polyacrylamide gel electro-
nology can be traced back to the 1950s. Whitfeld and col- phoresis (PAGE) can be used to extract the oligonucleotide
leagues used chemical degradation method to determine mixture from each group. The sequence of DNA nucleo-
branchless RNA sequence. In the mid-1960s, Robert and tides is read out directly.
colleagues completed the determination of 76 nucleotide As a result, humans have acquired the ability to explore
sequences of yeast alanyl-tRNA sequence for the first time the genetic information of living organisms and begun to
in 7 years by using small-segment overlap method [14]. In enter the era of genomics. With the continuous develop-
the early 1970s, Wu Rui, a Chinese molecular biologist, put ment of modern molecular biology technology, the classical
forward the strategy of location-specific primer extension. sequencing method of Sanger has been improved and opti-
In 1971, he successfully determined the 12 base sticky end mized and developed into an automated sequencing which
sequences of bacteriophage lambda for the first time [15]. has made great contributions to the Human Genome Project
This is the earliest DNA sequence analysis method recorded (HGP). In this period, new-generation sequencing methods
in the literature [16], but it is limited to the determination of such as shotgun method and sequencing by hybridization
short DNA sequences. In 1973, Gilbert and Maxam deter- (SBH) appeared, which provided strong support for DNA
mined the DNA sequences of 24 bases in the lac inhibi- sequence analysis.
tor binding region by chemical degradation method [17].
Sanger followed in 1975 to report a simpler addition-sub- 23.3.2.1 M axam-Gilbert Chemical Degradation
traction sequencing [18]. In 1977, Sanger established the Sequencing
end-of-deoxy-chain sequencing method [19] on the basis The Maxam-Gilbert chemical degradation sequencing
of the addition-subtraction sequencing method. In the same method, that is, radiolabeling the 5′ or 3′ ends of DNA frag-
year, Gilbert and Maxam co-founded the chemical degra- ments, then using specific chemical reagents to modify and
dation sequencing method [20] on the basis of the original cleave specific base sites, resulted in a series of DNA frag-
method. Although the principles of these two sequencing ment mixtures with common radiation starting points but
312 M. Wang
different lengths. The length and the arrangement of poly- electrophoresis and autoradiography and then inferring
acrylamide gel electrophoresis of these DNA fragments each other of base sequences of DNA chains. Sequencing
ending with a specific base were determined by the location system included DNA templates, sequencing primers, and
of the break point. Finally, the end-labeled molecules were DNA polymerase with dNTP for DNA synthesis and ddNTP
detected by autoradiography, and DNA base sequences were for chain termination. By optimizing the ratio of dNTP to
read directly from the bottom up. ddNTP in each sequencing reaction system, termination of
the new synthetic chain might occur at each A, T, C, and G
23.3.2.2 Deoxygenation Chain Termination position of the corresponding template chain to be tested.
Sequencing In the era of Sanger sequencing, owing to the demand
In the 1970s, Sanger shifted his attention from RNA sequence for high-quality DNA polymerase, specific oligonucleotide
research to DNA and, in 1975, together with Coulson, pro- primers, and cloning single-stranded templates, the related
posed the addition-subtraction sequencing method [18]. technology was difficult to grasp by ordinary laboratories and
Using this method, they completed the first genome, phage laboratory personnel. This method is affected by enzymatic
phi sequencing of 1,745,386 base sequences [21]. Addition reaction and may be affected by incorporation of incorrect
and subtraction sequencing are the first time to use specific bases into the synthesis of new strands. Especially for DNA
primers, using radionuclide-labeled dNTP as raw material, template rich in GC sequence, it is difficult to sequence, and
to conduct DNA chain extension reaction and base-specific the length of base sequence that can be measured is limited.
chain termination reaction under the action of DNA poly- A single terminal reaction can read about 500 single-stranded
merase. Some of the synthesized DNA products were used DNA sequences, while the length of a double-stranded DNA
for degradation in the “addition” system and others for syn- template that can be read is only 200 to 300 bases. Since
thesis in the “subtraction” system. Despite the two-system the advent of Sanger sequencing, after 40 years of continu-
mutually validated sequencing model, the results were still ous updating and improvement (such as fluorescent labeling
unsatisfactory. Because of the difference of reaction speed, instead of radioactivity labeling, capillary electrophoresis
some fragments may be more, and some fragments may be instead of traditional plate gel electrophoresis), its reading
less, which results in the phenomenon of rereading and omis- length can reach 1000 bp, and its sequencing accuracy is as
sion. The possible error of addition and subtraction sequenc- high as 99% [22]. The binding of EcoRI restriction endo-
ing method is 1/50 [18]. nuclease and single-stranded DNA bacteriophage vector
Based on addition and subtraction sequencing, Sanger could sequence the interrupted DNA fragments separately
and Coulson introduced dideoxynucleoside triphosphate and then splice into complete DNA fragments, which real-
(ddNTP) as chain terminator in the sequencing system. In ized the sequencing of DNA macromolecules and improved
1977, a more rapid and accurate end-of-chain termination the speed of sequencing [23].
sequencing method, also called as Sanger sequencing or Sanger sequencing is the basis of large-scale genome
enzymatic method, was established. This is a great break- sequencing. It is still the gold standard of gene sequencing
through in DNA sequencing. today. In the time of precision medicine, Sanger sequencing
The principle was to use DNA polymerase to catalyze the still plays a significant role as a validation method of real-
formation of 3′,5′-phosphate diester bonds between 5′-phos- time fluorescent PCR and high-throughput sequencing tech-
phate group of dNTP and 3’-OH end of primer under the niques. Sanger sequencing also opens the door to synthetic
guidance of oligonucleotide primers, using single-stranded sequencing and lays the foundation for the birth of Illumina
DNA as template and dNTP as substrate. New complemen- sequencing platform based on reversible terminal termina-
tary DNA chains could be obtained from 5′ → 3′ continuous tion sequencing technology.
extension through the formation of phosphodiester bonds,
ddNTP, as a chain terminator, infiltrated into the expanding 23.3.2.3 DNA Automated Sequencing
DNA chain through the 5′-triphosphate group. Owing to the In 1983, with the emergence of polymerase chain reaction
lack of a hydroxyl group at the 3′-position compared with (PCR) technology and the continuous expansion of its appli-
dNTP, ddNTP could not form a 3′,5′-phosphate diester bond cation scope, Sanger DNA termination sequencing gradually
with the subsequent dNTP, thus terminating the extension developed into thermal cycle sequencing or linear amplifica-
of DNA chain. The final four groups are terminated at each tion sequencing [24]. Based on the original Sanger sequenc-
A, T, C, and G position at the 3′ end of mixture of DNA ing method, Taq DNA polymerase was used to amplify DNA
fragments on it, respectively. Because the primers were pre- double-stranded template under the guidance of a single
labeled (later improved to fluorescein labeling for ddNTP), primer, using the efficient automatic circulation of the ther-
four sequencing reactions could be carried out in one tube; mal circulator. Thermal cycle sequencing laid the foundation
the DNA base sequence of the new chain could be directly for automation DNA sequencing. What really opened the
read out by high-resolution denaturing polyacrylamide gel door of DNA sequencing automation was the replacement of
32
P or 35S single radionuclide markers by fluorescent mark- second generation of high-throughput sequencing. Illumina
ers in the 1980s and the replacement of autoradiography by and Life Technologies launched Solexa high- throughput
fluorescent signal receivers and computer signal analysis sequencing system and SOLiD high-throughput sequencing
systems. system in 2006 and 2007 [27]. The emergence of these three
In 1986, Applied Biosystems (ABI) launched the first high-throughput sequencing systems marked the appear-
commercial automatic sequencer ABI Prism 370A, which ance of a new generation of high-throughput sequencing
could read 1000 bases per day. However, the sequencer technologies. The high-throughput sequencing technolo-
still used the plate gel as the substrate for electrophoresis gies also include the Ion PGM/Ion Proton sequencing sys-
[25, 26]. In 1995, ABI company introduced the first single- tem launched after Life Technologies acquired Ion Torrent
channel capillary electrophoresis sequencer ABI Prism 310, in 2010, as well as the BGIS EQ-1000/500 sequencing
using capillary electrophoresis to replace traditional poly- system based on CG platform launched by Huada Gene in
acrylamide gel electrophoresis. The time of sequencing was 2014 after its acquisition of CG in the United States. Since
shortened to 2.5 hours, and the time of PCR fragment size the third generation of sequencing, represented by single-
analysis and quantitative analysis was 10–40 minutes. Since molecule real-time sequencing and nanopore technology,
then, ABI has introduced DNA sequencers of 373, 377, 310, appearing after 2009, we usually turn the high-throughput
3700, 3100, 3730, 3130, and 3500. According to the num- sequencing technology which appeared in 2005 into the
ber of capillary channels (common channels are 4, 8, 16, second-generation sequencing technology. The core idea of
24, 48, and 96), the flux of the sequencer varied. The most the second-generation sequencing technology is sequencing
outstanding product is ABI’s 3730XL, which can perform by synthesis (SBS) or sequencing by ligation (SBL), that
96 sequencing reactions in 2 to 3 hours and read up to 900 is, to capture newly synthesized end markers to find DNA
bases. sequences. Its most notable features are high throughput and
The emergence of automated DNA sequencers has automation. Different from the first-generation sequencing
resulted in an exponential increase in the output of sequenc- technology, the template was cloned in vitro and reacted
ing data, which by 1986 had reached nearly 10 million in separately. The second-generation sequencing technol-
GenBank. The application of automatic sequencing has ogy interrupted the template DNA into small fragments
brought the Human Genome Project ahead of schedule. At and amplified the library by bridge PCR or emulsion poly-
present, DNA sequencing has realized the process automa- merase chain reaction (emPCR), At the same time, millions
tion from sampling to result report, greatly reducing the time of DNA templates were sequenced, so the second genera-
and labor intensity of sequencing. Although there are sig- tion of high-throughput technology was also considered as
nificant differences among different automated sequencing massively parallel sequencing (MPS). The emergence of the
systems, most of the reactions follow the Sanger sequencing second-generation sequencing technology made it possible
dideoxy chain termination principle. The main differences to sequence the genome and transcriptome of a species in
lie in the use of fluorescent markers, the labeling methods of depth. While maintaining high accuracy, it greatly lowered
reaction products, and the sequencing flux. the sequencing cost and improved the sequencing speed.
Here is a brief overview of five mainstream second-
generation sequencing platforms, 454 (Roche), Solexa
23.3.3 Next-Generation Sequencing (Illumina), SOLiD (Life Technologies), Ion Torrent (Life
Technologies), and CG (Beijing Genomics Institute).
Although the first-generation sequencing technology has
strength of long reading length and high accuracy, its high 23.3.3.1 Pyrosequencing
cost, time consumption, and low throughput make it unable Pyrophosphate sequencing is a novel enzyme cascade che-
to meet the requirements of such a deep sequencing and miluminescence sequencing technology. Real-time detection
repeat sequencing like large-scale sequencing. This has of bio-optical signals released in DNA synthesis is achieved,
prompted people to explore new and more efficient sequenc- which pioneers the process of sequencing while synthesiz-
ing techniques. ing. After annealing of primers and single-stranded template
Ronaghi and Uhlen established pyrophosphate sequenc- DNA, the polymerization of dNTP was catalyzed by DNA
ing in 1996. The biggest difference between pyrophosphate polymerase. If dNTP matched with the template, DNA poly-
sequencing and the first-generation sequencing technology merase would incorporate it into the primer extension chain
is side synthesis side sequencing. In 2005, 454 Life Sciences and release an equal amount of pyrophosphate (ppi). Inorganic
company introduced the Genome Sequencer 20 sequencing pyrophosphate was converted to ATP catalyzed by adenosine
system based on pyrophosphate sequencing principle, which triphosphate sulfatase. With the presence of ATP, luciferase
was a milestone event in the history of sequencing. It changed catalyzed the oxidation of fluorescein to produce signals,
the process of sequencing scale and became the pioneer of the which were detected by highly sensitive. ATP and undoped
314 M. Wang
dNTP were degraded by ATP diphosphatase, quenched light taneous light limits its greater flux, and the detection of
signal, and regenerated reaction system. The polymerization homopolymer is not accurate enough. The longer the homo-
reaction of dNTP was coupled with the release of optical sig- polymer is, the greater the error may be. In addition, com-
nal and corresponds to each other. Finally, the DNA template pared with other high-throughput sequencing platforms, the
to be tested could be sequenced accurately, quickly, and in cost of sequencing is much higher. In the fierce market com-
real time by reading the fluorescent signal and its peak value. petition, it has not played its leading edge. Roche announced
Pyrophosphate sequencing technology does not require any the formal closure of 454 sequencing business in 2013.
special fluorescent labeling and electrophoresis. It is easy
to operate and the speed of sequencing has been greatly 23.3.3.3 I on Torrent/Life Technologies
improved. The repeatability and accuracy of DNA sequenc- Sequencing System
ing are comparable to Sanger sequencing. The speed is 100 Immediately after Rothberg left 454 Life Sciences in 2007,
times faster. It is especially suitable for rapid sequencing of he founded Ion Torrent and developed a new generation of
known short DNA sequences (20–50 bp). revolutionary high-throughput sequencing platform based
on semiconductor chips. Rothberg is considered a biological
23.3.3.2 454/Roche Sequencing System legend because of its great contribution to high-throughput
The 454 system was the first commercial next-generation sequencing technology. Ion Torrent sequencing system is the
sequencing platform [28]. In 2005, Rothberg, an American, first high-throughput sequencing platform without optical
founded 454 Life Sciences, which was the first to introduce induction [28, 29]. The sequencing process was no longer
and commercialize Genome Sequencer 20 high-throughput to detect the light signal of biotin or fluorescein but to obtain
sequencing system based on the pyrophosphate sequencing the base information of the sequence by detecting the H+
principle. They combined pyrophosphate technology with released during the binding of dNTP. Ion Torrent chip was
emulsion PCR and optical fiber chip technology to develop a high-density semiconductor chip filled with small holes,
large-scale parallel pyrosequencing technology, achieving which used complementary metal-oxide-semiconductor
high throughput in the sequencing process [28]. The emul- (CMOS) technology. Each pore corresponded to a sequenc-
sion PCR technology was about to fix the single-stranded ing reaction cell with PH-sensitive field effect transistor
DNA with a joint on the captured magnetic beads. Then the (pHFET) built in. When dNTP is bound to DNA strands,
amplification reagent emulsified the magnetic beads to form the H+ released changed the pH of the reaction system. The
a mixture of oil and water, thus forming a lot of PCR micro- current of the crystal sensor changed accordingly. The sen-
reactors carrying a unique single-stranded DNA fragment. sor transformed the chemical signal into digital information
For each fragment, millions of copies were obtained after and completed detection. When the same continuous dNTP
amplification. This not only realized the parallel amplifica- is bound to the DNA strand, it released more H+. The sen-
tion of the whole DNA library but also ensured the specific- sor might be biased in sensing the current, so there was a
ity of the amplification. bias in judging the number of consecutive bases, which was
After 454 Life Sciences was officially acquired by Roche similar to the 454 sequencing system. The strong point of
in 2007, Genome Sequencer FLX System (GS FLX), a Ion Torrent system is that it uses natural dNTP without any
second- generation sequencing system with better perfor- modification, so it is more conducive to the enzymatic reac-
mance, was also introduced. The reading length of the sys- tion. It can produce a longer reading length (400 bp). The
tem was over 400 bp. And 1 million sequences could be read reagent cost is lower than other sequencing systems, and the
in 10 hours, 400 million to 600 million base information sequencing system does not need CCD scanning and fluo-
could be obtained, and the accuracy rate was over 99% [10]. rescence excitation light link. It can detect synthetic inser-
In the same year, the system was used to complete the first tion bases in a few seconds, greatly shorten the detection
individual genome sequence using high-throughput sequenc- time, and simplify the operation. All computer sequencing
ing technology. In 2008, GS FLX once again launched the can only be accomplished in 2–3.5 hours. It realizes that
Titanium series kit and software to improve the GS FLX Rothberg’s Ion Torrent needs users in a few hours at the
flux, read length, and accuracy. In 2010, a new generation of beginning of its establishment.
sequencer 454 GS Junior was introduced, which simplified After Life Technologies obtained Ion Torrent in 2010,
library preparation and was suitable for smaller laboratories. the Ion PGM sequencer was quickly introduced. The device,
454 high-throughput sequencing system has obvious advan- named personal genome machine (PGM), is the world’s first
tages in reading length, which makes the subsequent splic- DNA decoder based on silicon transistor, capable of accu-
ing work more efficient and accurate. It is the best choice rately reading 10 million genetic codes in 2 hours. Without
for genome ab initio sequencing, transcriptome analysis, labeling, laser, and imaging equipment, the price was much
and genome structure analysis. However, due to the use of lower than that of other sequencers at a price of only $50,000,
pyrophosphate sequencing principle, the detection of instan- which at that time was considered the smallest and cheap-
est gene decoder on the market. This economical and fast cutting was used. Single-end sequencing was cut off once,
sequencer was conducive to the popularization of sequenc- and paired-end sequencing was cut off twice. Solexa can
ing technology and brought hope for rapid gene detection read up to 2 × 75 BP by using paired module. However, com-
in clinic. With the next generation of Ion314-Ion316-Ion318 pared with 454 sequencing system, the computational com-
chips, the number of sensors increased from 1.2 million to 12 plexity and difficulty of subsequent splicing work are still
million, which increased the flux of Ion PGM by more than large. Because Solexa technology could only add one dNTP
100 times. In September 2012, a higher-yielding machine Ion at a time in the synthesis process, the accuracy of homopoly-
PGM was released, with an error rate of only 1.2%. It could mer determination was well solved.
be used to sequence the entire human genome for $1000. Illumina platform has dominated the second-genera-
Life Technologies was acquired by Thermo Fisher in tion sequencing market [10]. Genome Analyzer llx and
2003, and in September 2013, Ion S5/S5 XL of S5 series HiSeq high-throughput sequencer are the second-genera-
was released. The S5 series is easier to operate. It only takes
tion sequencers with the largest usage in the world. Since
24 hours from DNA sample preparation to data output when 2010, Illumina has launched one after another, HiSeq 2000
combined with Ion Chef library preparation kit and on-chip (2010), MiSeq (2011), HiSeq 2005 (2012), MiSeq DX
sample equipment. (2013), HiSeq X Ten (2014), NextSeq 500 (2014), HiSeq
X Five (2015), HiSeq 3000 (2015), HiSeq 4000 (2015),
23.3.3.4 Solexa/Illumina Sequencing System NextSeq 550 (2015), MiSeq FGx (2015), MiniSeq (2016),
Solexa launched Genome Analyzer (GA) in 2006. Its earli- and NovaSeq series (2017). Thus, Illumina short read-
est version could get 1GB data at one run, so it also had the ing length sequencing products cover from desktop low
meaning of 1GB Analyzer. Illumina bought Solexa for $600 throughput to large super high throughput, and there is a
million in 2007 and commercialized it. high degree of complementarity and crossover between
Solexa sequencing system still took sequencing by syn- platforms. The HiSeq X series launched in 2014 can gener-
thesis as its basic design concept and used bridge-type PCR ate more than 1800 human genome data with 30× coverage
and reversible end-to-end as its core technology. Bridge- in a year at most, making $1000 genome sequencing a real-
based PCR amplification referred to the complementa- ity. The NovaSeq series of instruments launched in 2017,
tion of the prepared single-stranded DNA library with the which run faster than 70% of the existing ones and can
single-stranded primers on the chip surface when it passed complete genome sequencing in only 1 hour, is the most
through the mobile pool. One terminal was fixed on the chip; powerful sequencer in Illumina so far, indicting the pos-
the other was randomly complemented with another primer sibility of a $100 genome sequencing era.
nearby, which formed a bridge. The bridge-type ssDNA was
amplified into bridge-type dsDNA, and then the bridge- 23.3.3.5 SOLiD/Life Technologies Sequencing
type dsDNA was deformed and released into complemen- System
tary single strands, anchored to the nearby solid surface to In the first generation of sequencing era, ABI had always been
form ssDNA. About 1000 copies of monoclonal DNA clus- the leader of the industry; its monopoly position cannot be
ters were formed after 30 rounds of amplification-denatur- shaken. From the early 370A to the fully automated 3730XL,
ation cycle, which reached the template of signal intensity ABI’s first-generation sequencer has been widely applied
required for sequencing reaction. Afterward, the amplifier in all aspects of genomics research. However, with the full
was linearized and sequenced while synthesizing. Based on speed development of the second-generation sequencing
Sanger sequencing, Solexa system used specific fluorescent technology, ABI started late and did not launch its SOLiD
labeling for four different dNTPs. Because the 3-OH ends of sequencing platform until 2007. In terms of flux, SOLiD is
these dNTPs contained chemically cleavable parts, only one revolutionary. A single run produces 50GB sequence data,
dNTP could be added to each round of the reaction. Other equivalent to 17 times the human genome coverage. Since
dNTPs, DNA polymerase and fluorescent groups that had then, SOLiD has been upgraded continuously. The sequenc-
not been combined were removed and a new round of reac- ing flux of SOLiD5 platform has reached 30GB/day, the cost
tions was initiated. These dNTPs were called reversible ter- is less than 60 US dollars/GB, and the accuracy is as high as
minators [30]. According to the captured fluorescence signal, 99.99% [10].
the sequence information of DNA was obtained by process- Sequencing by oligo ligation and detection (SOLiD) was
ing with a special computer software. On most Illumina plat- based on the ligase method. Four-color-labeled oligonucle-
forms, each dNTP is bound to a fluorescent group, so four otide ligation reaction replaced the traditional polymerase
different laser channels were needed. However, NextSeq and chain reaction. DNA could be prepared into fragment
MiniSeq used a double fluorescent group system [10]. In the libraries or mate-paired libraries according to different
sequencing process of Solexa, either single-end or paired- detection requirements. SOLiD was amplified by emulsion
end sequencing, the process of deliberately selective strand PCR. Because the SOLiD microspheres were only 1 micron,
316 M. Wang
each slide could accommodate microspheres with higher ogy are two unique sequencing technologies of the CG com-
density than 454 PTP plates, which could easily achieve pany. The accuracy of sequencing is 99.9998%, the market
high throughput. The SOLiD system did not use DNA price is low, and it has a considerable competitive advan-
polymerase, which was widely used in previous sequenc- tage. Huada Gene officially acquired CG in 2013. On July 2,
ing, but ligase. Chain fluorescent probes (3′-XXnnnzzz-5′) 2014, the China Food and Drug Administration (CFDA) first
were linked to a common primer (n) and matched with DNA passed the registration application of the second-generation
template chains according to the principle of base comple- gene sequencing diagnostic product BGISEQ-1000 (based
mentary pairing. The probes were labeled with fluorescent on CG sequencing platform). BGI launched BGISEQ-500
dyes of four colors, corresponding to numbers 3, 2, 1, and and BGISEQ-50 desktop high-throughput sequencing sys-
0, respectively. The single-stranded fluorescent probe with tem in 2015 and 2016.
eight bases was a double-base coding probe, in which the The CG platform used high-density DNB technology to
first and second bases (XX) were determined, the third to insert DNA nanospheres into the chip and then read the base
fifth sites (nnn) were random bases, and the sixth to eighth sequence by discontinuous and non-linked cPAL technol-
sites (zzz) were special bases that could be paired with any ogy. Four different color-labeled probes were used to read
base. When the fluorescent probe matched with the template the bases near the junction. At most 10 consecutive bases
chain and connected, it emitted fluorescent signals represent- were read at a time, and sequencing was independent of
ing the first and second bases. After recording the fluores- each other. That was to say, the sequencing results were not
cence signal, the fluorescence signal could be removed by affected by the sequencing results of the previous results, to
chemical cutting between the fifth and sixth base groups, so avoid the accumulation of errors. The anchoring sequence
that the next link could be made. Therefore, five bases were added in the sequencing was complementary to the junction.
added to each ligation reaction. After the last ligation reac- Then DNA ligase hybridized four different color-labeled
tion (7 ligation reactions in general fragment library and 5 probes to the corresponding base of the template and deter-
ligation reactions in double-ended sequencing library), the mined the base type by imaging the fluorescent group [31].
newly synthesized chain was denatured and eluted. Then the All probe-anchored molecule complexes were removed, and
second round of sequencing was carried out with primer n-1 new probe-anchored molecule complexes were combined
(the position of a base moved to the 3’end based on primer [10]. cPAL technology can greatly reduce the concentra-
n) until the fifth round of sequencing (one-way sequencing). tion of probes and enzymes, and unlike sequencing while
Finally, the base sequencing of all positions could be fin- synthesizing, each cycle of cPAL can read several bases at
ished, and the base of each position was measured twice. To a time, which greatly reduces the consumption of sequenc-
connect the color information corresponding to the template ing reagents and imaging time. At present, the reading length
sequence in the order of bits 0, 1, then 1, 2, and so on, the of the flux sequencing platform is 28–100 bp, which limits
original SOLiD color sequence consisting of “0, 1, 2, 3…” its role in the study of structural variation. At present, the
was needed. This was SOLiD’s unique two-base sequenc- bottleneck of high-throughput sequencing technology is that
ing method. SOLiD sequencing system detected all bases throughput and reading length cannot be perfect simultane-
of the target sequence twice, so the biggest advantage was ously. Flux determines the length and cost of sequencing,
high accuracy (99.999%) [10]. Because SOLiD system does while length of reading determines the difficulty of splicing
not use PCR reaction, it has great merits for samples with DNA fragments to restore the real situation of the genome.
high GC content. However, the maximum reading length of In addition, the second-generation sequencing technology
the technology can only reach 75 bp, which greatly reduces needs to build libraries through expansion, which is very
the operability of the genome splicing and structural varia- easy to generate perceived interference. In addition, errors,
tion research species. In the phase of fluorescence decoding, missing information, and sequence bias may occur dur-
because it is a double base to determine a fluorescent signal, ing the amplification process, resulting in the annihilation
it is easy to produce chain decoding errors once errors occur. of fragments with few copies in the originaly sample after
In addition, SOLiD technology is constrained by its indus- the amplification reaction, and some modification informa-
trial production and eventually withdraws from the second- tion in the original sequence may also be erased during the
generation sequencing without developing new instruments amplification process. These are the limitations of second-
under enormous market pressure. generation sequencing.
Using the second-generation sequencing technology,
23.3.3.6 C omplete Genomics/BGI Sequencing clinical laboratories can carry out disease targeting sequenc-
System ing, exome sequencing, and genome-wide sequencing. But
Founded in 2005, the US Complete Genomics company is the second-generation sequencing technology is much more
the world’s leading life science company to provide human complex than the traditional molecular detection technology.
genome sequencing services. DNB chip and cPAL technol- Selecting a suitable sequencer is essential to the second-
Table 23.1 Advantages and disadvantages of second-generation high- sequencing and nanopore sequencing eventually achieved
throughput sequencers
long reading length, single-molecule sequencing which once
Instrument Advantage Disadvantage again overturned the field of sequencing. Sequencing tech-
Illumina Cheap and easy to Limit to PE150; nology marked by non-amplified single-molecule sequenc-
MiniSeq operate expensive kit
ing and long reading length is called the TGS technology.
Illumina Medium price; low Compared with HiSeq,
MiSeq cost per Mb less reads and higher cost These technologies can read up to tens of thousands of base
sequencing; fastest per Mb sequencing fragments at one time, which greatly reduce the difficulty of
running time and splicing and, more importantly, greatly reduce the vulner-
longest reading length
abilities that could not be in the past. However, the current
among all Illumina
sequencer TGS technology still has a long way to clinical application
Illumina Easy to operate, Version 2’s new because it does not find a good solution about its high error
NextSeq 500 medium instrument sequencing technology rate.
and running cost does not achieve the
sequencing chemical
performance used by
23.3.4.1 P acific Bioscience SMRT Sequencing
MiSeq and HiSeq Technology
Illumina Low cost per Mb High cost of instrument; SMRT sequencing technology was proposed by Web and
HiSeq 2500 sequencing; high strict with personnel Craighead and further developed by Korlach, Turner, and
flexibility; two training; can’t run two
Pacific Bioscience [32, 33]. It launched as PacBio sequenc-
sequencing patterns; modes at the same time.
various possible ing platform in 2009. PacBio SMRT technology used a spe-
lengths cial flow cell (SMRT cell), which contained thousands of
Illumina Modeled flow through; Similar to HiSeq 2500, transparent picoliter wells-zero-mode waveguide (ZMW)
HiSeq 4000 low cost per Mb or per but a bit expensive and holes at the bottom. This was one of the key points of PacBio
read sequencing low flexibility
SMRT technology [32]. It can distinguish the reaction signal
Illumina Lowest cost per Mb or Massive data storage
HiSeq X per read sequencing at from the strong fluorescence background of the surround-
Five or Ten present ing free dNTP. Unlike short reading length sequencing while
Ion Low cost and simple More reading errors than synthetizing, which required the use of polymerase to bind
Torrent- instrument, three chips Illumina; compared with DNA and then to amplify along the template chain, PacBio
PGM with different readout MiSeq, longer manual
lengths operation time, cost more
SMRT technology fixed the polymerase at the bottom of the
and read less per Mb; a hole, and the DNA chain was detected by MW. In addition,
smaller user base the detection of base modification (methylation, etc.) can
Ion Similar price with Illumina; compared with be achieved by detecting the sequencing time between two
Torrent- MiSeq while PII and MiSeq, longer manual adjacent bases. If the base modification exists, the speed of
Proton PIII chips give more operation time, cost more
reads and read less per Mb; a passing through the polymerase will slow down, and the dis-
smaller user base tance between adjacent peaks will increase. One of the key
Ion More suitable price Less data analysis tools points of PacBio SMRT technology is that the engineered
Torrent-S5/ than MiSeq; best and approaches DNA polymerase used has high activity and can detect long
S5 XL application prospect
among all Ion Torrent
reading length. It can usually read 10 kb and some can reach
instrument 100 kb [34]. SMRT technology is quiet fast, and the flux can
reach 7 Gb per day, while the accuracy is only 85%.
generation sequencing. Table 23.1 shows the advantages 23.3.4.2 Oxford Nanopore Technologies
and disadvantages of common sequencers in the second- Nanopore Sequencing Technology
generation sequencing. The concept of nanopore sequencing was first proposed in
the 1980s [34, 35]. It was based on physical electricity and
used the change of local current when the DNA molecule
23.3.4 Third Generation of Sequencing passed through the nanopore to complete the determination
Technology of base sequence. Bayley founded the Oxford Nanopore
Technologies (ONT) company in 2005. MINION, the pro-
The ideal sequencing method should be direct and accurate totype of the first consumer-grade nanopore sequencer, was
sequencing of the original DNA template without the limi- born in ONT in 2014. Once it was launched, it attracted great
tation of reading length. As early as the 1980s, researchers attention of the scientific community and was regarded as
had started to work hard to achieve this goal. Although many the most promising single-molecule sequencer. The main
of these attempts failed, single-molecule real-time (SMRT) features of ONT sequencing are long reading length, fast
318 M. Wang
reading speed, high throughput, and portability. Because available sequencing technology are unable to satisfy the
of the advantages of nanopore itself, sample preparation is demands of practical application.
simple, and methylated C can be read directly without sulfite Sequencing technology in the future will surely move in
treatment, which helps directly the study of epigenetic cor- the direction of more precise, microscopic, high throughput,
relation phenomena at the genomic level. At the same time, and cheaper. Different generations of sequencing technol-
because the technology has more than 1000 independent sig- ogy will continue to coexist and develop for a long time,
nals, its error rate is also elevated. Modification of bases is striving to complement each other through their respective
also a challenge for ONT because the modified bases change function advantages. In addition, with the increasing demand
the voltage set by the original K-mer. Fortunately, a series of for human beings to explore the mysteries of life and the
recent improvements in reagents and algorithms have greatly deepening of research work, new sequencing principles and
improved the accuracy of ONT detection. technologies will continue to emerge to meet the application
needs of different disciplines.
23.3.5 Development and Prospect

of Sequencing Technology 23.4 Clinical Application
Over the past 40 years, sequencing platforms have been 23.4.1 Application of NGS in Noninvasive
changing. Before 1985, almost all sequencing was com- Prenatal Screening for Chromosome
pleted based on Angel sequencing method; by 2000, the Aneuploidy
automation of sequencing was achieved through four-
colored fluorescence labeling and capillary electrophoresis; It is well-known that the chromosomal aneuploidy in human
and since 2010, various high-throughput sequencing tech- oocytes and embryos would result in low implantation
niques (short reading length and long reading length) have and pregnancy rates. The discovery of cell-free fetal DNA
developed rapidly and gradually matured. Illumina HiSeq X (cffDNA) circulating in maternal blood [36] allows the devel-
series and ONT PromethION are striding toward the limit of opment of an early noninvasive prenatal diagnosis (NIPD)
sequencing cost and data output [10]. In 2004, the National based on analysis of maternal blood. However, this technique is
Human Genome Research Institute (NHGRI) launched the only performed after 11 weeks of gestation. The NGS technol-
Genome Sequencing Program (GSP) to rapidly cut down the ogy has the advantages of fast, accurate, and high throughput,
cost of genome sequencing and expand the application of which greatly accelerate the development and application of
genome information in medical research and medical ser- cffDNA sequencing [37]. Unlike PCR-RED, NGS can screen
vices. The cost of genome sequencing at the start of GSP all mutations in a single assay to rule out recurrence and diag-
in 2004 was still higher than $10 million. Since 2007, the nosis in cases de novo. It appears to be suitable for counting
focus of sequencing has shifted from a capillary electropho- genome representation and determining the overrepresented
resis platform based on anger sequencing to a second-gen- chromosomes of interest in the affected fetus [38, 39]. A short
eration sequencing platform, and the cost of sequencing has region at one end of each DNA molecule of maternal plasma
dropped to less than $1 million. From 2007 to 2012, the cost was sequenced using synthesis technology on an Illumina
of sequencing each base dropped by four orders of magni- platform and mapped against the reference human genome to
tude, exceeding “Moore’s law” effect in economics [34]. In decide the chromosomal origin of each sequence. The den-
2015, the cost of genome sequencing had basically dropped sity of sequenced tags from the chromosome of interest from
to $1000. The continuous development and integration of an aneuploid fetus was compared with cases of trisomy and
biological sciences, physical materials, and other disciplines euploid pregnancies (Fig. 23.5).
had brought a more powerful impetus to the development Using massively parallel shotgun sequencing to detect
of sequencing technology, making it continue to develop noninvasive fetal trisomies from maternal blood by analyz-
toward the trend of $100 genome sequencing. ing the relevant chromosomes in locus-independent assays
Although sequencing technology has shown an endless has demonstrated its possibility [39]. In addition, recent
trend of development, as far as the current research situa- studies also confirmed this finding [40]. Another way to
tion is concerned, on the road to ideal and perfect sequencing sequence a whole genome for noninvasive detection of fetal
technology, here are the key issues that need to be addressed. abnormalities is to use array capture to enrich the region of
First, the ability to sequence the human genome has vastly interest before sequencing [41]. After the successful large-
outstripped the ability to explain genetic variations. Second, scale application of NIPT in the detection of chromosome
whole genome sequence information being fully interpreted aneuploidies, a series of clinical studies began to focus on
still needs to take a long time. Third, the restrictions of detecting genomic diseases caused by microdeletions and
microduplications of chromosomes, such as abnormal copy
Fig. 23.5 Schematic representation of a framework for noninvasive prenatal testing (NIPT) of fetal chromosome aneuploidy using massively
parallel genome sequencing
number variations (CNVs), such as DiGeorge syndrome, cri implantation failure [47], and previous aneuploid concepts
du chat synthesis signs (5p-), 1p36 deficiency syndrome, etc. [48]. Preimplantation genetic testing is based on human-
[42, 43]. However, NIPT can only be used as a screening assisted reproductive technology, genetic analysis of gam-
item. When the NIPT result is positive, the fetal prenatal etes or embryos, diagnosis of gametes or embryos with
diagnosis must be confirmed by invasive prenatal diagnostic genetic defects, and then selection of abnormal embryos into
methods such as amniocentesis. the uterus, including PGD and PGS technologies. In 2013,
In conclusion, the use of NIPT to screen fetal aneuploidy Philadelphia used NGS technology for PGS and achieved
has extremely high sensitivity and specificity, which can be the first successful IVF delivery. Some studies demonstrated
widely used in clinical settings, greatly reducing the occur- that NGS is an effective and safe technique for preimplanta-
rence of unnecessary intervening materials and benefiting tion screening [49, 50]. Wells and colleagues reported the first
more pregnant women. high-throughput sequencing technology—successful cases of
PGS pregnancy [49–51]. NGS-PGS technology has gradually
become the most promising PGD/PGS technology due to its
23.4.2 Application of NGS in Preimplantation high throughput, high sensitivity, and high specificity [52–54].
Genetic Screening In clinical practice, the PGS process can be divided into
three steps.
Chromosome abnormalities or aneuploidies are the main
causes of embryo stagnation, implantation failure, repeated 1. DNA extraction and single-cell expansion. Take blasto-
abortions, and birth defects [44, 45]. The aneuploidy rate in mere single cells that develop to 3 days or take 3 to 5
in vitro fertilization (IVF) patients is extremely high, espe- blastocyst trophoblast cells for 5 days to extract DNA. And
cially in those with recurrent miscarriage [46], repeated perform single-cell whole genome amplification.
320 M. Wang
2. Database construction and sequencing. Library prepara- ing at a rate of 10–50 per year. Although the incidence of
tion of quality-qualified whole genome amplification prod-monogenic diseases is extremely low, the number of patients
ucts was performed and sequenced on a high-throughput worldwide has reached a million because of the wide cover-
sequencer. The reads obtained by sequencing are matched age of genes and the variety of diseases [57, 58]. For families
to the human genome by sequence alignment. suffering from monogenic diseases, the detection of single-
3. Data analysis and data interpretation. The sequencing gene disease has four aspects. First, as an auxiliary diagnos-
data is compared with the reference genome, and the tic method, patients with suspected monogenic diseases can
CNV algorithm is used to analyze the chromosome of the be detected to find the cause of the disease and assist clinical
embryo to be tested according to the analysis result, and diagnosis. The second, as a differential diagnosis method,
the bioinformatics is used to provide reference for select-
can determine the specific type of disease and facilitate
ing normal embryo transfer. early intervention and early treatment. The third, as a risk
assessment tool, can screen high-risk groups with a family
Using DNA barcode technology, NGS screening provides history of monogenic diseases and analyze the disease risk.
a unique method to evaluate multiple samples from multiple Finally, as a method of marriage and childbirth, genetic test-
patients on the same sequencing chip. These samples have ing can be performed on couples with family history or first-
different indications, such as recurrent miscarriage, repeated born children to guide prenatal and postnatal care, thereby
implant failures, and previous aneuploidy concepts. In a sin- effectively reducing the possibility of diseased offspring and
gle run, up to 96 samples can be analyzed using an upgraded improving the quality of the population. At present, there are
high-capacity NGS instrument (e.g., HiSeq). Furthermore, three methods for detecting single-gene genetic diseases by
NGS screening does not require co-hybridization of DNA high-throughput sequencing technology. According to their
control samples. As the number of global PGS cases increases detection regions, they are named WGS, WES, and specific
and the scale of platform production increases, it is foresee- exon region-targeted capture sequencing.
able that the NGS method may reduce the cost per patient in Beta-thalassemia is caused by the reduction (β+) or dele-
the near future [55], showing a cheerful prospect [49]. The tion (β0) synthesis of the globin chain of hemoglobin tetramer
cost and time of sequencing have been comparable to array [59]. The mutation of the HBB gene is located on chromo-
testing, and the cost of reagents for sequencing chromosome 11. When both parents are carriers of a thalassemia
somal abnormalities is currently less than $100. In addition, mutation, the risk of thalassemia in each pregnancy is 25%.
the cost-effectiveness of sequencing and the accuracy are Thalassemia can lead to severe anemia in childhood, and fail-
rapidly increasing due to continuous technological improve- ure to undergo regular blood transfusions may result in death
ments, including some benchtop sequencing platforms [56]. in the first year [60, 61]. The emergence of NGS makes it
Some limitations of NGS in preimplantation genetic testing possible to count millions of DNA molecules from a single
need to be addressed. First, although NGS-based compre- sample, greatly increasing the sensitivity of molecular detec-
hensive chromosomal screening brings significant benefits tion using cfDNA. Li X and colleagues [62] used overlap-
to many IVF-PGS patients, the approach is not applicable ping amplicon to target the four most common β-thalassemia
to all patients, especially those with reduced ovarian reserve. mutations and HbE in Southeast Asia and targeted sequenc-
It may not be possible for all IVF-PGS patients to achieve ing of 83 cases of parents with different β-thalassemia muta-
an increase in pregnancy rates, especially for older women tion carriers. Due to the presence of SNPs under the primers,
aged 40 or older. Further randomized larger clinical trials are the use of overlapping amplicons is essential to reduce the
required to confirm the clinical benefits for these patients. likelihood of false-negative results. This study showed that
Second, NGS cannot directly detect balanced chromosomal ND-derived NIPD can detect paternal-derived β-thalassemia
rearrangements due to the lack of total DNA content imbal- mutations with a sensitivity of 100%, a specificity of 92.1%,
ance. Finally, the WGA products were not defined for unbal- a positive predictive value of 94%, and a negative predictive
anced translocation break points [53, 54]. Hence, additional value of 100%. NGS can be used to screen for thalassemia
studies with the use of cell lines or WGA products are needed gene mutations in neonatal foot hemorrhage, which can not
for detection of imbalanced translocations. Overall, NGS- only effectively detect thalassemia gene types but also find
based PGS could be efficiently applied in clinical practice. new genetic mutations. The advantages of this method include
easy sample collection, accurate results, extensive clinical
diagnosis, and possible early diagnosis of thalassemia [63].
23.4.3 Application of NGS in the Detection Targeted sequencing using specificity for the mutation of
of Single-Gene Genetic Diseases interest means that the method is applicable to any condition
in which there is a point mutation or a small insufficiency.
A single genetic disease is a hereditary disease controlled by Compared to methods that rely on haplotype analysis of the
a pair of alleles, with more than 6600 species and increas- paternal allele, there may be no informational SNPs present
in nearby mutations of interest [64]. Therefore, NGS technol- In addition, NGS technology can advance tumor preven-
ogy is a direct, rapid, and effective strategy for discovering tion measures to the prevention stage. Numerous studies
pathogenic mutations in single-gene genetic diseases. It can have shown that the occurrence of certain tumors is closely
provide a basis for clinical diagnosis and genetic counseling, related to mutations in individual key genes. Hereditary
guide fertility, and has a great clinical application prospect. breast and ovarian cancer (HBOC) syndrome is usually
associated with genetic alterations in BRCA1 and BRCA2,
suggesting a screening and intervention to BRCA1/2 carriers
23.4.4 Application in the Detection of Tumors and their family members to reduce risk [71]. The BRCA1/2
gene is a type of tumor suppressor gene that can help repair
Since Ley and colleagues first reported the use of NGS tech- DNA damage and ensure the stability of the cell’s genetic
nology for genomic analysis of one case of acute myeloid material (DNA) and prevent cell growth mutation. However,
leukemia of normal karyotype in 2008, NGS has been widely when the BRCA1/2 gene is mutated, the cell loses this pro-
used in the field of tumors [65]. NGS breaks through the tective effect, the risk of developing cancer will be greatly
limitations of pedigree analysis and provides a convenient increased, and there is still family inheritance. NGS based
and effective method for detecting and diagnosing hereditary on clonal amplification coupled with massively parallel
cancer [66]. NGS technology achieves higher sensitivity and sequencing is a powerful DNA sequencing tool [72–74]. The
accuracy by repeatedly sequencing DNA fragments in the application of NGS technology in clinical diagnosis plays a
same area. At the same time, NGS has the advantages of high huge role in the diagnosis and treatment of tumor patients.
throughput and high degree of automation. It can complete Together, these findings implicated that NGS technology
the sequencing of tens of billions of bases in a short time, will play an important role in the field of tumor molecular
making it possible to complete the sequencing of the entire biology and improve the diagnosis and treatment of tumors
human genome in a few days. There are three main experi- with the improvement of gene sequencing throughput, accu-
mental techniques for high-throughput sequencing for tumor racy, and cost reduction.
gene mutation detection: WGS, WES, and targeted sequenc-
ing. Analytical strategies for high-throughput sequencing
data of tumor genes include SNV, small fragment inser- 23.4.5 Application of NGS in Tumor-Targeted
tion deletion, CNV, and SV analysis. With the deepening of Therapy
research, the current study found that the presence of free
nucleic acids can be detected in the peripheral blood of tumor With the development of personalized medicine and the
patients. These free nucleic acids are dominated by genomic concept of “precision medicine,” tumor-targeted therapy has
and mitochondrial DNA components and are also known as developed rapidly, and clinical studies have gradually discov-
circulating tumor DNA (ctDNA) [67]. Studies have found ered and confirmed more gene mutations related to targeted
that ctDNA can be detected in various human malignancies therapy. NGS uses massively parallel sequencing, capable
such as prostate cancer, lung cancer, breast cancer, colorectal of sequencing millions or even billions of DNA fragments
cancer, liver cancer, bladder cancer, cervical cancer, pancre- simultaneously. Therefore, under the condition of low cost
atic cancer, and ovarian cancer [68, 69]. There are genetic, and constant sample size, NGS can detect up to hundreds of
epigenetic, and transcriptomic differences between primary tumor-associated genes, whole exons, and the entire genome
and metastatic tumors. The use of ctDNA in peripheral blood at one time [75]. Large-scale application of this technology
can more accurately and efficiently assess tumor progres- in characterization of tumor molecule has led to the gen-
sion. If tumor cells have some kind of mutation, these muta- eration of databases that catalog the NGS cancer genome,
tions can be reflected in ctDNA. The mutation information including COSMIC database, ICGC, and TCGA database
of ctDNA was obtained by NGS sequencing, and the muta- [76–79]. Such targeted NGS groups for somatic mutation
tion information of tumor cells was obtained. Newman and detection can be used to guide treatment decisions [80–82].
colleagues [70] used NGS technology to detect ctDNA in Non-small cell lung cancer (NSCLC) is the most common
plasma and showed surprisingly high specificity and sensi- type of lung cancer, and targeted therapies are now available
tivity. The author named this method as cancer personalized or have been used in clinical trials [83]. Therefore, molecular
profiling by deep sequencing (CAPP-Seq). It was found that characterization of tumors using NGS technology has become
the detection sensitivity for stage II to IV non-small cell lung a key tool for the treatment decision-making and clinical
cancer (NSCLC) was 100% and that for stage I NSCLC was management of patients with NSCLC [84]. The use of NGS
50%; the specificity for all stages of lung cancer was 96%. allows simultaneous sequencing of thousands of short DNA
The above results show that CAPP-Seq is not inferior to the sequences in a massively parallel manner, providing a cost-
gold standard tumor biopsy in diagnosis and can accurately effective method for simultaneously detecting multiple genetic
determine the genotype of lung cancer. alterations in multiple samples. In addition, NGS achieves
322 M. Wang
sensitivity superior to traditional sequencing techniques and drug genes. Personalized cancer treatment allows for more
can detect mutations in a very low percentage of normal DNA targeted treatments, such as EGFR and ALK mutations car-
background, which is important for detecting somatic muta- rying in NSCLC and melanoma with BRAF mutations [91].
tions [85, 86]. NSCLC is a type of tumor with the most tar- The recent progress in this field also makes it possible to pre-
geted therapy. Genes with approved targeted agents include dict and monitor individual responses to specific treatments
EGFR, ALK, and ROS1 [84, 87]. In addition, specific genetic or drug concentrations. In addition, high-quality genomic
alterations in NSCLC are now considered to be the best tar- analysis is critical for personalized pharmacotherapy in
gets for agents approved in other tumor types. These drug tar- patients with cancer [92].
gets are genes that are frequently altered in NSCLC, such as In summary, as more and more targeted therapeutic drugs
KRAS, ERBB2, MET, and RET, which are recommended as enter clinical research, NGS detection of tumor multiple gene
emerging biomarkers for the NCCN guidelines. In addition, mutations and gene mutation detection of tumor-targeted
potential targets such as BRAF, PIK3CA, FGFR1, DDR2, and therapies have shown their broad application prospects [93,
PTEN are currently being verified in clinical trials [88, 89]. 94]. It is to be expected that NGS-based assays will help to
The NGS technology platform dedicated to researching is now overcome some of the current limitations of tumor-targeted
widely used, and the tumor genotype and its clonal structure therapy and personalized cancer medicine.
can be sensitively evaluated in less than 3 days, and even small
residual assessment and longitudinal sampling of the tumor
can be performed. Clinical trials that evaluate the treatment 23.4.6 Application of NGS in the Detection
of well-defined targets by targeting NGS in combination with of Pathogenic Microorganisms
other relevant biomarkers will accelerate the development of
drug and therapeutic strategies. Identification and characterization of microorganisms caus-
Pharmacogenomics focuses on genetic variations that ing infections are critical to successful treatment, recovery,
cause individual differences in drugs that not only alter and safety of patients. At present, the widely used immuno-
the pharmacokinetics of the drug but also alter the phar- logical methods and PCR technology can only detect a small
macodynamics of the drug. The clinical drug gene detec- number of samples each time and cannot detect unknown
tions are the content of the genetic variations of the target pathogens. The rapid development of NGS has greatly
gene in the metabolism, transport, and action of the drug promoted the development and application of metagenom-
and the changes of the expression level. The genetic vari- ics, making this technology quickly and widely used in the
ants associated with drug genes mainly include SNP, Indel, research of various environmental microorganisms, and
CNV, and SV. At present, with the reduction of the cost of has penetrated into medicine, agriculture, biology tech-
high-throughput sequencing technology and the shortening nology, and other fields. Using NGS could determine the
of sequencing time, it has also been successfully applied to DNA sequence of a complete bacterial genome in a single
clinical drug gene detection, including sequencing based sequence run, and information on resistance, virulence, and
on drug gene combination, WES, and WGS [90]. The com- information for typing is obtained. At present, NGS technol-
monly used analysis software for high-throughput sequenc- ogy has been applied in the field of clinical microorganisms,
ing of drug genes is shown in Table 23.2. The ultimate goal such as pathogen identification, genotypic resistance detec-
of clinical drug genetic testing is to guide the selection of tion, strain typing, epidemic traceability, outbreak monitor-
clinical medications and the adjustment of dosing doses. The ing, clinical specimen metagenomics research, and so on.
genes involved in clinical drug gene detection can be mainly The WGS detection process of pathogenic microorganisms
divided into four categories: drug metabolizing enzyme can be divided into two technical sections, namely, sample
gene, drug transporter gene, drug target gene, and other processing and sequencing and bioinformatics analysis of
the data. The definite steps can be seen in Fig. 23.6.
There are two main forms of NGS application in pathogen
Table 23.2 Common analytical software for high-throughput sequenc- identification, including rRNA gene sequencing and whole
ing data for drug genes
genome sequencing [95–97]. The rRNA gene sequencing
Function Software is commonly used to identify bacteria and fungi in clinical,
SNP analysis Atlas2, GotCloud, GATK
and it is also the basis of flora analysis. Bacterial 16S rRNA
Indel analysis Atlas2, Dindel, GATK
is called “molecular clock,” it is highly conserved in evo-
CNV analysis XHMM, CONSERTING
HLA allele analysis OptiType lution, and the sequence length is moderate, which is easy
Genotype UnifiedGenotyper to sequence. Therefore, 16S rRNA-targeted sequencing can
recognition quickly obtain bacterial species information, thereby achiev-
Variation annotation SnpEff, Variant Effect Predictor, ing rapid identification of bacteria. Because most viruses can-
PharmCAT not be cultured and are highly mutated, NGS is more efficient
Fig. 23.6 High-throughput WGS detection process for pathogenic microorganisms
than Sanger sequencing and gene chip methods and does not processes such as mitosis and meiosis in the absence of
require the design of specific probes. It is more suitable for changes in DNA sequences, resulting in changes in gene
virus identification in complex clinical specimens [98, 99]. In expression or phenotype. Epigenomics is a discipline that
2014, Wilson and colleagues [100] used Illumina to directly studies epigenetic modifications at the genome level and is
sequence cerebrospinal fluid specimens from patients with a new field of research in epigenetics. For the genome, not
recurrent meningitis to identify Leptospira. Follow-up PCR only the genetic sequence contains genetic information, but
and serological tests confirmed that the patient was infected also the modification can record genetic information [104].
with Leptospira. After targeted treatment based on the test There are many epigenetic phenomena, and the research
results, the patient recovered. In recent years, with the irreg- content mainly includes two types: one is the regulation of
ular use and even abuse of antibiotics, the drug resistance of gene-selective transcriptional expression, DNA/RNA meth-
bacteria has become increasingly prominent, and outbreaks ylation, gene imprinting, histone covalent modification, and
of drug-resistant strains have occurred in hospitals [101]. chromatin remodeling; the other is the posttranscriptional
Therefore, it is important to track and trace the epidemic regulation of genes, including regulation of noncoding RNA
bacteria. In 2012, Harris and colleagues [102] performed (ncRNA) in the genome, gene silencing, introns, and ribo-
type analysis of methicillin-resistant Staphylococcus aureus switches [105]. Understanding the epigenome is crucial due
(MRSA) outbreaks in hospitals through high-throughput to its purported involvement in myriad roles from individual
genome sequencing technology and confirmed that 26 hospi- diversity to development to cancer and other complex dis-
tal-related cases carried MRSA. Numerous research reports eases [106–108]. There are now a number of approaches to
have shown that the application of NGS technology in patho- profiling DNA methylation across a whole genome or large
gen typing and epidemic traceability has broad prospects and regions of a genome [109]. These methods bring the possibil-
is of great significance for controlling nosocomial outbreaks. ity of quantifying DNA methylation patterns and differences
However, further studies are required to improve the in DNA methylation [110]. Epigenomic research methods
workflow for NGS, in particular shorten the turnaround time based on high-throughput sequencing technologies include
for the library preparation and the runs on the NGS platforms whole genome bisulfite sequencing (WGBS), methylated
and further reducing costs. Faced with the huge amount of DNA coprecipitation sequencing (MeDIP-Seq), chromatin
sequence information obtained by NGS, how to classify, immunoprecipitation sequencing (ChIP-Seq), and various
store, analyze, and use these data is also a difficult problem RNA sequencing technologies. High-throughput sequencing
[103]. In short, NGS will undoubtedly become a powerful is used in epigenetic research. Currently, it focuses on DNA
tool for studying intestinal microorganisms and metagenom- methylation, histone modification, and noncoding RNA reg-
ics due to its large amount of data, low cost, and fast speed. ulation. The following describes the specific application of
NGS in epigenetics.
In general, the emergence of high-throughput sequenc-
23.4.7 Application of NGS in Epigenetic ing technology has led to the development of epigenetics,
Detection enabling people to study epigenetic modifications of DNA,
histones, etc. across the genome and to explore develop-
Epigenetics is the study of genetic information that can be mental and disease-related epigenetic variation providing a
transmitted between cells and individuals through genetic blueprint.
324 M. Wang
23.4.8 Application of NGS in Sequencing markers. The current clinical practice applications of tran-
of Immune Repertoire (IR) scriptome sequencing include gene expression pattern analy-
sis and detection of fusion genes. Compared to traditional
T lymphocytes and B lymphocytes are the most important expression profiling microarray technology, RNA-seq can
immune cells in the human body, responsible for cellular and detect the entire transcriptome of a research species without
humoral immunity, respectively. When the body is stimulated predesigning probes for known sequences. While analyzing
by an antigen, it can activate, divide, and proliferate, and a the structure and expression levels of transcripts, it is also
specific immune response occurs. At any given time point, the possible to simultaneously identify unknown transcripts and
sum of all specifically different T lymphocytes and B lympho- rare transcripts, accurately identify variable cleavage sites,
cyte clones in the individual constitutes the human IR [111]. detect fusion genes, and provide more comprehensive tran-
In recent years, NGS has greatly promoted the progress and scriptome information [119]. In addition, the applications
application of IR detection and analysis. Using NGS technol- of RNA sequencing analysis stretch further than character-
ogy for IR detection and analysis can simultaneously obtain ization of the transcriptome to differential gene expression,
millions or more of immunoglobulin (IG) and T cell receptor allelic imbalance [120, 121], eQTL mapping [122], RNA
(TCR) gene sequences from the same specimen, making true editing [123], RNA-protein interactions [124], and alterna-
omics analysis possible [112]. The immunological library is a tive splicing [125, 126]. RNA-seq offers sequencing of the
technique for studying the diversity of TCR or B cell recep- entire transcriptome, and ChIP-seq allows for sequencing the
tor (BCR)-encoding genes using high-throughput sequencing epigenetic architecture of the genome. Identifying genetic
technology, which can reflect T/B cell clonal changes and dis- variations in individuals can be used to predict disease risk,
eases [113]. TCR and BCR are composed of multiple peptide with the potential to halt or retard disease progression [127].
chains with antigen binding specificity, and the complemen- The main processes of RNA-seq include total RNA extrac-
tarity determining region (CDR) amino acid composition and tion, target RNA molecular enrichment, library construction,
arrangement order of each peptide chain are highly polymor- sequencing on the machine, and bioinformatics analysis.
phic [114]. At present, the emerging immune group library RNA-seq technology is a powerful tool for further study of
focuses on the diversity of CDR genes [115]. The improvement the complexity of the transcriptome and is now widely used
of IR detection and analysis technology using NGS provides a in biology, medical research, drug discovery, and so on.
good technical basis for more accurate analysis of IR diversity, The clinical application of transcriptome sequencing
accurate determination of sequence proportions, and realiza- mainly focuses on the study of disease-related gene func-
tion of highly sensitive clonality analysis. tion or the discovery of molecular markers. Transcriptome
In general, high-throughput sequencing technology has sequencing plays an important role in the study of disease
significantly reduced the cost of sequencing, while signifi- pathogenesis, as well as the diagnosis and prognosis of
cantly increasing the speed of sequencing, bringing revolu- diseases. At present, it is mainly divided into gene expres-
tionary advances in the research of immune libraries. The sion pattern analysis and detection of fusion genes. NGS
amount of data it obtains is closer to the true number of has proven effective in the analysis of retroviral biology in
human immunohistochemistry, making the study of immune relation to [128], for example, HIV infection and gamma-
pools into the era of big data. retroviral and lentiviral vector systems and has been applied
also in conjunction with anchored-type PCR enrichment to
determine positions of exogenous retroviral integration sites
23.4.9 Application of NGS in Transcriptomics in the DNA [129–133]. These newly discovered transcripts
not only help annotate the sequenced genome but also estab-
Transcriptomics refers to the process of studying a com- lish the basis for future functional research.
plete transcript of a given cell, tissue, or organism for a In summary, the use of NGS in transcriptome sequencing
given developmental stage or physiological condition. provides a more comprehensive transcriptome information,
The complete transcript is called the transcriptome and pushing RNA research to an unprecedented level.
includes mRNAs that encode proteins and ncRNAs (rRNA,
tRNA, and other ncRNAs). In recent years, transcriptome
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Digital PCR
24
Min Wang and Xianping Li
24.1 Overview Later, a method for qualitative analysis of the end point fluo-
rescence of PCR products was developed, but these tech-
In 1953, the DNA double helix structure and semi-reserved niques failed to achieve quantitative detection of target genes.
replication model proposed by Watson and Crick opened the In order to avoid the defects of conventional PCR and
curtain of modern life science and molecular biology. quantitatively analyze the expression level of PCR amplifica-
Polymerase chain reaction (PCR), molecular cloning, and tion products or genes, the “second-generation PCR” analy-
DNA sequence analysis are the foundation of modern molec- sis technology—real-time quantification PCR—was born in
ular biology research. Identifying unknown nucleic acid 1992. Real-time quantification PCR captures the fluorescent
sequences and examining target nucleic acid molecules qual- signal of a fluorescent probe-labeled target gene in the
itatively and quantitatively have become the core problems “extension” section of the polymerase chain reaction
to be solved in molecular biology research and application. “denaturation-anneal-extension” process. The copy number
In 1985, the PCR technology invented by American sci- of the target gene or the expression level of the gene is finally
entist Kary Mullis realized the desire of researchers to infi- determined by the relationship among the three parameters
nitely amplify nucleic acid fragments in vitro. PCR (fluorescence signal-Ct value-initial concentration of the tar-
technology makes DNA and RNA manipulation simple, get gene). The combination of real-time quantification PCR
while also enabling nucleic acid research to be detached and RT-qPCR enables quantitative detection of RNA and
from living organisms. Kary Mullis also won the 1993 Nobel gene expression analysis. Real-time quantification PCR
Prize in Chemistry. In 1988, Saiki and colleagues isolated a greatly expands the research and application of PCR technol-
hydrophilic bacterium from the hot springs of the Yellowstone ogy throughout life sciences in infectious diseases, tumors,
National Forest Park in the United States. A heat-resistant hereditary diseases, transplant matching, personalized medi-
DNA polymerase was extracted in its body. From then on, cine, and many other medical fields. Real-time quantification
researchers do not need to add extra enzymes to the reaction PCR has developed rapidly in areas such as clinical medical
system after each amplification reaction. This finding greatly testing and food safety testing and has become the gold stan-
improves the efficiency of PCR amplification. The PCR dard for the diagnosis of various pathogenic microorgan-
method has been widely used. PCR amplification technology isms. However, since the absolute quantitative analysis
laid the foundation for researching and solving the previous results of quantitative PCR ultimately depend on the Ct value
two core problems. and the standard curve, strictly speaking, “quantitative” is
In the more than 30 years since 1985, PCR analysis tech- only relative. Moreover, the detection sensitivity, accuracy,
nology has experienced three generations. The most tradi- and resolution of the low-copy target gene molecules and
tional “first-generation PCR” analysis technique uses gel template concentration are limited.
electrophoresis to conduct qualitative analysis and research With the increasing application requirements such as low-
on PCR products. However, it has the disadvantages of cum- abundance detection, rare mutation detection, and the devel-
bersome operation and high risk of cross-contamination. opment of microfluidic and MEMS technology, “the
third-generation PCR”—digital PCR—has emerged. Digital
PCR technology is an absolute quantification technique for
M. Wang (*) · X. Li nucleic acid molecules. It distributes a real-time PCR reac-
Department of Laboratory Medicine, The Second Xiangya tion system into a large number of tiny reaction vessels. One
Hospital of Central South University, Changsha, Hunan, or more copies of the target nucleic acid molecule are
e-mail: wangmin0000@csu.edu.cn
included or not included in each microreaction system.

330 M. Wang and X. Li
Digital PCR is accomplished by PCR amplification of a 24.2 Basic Principle

single-molecule template. After the end of the amplification,
the copy numbers of the target gene in the original sample 24.2.1 Statistics and Quantitative Principles
are calculated by the number of positive reaction units (deter- of Digital PCR
mined by the end point fluorescence signal) and statistical
methods. Digital PCR process contains at least three key links, namely,
Digital PCR allows accurate absolute quantitative detec- the sample dispersion, PCR amplification, fluorescence sig-
tion without relying on control samples and standard curves. nal acquisition, and data analysis. One sample is deliquated
In addition, since the digital PCR only judges two amplifica- and separated into hundreds or even millions of different
tion states of “yes/no” when interpreting the results, so there reaction units, so that each contains one or no copy of the
is no need to detect the intersection of the fluorescence signal target molecule (DNA template), and then independent par-
and the threshold line, completely independent of the Ct allel amplification reaction is performed. By counting the
value. Therefore, the influence of amplification efficiency on number of “positive” regions (where the sequence is
digital PCR reaction and result judgment is greatly reduced, detected) and “negative” regions (where the sequence is not
and the tolerance to PCR inhibitor is greatly improved. The detected), scientists can identify the number of DNA mole-
process of standard reaction system assignment in digital cule copies in the primitive sample (Fig. 24.1). After PCR,
PCR experiments can greatly reduce the concentration of the score of the amplification-positive partitions is utilized to
background sequences that compete with the target sequence. quantify the concentration of the target sequence, using sta-
Therefore, digital PCR technology is also particularly suit- tistical accuracy defined by Poisson statistics [1].
able for detecting rare mutations in complex backgrounds. Reaction unit containing target DNA amplification after
Digital PCR has superior sensitivity, specificity, and accu- the fluorescent signal strength reaches a certain level, the
racy over traditional real-time quantification PCR. The reaction unit can be seen as positive (red cell). DNA content
advantages have been widely proven in detecting trace of zero-response unit almost can hardly detect fluorescent
nucleic acid samples, detecting rare mutations in complex signal, regarded as the negative unit (gray). Digital PCR
backgrounds, identifying small differences in expression lev- reaction unit contains starting DNA copy number of x,
els, and detecting copy number variations. It also has broad according to the theory of mathematical statistics, x = k
application prospects in many aspects like gene expression (k = 0, 1, 2, 3…). The probability distribution function of p
research, microRNA, genomic copy number identification, in accordance with the type of Poisson probability model,
rare mutations detection, pathogenic microorganism identifi- the λ for reaction unit contains average molecular copy
cation, transgenic components identification, single-cell number:
gene expression, accurate quantification of NGS sequencing
ke
libraries, and validation of results. Px k (24.1)
From qualitative analysis of gel electrophoresis to “relative k!
quantification” of real-time quantitative PCR to absolute Therefore, getting the value of λ can achieve nucleic acid
quantitative analysis of digital PCR, three generations of PCR quantitative detection. Total reaction unit is n, and negative
analysis techniques are used to qualitatively and quantitatively cell number is b, using p as the ratio of negative cell. So,
detect target nucleic acid molecules. Digital PCR is undoubt- combining the Formula (Eq. 24.1), we can have the follow-
edly the most accurate and sensitive technology available. ing derivation:
Fig. 24.1 Principles of digital PCR

24 Digital PCR 331
b When we get the total volume (V) of the experimental

P p x 0 e (24.2) samples (each unit volume v), the condition of sample con-
n
centration c (copy/μL) can be calculated according to the fol-
Therefore, the average numbers of nucleic acid copies in lowing formula:
reaction units can only be determined by the proportion of
n
negative units in the digital PCR quantification, that is, to c (24.8)
say, the exact quantity of DNA can be achieved. Digital PCR V V /n v
principle determines that it can directly calculate the copy Statistically, the average λ of initial template concentra-
numbers of target sequence without depending on the control tion derived from the Poisson probability model from one
and absolute quantitative standard curve for accurate abso- test is probably not the true template concentration. The true
lute quantification. concentration may be in a range of λ value accessories.
According to Formula (Eq. 24.1) of Poisson distribution Except the average λ, the quantitative results of digital PCR
model, its statistical expectations μ and variance σ can have need to combine the confidence interval and confidence
the following derivation: level. In digital PCR detection, the confidence intervals of
sample are real concentration at a certain probability to fall

x 1
u x p x; e e e (24.3) around the measurement results of λ interval. The probability
x 0 x 1 x 1 ! is called the confidence level. The two ends of the confidence

interval are called confidence limit. The higher the confi-

2 x p x;
2 dence level is, the broader the confidence interval will be and
x 0 the fuzzier the result will be. Too high or too low confidence

level is disadvantageous to test results. Generally, it needs to
x x 0 2 1 x 2 p x;
x 0
calculate the confidence interval corresponding to 95% con-
fidence level in digital PCR [2].
x x 0 p x;0 2 1 xp x; 2 p x;
x 0 x 0 x 0
24.2.1.1 C onfidence Interval in Absolute
2

2 1 2
Quantification
When we analyze the absolute quantification, first consider a

special case: a large number of DNA molecules are com-
(24.4) pletely random distributed in an unlimited number and the
same reaction unit volume of microfluidic chip. Real con-
By the Formula (Eq. 24.3), the digital PCR reaction cell centration of target molecules is expressed with λ. The prob-
contains the target DNA molecules with average copy num- ability of positive cells is P. The λ and P are unknown
ber of λ; λ value can thus obtain senior quantitative detection parameters to population. Actual digital PCR experiments
of nucleic acid. P is the probability of positive results derived can be regarded as finite approximation of this situation.
from the distribution of negative and positive units, that is, In digital PCR, whether a cell contains the target nucleic
the possibility of the unit containing the target genes. acids or not is similar to the binomial distribution of flipping
Combining with Formula (Eq. 24.1) can have the following a coin in the experiment for many times—if the reaction unit
derivation: is positive, it can be seen as a positive event in toss of a coin.
Conversely, the coin is tails up. Therefore, we can analyze
P 1 p x 0 1 e (24.5)
this issue from the view of binomial distribution.
There are M DNA molecules distributed in n units; the
In practical operation, after dispersion, the total number probability of any molecule belonging to a particular unit
of unit is n, the number of negative unit is b, and the number will be 1/n. Thus, the probability of a certain unit which con-
of positive unit is h. Combining with Formula (Eq. 24.5), the tains at least one molecule is P. It can be represented by a
probability of positive results can be expressed as the follow- binomial distribution:
ing formula: M
1
b h P 1 1 (24.9)
P 1 1 e (24.6) n
n n

According to Formula (Eq. 24.6), the average template Because m = λn, and combining Formula (Eq. 24.6), can
copy number of each reaction unit can be calculated by the have the following derivation:
following formula: M M
1
ln 1 P (24.7) 1 P 1, 1, (24.10)
n M
The current digital PCR system has achieved the sample The problem of arbitrary sampling distribution can be
of a large number of discretization; the total number of units solved by means of the geometric interpretation of Fieller’s
n can come up to tens of thousands or even 1 million. For n theorem.
of this size, the right-hand side of Formula (Eq. 24.10) tends Suppose g1 1
and g2 2
are normal. For
to e-λ; then we can deduce the interaction between λ and P:
λ = ln(1–P).
1 and 2 , the ratio r 1 . can be expressed as the
In actual digital PCR experiments, if the number of the 2
total reaction units and positive units are n and h, P̂ can be slope of a straight line on the two-dimensional plane passing
used as an unbiased estimate of P and the expectation of P̂
equals to P. When the total number n is big enough, the

through the origin and the 2D point 1 , 2 . Luxburg shows
sample distribution function  P of P can be seen as nor-
how to construct a confidence ellipse on a two-dimensional
mal distribution. Therefore, the standard deviation σ̂ of P
plane. Thinking about two lines passing through the origin,
can be expressed by the following formula:
which is tangent to the ellipse, the intersection of these lines
with the vertical line at λ2 = 1 gives the required confidence
ˆ 1 P P n (24.11)
interval.

In the actual calculation of σ̂ , the P of Formula (Eq. 24.11)

The means of g1 λ1 ) and g2 2 are λ1and λ2, respec-
tively. The standard deviations are defined as σx and σy,
can be directly substituted by P̂ . The upper and lower limits respectively. For the given estimates λ1 and λ 2 , supposing
of PH confidence interval are PˆH , PˆL , respectively. According that distributions are normal, the analysis shows that the
to the reference, the limit of confidence interval can be given boundary of the confidence ellipse for a given confidence
as follows: interval Zc would be given by:
ˆ
PLow, High Pˆ  1.96
11Pˆ Pˆ (24.12) x y
2
2
1
2
n Zc2 (24.15)
x2 y2

Since the probability density functions in any given dif- So in a reasonable approximate, when we think it is nor-
ferential region, the probability remains the same under vari-
mal, the asymmetric distribution of the four quadrants of the
able change; the 95% confidence interval PˆL ,PˆH is directly
given as follows:
coordinate system is focused on the  , . Then the confi-
2 1
PˆL ln 1n PL (24.13) dence region is formed by an elliptic region of four
quadrants.
Let the asymmetric confidence intervals of the specified
PˆH ln 1n PH (24.14)

zc and the two concentrations be 1 H B ,1 HT and
2 WL ,2 WR
The Confidence Interval of Relative Quantification .
CNV is referred to the 500 bp length of the rearrangement If WR = WL = zcσx and HT = HB = zcσy, it is a symmetric
for the genome copy number increasing or decreasing. The case. Using simple algebra, it can be displayed with symme-
main manifestation is the absence and repetition of submi- try. The slopes of lines will be tangents to the ellipses of the
croscopic level. Whole-genome studies have confirmed that four quadrants of the union.
CNV is closely related to a variety of human genetic diseases
and has become an important application field of digital
PCR. rL
2

1 2 1 2 H B 2 1 WR 2 2
2
(24.16)
2
In CNV, our aim is to determine the exact concentration 2 WR 2

ratio of two genes (one is the reference gene, and the other is
the test gene) and the related confidence interval, which we
will complete in the next section.
r
2

1 2 1 2 HT 2 1 WL 2 2
2

Let’s test the distribution of sampling genes and reference H 2
(24.17)
2 WL 2

genes which will be g1 1 and g2 2 , respectively. If
these distributions are normal, we can use Fieller’s theorem. The formula listed above can be used as an approxima-
However, as described in the previous part, we cannot make tion. The numerical algorithm does not make any assump-
such hypothesis in summary. tions and is applicable to any sampling distribution.
24 Digital PCR 333
24.2.2 Experimental Principle of Digital PCR determined by studying the melting curve of each reaction.
They are also associated with very low-volume variability,
The strategy of digital PCR (dPCR) has been summarized as which contributes to shorten the uncertainty linked with
“divide and conquer”: dilute and divide a sample into hun- numerical counting analysis. However, for the purpose of
dreds or even millions of independent reaction units, each of minimizing chip size, the count of zones in various systems
which contains one or no copies of the related sequence. By is generally confined to a few thousand. Several dPCR sys-
counting the number of “positive” partitions (sequence tems with microchambers have been developed, of which the
detected) and “negative” partitions (not detectable Digital Array chip 12.765 (Fluidigm, South San Francisco,
sequences), scientists can accurately determine the copy CA, USA) can analyze 12 different samples at the same time,
number of a DNA molecule in the original sample. each of which is divided into 765 closed partitions with a
In digital PCR, the diluted template is dispersed into each volume of 6 nL or QuantStudio 3D (Thermo Fisher Scientific,
PCR reaction, and the amount of the original templates is Waltham, MA, USA), which can analyze 24 chips at the
measured by analyzing the positive amplification partition same time, each chip containing 20,000 partitions with a
number of the total number of templates. The distribution of 0.8 nL internal volume.
molecules throughout the entire reaction distribution is a ran- However, as the detection and dynamic ranges of low-
dom, independent process. The optimal template dilution abundance DNA point mutations need to be improved, more
generally ensures that just one target molecule is detected in digital segmentation platforms need to be provided.
each reaction partition when higher template concentrations Moreover, increasing the count of regions can also improve
are used; because some partitions contain more than one the accuracy, which can solve the concentration differences
template molecule, the actual number of molecules is under- among nucleic acid targets in the sample. This leads to the
estimated. This distortion can be rectified by Poisson statis- production of emulsion-type PCR or droplet dPCR (ddPCR).
tics to some extent. The presence or absence of specific PCR ddPCR is constitutive of water droplets diffused in oil for
products is detected using fluorescence chemistry. Finally, the isolation of PCR reactions, which make us imagine a lot
data interpretation is less dependent on complex bioinfor- of theoretically infinite separation space (resting with the
matics analysis. In some applications, the sequential proba- volume of the water droplets and the volume of available
bility ratio test can be given to detect the strength of evidence sample). The aqueous phase includes the target DNA and
that the allele distribution is different from the normal. After the reaction mixture. A number of systems have been
allele-specific PCR reaction with a fluorescent probe, each exploited, of which the QX200 system (Bio-Rad, Hercules,
fragment was tested separately, and the result of 1 or 0 was CA, USA) can analyze 96 wells at the same time, each well
obtained, indicating whether there was a copy of the target contains up to 20,000 droplets with a volume of 1 nL, and
DNA. With Poisson function = −ln(1–p), the positive and the RainDrop system (RainDance Technologies, Lexington,
negative event counts for each target are concerned with the MA, USA), which can analyze 8 wells at the same time,
target concentration in the sample, where k is the average each well contains up to 10 million droplets with a volume
number of copies per partition and p is the number of posi- of 5 pL. In the latest system, the Naica™ system (Stilla
tive partitions and total partition ratio. By knowing the reac- Technologies, Villejuif, France), the sample first flows
tive volume of each zone, the copy number of each zone can through a microchannel network and is divided into a large
be converted to the number of copies per liter, and the abso- two-dimensional array of 25,000–30,000 individual drop-
lute copy number of the initial target in the test can be lets. This array is called droplet crystal. Four droplet crys-
obtained without using a standard sample. When technical tals can be analyzed simultaneously. However, there are
replication is valid, all replications can be collected to some limits presenting in ddPCR technologies. First, since
acquire a onefold concentration estimate. Therefore, the the droplets are produced continuously, it takes minutes to
actional range of target DNA quantification can be expanded. hours to produce enough separations. Furthermore, the vol-
The first dPCR platform was implemented by microflu- ume uncertainty of a droplet system is a key issue that is
idic chip (Fig. 24.2), allowing real-time monitoring of the often underestimated. In fact, one of the key assumptions of
target molecule to infer false-positive reactions from the dPCR is that the assignment of DNA templates is wild, and
magnified curve of each single reaction. The dPCR method the possibility of each partition is equal. However, if the vol-
of microchip allows the reaction to take place in a micro- ume between partitions is different, the latter cannot be sat-
chamber, which is generally nano-sized. These platforms isfied; this is often the case during droplet formation. Ideally,
show many superiorities such as user-friendliness, occasion the volume changes of droplets should be viewed when cast-
for automated system injection of sample analysis response, ing k, especially in cases where accurate measurement is
and the possibility of real-time monitoring of fluorescence important, as per quantitative applications in the noninva-
signal situations, so that a false-positive reaction can be sive prenatal diagnosis (NIPD) field.
Fig. 24.2 Experimental
principle of digital PCR 1. Sample preparation
Target
Background
Target
Background
Hydrolysis probe assays
2. Partitionning
Chip Digital PCR Droplet Digital PCR
3. PCR amplification
Positive reactions
Target
Background
Target+Background
Negative
4. Counting & Analyzing

Target Target+Background
Fluorescence
Negative Background
Fluorescence
24 Digital PCR 335
24.2.3 Technology Development 24.2.3.2 Proposal of Digital PCR Concept

Vogelstein and Kinzler introduced this concept of digital
24.2.3.1 The Germination of Digital PCR PCR in 1999. They described a method to convert the PCR
In 1988, Kary Mullis, the inventor of PCR technology, pub- exponential analog properties into linear digital signals suit-
lished an article on Science called “Primer-Directed DNA able for the purpose. Individual molecules were isolated by
Enzymatic Amplification Using Thermostable DNA dilution and amplified separately via PCR; mutations of each
Polymerase.” This article pointed out that the β-globin gene product were then analyzed separately by fluorescent probes.
can still be amplified to a detectable amount after 40 PCR In the stool of patients with colorectal cancer, the feasibility
cycles when the gene sample was diluted from one copy in of this method was demonstrated by detecting mutant RAS
one genome to one copy in 106 genomes. This provided the oncogenes [6, 7].
theoretical and practical basis for single-hole single-copy The two steps of digital PCR are shown in Fig. 24.3. DNA
detection [3]. template was diluted to an average concentration of one copy
In 1990, Claiborne Stephens from Yale University per two reaction holes and amplified by PCR under opti-
Medicine Center used single-molecule dilution (SMD) PCR mized experimental conditions. Secondly, by adding fluores-
and biphasic polymerase chain reaction (booster PCR) to cent probes, the fluorescent signals of each hole were
study haploid DNA. This study further laid the theoretical analyzed to determine whether wild-type and mutant
foundation for single-molecule dilution in the quantitative sequences were present in the PCR amplification products
analysis of DNA [4]. and the corresponding proportional relationships [8].
In 1992, Sykes, from Flinders Medical Center in South Based on the theory and in combination with reality, the
Australia, described a data correction model based on lim- experimental conditions were optimized. In digital PCR, the
ited sample dilution, PCR, and Poisson distribution. Low- first step was PCR amplification from a single template mol-
abundance immunoglobulin heavy chain (lgH) mutant genes ecule. Nested procedures were used for amplification from a
from leukemia clones were detected in a complex back- small number of template molecules on most protocols that
ground using two-step PCR (nested PCR), and very fine use a set of primers to generate PCR products, because
quantitative studies were carried out. Although the method many applications of digital PCR require hundreds or thou-
used at that time was not named as “digital PCR,” they estab- sands of separate ones, and such nesting can be very ineffi-
lished the basic experimental procedure of digital PCR. Most cient. Convenient may cause pollution problems. Therefore,
importantly, they proposed an extremely important principle they sought the condition to achieve robust amplification
in the digital PCR “all-or-none end point” as a result inter- without nesting. The most important of these conditions is
pretation mark [5]. the use of a polymerase that can be activated only after heat-
Fig. 24.3 Experimental principle of digital PCR

ing and optimal concentrations of primers, dNTPs, tempera- asymmetric PCR. The DNA product hybridizes with two
ture, and buffer composition. The second step of digital fluorescent molecular beacons, respectively. The wild type
PCR involves detecting these PCR products. It is important and the mutant type were distinguished by fluorescent color
to modify the standard MB probe method to make it work and analyzed by the number and ratio of the number of dif-
effectively in digital PCR applications. In theory, a single ferent fluorescent reaction units [9].
MB probe can be used to detect each specific mutation. The Vogelstein and Kinzler’s original “digital PCR” method is
properties of the PCR products will be identified by includ- tedious, manual Digital PCR is a very powerful, reliable, and
ing one MB corresponding to the WT sequence and another accurate rare mutation detection method. But it is also a
MB corresponding to the mutated sequence. It is expected labor-intensive technology, so it is difficult to implement it
that some different mutations will occur in the same test widely.
sequence. They have tried to develop a single probe that
responds better to the WT sequence than any mutant 24.2.3.3 BEAMing Digital PCR
sequence in the test sequence. They found that the length of In 2003, Devin Dressman and colleagues proposed a solid-
the loop sequence, its melting temperature, and the length phase digital PCR method based on magnetic beads and
and sequence of the stem were critical to determining the microemulsion. Each DNA molecule is translated into a sin-
efficacy of these probes. Because of the variety of mutation gle magnetic particle. Such a single magnetic particle has
types and the simplification of the experimental process, thousands of DNAs identical to the original sequence. This
they designed two molecular beacon probes with different bead population then corresponds to a one-to-one represen-
fluorophores to hybridize with the PCR products, respec- tation of the starting DNA molecule. Fluorescently labeled
tively. One of the probes can be hybridized to the wild type particles can then be counted by flow cytometry to easily
and the mutant type as a control, and the other probe can assess variations within the original population of DNA mol-
only hybridize to the wild-type sequence. The number and ecules. The principle is illustrated in Fig. 24.4. BEAMing
ratio of alleles (wild type and mutant) of the same sample can be used to identify and quantify rare mutations and to
can be obtained by detecting the fluorescence signal in each study variations in gene sequences or transcripts in a specific
well. Statistical methods can be used to analyze significant population or tissue [10].
differences between samples. PCR products including wild BEAMing technology encapsulates DNA templates and
type and mutant type were obtained by a pair of universal magnetic beads attached to primers in droplets at very low
primers. A single-stranded DNA molecule is obtained by concentrations (such as a single copy). Droplets are units of
Fig. 24.4 Principle of BEAMing digital PCR technology

24 Digital PCR 337
nanoliters formed by oil-water two phases that rise to the extension products from the two different kinds of
skin. A PCR amplification reaction was carried out in these templates.
droplets. The amplified product is enriched on the surface of • The beads are purified with a magnet and the emulsions
the magnetic beads. After the demulsification is collected, are broken.
the product is tested and analyzed. The specific steps are as • After denaturation, magnetic beads are incubated with
follows: oligonucleotides that can distinguish between different
template sequences. The bound hybridization probe is
• Combine magnetic beads with streptavidin bound to bio- then labeled with a fluorescent-labeled antibody. After
tinylated oligonucleotides (oligonucleotides). appropriate laser treatment, the beads containing the PCR
• The intermediate compartments contain an average of product turn red or green.
less than one template molecule and less than one bead. • Flow cytometry is used to count red and green beads.
Red and green templates represent two template mole-
cules, the sequences of which differ by one or more Subsequently, Frank Diehl and colleagues adjusted the
nucleotides. BEAMing technology to meet the needs of low-abundance
• As in conventional PCR, microemulsions are temperature gene mutation detection and quantitative analysis. The spe-
cycled. If a DNA template and a bead are present together cific process is shown in Fig. 24.5.
in a single aqueous compartment, the bead-bound oligo-
nucleotides act as primers for amplification. The straight • Real-time PCR is used to determine the number of total
red and green lines connected to the beads represent gene fragments in the plasma sample (Fig. 24.5a, step 1).
Fig. 24.5 Experimental principle based on BEAMing technology

• BEAMing is used to transform the amplified DNA into 24.2.3.4 Microfluidic Digital PCR
beads (Fig. 24.5a, steps 2–4). With the development of nanofabrication technology, nano-
• Determine the mutation status of the extended beads by fabrication, and microfluidics, digital PCR technology has
single-base extension (Fig. 24.5b). encountered the best opportunity to break through the tech-
• Flow cytometer is used to measure Cy5, PE, and FITC nical bottleneck. In 1997, Kalinina, Brown, and Sliver
signals of a single bead simultaneously. obtained a US patent for monoclonal template PCR amplifi-
cation using a nanoscale chip. More importantly, this “elec-
BEAMing technology can remove unwanted fluorescent tric immersion” nano-upgrading chip makes microfluidic
probes by solid-liquid separation, thereby reducing signal digital PCR reveal the prototype. In 2006, Fluidigm launched
interference from background fluorescence. Moreover, com- the first commercial chip-based commercial digital PCR sys-
mon fluorescent probes can be used instead of high-cost tem. Fluidigm’s IFC platform uses a physical matrix strat-
molecular beacons and TaqMan probes to reduce the cost of egy. Using the company’s microfluidic expertise, their
the experiment. BEAMing has three major application qdPCR37K system “integrated flow path” distributes 48
advantages: first, it can evaluate (quantitatively analyze) tens samples one by one in 770 microreaction units. In order to
of thousands of DNA molecules under normal laboratory improve the accuracy, each sample was dispersed in a plural-
conditions. Moreover, this method can adjust the magnetic ity of sets of microreaction units. Up to 37,000 microreaction
bead concentration according to the experimental require- units can be divided into each sample. The “water-in-oil
ments to obtain higher detection sensitivity. Second, specific PCR” technology developed with the second-generation
mutations can be screened by flow cytometry for subsequent sequencing technology can generate tens of thousands or
analysis and research. Finally, identification and quantifica- even millions of nanoliters of individual water-in-oil drop-
tion can be provided for mutations that are rare in a particular lets. These water-in-oil microdroplets can be used as sample
tissue or population, as well as variations in overall gene dispersion carriers for dPCR.
sequences or transcripts. However, this method requires that Droplet digital PCR is derived from the emulsion PCR
a single target molecule is wrapped in a single droplet with a technique. The DNA template and the magnetic micro-
single magnetic bead. Because this operation is complicated spheres to which the primers are attached are wrapped in the
and difficult, a large number of conditional optimization oil-water phase at a very low concentration. The volume of
experiments are required. Moreover, the emulsification the droplets is nanoliter to skin upgrade. PCR amplification
method is rough, so the uniformity of the droplet size is poor. is subsequently performed. The amplified product is enriched
These factors limit the further improvement in detection on magnetic microspheres. Finally, the oil and water were
accuracy. separated and sequenced. The PCR reaction system in units
In general, the principle of digital PCR technology is of droplet is achieved by two-phase separation of oil and
quite simple. The basic principle is to achieve single- water. This reaction system is easier to achieve small volume
molecule nucleic acid amplification in each unit by sample and high throughput than microplates and IFC systems.
dispersion. Although digital PCR technology has broad Because of its simple system and low cost, it can be an ideal
application prospects, it is limited by the technical condi- digital PCR platform.
tions at that time. Early digital PCR mostly used 96-well QuantaLife has developed microdroplet digital PCR
plates and 384-well plates as carriers for dispersion reac- technology using water-in-oil droplet generation technol-
tions. However, the sensitivity and accuracy of digital PCR ogy. This is the first relatively mature digital PCR platform.
largely depend on the total number of reaction units. It basically meets the standard of commercialization in
Therefore, the analysis using ordinary 96/384-well plates terms of operating costs and stability of experimental
cannot be further improved not only in the degree of disper- results. In 2011, QuantaLife was acquired by Bio-Rad. The
sion but also in the volume of the data population. The high microdrop digital PCR was renamed the QX100TM model
cost of reagents in this method was also a major factor series and continued to be on the market. This type of instru-
restricting the development of digital PCR at the time. In ment has played a major role in promoting the popularity of
2008, Inostics of Germany introduced BEAMing dPCR the digital PCR concept and the expansion of its application
detection service based on magnetic bead emulsion amplifi- field. This company then launched its upgraded model
cation and flow detection. However, these methods cannot QX200TM in 2013.
achieve more detailed requirements in terms of the degree of In 2012, RainDance introduced the RainDrop™ digital
dispersion and the volume of the data population. Moreover, PCR device. This device translates its original second-
time and consumable costs severely limit the development of generation sequencing library preparation platform tech-
dPCR technology [11]. nology to the digital PCR technology platform. Each
24 Digital PCR 339
standard reaction system is divided into reaction emulsions target sequences, which enhances intra-batch stability and
containing from 1 million to 10 million picograde droplets inter-batch stability.
driven by a high-pressure gas. DrenLink, one of the com- • Analysis of gene expression can be realized based on high
pany’s founders, said the ultrahigh number of droplets pro- repeatability and accuracy of absolute quantification of
vides users with a higher detection dynamic range and is digital PCR, e.g., allelic expression imbalance and expres-
suitable for processing samples with larger concentration sion of microRNA.
differences. • Digital PCR performs better sensitivity in detection com-
In 2013, Life Technologies introduced the QuantStudio pared to other technologies, which has wide utilization on
3D digital PCR system. This system adopts high-density individualized medicine and liquid biopsy technique,
nano-level flow control technology. It distributes the sample detection of pathogenic microorganism, detection of
evenly into 20,000 but separate reaction wells. Samples genetically modified components, and food safety.
should be completely isolated from each other throughout
the workflow. The experimental design should try to prevent In summary, newly developed detection method and
cross-contamination of the sample, reduce the pipetting pro- quantitative molecular counting method endow digital PCR
cess, and simplify the operation steps. At the same time, this with many advantages, improving the sensitivity, accuracy,
chip design avoids the problem of pipeline blockage that precision, and repeatability of detection on a large margin. It
may occur in the microdrop system. has some indispensable values on life sciences including giv-
ing accurate analysis with very fine concentration difference
or extremely low content, detecting mutation sequence with
24.2.4 Clinical Application low frequency on the background of wild-type sequence
with high abundance, and calibrating certified reference
24.2.4.1 A pplication Characteristics materials of nucleic acid. The essence of digital PCR is a
of Digital PCR large scale of fluorescence PCR on single molecular level.
During the last 20 years of development and utility, real-time Nowadays, next-generation sequencing (NGS), DNA micro-
fluorescence quantitative PCR (qPCR) has been widely array, and gene-wide association study (GWAS) can obtain
applied on the field of industrial development and molecular large amount of information at a lower cost. However, digital
diagnosis. In the field of fundamental research, qPCR has PCR are able to verify this unselected information as far as
made magnificent achievement. However, big problems current technology permits. To a certain extent, the function
remain on its detection sensitivity, repeatability error, and of digital PCR is to confirm information on gene levels,
effective inhibition of nonspecific amplification. Since the which contributes to ample scope for its utility and unsubsti-
first concept of digital PCR being put forward, scientists are tuted status in the foreseeable future. Plenty of researches
expecting to realize absolute quantitative and low-abundance have been carried out based on digital PCR. This chapter will
sample detection through sample dispersion [12]. Due to conclude its application fields from perspectives of life sci-
technical bottleneck on technologies of automatic dispersion ences, individualized medication, food safety, environment
of samples, the real technique has not been well developed surveillance, and other fields related.
until recently. The major breakthrough from qPCR to digital
PCR is to detect nucleic acid molecules which were dis- 24.2.4.2 A pplication of Digital PCR
persed in several independent microunits, so as to achieve on Genomics
absolute quantification. Gene mutations are considered to be the focus of research,
Many researchers have accomplished detailed compari- and the rapid development of biomedicine has made momen-
son between qPCR and dPCR, whose advantages and disad- tous progress in the assessment of gene mutations. The
vantages can be summarized as follows [13]: advent of digital polymerase chain reaction (dPCR) has
raised the detection of gene mutations, especially in cancer-
• In terms of existing digital PCR equipment on the market, related genes, to unprecedented level of precision. dPCR has
qPCR takes great advantage on its linear range, which been used to detect tumor markers in cell-free DNA (cfDNA)
could detect different levels of abundance in various sam- samples from patients with different types of cancer, such as
ples. qPCR has realized automation under high-throughput cerebrospinal fluid, plasma, urine, and sputum, which are
conditions. conferred in clinical applications. dPCR is of significant
• Digital PCR could achieve absolute quantity at single value on basic research. Being a new technology, dPCR has
molecular level, completely taking off the trembles of specific limitations, such as the necessity of a large number
standard curve, while directly giving out copy number of of samples and high allele-specific probes.
24.2.4.3 A
pplication of Digital PCR below the detection limit. KRAS mutation is a predictor of
on Translational Medicine ineffective EGFR treatment for metastatic colorectal cancer
and Precision Medicine (MCRC). However, only half of non-mutated patients would
benefit from the prediction. Laurent-Puig and colleagues
Individualized Medicine and Liquid Biopsy Technique [15] discovered that in 136 samples of wild type, KRA,
Precision medicine is a new field of disease prevention and NRAS, and BRAF tumors were selected from two groups of
treatment. In January 2015, the US President Barack Obama MCRC patients. Tumor materials are sufficient for highly
proposed the concept of precision medicine, and digital sensitive pico-droplet-digital-PCR (dPCR) and 41 KRAS
PCR is listed among nine individualized tumor detection mutation tumors. All patients were treated with anti-
technologies. In September 2015, MIT Technology Review EGFR. Mutations in KRAS or BRAF could be detected by
published “Breakthrough Technology 2015,” and liquid digital PCR. Therefore, the performance of digital PCR is
biopsy is listed. Being a fast expanding field, liquid biopsy more suitable for formulating individual treatment strategies
plays important roles in multiple links in precision medi- for patients.
cine. It can be applied to early diagnosis, metastasis and
recurrence, risk assessment, real-time monitoring of effi- Application of Digital PCR on Drug Resistance
cacy, drug targets, and gene-resistant detection. Almost all Surveillance
body fluid could be served as samples including whole Genetic heterogeneity is one of the main mechanisms why
blood, plasma, serum, saliva, pleural effusion, taxes, cere- tumors are prone to acquired drug resistance. This heteroge-
brospinal fluid and urine, etc. The concept of liquid biopsy neity exists not only in the tumor itself but also in different
actually solves one crucial problem: the new samples. Based metastatic foci. Therefore, the targeted treatment of tumor
on cfDNA or ctDNA, we can integrally manipulate the sta- always goes through a short, successful initial stage, and
tus of cancer progression and eliminate spatial heterogene- then the therapeutic effect will be limited by the occurrence
ity. In addition, samples could be collected with few of acquired drug resistance. It is estimated that most patients
difficulties at almost any moment, which eliminates time with NSCLC take the first or second generation of EGFR-
heterogeneity. There have been quite a lot of researches ver- TKI for about 1 year, and some mutations lead to acquired
ifying the good correlation between ctDNA and tissue sam- drug resistance, of which 50% to 60% are EGFR-T790M
ples. In another way, ctDNA can reflect the level of tumor mutations. Therefore, it is not enough to rely solely on the
cells in tissues or human organs. This chapter aims to intro- results of a single biopsy to guide the treatment of patients,
duce the new breakthrough in individualized medicine and or some patients cannot remove the lesion again, and it is
liquid biopsy technique. impossible to perform multiple biopsies. The collection of
Digital PCR is a new technology which enables highly ctDNA from peripheral blood can be used as a noninvasive
quantitative analysis of nucleic acid targets with high sensi- method to evaluate the mutation status of tumors. ctDNA is a
tivity. Digital PCR combined with liquid biopsy can be used DNA fragment released from tumor cells into peripheral
to detect infinitely small nucleic acid samples and rare muta- blood, which can be detected in acellular serum or plasma. It
tions. It is especially suitable for sampling cancer patients is especially suitable for frequent sampling in drug resis-
who need repeated testing. Digital PCR can provide suffi- tance monitoring. A retrospective study of 135 patients with
cient genetic information in cancer diagnosis and recurrence advanced non-small cell lung cancer (NSCLC) was carried
monitoring. out by Wang and colleagues [16] with amplified refractory
Only when there is exon 21 L858R mutation or exon 19 mutation system (ARMS) and denaturing high-performance
deletion in EGFR, patients with non-small cell lung cancer liquid chromatography (DHPLC). The plasma EGFR-
(NSCLC) can benefit from gefitinib. Lee and colleagues [14] sensitive mutations and T790M mutations of 135 NSCLC
used PCR to detect L858R mutation and exon 19 deletion in patients before and after TKI were studied for a progression-
plasma cfDNA, which were consistent with tissue test free survival (PFS) of more than 6 months. The results
results, 87% and 86.2%, respectively. The great advantages showed that the detection limit of T790M mutation by dPCR
of dPCR in the detection of rare mutations are revealed. was about 0.03%. The frequency of detection of T790M by
Statistically, anti-EGFR therapies, such as cetuximab or dPCR was higher than that of plasma samples before TKI
panitumumab, are only effective in about 10–20% of colorec- (31.3% to 5.5%) and after TKI (43.0% to 25.2%). Therefore,
tal cancer patients, because ras/raf/mapk is the downstream the qualitative and quantitative detection of T790M in plasma
pathway of EGFR. Once a gene mutation occurs in the path- by digital PCR provides a sensitive and non-evasive method
way, it eventually affects anti-EGFR therapy. However, these for predicting the prognosis of EGFR-TKI.
mutations were not detected in a large number of patients, Acquired ESR1 mutation is the primary mechanism of
which proved to be insensitive to cetuximab. According to drug resistance in the breast cancer endocrine therapy. By
the existing technology, their mutation is most likely to be collecting plasma cfDNA regularly, we can monitor the drug
24 Digital PCR 341
resistance of breast cancer during endocrine therapy. Once In the investigation and safety evaluation of transgenic
detected, it can be replaced by other treatments in time to organisms, the way foreign genes integrated into receptor
ensure the effectiveness of the treatment. Guttery and col- genomes largely affect the expression of purpose genes and
leagues [17] developed a targeted 23-amplifier NGS group proteins. Genetic stability faces challenges, especially on its
for the detection of ESR1, PIK3CA, TP53, fibroblasts, copy number. Currently, qPCR and Southern blot are two
FGFR1, and FGFR2 in 48 patients with estrogen receptor- main methods to analyze foreign gene copy numbers. qPCR
alpha-positive metastatic breast cancer who received sys- relies on the standard curve and gene with known copy num-
temic therapy. The selected hotspot mutation was verified by ber. Southern blot, on the other hand, requires long working
digital PCR (ddPCR), and finally ddPCR analysis was more period and high operational skills and tends to perform badly
sensitive than NGS. on multiple copy genes. Digital PCR, as we mentioned
above, can achieve single-molecule amplification theoreti-
Prenatal Examination cally and calculate the concentration with end point PCR and
Digital PCR has been developed to detect low-abundance Poisson distribution.
nucleic acids, such as acellular DNA, which is an unprece- In order to ensure that GM food conforms to safety regu-
dented and sensitive alternative to traditional methods, espe- lations, it is objectively required to detect the exact number
cially in noninvasive prenatal diagnosis (NIPD). Carefully of copies of GM food. The limitations of real-time PCR
designed hydrolysis probes and primers should be consid- applications have brought about new developments in detect-
ered in any analytic methods. Because of the random frag- ing very few DNA targets, such as dPCR, which makes it
mentation and high fragmentation of cfDNA, dPCR could possible for accurate measurement of DNA copies. It is
only analyze very short cfDNA molecules. Due to this limi- according to the formula below:
tation, the primers and probes should be crucially selected GMO(%) = Transgenic content/Reference gene
which must target small sequences. Therefore, the length of content × 100%
the amplifier for NIPD should not exceed 120 bp [18]. A rare In the platform of digital PCR, transgenic content was
sequence per unit volume is detected by rare sequence detec- directly measured (X copies/ul) in FAM channel, while refer-
tion (RSD) using a single assay. ence gene content was measured in VIC channel.
Sillence and colleagues [19] investigated the sensitivity of Corbisier and colleagues [22] carried out researches on
the dPCR platform to the best and suboptimal samples and the amount of maize MON810. These copies of DNA were
reported that for suboptimal samples, qPCR could not detect extracted from certified seed powder, and the ratio of copies
a single-copy target (sry, rhd5, and rhd7) compared with was measured by digital PCR. The ratio of these absolute
dPCR. But dPCR, on the other hand, reached a sensitivity of copies determined by dPCR was the same as that determined
100% (95% confidence interval). Their research shows that by qPCR with plasmid DNA calibrator. These results show
digital PCR is more accurate than qPCR in fetal sex determi- that the copy number ratio of MON810 could be determined
nation and RHD genotyping, especially for suboptimal sam- by both methods. It is concluded that dPCR has a high accu-
ples with lower relative proportion of fetal DNA expression racy of measurement and can be used to prove the ratio of
(<2%). Svobodova and colleagues [20] confirmed the better gene copies in genetically modified field.
evaluated accuracy for digital PCR using quantitative Nowadays, qPCR is conventionally used to discover
standard. genetically modified organisms in food. However, its perfor-
dPCR RVD assays were previously reported in NIPD mance in trace DNA detection is unsatisfactory as in some
which include dominant conditions such as neurofibromato- complicated food and feed substrates. Morriset and col-
sis type1 and sporadic disorders such as achondroplasia [21]. leagues [23] measured the absolute numbers of MON810
In such cases, the method can be used to differentiate the transgene and hmg maize reference gene copies in DNA
fetus from unaffected (no sporadic or paternal mutations in samples using ddPCR duplex assay. The ddPCR system pro-
cfDNA) or affected (several sporadic or paternal mutations vides accurate target absolute and relative quantitation with-
in cfDNA). out the need for a calibration curve. The sensitivity of the
ddPCR assay (5 target DNA copies) was good compared to
24.2.4.4 A pplication of Digital PCR on Food the sensitivity of a single qPCR assay and the chamber of
Safety commerce digital PCR (cdPCR) method (5 target DNA cop-
Food safety detection is mainly aimed at determining detri- ies). Compared to qPCR, the ddPCR method is more repro-
mental elements in food, such as heavy metals or aflatoxin. ducible at a relatively low target concentration and more
Most of the food comes from animal and plant circles, which resistant to inhibitors. Ultimately, the amount and cost of
naturally exists distinguishing material-respondent abilities. ddPCR processing are advantageous for quantification of
It is a promising direction to ensure food safety by using the conventional transgenic organisms compared to
reaction of biomaterials and chemicals in food. qPCR. Therefore, ddPCR technology can be applied to both
routine quantitative analysis of genetically modified organ- between hosts, indicating limited lateral gene transfer of
isms and other fields where more accurate quantitative anal- these alleles despite of host proximity. It has been shown that
ysis of food and feed samples is required. microfluidic digital PCR does not require the cultivate host
or virus and provides a means to check for viral-bacterial
24.2.4.5 A pplication of Digital PCR interactions in various environments. There are certain kinds
on Environmental Microorganisms and amount of microbials and viruses in the water, soil, and
Environmental microorganisms are composed of bacteria, atmosphere. They are closely related to the spread of infec-
fungi, chlamydia, mycoplasma, spirochetes, and viruses. tious diseases, so their movements (genres, numbers, and
They stay in water, air, and soil and are spread through trends) must be checked in a timely manner. qRT-PCR was
human activities and development of social economy. used by Kim and colleagues [26] as a reference technique to
Environmental pollution, especially water pollution, is so evaluate the applicability of ddPCR as a quantification tool
intensifying as to affecting normal life of human beings. of soil DNA. Bacterial load tests indicate that ddPCR is more
They tend to bring about incalculable diseases and become distinct and sensitive than qRT-PCR. The two techniques
sources of public health incidents. To validate biological showed almost the same changes over time after monitoring
activity from pathogens environmentally bred, it is important DNA changes for three weeks. The linear test (y = a × x)
to detect their genetic materials with target nucleic acid mol- revealed excellent quantitative agreement between the two
ecules from low-concentration complex environmental sam- techniques (a = 0.98 in the MBT group, R2 = 0.97, a = 0.90 in
ples. Digital PCR, on one hand, could realize absolute the SphMD2 group, R2 = 0.94). These results indicate that
quantification with no need of standard curve or reference ddPCR is a promising technique for detecting the temporal
materials. On the other hand, the system of digital PCR is dynamics of microorganisms in complicated environments.
insensitive to the inhibitors of polymerase chain reaction, to
better overcome large amount of inhibitors from environ- 24.2.4.6 Other Applications on Digital PCR
mental samples including polluted water, mud, or soil, so as Digital PCR is a type of quantitative analysis technology vig-
to sensitively detect probable pathogens that existed and to orously developing. So far, several companies including
achieve super early warning. Fluidigm, Bio-Rad, and RainDance have promoted products
Traditional microbial identification methods are mainly on digital PCR. They have shown technical advantages and
based on studying its physicochemical and morphological application prospects in the field of early cancer diagnosis,
characteristics. Physicochemical characteristics include prenatal diagnosis, and single-cell analysis. Although dPCR
microscopic morphology and culture characteristics. has high sensitivity and precision, it remains a whole new
Morphological characteristics are composed of nutritional technology for the vast number of scientific researchers. As a
types, carbon and nitrogen utilization, metabolic reactions, matter of fact, some new applications are being opened up.
enzymatic reactions, and serological reactions. Traditional
methods can hardly meet the requirements of rapid and accu- Quality Control of Sequencing Library
rate detection of microbials from the environment. With the and Verification of Sequencing Result
rapid change of analytical chemistry technology, the potenti- Quantification for NGS sequencing is the basic premise for
ality of analytical methods has been cutting a striking figure high-efficacy utility. Quality control method based on digital
such as gas chromatography, gas chromatography-mass PCR can precisely control the sample size of sequencing,
spectrometry, and high-performance liquid chromatography. realizing equimolar mixing for homogeneous sequencing.
And progression of PCR, specific nucleic acid probe hybrid- The basic characteristic of digital PCR is product-recoverable,
ization, gene clip, biosensor, and immune technologies alto- which provides new technology for pre-amplification of
gether are opening a new era of microbial detection and sequencing library. Digital PCR has huge advantages on
identification. verification for sequencing results, especially on rare muta-
Ottesen and colleagues [24] used microfluidic dPCR to tions, CNV, and allele expression differences.
amplify and analyze a number of different genes obtained A study by the Fred Hutchinson Cancer Research Center
from a single bacterial cell harvested in nature, encoding in Seattle found that ddPCR can be used as an accurate tool
genes involved in key symbiotic interactions between ter- for quality control of NGS libraries. The quality of different
mites and their gut microbiota, which open up new opportu- types of NGS technology relies on homogeneous library
nities for environmental research in a complex ecosystem. molecules during processing. Whether it is too much or too
A microfluidic PCR method was used [25] to physically little will eventually lead to poor sequencing quality or
link individual bacterial cells from the natural environment wasted samples and reagents, and even sorting completely
to viral marker genes and applied the technique to microbial fails. Instrument manufacturers of NGS devices generally
communities that reside in the hindgut of termites. The diver- recommend qPCR for library quantification. Gel or capillary
sity of viral marker alleles revealed a restricted mix of alleles electrophoresis is used to determine the size range. In addi-
24 Digital PCR 343
tion, Qubit fluorescence assays and the Agilent Bioanalyzer itself is to better detect micro nucleic acid samples and iden-
are also available. All of these methods have some limita- tify small differences in expression under complicated back-
tions because they require a standard curve or have a bias ground. There are not many choices in mature commercial
caused by amplification. products or popularization in technique. Prices being the per-
In the study on ADAR regulating RNA editing, gene manent topic require prolonged discussion.
expression, and transcript stability [27], results revealed Since the proposal of the concept of digital PCR by
more than 60,000 A-to-G editing sites and thousands of Kenneth Kinzler and Bert Vogelstein in 1999, it has been
genes whose expression levels were affected by ADAR after widely applied on precision medicine, microbiological
sequencing and comparing the DNA and RNA of human B examination, and food safety. From the current point of
cells. Among these targets, 90% were identified. Twenty- view, NGS technology will hopefully become the fastest
four of the 25 sites were verified by Sanger sequencing, and growing part in nucleic detection regime. Conversely,
5 of the 6 sites were verified by ddPCR. The false discovery investigation on genetic variation, sequencing of human
rate (FDR) was about 6.5%. The combination of digital PCR genome, and precision medicine will put forward evolution
and NGS can reveal the unknown function of known genes, of new sequencing tools. In 2015 Annual Clinical Genetics
thereby promoting the improvement of gene function. Meeting, the combination of NGS digital PCR is regarded
as the future trend of application of gene sequencing. NGS
Gene Editing is to explore unknown nucleotide sequence, and digital
Gene editing techniques enable people to “edit” target genes, PCR can be performed as next-step confirmation and quan-
knock out, or knock on specific gene fragments for the fol- tification. The prior is definitely good at “batch processing”
lowing reasons. In the past few years, sequence-specific on the level of whole genomics, while the latter acts better
nuclease technologies, namely, the representative zinc finger at sensitivity to specific gene mutation sites. Combination
nuclease (ZFN) and transcription activator-like effector of these two technologies will become the standard process
nuclease (TALEN), have become the main methods of gene on gene detection. Aspects such as blood nucleic acid
editing. In early 2013, clustered regularly interspaced short screening and treatment of sudden infectious diseases are
palindromic repeats (CRISPR) have evolved into the third in urgent need of technology with higher closure, automa-
generation of fixed-point genome editing techniques. In the tion, high throughput, and speed, but digital PCR is not car-
field of preliminary research, more gene editing can be used ried out on them. We are looking forward to more
to confirm gene function, SNP, and multiple site mutations. applications of digital PCR on more aspects to better serve
In the clinical part, gene editing has been used to treat vari- human health.
ous diseases such as CCR5 for the treatment of HIV [28].
Currently, there are several ways to analyze gene editing
results. However, Sanger sequencing takes too much time, References
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Biosensor
25
Lei Zheng and Ye Zhang
25.1 Overview can be selected. Therefore, the biosensors can serve as a

receiver and a converter. As an interdisciplinary combination
In recent years, with the development of biological science, of bioactive sensitive materials and physicochemical trans-
information science, and materials science, biosensor tech- ducers, biosensors are not only indispensable monitoring and
nology has developed rapidly. Biosensor technology is the detection equipment for the development of biotechnology
product of the cross-combination of biotechnology and sens- but also the micro and rapid analysis methods for molecular
ing technology, which integrates multidisciplinary technolo- level [1]. Their common features are exploring and revealing
gies such as biology, physics, and chemistry, using bioactive the basic laws of the generation, storage, transmission, pro-
substances as identification components to test the remaining cessing, transformation, and control of information in the liv-
substances by physical and chemical transducers. The results ing system and exploring the basic methods applied to human
of the reactions are converted to identifiable and quantifiable economic activities. They share the common features: explor-
chemical or physical signals (such as optical, electrical, and ing and revealing the basic laws of the generation, storage,
acoustic signals) to be detected. The biosensors generally transmission, processing, transformation, and control of
consist of three parts: an identification system, a physical and information in the living system, as well as exploring the
chemical transducer, and a signal amplifying device. The basic methods applied to human economic activities.
identification component is generally an immobilized bio- For the traditional sensors, the signal receptor is com-
sensitive material, including biological substances such as posed entirely of nonliving matter. The largest difference
enzymes, antigens, antibodies, microorganisms, nucleic between biosensors and traditional physical sensors and
acids, cells, organelles, and tissues. When designing a biosen- chemical sensors is that biosensors contain vital substances
sor, it is of extreme importance to select an identification in the receptors. For example, using certain plant cells or ani-
functional substance suitable for the measurement object. It is mal cells as sensory sensors, various cell sensors can be
necessary to take into account the properties of the resulting made; tissue sensors (or tissue electrodes) can be made using
composite. The physical and chemical transducers usually biological tissue as a sensor; and some specific organelles
include electrochemical electrodes, gas-sensitive electrodes, can be separated from the cells, which can be made into an
photosensitive tubes, piezoelectric electrodes crystals, field organelle sensor; microorganism can also be used as a sus-
effect transistors, surface plasmon resonators, etc. It is another ceptor to make a biosensor; and the biomolecules such as
important step to select a transducer based on chemical or protein and nucleic acid serving as a susceptor becomes a
physical changes caused by sensitive components, which are mainstream of contemporary biosensor development.
mainly based on the molecular recognition functional sub- Biosensors appeared in the mid-twentieth century. As early
stances in the development of biosensors of high qualities. as in 1962, Professor Clark from the United States reported a
Corresponding changes occur during the generation or con- sandwich-foil device at the annual meeting of the New York
sumption of light, heat, and chemicals in sensitive compo- Academy of Sciences, in which a glucose oxidase film was
nents. Based on these variations, an appropriate transducer fixed between two plastic dialysis membranes and then an
oxygen electrode was installed together to quantitatively
determine the concentration of the glucose. Professor Clark
L. Zheng (*) · Y. Zhang called it “enzyme electrode,” which marks the birth of this
Department of Laboratory Medicine, Nanfang Hospital,
Southern Medical University, Guangzhou, Guangdong,
biosensor [2, 3]. Subsequently, with the integration and
People’s Republic of China development of bioelectronics, microbiology, and biotech-
e-mail: nfyyzhenglei@smu.edu.cn

346 L. Zheng and Y. Zhang
nology, biosensors are not based on the electrochemical proenzyme, which analysis accuracy can reach five
cesses in biological reactions but include various information thousandths.
generated by biological reactions such as optical effects,
thermal effects, field effects, and quality changes to design a 25.1.1.3 High Specificities
variety of sophisticated equipment for detection. After the Biosensors only work on specific substrates and are not
1990s, bioaffinity sensor technology based on biochips and affected by turbidity and color. They are highly resistant to
surface plasmons became another climax in biosensor tech- interference and generally do not require pretreatment of
nology development. The first enzyme biosensor, glucose samples.
sensor designed in the 1960s, was called the first-generation
biosensor. Since then, with the rapid development of biosen- 25.1.1.4 Wide Range of Applications
sor technology, the second generation of biosensors has been Some biosensors can reliably indicate the oxygen supply
more widely used. The second generation of biosensor conditions and the production of by-products in the micro-
includes immune, microbial, cellular, and enzyme immuno- bial culture system, and many kinds of physicochemical
sensors [1]. After more than 30 years of development, bio- interaction information can be obtained during the produc-
sensor technology has broad application prospects in tion control. At the same time, they can also indicate the
industrial development, food and drug analysis, clinical direction of increasing the product acquisition rate.
diagnosis, biotechnology, biochips, and environmental pro- Nowadays, biosensors have been applied in various fields
tection. Nowadays, biosensors are mainly used for monitor- such as food monitoring, environmental monitoring, fermen-
ing clinical diagnosis, examination, and treatment in the tation industry, healthcare, and precision diagnosis.
medical field, playing an important role in the study of chal-
lenging diseases. 25.1.1.5 S imple Operation and Automatic
When the biosensor is working, the substance to be tested Analysis
is diffused into the biologically active substance, and after For the surface plasmon resonance biosensor commonly
the molecular recognition, the biological reaction occurs, used in drug analysis, it possesses the advantages of small
and the generated information and results are converted into volume, high sensitivity, high precision, wide measurement
the quantifiable and measurable signals such as light, elec- range, simple operation, complete function, and low cost,
tricity, and sound by the corresponding physical or chemical which is a modern scientific instrument of practicality and
physical and chemical transducer. As the signal intensity is innovativeness.
proportional to the concentration and quality of the analyte,
the detection signal is amplified and output by the instru-
ment, and the concentration of the substance to be tested can 25.1.2 Classifications
be obtained.
The classification methods for biosensors are diverse from
different aspects, and currently there are three main
25.1.1 Characteristics methods.
According to bioactive substances and molecular recogni-
25.1.1.1 Low Cost and High Speed tion components applied in the biosensors, sensors can be
Immobilized enzyme biosensing analyzers, which appeared classified into enzyme sensors, cell sensors, immunosensors,
initially and possessed the highest accurate biosensors, have microbial sensors, tissue sensors, and DNA sensors. The
evolved into a class of reliable precision analyzers. Due to enzyme sensor is mainly composed of an immobilized
the use of immobilized enzyme membranes as analytical enzyme membrane and a transducer: the immobilized
tools, enzymatic reagents can be used thousands of times enzyme membrane can selectively recognize the detected
repeatedly, and the cost of analysis is only one-tenth of that substance and catalyze a chemical reaction associated with
of palm-type blood glucose analyzers. More importantly, its the substance; the variables from the substrates or products
analysis speed is quite fast, less than 20 seconds, which is are converted into electrical signals which are then displayed
very important for a number of critical care patients, clinical by the instruments. The blood glucose tester is used to test
emergency rooms, and many other occasions. blood glucose, which has the advantages of high sensitivity
and high speed. Immunosensor is a kind of sensor in which
25.1.1.2 High Precision and Stability an antibody or an antigen is bound to biologically active
The high-precision blood glucose analyzer on the market material and the specific recognition and reaction between
currently is a biosensing analyzer using an immobilized the antigen and the antibody are utilized to determine the
25 Biosensor 347
Sample to be Sensitive Conversion Electronic

tested component element equipment
Fig. 25.1 The basic compositions of a biosensor
concentration of the corresponding antigen or antibody in 25.2 Basic Principle

the sample [4]. Some researchers have reported that the
application of immunosensors can detect cholera toxins at The basic principle of biosensor is shown in Fig. 25.1. It usu-
concentrations below 1 × 10−8 mol/L and lower concentra- ally consists of a biosensing element and its associated phys-
tions of pathogenic microorganisms such as staphylococci icochemical sensor called transducer. Biosensing elements,
and botulinum in about 30 minutes. In the microbial sensors, also known as martial-sensitive elements, include biological
the microorganism is fixed on the biologically active carrier. substances such as enzymes, antibodies, nucleic acids, cells,
The oxidative respiration reactions of the microorganism or tissues, and receptors, which are used to convert a test sub-
the biologically active enzyme contained in the microorgan- stance into a chemical quantity or a physical quantity. The
ism are utilized to detect the concentration or content of the physicochemical sensor, also known as the transducer, is
substance to be tested. The microbial sensor can detect used to convert the chemical or physical quantity converted
pathogens in the sample to be tested within a few minutes, by the biometric component into an electrical signal that can
such as Escherichia coli O157. A tissue electrode sensor is be calculated by a computer [6]. According to the signal con-
an electrochemical sensor directly using an animal and plant version component, it can be divided into several types, such
tissue sheet as a sensitive component. The tissue sensors as electrochemical biosensors, metering biosensors, and
exploit the catalytic reactions of the multi-enzyme systems piezoelectric biosensors. In the realization of the actual bio-
in animal and plant tissues. They possess the advantage that sensor, the biosensor elements must be well coupled with the
the enzyme activity and stability are much higher. Besides, transducer. And the immobilization technologies, such as
the material is easy to obtain and the preparation is simple. lipid membrane embedding, physical absorption, base value
There are still some shortcomings in terms of selectivity, adsorption, and covalent bonding, can improve the stability
sensitivity, and short responsive time. Animal tissue elec- of biosensor elements by the realization of coupling.
trodes mainly include renal tissue electrodes, liver tissue Different types of sensor and different sensor interface mate-
electrodes, intestinal tissue electrodes, muscle tissue elec- rials may require different immobilization techniques.
trodes, thymus tissue electrodes, and the like. Plant tissue Basically, the ideal immobilization method should not
electrode sensing elements are available in a wide range of destroy the activity of the biological material and prolong the
materials, including roots, stems, leaves, flowers, and fruits activity of the material.
of different plants. Plant tissue electrode preparation is sim-
pler, less expensive, and easier to store than animal tissue
electrodes [5]. 25.2.1 Principles of Electrochemical Biosensor
Biosensors are classified according to the physical and
chemical transducers used, including electrochemical bio- Electrochemical biosensors use electrodes as conversion ele-
sensors or bioelectrodes, thermal biosensors, photobiosen- ments and immobilization carriers to immobilize bio-
sors, sonic biosensors, microcantilever biosensors, sensitive substances (such as antigens, antibodies, enzymes,
conductance/impedance biosensors, semiconductor biology hormones) or the organism itself as sensitive elements on the
sensors, FET biosensors, etc. electrodes. The target molecule and its reaction signal are
According to the interactions between bio-sensitive sub- converted into electrical signals (such as capacitance, cur-
stances and the types of reactions, biosensors can be divided rent, potential, conductivity, etc.) through the specific recog-
into three types: affinity type, metabolite type, and catalytic nition between biomolecules to achieve qualitative or
type [1]. Bioaffinity biosensors exploit the nature of specific quantitative detection of the target analyte [7].
recognition between antigens and antibodies to detect anti- Electrochemical biosensors are classified into the following
gens, haptens, or antibodies, which are the result of thermo- types depending on the sensitive components.
dynamic equilibrium. The catalytic biosensors utilize the
catalytic and specificity of the enzyme to detect the substrate 25.2.1.1 Electrochemical Immunosensor
of the enzyme under the conditions of room temperature and The binding of antibody and antigen is specific. Based on
neutrality, which detect the total effect during the entire reac- this principle, electrochemical immunosensor immobilizes
tion kinetics process. the immune substance on the surface of the electrode as a
Hybridization of Hybrid indicator

Fixation of ssDNA dsDNA embedding
Fig. 25.2 The major steps of DNA electrochemical biosensor fabrication
sensitive element and converts the biological reaction signal DNA by detecting the change of the electrochemical signal
into the electrical signal outputted through the electrode after of the modified electrode in the solution.
the antigen/antibody reaction reaches equilibrium [8]. The target DNA detection by this type of biosensor
Electrochemical immunosensors are classified into direct (shown in Fig. 25.2) includes ssDNA immobilization,
type and indirect type by the difference of whether a label is hybridization, hybridization instructions, and detection of
used. Direct electrochemical immunosensors rely directly on electrochemical signals.
antibodies or the electrochemical changes of their large
amounts of charge in the process of immunological binding, 25.2.1.4 Electrochemical Cell Sensor
thereby measuring changes in parameters such as imped- The cell sensor uses the immobilized or unimmobilized liv-
ance, ion permeability, and conductivity. Indirect electro- ing cells as sensitive identification elements, combined with
chemical immunosensors require the use of markers to electrodes or other signal elements, to qualitatively or quan-
amplify the signal of immune response and then determine titatively detect the basic information of the cells and the
the concentration of the immunological substance properties of the substance to be tested. In the cell sensor,
indirectly. information recognition mainly includes two parts: cells for
primary signal transmission and a secondary converter with
25.2.1.2 E lectrochemical Enzyme Electrode signal conversion. When living cells specifically bind to a
Sensor toxin or pathogen, the resulting information is converted into
After the chemical change of the biomolecule under the a processable signal by a transducer to qualitatively or quan-
catalysis of the immobilized enzyme, the electrochemical titatively detect the properties of the toxin or pathogen. In
enzyme sensor records the change through the transducer to addition, if the same cell line or cell type combines with dif-
indirectly detect the concentration of the analyte. The earli- ferent secondary converter, different detection results can be
est proposed principle of biosensor is to use glucose oxidase produced.
combined with oxygen electrode to detect glucose.
It uses oxygen in nature as electron transporter to com- 25.2.1.5 Cell Receptor-Based Cell Sensor
municate the electron channel between electroactive center The cell receptor-based cell sensor mainly comprises a sensi-
of enzyme and electrode, thereby directly detecting the tive recognition component and a signal conversion compo-
reduction of the substrate of enzyme or the production of nent. The receptor on the cell surface is used as a sensitive
product. And the subsequent research on enzyme sensing recognition component to specifically bind to the substance
technology has developed into an interdisciplinary compre- to be tested. When the substance is combined with the cell
hensive technology gradually, which has been widely used surface receptor, a series of cascade reactions occur within
in food testing, environmental monitoring, drugs, and the the cell, and these reactions can be converted into detectable
clinic. signals by signal conversion elements to provide functional
information on these reaction mechanisms.
25.2.1.3 Electrochemical DNA Sensor Most cell sensors use cellular receptors in the initial
The design principle of the DNA electrochemical biosensor stages of biorecognition, so cell receptors are critical for the
is to fix the single-stranded DNA as a sensitive element on development of novel cell sensing systems in the future. Cell
the surface of the solid electrode and add an electroactive receptors associated with cell sensors generally include car-
substance to indicate the hybridization information and bohydrate receptors, cell adhesion receptors, and membrane
finally determine the concentration or sequence of the target protein receptors, which can be used as ligands for various
25 Biosensor 349
toxins or pathogens and are used in biochips and cell sensors no excitation source, an optical signal as an excitation source,
to test various toxins or pathogens. and an electrical signal as an excitation source.
25.2.1.6 Cellular Lesion-Based Cell Sensor 25.2.2.1 Passive Optical Sensor

The cytopathic sensor, like the cell sensor, consists of two The passive optical biosensor is tested by using the light
parts: the sensitive recognition element and the signal con- radiation generated by the biochemical reaction of the test
version element. Different toxins or pathogens have a certain substance and the sensing layer in combination with the sen-
specific relationship to the destruction of physiological func- sor layer without any energy excitation. There are two main
tions or structures of host cells. types of luminescent systems, one is chemiluminescence,
Such cell sensors reflect cell damage caused by toxin or and the other is bioluminescence. Chemiluminescence and
pathogen-mediated cytotoxicity by detecting the damage and bioluminescence are characterized by high luminous effi-
extent of the target host cell or tissue. In addition, it can dis- ciency and sensitivity, simple equipment, and suitable for
tinguish different toxins or pathogens based on different flow injection analysis. They are hot areas of biosensors and
types of damage to the animal or human cells caused by the have shown great potential for application in the fields of life
toxin or pathogen and can analyze the death of various cells sciences, clinical medicine, and environmental sciences.
through the cytopathic effect.
25.2.2.2 Photoinduced Optical Biosensor
25.2.1.7 C ell Sensor Integrating Optical The use of an excitation source not only ensures a stable test
Measurement Technology light signal, but the wavelength of the excitation light can
and Electronic Measurement also be selected over a wide range to suit different test envi-
Technology ronments. Depending on the structure of the sensing layer,
Although the morphology, size, and number of cells can be such sensors can be classified into fluorescence-label sensors
observed by microscopy, small changes in cellular and label-free sensors. A fluorescence-label sensor refers to
responses caused by some toxins and pathogens cannot be an indirect analysis method that requires labeling of a living
observed with conventional microscopy techniques. body with a fluorescent substance and uses secondary light
Therefore, cell sensors with optical detectors or integrated excited by incident light as a detection signal. The label-free
microelectrodes can quantify the degree of damage to sensor does not need to introduce a fluorescent marker, and
cells. Adherent growth is one of the important features of the primary light, such as reflected light and scattered light
mammalian cells. Changes in the state of motion and cell after the incident light itself passes through the sensing layer,
state of adherent growth on the electrodes will cause is used as a detection signal. The optical principles utilized
changes in the impedance of the adherent interface. are surface plasmon resonance, reflection interference spec-
According to this feature, researchers have built cell sen- trum, and various fiber optic technologies such as resonator
sors that integrate optical measurement technology and mirrors, grating couplers, and the like.
electronic measurement technology.
25.2.2.3 Electro-optical Biosensor
Such sensors are based on electrochemiluminescence. Unlike
25.2.2 Principles of Optical Biosensor the above sensors, a working electrode needs to be added to
its sensing layer. The working electrode is generally made of
The optical biosensor generally consists of three functional different materials such as platinum, ITO-coated glass,
modules: a biological recognition layer, an optical signal graphite, and gold. When a certain voltage is applied to the
transducer, and an amplifier, as shown in Fig. 25.1. The opti- working electrode, an electrochemical reaction occurs on the
cal biosensor uses the optical signal generated by the reac- electrode, and the intermediate substance generated by the
tion of the test substance and the detection reagent as a basis reaction reacts with the test substance to release an optical
for detection. The sensing layer is a “receptor” that has high signal.
selective recognition ability for the substance to be mea-
sured. It is a core component of optical biosensors that
extracts light information related to various measured 25.2.3 Principles of Piezoelectric Biosensor
biomass.
Optical biosensors use light as a detection signal. The Piezoelectric biosensors are new biosensors that use piezo-
illuminating form of the sensing layer, the intensity of the electric materials as transducers. In recent years, piezoelec-
optical signal, and its stability are at the heart of this type of tric sensors using quartz crystals as converters have developed
sensor. Therefore, according to the manner in which the light rapidly and have been widely used in the field of medical
signal of the sensing layer is generated, it can be divided into diagnosis.
In 1880, Jacques Curie and Pierre Curie first discovered tion of big data and the Internet, new technologies and new
the phenomenon of piezoelectricity. The theory of crystal business models have brought disease prevention, diagnosis,
piezoelectricity was first proposed by Jacques Curie and treatment, and control into the era of intelligence. As an
Pierre Curie in 1880. In 1959, Sauerbrey first studied the intelligent terminal, biosensor, physiological sensor system,
relationship between the surface load mass variation Δm and and mobile phone will become an irreplaceable data source
the frequency shift Δf of the quartz crystal in the gas phase. for healthcare data. By accepting, storing, managing, and
For the AT quartz crystal oscillating in the thickness-shear processing these data, we can summarize and analyze the
mode, assuming that the thickness of the precipitated sub- public health status and the law of disease occurrence, so as
stance is very small and there is no elasticity, the precipitated to provide better disease prevention and control strategies.
substance vibrates together with the particle on the surface of Wearable sensor system mainly consists of the following
the crystal, demonstrating that the linear relationship between parts: sample collection part, target material separation and
the frequency change and the mass change is Δf = −2.26 × extraction part, signal amplification and conversion part, and
10–6 f02△m/A (where Δm is the mass change of the bound signal collection and analysis part. The whole process is as
material of the metal electrode; f0 is the natural frequency of follows: at first, the sample collection part will collect the
the quartz crystal; Δf is the change of the crystal resonance required samples, such as tears, saliva, or sweat. They are all
frequency caused by the mass change; A is the surface area readily available samples of body fluids. Then the separation
of the electrode; and negative sign indicates an increase in and extraction part of the target substance will work, which
mass resulting in a decrease in frequency). Thus, the theo- will separate, extract, or concentrate the target substance in
retical basis of the piezoelectric quartz crystal resonance the obtained samples, such as general qualitative filter paper
measurement technology is established. method commonly used in the past and the newly developed
When some electrolyte crystals are deformed by an exter- FTA test paper method for extracting nucleic acid. And then,
nal force, an odd-numbered polarization charge appears on the signal conversion and amplification can amplify the con-
the surface. This phenomenon of no electric field is simply centration information of the target. The signal amplification
due to strain or stress, and the phenomenon of polarization in in these methods also depends on the body surface tempera-
the crystal is called piezoelectric effect. The piezoelectric ture, which can be described as “adapting to local condi-
effect is divided into a positive piezoelectric effect and an tions.” After amplifying the signal, it can be converted into
inverse piezoelectric effect. The so-called positive piezoelec- the fluorescence signal or electrochemical signal. Some
tric effect means that when the crystal is subjected to an wearable sensors do not amplify the signal but directly detect
external force in a certain fixed direction, an internal polar- it. For example, the common blood glucose meters on the
ization phenomenon occurs, and at the same time, opposite market make use of electrochemical method to test the blood
charges are generated on certain two surfaces, that is, glucose level. The electrons produced by the enzyme react-
mechanical energy is converted into electric energy. The ing with glucose are then converted into glucose concentra-
inverse piezoelectric effect refers to the phenomenon that the tion readings using current counting facilities to read the
alternating voltage is applied to the crystal to cause mechani- number of electrons. Finally, the signal collection and analy-
cal deformation of the crystal, that is, the electrical energy is sis system is used to analyze the collected signals and draw
converted into mechanical energy, which is also called elec- conclusions [9].
trostrictive effect. Quartz crystal resonance measurement At present, wearable sensor systems for physiological
technology is the application of the inverse piezoelectric indicators such as body temperature, pulse, blood pressure,
effect. In medical diagnosis, piezoelectric immunosensors and respiratory frequency have begun to popularize. These
are mainly used to detect some pathogenic microorganisms indices can be directly measured by physical sensors.
such as bacteria, viruses, chlamydia, and the like.
25.3.2 Molecular Biosensor and Imaging

25.3 Technology Development
Molecular biosensors are biosensors composed of DNA or
25.3.1 Wearable Biosensor protein and other biological macromolecules through gene
recombination or DNA synthesis technology. They are espe-
Wearable biosensor has many advantages, such as comfort- cially suitable for detecting intracellular molecular events.
able wearing, even insensitivity, noninterference in normal At present, there are four kinds of molecular sensors widely
life, monitoring, and evaluation of physical condition conve- used: molecular beacon (MB), fluorescence energy transfer
niently at anytime and anywhere. The data to medical data system (FRET), bioluminescence energy transfer system
center are conducive to patient home monitoring and indi- (BRET), and bimolecular fluorescence complementary sys-
vidualized medical treatment. With the cross-border integra- tem (BIFC). They indicate the location, movement, and dis-
25 Biosensor 351
tribution of target biomolecules in living cells, interactions sensitivity of different sensors for the same gas are different.
between molecules, conformational changes of molecules, The response of each sensor to the mixed gas can be synthe-
detection of enzyme activity, and response of cellular and sized, and the response degree of the corresponding gas can
subcellular structures to environmental changes and exoge- form various patterns. Because the response patterns of the
nous compounds by their conformational changes, photore- whole sensor array are different for different kinds of odors,
actions, and changes in optical activity. The combination of the system can recognize different types of odors according
molecular biosensor and super-resolution microscopy sys- to the response patterns of the sensors. More and more stud-
tem can realize imaging detection of single molecule events, ies have shown that the use of electronic nose technology for
which is beyond the reach of traditional biosensors and is of odor analysis and detection can quickly, objectively, and
great significance to life science research. accurately evaluate odor and has excellent repeatability. It
can continuously and real-time monitor the odor situation in
a specific place for hours, days, or even months. This is
25.3.3 Biological Function Simulation Sensor beyond the reach of human and animal noses.
With people’s in-depth research on “electronic nose,” the
25.3.3.1 Odor Sensor application fields of “electronic nose” are also expanding,
The odor sensor is now called “electronic nose.” The so- such as food industry, pharmaceutical industry, environmen-
called electronic nose is actually an electronic system capa- tal testing, healthcare and military, and other fields. Among
ble of sensing and recognizing odors. Its basic principle is to them, it is most widely used in food industry, such as detect-
simulate human olfactory organs to sense, judge, and ana- ing freshness of meat, fruits, vegetables, and fish; classifying
lyze odors. This concept was first proposed by Professors cereals; checking pathogens of poultry meat; monitoring
Persaud and Dodd of Warwick University in England in cooking, storage, and fermentation in food production pro-
1982. By imitating the structure and mechanism of mamma- cess; and evaluating the quality of fruits, meat products, and
lian olfactory system, they carried out species analysis of wine. In addition, it can also analyze the interaction between
several organic volatile gases, which is an instrument devel- packaging materials and products. If we can solve some
oped by integrating computer science, chemistry, and other problems in the development of electronic nose technology,
disciplines. such as the miniaturization and practicality of sampling and
Odor sensor is mainly composed of odor sampling ele- concentration device, the improvement of sensitivity and
ments, gas sensor array, and signal processing system. The speed of gas sensor, and the reduction of cost, it will make
working process is as follows: firstly, the air sample is the application scope of electronic nose technology spread
absorbed into the chamber equipped with the gas sensor rapidly.
array, so that the sensor array is exposed to the air sample.
The instantaneous response can be generated when the gas 25.3.3.2 Taste Sensor
contacts with the active and sensitive material on sensor sur- Human beings have five basic taste indicators: sour, sweet,
face. Then the response is recorded and transmitted to the bitter, salty, and fresh. Human taste is composed of the
signal processing system for processing. Finally, the specific above five basic taste indicators. In taste sensors, acid can be
analysis results are obtained and determine odor types. supplied by hydrochloric acid, sweetness by glucose, bitter-
Before entering the next round of measurement, the sensor ness by caffeine and manganese chloride, salty mainly by
should be initialized again and cleaned with reference gas to sodium chloride, and delicious by sodium glutamate in fish
achieve the reference state. and meat.
Air samples are often a mixture of many gases, so the Taste sensor-sensitive active materials and their arrays are
“electronic nose” usually uses a gas sensor array consisting the core of artificial intelligence taste system. When the taste
of several gas sensors with different selectivity. By means of substance contacts with the active material on the sensor, the
its cross-sensitivity to various gases, the functions of differ- potential of the taste substance can be changed, and finally
ent kinds of odor molecules on their surfaces are transformed the electric signal can be output. The response patterns of
into time-dependent measurable physical signal sets, which substances with different taste characteristics are similar. For
are convenient for calculation, and the analysis of mixed example, the response modes of sweet substances, acid sub-
gases is realized. For example, assuming that air sample A is stances, and salty substances are different, while KCl, NaCl,
a mixture of four gases, such as a, b, c, and d, sensor number and KBr are salty substances, and they have similar response
1 is most sensitive to c. It can also measure three other gases, modes. At the same time, even for substances with similar
a, b, and d. Sensor number 2 is most sensitive to gas a, but it taste characteristics, their response patterns are not com-
can also measure three other gases, b, c, and d. Sensor num- pletely consistent. Therefore, the taste sensor can distinguish
ber 3 is the best one for gas b, but it can also measure the each type of taste according to its characteristic response
other three gases a, c, and d. That is to say, the specificity and mode, and the standard deviation can be less than 1%.
Similar to odor sensors, taste sensors mostly use array ciplines (acoustics, optics, magnetism, nanomaterials, etc.),
structure in order to obtain rich, stable, and reproducible sys- biosensor chips with different functions and applications
tem information. Arrays are arranged in different ways. The have been developed. Common biosensor chips mainly
general requirements are easy to fabricate, moderate size, include DNA sensor chips, surface plasmon resonance sen-
high signal-to-noise ratio, durability, and no interference sor chips, microfluidic sensor chips, piezoelectric crystal
between working electrodes. Generally, the number of sen- sensor chips, etc. [13], which can be used for various bio-
sors in a sensor array ranges from 8 to 40. If the number of logical macromolecules such as proteins, nucleic acids, etc.,
sensors is too large, the structure of sensor array is complex, the determination of metal ions, the detection of pathogens,
and the redundancy of data information is large. At the same and the screening of drugs.
time, it brings some difficulties to data analysis. If the num- The main components include (i) recognition elements
ber of sensors is too small, the information obtained is not (e.g., DNA, enzymes, antibodies, etc.) that can bind (or iden-
rich enough. So the number of sensors in the sensor array tify) analytes under investigation. (ii) Transducer or detector
should be appropriate. elements (working physicochemically, such as optical,
Taste sensors are widely used in the identification of mineral piezoelectric, electrochemical, etc.) convert signals gener-
water quality. In recent years, more and more people require ated by the interaction of analytes with biological elements
safe and healthy mineral water to drink. Especially in the case into signals that are easy to measure. (iii) Biosensor display
of more and more serious environmental pollution, the detec- devices and associated signal processors are primarily
tion of water quality is crucial. But at present, there is no simple responsible for displaying the results of the analysis.
and convenient measuring system for the quality evaluation of Take the electrochemical biosensor chip detection process
drinking water. People feel weak about the taste of mineral as an example: (1) mounting a molecular recognition probe
water; it is difficult to distinguish different brands of mineral on the surface of the electrode chip, (2) hybridization reac-
water. However, taste sensors are very sensitive to different tion between the target molecule and the recognition probe,
types of response from hard water to soft water. Although min- (3) generation of electrochemical signals; and (4) the signal
eral water contains only low concentration of taste substances, processor detects electrochemical signal parameters such as
due to the high sensitivity of taste sensors to inorganic ions, conductance, resistance, potential, etc. and calculates the
different brands of water can be distinguished. concentration of the target molecule [14].
Taste sensor can also be applied to environmental detec- Researchers at Stanford University have developed a new
tion of water quality. As mentioned above, conventional biosensor chip that can significantly accelerate drug develop-
chemical analysis methods are better but time-consuming. ment. The chip can analyze how proteins bind to proteins,
Therefore, it is necessary to develop a simple and fast water helping to assess the potential efficacy of drug treatment and
quality detection device in daily life. Taste sensor can achieve possible side effects. Each square centimeter array on this
this goal, because the sensor can simultaneously respond to a nanosensor can simultaneously and continuously detect
variety of different chemicals with high sensitivity, so we thousands of protein binding reactions. This new sensor chip
only need to know the safe range of response mode of sensor not only has higher sensitivity of existing chips but also can
output to know whether drinking water is safe. Nowadays, provide test results more quickly. The biosensor chip has
the application of taste sensor in water quality monitoring many advantages, such as small in size, enabling microport-
has been continuously studied. ability of the device, and reagent sample elimination. The
function of microchemical biosensing chip is highly inte-
grated, realizes the function of “chip lab,” and can complete
25.3.4 Biosensor Chips the experimental operation automatically and efficiently.
Currently, the technology can monitor interactions in less
Biosensor chips are a new class of biochips that combine than 4 hours at the same time, and the process can be com-
chip technology with biosensing technology. Biosensor chip pleted in only a few hours.
is based on the principle of specific interaction between bio-
molecules; the biochemical analysis process is integrated
into the surface of the chip, so as to achieve high-throughput 25.4 Clinical Application
and rapid detection of DNA, RNA, polypeptide, protein, and
other biological components [10]. With the advancement of Biosensors are devices that are sensitive to target substances
chip preparation technology, people have realized an inte- and transform their information to electrochemical or optical
grated platform with integrated functions of mixing, separa- signals for detection. They have been widely applied in clini-
tion and capture, reaction, detection, and other laboratory cal diagnosis and prognosis, and more biosensors that are
operations on a few square centimeters of chips, with minia- highly accurate, time-saving, and cost-effective are being
turization, integration, automation, and so on [11, 12]. At developed. Table 25.1 summarizes several most recent
present, by combining chip technology with a variety of dis- biosensors.
Table 25.1 Examples of biosensors for clinical diagnosis, prognosis, and treatment
Disease Analytes Sample Detection technology Application field Reference
Synthetic materials/serum/plasma/urine/SCF...
Neurodegenerative disease Neurotransmitters Neural stem cells Core-shell-shell UCNPs Diagnostics [15]
25 Biosensor
β-Amyloid Plasma/serum OWLS Diagnostics [16]

Cu2+/pH CSF FSCV Monitoring/therapeutics [17]
Glutamate Synthetic materials ELC Diagnostics [18]
Chromosome 21 Blood Electrical detection Diagnostics [19]
Dopamine Blood PTR Diagnostics [20]
GFAP Synthetic materials Electrical detection Diagnostics [21]
Cu + ions/pH Mouse model ECL Diagnostics [22]
Cancer VOCs Gas ICS Diagnostics [23]
HEGFR-2 Breast cancer cells OECTs Prognosis [24]
PSA glycan structure Serum ELC Diagnostics [25]
MicroRNA-10b Plasma Dual-SERS Diagnostics [26]
PSA Serum EIS Diagnostics [27]
MicroRNA let-7a Synthetic materials PEC Diagnostics [28]
HER2+ Breast cancer cell Colorimetric Diagnostics [29]
MicroRNA Lung cancer cells ELC Diagnostics [30]
DNA Tumor cells ELC Diagnostics [31]
Metamaterials Cancer cells EIT-like plasmonic resonance Diagnostics [32]
CA125 II Synthetic materials Fluorescent detection Diagnostics [33]
Survivin-encoded DNA Synthetic materials FRET Diagnostics [34]
CEA/HER2 Serum Electrical detection Diagnostics [35]
MGMT Serum/tissue ECL Diagnostics [36]
BRCA1 Synthetic materials Electrical detection Diagnostics [37]
Proteins Synthetic materials Colorimetric detection [38]
DNA methylation Synthetic materials Magnetoresistive (GMR) Diagnostics/prognosis [39]
DNA methylation Synthetic materials ECL Diagnostics [40]
Physiological disfunction DNA Serum Fluorescent detection Diagnostics [41]
IL-6 Breath Electrical detection Diagnostics [42]
Cortisol, DHEAS, NPY Serum Electrical detection Diagnostics/treatment [43]
DNA Synthetic materials Electrical detection Diagnostics [44]
DNA Synthetic materials FRET Diagnostics [45]
Rac1 GTPase Living cells FRET Diagnostics [46]
VEGFR Living cells Bioluminescence microscopy Diagnostics [47]
Glucose/lactate/triglycerides Serum Electrical detection Diagnostics [28]
Glucose Synthetic materials Electrical detection Diagnostics [22]
Glucose In vivo human clinical trials ECL Diagnostics [42]
Hydrogen peroxide Synthetic materials Luminescent detection Diagnostics [48]
DNA Synthetic materials ECL Diagnostics [49]
Avian influenza virus Oral swab/feces ECL Diagnostics [50]
(continued)
353
Table 25.1 (continued)
354
Disease Analytes Sample Detection technology Application field Reference

Virus infection M13 virus Synthetic materials Electrical detection Diagnostics [51]
Influenza virus (H5N1) Synthetic materials IRAS Diagnostics [52]
Heme Blood IMBEDs Diagnostics [53]
Gastrointestinal disease
PKA Living cells FLINC–AKAR1 Diagnostics [54]
Metabolic disease Sweat-alcohol/ISF glucose ISF ECL Diagnostics [55]
Protein Blood p-BNC Diagnostics [56]
Cardiovascular disease
Lysozyme and lactoferrin Tear LSPR Diagnostics [57]
Chronic dry eye
CRP/bBSA Synthetic materials PTR Diagnostics [58]
Inflammatory diseases IL-3 Serum/plasma ECL Diagnostics [23]
8-OHdG Serum ECL Diagnostics [59]
L. Zheng and Y. Zhang
25 Biosensor 355
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Microfluidic Chip
26
Xueen Fang
Along with the development of science and technology, there enough information from rare and precious samples.
are more and more detecting methods in clinic. Microfluidic Moreover, it produces less waste [3, 4]. In the microtube, it
chip is one of them. Microfluidic chip, a technique to control was fast to dissipate heat and to pass analytes than other rou-
tiny amounts of liquid, has fast development in the past two or tine systems. So, the time of reaction and assay is reduced.
three decades. It has already been applied to detect nucleic The additional advantage of microfluidic chip is that because
acid, proteins, cell culture, cell selection and drug screening, several technologies are integrated into a tiny controllable
and so on. It provides us an accurate, high throughput, and platform, microfluidic chip has an integral, closed system
easy integrated platform for biomarker detection and research. which makes it save space and reduce contamination. It is
possible all the equipment in the laboratory can be developed
into miniaturization and domestication.
26.1 Overview The development of microfluidic chip promotes the
appearance and development of the concept of point-of-care
Microfluidic chip is also named Lab-on-a-chip. It has become testing (POCT) [5]. POCT refers to the inspection by the
a hotspot and development frontier in the field of Miniaturized bed. It needs the machine for testing to be portable, easy to
Total Analysis (μTAS) since the concept of μTAS debut in operate. It also requires the detection to be quick and the
1989 by Manz and his colleagues [1]. In 1998, lab-on-a-chip result to be accurate. It is better if non-laboratory technicians
was introduced by Burns and his colleagues. It was supposed or even patients can operate. Then it can reduce the work of
to integrate many bioassays and chemical analysis into a laboratory technicians. However, there are some limitations,
chip (Fig. 26.1). It was a good prospect of applying microflu- such as the complexity in the producing process and the
idic chip to clinical detection and precision medicine [2]. absorption of chemical component by the chips, to spread
Microfluidic chip refers to a technique that controls the microfluidic chip. But with the development of new material,
microfluid in 5–500 μM microtube. It is a new cross-subjects new technique, and new discovery, there are researchers
research field that integrates physics, microelectronics, mate- from different fields to study and improve microfluidic chip.
rial, chemistry, biology, medical, and other subjects. It makes There is a considerable potential that microfluidic chip will
sample preparation, reaction, separation, detection, cell cul- be used in clinical test and basic medical research. The
ture, selection, lysis, and other basic operations on a single industrialization of microfluidic chips will promote its devel-
tiny chip. The microtube forms network in it, and the con- opment into miniaturization, rapidness, high throughput, and
trollable fluid becomes involved in the system. It can realize automation. At present, there are several applications of
many functions in chemical laboratory and biology labora- microfluidic chip in clinical detection, such as detection of
tory. Compared with other regular experimental techniques, nucleic acid, immunoassay, and drug-resistant test.
by using microtubes and manipulating microfluid, microflu-
idic chip has the potential to enhance biomedical research in
diverse ways by simplifying complex assay protocols, reduc- 26.2 Basic Principle
ing reagent costs, decreasing sample volumes, and getting
Microfluidics integrates several function units such as sam-
ple preparation, reaction, separation, and detection in bio-
X. Fang (*) logical, chemical, environmental, and medical analysis
Department of Chemistry and Institutes of Biomedical Sciences, processes onto a micrometer-scale chip to make the whole
Fudan University, Shanghai, People’s Republic of China
process automatic. Owing to its great potential in the fields
e-mail: fxech@fudan.edu.cn

358 X. Fang
Fig. 26.1 The concept of the microfluidic chip
of biology, chemistry, medicine, etc., it has developed into a channel surface, magnetic beads, or electrode surface to real-
new research field of biology, chemistry, medicine, fluids, ize the on-chip sample separation and detection [9].
electronics, materials, machinery, and other disciplines. Another technology like gel electrophoresis was also
widely used for sample separation and biomarker analysis.
For example, Shameli and colleagues developed a microflu-
26.2.1 Separation idic chip based on PDMS-glass to separate proteins by com-
bining TGF with sodium dodecyl sulfate-polyacrylamide gel
Sample preparation and sample separation are key steps for electrophoresis [10]. Another research group reported a
analytical testing applications. A lot of studies have made microfluidic chip for stacking, separation, and extraction of
efforts to use the microfluidic chip for the sample prepara- multiple DNA fragments; this chip utilized isotachophoresis
tion and separation. For biomarker detections, the antibody- hyphenated gel electrophoresis to separate DNA fragments
based strategies were widely used. The antibody has ultrahigh on-chip, which showed better performance than traditional
specificity and affinity toward the relevant antigens, which methods [11].
was very suitable to capture desired molecules in microfluid- For some samples lacking biological activity like nanopar-
ics. For example, Eletxigerra and colleagues used monoclo- ticles, they can be separated by their own characteristics
nal anti-TNFα to capture and separate this classic immune including the size, density, magnetic properties, and electric
cytokine in the channel surface of the microfluidic chip, properties (Fig. 26.2). Based on the above different proper-
which realized accurate on-chip detection with good detec- ties, the samples could be divided by external fields, such as
tion limit at 4.1 ng/mL [6]. The specific antibody also can be electronic field, centrifugal force, magnetic field, or ultra-
conjugated on the surface of nanoparticles or magnetic sonic sound wave [12].
beads, which could capture the desired antigen in the chan-
nels and realize sample separation. This method was suc-
cessfully performed to the quantitative and sensitive detection 26.2.2 Detection
of anthrax biomarker PGA in solution [7] and detection exo-
somes from human ovarian cancer patients’ serum [8]. Various detection strategies could be realized on the micro-
Similar to antibodies, aptamer has higher affinity and speci- fluidic chip, which mainly include methods as follows.
ficity for target and widely used in sample separation on the First is the electrical detection. As a typical type of sensor,
chip. The aptamer could be immobilized at the microfluidic electrochemical sensors measure (biological) chemical reac-
26 Microfluidic Chip 359
Fig. 26.2 Nanoparticle separation using ion concentration polarization low electric field produces a small repulsion distance, while a high elec-
with a Nafion nanojunction, which generates a repulsion distance, tric field traps the nanoparticles in the ion depletion region
depending on the electric field strength in the ion depletion region. A
tions through digital electronic signals converted by transducers. detection ability [15]. For example, fluorescence- and
As a fixed and transduction platform, electrodes have been luminescence-based methods were fundamentally technical
used in most electrochemical detection. There are many elec- deeply intergraded with microfluidics. Li achieved analysis
trochemical sensors combined with microfluidics for multiple protein or DNA on paper-based microfluidics by using
types of biomarker analysis. There are several electrochemical FRET-based optical detection [16]. Chen used LSPR micro-
methods, including amperometry, differential pulse voltammo- array integrated with microfluidics with eight parallel chan-
grams, chronoamperometry, cyclic voltammetry, impedance nels, which showed high sensitivity and quantitatively
spectroscopy, and electrochemiluminescence. The above measured cytokines from raw serum samples with very low
detection methods were successfully used in microfluidics to concentration [17].
analyze a wide type of samples, including protein, nucleic The third is the acoustic waves-based method. This is a
acids, glucose, lactate, cancer markers, pathogens [13]. noninvasive method which was widely used for cell isola-
Second is the optical detection. This category has a wide tion, sorting, fusion, and interaction studies based on micro-
variety of methods and is widely used, which mainly includes fluidic chips [18]. Acoustic forces for manipulation of cells
fluorescence absorption and optical luminescence detection, and molecules in microfluidics offer rapid, noninvasive, and
surface plasmon resonance-based detection, surface- highly efficient techniques which are of great interest in bio-
enhanced Raman scattering-based detection, and optical logical and chemical tests. Other detection methods such as
waveguide-based detection [14]. Optical detection has enor- mass spectrometry, flow cytometry, and DNA sequencing
mous advantages such as less interference, ultrahigh speci- were also combined with microfluidics for multiple target
ficity and sensitivity, low background signal, and multiplexing analyses.
360 X. Fang
26.3 Technology Development 26.3.2 Polymers
The material for fabricating microfluidic chip dominates the Compared with silicon-glass materials, polymers are easier
functions of microfluidics. In the past decades, many kinds to obtain and cheap. As a result, they become one of the most
of materials have been introduced in this field, including con- widespread materials in microfluidics. Polymers in microflu-
ventional material silicon and glass, plastics, hydrogel, idics are fallen into three categories: elastomers, thermosets,
paper, and droplet (Fig. 26.3). and thermoplastics.
One of the most feasible elastomers for microfabrication
is polydimethylsiloxane (PDMS). It is easy and low cost of
26.3.1 Silica and Glass microfabrication. Another superiority of PDMS is its high
elasticity. It is also gas permeable and compatible with cell
In the 1970s and 1980s of the last century, a miniaturized culture. So, PDMS-based equipment is widely used in bio-
analytical device, gas chromatograph, was fabricated on sili- materials research, such as cell culture and screening, bio-
con. It could be used to separate a simple mixture of com- chemical tests, and so forth [10, 23]. It also has limitations.
pounds rapidly [19]. At the end of the last century, the The porous matrix of PDMS was composed of Si-O back-
silicon-based analyzers were presented with a miniaturized bones covered with alkyl groups. Therefore, it is incompati-
open-tubular liquid chromatography on a silicon wafer [20]. ble with organic solvents [24].
At the same time, the concept “miniaturized total chemical The fabrication technology of the polymer microfluidic
analysis system” or μTAS was proposed. In this silicon chip chip is quite different from that of the glass chip. The fabri-
analyzer, sample pretreatment, separation, and detection were cation techniques used include hot pressing, molding, injec-
incorporated [1]. Silicon-glass microfluidics has provided tion molding, LIGA, and soft etching [4].
optical accessibility, unprecedented compatibility, and record Hot pressing is a chip-making technology that is widely
bonding strength, but the fabrication of silicon-glass is expen- used to rapidly replicate microstructures [5]. The polymer
sive. To reduce the cost, chip area was reduced and high-pres- substrate and the mold are aligned and heated to apply a cer-
sure and temperature fluid connectivity was achieved. Finally, tain pressure to obtain a micro-structured chip. The mold for
the price for each chip is USD5. So the silicon-glass microflu- the simple hot pressing method may be a wire having a diam-
idics are disposable for many applications [21, 22]. eter of 50 μm or less or a positive film of a microchannel
wafer having an embossed protrusion. The simple micro-
channels are intelligently fabricated using the wire as a mold.
And the channel intersections are pressed into irregular
shapes in the same plane, which adversely affects the injec-
tion and separation. The microfluidic chip is fabricated by
etching a microchannel silicon positive film to obtain a com-
plex microchannel, and the channel junction has a satisfac-
tory structure.
Molding is the main method for producing polymer chips,
mainly by using a mold such as a photoresist and curing a
liquid polymer on a mold to obtain a microstructured chip.
Epoxy SU-8 photoresist, AZ positive photoresist, silicon
material, or glass are commonly used molds [6]. Whitesides
proposed a soft lithography technique to design polydimeth-
ylsiloxane (PDMS) devices in 1998 [22], aiming to investi-
gate the fluid behavior at laminar flow in narrow well-defined
channels, which is referred to as microfluidic. Thus microflu-
idics based on PDMS has become popular devices for mic-
roparticle production. Nevertheless, their 2D characteristics
and incompatibility with several organic solvents limit
applications.
Injection molding is a method in which raw material is
placed in an injection machine [25], heated to become a fluid
into a mold, and cooled to release a mold. In the process of
Fig. 26.3 Various materials for the fabrication of microfluidic chip injection molding, the mold is complicated to manufacture,
the technical requirements are high, and the cycle is long, decade, starting with the groundbreaking work of a single
which is a key step in the whole process. Usually a mold can laboratory and becoming a business involving dozens of
produce 300,000–500,000 polymer chips, with good repeat- independent groups in academia and industry. The practica-
ability, short production cycle, and low cost, which is suit- bility of these methods, as well as the material sciences that
able for the production of formed chips. govern their operations, the richness of chemistry and phys-
LIGA is the abbreviation of German lithographie galva- ics, may continue to generate interest in the field in the com-
noformung abformung. It consists of three parts: X-ray ing years.
deep lithography, micro-electroforming, and micro-replica- Sim and colleagues fabricated microparticles by introduc-
tion. It is mainly used to make high-amplitude microfluidic ing a novel double-layered PDMS substrate [27], combining
chips. The first step is synchrotron radiation X-ray deep conventional photolithography and multilayer soft-
lithography. A few millimeters thick X-ray sensitive photolithography. Two straight control channels for pneumatically
sensitive material (usually PMMA) is coated on a layer of actuating the membrane to mold and release the particles are
highly conductive metal film, using X-rays of synchrotron located on the top layer, while a T-shaped joint channel for
radiation source with good parallel performance and high the floating flow is integrated into the bottom layer.
radiation intensity. The pattern on the film is transferred to Interestingly, by controlling the pressurization in the top
the photoresist layer, typically at lithography depths of a channel, the film is deformed to confine the suspension to the
few hundred microns. The second step is electroforming, shape of the design, and then the particles obtained by UV
that is, depositing a metal in the gap of the photoresist pat- irradiation or by release are restored to their original shape.
tern after development, and electroplating can be used, and Finally, the particles can be discharged through the bottom
the metal film of the lower layer of the photo-adhesive is channel. Of course, there are some limitations in the applica-
directly used as an electrode for electrolysis. In the third tion of soft lithographies, such as 1% shrinkage deformation
step, the electrophoresis chip is copied by injection mold- after PDMS curing, and a certain expansion of the aspect
ing using a thermoplastic polymer material such as PMMA ratio under the action of toluene and ethane. The elasticity
and PC. and thermal expansion of PDMS makes it challenging to
Soft lithography is a new micro-pattern replication tech- obtain accuracy, and soft lithography has been limited in the
nology that emerged in the 1990s [22], with advantages of processing of multiple layers. Since the elastic film is too
soft, elastomeric elements for pattern formation. The earliest soft, a large aspect ratio cannot be obtained. The ratio of
soft-lithographic method is a form of contact printing that depth and width will cause distortion of the microstructure
uses a high-resolution elastomeric stamp with a chemical ink after deformation.
capable of producing a self-assembled monolayer on a sub-
strate [10], and then material deposition or removal from the
substrate forms desired patterns. Interest in this technology 26.3.3 Hydrogel
stems from its ability to form structures with dimensions
deeper into sub-micron systems without the use of common Hydrogel is a kind of natural or synthetic polymer material.
chemical laboratory equipment. The proposition of the It is microscopically a three-dimensional network structure
micro-contact printing method initiated a series of related composed of hydrophilic polymer chains. The network gap
work, initially led by Whitesides group, which explored is usually from nanometer to several hundred nanometers.
these elastomeric components as seals for printing as well as This type of material normally includes polyethylene glycol
imprinting [24, 26]. These technologies are used in laborato- (PEG), polyvinyl alcohol (PVA), and poly hydroxyethyl
ries across the world, ranging from photonics and biotech- methacrylate (PHEMA), or agarose gel, gelatin, sodium algi-
nology to microfluidics and electronics. The complexity and nate gel, and hyaluronic gel.
performance of soft lithography patterning methods are rap- Hydrogels have a range of bulk and surface properties
idly evolving. The soft lithography process can be divided that are different from other materials. The special bulk prop-
into two parts: the fabrication of elastomeric components erties are high water content, high permeability, structural
and the use of these components to pattern the features of the stability, and mechanical elasticity. In addition, some hydro-
geometry defined by the relief structure of the component. gels also have excellent optical properties such as transpar-
Elastomeric poly(dimethylsiloxane) or PDMS (Sylgard 184, ency. The surface properties of hydrogels are mainly
Dow Corning) is commonly used for this purpose. This man- manifested by the overall hydrophilicity. Hydrogel material
ufacturing sequence has a very high fidelity through opti- can be synthesized in situ from a monomeric aqueous
mized materials and chemical composition. In fact, recent solution by a free radical reaction initiated by ultraviolet or
work has shown that reliefs with nanometer depth, and both visible light. The basic process of hydrogel synthesis includes
nanometer depth and width, can be accurately reproduced. (1) adding an appropriate amount of photoinitiator to an
The field of soft lithography has grown rapidly over the past aqueous solution containing a double bond monomer and (2)
362 X. Fang
photoinitiating the photodegradation to generate a primary drug delivery, and cell survival environments. A hydrogel
radical under illumination conditions and further initiating system can be thought of as a network of overlapping chains
polymerization of the monomer and crosslinking reaction to and cross-linked chains, where pore size or mesh size can be
form a gel. The micron-scale hydrogel structure can be pre- a defining parameter. The size of the microstructure-like
pared by using the photolithographic polymerization princi- pores leads to molecular diffusion, directional migration, and
ple described above by photolithography. water swelling. These properties can be transformed and
Various hydrogels are often utilized in applications of understood by different parameters, such as pore size.
microfluidics, such as tissue engineering [15], and bio- Hydrogels have high permeability to oxygen, nutrients, and
chemical research [20], because of desirable biocompati- other water-soluble metabolites. For example, molecules
bility, stiffness, and degradation. Hydrogels are one kind of having a molecular weight of less than 1 kDa and a Stokes
materials showing hydrophilic polymer chain networks, radius of less than 1 nm can be freely diffused into the gel
typically using water as the dispersion medium. Some rep- system.
resentatives of naturally derived hydrogels are collagen, Hydrogel with basic bulk properties of permeability and
agarose, alginate, chitosan, gelatin, fibrin, and hyaluronic biocompatibility is beneficial for stereo micropatterns as
acid. Synthetic- derived hydrogels such as poly(acrylic well as cell biology applications, because cell culture has
acid) and poly(ethylene oxide) have also been widely used been influenced by surrounding environment chemically and
[28]. Hydrogels have a variety of properties (e.g., swelling, physically. Recently, microfluidic technology makes it real-
mechanics, rheology, and adhesion) by additional chemical istic to culture cells in vitro through a microenvironment.
chelating agents, UV irradiation, or temperature crosslink- Hydrogels set diffusion of chemical molecules free, mini-
ing [29]. mizing flow effects for high resolution, time-cse cell activity
The main merits of hydrogels in microfluidics are as fol- imaging. Hydrogel integrated systems can easily introduce
lows. (1) Free diffusion of small molecules. Most cytotro- multiple cell types in a system and provide the creation of
phins and growth factors are diffusible in hydrogels, so biochemical gradients in two- or three-dimensional
hydrogel-based devices can easily create diffusion mem- microfluidics.
branes for different applications. (2) Optical characteristics. Cheng and colleagues prepared a microfluidic based on
This property of the hydrogel makes it possible to observe hydrogel capable of forming steady and long-term chemical
the diffusion of fluorescent molecules and the behavior of the gradient in a microenvironment [20]. The responses of wild-
cells within the gel structure under a microscope. (3) Easy to type bacterial and HL-60 cells line were recorded and col-
get. Hydrogels, such as agarose, are inexpensive and com- lected for analysis, which was more comparable due to stable
mercially available. (4) Quantitative manufacturing. The gel and reliable chemical gradients and zero flow conditions
has a variety of designs and plasticity for precise dimen- within the bacterial or cell area. Similar gel integration meth-
sional alignment. (5) Cell biocompatibility. Most of the ods for cell migration have been widely used in research,
hydrogels with Young’s modulus comparable to cells are such as high-throughput bacterial chemotaxis studies and
non-toxic. competition for multiple chemoattractants.
On account that hydrogels-based microfluidics has many The usage of hydrogels for 3D cell culture or tissue engi-
merits, many reports have been investigated to integrate neering is not only a research topic for scientists but also a
hydrogel into the microfluidics. But a well-defined hydrogel- research topic for medical doctors. We believe that the devel-
based microfluidic chip largely depends on several variables, opment of different hydrogels, integrated methods, and
such hydrogel preparation, mechanical properties, mass dif- applications will further the advancement of basic science
fusion properties, and biocompatibility. and clinical applications.
Hydrogels can be crosslinked primarily by three methods:
temperature, ultraviolet (UV), and chemical chelating agents.
Ionic or covalent crosslinking will bond the hydrogel homo- 26.3.4 Paper
polymer, copolymer, or macromonomer to form an insoluble
polymer matrix. Therefore, the nature of the matrix depends The paper-based microfluidics is a microfluidic chip which
primarily on the polymer chain, the type of crosslinking mol- uses paper (such as filter paper, chromatography paper, and
ecule, and the crosslinking density. nitrocellulose membrane) as a chip manufacturing material
Elastic modulus is a very important indicator of materials, and a biochemical analysis platform [30]. The paper chip
especially for biological systems. The softness or hardness system can also integrate basic operations such as sample
of hydrogel may not only limit the size of the structure but preparation, biochemical reaction, separation, and detection.
also limit the life pressure of cell growth and exercise. The microchannel forms a network, and the controllable
The diffusion of molecules in the system is critical to fluid runs through the entire system to realize various func-
understanding and controlling chemical gradients, small tions of the conventional laboratory. Paper chips have many
significant advantages, such as simple production, low cost, The plotting method was originally proposed by the
small size, lightweight, easy storage and transportation, good Whitesides group [31]. They used a plotter to plot the PDMS
biocompatibility, low sample reagent consumption, and fast dissolved in n-hexane onto a common filter paper to form the
analysis speed, and can transport samples without their own desired pattern, which was then cured to give a paper path.
porous structure without external driving force. Compared There are two modes of printing: one is to soak the entire
with the traditional test strip, the paper chip has the follow- sheet of paper with a hydrophobic material, and the solvent
ing advantages: (1) the paper-based microfluidics form a net- is inkjet-printed to dissolve the hydrophobic material, expos-
work with microchannels, and the controllable fluid runs ing a hydrophilic paper path on the paper. The other is to
through the whole system, and have the advantages of quan- print a hydrophobic material directly on the paper to form a
titative, constant speed, fluid uniformity, multiple simultane- hydrophilic/hydrophobic phase-to-phase region to form a
ous detections, and high throughput; (2) microchannel paper channel. In 2008, Abe and colleagues used a printing
minimum width of paper-based microfluidics is up to method to prepare paper chips [32]. The filter paper was
100 μm. It only needs a small dosage of reagents and sam- soaked in a 1% polystyrene toluene solution and placed in an
ples and can achieve micro-analysis, further reducing the inkjet printer after drying. The ink cartridge in the inkjet
cost of detection; (3) paper-based microfluidics can also be printer is replaced with a toluene solution, and the pre-
assembled into a three-dimensional structure that allows designed pattern is printed on the filter paper by inkjet print-
multiple steps of separation, purification, and detection in a ing. The toluene printed from the printing nozzle dissolves
single injection. Paper-based microfluidics technology has the polystyrene and exposes the fiber of the paper. Thus, a
been increasingly involved in the fields of clinical diagnosis hydrophilic paper channel is formed.
and food safety and has important practical significance and In order to achieve a multi-step ordered chemical reaction
practical value. or multiple pretreatment steps on a single chip and to increase
Paper-based microfluidics is a new technology that has the speed and efficiency of the analysis process, the staff has
emerged in recent years. In 2007, the Whitesides group first studied the manufacturing method of 2D μPAD-based 3D
proposed this concept and successfully produced paper chips μPAD. Recently, two methods for 3D μPAD fabrication have
capable of simultaneously detecting proteins and glucose been reported: stacking and origami.
[21]. Paper chips have attracted much attention since they The 3D μPAD has a stereo channel, which makes the 3D
became research hotspots. In 2011, Harvard University and μPAD better than the 2D μPAD. The 3D μPAD has a higher
DFA’s Bill Gates Foundation and the UK Department for flow rate than the 2D μPAD because length in the z-direction
International Development sponsored a project to use paper is shorter than the length in the x–y plane. And 3D μPAD
chips to detect aflatoxin in bacteria and corn in milk. contains simultaneous experiments. Each layer of 3D μPAD
The material of the paper chip is divided into two types: can be used to implement different functions. For instance,
hydrophobicity and hydrophilicity, such as photoresists, the first layer can be used as a filter layer.
polydimethylsiloxanes, waxes, polystyrenes, and alkyl In addition, 3D μPAD is based on 2D μPAD, so the pro-
ketene dimers. The hydrophilic material is a matrix material duction method here only involves how to convert 2D μPAD
of a paper chip, such as filter paper, nitrocellulose mem- to 3D μPAD.
brane, and cotton cloth. At present, methods for producing The paper-based microfluidics detection includes colori-
paper chips include photolithography, drawing, and metric, electrochemical, chemiluminescence, electrochemi-
printing. luminescence detection, and various detection technologies
Whitesides team first proposed the application of photoli- related to an immune reaction. These developing methods
thography to the production of paper chips in 2007 [21]. are appropriate for fast diagnostic in terms of cost and
They used the chromatographic filter paper as the substrate volume.
to complete the curing of the photoresist on the paper by Visually observing the color change produced by the
traditional photolithography and obtained various design reaction of the analyte and the reagent is a rapid and simple
patterns with a channel width of 1 mm. Typically, the filter method for qualitative colorimetric detection. And it is also
paper is immersed in a SU-8 photoresist dissolved in cyclo- the first detection method commonly used in paper chips.
pentanone, then spin-coated at 2000 rpm/min, dried to Whitesides group firstly used paper-based microfluidic to
remove cyclopentanone, and immersed in photoresist. The qualitatively analyze glucose and BSA in 2007 [21]. Glucose
filter paper and the mask are exposed to ultraviolet light. oxidase and horseradish peroxidase catalyze the production
After dried in the oven, the photoresist is polymerized and of hydrogen peroxide. Iodide was oxidized to produce
immersed in propylene glycol monomethyl ether and washed iodine, radically producing a color change. After combina-
with isopropyl alcohol to remove unpolymerized photoresist. tion of ionized tetrabromophenol blue and protein, the color
Then the whole piece of chromatography enhanced its was changed from yellow to blue, with a detection limit of
hydrophilicity by oxygen plasma exposure. 0.3 μmol/L and 2.5 mmol/L, respectively.
364 X. Fang
Electrochemical detection is a method of converting a paper-based microfluidics enables in-sample response,

chemical signal of the substance into an electrical signal by which will be an important step in improving POCT.
an electrode in a solution. The electrochemical detection in
the microfluidic chip has the advantages of high sensitivity,
good selectivity, simple device, and easy electrode prepara- 26.3.5 Droplet
tion. It is one of the easy methods to realize the miniaturiza-
tion of paper-based microfluidic detection. The droplet microfluidic chip is also a new technology for
In 2011, Yu and colleagues firstly reported a chemilumi- manipulating tiny volume liquid [35]. The existing droplet
nescence detection method for paper chips [33]. They immo- microfluidics can be divided into two types: droplet moving
bilized uricase in a paper channel made by cutting, and then in the microchannel and digital droplet moving in the plane
added 20 μL of the uric acid sample. The uric acid was cata- based on the surface electrowetting effect. The basic process
lyzed by uricase to form hydrogen peroxide, and the gener- of channel droplets is using two immiscible liquids, one of
ated hydrogen peroxide migrated to the detection zone to which is the continuous phase and the other as the dispersed
react with the target substance. The illuminating analyzer phase. The dispersed phase in the continuous phase to form
detects the light intensity, and the data is processed by com- droplets moving within the channel is in a tiny volume
puter recording. After 10 weeks of dry storage at 4 °C, the (10−15–10−9 L). The formation of droplets on a microfluidic
chemiluminescence intensity was 97.8% of the initial lumi- chip is similar to the emulsification process. In a conven-
nescence intensity. tional emulsification process, two immiscible liquids, such
In 2011, Delaney used the electrochemiluminescence as oil and water, are divided into two layers in a container.
detection method for the detection in paper chips [34]. They When a suitable surfactant is added and strongly with stir-
used terpyridine pyridinium as an electrochemiluminescent ring, the oil is dispersed in water to form a milky droplet.
reagent to detect the contents of the co-reactant nicotinamide Droplet microfluidics systems create discrete volumes
dinucleotide, and 2-N-dimethylaminoethanol and the detec- using immiscible phases instead of continuous flow systems.
tion limits were 72 μmol/L and 0.9 μmol/L, respectively. Microfluidics is characterized by low Reynolds number flow
The immunoassay technology lies in specific binding of conditions that require that fluid flow be substantially lami-
antigen to antibodies. This binding can also occur in vitro. nar. Continuous flow-based systems use this phenomenon to
After the recognition of antigen and antibody, different tags produce many novel microenvironments. Layer prevalence
were labeled for the naked eye or instrument detection. also allows for the generation of precise concentration gradi-
Immunoassay technology is one of the most widely used bio- ents that have been used in cell migration studies. While con-
logical detection methods. It has the advantages of good ver- tinuous flow devices provide fine control overflow
satility and specificity. It is applied in the fields of disease characteristics, amplification is a challenge as device size is
screening and diagnosis, drug concentration monitoring, almost linearly proportional to the number of parallel experi-
environmental monitoring, and video security testing. The ments. However, various reactions can be conducted on
immunoassay technologies implemented on paper chips droplet microfluidics without increment of device size. In
include enzyme-linked immunoassay, gold-labeled immuno- addition, recent discoveries have demonstrated that droplet
assay, electrochemical immunoassay, chemiluminescence microfluidics was applied for Boolean logic functions [36], a
immunoassay, and electrochemiluminescence key step in implementing microfluidic computer chips.
immunoassay. The droplets can be divided into two types depending on
Paper-based microfluidic are broad-spectrum used for the dispersed phase and the continuous phase, that is, W/O
point-of-care testing (POCT) diagnostics owing to their low type droplets and O/W type droplets. Among them, the W/O
cost and ease of replication in manufacture. Through the type droplets have a water phase as a dispersed phase and an
interface between materials science and biochemical engi- oil phase as a continuous phase. The O/W type droplets have
neering, paper-based analysis becomes convenient, sensitive, an oil phase as a dispersed phase and an aqueous phase as a
accurate, and functional. Combining paper diagnostics with continuous phase. The most common application of droplet
mobile-based optical inspections, telemedicine plays an microfluidics is as a microreactor to investigate reactions on
important role in improving healthcare services in resource- microscale during processes. The reactions here generally
constrained environments. However, the potential for paper refer to various chemical reactions, biochemical reactions,
diagnostics is not exerted to the uttermost, unless some and various processes involving phase transitions such as the
aspects of POCT diagnosis would be achieved, such as sam- synthesis of nanoparticles.
ple pretreatment, plasma separation, nucleic acid isolation, Droplets and particles as important tools have the poten-
and nucleic acid amplification. These aspects are more tial for drug delivery and chemosensing. In order to work
advantageous than traditional immunoassays in specificity properly, the proper dosage, as well as manufacturing, must
and sensitivity. It can be envisaged that a fully integrated be ensured. Because the biological and chemical characteris-
tics of the microparticles are strongly influenced by size and trodes on the chip can be fabricated on a 2D array with a
morphology, it is necessary to be able to produce these struc- common electrode design, so a single platform can be recon-
tures in a defined volume and composition. Conventional figured for a variety of applications. The droplet-based DMF
top-down emulsion and particle formation methods, such as allows parallel operation on the platform, increasing the
direct agitation of the immiscible fluid and grinding of the speed of the analysis processing steps. In addition, signifi-
polymeric material, respectively, give rise to a broad size dis- cant precision in sample preparation can be achieved on
tribution. Additionally, droplet-based microfluidics has been these systems. As a result, DMF has the potential to develop
shown to produce highly monodisperse droplets with a equipment for mass production and can be incorporated into
dimensional change of less than 1% [35]. the preferred equipment for POCT.
In a T-joint structure, the inlet containing the dispersed Sista and colleagues proposed a portable DMF system
phase intersects perpendicularly with the main channel con- that enabled immunoassays, enzyme assays, and DNA
taining the continuous phase. The two phases form an inter- amplification via chemiluminescence and fluorescence assay
face at the junction. As the fluid flows, the dispersed phase [38]. It had a PCB chip and an integrated reservoir for storing
enters the main channel. The sheer force produced by con- samples and removing waste buffer. The magnetic and heat-
tinuous phase and subsequent pressure gradient causes the ing segments required for the measurement were embedded
head of the dispersed phase to elongate into the main pas- in the DMF chip. The electrical system unit is integrated into
sage, till the dispersed phase becomes thinner and eventually the device to control the on-chip process. The study showed
breaks the fluid into droplets. The size of the droplets can be an attempt to successfully develop a DMF-based portable
varied by varying the fluid flow rate, the channel width, or device for POCT.
relative viscosity between two phases. There may be many
inlets in T-junctions, which have been used to implement
chemical reactions and compose droplets alternatively. 26.4 Clinical Application
In a flow-focusing configuration, continuous phases and
the dispersed phase are forced through designed regions in Since microfluidic technology was established, it has been
microfluidics. This design uses continuous symmetrical applied in various clinical applications, including diagnosis,
shear on the dispersed phase, which enables more controlla- biomarker detection, therapeutic drug monitoring, disease
ble and stable droplet generation. The extension of the flow classification, prognosis assessment, etc. (Fig. 26.4). This
is shear focus, the purpose of which is to produce the highest technology demonstrates strong analytical performance in
shear singularity that exists in the nozzle region. This singu- clinical applications, significantly promoting the level of
larity ensures that the breakout of the droplet from the fluid clinical medical research and disease diagnosis and treat-
stream always occurs at this point, forming a uniform drop- ment [39]. The specific applications are as follows:
let. And the size of droplets can be changed by manipulating
the flow rate of the continuous phase.
Dielectrophoresis (DEP) can be used to create uniform 26.4.1 Capillary Electrophoresis
droplets by pulling droplets from liquid reservoirs [37]. It
differs from electroosmosis because fluid can be electrically Capillary electrophoresis (CE) is a liquid phase separation
neutral and the force exerted on the body without the current technology with capillary as separation channel and high
is caused by a non-uniform electric field. The operating prin- voltage direct current electric field as a driving force. In fact,
ciple behind DEP for droplet formation is based on the phe- capillary electrophoresis involves electrophoresis, chroma-
nomenon that a polarizable fluid will be attracted to a region tography, and its cross-cutting content, which moves analyti-
of higher electric field strength. The electrowetting on dielec- cal chemistry from micro-level to nano-level, carries out
tric (EWOD)-based droplet platform utilizes the wetting-like single-cell analysis, and even makes single-molecule analy-
force, which acts by three main forces: the droplet, the wet- sis possible. Various studies integrated capillary electropho-
ting force on the interface between the surrounding medium; resis with microfluidics for different types of biomarker
the two-fluid interfaces force; and physical strength due to analysis. There are several capillary electrophoresis modes,
pressure gradients in fluid. The size and uniformity of the including capillary zone electrophoresis, capillary isoelectric
droplets depend on the voltage magnitude and frequency. focusing, capillary isotachophoresis, and micellar
Digital microfluidics (DMF) is a droplet-based microflu- electrokinetic chromatography gel electrophoresis. The

idic system with a planar geometry that can be processed by above strategies could be used with microfluidics for protein
photolithography [37]. or nucleic acid detection purposes.
Fluid mechanisms on these systems do not require exter- Specifically, Guo and colleagues developed a microflu-
nal modules or complex geometries such as pumps or valves. idic chip-CE based glycated hemoglobin rapid separation
Another unique feature of DMF is that the actuation elec- detection method, which used the specific absorption peak of
366 X. Fang
Fig. 26.4 Biochemical analysis integrated on microfluidic chip for different applications
glycosylated hemoglobin at 415 nm for detecting the level of applications. Facing this problem, many studies that utilized
glycated hemoglobin in healthy people and high-risk groups microfluidic technology successfully achieved the above
with a low dosage, good specificity, high precision, and high requirement. The specific application cases are as follows.
accuracy [40]. Similarly, Redman and colleagues also estab-
lished a microfluidic CE-ESI devise for rapidly assessing 26.4.2.1 Polymerase Chain Reaction
hemoglobin and human serum albumin glycation with mini- Polymer chain reaction (PCR) is the most popular nucleic
mal sample preparation from raw blood. This method has the acid analysis technology. Many studies integrated PCR with
potential to provide much more information by using a sin- microfluidics, which realized high-throughput, fast, easy,
gle and easy step than traditional methods and holding good and accurate nucleic acid detection. For example, Despina
clinical application prospects [41]. and colleagues proposed a reaction device with three inte-
grated resistance copper heaters. The reaction liquid flows in
the channels and undergoes different temperatures as it
26.4.2 Detection of Nucleic Acid passes through different regions, achieving a cycle of dena-
turation, annealing, and extension (time ratio is 1:1:2); the
The nucleic acid is one of the most basic substances in life. It device was designed for a total of 30 cycles to complete the
is also the main material basis for storing, reproducing, and PCR reaction. The whole reaction device is made of plastic,
disseminating genetic information. Nucleic acid biomarkers which guarantees the low cost, easy to handle after one-time
were critical in the clinic, and it has great significance in sev- use, and can effectively prevent cross-contamination between
eral diseases’ progress such as cancer, infectious disease, detections [42]. Centrifugal-driven disc-type microfluidic
and hereditary disease. Therefore, achieving fast and accu- chips are also used in the construction of integrated
rate nucleic acid analysis is an important goal for clinical microfluidic-PCR. Strohmeier and colleagues reported a
low-cost, ready-to-use, primer, probes, and reaction buffer The integration of microfluidics and PCR effectively
embedded centrifugal chip. The diameter is only 130 mm, overcomes the shortcomings of traditional PCR technology
and each reaction zone has an inlet hole for the sample to be like the complex operation steps and high environmental
tested. The inlet hole is connected to a bridge channel having requirements. The microfluidic-PCR platform holds great
eight equal volumes and further connected to the final eight potential in future clinical use, especially in the application
amplification reaction wells. After loading, the chip is placed of point-of-care settings.
in a centrifugal-thermal cycle integrated detector to achieve In addition, as an alternative to PCR, a large class of iso-
simultaneous, rapid, and accurate detection of multiple thermal nucleic acid amplification detection techniques
KRAS gene mutations [43]. Gan and colleagues invented a have been established. These amplification methods are car-
chitosan-modified filter paper embedded microfluidic device ried out at a constant temperature, freeing the requirements
that can efficiently capture and enrich DNA and in situ PCR of PCR for thermal cycler. Compared with classic PCR,
amplification of raw clinical samples. The method utilizes a these methods are simpler and more convenient. By inte-
solid phase paper-based microfluidic to integrate nucleic grating with microfluidic technology, high-throughput accu-
acid extraction and amplification into a reaction well and can rate and simple nucleic acid analysis can be realized. For
directly detect trace targets in blood samples on-chip [44]. example, Fang and colleagues described an octopus-like
Besides the above traditional PCR method, microfluidic multiplex microfluidic LAMP assay for rapid and multiple
was also used to construct the digital PCR platform for the analysis of gene targets in the point-of-care settings, which
absolute quantification and single-copy level nucleic acid provide a robust approach for accurate and efficient diagno-
detection. Through the droplet generation, microporous sepsis viruses. This chip has ten microchamber amplification
aration, or channel separation strategies, the PCR reaction system in a PDMS-glass format. Each microchamber was
system can be diluted indefinitely on the microfluidic chips, coated with different probes which may achieve direct
which can guarantee that a micro-reaction system contains at naked-eye and simultaneous determination of the result for
most a single copy of the target sequence. These methods are each target. The assay has the ability of qualitative and
also applied in a variety of clinical applications like ALK quantitative analysis of multiple genes, high specificity,
gene detection [45], Trypanosoma cruzi detection [46], and simple operation, and cost/time effectiveness (Fig. 26.5). At
Alu gene methylation status detection [47]. the same time, this chip has the advantages of isothermal
Fig. 26.5 Fully automatic microfluidics for nucleic acids

368 X. Fang
nucleic acid detection and microfluidic technology [48]. The use of microfluidics may solve the above problems
Further, Fang and colleagues developed a portable inte- [53]. Wei and colleagues reported a microfluidic chip using
grated microchip of LAMP which combined rapid DNA micro trench chip stacked on a glass slide, which has a sam-
release, exponential signal amplification, and naked-eye ple inlet hole and an air chamber. The microfluidic channels
result read-out on the chip. This novel system also success- on the chip can reduce the DNA-DNA hybridization time
fully applied for point-of-care diagnosis of bacteria with from 18 h to 500 s using 1 μL DNA sample, which greatly
good detection performance [49]. Similarly, Yuan and col- improved the reaction speed [54]. Zhang and colleagues
leagues introduced a LAMP method based microfluidic chip developed an integrated microfluidic chip, which realized
for detecting three allergens at the same time. The chip is multiplex SNP analysis from whole blood. The chip surface
made of PMMA and the diameter is 80 mm. The main struc- was 32 mm by 30 mm and had blood lysis chamber, a cross-
ture on the chip, including a sample well, a vent, and eight flow filter, a T-junction mixer, and two single quantitative
reaction wells, may realize automated multiplex nucleic PCR micro-reactors. This chip realized the automated and
acid detection. The accuracy of this novel method was com- simultaneous detection of two SNPs, which could be
parable with traditional methods [50]. These microfluidic- accepted by the clinic and POCT demands as well as pro-
isothermal nucleic acid detection platforms have a broad mote precision medicine [55].
application foreground in promoting the automated, porta-
ble, and high-precision analysis. 26.4.2.3 Genotyping Detection
Genotyping is a technique for determining individual geno-
26.4.2.2 Detection of Gene Mutation types using biological assays. Traditionally technology for
Gene mutation refers to the change of base-pair composition genotyping includes restriction fragment length polymor-
or gene sequence in structure. Gene mutations can occur at phism (RFLP), terminal restriction fragment length poly-
any developmental stage, usually at the stage of DNA repli- morphism (T-RFLP), amplified fragment length
cation, that is, cell division intervals, including mitotic and polymorphisms (AFLP), and multiplex ligation-dependent
meiotic intervals. Gene mutations are closely associated with probe amplification (MLPA). Genotyping detection was
DNA replication, DNA damage repair, cancer, and aging, important for many genetic variations like microsatellite
which play a vital role in many biological function regula- instability, trisomy, aneuploidy, and loss of heterozygosity
tions. The detection of gene mutation is important for under- which may cause various diseases like cancer and Down syn-
standing the biological behavior and the development of drome. When using microfluidics to detect genotyping, it
diseases. The types of genetic mutations include substitu- may show some obvious advantages.
tion, translocation, deletion, and insertion, which may cause Kukhtin and colleagues established a lab-on-a-film sys-
the same sense mutation, missense mutation, and nonsense tem for genotyping of multidrug-resistant Mycobacterium
mutation. Until now, a lot of researchers used microfluidic tuberculosis in the sputum sample. Amplification, hybridiza-
technology for the detection of gene mutation and made tion, and washing were all performed on the chip, which
great results. consists of a total of 203 hemispherical gel elements on the
Wu and colleagues established a microfluidic chip using substrate. This disposable chip could automatically and sen-
SERS analysis of KRAS mutation. The extracted DNA of sitively detect MDR-TB markers in clinical samples, which
wild-type KRAS could open the loop probe of SERS attached have important clinical significance [56]. Jung and col-
on the microfluidic and made the SERS signal off. Oppositely, leagues developed a novel three-dimensional microfluidic
the mutant KRAS can’t open the loop and made the SERS hydrogel array for detecting multiplexed genotyping from
signal remained on. This method showed great detection per- clinical samples. The chip was designed of 1*11 hydrogel
formance and was useful for tumor diagnosis and personal- array and contained two T-shaped channels. Each channel
ized therapy [51]. Cao and colleagues developed a PNA-LNA has different probe DNA and realized multiplexed detection
based isothermal amplification method on a CD-like micro- with good detection limit and high sensitivity [57]. Zhi and
fluidic chip for rapid detection of CALR type1 and 2 muta- colleagues reported a novel HBV genotypes method based
tions. The CD-like chip has four detection zones, and each on a microfluidic chip combined with loop-mediated isother-
zone has eight reaction chambers which could simultane- mal amplification. This chip was low cost, having good
ously implement eight-target detection and greatly improve detection performance, which was quite suitable for HBV
detection throughput. This method showed perfect sensitiv- diagnosis in underdeveloped countries [58].
ity, specificity, and stability [52].
Single nucleotide polymorphism (SNP) is also a com- 26.4.2.4 DNA Sequencing
mon form of gene mutation in humans, which is closely DNA sequencing technology could provide the base
related to many diseases. Conventional methods for SNPs sequence of a specific DNA fragment. DNA sequence infor-
detection were limited by their sensitivity and throughputs. mation has become an indispensable part of many applica-
tions, such as diagnosis, biotechnology, forensic biology, in which HV is applied, and a gate storage device where volt-
and biosystems. Various studies integrated sequencing and age alternates between the ground and the float. This device
microfluidic technologies to realize single copy, single could provide continuous monitoring of the amino acid
molecular, and single-cell analysis and got great success. change for 2 h, and 10 amino acids were resolved with the
Using droplet method, it can establish a low-cost, high- detection limit ranging from 1 to 20 nM [62].
throughput analysis platform by encapsulating thousands of Batalla and colleagues used electrochemical microfluidic
cells into the individual drop. Unique barcode RNA traps strategy to separate and detect d-methionine (d-Met) and
particles and cells through microfluidic devices in droplets, d-leucine (d-Leu). The detection strategies include electro-
where they undergo cell lysis and RNA hybridization. After injection of enzymes and d-AAs, electro-focusing of
splitting droplets and collecting hybridized particles, reverse enzymes and d-Aas, separation of d-AAs, intra-channel
transcription, PCR, and sequencing can be performed in a enzymatic reaction, and detection of hydrogen peroxide. The
single reaction, generating data from thousands of single-cell method has been proved feasible, which could be used for
transcriptomes. This is the basic principle for single-cell cholera-related diseases in point-of-care settings [63].
sequencing using droplet-microfluidics [59].
Nilsson and colleagues designed a microfluidic plat-
form for automated and multiplexed in situ sequencing 26.4.4 Peptide and Protein Analysis
purposes. The main operation steps of this chip include
reverse transcription, padlock probes ligation, rolling cir- Recently, with the development of micro- and nanotechnolo-
cle amplification, and sequencing by ligation. This tech- gies, microfluidics technology has supported the analysis of
nique has high efficiency, fast turnaround time, and lower the peptides and proteins [64–67]. Particularly, microfluidic
consumption of reagents. It could get the exact nucleic protein analysis has made a great impact in the field of POC
acid sequence information directly in the fixed cells, which diagnosis [68].
would benefit the cancer diagnostic ability and guide the
effective therapies [60]. 26.4.4.1 Sample Manipulation and Detection
Recently, Kong and colleagues developed a new method Microfluidics is capable of separating, concentration, and
for sequencing transcripts and targeting genome regions mixing solutions of proteins and their complexes effectively
(CORTAD-seq) simultaneously within the same single cell and quantitatively. It offers advantages of precise control,
on an automated microfluidic platform. This approach also low sample consumption, and fast sample processing, hold-
achieves single-cell level analysis and has the ability to ing great potential in basic research and clinical applications
assess the relationship between genetic and transcriptomic [4]. We discuss some developments relating to sample
changes in a single cell [61]. This research also indicated the manipulation and detection based on the microfluidic sys-
great application prospect of microfluidic technology with tem. Currently designed microfluidic biochips generally
sequencing. integrate functions such as liquid mixing, biological separa-
tion, and specific detection. An integrated blood barcode
chip (IBBC) is designed for sample separation and in situ
26.4.3 Amino Acid Analysis multiple plasma protein analyses. This chip was demon-
strated to sensitively and rapidly sample a large panel of pro-
Amino acids are the basic substances that make up the proteins in a wide concentration range from clinical samples
tein. Amino acids have series of metabolic pathways in the [69]. In addition, the IBBC system was also applied to detect
body, including synthesis of tissue proteins; transformation plasma proteins from a finger prick of whole blood. A bio-
into acid, hormones, antibodies, creatine, and other ammo- conjugated magnetic nanochains (Magchains) microfluidic
nia-containing substances; transformation into carbohy- biochip (MiChip) was developed, which was designed for
drates and fats; and oxidation into carbon dioxide, water, and multiplexed detection of protein biomarkers and specific
urea, producing energy. Under the disease state, certain bacteria based on Magchains and surface-enhanced Raman
amino acid metabolism patterns are characteristically scattering (SERS) nanoprobes using trace samples (~1 μL)
changed, which could be analyzed and benefit for disease within 8 min. Another magnetic microfluidic device was
diagnosis and treatment. Many studies also use microfluidic developed to detect prostate-specific antigen (PSA) employ-
technology to analyze small molecules such as amino acids, ing reduced graphene oxide-functionalized BiFeO3 (rGO-
and the specific examples are as follows. BiFeO3) as the photoactive material and target-triggered
Wang and colleagues developed a microfluidic device for hybridization chain reaction (HCR) for signal amplification
measuring the kinetics of amino acid secretion in Langerhans [70]. Based on EDTA-treated cotton-thread, researchers pro-
cells. All chip designs tested had three grounded sample posed a microfluidic device for one-step whole blood plasma
pools (islet, CN–, and NDA), a separate waste storage device, separation and analysis [71].
370 X. Fang
26.4.4.2 Protein-Ligand Interaction Analysis the needs of the biological, clinical, and medical fields [84–
Protein-protein interaction systems play an important role in 86]. Immunoassay greatly benefits from the microfluidic sys-
understanding the mechanism of biological processes on a tem, because microfluidic immunoassays possess remarkable
molecular level [72, 73]. Microfluidic devices have been advantages over traditional methods such as much higher
demonstrated to significantly miniaturize and automate the surface area to volume ratios, multiple detections, and auto-
study of protein-ligand interaction. For example, researchers matic assay.
described droplet-based microfluidic system integrated with Various techniques have been exploited to achieve micro-
fluorescence anisotropy [74] and FRET [75] for the study of fluidic immunoassay like centrifugal microfluidic [86],
protein-ligand interactions, which enabled to automatically paper-based microfluidic [85, 87, 88], capillary microfluidic
handle and assay up to 48 protein samples and 48 ligands in [89], microbead-based microfluidic [90], and digital micro-
one experimental run. A microfluidic droplet platform with fluidic [91]. These microfluidic strategies enable efficient
an 18 × 18 droplet array facilitated high-throughput screen- implementation of various types of complex assays, toward
ing of small-molecule protein ligands and low sample con- realizing miniaturized high-performance immunoassays. For
sumption (only needed 5 nL), which shows great potential in example, a microfluidic immunoassay chip was designed for
screening rare proteins and disease-related proteins [73]. diagnostics of infectious diseases. This chip integrated mul-
tiple procedures into an easy-to-use POC assay that signifi-
26.4.4.3 Mass Spectrometry-Based Proteomics cantly simplified the steps of ELISA, serving as a miniaturized
Microfluidic devices have greatly improved the utilization of ELISA enabling multiple targets detection for various types
mass spectrometry (mchip-MS) in the field of proteomics. of sample (whole blood, plasma, and sera). In particular, the
Conventional proteome researches require complex proce- chip is useful for screening infectious diseases like HIV and
dures and involve many hours of labor-intensive work. syphilis in the developing world because of the timely diag-
Microfluidics can address these problems and accelerate the nosis [92]. Researchers also developed a digital microfluidic
whole procedure, because it is capable of shortening analysis system containing inexpensive, inkjet-printed digital micro-
times, reducing reagent consumption, and increasing high- fluidic cartridges for serological immunoassays [91].
throughput ability by automated sample introduction. Up to Researchers reported a fluorescent microbead-based micro-
date, microfluidic devices based on electrospray ionization fluidic immunoassay chip allowing on-chip immune cell cul-
(ESI) and matrix-assisted laser desorption/ionization ture, biochemical stimulation, isolation, biosample
(MALDI) have been developed and applied to proteomic processing, and importantly cell cytokine secretion quantifi-
analysis [65, 66]. cation [90]. Lately, SERS has received great attention for the
Typical microfluidic platforms such as analog microfluid- detection of biomarkers owing to its inherent properties. A
ics, droplet microfluidics, or digital microfluidics (DMF) SERS-based microfluidic immunoassay platform was
have been typically employed in mchip-MS systems, which explored to detect biomarkers with high sensitivity [93].
are frequently used for protein digestion [76] and separation
[41, 77] (e.g., liquid chromatography (LC), capillary electro-
phoresis (CE), and affinity extraction). Droplet-based sys- 26.4.6 Cell Culture and Analysis
tems provide the benefit for the processing and manipulation
of discrete samples with precise control. By now, proteomic For over two decades, microfluidic technologies have been
mchip-MS systems have been reported for the early diagno- regarded as an ideal platform for in vitro cell analysis, includ-
sis of diseases [78–81]. For example, researchers present ing cell manipulation, isolation and analysis, 2D/3D cell cul-
microarray MALDI plate to detect protein phosphorylations ture, cell-cell communication analysis, and analysis and
[78]. A prototype microfluidic system is reported for the single-cell analysis, as it offers an excellent vivo-like envi-
quantitative detection of amino acids in dried blood spots by ronment [94].
tandem MS [81].
26.4.6.1 Cell Manipulation
Microfluidics is widely used in cell-related handling proce-
26.4.5 Immunoassay dures for various biochemical analyses [95]. Particularly,
microfluidic methods have been effectively used for precise
Immunoassay provides high reliability, excellent sensitivity, cell sorting [96]. Numerous microfluidic technologies have
and specificity, which is broadly employed in biological been successfully applied to isolate circulating tumor cells
research and clinical field [82, 83]. In recent years, microflu- (CTCs) from clinical samples through their unique biologi-
idic immunoassay chip has been extensively explored due to cal and/or physical properties [97].
26.4.6.2 C ell Culture and Cell-to-Cell 26.4.7 Drug Assay and Screening

Interaction Analysis
The culture of cells using various microfluidic devices has It is of great importance to develop novel drugs and new
considerably changed the experimental way of cell biology. A strategies for efficient delivery, thereby improving treatment
great number of two-dimensional (2D) and three-dimensional outcomes while minimizing toxicity [111]. Miniaturized
(3D) microfluidic-based devices are emerging for cell culture, microfluidic reactors contribute a lot to drug discovery and
especially with the aim of investigating the development and screening automation, addressing several drawbacks resulted
progression of cancers and their treatment [94, 98, 99]. by conventional bulk methods.
Compared to conventional culture, microfluidic devices
can be designed with great flexibility to meet the needs of 26.4.7.1 Drug Delivery System
different cell types and achieve cellular co-cultures. The Microfluidic systems work as effective drug delivery sys-
microfluidic device integrates other analytical biosensors, tems (DDS) to precise handling and transport of small liquid
which can detect the physiological response of cells in time volume [112]. These microfluidic-based drug delivery sys-
[100]. Microfluidic system also makes it possible to explore tems can be classified into self-assembled drug delivery sys-
complicated cellular behavior in vitro, as it can mimic the tem [113], droplet-based carriers [114, 115], non-spherical
real living microenvironment with definition and reproduc- drug delivery systems, and nucleic acid delivery systems
ibility [101]. Researchers reported a droplet-based 3D cell [111, 116, 117]. Self-assembled drug delivery allows for
culture model using a microfluidic sidewall-attached droplet high drug content, flexibly releasing, while it fails to incor-
array, enabling a series of 3D cell assay operations, including porate amphiphilic drugs, resulting in the requirement of fur-
droplet generation, drug treatment, cell staining, and in situ ther chemical modification. Droplet-based microfluidics is
observation [99]. regarded as the most powerful carrier production strategy,
Microfluidic systems are crucial for understanding cell- which possesses lots of properties, including the generation
to-cell interactions, providing vivo-like physiological mod- of monodispersed and size-controlled particles, the stable
els to mimic cell-to-cell interactions [102]. On-chip models encapsulation ability, and high drug loading dose.
were established to investigate the interactions between can- Applications in gene carrier fabrication and DNA synthesis
cer and immune cells [103, 104]. A 3D microfluidic-based have been developed based on microfluidic techniques to
assay was performed to mimic the tumor-vascular interface, greatly reduce the cost. Microfluidics has promoted the qual-
allowing for in vitro study of endothelial barrier function ity of nucleic acids delivery systems; however, scaling-up
[105]. Another 3D microfluidic device was established to clinical application is still a challenge and remains further
mimic the development of breast cancer [106]. verification.
26.4.6.3 Single-Cell Analysis 26.4.7.2 Drug Screening

Today, single-cell analysis is extremely popular in the scien- In order to build the next generation drug screening tool
tific community. Researchers from different fields contribute moving toward advanced biomimetic, miniaturized micro-
to single-cell analysis. The smallest organizational system fluidics devices are employed as in vitro models of the human
represents life and maintains a highly complex and hierarchi- body to drug screening in tiny volumes, thereby reducing the
cal structure of interconnected molecular networks. As an use of animal models [118, 119]. Importantly, “organ-on-a-
extension of cell analysis, single-cell analysis allows people chip” microdevices have been proved to build functional bio-
to understand such a complex system and learn more about mimetic microsystems such as gut, heart, lung, kidney, and
the function of organs and explore new ways to treat diseases blood vessel for precise drug screening [120]. Here, we
and develop personalized medicine. Microfluidic techniques introduce the recent advances in organs-on-chips with great
have evolved as a reliable tool to meet strict experimental significance for drug screening and discovery.
conditions and requirements of single-cell analysis for Researchers develop a dynamic microfluidic BBB (blood-
manipulating fluid ranging from nanoliters to femtoliters, fit- brain barrier) BBB-on-a-chip model to mimic in vivo BBB
ting to the size of individual cells, and allowing the integra- integrity and compound permeability, which helps to
tion of multiple liquid handling operations [107]. For investigate central nervous system diseases and test brain
example, a high-throughput generation of durable microdro- drug permeability studies [121]. Lung-on-a-chip devices
plets platform was developed for single-cell encapsulation were designed to provide a biomimetic microsystem, exam-
and monitoring [108]. Researchers presented a dynamic ine the toxicity of some nanoparticles, analyze inflammatory
microfluidic cytometry for single-cell cellomics analysis, responses of the lung, and study the pulmonary diseases
enabling cell trapping automatically, stimulating, and releas- [111, 119]. A microfluidics platform is developed for drug
ing [109]. It was reported that single-cell RT-PCR could be screening directly on cancer patients’ biopsies [122].
realized via droplets-based microfluidic device [110]. Microfluidic organs-on-chips provide a promising tool for
372 X. Fang
disease modeling, drug discovery and screening, and toxicol- detecting nucleic acids at the single-molecule level.
ogy research. More effort is still needed. For example, future Researchers developed an agarose droplet microfluidic chip
advances in microfluidic systems are required to handle for efficiently performing single-molecule emulsion PCR
more complex samples. Also, the cost and productivity of [134]. In subsequent work, researchers further demonstrated
microfluidic platform should also be taken into consideration that the agarose droplet microfluidic method could be
in the next step. employed for single RNA molecule detection via reverse
transcription-PCR [135]. Researchers also proposed a micro-
fluidic platform which is capable of generating monodis-
26.4.8 Enzymatic Analysis perse agarose-in-oil droplets for single-copy DNA
amplification [136]. A report described a novel method for
Enzymatic reactions play an essential role in our world. separating and measuring individual enzyme molecules via
Enzymatic assays are vital in various applications, such as picoliter polymer microwell arrays [137].
monitoring of glucose in serum described elsewhere [123].
In this extensive research field, enzymatic biosensing is
among the most popular and most studied areas, particularly References
when combined with microfluidics [124]. The combination
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Liquid Biopsy
27
Jianyu Rao, Weibo Yu, Teresa Kim, and Thomas Lee
27.1 Overview Defined liquid biopsy as a test to find cancer cells from a
tumor or pieces of DNA from tumor cells those circulating in
Over the years, precision medicine has revolutionized the the blood. The extraction and analysis of circulating tumor
management strategies for cancer, with the aim of achieving cells (CTCs), circulating tumor DNA (ctDNA), and other
early diagnosis and individualized treatment. This requires tumor-derived material generate tremendous information
effective integration of a large amount of molecular informa- available for cancer detection. The major advantage of the
tion derived from tumors and patients. Treatment choice liquid biopsy approach is its noninvasive nature compared to
should take tumor-specific molecular profiles into consider- tissue biopsy. Liquid biopsy allows for convenient, real-time
ation. Patient screening and selection have become increas- cancer surveillance and can easily be repeated as often as
ingly important for successful targeted treatment. necessary to monitor disease progression. Liquid biopsy has
Conventionally, the diagnostic routine is based on sampling the unique potential to detect early subclinical disease and
primary tumors. However, in the era of precision medicine, minimal residual disease after resection. Nowadays, relying
this approach has increasingly demonstrated its limitations. on high-throughput, high-sensitivity next-generation
First, tumor sampling involves invasive procedures, such sequencing (NGS) technology, liquid biopsy provides a sim-
as needle aspiration, endoscopic approach, excision, and ple, robust, and sensitive method to obtain diagnostic infor-
other surgical techniques, to extract specimens from the mation from blood samples. However, it must be realized
body. Anesthesia and hospital stay may also be required. that liquid biopsy is not a replacement for conventional tis-
Furthermore, a series of complications, including hemor- sue biopsy. Compared to the number of hematological mol-
rhage, infection, and prolonged recovery can occur. Second, ecules, CTCs or ctDNA are very rare, found in a blood
multilevel heterogeneity in a tumor presents a major chal- sample. To regard the detection ability of these tests, there
lenge to capturing whole genomic and epigenetic alterations are numerous challenges in both technical and clinical
through a single-point sampling at one time. Moreover, settings.
under endogenous and exogenous pressures, the genomic In this section, we discuss the current understanding of
landscape of each individual tumor continues to evolve and ctDNA, CTCs, and exosomes as a tool in cancer detection
change over time. Therapeutic stress, including chemother- and compare their analytical technologies and clinical utili-
apy and targeted therapy, can dynamically modify tumor ties (Fig. 27.1).
genomic profiles and suppress or improve the growth of spe-
cific cell clones. Thus, longitudinal surveillance of this
dynamic landscape is critical for the precise management of 27.2 Basic Principle
disease. However, by conventional tissue sampling
approaches, this cannot be effectively achieved. In addition 27.2.1 Circulating Tumor DNA
to these main limitations, sampling cost, technical demand,
and concerns about tumor seeding are also considered as Small fragmented cell-free DNA (cfDNA) was first reported
drawbacks in healthcare practice. in the plasma in 1948 with unidentified clinical significance.
In 1977, Leon and colleagues developed a radioimmunoas-
say for measuring the quantity of free DNA. Based on this
J. Rao (*) · W. Yu · T. Kim · T. Lee method, the serum-free DNA level was determined in 173
Department of Pathology and Laboratory Medicine, University patients with various types of cancer and in 55 healthy indi-
of California at Los Angeles, Los Angeles, CA, USA
viduals. Compared to patients without metastatic disease,
e-mail: jrao@mednet.ucla.edu

378 J. Rao et al.
Fig. 27.1 Current strategies of blood-based liquid biopsy
this study demonstrated significantly higher DNA levels in patients with hepatocellular carcinoma and controls [4]. In
the serum of patients with metastatic disease. After radiation the size distribution plot of each subject, the most promi-
therapy, the serum DNA level decreased in a majority of can- nent peak was observed at 166 bp. This conclusion suggests
cer patients [1]. This was the first study showing the potential that most of the circulating DNA molecules are derived
significance of cell-free DNA in cancer detection. The con- from apoptosis. Similar results were observed in mice mod-
cept of circulating tumor DNA (ctDNA) was further devel- els with human colorectal cancer xenografts. This study
oped when Sorenson and colleagues reported the detection identified that mononucleosome-derived fragments in
of specific sequences of the cystic fibrosis and K-ras genes in xenografted animals’ plasma are predominant, and it also
plasma DNA. This study identified that mutated K-ras demonstrated apoptosis as the main source of ctDNA [5].
sequences in the plasma of pancreatic carcinoma patients Necrosis is another important mechanism of plasma ctDNA
contained mutated K-ras genes [2]. It also provided the ini- release because large DNA fragments, more than 10,000 bp,
tial evidence that serum DNA fragments bear the unique are frequently observed in cancer patients [6]. In contrast
genetic features of the primary tumor. with these enormous fragments, cell-free mitochondrial
The molecular origin and release mechanisms of ctDNA tumor DNA has also been found in the plasma of cancer
are still not fully understood. Most studies indicated cell patients. The mitochondrial genome is composed of a cir-
apoptosis as the major source of plasma DNA fragments. cular piece of DNA, measuring 16.5 kb in length, and
The activation of endogenous endonucleases is a character- mutations in mitochondrial DNA are associated with vari-
istic of apoptosis. Subsequently, nuclear DNA splits up into ous malignant diseases [7]. It should be noted that all cells,
internucleosomal fragments of roughly 180 base pairs and from diverse parts of the body and tumor microenviron-
multiples thereof, which correspond to a DNA fragment ment, are capable of releasing DNA fragments into plasma.
wrapped around a nucleosome and the DNA stretch on his- Therefore, the cell-free DNA in a patient’s blood sample is
tone H1. Under agarose gel electrophoresis, apoptotic DNA a mixture of various DNA fragments from different tissues
fragmentation demonstrates a ladder pattern at 180 base through different mechanisms, which significantly increase
pair intervals [3]. A study investigated the full-size profile the difficulty in detecting tumor-associated genetic altera-
of tumor-derived and non-tumor-derived plasma DNA in tions [8].
27 Liquid Biopsy 379
27.2.1.1 Sample Collection and DNA Extraction 27.2.2 Circulating Tumor Cells

ctDNA has a very short half-life and a very low concentra-
tion in blood ranging from less than 10 ng/mL to more than CTCs represent a heterogeneous population of cancer cells
100 ng/mL. Standardized sample collection and DNA extrac- that are detached from a primary tumor or a metastatic site
tion are crucial to acquiring clinically relevant genetic muta-and may contribute to the establishment of distant meta-
tions. Column-based and magnetic bead-based approaches static lesions. CTCs provide an ideal model for the study of
are currently provided commercially for cell-free DNA cancer development and progression. Previous studies were
extraction. mostly focused on CTC enumeration for predicting progno-
sis and disease surveillance. Current CTC molecular charac-
27.2.1.2 ctDNA Detection terization and gene profiling have generated a powerful
approach to assess the cancer development at an early stage
PCR-Based Gene Analysis and decipher cancer biological features without invasive
The PCR-based method is the simplest and most cost- procedures.
effective approach to detect gene point mutations with high CTCs have very low concentrations in the blood. It is esti-
sensitivity. However, this method only focuses on a few mated that there could be one CTC per 106–107 leukocytes.
known gene alterations and is not able to capture related Therefore, for the purpose of enhancing their sensitivity and
genetic changes within a large range. specificity, CTC assays lean upon a combination of different
enrichment and detection steps. CTC detection platforms
Sequencing-Based Gene Mutation Analysis must be repeatable, reliable, rapid, cost-effective, and suited
Sequencing-based gene mutation testing has been developed to the large-scale production and use.
to provide various diagnostic panels according to different Current approaches for detecting CTCs can be simply
clinical demands. This method is more sensitive to detect tar- divided into two ways: (1) CTC detection technologies with-
get genetic alterations and covers multiple genetic points. out enrichment and (2) CTC detection technologies with
enrichment (Fig. 27.2).
Whole Genome Sequencing Analysis
The most advanced gene detection method is whole genome 27.2.2.1 CTC Detection Technologies Without
sequencing, which comprehensively covers the whole gene Enrichment
landscape. Because of the long turnaround time, sophisti- CTC detection without an enrichment step is also called
cated processes, and relatively high cost, this approach is direct detection of CTCs. The reported technologies include
mostly used for patients with inaccessible tumors. (1) line confocal microscopy and (2) SERS.
By line-
Confocal
Microscope
Without
Enrichment
By SERS EPCAM,CK,
Positive
MUC-1, HER2,
Enrichment
etc.
CTC Detection SP70 Targeted

Strategy Flow Cytometry Nonspecific Negative
CD45
Enrichment Enrichment
SP70 Targeted
Molecular Specific
Tumor Cell
Marker-based Enrichment
With Enrichment
Enrichment
Size, Density,
Physical Deformability,
Property-based Electric Charge
Microfiltration,
Microfluidics
Fig. 27.2 Current approaches for detecting CTCs

380 J. Rao et al.
27.2.2.2 CTC Detection Technologies commonly use centrifugation as an initial enrichment step
with Enrichment prior to additional processing using alternative strategies.
Microfluidic-assisted cell enrichment is a novel emerg-
Molecular Marker-Based Cell Enrichment ing approach to address the need to generate specific cell
Utilizing molecular marker to capture CTCs based on the populations from bodily fluids for molecular analysis.
unique antigen expression is one of the most widely used Microfluidics is the technology platform containing ele-
CTC isolation techniques. ments constructed within the micrometer scale, which oper-
ates on and processes fluid, essentially providing a
Negative Enrichment miniaturized analysis approach. The current development of
One is the so-called negative enrichment, which aims to cap- this technique is mainly focused on concentrating rare cells
ture nontarget cells, such as leukocytes and elute target cells from patient specimens with large volumes, which include
(i.e., CTCs). but are not limited to blood, urine, pleural, and peritoneal
The advantages of the CTC detection technologies with fluid, efficiently preparing small volume samples for multi-
negative enrichment include the following: (1) they do not ple assays, preparing samples with high cellularity, auto-
rely on the biomarker expression of CTCs; (2) they can col- mating multistep sample preparation, and obtaining high
lect the CTCs in an intact form, which is viable for following purity for molecular assays [9].
studies such as cellular and molecular detection. Limitations Although many methods, including size-based filtration,
are also observed: (1) because the CTCs are very rare in immunomagnetic separation, and microfluidic enrichment,
blood, even a high capture rate of blood cells cannot lead to have been technically mature, more carefully designed and
a high purity of CTCs; (2) the identification of the obtained large-sample clinical trials are still needed to establish stan-
CTCs with low purity needs further complicated analysis dard procedures and validate their clinical correlations.
procedures; (3) a large amount of antibodies are required to
capture the blood cells, which need a high cost.
27.2.3 Exosomes
Positive Enrichment
Another is called positive enrichment, which captures CTCs The concept of liquid biopsy extends beyond ctDNA and
and elutes healthy blood cells. During positive selection, CTCs. At a sub-cell level, exosomes, which contain various
tumor-associated cell surface antigens, such as EPCAM, are markers of original cells, such as proteins, messenger RNAs,
targeted. microRNAs, DNA, and bioactive lipids, have emerged as
One of the challenges to positive selection method is the another exciting target for cancer detection. Exosomes are
uncertainty of definition of CTCs. The heterogeneous array protective and inherently more stable than plasma genetic
of CTCs’ surface markers has made it impossible to identify markers.
a universal CTC-specific antigen. EPCAM is not a universal Exosomes are considered to be the smallest particle of
marker for CTCs, especially for cells of mesenchymal ori- extracellular vesicles (EVs) discharged by most cells. Their
gin, including soft-tissue tumors and lymphomas, or cells diameters range from 40 to 100 nm. Therefore, isolation and
undergoing epithelial-to-mesenchymal transition. Physically, identification of these nanometer-scale particles have been
CTCs are derived from primary or metastatic cancer lesions the biggest challenges in exosome study.
and are generally larger and heavier than blood cells.
27.2.3.1 Isolation
Physical Property and Non-affinity-Based Conventionally, they can be isolated by a series of ultracen-
Microfluidic Cell Enrichment trifugation processes. Larger cell components, vesicles, and
Most of current existing physical property-based cell protein contaminants are separated through the different spin
enrichment-based CTC capture methods take advantage of speed. However, this method is quite time-consuming and
the inconsistency of physical property (size, density, inefficient. Protein contamination is common and affects
deformability, and electric charge) between tumor cells and downstream analysis.
normal blood components. Some commercially developed exosome isolation kits use
Unlike molecular marker-based methods, CTCs captured reagents to induce exosome precipitation. This approach
using label-free methods are not “tagged” with an antibody. only involves standard centrifugation processes and signifi-
As centrifugation is reliable and inexpensive, it has been cantly decreases isolation time. But non-vesicular contami-
widely used for CTC isolation. However, even in the most nants can also be precipitated, and the precipitation reagents,
advanced centrifugation systems, the capacity of eliminating such as polymer material, may affect the subsequent analy-
leukocyte contamination is limited. As a result, researchers sis. There were many studies exploring specific biomarkers
of exosomes and developing antibody-binding-based meth- tions of the EGFR gene. DNA isolated from formalin-fixed
ods for exosome collection. paraffin-embedded tumor tissue (FFPET) or circulating-free
However, most exosome protein markers are cancer- tumor DNA from plasma is used to detect defined EGFR
specific. Therefore, biomarker-based approaches are more mutations. The test uses selective oligonucleotide probes for
suitable for the subpopulation isolation of exosomes. Like each targeted mutation, which is labeled with a fluorescent
CTCs, microfiltration has also been used in exosome col- reporter dye as well as a quencher molecule that absorbs
lection. Ciliated micropillars for multi-scale filtration can fluorescent emissions from intact probes [10].
also be combined with a microfluidic device, in which Epi proColon. Epi proColon was approved in 2016 by the
exosome-like lipid vesicles are trapped while smaller pro- US FDA for the detection of methylated Septin 9 DNA in
teins and larger nanoparticles with cellular debris are EDTA plasma [11]. In the promoter region of the SEPT9
unhindered. Finally, by dissolving the porous silicon transcript, methylation of the target DNA sequence has been
nanowires, the captured lipid vesicles are released to obtain found to be associated with the occurrence of colorectal can-
intact exosomes [8]. cer, with an overall sensitivity of 90% and specificity of 88%
In recent years, for exosome isolation and analysis, multi-for detecting disease at all stages [12]. Epi proColon used a
farious microfluidic systems have been reported. These plat- real-time PCR with a fluorescent hydrolysis probe to detect
forms integrate a range of exosome isolation approaches, the specific methylation of the Septin 9 DNA target.
such as immunoaffinity, membrane-based filtration, trapping CancerSEEK. CancerSEEK developed two approaches
on nanowires, acoustic nanofiltration, and deterministic lat- that enabled the detection of the rare mutations expected to
eral displacement sorting, and have demonstrated promising be present in plasma ctDNA. First, it used multiplex-PCR to
results [9]. These technological advancements are a signifi- directly and uniquely label each original template molecule
cant step to making exosome-based routine analysis with a DNA barcode. This design minimizes the errors inher-
feasible. ent to massively parallel sequencing and makes efficient use
of the small amount of cell-free DNA present in plasma.
27.2.3.2 Analysis of Exosomes Additionally, it divided the total amount of DNA recovered
There are typically two different types of analysis performed from plasma into multiple aliquots and performed indepen-
on the isolated exosome, including physical and chemical/ dent assays on each replicate. In effect, this decreases the
biochemical/compositional analysis. Physical analysis, number of DNA molecules per well; however, it increases
which gives insight to particle size and concentration, is the fraction of each mutant molecule per well, making the
done using nanoparticle tracking analysis (NTA), dynamic mutants easier to detect. Because the sensitivity of detection
light scattering (DLS), electron microscopy, and tunable is often limited by the fraction of mutant alleles in each rep-
resistive pulse sensing (tRPS). The chemical/biochemical/ licate, this partitioning strategy made it to increase the
compositional analysis is typically done via staining, immu- signal-to-noise ratio and identify mutations present at lower
noblotting, or proteomic analysis and gives information prevalence than possible if all of the plasma DNA was evalu-
regarding the content of the isolated exosomes. ated at once [13].
However, PCR-based technology has limited capabilities,
such as only interrogating a discrete set of previously defined
27.3 Technology Development mutations in limited number of oncogenes. Therefore, rare
but nonetheless clinically relevant mutations can be missed.
27.3.1 Circulating Tumor DNA Compared with the PCR-based methods, a NGS multiplex
panel is preferred and recommended as it can detect not only
There are two groups of ctDNA analysis techniques: PCR- common mutations but also a spectrum of alterations.
based techniques and sequencing techniques.
27.3.1.2 Sequencing Techniques
27.3.1.1 PCR-Based Techniques
For now, numerous PCR-related methods are utilized to NGS in Liquid Biopsy
interrogate circulating DNA, such as end-point measurement NGS has been developed over the past two decades follow-
PCR, amplification-refractory mutation system (ARMS- ing the previous fragment-cloning-based Sanger sequencing.
PCR), digital PCR, qPCR, BEAMing, etc. Furthermore, sev- The basic principle of NGS involves the massive parallel
eral commercial companies begin to afford their own sequencing of millions of different DNA strands. NGS yields
solutions. substantially high throughput and captures a broader spec-
Cobas EGFR Mutation Test v2. It is a real-time PCR sys- trum of gene variations, including insertions and deletions in
tem. In patients with non-small cell lung cancer (NSCLC), it small fragment, deletions of exon or whole genes, and rear-
can be used for the qualitative detection of the defined muta- rangements (inversions and translocations) in large genomic.
382 J. Rao et al.
NGS technology has been rapidly implemented in the study ations associated with disease progression and drug resis-
of liquid biopsy and has tremendously empowered research- tance [14]. Tumor clones keep evolving with a branched
ers to look for insights into blood ctDNA (Fig. 27.3). evolution and confer growth advantages within a specific
Whole exome sequencing (WES). WES in liquid biopsy individual microenvironment. These dynamic changes
was investigated to show proof of principle to complement within one tumor or among a primary tumor and metastatic
current invasive biopsy techniques and to identify gene alter- lesions are described as genomic heterogeneity. Whole
Targeted gene panel
Bulk RNAseq
Whole exome sequencing
Microdissected RNAseq
Whole genome sequencing
10X Genomics RNAseq
Fig. 27.3 Next-generation sequencing

exome sequencing provides a high-yield approach to study positions of microcolumn. The capture efficiency of this
the temporal and spatial heterogeneity of tumors. Current approach achieved 92%, and the purity reached 82% with
whole exome sequencing is mostly being investigated to blood samples [16].
evaluate mutation profiles of tumors without accessible biop-
sies and to determine tumor mutation load, which is a critical 27.3.2.3 ISET
marker for patient screening and response prediction in can- A simple density-based gradient centrifugation may help the
cer immune therapy. Whole genome sequencing (WGS) pro- separation process. In 2000, Giovanna and colleagues
vides more comprehensive genome coverage than whole reported a microfiltration method to collect CTCs with
exome sequencing, including all protein coding regions as downstream immunomorphological and molecular charac-
well as intronic and other noncoding regions. Presently, there terization [17].
is no longer a cost barrier to using WGS in liquid biopsy
practice. However, the lack of clinical evidence and under- 27.3.2.4 MEMS
standing of the data still exists. How patients will benefit A recently developed microelectromechanical system
from WGS is uncertain. Building an up-to-date, comprehen- (MEMS) further improved the effectiveness of the mem-
sive and clinically relevant genomic database is important to brane microfilter device. It was reported that this approach
improve WGS data interpretation and ultimately pave the can achieve CTC capture on filter within 10 min, and the
way for WGS to be used in routine, noninvasive liquid recovery is approximately 90% [18].
biopsy.
10X GemCode technology. A novel single cell sequenc- 27.3.2.5 FMSA
ing platform has been developed. The laser-captured micro- Another flexible micro spring array (FMSA) device was
dissection technique is an effective approach to acquire reported to enable viable enrichment of CTCs directly from
single cells from tissue samples. But the whole procedure is whole blood without sample processing. Notably, this tech-
time-consuming, low throughput, and not suitable for liquid nique minimized the operation pressure and reduced con-
biopsy study. Using the 10X GemCode technology, a pool of centrated stress on cells during enrichment to better
750,000 barcodes were sampled, and each cell’s transcrip- maintain cell viability. A study showed that at least 1 CTC
tome was indexed. To achieve that, thousands of cells were in 16 out of 21 clinical samples can be detected by FMSA
partitioned into nanoliter-scale Gel Bead-In-Emulsions, and (76%) [19].
then a common 10X barcode was shared by all generated
cDNA. cDNA libraries are generated and sequenced. 27.3.2.6 Vortex
Meanwhile, the 10X barcodes are applied to associate indi- One of the representative microfluidic technologies is the
vidual reads with individual partitions. enrichment of CTCs with the Vortex platform. By utilizing
the unique physical phenomena of fluids flowing through
microchannels and microstructure, the Vortex device inte-
27.3.2 Circulating Tumor Cells grates a microfluidic chip functioning as a high-throughput
benchtop centrifuge and concentrates rare cells of interest,
27.3.2.1 CellSearch System including malignant cells and mesothelial cells, from a large
Epithelial cell adhesion molecule (EPCAM) is detected on background of cells in diluted blood. This approach also
80% of solid cancers and is regarded as a common CTC bio- showed a fast processing ability; cell enrichment can be
marker. Based on EPCAM capturing, the CellSearch system completed within 20 min for 7.5 mL of blood. Most impor-
was developed and obtained US FDA approval for breast tantly, this is a label-free method, facilitating any desired
cancer, prostate cancer, and colorectal cancer detection. In downstream morphology and molecular and gene mutation
this platform, magnetic particles coated with anti-EPCAM analysis [20]. Because of the close relevance of CTCs and
are used to positively collect potential CTCs. These cells can primary or metastatic tumors and the noninvasive assessment
be further verified by immunostaining with an epithelial of CTCs, the development of next-generation CTC isolation
marker cytokeratin(+) and a leukocyte marker CD45(-) to and purification platforms has attracted much attention in the
confirm their epithelial origin. field.
27.3.2.2 MagSweeper 27.3.2.7 SP70-Targeted Tumor Cell Enrichment

MagSweeper used the same immunomarker to select for epi- Recently, immunomagnetic separation, which is stable
thelial cells circulating in the blood [15]. To improve effi- and has a large surface to volume ratio, is routinely used
cacy, the EPCAM antibody was further integrated into a for cancer cell enrichment with improved sensitivity [21,
hydrodynamic gradient chip system. According to the 22]. However, most, if not all, immunomagnetic separa-
expression level of EPCAM, CTC was captured at different tion uses pan-epithelial targets that are not necessary can-
384 J. Rao et al.
cer-specific antigens, so the cells identified or enriched 2. Application examples:

may only represent epithelial cells rather than cancer (a) Tumor cells were enriched with immunomagnetic
cells. A monoclonal antibody designated NJ001 was bead capturing kit according to the manufacturer’s
developed from homemade cancer monoclonal antibody instruction (Code Biotech, Jiangsu, China).
library [23], which targets tumor-specific antigen SP70. (b) Further after Pap staining, microscopic examination
Biochemically functionalized paramagnetic beads, which was performed.
are coated with NJ001 can recognize malignant cells (c) DNAs of CTC captured by immunomagnetic beads
through the surface antigens. SP70-positive cells are then were then used for NGS.
isolated from other cells using a magnetic field. Then, its 3. Results:
morphological and genetic changes were analyzed SP70-positive cells are attached with brown immunomag-
(Fig. 27.4). netic beads (Fig. 27.4).
A two-center study in the First Affiliated Hospital of Individual samples of 111 malignant cases examined showed
Nanjing Medical University (NMU) and Department of at least 1 detectable alteration (Fig. 27.5).
Pathology and Laboratory Medicine, University of
California, Los Angeles (UCLA), indicated that SP70- 27.3.2.8 SP70-Targeted Flow Cytometry
targeted liquid biopsy could significantly increase the One of the challenges to positive enrichment method is the
accuracy of routine cytology diagnosis of cancer in body ever-changing definition of CTCs. The surface markers of
fluid samples (82.1% vs. 55.7%). Further, the captured CTCs were heterogeneous, and thus the identification of a
cells can be utilized to perform NGS-based molecular universal CTC-specific antigen is impossible, just as EPCAM
analysis. CTC-oriented DNA (captured with SP70-targeted is not a universal marker for CTCs. Capturing blood cells
developed method) can be used for decreasing the depth on using the negative enrichment technologies needs a large
NGS. That is crucial for the precision care of cancer amount of antibodies and is costly. Furthermore, most of the
patient. process is difficult to automate, including CTCs capture,
enrichment, and detection.
1. Experimental process: Tumor-specific antigen SP70 is highly specific expressed
(a) Sample preparation on the cytomembrane of tumor cells. Monoclonal antibody-
(b) Immunomagnetic bead capturing designated NJ001 was developed from homemade cancer
(c) SP70-targeted tumor cell collection monoclonal antibody library, which targets tumor-specific
(d) Cytology and genetic change analysis antigen SP70 [23]. Utilizing fluorescent-conjugated NJ001
a b c
SP70 NJ001
Genetic Changes
Analysis
d
SP70 Targeted
Blood and Tumor cell
Body Fluids Enrichment
Magentic
beads
conjugated
with NJ001 Morphological
Analysis
Fig. 27.4 SP70-targeted tumor cell enrichment. (a) Flowchart of SP70 expression captured by NJ001-coated magnetic beads. (d) SP70-
SP70-targeted tumor cell enrichment. (b) Pictures of magnetic beads positive cells are attached with brown immunomagnetic beads (400×)
coated with NJ001 in transmission electron microscopes. (c) CTCs with
Fig. 27.5 The percent of gene alterations in 111 patients. This bar graph represents the percent of all molecular alterations in all patients. TP53
alterations were observed in the most number of patients, followed by KDR, EGFR, and MET
based on flow cytometer, we developed a rapid and (c) Wash and suspend the cells: after lysing, cells were
quantitative direct detection method to detect CTCs in the centrifuged at 155 × g for 8 min and washed with
peripheral blood (Figs. 27.6 and 27.7). PBS twice. Cells were suspended with PBS.
(d) Gate strategy on FACSCalibur (BD Biosciences,

1. Experimental process: USA): region (R1) in dot plot 1 (FSC-H vs. SSC-H)
(a) Sample and stain. was set around all nucleated cell (WBC and CTCs),
(b) Erythrocyte lysis. and a minimum threshold value of 100 was set on
(c) Wash and suspend the cells. FL1 to exclude the debris. Region (R2) in dot plot 2
(d) CTCs count by flow cytometer. (FL1 vs. SSC-H) was set around adjusted positive
(e) Data analysis. controls alone (Fig. 27.8).
2. Application examples: (e) CTC count: acquisition was terminated in 5–10 min
(a) Sample and stain: 100 μL of EDTA-anticoagulated when 200,000 cells were counted within region R1,
whole blood was stained with optical concentration the number of fluorescent events in region (R2) was
of fluorescent antibody NJ001-Mix-n-Stain™ defined as number of SP70(+) CTCs.
CF™488A (Code Biotech, Jiangsu, China) at room (f) Data analysis: the number of leukocytes was ana-
temperature for 20 min in the dark. lyzed by hematology analyzer. Further, the number of
(b) Erythrocytes were subsequently lysed in 1000 μL SP70(+) CTCs was converted to the concentration of
1 × FACS™ lysing solution. CTCs in peripheral blood by the formula (Eq. 27.1).
Concentration of CTCs ( / mL ) = Number of CTCs ´ correction coefficient

The number of leukocytespermilliliter of the peripheral blood (27.1)
Correction coefficient =
2 ´ 10 5

3. Results under the curve (AUC) for SCLCC was 0.825 (95%CI
A study in the First Affiliated Hospital of Nanjing Medical 0.756–0.894), with a corresponding sensitivity of 62.50%
University (NMU) showed that in non-NSCLC controls and a specificity of 89.55%, which indicated the most
(including benign lung diseases and healthy controls), the optimal diagnostic performance. Concentration of CTCs
median concentration of CTCs in peripheral blood was of patients with NSCLC was obviously higher than those
214 (151–273)/mL, while in NSCLC patients was 335 of non-NSCLC. Forty-six percent (33/72) NSCLC patients
(274–440)/mL. When the base value was 309/mL, the area were classified as stage IIIB-IV.
386 J. Rao et al.
Fig. 27.6 Basic principle of

CTC detection
Fig. 27.7 The flowchart of

CTC detection
Among all NSCLC patients, 81% (58/72) were adeno- levels in patients with chemotherapy and surgery. Taken
carcinoma pathologically. Concentration of CTCs in 48 together, dynamic changes of peripheral blood concentra-
patients with lymph node metastasis was higher than those tion of CTCs in NSCLC patients could monitor the treat-
who did not have lymph node metastasis (359/mL vs. 312/ ment efficacy and provide more valuable clinical guidance
mL, P = 0.042). Concentration of CTCs markedly decreased for doctors timely. These findings highlighted that concen-
(P < 0.001) after the first chemotherapy cycle in 16 patients. tration of CTCs is a sensitive indicator of early diagnosis of
Concentration of CTCs strikingly decreased (P = 0.004) NSCLC and can be used to evaluate the therapeutic efficacy
after surgery within 1 week in 21 patients. Unfortunately, of NSCLC patients that underwent chemotherapy or
there were no significant induction in CEA and CYFRA21-1 surgery.
388 J. Rao et al.
a 1000 Plot 1 Plot 2
1000
800
800
600
600
SSC-HEIGHT
SSC-HEIGHT
R1 R2
400
400
200
200
0.00%
0
0
0 200 400 600 800 1000 100 101 102 103 104
FL1-HEIGHT FL1-HEIGHT
b
Plot 1 Plot 2
1000
1000
800
800
600
600
SSC-HEIGHT
SSC-HEIGHT
R2
400
400
200
200
99.9%
0 0
0 200 400 600 800 1000 100 101 102 103 104
FL1-HEIGHT FL1-HEIGHT
Fig. 27.8 (a) Negative control. (b) Positive control
27.3.3 Exosomes lular debris and larger particles were pelleted out from the
matrix. Then, the larger exosomes and apoptotic bodies were
27.3.3.1 Isolation of Exosomes removed after 0.22 μm filtration and 10,000 × g steps.
Finally, 100,000 × g centrifugation steps were used to pellet
Ultracentrifugation Techniques out and wash the exosomes.
Ultracentrifugation techniques can be divided into two major Density gradient centrifugation. Separation is still based
types: differential ultracentrifugation and density gradient on size and density with the presence of a preconstructed
ultracentrifugation. density gradient that is typically made of sucrose or iodixa-
Differential ultracentrifugation. Depending on density, nol, in the centrifuge tube. The sample is placed at the top of
size, and shape, the larger and more dense particles were the gradient, where the density is lowest. When centrifugal
sedimented out first [24]. With the help of 500 × g step, cel- force is applied, the particles in the sample will pass through
the gradient at unique rates to complete the separation. By tetraspans or the transmembrane 4 superfamily (TM4SF),
fractionation collection, the exosomes can then be collected are regarded as common exosome markers. Specifically, at
typically in the density range of 1.1 and 1.2 g/mL. least one transmembrane or lipid-bound extracellular pro-
tein, CD9, CD63, or CD8, should be noted in experimental
Size-Based Techniques exosome models. The other exosome protein markers include
Ultrafiltration. Ultrafiltration is one of the most common glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
size-based techniques for the isolation of exosomes. Basing membrane transporters (GTPases), and cell surface proteo-
on the size and molecular weight cutoff (MWCO) of the glycan (GPC1). The main limitation in the development of
membrane, ultrafiltration has the same principle with con- this method is protein/antigen used to capture the exosomes.
ventional membrane filtration [25]. That is, particles larger Since the antibody will not be able to capture an antigen
than the MWCO of the particular filter are retained by the enclosed within the vesicle, the protein/antigen must be
filter, but the smaller will pass through the filter into the expressed on the surface of exosomes [25].
filtrate.
Sequential filtration. In this approach, the isolation of dif- Precipitation
ferent particles is mainly dependent on their molecular Polymers can be used for coprecipitate hydrophobic proteins
weight and size. By using membrane filters with different and lipid molecules. The PEG-based method could be modi-
size or molecular weight, exosomes can thus be separated. fied for exosome isolation. Lately, easy-to-use commercial
Firstly, cells, cell debris, and exosomes are removed by nor- kits (e.g., Exo-spin, Total Exosome Isolation, ExoQuick)
mal prefiltration, in which a 0.22 mm membrane filter usu- have been developed based on coprecipitation to concentrate
ally will be used. Then, free proteins are filtered out by using and purify small exosomes. Usually, the sample (culture
a dialysis bag with 500 kDa molecular weight. Finally, the supernatant or other biofluids) should be treated to remove
condensed samples are filtered, and a 100 nm membrane fil- cells and cellular debris and then incubated with PEG solu-
ter is used. tion at 4 °C for 14 h. After centrifugation at low speed, the
Size exclusion chromatography (SEC). SEC, by filtration bottom of the centrifugation tube has precipitated EV prod-
through a porous stationary phase, is a technique based on ucts. Because it is convenient and does not require prolonged
particle size. Generally, the porous stationary phase is com- ultracentrifugation, many researchers use this method to
posed of spherical gel beads with many particular size pores. separate exosomes. Although coprecipitation can directly
When passing through the stationary phase, small particles isolate exosomes in a few steps, the relatively low through-
can pass through the pores, while large particles cannot [26]. put and chemical contamination limit its application.
As a result, particles that exit the column earlier are larger
ones. Microfluidic-Based Isolation Techniques
Flow field-flow fractionation (FFFF). Sample is injected The development of microfluid-based exosome isolation
into a chamber first; then, it will be subjected to parabolic method is to solve the problems of more traditional methods
flow reaching the length of the chamber. At the same time, a and make the use of exosomes more feasible in the clinical
crossflow (a flow perpendicular to the parabolic flow) is used environment. The main advantage of microfluidic techniques
to separate the particles in the sample. Larger particles are is the ability to separate exosomes according to both their
more susceptible to the crossflow. They are pushed closer to physical and biochemical characteristics [25]. In addition,
the walls of the chamber with slower parabolic flow. Thus, microfluidic isolation methods are usually fast and effective
the larger particles elute later than the smaller particles, and require a small starting volume (10–100 s of μL). It also
which are less affected by the crossflow, remaining in the allows the development of innovative isolation mechanisms,
center of the parabolic flow [27]. such as acoustic, electrophoretic, and the electromagnetic
Hydrostatic filtration dialysis (HFD). For hydrostatic pres- characteristics of exosomal vesicles [29, 30].
sure, sample is forced through a dialysis tube with a 1000 kDa
MWCO. The solvent and small solutes pass through the tube 27.3.3.2 Analysis of Exosomes
easily. The larger particles, such as exosomes, will remain in
the tube and can be collected there [28]. Physical Analysis
Nanoparticle tracking analysis (NTA). Nanoparticle tracking
Immunoaffinity Capture-Based Techniques analysis, or NTA, can determine both particle size and con-
Immunoaffinity capture-based techniques rely on the use of centration. The size of the particles is estimated using the
an antibody to capture exosomes that express a specific anti- Stokes-Einstein equation, where the diffusion coefficient is
gen on the surface. Antibodies especially for a specific anti- based on the Brownian motion of particles in the cavity.
gen of interest can be attached to the plate, magnetic beads, When the laser light interacts with the particles (under
resins, and microfluidic devices. Tetraspanins, also called Brownian motion) in the chamber, the laser light is scattered,
390 J. Rao et al.
and the scattered light is collected by a microscope fitted approaches are frequently used. ctDNA-based liquid biopsy
with a camera. The camera on top of the microscope captures provides an alternative and noninvasive solution. A study
the motion of particles in a video, which is then used by the from Japan evaluated noninvasive genotyping of cell-free
NTA software to estimate the size and concentration of the plasma DNA as a marker for outcome prediction of EGFR
particles [31]. NTA is able to determine particle size between TKI osimertinib therapy. In this study, genotyping of cell-
10 and 1000 nm in diameter. This particle size is within the free plasma DNA was focused on EGFR T790M, which was
size of exosomes known to be between 50 and 150 nm. performed by using BEAMing. The sensitivity of plasma
However, the challenge with NTA is that it requires sample genotyping to detect T790M was 70%. It was concluded that
volumes of ~0.5 mL, optimizing data collection and analysis patients with positive results of T790M in plasma have out-
parameters. comes after osimertinib treatment that are equivalent to posi-
Dynamic light scattering (DLS). DLS uses the scattered tive patients by tissue-based assays. The author also indicated
light generated by Brownian motion of particles to estimate that considering a 30% false-negative rate of plasma geno-
particle size and concentration and uses the fluctuations in typing, patients with negative plasma results still need tumor
the intensity of the scattered light to estimate the size of par-
biopsies to determine EGFR T790M status [36].
ticle. DLS requires very little sample volume (70 μL), which In a multicenter, randomized study, 217 advanced NSCLC
is easy to use and requires few parameters to optimize [32]. patients who had previously tested positive for EGFR exon
Electron microscopy. Transmission electron microscopy 19 deletions or exon 21 L858R point mutations accepted fur-
(TEM) and scanning electron microscopy (SEM) are the two ther serum ctDNA-based testing with the cobas EGFR
common types of electron microscopy, which are used to Mutation Test v2. The results demonstrated that the liquid
evaluate the morphology of exosomal vesicles. biopsy method showed mutation positivity in 76.7% of
tissue-positive specimens and mutation negativity in 98.2%
Chemical, Biochemical, and Compositional Analysis of tissue-negative cases [10, 37]. This indicated that the
With the development of research, various cancer-related cobas is a highly specific companion diagnostic tool for the
proteins and protein receptors are found in exosomes. administration of erlotinib-targeted therapy. When this blood
Therefore, the analysis and quantification of exosome pro- test is positive, patients can avoid invasive tissue sampling
tein is crucial for the development of new diagnostic mark- for EGFR assay.
ers. The presence of known proteins in exosomes has been A prospective multicenter study compared the perfor-
analyzed by mass spectrometry, flow cytometry, enzyme- mance of the Septin 9 DNA methylation test with the fecal
linked immunosorbent assay (ELISA), Western blotting immunochemical test (FIT) for colorectal cancer screening.
(WB), and other analytical methods. The sensitivities of Septin 9 and FIT for colorectal cancer
Exosomal nucleic acids have received more attention than detection were 73.3 and 68.0% in all patients. The specifici-
exosomal proteins because they are directly or indirectly ties of Septin 9 and FIT were 81.5 and 97.4%. With a sensi-
involved in the development of disease and tumor progres- tivity of 72%, the Epi proColon test was shown to be
sion. Currently, the distributions and functions of miRNAs in statistically non-inferior to FIT. With negative predictive val-
different cell lines are widely studied. ues of 99.8%, both methods were the same in excluding
colorectal malignancy [38]. Although it has a lower specific-
ity compared to FIT, blood-based testing provides an impor-
27.4 Clinical Application tant alternative for patients who are reluctant to undergo
conventional screening. A study investigated whether
27.4.1 Circulating Tumor DNA patients were willing to use noninvasive stool or blood-based
screening tests after rejecting colonoscopy. Among 106 sub-
Several ctDNA-based tests have been implemented in clini- jects who refused colonoscopy and accepted an alternative
cal practice. Since an EGFR mutation was identified as a key noninvasive method, 90 subjects chose the Septin 9 blood
driver event in the development of lung adenocarcinoma, a test (83%) and 16 subjects chose a stool test (15%) [39]. This
few EGFR tyrosine kinase inhibitors (TKIs), including erlo- finding indicated that offering a noninvasive blood-based test
tinib and gefitinib, have been developed and verified as an might significantly increase compliance and effectiveness of
effective targeted treatment choice [33, 34]. In clinical prac- colorectal cancer screening [14].
tice, the relevant EGFR-activating mutation status needs to
be confirmed before the treatment of EGFR TKIs [35]. Thus,
obtaining DNA samples from tumor tissue was considered as 27.4.2 CTCs
a critical step in patient management. However, most patients
with advanced disease are not amenable to surgical treat- In a preliminary report, they found that a CTCs count >25
ment. Other image-guided needle biopsy or cytology could help distinguish lung cancer from benign lesions in
patients with abnormal lung imaging [40]. Another study targeted liquid biopsy could significantly increase the accu-
using the microfluidic NanoVelcro CTC chip showed that racy of routine cytology diagnosis of cancer in body fluid
CTCs have a good biomarker function in the diagnosis and samples. SP70-targeted cytology had a higher interrater
staging of pancreatic ductal adenocarcinoma (PDAC). They agreement (Kappa = 0.593) compared to malignant diagno-
found that a cutoff of ≥3 CTCs in 4 mL venous blood was sis, which was superior to cytology (Kappa = 0.272). In addi-
able to discriminate between local/regional and metastatic tion, SP70-targeted cytology had a larger AUC (0.836) for
disease (AUROC = 0.885; 95% CI = 0.800–0.969; and malignance diagnosis, which was superior to cytology
P < 0.001) [41]. Cristofanilli and colleagues confirmed that (AUC = 0.707) (p < 0.001). A combination of two methods
CTC enumeration should be used for the prognostic stratifi- can elevate diagnostic efficacy (AUC = 0.857). Further, the
cation of metastatic breast cancer (MBC) in two defined captured cells can be utilized to perform NGS-based molecu-
groups of patient identified as stage IVindolent and stage lar analysis. CTC-oriented DNA (captured with SP70-
IVaggressive [42]. Minzhi Hou et al. demonstrated that gesta- targeted developed method) can be used for decreasing the
tional choriocarcinoma (GC) patients with <6 CTCs at both depth on NGS. That is crucial for precision care of cancer
time points had longer PFS than those with ≥6 CTCs at patient.
either time point. CTC count assessment could be used to
evaluate whether patients are benefiting from a current che-
motherapy regimen. They suggested that CTC count in GC 27.4.3 Exosome
patients remains or becomes ≥6 after one cycle of chemo-
therapy; an alternative regimen may be essential [43]. A trial A study compared the different results of using next-
in castration-resistant prostate cancer (CRPC) showed that generation sequencing to profile miRNA in various blood
CTC number and lactate dehydrogenase (LDH) level were a components. They demonstrated that exosome RNA is pro-
surrogate for survival at the individual-patient level in a trial tected by RNaseA treatment and exosomes provide a consis-
of abiraterone acetate plus prednisone versus prednisone tent miRNA source as a disease detection biomarker [46].
alone for patients with metastatic CRPC [44]. A study Another study verified the presence of dsDNA in exosomes
showed that early assessment of response to chemotherapy representing the whole genomic DNA. The authors detected
using a combined approach of PET-CT and CTCs analysis at exosome DNA isolated from various cancer cell lines with
4–6 weeks after starting chemotherapy has prognostic and known driver mutations. The mutation status, including
probably predictive significance in the first-line treatment of BRAF and EGFR gene mutations in original cells, was
patients with metastatic colorectal cancer (mCRC). A novel exactly reflected in exosome DNA [47]. Furthermore, a few
dual-response criterion has potential application in the evalu- tumor-derived exosome markers have been investigated.
ation of new drugs and in influencing treatment decisions at Using mass spectrometry analyses, Sonia and colleagues
earlier time points during chemotherapy [45]. identified a cell surface proteoglycan, glypican-1 (GPC1),
A preliminary study among 72 NSCLC patients utilizing which is enriched on cancer cell-derived exosomes. A spe-
SP70-targeted flow cytometry in the First Affiliated Hospital cific KRAS mutation was carried in GPC1-positive exo-
of Nanjing Medical University (NMU) showed that concen- somes. Further study found that the GPC1 level on exosomes
tration of CTCs in 48 patients with lymph node metastasis correlated with tumor burden and survival in pancreatic can-
was higher than those without lymph node metastasis (359/ cer patients with surgical treatment [48]. Another study iso-
mL vs. 312/mL, P = 0.042). Concentration of CTCs mark- lated exosomes from the plasma of 40 patients with head and
edly decreased (P < 0.001) after the first chemotherapy cycle neck squamous cell carcinoma and found that PD-L1 expres-
in 16 patients. Concentration of CTCs strikingly decreased sion levels on exosomes were correlated with disease activ-
(P = 0.004) after surgery within 1 week in 21 patients. ity, stage, and lymph node status. In contrast, plasma levels
Unfortunately, there were no significant induction in CEA of PD-L1 did not correlate with any specific clinicopatho-
and CYFRA21-1 levels in patients with chemotherapy and logic features [49].
surgery. Taken together, dynamic changes of peripheral
blood concentration of CTCs in NSCLC patients could mon-
itor timely the treatment efficacy and provide more valuable 27.4.4 Comparison Between CTCs, ctDNA,
clinical guidance for doctors. These findings highlighted that and Exosome Tests in Cancer Detection
concentration of CTCs is a sensitive indicator of early diag-
nosis of NSCLC and can be used to evaluate the therapeutic Currently, CTCs and ctDNA are the most advanced and read-
efficacy on NSCLC patients that underwent chemotherapy or ily applicable markers. Because of relatively high levels and
surgery. A two-center study in NMU and Department of the abundant amounts of genomic and protein information
Pathology and Laboratory Medicine, University of they contain, exosomes have been another powerful tool in
California, Los Angeles (UCLA), indicated that SP70- liquid biopsy. The potential application of each approach is
392 J. Rao et al.
slightly different [50]. ctDNA carries multilevel genetic 27.4.5 Challenges and Outlook
information and has the potential to fully recapitulate spatial
and temporal tumor heterogeneity. However, since all of the The US Centers for Disease Control and Prevention’s
tumors were not sufficient in DNA amounts released into Public Health Genomics Office developed the ACCE
peripheral circulation for detection and even the most sensi- framework that includes collection, assessment, interpreta-
tive assay appears to achieve approximately 85% sensitivity tion, and reporting data about genetic-related testing for
in advanced disease stage, it is important to be aware of the diseases in a format that allows policy-makers to obtain up-
possibility that a false-negative result might come from the to-date and reliable information for decision-making. The
liquid biopsy. In contrast, CTCs provide the opportunity to key components to determine whether a diagnostic test is
study cell morphology, mechanical features, and function. It ready for the clinic include analytical performance and val-
was also reported that CTCs demonstrated cellular PD-L1 idation, which is focused on technical performance, such as
expression as a biomarker for stratification and monitoring sensitivity, specificity, limit of detection, and sample pro-
of cancer patients undergoing immune checkpoint blockade cessing; clinical validation, which is how the test interprets
[51]. CTCs contain the whole proteomic information, which outcomes compared to the current gold standard test; clini-
is unique for cancer proteomic analysis. This could also be cal utility, which evaluates the real value of the test for the
achieved by the analysis of exosomes, which transfer genetic individual and how likely the test lead to an outcome; and
information and proteins from tumor cells to recipient cells the ethical, legal, and social implications that may be
in the tumor microenvironment. A study on glioblastoma related to the risks, benefits, and cost-related issues. In the
detected tumor-specific EGFRvIII in serum exosomes from liquid biopsy research field, to meet the criteria of the
7 out of 25 glioblastoma patients [52]. CTCs and exosomes ACCE framework, more evidence is still needed to explore.
are both suitable for RNA expression analysis. This is critical For cancer-screening markers, the National Cancer Institute
for characterizing tumor behavior and determining tumor Early Detection Research Network (EDRN) also defined a
origin. formal structure to guide the process of biomarker develop-
Compared to the tissue biopsy, CTCs, ctDNA, and exo- ment [54]. Five consecutive phases exist: preclinical explo-
some approaches are all capable of detecting somatic muta- ration for identifying promising directions, clinical assay
tions, copy-number alterations, and gene fusions, as well as development and validation for disease detection, retro-
evaluating methylation patterns. In 2018, the American spective longitudinal repository studies to evaluate diag-
Society of Clinical Oncology and the College of American nostic capacity and define positive criteria, prospective
Pathologists conducted a joint review to analyze the assess- screening studies for the extent and characteristics of dis-
ment of genomic variants in blood ctDNA for clinical test- ease detected by the test, and cancer control studies to
ing [53]. The significant overlap between tissue and ctDNA quantify the impact of the test on reducing the burden of
genotyping in advanced cancer was confirmed. Most disease on the population.
patients have concordant tissue and plasma genotyping As we discussed, the development of most liquid biopsy
results; however, tumor-positive and ctDNA undetected approaches is still in phase I and phase II stages. For exam-
gene mutations are also relatively common. Undetected ple, even the most readily applicable ctDNA test presents a
tumor and ctDNA gene alterations are relatively rare but can variety of standardization issues which significantly affect its
be more common in acquired, resistant mutations with high clinical implementation [50]. At the preanalytical level, sam-
heterogeneity and in mutations found in clonal hematopoi- ple stabilization, time period between blood collection and
esis. To increase the diagnostic sensitivity and improve processing, quantification methods, and ctDNA isolation
identification of the underlying tumor tissue of origin, a protocols need to be standardized. At the analytical level,
combination of approaches has been investigated. intrinsic PCR errors, nonuniform genomic coverage, and
CancerSEEK, a multiplex blood test system, which inte- technological errors need to be addressed. At the biological
grates cell-free DNA with conventional protein marker tests, level, because of the variability in tumor types and stages,
was developed for early cancer detection and localization. different sample processing methods, and different genetic
CancerSEEK tested 16 driving genes and 8 protein markers alteration targets, it is challenging to determine the sensitiv-
(CA125, CEA, CA19-9, PRL, HGF, OPN, MPO, or TIMP1) ity of the ctDNA test. The other obstacles, including the lack
and was used to study 1005 patients who had been diag- of a tumor locator to determine which organ system the
nosed with stage I to III cancers of the ovary, liver, stomach, ctDNA is derived from and differentiating somatic variants
pancreas, esophagus, colon, rectum, lung, or breast. With an in healthy people, such as clonal hematopoiesis of indetermi-
overall sensitivity of 70% and corresponding specificity of nate potential, are limiting its clinical implementation. The
99%, this combination method demonstrated promising American Society of Clinical Oncology and the College of
advantages in early cancer detection. American Pathologists 2018 joint review concluded that
some ctDNA tests have demonstrated clinical validity and 11. Lamb YN, Dhillon S. Epi proColon((R)) 2.0 CE: A blood-

based screening test for colorectal cancer. Mol Diagn Ther.
utility for certain types of advanced cancer. However, there is 2017;21:225–32.
not enough evidence to apply this conclusion to the majority 12. Warren JD, Xiong W, Bunker AM, et al. Septin 9 methylated DNA
of ctDNA assays in advanced cancer. Discordance between is a sensitive and specific blood test for colorectal cancer. BMC
the results of ctDNA assays and genotyping tumor speci- Med. 2011;9:133.
13. Cohen JD, Li L, Wang Y, et al. Detection and localization of sur-
mens exists. There is currently no evidence for the clinical gically resectable cancers with a multi-analyte blood test. Science
utility and little evidence for the clinical validity of ctDNA (New York, NY). 2018;359:926–30.
assays in early-stage cancer, treatment monitoring, or resid- 14.
Issa IA, Noureddine M. Colorectal cancer screening: An
ual disease detection. At the present time, techniques are updated review of the available options. World J Gastroenterol.
2017;23:5086–96.
continually evolving, and new evidence is constantly emerg- 15. Talasaz AH, Powell AA, Huber DE, et al. Isolating highly enriched
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Looking ahead, better knowledge of the dynamic biology of and antigen- based profiling of circulating tumor cells using a
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Molecular-Targeted Imaging
28
Fang Wang, Jian Xu, and Wenying Xia
Molecular-targeted imaging is a new subject initiated by the used radiotracers combined with growth hormone receptors
combination of medical imaging technology and molecular to image endocrine tumors [3]. Fischman used 111In-labeled
biology in the twenty-first century. Molecular imaging is one chemotactic peptide to detect inflammatory lesions.
of the ten frontier medical sciences with potential in the Meanwhile, with the development of high-efficiency and
future by the American Medical Association. Over the last high-sensitivity imaging equipment and molecular biology
decade, molecular-targeted imaging of living subjects has technology, the field of molecular imaging research has been
evolved considerably. It has seen spectacular advances in expanding.
chemistry, engineering, and biomedical applications. It is widely understood that MTI is an emerging frontier
Comprehensive molecular-targeted imaging centers have of biomedical research with important potential ramifica-
been established in many countries in a short period of time. tions for medical diagnosis and therapy. Techniques of
They are increasingly integrated into basic sciences and MTI are revealing new knowledge about normal and
translational networks. This chapter will introduce you to the abnormal structure and function at the cellular and molec-
principles and applications of molecular-targeted imaging. ular levels and are providing promising mechanisms for
guiding the delivery of new therapies to aberrant cells and
tissues. MTI aims to study molecular or cellular events in
28.1 Overview the living animal or human. These events can be location(s)
of a specific cells or levels of a given protein receptor on
Molecular-targeted imaging (MTI) is a discipline developed the surface of cells.
on the basis of nuclear medicine. The development of nuclear MTI is widely used in the diagnosis and treatment of
medicine has greatly promoted the birth of MTI. In 1974, tumors, cardiovascular diseases, central nervous system dis-
Goldenberg and colleagues scanned GW-39 tumor-bearing eases, and autoimmune diseases. The purpose of detecting or
mice with 125I-labeled IgG and detected specific targets of imaging various molecular targets is to understand as much
tumors by the principle of specific binding of antibodies to as possible the disease processes associated with one or more
carcinoembryonic antigen [1]. In 1978, they used radionu- of these molecular targets. Another very important reason to
clide markers for imaging human tumors [2]. In 1983, study molecular targets is to help dissect complex underlying
Eckelman and Reba successfully performed single-photon biology.
emission computed tomography (SPECT) imaging of human Specificity of MTI has always been its advantage. MTI has
brain receptors for the first time, and Wagner successfully been widely used in various basic and clinical research fields,
performed positron emission computed tomography (PET) such as life science, medical research, and drug development,
imaging of human brain receptors in the same year. In 1987, and has broad application prospects. Modern medical imag-
the FDA approved the first radioactive drug 123I-IMP, for ing, with molecular imaging as its core feature, will have a
cerebral blood perfusion. In 1990, Lamberts and colleagues direct and far-reaching impact on future medical models and
play an increasingly important role in life science research
and clinical practice. Nevertheless, the field of MTI still needs
to be mature and improved. Therefore, more new generation
F. Wang (*) · J. Xu · W. Xia of detection technology is needed to guide us to deepen our
Department of Laboratory Medicine, The First Affiliated Hospital understanding of biology and pathology and constantly
of Nanjing Medical University, Nanjing, Jiangsu, improve the clinical diagnosis and treatment of patients.

396 F. Wang et al.
28.2 Basic Principle outside and then displaying the target molecule through an
imaging instrument. This molecular marker is called an
MTI is a noninvasive real-time measurement of the internal imaging probe. Recently, more than 100 imaging probes
processes in vivo at the molecular level. It can obtain molec- have been successfully developed before clinic, but only a
ular information by introducing extremely small amounts of few have been applied in clinic.
molecular probes into the human body and then combining The most straightforward route to design an efficient
them specifically with imaging targets, using a series of imaging reporter in PET studies is to directly label small
advanced imaging technology to image the interaction endogenous molecules with F-18 or C-11 isotopes, such as
between molecular probes and target molecules. It can trace in the case of [18F]fluoro-16α-fluoroestradiol (18F-FES, estro-
specific molecular behavior in the body, especially the key gen receptor ligand) [4] or in [18F]fluoroethyl-L-tyrosine
target molecule to determine the course of disease (Fig. 28.1). (18F-FET) and [11C]-methyl-L-methionine (11C-MET, amino
In general, MTI must satisfy the following four key condi- acid transporter tracers), respectively.
tions or elements in order to achieve specific molecular imag- Magnetic resonance imaging (MRI) contrast agents make
ing in vivo: reasonable pharmacokinetics, high-affinity and use of local changes in the relaxation times of tissue water.
high-specificity imaging probe; imaging probes must over- The limited sensitivity of MRI requires amplification. Efforts
come biological barriers such as blood vessels, stroma, and still have to be devoted to seeking real breakthroughs in
cell membranes; high-efficiency and suitable signal amplifica- high-relaxivity systems.
tion technology; and sensitive and high-resolution imaging Optical imaging probes possess high sensitivity, while it
system. has the drawback of limited penetration of light through bio-
logical tissues. Improvements can be foreseen with the use of
time-resolved fluorescence-based approaches.
28.2.1 Probes
High-affinity and high-specificity probes are prerequisites 28.2.2 Molecular-Targeted Imaging Strategy
for MTI, signal amplification, and high-sensitivity detection.
Usually, molecular imaging requires introducing a molecular Molecular imaging can be divided into three strategies:
marker that can specifically bind to the point in vivo from direct imaging, indirect imaging, and substitute imaging.
Fig. 28.1 Molecular-targeted
imaging research chain
28 Molecular-Targeted Imaging 397
Direct imaging is a relatively simple method that uses spe- Substitute imaging is generally used to monitor the down-
cific imaging probe to directly react with the corresponding stream effects of specific endogenous molecular-genetic pro-
imaging point to image the point. In contrast, indirect imag- cesses that occur in diseases such as cancer or to assess the
ing is more complex and involves many factors, but it is more therapeutic effects of diseases by monitoring the downstream
widely used than direct imaging. results of these genetic changes. STI571 (Gleevec) causes
Direct imaging is the insertion of molecular probes into changes in glucose uptake in gastrointestinal stromal tumors
cells. When a molecular probe encounters a specific mole- (GIST). It is generally believed that the activity of cKIT
cule or a specific gene product, it sends out signals. The sig- receptor is inhibited. Therefore, 18F-FDG PET imaging can
nals are recorded by imaging equipment (such as PHT, MRI, detect changes in glucose metabolism, monitor the activity
or infrared) and converted into molecular images or meta- of signal transduction pathway mediated by cKIT receptor,
bolic images. Probe location, image intensity, and brightness and evaluate the efficacy of Gleevec in GIST.
are directly related to probe, target molecule, antigen epitope
or enzyme, etc. Halbhuber and colleagues used radiolabeled
glycated peptide containing RGD sequence to image integrin 28.2.3 Molecular-Targeted Imaging Type
and obtained satisfactory results [5]. Blend and colleagues
used specific ligands labeled with radionuclides or gadolin- MTI technology plays an important role in noninvasive
ium chelates to image human epidermal growth factor recep- research of receptors, antigens, and gene expression. At pres-
tor-2 (HER-2) on the surface of breast cancer cells [6]. In ent, the common types of molecular imaging are protein
addition, a specific antisense oligonucleotide labeled by molecular imaging and gene expression imaging. Among
radionuclides can be used as a probe, and the corresponding them, protein molecular imaging includes receptor imaging,
target genes and proteins in vivo can also be directly imaged immunoimaging, and other protein imaging. Gene expres-
by the principle of antisense complementarity. Others, Bulte sion imaging mainly includes antisense imaging and reporter
used SPIO cell markers for imaging, also belong to the cat- gene expression imaging. By understanding the principles
egory of direct imaging [7]. and conditions of these imaging types, we can use molecular
Indirect imaging is a real molecular imaging technology, probes to directly detect the changes of the quantity and
which indirectly realizes the imaging of the target of interest quality of receptors under physiological and pathological
through the interaction between reporter probe and reporter conditions, the distribution of antigen molecules, the changes
gene expression products. Reporter gene imaging has of protein molecules, and gene expression levels in vitro, so
become the most common indirect imaging, which is widely as to provide early, reliable, and in vivo imaging data for the
used in molecular imaging fields such as radionuclide imag- diagnosis and treatment of diseases.
ing, MRI, and optical imaging. It is mainly used to monitor Receptor imaging is based on the principle that ligands
endogenous gene expression and gene therapy process. labeled with imaging agents bind specifically to receptors in
Reporting probes and reporter genes are required for reporter target tissues. Receptor imaging introduces specific ligands
gene imaging. Reporting probes can be known or new labeled by imaging agents into or in vivo and displays spatial
radionuclide-labeled probes, MR molecular imaging probes, distribution, density, quantity, and affinity of receptors
and so on. The products encoded by the reporter gene may be in vitro using imaging instruments. It is a noninvasive in vivo
enzymes, receptors, or transporters. functional imaging method that integrates ligand-receptor-
HSV1-tk reporter gene is the most commonly used specific binding tracer technology with high sensitivity.
reporter gene in nuclear medicine. It is helpful to under- The principle of immunoimaging is to use antibodies as
stand the principle of reporter gene imaging. HSV1-tk carriers, through the specific combination of antibodies and
reporter genes need to be transfected into cells by means of antigens (such as tumor tissues), to guide the imaging agent
vectors. At present, retroviruses, adenoviruses, adeno-asso- into the target, and to detect the distribution of antigen mol-
ciated virus, lentiviruses, and liposomes are commonly ecules in vivo directly by high-sensitivity imaging equip-
used vectors. ment in vitro, so as to realize the targeted enhancement of the
Substitute imaging, or biomarker imaging, refers to the target and to provide reliable imaging information for early
downstream effects of one or more endogenous molecular- diagnosis and treatment of diseases. At present, the antibod-
genetic processes reflected by the use of alternative marker ies labeled by imaging agents include polyclonal antibodies,
probes. Substitute imaging is simpler than direct imaging monoclonal antibodies, antibody fragments, and so on.
and reporter gene imaging. It uses existing tracers, contrast However, due to the high molecular weight of antibodies,
agents, and imaging methods to image downstream physio- slow clearance rate in the blood, and weak penetration of
logical or pathological effects of endogenous molecular or molecules, it is not easy to reach the lesion site, and with the
genetic processes such as signaling pathways, rather than strong immunogenicity of xenogenic antibodies, it is easy to
specific interactions between molecular probes and targets. produce hypersensitivity, so the miniaturization of antibod-
398 F. Wang et al.
ies and the preparation of humanized antibodies have become evaluating therapeutic effect after treatment. Reporter gene
the research hotspots in this field. imaging is to detect the expression of reporter gene by using
A starting point of protein imaging is the imaging target the protein expressed in the reporter gene reacting or specifi-
itself—many of the original molecular imaging approaches cally binding with the reporter probe labeled by the imaging
are based on the ability to detect specific proteins, which agent. Fusion protein approaches have been used to generate
include enzymes (e.g., hexokinase, thymidine kinase [tk]), reporter genes that function in a variety of imaging modes,
receptors (for neurotransmitters, growth factors, hormones, for example, fluorescence, bioluminescence, and PET imag-
etc.), tissue or differentiation markers, and other specific pro- ing [8–10]. The use of reporter genes in conjunction with
teins. Once a key molecular target has been identified, gene therapy or cell-based therapies will provide an impor-
molecular imaging needs to provide two functions: specific- tant window for assessment of these therapies in living
ity, to single out the target of interest, and signal generation organisms, including patients [11–13]. These concerns have
(sensitivity), such that activity can be monitored noninva- prompted development of additional reporter genes of human
sively in living subjects. Again, proteins can provide key origin, to reduce the possibility of an immune response in
starting points. For example, antibodies represent a class of patients [14].
proteins whose natural purpose is to recognize target mole-
cules with exquisite specificity. Adaptation for use in molec-
ular imaging requires engineering to optimize their affinity if 28.3 Technology Development
needed, as well as modification of pharmacokinetics and
reduction of immunogenicity. Finally, a means for detection MTI has been developed on the basis of existing medical
needs to be incorporated—by conjugation or fusion to signal- imaging techniques. It aims to detect abnormalities at the
generating moieties. At present, great progress has been molecular and cellular levels. Modern medical imaging tech-
made in protein molecular imaging, especially in apoptotic nologies such as nuclear medical imaging (PET, SPCT),
imaging. MRI, optical image (OI), ultrasound (US), computed tomog-
Gene expression imaging is a noninvasive imaging raphy (CT), and so on are the basis of molecular imaging
method that uses imaging agent-labeled probes to display the (Fig. 28.2).
functional changes of genes and their expression products at
the DNA, RNA, or protein levels, thus providing a method
for clinical diagnosis or therapeutic evaluation. Gene expres- 28.3.1 PET/CT
sion imaging has important clinical application value. It is
the main means of predicting therapeutic effect before gene The majority of PET/CT-installed base is in routine clinical
therapy, monitoring gene expression during treatment, and operation. Many literatures approve the accuracy of staging
Fig. 28.2 Typical molecular

imaging instruments and
images representative of each
modality. (a) MRI, (b)
computed tomography, (c)
positron emission
tomography, (d) single- b
photon emission computed
tomography, (e) optical
imaging, (f) ultrasound
a
c
d
f
e
or restaging with PET/CT compared with either CT or PET logic imaging. They can be identified by PET, leading to an
separately [15]. Many publications clearly support the sig- improvement of this morphologic reference standard. The
nificant improvements in specificity and sensitivity and espe- weakness of morphologic lymph node staging is the lack of
cially in early detection of cancer recurrence. The reliable criteria, since assessment can be made only on the
improvements are incremental that alone demonstrates high basis of size [22]. In the scientific literature, compared to
levels of sensitivity and specificity for many diseases. visual correlation of the images, the simultaneous acquisi-
Improved accuracy has been documented for cancers includ- tion of co-registered molecular/biochemical and anatomic/
ing head and neck, thyroid, lung, breast, esophageal, colorec- morphologic information has been shown to improve diag-
tal, and melanoma [16–20]. There is also evidence that PET/ nostic accuracy of the cancer staging. It allows the discrimi-
CT improves accuracy in lymphoma1 and solitary pulmo- nation of variable physiologic radiotracer uptake (brain,
nary nodules, in spite of the fact that in lymphoma the accu- thyroid gland, fat, striated muscle, myocardium, digestive
racy of PET alone is very high [21]. tract, bone marrow, and genitourinary tract) that can mimic
metastatic lesions from pathological uptake and helps to
avoid potential false-positive interpretations [16, 23–26].
28.3.2 SPECT The fusion of PET with MRI can compensate for their sepa-
rate disadvantages and therefore offers several combined
There are various pros and cons for the use of SPECT in advantages in comparison to PET or MRI alone.
comparison with studies performed using PET. Compared
with PET system, one of the advantages of SPECT system is
low equipment cost. Therefore, it is widely used in clinical 28.3.4 CT
practice. In preclinical small animal imaging, the spatial
fraction of microSPECT is higher than that of CT has the advantages of high spatial resolution, but low
microPET. SPECT can perform multi-nuclide imaging. With resolution, radioactivity, and low imaging sensitivity for soft
the development of microPET, its super-high resolution and tissue. Therefore, it is often combined with other imaging
detection efficiency have been enhanced. SPECT continues methods such as SPECT and PET in molecular imaging. In
to be widely used to complement planar nuclear medicine addition, in recent years, with the development of new CT
studies, being the method of choice in an increasing number contrast agents, it has brought new hope for CT molecular
of applications (e.g., heart, brain). The ease of access to imaging. Researchers used nanoparticle N1177 to image
single-
photon emitting radionuclides with good imaging giant cells in rabbit models of non-atherosclerosis. The
properties and the wide range of suitably labeled radiophar- results showed that N1177 could be used to detect macro-
maceuticals guarantees continued use. Continuing develop- phages in atherosclerotic plaques, which improved the
ments in instrumentation and reconstruction and the enhancement effect of CT imaging.
availability of combined SPECT/CT units suggest an increas-
ing utility in both clinical practice and research.
28.3.5 Ultrasound
28.3.3 PET/MR Compared with other molecular imaging methods, ultra-

sound has the advantages of high spatial resolution and time
Over the past decade, there have been great technical resolution, real time, noninvasive, relatively low cost, non-
improvements in PET and MRI. The combination of these radiation, and so on. It is an imaging technology with both
two excellent diagnostic imaging modalities into a single imaging and therapeutic effects. With the development of
scanner offers several advantages and improves diagnostic new ultrasound contrast agents and the continuous innova-
accuracy by facilitating the accurate registration of molecu- tion of new ultrasound technology, ultrasound molecular
lar aspects and biochemical alterations of disease with exact imaging has made great progress and played an important
correlation to anatomic information and morphologic find- role in the field of molecular imaging. Ultrasound molecular
ings. PET facilitates the evaluation of molecular aspects and imaging technology is a real-time, dynamic detection and
biochemical alterations. They are fundamental to the detec- imaging method using ultrasound molecular probes at the
tion of tumor and assessment of tumor stage, therapeutic molecular and cellular levels. If targeted ultrasound micro-
response, and tumor recurrence. In general, increased radio- bubbles are combined with genes or drugs, targeted therapy
tracer accumulation can be studied before anatomic/morpho- can be achieved while imaging, which opens up new ideas
logic structure changes. Another advantage of PET is the for the diagnosis and treatment of diseases. The idea that bio-
sensitive detection of smaller than 10 mm lymph node colloids consisting of microbubbles (MBs) or nanoparticles
metastases. They are not assessable with anatomic/morpho- could be coupled to ligands for specific endothelial cell sur-
400 F. Wang et al.
face proteins and imaged to report molecular targets was eases. Some achievements have been made in drug screen-
developed in the late 1990s in the laboratories of Lanza [27, ing, efficacy evaluation, angiogenesis, and cell apoptosis.
28], Unger [29], and Lindner [30, 31]. Researchers have
shown that MB ultrasound contrast agents can be selectively
targeted to intravascular molecular markers of various dis- 28.4.1 Cancer
ease processes, including inflammation [32–35], thrombosis
[36, 37], and tumor angiogenesis [38–42]. Diagnosis, staging, treatment planning, therapeutic monitor-
ing, and surveillance are the key processes in evaluation of
appropriate management of patients with cancer. Because of
28.3.6 Optical Imaging its noninvasive nature, imaging plays a key role in all these
phases of cancer evaluation. It is currently highly dependent
At present, bioluminescence imaging, fluorescence imaging, upon anatomical imaging techniques, particularly including
and near-infrared imaging are the three main techniques for CT and MRI. As a glucose analog, FDG is used for the vast
optical molecular imaging in vivo. In addition, with the majority of clinical PET studies performed for the evaluation
development of technology, new methods of optical imaging of suspected or confirmed malignancy. Using a range of
continue to emerge. Photoacoustic imaging, multispectral imaging devices, studies found that PET had a relatively high
imaging, autoluminescent fluorescence imaging, Raman accuracy with the majority of FDG-avid lesions being malig-
microscopy, and tomographic fluorescence systems are nant and the vast majority of non-avid lesions being benign
developing. Compared with other in vivo imaging technolo- [45–47]. Similarly, some rationale may exist for a screening
gies, optical molecular imaging technology has many unique PET study in patients with a high genetic predisposition to
advantages, such as no radiation, high sensitivity, relatively cancer. There is a strong rational basis for the use of molecu-
simple imaging process, real-time monitoring, and low cost. lar imaging and particularly FDG PET/CT for cancer stag-
At present, this technology has been widely used in various ing, response assessment, and oncologic drug development
biological researches, including oncology. The limitation of [48–53].
optical imaging technology lies in its limited penetration, The medical profession needs to develop an increased
which is from millimeters to centimeters, so it is mostly used sophistication in the use of the complex information pro-
in the study of small animal disease models. In addition, vided by FDG beyond simply counting lesions and assessing
fluorescence imaging has some disadvantages, such as high the locations. Challenges for the future include refining the
background signal caused by tissue autofluorescence and information provided by the multidimensional signals now
limited stability of fluorescence of many small molecules. available from previously exclusive anatomic imaging tech-
Although optical imaging has some limitations in deep tissue niques with the biologic information obtained from PET
imaging, these limitations can be overcome by using it in tracers. More challenges for the future include determining
conjunction with other technologies. Optical coherence how and when to integrate new tracers into evaluation para-
tomography (OCT) can be used in ophthalmology, gastroin- digms as these agents will almost certainly struggle to com-
testinal tract, and vascular imaging if noninvasive or mini- pete with the excellent overall performance of FDG and will
mally invasive instruments such as handheld probes, need to be used in niche applications and to address either
catheters, endoscopy, and laparoscopy are used. Integrating the recognized limitations of FDG or to provide unique bio-
with other imaging techniques such as MRI or PET, optical logic characterization of lesions of greatest relevance to the
imaging will have a broad application prospect in clinical therapy being considered.
application [43, 44]. In recent years, CT enterography (CTE) and capsule
endoscopy (CE) have good diagnostic value for small intes-
tinal bleeding. Both CTE and CE can effectively detect small
28.4 Clinical Application intestinal lesions, but CE is superior to CTE in diagnosing
Crohn’s disease (CD) patients. For patients with unexplained
In recent years, with the development of high-throughput abdominal pain, it is often necessary to use combination
screening technologies such as genomics, proteomics, and therapy to make a more accurate diagnosis.
chip technology, more and more molecular imaging targets Tumor-specific protein 70 (SP70) is a novel tumor marker
have been developed. Successful development of small ani- of non-small cell lung cancer (NSCLC), especially lung ade-
mal imaging equipment, the establishment of transgenic ani- nocarcinoma [54]. It was proved that SP70’s monoclonal
mal models, and the use of new highly specific imaging antibody NJ001 could specifically recognize and react to
probes have promoted the rapid development of MTI and lung adenocarcinoma cells [55]. Pan demonstrated the feasi-
made it useful in early diagnosis and gene imaging of dis- bility of SP70-targeted imaging with NJ001-conjugated
nanomagnetic beads (immuno-nanomagnetic beads) on lung ture, and valvular function. Several potential imaging modal-
adenocarcinoma and the potentials for its real-time monitor- ities, either as single or fusion technologies, have become
ing and early detection. available for molecular imaging of the failing heart. The abil-
Three kinds of in vivo molecular imaging techniques ity of nuclear imaging techniques to detect cellular and bio-
were developed in Pan’s study: BLI based on the activity of logical processes may be combined with excellent anatomical
luciferase that catalyzes the substrate luciferin in transfected and functional information provided by CT or MRI [56, 57].
cells, targeted fluorescence imaging with CF750-NJ001, and Moreover, molecular imaging approaches that may be suit-
targeted micro-CT with NJ001-conjugated nanomagnetic able to image various cellular processes have been developed
beads. Since bioluminescence signal intensity positively cor- to be combined with established functional cardiac imaging
relates with tumor volume, they can make a quantitative techniques, such as echocardiography and MRI [58, 59].
assessment of tumor growth in vivo. Furthermore, it was In conjunction with high-resolution hardware detection
found that lesion in orthotopic lung models could be detected systems, new molecular imaging agents for MRI, nuclear
by SP70-targeted fluorescence imaging at the third week, imaging, OI, CT, and ultrasound imaging show substantial
meanwhile positive signals were found at the sixth week by capabilities for atherosclerotic vascular disease [60]. Several
conventional micro-CT. It indicates that SP70-targeted fluo- imaging methods such as X-ray, ultrasound, CT, and MRI
rescence imaging is much more sensitive than traditional have been used to various degrees of success in the diagnosis
imaging techniques. Therefore, SP70 acts as an oncogenic of thrombosis; however, each suffers from limitations.
protein and could be a target of molecular imaging, even for Besides that, MTI will likely play an increasingly important
micrometastasis. Because of weak penetration ability in role in the current phase of stem cell development. Techniques
human body, SP70-targeted fluorescence imaging might be will be needed to image the survival, differentiation, func-
only used to locate lung adenocarcinomas and identify position, interaction, and movement of stem cells in the myocar-
tive margins during surgery. dium serially over time.
In Pan’s study, the diameter of the nanomagnetic beads
was 180 nm, so they can easily distribute into cancer tissue.
NJ001-conjugated nanomagnetic beads could specifically 28.4.3 Central Nervous System Diseases
identify lung adenocarcinoma cells, molecular-targeted
enhancement significantly improving the sensitivity of MTI modalities used to study the human central nervous sys-
micro-CT imaging and monitoring tumor growth. Lesions tem (CNS) are largely limited to nuclear medicine techniques
were detected at the fourth week in SP70-targeted micro-CT and, to a lesser extent, to those that rely on MR. Optical
group, 2 weeks earlier than groups injected with normal molecular imaging, widely used in small-animal studies,
saline control and bare nanomagnetic bead control currently has few applications in humans due to the limited
(Fig. 28.3). Considering 1/3 time in advance for tumor detec- depth of penetration of emitted photons, relative paucity of
tion in mice, it was inferred that SP70-targeted imaging suitable probes, and lack of readily available tomographic
could greatly shorten the period from tumor onset to diagno- imaging systems. Currently, PET and SPECT account for the
sis, and therefore this technique could be used for the early majority of molecular imaging applications in the evaluation
detection of lung adenocarcinoma. of CNS pathology such as neurodegeneration, neuropsychi-
In summary, many molecular imaging techniques are still atric diseases, and malignancy [61–63]. The main advantage
in the experimental stage in vivo. SP70-targeted imaging can of the nuclear medicine techniques is the high sensitivity.
be used in clinic and can significantly improve the detection PET and SPECT have been used to demonstrate signifi-
ability of lung adenocarcinoma. Molecular-targeted imaging cant abnormalities in brain function even in the absence of
with NJ001-labeled probe may have precision medical appli- detectable or specific structural abnormalities [64, 65].
cations for early diagnosis of lung adenocarcinoma. Although these technologies have proven to be clinically
useful in the diagnosis and management of several neurode-
generative diseases, the full potential of functional imaging
28.4.2 Cardiovascular Diseases in neurodegeneration remains to be studied. New functional
imaging targets and agents that are more disease and
Noninvasive imaging has several implications in the diag- pathology specific, such as amyloid imaging in Alzheimer’s
nostics and management of patients with heart failure. disease (AD), have provided a tantalizing view of what the
Echocardiography is considered as the single most useful future of MTI may contribute to the diagnosis and treatment
test for evaluation of a patient with heart failure that allows monitoring of neurodegenerative disorders in clinical
assessment of systolic and diastolic function, chamber struc- practice.
402 F. Wang et al.
Fig. 28.3 The early diagnosis of lung cancer in nude mice after immunomagnetic bead solution injection
28.4.4 Autoimmune Diseases lead to joint inflammation and destruction. In the future, it
will help us to refine our understanding of RA. Furthermore,
Currently, nuclear medicine techniques are not routinely MTI will be instrumental in monitoring treatment response
used for the diagnosis of chronic inflammatory diseases but and efficacy in the clinical setting [66, 67].
largely contribute to the management and prognosis. Nuclear
medicine techniques are the most sensitive diagnostic meth-
ods for the evaluation of the state of activity of the disease. In 28.4.5 Drug Development
most cases, therapeutic options are available, and provoking
the start of treatment may lead to disease prevention or delay MTI technology has become an important tool to break the
of complications. bottleneck of new drug development. MTI can dynamically,
Nowadays, researches had given us a remarkable amount continuously, and repeatedly observe drugs in living animals
of mechanistic insight into the pathogenesis of rheumatoid or animal models. It is a real in vivo evaluation. It provides a
arthritis (RA). This is supported by the success story of new technology platform for new drug research and develop-
newly available targeted therapies that have opened up new ment and plays a wide role in drug screening, pharmacody-
possibilities in the management of RA. MTI has been essen- namics evaluation, and pharmacokinetics research. The
tial in revealing these cellular and molecular processes that application of MTI can not only greatly shorten the time of
drug development but also greatly reduce the cost of drug 11. Yaghoubi S, Barrio JR, Dahlbom M, et al. Human pharmacokinetic
and dosimetry studies of [(18)F]FHBG: a reporter probe for imag-
development. After labeling the drug molecular probes by ing herpes simplex virus type-1 thymidine kinase reporter gene
MTI, the drug was injected into the experimental animals. expression. J Nucl Med. 2001;42:1225–34.
MTI was used to monitor the situation of the drug in the 12. Penuelas I, Mazzolini G, Boan JF, et al. Positron emission tomog-
animals and to judge whether the drug could reach the target raphy imaging of adenoviral-mediated transgene expression in liver
cancer patients. Gastroenterology. 2005;128:1787–95.
area accurately. If the recognition could not reach the desig- 13. Traversari C, Marktel S, Magnani Z, et al. The potential immunoge-
nated day target, the drug would not need further clinical nicity of the TK suicide gene does not prevent full clinical benefit
trials, thus eliminating a large number of invalid drugs accu- associated with the use of TK-transduced donor lymphocytes in
rately and efficiently, so as to improve the efficiency of the HSCT for hematologic malignancies. Blood. 2007;109:4708–15.
14. Serganova I, Ponomarev V, Blasberg R. Human reporter

test. Furthermore, MTI can be used to track the interactions genes: potential use in clinical studies. Nucl Med Biol.
between labeled drugs and the body. The process and phar- 2007;34:791–807.
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to obtain reliable pharmacodynamic and pharmacokinetic lung cancer with integrated positron-emission tomography and
parameters, which greatly shortened the time of animal computed tomography. N Engl J Med. 2003;348:2500–7.
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ments, and reduced the cost of drug development. In the field (stage IIIa) non-small cell lung cancer after neoadjuvant chemo-
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and may explain the mechanism of drug action, so as to help 2-[F-18]fluoro-D-glucose positron emission tomography and inte-
develop drugs with better efficacy and less toxic side effects. grated PET/CT in restaged breast cancer patients. Mol Imaging
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Fluorescence In Situ Hybridization
29
Min Hu and Weimin Wu
Fluorescence in situ hybridization (FISH) is a method of tively high. (c) FISH is convenient. (d) FISH can use differ-
using fluorescent probes to detect specific nucleic acid ent probes at the same time and can be combined with other
sequences within cells and their location. It can be used to detection methods, with good scalability.
detect a variety of cytogenetic variations, including chromo-
somal deletions, amplification, and translocation. As a clas-
sical cytogenetic method, FISH is widely used in prenatal 29.2 Basic Principle
diagnosis as well as the detection of tumors and infections.
FISH is a combination of cytogenetics and molecular biol- There are many methods derived from in situ hybridization,
ogy. It is a bridge between cytogenetics and molecular biol- but the basic principles are the same (as shown in Fig. 29.1).
ogy and belongs to molecular cytogenetics. FISH is fast, All in situ hybridization techniques utilize the principle of
safe, with high sensitivity, and is easy to use. base pairing, although the methods of labeling and detection
are different. FISH is developed on the basis of radioactive in
situ hybridization, but the detection method is not autoradi-
29.1 Overview ography. The procedures of FISH mainly include preparation
and pretreatment of chromosome specimens, preparation
In classical cytogenetic testing, Giemsa-trypsin banding or and labeling of probes, denaturation of nucleic acids in spec-
fluorescent banding is widely used for chromosomal abnor- imens, hybridization, and fluorescence microscopy and
malities, such as circular chromosomes, chromosome dele- results interpretation.
tions, and so on. However, the resolution of these methods is Here, we selected the CTC (circulating tumor cell) detec-
very low, limiting their further clinical application. tion used in our laboratory as an example to clarify the basic
In 1969, Gall and Pardue invented a method of in situ principles of FISH.
hybridization that successfully detected human DNA in the
nucleus of Xenopus laevis using a radioisotope-labeled DNA
probe [1]. In 1980, fluorescein-coupled RNA probes were 29.2.1 Preparation of the Slides
first applied to in situ hybridization [2]. Subsequently, the
researchers found that hapten-modified nucleotides such as The samples used mainly include peripheral blood, paraffin
biotin and digoxigenin can be incorporated into nucleic acid sections, and frozen sections. First, cells in the interphase or
probes and recognized by fluorescein-labeled avidin or anti- metaphase are immobilized on glass slides. If the specimen
digoxigenin antibodies. With the maturity of fluorescent is peripheral blood, it is generally necessary to lyse the red
dyes, fluoresceins quickly replace radioisotopes and become blood cells. If the specimen is a paraffin section, it needs to
the first choice for in situ hybridization. FISH has the follow- be dewaxed. If chromosomal variation needs to be detected,
ing characteristics: (a) FISH is safer than in situ hybridiza- the specimen usually needs to be treated with RNase and
tion using isotopes. (b) FISH can increase sensitivity by the pepsin. Then, the DNA or RNA in the sample needs to be
biotin–avidin amplification system, and its sensitivity is rela- denatured to make the DNA or RNA into single strands.
In this CTC example, blood samples and body fluid sam-
M. Hu (*) · W. Wu ples could be used. Blood samples (4 mL) were collected
Department of Laboratory Medicine, The Second Xiangya using ACD tubes. The amount of pleural fluid, ascites, cere-
Hospital of Central South University, Changsha, Hunan,
brospinal fluid, and lavage fluid collected was 10 mL. For
e-mail: huminjyk@csu.edu.cn blood specimens, firstly, we used PBS (including BSA) to

406 M. Hu and W. Wu
Fig. 29.1 Schematic representation of FISH procedure. There are two probe nucleic acid fragments, and then hapten is recognized using anti-
preparation methods for the probe, direct and indirect. The direct bodies coupled with fluorescein. The basic principle of FISH is base
method is to directly label probe nucleic acid fragments using fluores- pairing
cein. In the indirect method, haptens were firstly incorporated into the
replace the plasma and then lysed the red blood cells with The preparation methods of probes mainly include gap
ammonium chloride solution. The cells were further sub- translation, random primer method, PCR, and oligonucle-
jected to negative selection using magnetic beads coated otide end labeling.
with anti-CD45 antibodies. Cells that did not express CD45 The labeling of fluorescein to probes mainly includes
were obtained and then fixed on slides. Similarly, CD45 neg- direct method and indirect method. In the direct method,
ative cells in pleural fluid, ascites, cerebrospinal fluid, and fluorescein is directly coupled to the nucleotide sequence
lavage fluid were fixed on slides. of the probe. Indirect labeling probes are relatively com-
plex, using either biotin or digoxigenin-labeled nucleotide
sequences, and then fluorescein-loaded avidin or antibod-
29.2.2 Preparation of the Probes ies to detect biotin or digoxin was used, respectively. In
clinical practice, two or more probes carrying different
The choice of the probe is the key to FISH. A suitable probe fluoresceins can stain one sample simultaneously. The
can ensure the sensitivity and specificity of the FISH detec- advantage of the direct method is simple, and the back-
tion, and can finally ensure its diagnostic efficiency. FISH ground is low. The disadvantage is that the fluorescent sig-
probes consist of two parts, nucleic acid fragments, and nal is weak. The advantage of the indirect method is that
labels (fluorescein or hapten). The nucleic acid fragment on the signal is strong, and the disadvantage is that there are
the probe shows cytogenetic variation by specifically bind- many steps and high background.
ing to the corresponding nucleic acid fragment in the sample. Clinically, the FISH probes can be roughly classified into
The nucleic acid fragments on the probe typically contain the Chromosome Enumerating Probe (CEP probe), the
15–30 bases. There are many types of probes, and there are Locus Specific Identifiers Probe (LSI probe), and the
various methods for labeling. In clinical situation, different Chromosome Painting probe (CP probe).
fluorescent probes are selected depending on the purpose of
the test and the sample. In general, there are two main types 1. The CEP probe consists of 26 pieces, which combine the
of probes, one is a nucleic acid fragment (RNA/DNA) in alpha satellite sequence of a specific chromosome centro-
which a hapten is incorporated, and the other is a nucleic mere region to detect the copy status of chromosomes.
acid fragment directly labeled with fluorescein. The nucleic 2. Multiple types of LSI probes that could detect amplifica-
acid fragment of probes could derive from cloned DNA (cos- tion or deletion of a gene by site-specific binding.
mid, plasmid, phage, BAC, PAC, YAC), somatic hybrid 3. Chromosome painting probes, including whole or arm-
DNA, flow cell sorting chromosome, chromosome microdis- specific probes, to detect chromosomal translocations, or
section metaphase chromosome, or PCR products. to label chromosomes.
29 Fluorescence In Situ Hybridization 407
Among them, LSI probes are mainly divided into the fol- 2 . Sensitivity and specificity of the probe.
lowing categories: 3. The number of fluorescent signals after hybridization is
correct to ensure no pollution and degradation.
1 . Single color probe (SC), such as CEP8 4. Low fluorescent background.
2. Dual color probe (DC), such as HER2 + CEP17 5. Each batch of probes has a reference range in the labora-
3. Triple color probe (TC), such as X + Y + CEP18 tory, even if the reference range is provided in the probe
4. Dual color separation probe (Dual color, break apart
manual.
probe, DC, BCR), their separation represents genetic 6. If the experimental results are not in the reference range,
abnormality repeat the experiment or find out the reason.
5. Two-color single fusion probe (dual color, single fusion, 7. Have maintenance and calibration records for all instru-
DC, SF), their fusion represents genetic abnormality ments and equipment, at least twice a year.
6. Extra signal (ES), extra signals represent genetic abnor-
mality, such as the presence of BCR/ABL fusion gene
7. Dual color and dual fusion probe (DC, DF Trans) 29.3 Technology Development
In this CTC example, our laboratory uses directly labeled In recent years, a lot of technology development has emerged
probes (CEP7 and CEP8). in the field of FISH. Several common FISH derived-methods
are listed below.
29.2.3 Fluorescence In Situ Hybridization

29.3.1 Immuno-FISH
The probes and the immobilized chromosomes on slides
need to be denatured and annealed. During nucleic acid Immuno-FISH is a method that combines immunofluores-
annealing, the fluorescently labeled probe binds to the DNA cence and FISH. Proteins and nucleic acids can be detected
or RNA of interest in the sample. Then, by washing, the simultaneously in the same cell using Immuno-FISH. Immuno-
unbound fluorescent probe on the sample is removed. Probes FISH can determine the relative position and quantity of the
that use hapten-labeled probes require further incubation protein of interest and the nucleic acid of interest, and can also
with fluorescein-conjugated avidin or digoxigenin antibody be used to determine which cell the nucleic acid fragment of
before detection. After the probe binds to the DNA/RNA of interest is present when a plurality of cells exists.
interest, it shows fluorescence at the position of the nucleic
acid sequence of interest and is detected by fluorescence
microscopy. When there are multiple cell types in the sample 29.3.2 Multiplex-FISH (M-FISH), Spectral
and cannot be accurately distinguished by morphological Karyotyping (SKY) and RxFISH
methods, another fluorescein-labeled antibody can be used to
bind to the cellular protein to distinguish the type of FISH- M-FISH, SKY, and RxFISH are karyotyping techniques that
positive cells (Immuno-FISH). analyze all chromosomes at the same time for complex chro-
In the CTC example test, the slides with specimens were mosomal rearrangements. All three methods are based on
pre-fixed, washed, and dehydrated, and hybridized with Multicolor-FISH. Multicolor-FISH uses multiple colors to
fluorescein-labeled CEP7 and CEP8 probes (denaturation at simultaneously label chromosomes. Because of the limited
76 °C for 5 min, then hybridization at 37 °C for 1.5 h). After variety of fluorescein, each chromosome cannot be individu-
hybridization, the cells were stained with anti-CD45-AF594 ally labeled by one fluorescein, so a limited number of fluo-
antibody and DAPI. Finally, the entire slide was microscopi- rescent dyes are used to encode the chromosomes. The
cally examined using a fluorescence microscope. CTCs were procedures of M-FISH and SKY are very similar; the
those cells that FISH signal >2 and CD45 negative. differences are in the data acquisition and analysis. The
RxFISH probe is derived from gibbons and is a cross-species
FISH method.
29.2.4 FISH Quality Control
FISH testing is involved in clinical diagnosis and prognosis, 29.3.3 RNA-FISH

and the reliability of the results is of great significance.
According to the American Cytogenetics Laboratory Guide RNA-FISH is used for RNA detection. Different cell types
[3], FISH quality control mainly includes: express different kinds of RNA, and cells can produce viral
RNA after being infected. Appropriate RNA expression indi-
1. Determine the validity of the probe and the position indicates normal cell function, while abnormal RNA expression
cated by the probe is correct. indicates a problem with cell function. Although RT-PCR or
408 M. Hu and W. Wu
Real-time RT-PCR can measure the expression level of RNA,

RNA-FISH can analyze the expression level and location of
specific RNA without destroying the basic morphological
structure of the cell. RNA-FISH often uses fluorescein-
labeled RNA probes to detect the expression of specific RNA
in cells. Because, compared to DNA probes, RNA probes
work better. Nowadays, RNA-FISH was further evolved to
detect single-molecule RNA in situ.
29.3.4 Three-Dimensional FISH (3D-FISH)
Three-dimensional FISH uses a special method to retain the

3D structure of the nucleus, and thus also fixes the relative
position of nucleic acid fragments in the nucleus. Then, a
plurality of fluorescent probes is used to determine the rela-
tive spatial positions of the designated nucleic acid frag-
ments by binding them to the nucleus. When 3D-FISH is
combined with Immuno-FISH, the relative positions of tar-
get nucleic acid fragments and target proteins in the nucleus
can also be analyzed. Compared to conventional FISH,
3D-FISH can be used for nuclear localization of nucleic
acid.
29.3.5 Fiber-FISH
Fiber-FISH uses high-quality linear DNA fibers for FISH to

obtain extremely fine chromosome maps. Fiber-FISH is the
highest resolution FISH currently known, with resolutions
from 1 to 500 kb. Because DNA fibers are linearized, Fiber-
FISH can be used to analyze the base distance between two Fig. 29.2 Schematic representative of array-CGH
nucleotide fragments.
genomic DNA probe and the normal reference DNA probe
carrying different fluorescein are hybridized with the chip,
29.3.6 CGH (Comparative Genomic and the DNA abnormalities in the tumor genome are ana-
Hybridization) lyzed by the intensity of the two fluorescences [5].
In this method, tumor genomic DNA and normal DNA (for

reference) are used. Firstly, both DNA samples were labeled 29.3.8 Flow-FISH
with different fluorescein and equally mixed. Then, These
DNA samples were hybridized with normal human meta- Flow-FISH is a combination of FISH and FACS. The target
phase chromosomes, and the gain or loss of tumor genomic chromosome fragment was labeled by FISH and detected by
DNA fragments was detected according to the ratio of differ- flow cytometry. Flow-FISH can be used for telomere length
ent fluorescence on the chromosome [4]. measurement. The principle of Flow-FISH for telomere
length measurement is fluorescein-labeled telomere
sequence-specific peptide nucleic acid probe (CCCTAA)
29.3.7 Array-CGH hybridization and then detected by FACS. The fluorescence
intensity indicates the length of the telomere [6, 7]. Compared
Array-CGH is derived from CGH, except that it does not with the traditional FISH method, Flow-FISH is simple and
hybridize to normal human meta-chromosomes but to gene convenient, and easy to repeat.
chips (as shown in Fig. 29.2). The gene chip is pre-coated Similarly, Flow-FISH could also detect chromosomal
with a large number of nucleotide fragments, and the tumor repeats and satellite sequences for the analysis of cellular
homology and satellite sequence instability [8]. Also, Flow- reached almost 100%. The MultiVysion PGT kit from
FISH can be used to detect RNA and to analyze the expres- Abbott-Vysis is available for pre-implantation diagnosis.
sion of RNA in cells using flow cytometry [9]. The 13q14 and 21q22 site-specific probes could detect the 13
and 21 trisomy. The 18, X and Y centromere probes could
detect trisomy 18 and sex of fetuses. The 22q11 site-specific
29.4 Clinical Application probe could detect congenital heart disease caused by
microdeletions.
FISH is a method to detect specific cytogenetic variations. According to the 2016 edition of the Expert Consensus on
Compared to other methods, it retains the basic morphology the Application of FISH in Prenatal Diagnosis, FISH is
of the cells and can be used for nucleic acid localization. As mainly applicable to the following situations in prenatal
a complement to traditional histopathological methods, diagnosis:
FISH can be used for clinical diagnosis. Conventional band-
ing karyotyping remains the preferred technique for chromo- 1. High-risk pregnant women identified by Down syndrome
somal abnormality screening in hematologic malignancies. serological prenatal screening.
However, it should be noted that the interpretation of the 2. High-risk pregnant women identified by Non-invasive
FISH results requires a comprehensive analysis by the clini- prenatal testing (NIPT).
cian in combination with the patient’s clinical presentation 3. For fetuses with invasive diagnostic indicators, FISH
and other results. could be combined to analyze abnormal chromosome
numbers.
4. For those fetuses that cytogenetic prenatal diagnosis can-
29.4.1 Prenatal Diagnosis not be performed, FISH could be used as a rescue
measure.
At present, the karyotype analysis of G-banding is still the 5. Combined with other molecular genetic diagnostic tech-
“gold standard” for prenatal diagnosis of cytogenetics. niques to further confirm the cytogenetic status of the
However, this technique takes a long time and requires cell fetus.
cultivation. FISH is widely used in the field of prenatal diag-
nosis. The outstanding feature of FISH in the field of prena-
tal diagnosis is rapid. In 1993, the American College of 29.4.2 Hematopoietic Diseases
Medical Genetic (ACMG) allowed FISH for prenatal diag- and Lymphoma Related Tests
nosis, which can be used for aneuploidy abnormalities detec-
tion in common fetal chromosomes (13, 18, 21, X, Y), as In the clinical diagnosis of hematopoietic diseases and lym-
well as birth defects and mental retardation [10]. The dis- phoma, FISH is mainly used to find fusion genes, chromo-
eases caused by abnormalities of 13, 18, 21, X, and Y chro- some deletions, detection of minimal residual lesions, and
mosomes take up 85–90% of neonatal chromosomal implantation status monitoring of hematopoietic stem cell
disorders. Using FISH, Down syndrome, Patau syndrome, transplantation [11]. There is a thorough review covering
Klinefelter syndrome, Edward syndrome, and Turner syn- the FISH probes used for hematopoietic and lymphoid
drome could be diagnosed. tumors [12].
In 2010, ACMG developed detailed technical pathways,
analytical standards, and quality control requirements for
FISH technology [3]. FISH is highly sensitive and specific 29.4.3 Application of FISH in Pathogen
for prenatal diagnosis. A large sample comparison with Detection
karyotyping found that the false-positive rate was 0.019%
and the false-negative rate was 0.049%. Because that cell The gold standard for pathogen detection is cultivation.
culture is not required, FISH can use amniocytes, villus cells, However, in clinical situations, many pathogens are difficult
fetal nucleated red blood cells, and other specimens, and the to culture. Moreover, some pathogens, even if they could be
test could be completed within 1–2 days. cultivated in vitro, the long time used seriously hindered the
The AneuVysion multi-color DNA probe kit manufac- timely diagnosis of infectious diseases. FISH is an important
tured by Abbott-Vysis can detect abnormalities in chromo- method for pathogen detection that could be used to detect
somes 13, 18, 21, X, and Y simultaneously. The correct rate pathogens, such as Mycobacterium tuberculosis and
for the detection of chromosomal abnormalities is 99.9%, Treponema pallidum [13]. FISH could also be used for virus
and the correct rate for normal specimens of chromosomes detection, such as HIV and EBV [9, 14].
410 M. Hu and W. Wu
29.4.4 FISH for Detection of Circulating Tumor therapy, and paclitaxel chemotherapy and high-intensity
Cells (CTC) anthracycline regimens should be used. Patients with HER2
gene amplification are suitable for molecular targeted ther-
CTC stands for circulating tumor cells and is a general term apy with Trastuzumab. The PathVysion HER2 DNA Probe
for various tumor cells in peripheral blood. During the Kit detects the HER2 gene and chromosome 17 and can
development and progression of tumors, part of the tumor determine whether the HER2 gene is amplified by the ratio
cells will enter the bloodstream to form CTC. CTC is very of the two in the specimen. For gastric cancer, all patients
rare, and most CTCs undergo apoptosis soon after entering with gastric and gastroesophageal junction cancer need to
the peripheral blood or killed by phagocytic cells in the detect the HER2 status of the tumor when first diagnosed.
blood. Only a few CTCs can reach distant places and form HER2 gene amplification in gastric cancer suggests a poor
tumor metastases. Previously, tumor detection relied mainly prognosis and more progressive status. HER2 gene amplifi-
on pathological biopsy. Pathological biopsy is traumatic cation testing is a prerequisite for targeted therapy screening
and difficult to sample multiple times. CTC testing is an and efficacy prediction. In 2010, Europe and the USA have
important means of liquid biopsy. CTC testing can be used approved chemotherapy combined with trastuzumab to treat
to assess the progression of tumors in patients, and to ana- HER2-positive gastric and gastroesophageal junction
lyze the phenotype of tumors in patients [15, 16]. An cancer.
important method for detecting CTC is based on the fact HER2 gene amplification FISH interpretation criteria are
that tumor cells often exhibit chromosomal variations, as follows. (HER2/CEP-17 means the total number of HER2
especially changes in the number of chromosomes. gene points in 20 tumor cells divided by the sum of CEP-17
Therefore, after the purification of CTC in peripheral blood, points)
the number of chromosomes in the cell is determined by
CEP probes, which can be used to define tumor cells [17]. 1. HER2/CEP-17 ≥2.2, HER2 gene has amplified.
When FISH is used to detect chromosomal variation in 2. HER2/CEP-17 <1.8, HER2 gene is not amplified.
CTC, CEP8 probes are often used to detect lung cancer, 3. HER2/CEP-17 1.8–2.2, uncertain results. (If the result is
CEP8 and CEP7 are often used to detect esophageal cancer, uncertain, a further count of 20 cells or repeat detection is
CEP8 and CEP17 are used to detect breast cancer and gas- required. If the result is still uncertain, measurement of
tric cancer [15–17]. The CTC FISH testing was used as an HER2 protein expression via IHC is recommended.)
example to elucidate the procedure of FISH in this chapter
(see above). As to the diagnosis of bladder cancer, UroVysion can
simultaneously detect chromosome 3, 7, and 17 aneuploidy
and 9p21 (P16 tumor suppressor gene) deletion using exfoli-
29.4.5 Application of FISH in Solid Oncology ated cells in urine for [18]. For urothelial cancers, UroVysion
has a sensitivity of 72% (69–75%) and a specificity of 83%
FISH is widely used to detect tumor cell chromosomal varia- (82–85%) [19].
tions, such as breast cancer, bladder cancer, cervical cancer, The ALK two-color separation probe is capable of detect-
and lung cancer, including early diagnosis, efficacy monitoring all rearrangements of the ALK gene and is of great sig-
ing, and chemotherapeutic drug sensitivity test and progno- nificance for the diagnosis of non-small cell lung cancer.
sis prediction. Moreover, FISH could be used for the diagnosis of syno-
Whether patients have corresponding cytogenetic varia- vial sarcoma, liposarcoma, gastrointestinal stromal tumors,
tions has major impacts on the choice of the treatment regi- prostate cancer, neuroblastoma, glioma, melanoma, cervical
men. For example, the expression of the HER2 gene cancer and bladder cancer, etc.
determines the treatment regimen of breast cancer patients. There are some limitations to the use of FISH in clinical
Traditional immunohistochemical methods are susceptible diagnosis. Firstly, the number of excellent fluorescent probes
to protein instability and subjectivity of results interpreta- is very limited, and only partial cytogenetic abnormalities
tion, especially for those patients with immunohistochemis- can be detected. Secondly, FISH is detected by fluorescence
try results. Overexpression of HER2 protein is often microscopy. Subjective factors of the pathologist will influ-
accompanied by the amplification of the HER2 gene. So, ence the judgment of results, especially when the number of
compared to HER2 protein measurement, detection of HER2 target nucleotide fragments in the sample is sparse. Thirdly,
gene amplification is better to guide treatments directly. the FISH procedure is numerous and requires a large amount
Patients whose HER2 gene was amplified have poor progno- of manual processing, and the stability of the results and the
sis, shortened disease-free survival and overall survival, high high throughput processing capacity are also limited.
lymph node metastasis, and high risk of recurrence. Patients Although FISH has been rapidly developed in clinical diag-
with HER2 gene amplification are resistant to endocrine nosis, the current application is mainly focused on prenatal
diagnosis, leukemia, and breast cancer. As more and more 8. Brind’Amour J, Lansdorp PM. Analysis of repetitive DNA in chro-
mosomes by flow cytometry. Nat Methods. 2011;8:484.
commercial fluorescent probes are put into use, as well as 9. Baxter AE, Niessl J, Fromentin R, et al. Multiparametric character-
increased sensitivity and specificity, FISH will be more ization of rare HIV-infected cells using an RNA-flow FISH tech-
widely used. nique. Nat Protoc. 2017;12:2029–49.
10. American College of Medical Genetics. Prenatal interphase fluo-
rescence in situ hybridization (FISH) policy statement. Am J Hum
Genet. 1993;53:526–7.
References 11. Van der Burg M, Poulsen TS, Hunger SP, et al. Split-signal FISH
for detection of chromosome aberrations in acute lymphoblastic
1. Gall JG, Pardue ML. Formation and detection of RNA-DNA hybrid leukemia. Leukemia. 2004;18:895–908.
molecules in cytological preparations. Proc Natl Acad Sci USA. 12. Cui C, Shu W, Li P. Fluorescence in situ hybridization: cell-based
1969;63:378–83. genetic diagnostic and research applications. Front Cell Dev Biol.
2. Bauman JG, Wiegant J, Borst P, et al. A new method for fluores- 2016;4:89.
cence microscopical localization of specific DNA sequences by 13. Prudent E, Raoult D. Fluorescence in situ hybridization, a comple-
in situ hybridization of fluorochromelabelled RNA. Exp Cell Res. mentary molecular tool for the clinical diagnosis of infectious dis-
1980;128:485–90. eases by intracellular and fastidious bacteria. FEMS Microbiol Rev.
3. Mascarello JT, Hirsch B, Kearney HM, et al. Section E9 of the 2019;43:88–107.
American College of Medical Genetics technical standards and 14. Frickmann H, Zautner AE, Moter A, et al. Fluorescence in situ
guidelines: fluorescence in situ hybridization. Genetics in medi- hybridization (FISH) in the microbiological diagnostic routine
cine: official journal of the American College of Medical. Genetics. laboratory: a review. Crit Rev Microbiol. 2017;43:263–93.
2011;13:667–75. 15. Lin E, Cao T, Nagrath S, et al. Circulating tumor cells: diag-
4. Zou H, Kang X, Pang LJ, et al. Xp11 translocation renal cell car- nostic and therapeutic applications. Annu Rev Biomed Eng.
cinoma in adults: a clinicopathological and comparative genomic 2018;20:329–52.
hybridization study. Int J Clin Exp Pathol. 2014;7:236–45. 16. Dive C, Brady G. SnapShot: circulating tumor cells. Cell.

5. Bejjani BA, Shaffer LG. Application of array-based comparative 2017;168:742–e741.
genomic hybridization to clinical diagnostics. J Mol Diagn JMD. 17. Li Y, Ma G, Zhao P, et al. Improvement of sensitive and specific
2006;8:528–33. detection of circulating tumor cells using negative enrichment
6. Baerlocher GM, Vulto I, de Jong G, et al. Flow cytometry and and immunostaining-FISH. Clin Chim Acta Int J Clin Chem.
FISH to measure the average length of telomeres (flow FISH). Nat 2018;485:95–102.
Protoc. 2006;1:2365. 18. Halling KC, Kipp BR. Bladder cancer detection using FISH

7. Rufer N, Dragowska W, Thornbury G, et al. Telomere length (UroVysion assay). Adv Anat Pathol. 2008;15:279–86.
dynamics in human lymphocyte subpopulations measured by flow 19. Hajdinjak T. UroVysion FISH test for detecting urothelial cancers:
cytometry. Nat Biotechnol. 1998;16:743. meta-analysis of diagnostic accuracy and comparison with urinary
cytology testing. Urol Oncol. 2008;26:646–51.
Circulating DNA Quantification
30
Min Hu and Zeyou Wang
Circulating DNA, also named cell-free DNA (cfDNA), is a unteers (approximately 10–15 ng per milliliter, on average)
series of highly fragmented DNA that is present in the blood [6]. But some events can increase the levels in patients, such
circulation and other body fluids of human and is free of as exercise, inflammation, myocardial infarction, or tissue
extracellular. In certain situations, such as cancer patients, injury [7–10]. Up until now, the most widespread transla-
pregnant women, patients undergoing organ transplants, etc., tional researches about circulating DNA focus on trauma
a small number of circulating DNA from “heterologous” injury severity assessment, cancer diagnosis, and prenatal
cells can be used as markers for genetic testing [1]. Large assessment [11, 12].
quantity of autologous cell-free DNA reflects abnormal cell Circulating tumor DNA (ctDNA) is a group of circulating
death. Autologous cell-free DNA quantification can be used DNA deriving from tumor cells. In 1977, Leon SA observed
for the diagnosis and monitoring of related diseases. The that the levels of circulating DNA in serum of cancer patients
“liquid biopsy” technology based on circulating DNA detec- were elevated when compared with the healthy control group.
tion has received great attention in many aspects such as pre- The quantity of circulating DNA was much higher in those
natal diagnosis, tumor screening, early diagnosis, and with metastatic diseases and reduced after radiotherapy [6].
treatment monitoring, and prognosis evaluation. Because of Shapiro B’s study determined that elevated DNA levels act as
its extremely low content, a very large number of studies a useful diagnostic marker in couple with high circulating
have been done to develop various methods for quantitative carcinoembryonic antigen (CEA) in gastrointestinal tract
detection [1, 2]. At the moment circulating DNA quantitative cancer [13]. In 1989, Stroun M and colleagues showed that
detection still faces challenges such as the lack of specifica- the circulating DNA collected from the plasma of cancer
tion, precision, and standardization in detection processes. patients was identical to that in the corresponding cancer cells
[14]. The current research indicates that ctDNA released into
circulation comes from a wide variety of sources, including
30.1 Overview apoptosis, necrosis, lysis of circulating tumor cells, and active
secretion from the tumor [15]. Subsequently, it has been iden-
Circulating DNA is a group of double-stranded, highly frag- tified that the DNA mutations in ctDNA are associated with a
mented DNA that is free-floating and not within cells, which variety of cancer subtypes, as well as microsatellite altera-
can play a vital role in life. Mandel and Métais described it tions. DNA extraction and PCR techniques are two key
in blood plasma for the first time in 1948 [3]. This innovative aspects of ctDNA research. Now the importance of ctDNA
work is drawn little attention at the beginning. Until 1966, has been established in tumor diagnosis and treatment with
Tan and colleagues [4] demonstrated the presence of circu- the huge improvements of these technologies. Accumulating
lating DNA in patients with systemic lupus erythematosus. evidence is found that ctDNA carries specific mutations
Circulating DNA is 150–200 base pairs (bp) in length which may act as diagnostic markers. And ctDNA is becom-
approximately due to cell apoptosis or necrosis and it can be ing increasingly important in early diagnosis, the detection of
released into the bloodstream [5]. Circulating DNA can be minimal residual disease (MRD), and the assessment of ther-
found in blood but usually of small quantity in healthy vol- apeutic response and resistance [1, 2]. Furthermore, besides
its noninvasive nature, we begin to recognize these specific
M. Hu (*) · Z. Wang mutations carried by ctDNA from a blood sample test and
Department of Laboratory Medicine, The Second Xiangya establish personalized oncological therapies [16].
Hospital of Central South University, Changsha, Hunan, Circulating fetal DNA is cell-free fetal DNA (cffDNA) in
the blood of pregnant women. In 1997, Lo and colleagues
e-mail: huminjyk@csu.edu.cn

414 M. Hu and Z. Wang
first reported circulating fetal DNA which could be detected What is more, these new methods are more sensitive, more
in maternal circulation [17]. Since then, more and more practical, and easier for patients to accept.
researchers investigate the properties and potential utilities According to the present research, ethylenediaminetet-
of circulating fetal DNA. Approximately 10–15 percent of raacetic acid (EDTA) is the best preservative choice.
the circulating fetal DNA in maternal plasma originates from Circulating fetal DNA can be stored with high stability and
the placenta; the remainder derives from maternal hemato- quality in EDTA anticoagulated blood [26]. Besides, the
poietic cells undergoing apoptosis and, in obese women, other two crucial variables are the storage temperature and
necrosis and apoptosis of adipocytes and stromal cells [18]. the time-around-time. DNA extraction is performed within
Circulating fetal DNA fragments are approximately 200 bp 6 hours after blood collection. The blood samples can be pre-
in length. They are much smaller than maternal DNA frag- served at either 4 °C or room temperature. It will not influ-
ments [19]. The discovery of cffDNA is a milestone event, ence the final concentration of circulating DNA [27]. In
which opens up new horizons in blood-based noninvasive addition, ultra-low temperature freezer (−80 °C) can be used
prenatal testing (NIPT). It only takes blood from the preg- to store plasma samples with high stability and quality for
nant woman and will not cause any harm to the fetus. The two weeks. If circulating DNA is isolated from plasma, it can
chromosomal abnormalities in the fetus can be detected by be stored for a longer time at −80 °C (up to 3 months) [28].
testing the maternal blood. Since late 2011, circulating fetal
DNA-based noninvasive prenatal screening (NIPS) for fetal
aneuploidy has been widely carried out in clinical practice 30.2.2 Isolation of Circulating DNA
with an unprecedentedly rapid [20]. Compared to traditional
invasive tissue sampling methods, such approaches are more Because the density of circulating DNA in plasma is very low,
safe, comfortable, and acceptable. Thus far, NIPT has been it is a challenge to extract it. The key points for reproducible
applied for detecting chromosomal disorders due to an extra and accurate assays of circulating DNA are the amount and
or missing copy (aneuploidy) of a chromosome [21–24], quality. Scientists have established multiple techniques to iso-
such as fetal RhD blood group genotyping, fetal sex determi- late the circulating DNA, including laboratory- developed
nation to identify sex-linked disorders, and so on. In particu- methods and commercial kits. But we still need to explore
lar, more than 90 countries have performed NIPT for clinical more accurate, efficient, and convenient methods to isolate
service to analyze common chromosomal of pregnant women circulating DNA in blood serum or plasma, particularly with
worldwide [25]. small volumes. Unfortunately, there are still no standard
methods of separation or quantification of circulating DNA in
blood serum or plasma until now [29].
30.2 Basic Principle Circulating tumor DNA in the blood has two main charac-
teristics: one is the low levels, and another is the short half-
30.2.1 Sample Selection of Circulating DNA life. Scientists studying ctDNA must pay attention to how to
Testing keep ctDNA stable. Another key point is how to avoid the
potential for contamination with normal DNA from the lysis
Circulating tumor DNA is found both in plasma and serum of normal blood cells. To minimize these effects, appropria-
of cancer patients, and has been assessed both in them. tive phlebotomy tubes are used to collect blood for ctDNA
Different from ctDNA, circulating fetal DNA testing focuses isolation. Then centrifugation and separation of plasma must
on plasma for consistent collection. In general, circulating typically be performed as quickly as possible after collection
DNA is stable at room temperature. It can be preserved for (within 1–4 h). The concentration and purity of ctDNA could
3 days (up to 72 h) before extraction. Moreover, the antico- be different and fluctuant during processing times, which
agulant is used for the preservation of whole blood, it can cause the potential for pre-analytic variability. So rapid pro-
prevent blood clots. So, plasma has less circulating DNA cessing is an enormous challenge for ctDNA research [27,
compared with serum. On the other hand, blood coagulation 30]. Fortunately, Scientists have found some ways to cope.
can cause trapped leukocytes to release DNA in serum. The Special fixatives have been found and applied to stabilize
researchers should pay more attention to this part of circulat- both circulating fetal DNA and intact cells. They are added
ing DNA which can interfere in circulating DNA detection. to the collection tubes and will stable and prolong the half-
As a result, centrifugation and filtration of contaminant com- life of ctDNA. ctDNA remains relatively stable as long as
ponents, such as white blood cells and circulating tumor 14 days at room temperature after separation from the body.
cells, are beneficial and necessary before cryopreservation. It is helpful and valuable for shipping, storage, and batched
Compared to other early detection methods which require or centralized processing [31].
large amounts of blood (up to 10 mL), current ctDNA assays The isolation of circulating fetal DNA is even more com-
require little volumes of plasma or serum (less than 2 mL). plicated. In fact, a large proportion of fetal DNA originates
30 Circulating DNA Quantification 415
from the mother. On behalf of only a small portion of the or specially-treated polypropylene plastic-ware. Available
total circulating DNA in plasma of the pregnant woman, data suggest that they are suitable for DNA storage, and they
Circulating fetal DNA is from the fetus. Several approaches can maximize nucleic acid recovery during preanalytical
for enrichment circulating fetal DNA have been suggested, stages [39].
and separating circulating fetal DNA by size is the most
obvious approach [32]. Some other approaches also are 30.2.3.2 Q uantitative Polymerase Chain
explored to enrich the circulating fetal DNA and showed Reaction, PCR
some success, such as enrichment by the different methyla- Polymerase chain reaction (PCR) is a classical method
tion pattern or using gel size fractionation [33, 34]. widely used in molecular biology and clinical molecular
diagnosis. We can get many copies of a specific DNA seg-
ment using PCR. With this technology or approach, a single
30.2.3 Analysis Platform of Circulating DNA copy (or more) of a specific DNA sequence can generate mil-
lions of copies. Now it is a common and often indispensable
Owing to the low level of circulating DNA in body fluids, technique. You can see it everywhere in medical laboratories
detection of individual point mutations of circulating DNA is and clinical laboratory researches.
even more complicated. Scientists have explored several The basic principle of PCR relies on thermal cycling. It
techniques for the detection of these low abundance individ- involves the primer-mediated enzymatic amplification of
ual point mutations, based on polymerase chain reaction DNA. Primers and DNA polymerase are the two main
(PCR) analysis. BEAMing (beads, emulsion, amplification, reagents of the PCR reaction. The first step of PCR is DNA
and magnetics) and droplet digital PCR (ddPCR) analysis melting. The DNA double helix is physically separated into
are two familiar and widely used mutation-specific tech- two single strands at a high temperature. In the second step,
niques. With the assistance of these technologies, the altera- when the temperature drops, the primers can bind to their
tions in circulating DNA that present at allele frequencies of target DNA sequences. The two single strands become tem-
0.01% or even more less can be identification and quantifica- plates. A new DNA strand is assembled and elongated in the
tion [35]. 5′ to 3′ direction by DNA polymerase using the template and
free nucleotides. As the PCR reaction proceeding, it gener-
30.2.3.1 Massive Parallel Sequencing ates a series of DNA products. Then new DNA products can
Massive parallel sequencing is also known as second- be used as template for replication in the next PCR cycle. As
generation sequencing. And it is often referred to next- a result, the original DNA template is exponentially
generation sequencing (NGS). Because of sequencing DNA amplified.
with the concept of massively parallel processing, it is a A real-time polymerase chain reaction (Real-Time PCR)
high-throughput approach. It has dominated the field of is widely used in scientific research and clinical molecular
DNA sequencing since its development. diagnosis. It is a laboratory technique based on PCR, also
Several NGS platforms are commercially available for the known as quantitative polymerase chain reaction (qPCR).
researcher to sequence DNA now. The processes of different The conventional PCR monitors the amplification of the tar-
types of platforms for DNA sequencing are various. They are get DNA sequences only when the reactions stop, while
generally conducted with the following steps [36]. First, qPCR can monitor the amplification reaction in real time
clonal amplification generates the DNA sequencing libraries during the PCR progresses. There are two common methods
using PCR. Second, it is to sequence the DNA by synthesis. in clinical and scientific research for monitoring the amplifi-
Next, it is the sequencing step. NGS sequencing need not cation of the target DNA sequences in qPCR. One method is
separate and purify the DNA templates. The platform can to use non-specific fluorescent dyes. These fluorescent dyes
segregate, amplify, and sequence DNA templates simultane- can intercalate with any double-stranded DNA, target DNA
ously in a massively parallel fashion. sequence, or non-specific amplification products. With the
The most common, fastest, and labor-saving method to progress of amplification of Real-Time PCR, all the double-
analyze circulating DNA is NGS. It can analyze the whole- stranded DNA sequences are bind by the dyes. Along with
genome or whole-exome of circulating DNA. It can also ana- the increase of DNA products, the fluorescence of the dyes
lyze the targeted sequencing of a limited gene [37]. increases. Then we measure the intensity of the fluorescence
Circulating DNA can be stored for extended periods of time to a specific point in time of each cycle during PCR. DNA
after extraction at an ultra-low temperature refrigerator synthesis can be monitored as an increase in fluorescent sig-
(−80 °C). And multiple freeze-thaw cycles should be nal. Another is to use sequence-specific DNA probes, which
avoided, which will cause circulating DNA fragmentation only detects the target DNA products. The sequence-specific
[28, 38]. Additionally, scientists have made some attempts to DNA probe is a fragment of DNA that contains a nucleotide
avoid circulating DNA fragmentation by using polyallomer sequence specific for the gene or chromosomal region of
interest. A fluorescent reporter labels this nucleotide (C) Duplex real-time PCR amplification
sequence. When the probe hybridized with the targeted DNA ( D) Quantification analysis and calculation
sequence, the fluorescence of the probes can be detected.
Therefore, the specificity of this method is significantly
higher than the method using non-specific fluorescent dyes. Application Examples
Owing to the use of sequence-specific DNA probes, the PCR
reaction can perform well even in the presence of other non- (A) Plasma DNA Quantification:
specific double-stranded DNA. Duplex real-time quantitative PCR with a 50 μL reac-
tion volume is carried out using the Taq R-PCR system
(Takara). PCR condition: 95 °C for 5 min (1 cycle);
30.3 Technology Development 95 °C for 15 s, 56 °C for 30 s (total 50 cycles); 72 °C for
40 s (1 cycle). After amplification, set the baseline value,
In recent years, there are a number of methods used to inter- analyze with linear regression, and export normalized
rogate circulating DNA. These methods can be broadly sepa- reporter signals (Rn) data. They can obtain the average
rated into two basically distinct categories: candidate gene amplification efficiencies of the targeted gene and inter-
analysis approaches and deep sequencing analysis nal standard. The amplification plots of the β-actin gene
approaches. The sensitivity of these candidate gene analysis and internal standard all use the same threshold. The
approaches is similar. They are generally more economical plasma DNA concentrations (copies/mL) are calculated
and do not rely on bioinformatics analysis. However, such by using 3.3 pg of single-copy human genomic DNA as
approaches can only identify the known mutations. The a conversion factor with the equation, as follows:
throughput of these detection approaches is very low.
1 EIS
Ct IS
Correspondingly, deep sequencing analysis approaches are C CIS

1 E
Ct
high-throughput and enable monitoring of unknown muta-
tions. But they are usually expensive. The resulting data
should be analyzed by bioinformatics. And it is the most C: β-actin gene concentration in plasma (copies/
effective analysis approach to discover a candidate gene that mL);
can easily be applied to clinical practice. E: average amplification efficiency;
β: the β-actin gene;
IS: the internal standard.
30.3.1 Duplex Real-Time PCR for Total Plasma (B) Sampling: Use EDTA-K2 anticoagulation vacuum blood
DNA Quantitation collection tube to collect 2 mL of peripheral venous
blood. Extract plasma circulating cell-free DNA imme-
Accumulating evidence suggests that the concentration of diately or place the blood samples at room temperature
circulating DNA varies with the physical condition [40–42]. for no more than 12 h or at 2–8 °C for no more than
However, the quantitative analysis of circulating DNA in dif- 48 hours. Hemolysis will affect the test results, and
ferent laboratories varied significantly. It becomes a consid- blood samples should be re-collected.
erable pitfall and hampers its clinical application. Here we (C) Plasma Separation: At room temperature, centrifuge
describe a newly developed duplex real-time PCR assay for peripheral venous blood samples were collected by
circulating DNA quantitation, which was used to test 200 EDTA-K2 anticoagulation vacuum blood collection tube
trauma patients and 5442 healthy control individuals [43]. at 1600 g for 10 min. Pipette 400 μL of upper plasma
Human β-actin gene is selected as the amplification target carefully (please do not touch middle white blood cells),
region, and a synthetic DNA fragment designed by ourselves and transfer to another 1.5 mL of DNase-free microcen-
that has no homology to the human genome is used as the trifuge tube. After centrifuge (4 °C, 16,000 g, 10 min),
internal standard. In prior to amplification, internal standard pipette 195 μL of upper plasma (please do not touch
with setting quantity is added into human plasma samples bottom blood cells) and transfer to a new 1.5 mL DNase-
which will be processed and amplified synchronously with free microcentrifuge tube.
cfDNA, and then use the internal standard as reference to (D) Add Internal Standard: At room temperature, take out
calculate the content of cfDNA in measured plasma internal standard, after thaw, vortex 10 s, and then quick
[40–44]. spin. Add 5 μL of internal standard for each sample or
quality control.
Experimental Process (E) Plasma Preservation: Isolated plasma sample which
cannot be extracted immediately can be stored at 2–8 °C
( A) Plasma DNA quantification within 7 days or long-term preservation below −20 °C,
(B) Plasma DNA extraction avoid repeated freezing and thawing.
(F) Circulating DNA Extraction: The commonly used stringent conditions. So, it is successful to amplify the spe-
nucleic acid extraction kit can be used, and the verified cific SNP in the DNA sequence with the presence of an
QIAamp DNA Blood Mini Kit (product No.: 51104) is SNP-specific primer.
recommended.
(G) Real-Time PCR Amplification: 95 °C for 10 min
30.3.2.2 Competitive Allele-Specific TaqMan
(1 cycle); 94 °C for 30 s, 56 °C for 30 s, 72 °C for 40 s PCR (CAST PCR)
(total 40 cycles). Competitive allele-specific TaqMan PCR referred to as
(H) Quantification Analysis and Calculation: CAST PCR, is a modification of AS PCR. It is a TaqMan
a. If the PCR reaction amplification curve shows S type probe-based assay. In the same PCR reaction, it contains a
in both FAM and JOE channels, and Ct (Cp) value in mutant allele-specific primer (ASP) and a wild-type allele-
the FAM channel is ≤38, then the test result of the specific blocker (ASB). The amplification of the wild-type
clinical sample can be calculated. allele is completely suppressed by ASB, while the ASP can
Calculate the content of cfDNA (ng/mL) accord- bind sequence in the mutant-type allele to initiate the PCR
ing to the formula below: amplification. So that the amplification of the mutant allele
cfDNA concentration (ng/mL) = 825 × 1.9s ÷ 2t will not be interfered. The CAST PCR can greatly improve
Note: s is the Ct (Cp) value in the FAM channel, the sensitivity and specificity of the amplification.
and t is the Ct (Cp) value in JOE channel.
b. Other situations that may be encountered: Experimental Process
There is S type amplification curve in the FAM
channel, and Ct (Cp) value in FAM channel is ≤38: ( A) DNA extraction and purification
If the test sample dose has not S type amplification (B) PCR amplification
curve or Ct (Cp) value in the JOE channel, it indi- (C) Data collection and analyzation
cates that there are errors in the sample processing or
sample process, and it is necessary to resampling,
pretreatment, and testing. Application Examples
There is S type amplification curve in the FAM
channel, but Ct (Cp) value in the FAM channel is ( A) Extract DNA with phenol chloroform.
≤38, or it does not show S type amplification curve (B) Purify DNA using GENECLEAN® II kit (Vista).
in the FAM channel: Whether the JOE channel has (C) PCR amplification: add 10 ng of DNA samples into the
an amplification curve or not, it indicates that there reaction well, and carry out the reaction in ABI PRISM
are errors in the process of adding internal standard, 7900HT Sequence Detection System (Life
sample pretreatment or adding the sample. It is nec- Technologies). The PCR condition: 95 °C for 10 min
essary to resampling, pretreatment, and testing. (1 cycle); 95 °C for 15 s, 58 °C for 1 min (total 5 cycles);
Reference interval: Male: ≤50.0 ng/mL, Female: 95 °C for 15 s, 60 °C for 1 min (total 40 cycles).
≤ 45.0 ng/mL. (D) Collect the real-time data and analyzed using the

software.
30.3.2 Candidate Gene Approach Results: (Fig. 30.1)
30.3.2.1 Allele-Specific PCR (AS PCR) 30.3.2.3 Coamplification at Lower

Allele-specific PCR (AS PCR) technique, also referred to Denaturation Temperature PCR
“PCR allele-specific amplification” (PASA) or “amplifica- (COLD-PCR)
tion refractory mutation system PCR (ARMS PCR), is a Coamplification at lower denaturation temperature PCR
convenient and low-cost approach (Fig. 30.1). It is com- (COLD-PCR) is a candidate PCR approach to gene analysis.
monly applied for the speedy detection of mutation in clin- Circulating DNA is a mixture, and consists of wild-type and
ical specimens. This is a two-step nested PCR. It is based mutation sequences. When performing COLD-PCR, we
on the observation that Taq polymerase will extend prim- preferentially enrich “minority alleles” from circulating
ers with a 3′ mismatch less efficiently than perfectly DNA, irrespective of where an unknown mutation lies
matched primers [45]. By choosing a primer that is com- (Fig. 30.2). Consequently, the PCR products of COLD-PCR
plementary at its 3′ end to a point mutation, selective amplificated from the circulating DNA contain high percent-
amplification of a mutant allele can be achieved. When a ages of variant alleles.
mismatch between the template and primer is present, Each DNA sequence has different melting temperature
PCR amplification will be much less efficient under such (Tm). This temperature Tm is dependent on the DNA
a b
Fig. 30.1 One example of CAST PCR
167 bp TP53 exon 8 sequence

a REGULAR PCR COLD-PCR b
(94 ºC denaturation) (denaturation at Tc = 86.5 ºC)
Mutation not visible
Lung tumor 64 Lung tumor 64
80 90
Reverse seq. No mutation visible
A C A A A C A N G C A C C T C A A A REGULAR PCR
Reverse seq. G→A mutation visible
TP53 exon 8
70 80 Independent verification Lung tumor 64 A C A G C T G A G T G C G A G T
T T T G A G G T G C G T G T T T
Forward seq. No mutation visible via RFLP G→A mutation verified
Reverse seq.
Codon 273 TP53 mutation
Mutation clearly visible
Colon tumor 20 Colon tumor 20 G→A mutation visible
No mutation visible
COLD-PCR
Reverse seq. Independent verification

via RFLP G→A mutation verified A C A G C T G A G T G C G A G T
Fig. 30.2 One example of COLD-PCR
sequence itself. During COLD-PCR, another temperature, (C) PCR amplification

the critical denaturation temperature (Tc), for each DNA (D) Downstream assays
sequence could be determined. The temperature Tc is a lower
temperature than the melting temperature (Tm) of the target Application Examples
sequence. When the temperature is below, PCR efficiency
drops abruptly. When the PCR denaturation temperature is (A) Extract plasma circulating DNA with the DNeasy Tissue
set to the temperature Tc, each amplicon has substantially Kit (Qiagen).
different amplification efficiencies owing to the single nucle- (B) Critical denaturation temperature identification.
otide variation. As a result, we can enrich mutations at any (C) COLD-PCR amplification: First amplify larger DNA
position along the sequence during the amplification, with fragments. Then perform and monitor COLD-PCR in
the help of fixing the denaturation temperature to Tc. real time using Smart Cycler I machine (Cepheid).
(D) Downstream assays: COLD-PCR and regular PCR

Experimental Process amplicons sequence analysis: MALDI-TOF genotyp-
ing, pyrosequencing, Sanger sequencing are performed
( A) DNA extraction and purification to analyze.

(B) Primer design and critical denaturation temperature
identification Results: (Fig. 30.2)
30.3.2.4 P eptide Nucleic Acid-Locked Nucleic designed in the PCR system. The microfluidic system can
Acid PCR (PNA-LNA PCR) completely separate the DNA molecules in the compartment.
Peptide nucleic acid-locked nucleic acid PCR (PNA-LNA To perform thousands of reactions simultaneously, the reac-
PCR) is a fast and sensitive method to detect DNA mutation volume for each compartment is reduced. Normally it is
tions. The presence of wild-type sequences (to 1000) as a nano/picolitre-scale. Each compartment is an individual
background does not interfere with the detection of DNA reaction chamber. It can avoid cross-contamination between
mutations. PNA clamping PCR is a simple and high sensitiv- neighboring compartments. This series of measures is taken
ity method. Owing to the independent of fluorescent probes, to ensure accurate quantification of targets in each sample.
it can detect the low-frequency mutations with high cost-
effectiveness. In the PNA/LNA PCR system, a specific 30.3.2.7 “ Beads, Emulsion, Amplification,
sequence is used to block the wild-type allele amplification. Magnetics Digital PCR” (BEAMing)
This specific sequence can improve the detection of low “Beads, Emulsion, Amplification, Magnetics digital PCR”
abundance mutations. There are mismatches between (BEAMing) is an alternative approach with strong sensitivity,
mutant-type DNA and wild-type DNA. These mismatches specificity, and reproducibility. The provided frequency from
can change the melting temperature. Even a single base mis- molecular information about mutations is 1 over 10,000.
match is enough to discriminate amplification. As a result, BEAMing is built upon ddPCR. But the mutations of target
the single nucleotide polymorphisms or mutations can be DNA copies are detected and quantified by flow cytometry
identified by melting curve analysis. When point mutations [48]. According to different studying objects and purposes,
or small deletions at fixed positions are known and needed to gene regions of interest are analyzed and chosen. Then the
be detected sensitively in a short time, this method is appli- primers targeted these gene regions are designed and used to
cable to detect these mutations. perform BEAMing after circulating DNA is extracted. After
the amplification step, a magnet is used to separate the DNA-
30.3.2.5 Droplet Digital PCR (ddPCR) bound beads. The single DNA molecule copies are coated on
Droplet digital PCR (ddPCR) is the most common digital the surface of beads by thousands. Next fluorescent oligonucle-
PCR method (Fig. 30.3) [46]. It can discretize DNA template otides are specifically denatured and hybridized with the DNA
in single emulsion droplets and amplify the template indepen- on the beads. Then flow cytometry or optical scanning instru-
dently. The water–oil emulsion technique divides PCR solu- ments is used to analyze them. The DNA isolated can also be
tion into smaller reactions. In each droplet, the individual used for further sequencing analysis or other studies [49, 50].
PCR occurs with selected primers on regions of circulating
DNA. Each 20 μL sample can generate approximately 20,000
oil droplets. Droplet digital PCR allows detection of allele 30.3.3 Whole-Genome Sequencing Methods
and mutant frequencies in circulating DNA with high sensi-
tivity, specificity, and detection efficiency. Compared to other Whole-genome or exome sequencing typically is performed
methods, much more fluorescent probes (up to 5 probes) can by high-throughput DNA sequencing technologies. The new
be used in one assay, which is limited to the ddPCR method. mutations in circulating DNA may be discovered using
The sensitivity of the assay, around 1 in 10,000, can be varied whole-genome or exome sequencing [51]. Particularly
depending on the amount of DNA analyzed. whole-exome sequencing only can identify variants that
exist in the exome. These variants which are located at the
30.3.2.6 Microfluidic Digital PCR coding region of the gene can influence protein function.
Microfluidic digital PCR is one type of digital PCR based on Whole-exome sequencing can only cover about 1% of the
the microfluidic system [47]. A mass of compartments is human genome, and 99% of which remains uncovering. This
Fig. 30.3 Digital Droplet PCR

Fig. 30.4 Tagged AMplicon

a b
deep Sequencing
Pool and sequence
99% of the human genome is the non-coding regulatory sequencing is performed to generate up to 30 million reads
regions. So whole-exome sequencing loses the information per lane. This technique has been successfully applied to
about mutations in these non-coding regions. On the other molecular diagnosis in clinical practice. Circulating DNAs
hand, it can decrease expense and increase the speed of of advanced cancer patients are extracted and successfully
genome sequencing analysis [52]. Whole-genome sequenc- identified mutations scattered in the TP53 gene which is the
ing is a full genomes-sequenced technology with the high most eye-catching tumor suppressor gene. TAM-Seq may be
cost and long time. Presently, it is rarely practiced in the widely applied in clinic fields with promising applications in
clinical context. Remarkably, they play an important role in the future (Fig. 30.4).
discovering the initial mutation, which can subsequently be
used for clinical diagnosis and treatment of the disease. 30.3.4.2 Safe-Sequencing System (Safe-SeqS)
Safe-Sequencing System (Safe-SeqS) is an approach for
identifying rare variants. It is able to substantially enhance
30.3.4 Targeted Deep Sequencing the sensitivity of massively parallel sequencing [54]. It
achieves this as follows. First, each DNA template is added
30.3.4.1 T agged AMplicon Deep Sequencing with a unique identifier (UID) sequence. Second, the added
(TAM-Seq) UIDs are used to amplify the DNA, and then to sequence the
Tagged AMplicon deep Sequencing (TAM-Seq) allows to DNA. It can generate many daughter DNA molecules. These
amplify and sequence of circulating DNA. The length of tar- daughter DNA molecules belong to a UID family. They are
geted regions can be from single copies to large genomic. all labeled with the identical sequence. And the mutation
The low-level mutations in the plasma of high-grade carci- preexisted in the template molecule can be present in every
noma patients can be identified by TAM-Seq [53]. At the daughter DNA molecule. The amplicon is analyzed to find
beginning, the genome regions of interest are analyzed. the “super-mutant,” which is considered as if 95% of the
These regions tile in short segments (from 150 to 200 bases), sequenced reads are in agreement. What need to remind is,
and primers targeted these regions are designed and used to new mutations can be introduced during the amplification, or
amplify the copies of these regions. This is a general ampli- by incorrect base assignments. These mutations not occur-
fication step. Then, each amplicon is attached to adaptors ring in the original templates, do not give rise to super-
with a unique identifier with the help of microfluidics sys- mutants. As a result, the true mutations of the circulating
tem. And the DNAs are further amplified in parallel single- tumor with the UID will be separated from these methodol-
plex reactions. The barcode sequencing primers are used to ogy errors. The sensitivity of Safe-SeqS is 9 molecules in
amplify an additional 10 cycles. At last, 100-base single-end 1,000,000 [55] (Fig. 30.5).
30.3.4.3 Duplex Sequencing

Duplex Sequencing (DS) is another common next-
generation sequencing technology. This technique is an
improvement by the Safe-Seq, in which the single UIDs are
added. A single mutation can be detected among 10,000,000
wild-type DNA sequence reads [56]. Thereby DS is exten-
sively applied in accessing very-low-frequency and rare
genetic variants and heterogeneous populations. In duplex
sequencing, each randomized fragmented DNA molecules
are labeled by degenerate molecular tags. Then they are
attached to an invariant spacer sequence. The adapters of the
sample DNA are ligated to α and β tags which are all a
unique 12 nucleotides tag sequence. These series of opera-
tions result in two unique templates on both ends of the mol-
ecule for PCR. There is α tag on the 5′ end of one template,
and a β tag on the 3′ end. Different from this template, the
other is marked with a β tag on the 5′ end and α tag on the 3′
end. Then primers targeted these invariant nucleic acid tags
are used to perform PCR for amplification. PCR products
are sequenced and analyzed. If and only the mutations are
presented at the same position in both these two types of
DNA products, they will be considered as true mutation and
scored. If not, those will be the false mutation and not
scored. The sensitivity of DS to discover mutants is 1 mol-
Fig. 30.5 Safe-Sequencing System ecule in 10,000,000 (Fig. 30.6).
a c
Fig. 30.6 Duplex sequencing

30.3.4.4 C ancer Personalized Profiling by Deep may up to months [61]. What is more important, the level of
Sequencing (CAPP-Seq) blood ctDNA is lower in patients who have non-metastatic
Circulating tumor DNA (ctDNA) harbors the mutations of tumors. It is much higher in patients with metastatic tumors.
the original tumor and has cast new lights on a promising When ctDNA reemerges or increases significantly in plasma
biomarker finding in cancer liquid biopsy. Among existing of cancer patients during drug therapy, it strongly represents
ctDNA detection methods in clinical practice and research, radiological/clinical progress [10, 53, 57]. The improved
CAPP-Seq is one of the most ultrasensitive and economical clinical condition and a better therapeutic can be indicated
methods for the circulating DNA quantification of cancer by a reduction of the amount of ctDNA. Further, ctDNA lev-
patients [57]. It is commercially available off-the-shelf to els are much higher in patients expecting a relapse than those
most cancer patients of the given type. CAPP-Seq can exam- who are disease-free [62]. The outcome of these clinical tri-
ine all major types of mutations, concerning single nucleo- als has shown the future of circulating tumor DNA as a mol-
tide variants, small insertion and/or deletion of nucleotides, ecule biomarker for the assessment of disease progression in
rearrangements of sequences, and copy number variations, clinical practice. And circulating DNA quantification has
and so on [57]. great potential prognostic value in the management of the
First of all, according to the publicly available cancer disease.
databases, a multi-phase bioinformatics is used to analyze
and obtain recurrent mutations in cancer, then to design and
construct a universal probe library against these mutations. 30.4.2 Diagnosis Value
Then probes labeled by biotinylated oligonucleotide are used
to detect the DNA relevant to ctDNA by targeting the ctDNA Detection of circulating DNA from blood (plasma/serum) in
sequences [57]. The level of observed ctDNA is low. They the patients of cancer is an important component of cancer
are enriched with the optimized protocol. Then deep sequenc- precision medicine. Blood-based circulating DNA quantifica-
ing is performed to sequence the isolated DNA with increased tion offers an easily handleable and noninvasive way for early
sensitivity. CAPP-Seq allows detecting hundreds of DNA diagnosis and molecular typing of the disease. In human
regions simultaneously. With the present study, the sensitiv- malignancies, the concentration of circulating DNA appears
ity of CAPP-Seq is 2.5 molecules in 1,000,000 [58]. to increase as the disease progresses [63]. Circulating DNA
quantification can be used for the early diagnosis of the pri-
mary disease. While very little circulating DNA can be
30.4 Clinical Application detected in the early stage of human malignancies, unlike in
the late stage. Initial studies have been performed to explore
Numerous studies and trials have demonstrated that circulat- circulating DNA quantification in the early cancer diagnosis
ing DNA is involved in multiple stages of clinical practice, or early detection of the primary disease. Gormally E and col-
including disease diagnosis and prognosis, detection and leagues performed a particular prospective study, in which
identification of resistance mechanisms at disease relapse, they detected circulating DNA mutations in the plasma of
detection of minimal residual disease (MRD), noninvasive healthy individuals and performed continued follow-up for
prenatal testing (NIPT), injury assessment and so on. With up to 100.8 months. They found that the healthy individuals
the development of research, the clinical application of cir- who carried KRAS2 and P53 mutations had a higher risk of
culating DNA quantification will be more and more developing clinically detectable bladder cancer within 6 years
extensive. [64]. In 2014, Bettegowda and colleagues detected circulat-
ing tumor DNA by ddPCR in numerous early and late stage
malignancies [63]. As shown in this study, the detection rate
30.4.1 Prognostic Value in localized colorectal cancer (73% of patients) is much
higher than that in the patients with localized gastroesopha-
Circulating DNA represents a promising molecular marker geal (57%), pancreatic (48%), and breast cancer (50%)
in predicting disease prognosis and clinical treatment respectively. These data provided some impetus for the early
response. The abundance of circulating tumor DNA is diagnosis of colorectal cancer using circulating tumor
dynamic changes in patients with cancer. The proportion of DNA. Cohen and colleagues [65] reported a blood test, called
ctDNA correlates with clinicopathological characteristics of CancerSEEK, with the ability of detecting eight common
tumor such as tumor stage, tumor volume, and, indirectly, kinds of cancer simultaneously. The evaluation indicators of
with time to progression after chemotherapy and burden [59, CancerSEEK are the levels of circulating proteins and muta-
60]. Currently, accumulating evidence has shown that the tions in circulating tumor DNA. CancerSEEK tests are posi-
early changes of circulating tumor DNA levels can predict tive with a median of 70% in these eight common kinds of
progression prior to radiographic progression. The lead time cancer. The specificity of CancerSEEK is over 99%. The sen-
sitivity of CancerSEEK ranges from 69 to 98%. While circu- cholangiocarcinoma (ICC) is performed [79]. All the FGFR2
lating DNA-based early diagnosis for cancer may not yet get mutations found in individual resistant clones can lead to
ready for clinical application, it still gives us great hope for BGJ398 resistance. It strongly suggests that the FGFR path-
early cancer diagnosis. On the other hand, circulating DNA way probably is a rational therapeutic target with a great
quantification also can be used to stratify the molecular clas- development prospect. Clinical adoption of circulating DNA
sification of cancer in the late stage and guide therapy, includ- quantification requires clear and actionable mechanisms of
ing targeting therapy, adjuvant therapy [66], and endocrine resistance. Timely identification of resistance mechanisms
therapy [67]. While it is difficult to obtain biopsy samples to allows earlier clinical intervention.
detect EGFR hotspot mutations from NSCLC [68], the circu-
lating DNA quantification is a good choice. Studies have con-
firmed that tumor and plasma mutations have high correlation 30.4.4 Detecting Minimal Residual Disease
[69, 70]. And it will accelerate the clinical implementation of
circulating DNA testing. Recurrence of a disease is a thing that makes people worry
much, especially for the patients who have done the radical
operation or got complete remission. The most common rea-
30.4.3 Detecting Resistance Mechanisms son for recurrence is the presence of previously undetected
MRD [80]. The highly sensitive and specificity methods
Cancer remains one of the most intractable medical prob- described above for circulating DNA quantification have
lems for a long time in the world. Cancer recurrence and also been explored to detect MRD. Recently, extensive stud-
therapeutic resistance are currently the most important chal- ies of circulating DNA indicate that circulating DNA can
lenges in cancer treatment [71]. Our deficient knowledge on serve as a prognostic marker for MRD in multiple cancers
drug resistance mechanism is one of the prime reasons for [81–83]. It shows a high predictive power for the early detec-
treatment failure. Furthermore, accumulating evidence sug- tion of recurrence. In a prospective clinical trial of NSCLC,
gests that genetic mutation is closely related to drug resis- circulating DNA quantification using patient-specific NGS
tance, and circulating DNA quantification can be used to analysis is performed for MRD prediction. Compared with
screen the known mutations [71–73], search for novel muta- computed tomography (CT) imaging, ctDNA detection is a
tions [74], and monitor the acquisition of resistance [71–74]. new approach with high sensitivity (92.3%) and specificity
Because of its noninvasiveness, serial sampling can be per- (100%), with a median lead time of 70 days [11]. What is
formed in real time. It increases the chance to identify resis- more, ctDNA-based detection of metastasis precedes clinical
tant clones ahead of clinical progression [75, 76]. Compared detection with an average lead time of 11 months in 86% of
with invasive tissue biopsy, the results of screening the muta- patients. On the other hand, ctDNA is undetectable in post-
tions with circulating DNA are similar to that of invasive tis- operative patients in a permanent disease-free circumstance
sue biopsy [75, 76]. Increasing tumor patients benefit from [84]. Tie and colleagues [78] carry out a research to assess
it. Cetuximab is the first-line agent for anti-EGFR treatment the ability of ctDNA for the detection of MRD using NGS-
in patients with colorectal cancer. Recently, increasing evi- based assays. They analyze 1046 plasma samples separated
dence suggests KRAS mutation is causally associated with from 230 patients with resected stage II colon cancer. They
cetuximab resistance acquirement. It is necessary to rou- find that ctDNA performs better than carcinoembryonic anti-
tinely monitor KRAS mutations in circulating DNA for the gen (CEA) for early prediction of recurrence. Another
cetuximab-treated patients. It is up to 10 months ahead of research in diffuse large B-cell lymphoma reaches a similar
radiographic documentation of disease progression [77]. In conclusion. Compare with clinical evidence, monitoring
metastatic breast cancer (MBC), Estrogen receptor α (ESR1) circulating tumor DNA had confirmed the risk of recurrence
mutations play an important role in promoting ligand- at a median of 3.5 months, with desirable a positive predic-
independent receptor activation and acquiring estrogen- tive value of 88.2% and a negative predictive value of 97.8%
deprivation therapy resistance. ESR1 mutations (Y537S and [85]. Notably, ctDNA analysis can predict clinical relapse
D538G) are common in the circulating DNA in the MBC and poor outcomes in pancreatic cancer patients after resec-
patients treated with ER-positive aromatase inhibitor [78]. In tion desirably. It is earlier than CT imaging with a lead time
recurrent high-grade serous ovarian cancer (HGSC), rever- of 6.5 months [86]. However, varying platforms are used in
sion of BRCA1/2 germline mutations can be tested by an circulating DNA analysis for the detection of MRD, includ-
unbiased analysis of circulating DNA [71]. This suggests ing targeted NGS, low-coverage WGS, digital PCR, and so
that screening circulating DNA for BRCA1/2 germline on. The standardization is lagging, making clinical applica-
mutations can induce momentous clinical value in predicting tion full of challenges. With increasing sensitivity and com-
chemotherapy response [71]. Serial analysis of circulating prehensive standardization, broader panels are available for
DNA in patients with FGFR2 fusion-positive intrahepatic clinical implementation in the near future.
30.4.5 Noninvasive Prenatal Testing (NIPT) levels in patients with organ injury are much higher than that
in the healthy controls in the early stage of injury, with
Due to the sequence analysis of circulating DNA in maternal median level higher up to five times. In addition, circulating
blood, along with the translation of this method into screen- cell-free DNA quantification could be used in cancer detec-
ing for fetal chromosome abnormalities, noninvasive prena- tion, monitoring, and prognosis.
tal testing (NIPT) is becoming more promising [87, 88]. Hepatitis B, a prevalent public health problem in China,
Especially in recent years, with the development of mas- can also be diagnosed and treated by circulating DNA
sively parallel sequencing, the analysis of prenatal circulat- quantification. Circulating DNA quantification combined
ing DNA has unprecedented sensitivity and precision. with other serum biomarkers have been used for liver
Prenatal circulating DNA testing has progressed from small, injury assessment in hepatitis B patients. It is able to
proof-of-principle studies to a global transformation of pre- increase the accuracy of assessing liver injury, which is
natal care. Until now, millions of pregnant women have essential for clinical evidence-based treatment. As shown
detected their circulating DNA to screen for fetal aneuploidy in the study, hepatitis B patients with higher degree of liver
[89]. In 1997, it was first reported Y chromosome-specific injury have a significantly higher level of circulating DNA,
DNA sequences could be identified in maternal plasma [17]. HBV DNA, serum alanine aminotransferase (ALT), aspar-
In 2011, NIPT using prenatal circulating DNA has become tate aminotransferase (AST), and bilirubin than the control
commercial. It provides noninvasive detection for common group. And these indexes are dramatically higher in the
aneuploidy. Since then, with the development of detection patients with marked severe inflammation, compared with
technology, the detection rate is much higher and the false- those with mild-moderate inflammation. There is a statisti-
positive rate lowers [90], particularly for down syndrome cally significant relevance between hepatocyte inflamma-
screening (99.7%) [91]. It can be done any time as early as tion severity and circulating DNA concentration. The
the 10th week of pregnancy [90]. As a result, NIPT is being combination of circulating DNA, serum ALT, and bilirubin
used more and more widely. While the detection rates for is regarded as a candidate promising liquid biopsy targets
other common aneuploidy (trisomy 18, 13, and monosomy for noninvasive liver injury assessment in hepatitis B
X) are a little lower (98.2, 99.0 and 95.8% respectively) [91, patients [44].
92]. Nothing is perfect, and NIPT is no exception. Currently,
it is not recommended for pregnant women to do aneuploidy
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DNA Methylation Detection Techniques
31
Shiyang Pan and Jiexin Zhang
Gene expression can be mediated by DNA methylation, 5-methyl-cytosine (5mC). Methyl restriction-based assays can
modifications of histones, and positioning of nucleosome be combined with enrichment methods to improve the detec-
along the DNA in which DNA methylation is intensely stud- tion of unmethylated cytosines. Enrichment-based methods
ied. There are many methods designed to be applicable. require 50–100 million sequence reads per sample. Two main
strategies for library construction have emerged for bisulfite
sequencing: (1) a genomic library with adapters added is sub-
31.1 Overview jected to bisulfite treatment; (2) bisulfite-converted genomic
DNA is subjected to library construction. For conversion
Conrad Waddington firstly used the term “epigenetics” in methods 1 billion 100 nt sequence reads are generated for each
1942 [1]. The modern definition of epigenetics is: modifica- sample and library construction methods introduce distinct
tions of DNA or associated factors that have information library-specific biases in genome coverage [22].
content, other than the DNA sequence itself, are maintained
during cell division, are influenced by the environment, and
cause stable changes in gene expression [2, 3]. 31.2.2 ChIP-Seq
DNA methylation was discovered in mammals in the
1940s [4, 5]. In the 1980s, it was demonstrated to regulate ChIP-seq is applied to detect genomic locations of modified
genes as well as cell function [6, 7]. DNA methylation typi- histones. Typically, 25–50 million immunoprecipitated frag-
cally involves covalent addition of a methyl group (–CH3) at ments are sequenced for a histone mark. Nucleosomes that
the 5′ position of the cytosine ring within the 5′-CpG- are released from chromatin are often associated with 100–
3′dinucleotides to create a 5-methylcytosine (5-mC) in 300 bp genomic fragments [22].
human. DNA methyltransferases (DNMTs), considered as
the “epigenetic writers,” catalyze this reaction with
S-adenosyl-methionine [8–11]. Most genes have short 31.2.3 Mapping Open Chromatin
(approximately 1 kb) CpG-rich regions in CpG islands (CGIs)
[12–16]. Promoters containing CGIs are DNA methylated Genomic DNA fragments sequencing is a method that can
[17–19] followed by gene expression regulation [20, 21]. locate genomic positions of open chromatin regions which
are released from chromatin either by transposon insertion or
enzymatic digestion. This protocol contains three main steps:
31.2 Basic Principle (1) chemically cross-links chromatin; (2) uses sonication to
release non-cross-linked regions; (3) fragments enrichment
31.2.1 DNA Enrichment Methods via phenol-chloroform extraction. Sequencing requirements
depend on experimental parameters and resolution require-
Enrichment methods distinguish oxidative derivatives (e.g., ments and range from 10s to 100s millions of fragments per
hmC) to provide qualitative measures of Genome-wide sample [22].
S. Pan (*) · J. Zhang

e-mail: sypan@njmu.edu.cn

428 S. Pan and J. Zhang
31.2.4 Chromosome Conformation Capture there are other principles of detection methods, such as the
simple TLC method and the Nearest Neighbor TLC, based
This technology provides the location of DNA fragments on anti-5mC immunological techniques, SssI methyl
that interact together due to their proximity positions in Acceptance Assay, on the basis of bisulfite treatment of
three-dimensional (3D) space. It typically requires 500 mil- Chloroacetaldehyde reaction method and Enzymatic
lion sequence reads to measure chromatin interactions Regional methylation Assay (ERMA). To notice, although
genome-wide in each experiment [22]. these methods can clearly detect the CpG methylation status
of target sequences, they fail to locate each CpG site.
31.3 Technology Development

31.3.2 Methylation Detection of Specific Sites
DNA methylation detection techniques can be divided into
two categories: methylation detection of specific sites and 31.3.2.1 Bisulfite Sequencing (BSP)
genome-wide methylation analysis, and the latter is also This method was used to be the gold standard for DNA meth-
known as methylation profiling. ylation analysis and was based on Methylation Specificity
PCR (MSP) to further study the methylation of CpG islands.
When PCR amplification (primers are designed to avoid
31.3.1 Genome-Wide Methylation Analysis CpG as much as possible to avoid the influence of methyla-
tion factors) is performed, uracil is completely converted to
31.3.1.1 High-performance Liquid thymine. Finally, PCR products are sequenced and compared
Chromatography with untreated sequences to determine whether CpG sites are
High-performance liquid chromatography (HPLC) is a rela- methylated.
tively traditional method for isolation of DNA or protein
according to their molecular weight and conformation, and 1. Experimental process:
for quantification according to the light absorption of mole- (A) DNA extraction.
cules in the dynamic phase and the static phase. As the pres- (B) Bisulfite treatment.
sure of the system increases, the resolution is optimized. (C) DNA purification.
Therefore, the whole level of DNA methylation can be mea- (D) DNA desulfonyl reaction.
sured quantitatively. This method was first reported in 1980. (E) PCR amplification.
The process is to hydrolyze the DNA sample into bases (F) PCR products recovery and purification after agarose
through hydrochloric acid or hydrofluoric acid, and the electrophoresis, followed by connection to plasmid
hydrolysis product is compared with the standard product vector for cloning and sequencing.
through chromatographic column. The absorption peak and 2. Application Examples:
its amount are determined by ultraviolet light, and the whole (A) Extract genomic DNA.
genome methylation level is calculated via the integral area (B) Prepare DNA with sodium bisulfate.
of 5 mC/(5 mC + 5 C). (C) Design BSP primers by the online MethPrimer
software.
31.3.1.2 High-performance Capillary (D) PCR amplification including genomic DNA, MSPF/
Electrophoresis MSPR primer, and enzymes.
It is applied for separating different chemical components (E) Subclone products.
from the complex by using a narrow pore fused silica capil- (F) Send positive clones for sequencing.
lary. It is based on the separation of different molecules due (G) Sequences analysis.
to their different charges, sizes, structures, and hydrophobic- 3. Deficiencies:
ity under strong electric fields. It is simple, economical, and (A) More workloads of cloning and sequencing resulting
sensitive to determine the level of 5mC by hydrolyzing DNA in high costs.
hydrolysates by High-Performance Capillary Electrophoresis (B) No information at a single molecular level.
(HPCE). (C) Possible PCR bias (no detectable).
On the basis of these two methods, new methods have (D) Considerable noise in sequencing results [23].
been continuously improved, including denaturing high-
performance liquid chromatography (DHPLC), reverse high- 31.3.2.2 Methylation Specificity PCR
performance liquid chromatography (Reversed-phase This method is economical and practical without special
HPLC), and thin layer chromatography (TLC) combined instruments, so it is the most widely used method at present.
with HPLC-TLC method. In addition to the above methods, MS-PCR can be carried out after bisulfite treatment. In
31 DNA Methylation Detection Techniques 429
Fig. 31.1 Experimental process of methylight
t raditional MSP methods, one pair of MSP primers amplifies complementary to the site is designed. Then, real-time quan-
DNA template after bisulfite treatment, and the other pair titative PCR was carried out. Two methylation-sensitive
amplifies unmethylated fragments. If the primers can TaqMan® probes and two methylation insensitive PCR
amplify, it indicates that methylation exists on the detection primers. Only FAM-labeled bisulfite-transformed methyl-
site. This method is highly sensitive and can be applied to ated DNA-specific TaqMan® probes or VIC-labeled
paraffin-embedded samples and is not limited by bisulfite-transformed unmethylated DNA-specific TaqMan®
endonucleases. probes can hybridize with the different methylation status of
target sequences. If the probe hybridized with DNA, the fluo-
1. Experimental process: rescence signal will be released. The degree of methylation
(A) DNA extraction. of the sample can be calculated (Fig. 31.1). The greatest
(B) Bisulfite treatment. advantage of this method lies in its high throughput and sen-
(C) DNA isolate. sitivity, and it does not require post-PCR electrophoresis,
(D) DNA desulfonyl reaction. hybridization, and other operations, reducing contamination
(E) Methylation-specific PCR reaction. and operational error [24].
2. Application Examples:
(A) Bisulfite-convert DNA. 1. Experimental process:
(B) Primers design. (A) DNA Isolation.
(C) PCR amplification. (B) Bisulfite conversion of isolated DNA.
(D) Run 2% agarose gel electrophoresis and analyze
(C) RT-PCR for methylated DNA amplification.
PCR products. (D) Hybridization between methylated amplicon and

3. Deficiencies: methylation-specific TaqMan probe.
(A) It is very important to design good primers. (E) Detection of fluorescent signal of TaqMan® probe.
(B) If there is an incomplete treatment of bisulfite, it may 2. Application Examples:
be false. (A) DNA isolation from breast tissue paraffin sections
(C) Due to methylated specific primers for PCR, the (10 μm).
method can only detect one site at a time and can (B) Determine DNA concentration.
only be qualitatively determined. (C) Sodium bisulfite treatment.
(D) False-positive results (no easily detectable). (D) PCR probes synthesis.
(E) Two separate tube reactions for assay. (E) RT-PCR application.
3. Deficiencies:
31.3.2.3 Methylight (A) No information at single-molecule level unless using
This method uses TaqMan® probe and PCR primer to distin- digital MethyLight.
guish methylation and non-methylation of DNA. DNA frag- (B) No information of methylation at CpG resolution
ments were first treated with bisulfite, and a probe level, unless usage of CpG specific probes.
31.3.2.4 High-resolution Melting (HRM) 1. Application Examples:

It detects gene mutation, genotyping, and methylation in (A) Collected oocytes were divided into PCR tubes.
recent years. The basic principle is that following bisulfite (B) Samples were digested and denatured.
treatment, methylated and unmethylated DNAs will have (C) Add low-melting point agarose and mix.
sequence differences, which can be detected by melting (D) Chill the mixtures for beads formation.
curve analysis. Because methylated DNA contains more GC, (E) Add bisulfite solution.
it is relatively more difficult to melt. According to the change (F) Cover with mineral oil and incubation avoid light.
of melting temperature and peak shape, complete methyla- (G) Cease reaction and repeat incubation in TE.
tion, complete non-methylation or hybrid methylation can be (H) Desulfonation.
easily distinguished. HRM technology can distinguish (I) Nested PCR.
extremely small differences. Methylation analysis using this (J) Run agarose gel electrophoresis.
method requires only one pair of primers, which is more 2. Deficiencies:
rapid, simple, and accurate than previous methods. Only the methylation of specific enzyme digestion sites
can be obtained leading to false-negative results.
1. Experimental process:
(A) Methylation primer design for candidate sequence 31.3.2.6 Pyrosequencing
fragments or sites (less than 300 bp). As a new sequence analysis technique, it can quickly detect
(B) Preparation of standard products using methyltrans- methylation frequency, and can achieve qualitative and
ferase modified DNA and non-methylation modified quantitative detection of methylation sites in samples,
DNA (gradient HRM standard curve: 0%, 1%, 5%, which provides a new way for methylation research.
10%, 20%, 50%, 100%). Pyrophosphate sequencing itself can detect subtle changes
(C) Bisulfite treated sample DNA. in methylation levels by accurately quantifying methyla-
(D) On-board testing. tion frequencies at a single continuous CpG site. In the
(E) Data processing. sequence extension process, the C–T ratio of single locus
2. Application Examples: was quantitatively determined according to the amount of
(A) PBMC extraction. C and T incorporation. Therefore, the methylation variation
(B) DNA extraction. of different loci can be accurately detected. Because pyro-
(C) Sodium bisulfite treatment. sequencing provides real sequence data, methylation status
(D) Methylation analyses by RT-PCR. is also shown in sequence.
3. Deficiencies: The technique is an enzyme cascade chemiluminescence
(A) The requirements for instruments are quite high.
reaction containing 4 enzymes (DNA polymerase, ATP sul-
Fluorescent quantitative PCR instrument with HRM furytase, luciferase, and Apyrase) catalyzed in the same reac-
module is needed. At present, there are many PCR tion system. Only one deoxyribonucleotide three phosphate
instruments on the market to meet the requirements. (dNTP) is added to each sequencing reaction system. If the
(B) No information of methylation at CpG resolution dNTP is paired with the template, a molecule of pyrophos-
level. phate (PPi) will be released. The amount of dNTP and PPi
(C) No information at single molecule level, unless using released is equal. CpG methylated pyrophosphate sequenc-
digital MS-HRM [25]. ing method based on primer extension of the Pyrosequencing
technology, by the chain synthesis process released in the
31.3.2.5 C ombined Bisulfite Restriction pyrophosphate (PPi) into optical signals to monitor the syn-
Analysis (COBRA) thesis process. The methylation degree of the target site was
Followed by bisulfite treatment and PCR amplification, the quantitatively analyzed by detecting the ratio of C/T infiltra-
purified product is digested by restriction endonuclease tion at the corresponding sites of CpG [28].
(BstUI). If C in the recognized sequence is completely meth-
ylated (5mCG5mCG), it will be retained as CGCG after 1. Application Examples:
PCR amplification, and BstU I can recognize and cleave it; if (A) Prepare bisulfite-converted genomic DNA
C does not methylate in the detected sequence, it will be (B) Design primers
transformed into TGTG after PCR, and BstUI recognition (C) Purify DNA strands
site will be lost, so it cannot be cleaved. In this way, the ratio (D) Pyrosequencing
of methylation in the original sample can be obtained by
electrophoresis separation, probe hybridization, and scan- 31.3.2.7 A nalysis of DNA Methylation Map
ning quantitative analysis. This method is relatively simple, Based on Chip
can quickly quantify the methylation of several known CpG As far as methylation map analysis is concerned, the popular
sites, and requires a small sample size [26, 27]. analysis method is chip. Several companies have provided
this tool, including Agilent, Illumina, Roche NimbleGen, experts, the biggest advantage of its product is the resolution
and Affymetrix. During Agilent and NimbleGen’s analysis, of a single CpG site. Other analyses locate methylation in a
genomic DNA was divided into two parts, one for MeDIP region, and Illumina analysis can accurately determine the
and the other for control. Both samples are labeled with fluo- level of methylation at a CpG site [29].
rescence (the enriched samples were labeled with Cy5 and
the control with Cy3), and then are hybridized with the chip. 1. Application Examples:
The Cy5/Cy3 intensity ratio of each probe on the chip shows (A) Prepare genomic DNA.
the degree of methylation in the region. Agilent’s 244,000 (B) Bisulfite conversion.
element array covers more than 27,000 CpG islands and the (C) Chip-based genome-wide DNA methylation screen.
undermethylation region (UMR). NimbleGen’s Hauman (D) Data analysis.
2.1 M Deluxe Promoter array also covers more than 27,000
CpG islands and includes more than 27,000 promoter 31.3.2.8 S ingle Molecule Real-Time (SMRT)
regions, all at 100 bp resolution. Neither platform can report Sequencing Technology
methylation status at a single nucleotide resolution, but Single-Molecule Real-time (SMRT) is a method of real-time
Illumina’s Infinium and Golden Gate analyses do. The monitoring the working state of DNA polymerase. The
Infinium HumanMethylation27 BeadChip chip of Illumina incorporation of nucleotides is detected as a fluorescence
covers 27,578 CpG sites. The analysis used two site-specific pulse and the nucleotide is identified according to its color.
probes to interrogate these chemically different sites. The When the polymerase cuts off the fluorophore attached to the
product is replaced by Infinium HumanMethylation450 terminal of the nucleotides, the pulse terminates (Fig. 31.2).
BeadChip chip. It covers more than 450,000 methylation
sites in each sample with a single base resolution. Its high 1. Application Examples:
coverage, high throughput, and low price make it an ideal
choice for screening GWAS population. VeraCode 31.3.2.9 Flight Mass Spectrometry
GoldenGate methylation analysis is more suitable for high MassARRAY® EpiTYPER™ DNA methylation analysis
throughput validation studies than for more information on technology combines base-specific enzyme digestion reac-
the Human Methylation 450 BeadChip chip, which analyzes tion and MALDI-TOF detection principle to achieve multi-
48 to 384 user-specified CpG sites in solution form. First, ple CpG analysis and detection. Base specific enzyme
bisulfite-treated genomic DNA was mixed with analytical digestion (MassCLEAVE) experiment is started by bisulfite
oligo, which complemented the U of unmethylated sites or treatment of DNA. The 5′ terminal primers with T7-promoter
the C of methylated sites. After hybridization, primers extend are used for PCR amplification, and the products are treated
and connect the superposition specific oligo to produce uni- with SAP (shrimp alkaline phosphatase) for base-specific
versal PCR templates. According to Illumina’s product enzyme digestion. The size and molecular weight of DNA
Fig. 31.2 One example of SMRT sequencing

fragments after digestion depend on the base changes after (B) DNA bisulfite convertion.
sulfite treatment. The molecular weight of each fragment can (C) PCR.
be measured by flight mass spectrometry. The corresponding (D) MALDI-TOF measures DNA methylation levels.
software EpiTYPER can automatically report the methyla-
tion degree of each fragment. Mass ARRAY methylation
assay does not require any fluorescent markers. Each reac- 31.4 Clinical Application
tion covers up to 500 BP of CpG sites, and is highly sensitive
to detect methylation levels as low as 5% [30]. DNA methylation changes are known to be involved in many
life events or disease pathogenesis, for instance, aging, car-
1. Application Examples: diovascular disease, diabetes mellitus, and cancer (Fig. 31.3).
(A) Genomic DNA preparation. Table 31.1 summarizes several mostly studied biomarkers.
Fig. 31.3 Interest of DNA methylation biomarker in cancer diagnosis. The relative importance of DNA methylation-based biomarkers regarding
clinical need is bolded (high interest) or not (modest interest)
Table 31.1 Examples of DNA methylation biomarkers for cancer diagnosis, prognosis, and treatment/chemotherapy sensitivity
Tumor type Gene Sample Detection technology Application field References
Bladder CDH1 Tissue MSP Diagnosis [31]
Tissue MSP Prognosis [32]
DAPK Tissue MSP Diagnosis [33]
Tissue MSP Treatment/Chemotherapy sensitivity [35]
RASSF1 Tissue MSP Diagnosis [36]
Tissue qMSP Prognosis [37]
RUNX3 Urine Sediments MSP Diagnosis [38]
Tumor type Gene Sample Detection technology Application field References
Breast APC FFPE MS-MLPA Diagnosis [40]
Tissue qMSP Diagnosis [41]
FFPE MSP and Methyl Light Prognosis [42]
BRCA1 Tissue MSP Diagnosis [43]
Serum MSP Treatment/Chemotherapy sensitivity [45]
CDH1 Tissue qMSP Diagnosis [41]
ESR1 Tissue MSP Prognosis [47]
Plasma qMSP Treatment/Chemotherapy sensitivity [48]
GSTP1 Tissue MSP Diagnosis [43]
Tissue Methyl Light Prognosis [49]
Tissue qMSP Treatment/Chemotherapy sensitivity [50]
MGMT Tissue MS-HRM Diagnosis [51]
Tissue Pyrosequencing Diagnosis [52]
RARβ Serum MSP Diagnosis [54]
RASSF1 Tissue MSP Diagnosis [43]
Serum MSP Diagnosis [54]
TWIST FFPE MSP Diagnosis [56]
FFPE MSP/Methyl Light Prognosis [42]
Colorectal APC Tissue MSP Diagnosis [57]
Tissue Pyrosequencing Diagnosis [58]
Tissue MS-MLPA Diagnosis [59]
Tissue Pyrosequencing Prognosis [60]
CDH1 Tissue MSP Diagnosis [57]
Abdominal lavage fluid qMSP Prognosis [61]
hMLH1 Tissue MSP Diagnosis [57]
Tissue MS-MLPA Prognosis [62]
MGMT Tissue MSP Diagnosis [57]
FFPE qMSP Diagnosis [63]
FFPE MSP Treatment/Chemotherapy sensitivity [64]
RASSF1 FFPE qMSP Diagnosis [63]
Blood MSP Treatment/Chemotherapy sensitivity [65]
TWIST Faece Droplet digital PCR Diagnosis [66]
FFPE MSP Prognosis [67]
FFPE formalin-fixed paraffin embedded tissue, MS-HRM methylation-sensitive high-resolution melting, MS-MLPA methylation-specific multi-
plex ligation-dependent probe amplification, MSP methylation-specific PCR, qMSP quantitative methylation-specific PCR
5. Avery OT, MacLeod CM, McCarty M. Studies on the chemical

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Part IV
Disease Presentation and Clinical
Laboratory Procedures
Immunological Disorders
32
Hong Mu, Chunlei Zhou, Ling Fang, Feng Xie, Yan Zhang,
and Huanhuan Chen
32.1 Allergic Disease processed and presented to T cells by APC. Activated T cells

stimulate the transformation of B lymphocytes into plasma
Hong Mu and Chunlei Zhou cells, which synthesize IgE. IgE binds to high-affinity recep-
tors on the surface of mast cells and basophils, thereby com-
pleting the body’s sensitization process to allergens. When
patients re-exposed to allergens, mast cell degranulation can
32.1.1 Overview be induced by specific IgE, releasing inflammatory media-
tors, leading to local tissue inflammation. This is the early
Allergic diseases are one of the most common chronic dis- phase reaction [2].
eases, including asthma, atopic eczema/dermatitis (AD), The delayed phase reaction occurs 4–6 h after the allergen
allergic rhinitis (AR), allergic conjunctivitis (AC), food exposure. Mast cells and eosinophils release substances from
allergy, and drug allergy. They often start in early childhood the granules. Th2 cells release cytokines and chemokines.
and continue throughout adulthood. Allergic diseases cause a Inflammatory mediators activate vascular endothelial cells
considerable burden on individuals and lead to a decline in and increase the expression of endothelial cell adhesion mol-
the quality of life. At the social level, they can lead to addi- ecules, proinflammatory cytokines, and chemokines. Under
tional costs, especially occupying health care resources, the combined action of chemokines and cytokines, eosino-
reducing economic productivity [1]. In recent years, with the phils, neutrophils, basophils, T cells, and macrophages infil-
continuous improvement of the research level in the field of trate, causing local tissue inflammation. The delayed phase
life sciences, people’s understanding of the pathogenesis of reaction can be stimulated by mast cells or T cells. The dif-
allergic diseases has gradually deepened, which provides new ference is that the former depends on the role of IgE, while
targets for the diagnosis and treatment of such diseases. the latter does not depend on IgE, but is mediated by the
major histocompatibility complex (MHC) [2].
32.1.2 Pathogenesis 32.1.2.1 Immune Cells in Allergy

CD4+ T cells play a key role in allergic reaction. They can
Allergic diseases are a group of diseases mediated by IgE not only recognize the antigen peptide presented by APC,
and caused by the body’s destructive immune response to and then activate B cells, but also regulate inflammation by
various allergens. When allergens enter the body, they are releasing a series of cytokines. CD4+ T cells can differenti-
captured by antigen-presenting cells (APC). Allergens are ate into four subgroups: Th1, Th2, Treg, and Th17. Th1 cells
mainly secrete IL-2, TNF, and IFN, which mediate cellular
H. Mu (*) · C. Zhou immune response and inhibit the differentiation of Th2 cells.
Tianjin First Central Hospital, Tianjin, People’s Republic of China Th2 mainly secretes IL-4, IL-5, IL-13, IL-25, and other cyto-
L. Fang · F. Xie (*) kines, promotes B cell activation and proliferation, promotes
Department of Laboratory Medicine Center, China-Japan Union IgE immunoglobulin synthesis, and induces eosinophil acti-
Hospital of Jilin University, Changchun, Jilin, vation. Treg can actively inhibit the function of other immune
People’s Republic of China cells, inhibit the immune response, and alleviate allergic
Y. Zhang (*) · H. Chen reactions. Th17 can promote immune inflammation by
Department of Laboratory Medicine, The First Affiliated Hospital secreting IL-17. Studies have shown that the expression of
People’s Republic of China IL-17 is increased in allergic rhinitis, allergic asthma, aller-

440 H. Mu et al.
gic dermatitis, allergic conjunctivitis, food allergy, and other of cytokine. Many cytokines can regulate the expression of
diseases [2, 3]. the same cytokine receptor and cross-regulate the expression
Most mast cells are activated by cross-linking of FcεRI, of specific receptors. Among them, the major ones involved
the high-affinity IgE receptor, and some are activated by in regulating anaphylaxis are IL-4, IL-5, IL-13, and IL-29.
Neurokinin, Toll-like receptor, complement receptor, and so IL-5 can regulate the proliferation of B cells and eosinophils.
on. After activation, mast cells release heparin, histamine, IL-4 and IL-13 are the most important inducers of IgE. They
tryptase, and chymotryptase, which can also lead to the syn- initiate gene transcription in the conserved region of immu-
thesis and release of prostaglandins and leukotrienes, and noglobulin heavy chain. IL-3 promotes granulation of imma-
then promote the secretion of chemokines and cytokines ture mast cells. IL-4, IL-3, IL-10, and SCF can stimulate the
such as IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, Monocyte che- proliferation of mast cells. IL-29 can induce and regulate the
moattractant protein (MCP), regulated upon activation nor- release of many cytokines from mast cells, eosinophils, neu-
mal T cell expressed and secreted (RANTES), TNF-α, and trophils, monocytes, and T cells.
so on. Complement system is an important part of the immune
Neutrophils are chemotactic to IL-8, leukotrienes, IL-17, system and also participates in the pathogenesis of allergic
and TNF and release the substances in their granules, such as diseases. C3a binds to C3a receptors on the surface of inflam-
myeloperoxidase (MPO), metalloproteinase (MMP), lacto- matory cells such as mast cells and eosinophils and mediates
ferrin (LF) elastase (NE), acid phosphatase, lysozyme, alka- the release of inflammatory mediators from inflammatory
line protease, TNF, IFN-gamma, IL-1, IL-6, IL-8, cells. C3b can bind to the C3b receptor on the surface of
platelet-activating factor, and reactive oxygen species (ROS), neutrophils, play a regulatory role, and further activate C5 to
which lead to mucosal edema and increased vascular initiate the next stage of complement activation. C3a, C4a,
permeability. and C5a are allergic toxins that cause degranulation of mast
Eosinophils express a variety of receptors, such as adhe- cells and release histamine and other components.
sion molecule receptor, immunoglobulin receptor FcεRI, Substance P (SP) originates from nerve tissue. In allergic
complement receptor, cytokine receptor, chemokine recep- diseases, it stimulates mast cells to release histamine and
tor, and so on. After activation, eosinophil cationic protein various mediators, causing local vasodilation, plasma extrav-
(ECP), major basic proteins (MBP), eosinophil peroxidase asation, and inflammatory cell infiltration. It promotes the
(EPO), and eosinophil neurotoxin (EDN) can be secreted [2]. expression of TNF-α in mast cells and stimulates mast cells
to release histamine and various mediators, resulting in local
32.1.2.2 Inflammatory Molecules in Allergy vasodilation, plasma extravasation, and inflammatory cell
Histamine-mediated allergic inflammation mainly causes infiltration, and then local congestion and edema. SP can
mucosal congestion and edema by binding with H1 receptor, promote the phagocytosis and chemotactic migration of
and also can combine with H1 and H2 receptor, which can mononuclear macrophages.
induce smooth muscle contraction, telangiectasia, and Platelet-activating factor (PAF) is the most potent lipid
increase vascular permeability. In addition, histamine can mediator for platelet activation. The main role of PFA in the
promote the maturation of dendritic cells, release inflamma- pathogenesis of allergic diseases is the chemotaxis of eosino-
tory factors from macrophages, enhance the activity of basophils, which releases alkaline proteins, eosinophil cationic
phils and eosinophils, and increase the expression of cell proteins, and peroxidase.
adhesion molecules and class II histocompatibility antigens. Tryptase is secreted by mast cells and plays its role by
Leukotriene (LT) is one of the metabolites of arachidonic binding with tryptase receptor PAR2. The main function is to
acid. LTB4, a member of LT family, is a strong inflammatory stimulate the release of IL-8, up-regulate the expression of
factor and a highly active chemokine, which has strong che- Intracellular adhesion molecule-1 (ICAM-1) on the surface
motaxis to neutrophils, eosinophils, and monocytes. LTB4 of epithelial cells, and stimulate the release of mediators
promotes the adherence of leukocyte to vascular endothelial from adjacent mast cells to promote inflammation [2].
cells, resulting in increased leukocyte-dependent vascular
permeability. It can promote the activation of mast cells and
strengthen the role of mast cells. Together with IL-4, it also 32.1.3 Atopic Dermatitis
promotes IgE synthesis in plasma cells.
Cytokines are small molecular polypeptides involved in 32.1.3.1 Overview
immune response, including interleukin, interferon, colony- Atopic dermatitis (AD) is a chronic, multifactorial, and
stimulating factor, tumor necrosis factor, chemokine, trans- inflammatory skin disease with a main manifestation of pru-
forming growth factor, and so on. Cytokines have multiple ritus in conjunction with erythema, edema, xerosis, erosions/
regulatory and overlapping immune functions. One kind of excoriations, crusting, oozing, and lichenification. AD is one
cytokine induces or inhibits the production of another kind of the most common non-communicable skin diseases. AD is
32 Immunological Disorders 441
generally present in 15–30% of children, with 85% of cases pityriasis alba, ichthyosis vulgaris, palmar hyperlinearity,
manifesting before age 5. And the prevalence of AD varies Ocular/periorbital changes, Perifollicular accentuation/
from 2 to 10% in adults. At present, the pathophysiology of lichenification/prurigo lesions. Severity of AD can be deter-
AD is not completely clear. It is a multifactorial disease mined by Scoring Atopic Dermatitis (SCORAD) index
caused by a series of genetic mutations, immune dysregula- developed by the European Task Force of Atopic Dermatitis
tions, allergic reaction, environmental triggers, skin barrier (ETFAD), with possible scores from 0 to 83, with higher
dysfunctions, psychic factors and skin and gastrointestinal scores indicating more severe AD [7].
dysplasia [4]. A study of AD in childhood revealed a higher
AD genetic risk score was associated with an increased risk 32.1.3.3 Laboratory Diagnosis
for persistent AD, together with paternal asthma, paternal
AD, and higher social circumstances [5]. Serum Total IgE
Allergen-specific IgE detection: Serum total IgE and
32.1.3.2 Clinical Appearance allergen-specific IgE detection are important indicators for
The diagnosis of AD is based on age-specific clinical mani- judging allergic diseases. It has been reported that the total
festations, including pruritus and chronic or relapsing spon- IgE level is positively correlated with the severity of
giotic dermatitis involving the face, trunk, and/or extensor AD. Serum IgE testing is obtained by simple blood work,
extremities in infants, flexural surfaces like the wrists/ankles and the physician must specify each specific antibody to be
and antecubital/popliteal fossae in children, or the hands in tested and will be run with other similar allergens in panels.
adults [6]. Generalized xerosis is a common feature, usually High levels of IgE do not necessarily correlate with the
accompanied by ichthyosiform scales and palmoplantar severity of the reaction. The results of serum tests need to be
hyperlinearity. The most typical skin lesion is diffuse rash analyzed with the patient’s history and clinical exam context,
accompanied by pruritus. In addition, AD is also character- and cannot be strictly diagnosed with just the IgE levels [9].
ized by spacing flares, often without obvious trigger. Acute
flaring AD is characterized by erythema, edema, and scales, Detection of Eosinophils in Peripheral Blood
often accompanied by multiple and widespread excoriations. Eosinophilia proliferation is one of the reference indicators
The change of Papular follicular is more obvious in dark of anaphylaxis.
skin. In more severe cases, fine vesicles/papules are evident,
accompanied by serosal drainage and scab formation. Serum Eosinophil Cationic Protein (ECP)
Chronic clinical changes, especially in dark skin, include ECP is considered as a marker of eosinophil activation,
lichenification and dyspigmentation. Common comorbidi- reflecting the degree of eosinophil activation. It is an effec-
ties include sleep disorders, mental and emotional disorders, tive and reliable index to reflect the changes of the condition
asthma, allergic rhinitis, and allergic conjunctivitis. of AD patients.
Eosinophilic gastroenteritis and celiac disease are also com-
mon complications of AD. In addition, clinical manifesta- Allergen Skin Patch Test or Prick Test
tions associated with AD include Danny Morgan and anterior Allergen skin test is helpful in screening triggers of atopic
neck wrinkles, itching during sweating, skin leukoplakia, dermatitis, but positive allergen skin test cannot prove that a
skin infection tendency, wool intolerance, food intolerance, specific food or inhalation allergen has clinical significance
and food allergy [5]. in the pathogenesis of AD, but only indicates that the aller-
There are many diagnostic guidelines for AD [4], among gen is allergic [9].
which the classical Hanifin and Rajka criteria are still the
most widely used criteria worldwide [7]. Among the Basophil Activation Test (BAT)
improved diagnostic criteria in recent years, the one pub- BAT is an experiment to detect the function of basophils, i.e.,
lished by Eichenfield et al is more famous [8]. There are the degree of activation of basophils under allergen-induced
three parts in this guideline: essential features, important fea- conditions. The basic principle of BAT is as follows: aller-
tures, and associated features. Essential features include as gens and specific IgE antibodies bound to the high-affinity
follows: Pruritus, Typical morphology (e.g., facial, extensor IgE receptor (FcεRI) on the surface of basophils, that is,
in infants, flexural in older children) and waxing and waning basophils are sensitized by antigen specific IgE. When anti-
history. Important features include as follows: Early onset, IgE antibodies or corresponding antigens bound to IgE and
Personal or family history of atopic conditions (e.g., asthma, lead to cross-linking of IgE, activation markers on the sur-
allergies, hay fever), Immunoglobulin E hyperreactivity, and face of basophils will be upregulated, which can be detected
Xerosis. Associated features include as follows: Upper lip using flow cytometry. There are numerous different markers
cheilitis, Nipple eczema, Atypical vascular responses (cen- which can be used to identify basophils (such as CCR3,
trofacial pallor, white dermographism), Keratosis pilaris, CD203c, CD123, IgE, and CRTH2) and to quantify their
442 H. Mu et al.
activation (such as CD63, CD107a, CD107b, CD13, CD164, affecting all age groups. In recent years, the prevalence of
and CD69) by flow cytometry. Study observed a lower diseases in developing countries has shown a marked upward
expression of FcγRIIIB on basophils from AD patients com- trend.
pared to patients with allergic rhinitis, asthma, or chronic Allergic asthma is one of the most common types of
urticaria [10]. asthma, which is mainly caused by inhalation allergens.
Whole-gene scanning and linkage analysis revealed that Allergic Asthma has a variety of different inflammatory phe-
AD-related susceptibility genes on chromosomes included notypes, such as eosinophilic, neutrophilic, mixed, and oli-
3p26–22, 3q13–21, 15q14–21, 17q21–25, 18q11–12, and gocystic. Persistent airway inflammatory reaction is the main
11q13–5 (rs7927894). Moreover, 18q21, 4p14–15, and pathological change of allergic asthma. These changes
3p24–26 are also associated with elevated levels of IgE in include bronchial mucosal swelling, increased secretion, air-
AD [11]. way inflammatory cell infiltration, airway smooth muscle
SNPs of the filaggrin gene (FLG) have been considered to spasm, etc.
be the main cause of keratinization disorders including AD Factors associated with the risk of asthma include host
in Western populations. The filaggrin gene is located in an factors and environmental factors. However, the mechanism
area called the epidermal differentiation complex (EDC) on of these factors affecting the occurrence and development of
the 1q21 chromosome. The EDC is involved in the formation asthma is complex and interactive. Host factors include
of the stratum corneum, which is the outermost layer of skin Genetic (e.g., genes predisposing to atopy, airway hyperre-
and acts as a barrier [12]. The SNPs of FLG are very differ- sponsiveness, airway inflammation), Sex, Obesity, Pre-term,
ent between Asian populations and European populations. In or with small size for gestational age. Environmental factors
the AD patients of South Korea, FLG 3321delA, S2889X, include as follows: allergens (Indoor: furred animals, domes-
S3296X, and K4022X heterozygote mutations can be tic mites, fungi, cockroaches, Outdoor: pollen, molds), occu-
detected (AD2). In the AD patients of Indonesian, 5 SNPs of pational sensitizers and allergens (e.g., laboratory rodents,
the FLG gene mutation were found; they are rs61816761, flour, paints), microbiome, infections (predominantly viral),
rs121909626, rs797045090, rs74129447, and rs558269137. exposure to tobacco smoke (passive and active smoking),
In European populations, The R501X (Arg501Stop) and outdoor or indoor air pollution, diet [15, 17].
2282del4 mutations are most commonly found [13].
Polymorphisms of cytokine genes are also associated 32.1.4.2 Clinical Appearance
with AD. Univariate analysis showed a significant negative Asthma is characterized by variable symptoms such as
association of the TNFα −308G>A polymorphism with wheezing, shortness of breath, chest tightness, and/or cough-
atopic asthma, atopic dermatitis, asthma and skin symptoms ing with reversible limited airflow. Symptoms and airflow
and positive skin prick test [14]. IL-13 can induce Th2 cell limitations vary with time and intensity. These changes are
differentiation and IgE synthesis, and it plays an important usually induced by exercise, allergens and stimuli, weather
role in AD. 2 gene Polymorphisms of IL-13, C/TSNP, and changes, or viral respiratory infections.
Argl3OGln are associated with elevated serum IgE levels The diagnosis of asthma is based on the history of charac-
and IL-13 [2]. teristic respiratory symptoms and evidence of variable expi-
Serine protease inhibitor kazal-5 gene (SPINK5) G1258A ratory airflow limitation [15]. There are many methods for
mutation can lead to the defect of skin barrier function, assessing airflow limitation, two of which have been widely
which is obviously related to the pathogenesis of AD. The used in patients over 5 years old. The most widely used mea-
SNK of TLR2-16934 of Toll-Like Receptor (TLR) is related surements are the forced expiratory volume in 1 s (FEV1)
to the severity and SCORAD score of AD [2]. and forced vital capacity (FVC) and their ratio (FEV:/FVC),
and the measurement of peak expiratory flow (PEF) [15].
32.1.4 Allergic Asthma 32.1.4.3 Laboratory Diagnosis
32.1.4.1 Overview Allergic Skin Prick Test and Serum IgE Test

Asthma is an allergic disease of the airways marked by These tests can identify patients who are susceptible to aller-
repeated attacks of dyspnea and wheezing, affecting both gens and identify specific allergens. These experiments are
children and adults. Asthma affects an estimated 300 million valuable in the diagnosis of atopic diseases such as atopic
individuals worldwide; the prevalence in children aged cough and allergic rhinitis. About 60–70% of cough variant
13–14 years is 1.5–15.6% in different countries [15, 16]. asthma patients and 30% of Eosinophilic bronchitis patients
Allergic asthma is a serious health problem worldwide, are predisposed to allergen sensitization.
Induced Sputum Test and adults from several European countries revealed that a
This is a safe, non-invasive, and well-tolerated test for the SNP located at the promoter region of ZPBP2 (rs11557467)
diagnosis of chronic cough and airway inflammation. is the most significant factor associated with asthma. But it is
Eosinophilic bronchitis is indicated by the presence of different in the expression level of ZPBP2 and GSDMB
eosinophils in induced sputum, and cough variant asthma between European and African populations. Another signifi-
patients also have this symptom. Induced sputum cytology can cantly related locus is the SNP rs2952156 located at the Erb-
be used to monitor the response of patients with chronic cough B2 Receptor Tyrosine Kinase 2 (ERBB2) gene, whose G
after inhaled corticosteroids. It is recommended to use 3% allele was associated with protection for asthma in ethnically
hypertonic saline through an ultrasonic nebulizer, but repeated diverse populations [19]. A polymorphisms of cytokine
induction of the sputum test within 48 h should be avoided. genes revealed a significant negative association of the TNFα
−308G>A polymorphism with atopic asthma, atopic derma-
Fractional Exhaled Nitric Oxide (FeNO) Measurement titis, and so on [14].
This is a novel non-invasive technology for the diagnosis of The most significant variants identified by the genome-
airway inflammation. FeNO is the continuous production of wide association studies of asthma susceptibility can be
NO by airway epithelial cells induced by inflammatory fac- summarized as follows [19]:
tors, especially eosinophil-mediated airway inflammation,
which significantly increases FeNO. Therefore, FeNO can • rs1420101 is located at position 102957716 in
reflect the degree of eosinophil-mediated airway inflamma- Chromosomal region 2q12 with an effect allele “T,” and
tion and has the advantages of non-invasive, effective, safe, the nearest gene is IL1RL1.
and easy to operate, which can provide valuable help for the • rs10455025 is located at position 110404999 in
diagnosis of airway hyperresponsiveness. Asthma mediated Chromosomal region 5q22 with an effect allele “C,” and
by non-eosinophils does not cause an increase in FeNO, so the nearest gene is TSLP.
FeNO can be used to identify different types of airway • rs20541 is located at position 131995964 in Chromosomal
inflammation, which is beneficial to individualized treat- region 5q31 with an effect allele “G,” and the nearest gene
ment. However, the sensitivity of this test is not very high is IL13.
when it is used for screening of eosinophilic inflammation. • rs7705042 is located at position 141492419 in
Approximately 40% of patients with increased numbers of Chromosomal region 5q31 with an effect allele “A,” and
eosinophils have normal FeNO [18]. the nearest gene is NDFIP1.
• rs9272346 is located at position 32604372 in
BAT Chromosomal region 6p21 with an effect allele “A,” and
BAT has been proved more sensitive and able to diagnose the nearest gene is HLA-DQA1.
IgE-mediated allergy despite the absence of allergen-sIgE • rs2325291 is located at position 90986686 in
system proof. Moreover, BAT has been used to detect baso- Chromosomal region 6q15 with an effect allele “A,” and
phil activation in allergic asthma and allergic rhinitis. It has the nearest gene is BACH2.
been confirmed that BAT is associated with nasal stimulation • rs992969 is located at position 6209697 in Chromosomal
titers and bronchial stimulation thresholds [10]. region 9p24 with an effect allele “G,” and the nearest gene
is IL33.
Gene Detection • rs7927894 is located at position 76301316 in
Several studies have confirmed the genetic contribution to Chromosomal region 11q13 with an effect allele “T,” and
asthma predisposition, called heritability, which is estimated the nearest gene is LRRC32.
to be as high as 55–74% in adults and as high as 90% in • rs167769 is located at position 57503775 in Chromosomal
children. Genome-wide association studies (GWAS) revealed region 12q13 with an effect allele “T,” and the nearest
genome-wide significant associations with asthma. 14 loc gene is STAT6f.
has been validated their association previously with asthma • rs2033784 is located at position 67449660 in
susceptibility. The well-known 17q21 asthma locus is the Chromosomal region1 5q22 with an effect allele “G,” and
most repetitive signal, although the main driver of this asso- the nearest gene is SMAD3.
ciation has not yet been resolved. Several studies have con- • rs2952156 is located at position 37876835 in
firmed that the gene encoding zona pellucida binding protein Chromosomal region17q12 with an effect allele “G,” and
2 (ZPBP2) is a common locus of asthma in children and the nearest gene is ERBB2.
adults. The results of this study are supported by either SNP • rs17637472 is located at position 47461433 in
in intron or variants located within the intergenic region of Chromosomal region17q21 with an effect allele “A,” and
ZPBP2 and GSDMB. A meta-analysis of 13,556 children the nearest gene is ZNF652-PHB.
444 H. Mu et al.
• rs200567451 is located at position 37902883 in increasing year by year. The global AR patients are conser-
Chromosomal region 17q21 with an effect allele “G,” and vatively estimated to be more than 500 million, accounting
the nearest gene is GRB7f. for 10–20% of the world’s population, although AR has no
• rs12946510 is located at position 37912377 in lethal effect on individuals [20, 21]. It has seriously affected
Chromosomal region 17q21 with an effect allele “T,” and the quality of life of individuals and has a great impact on
the nearest gene is GRB7-IKZF3. individuals and society as a whole and has developed into a
• rs907092 is located at position 37922259 in Chromosomal global public health problem.
region 17q21 with an effect allele “A,” and the nearest According to Joint Task Force guidelines set forth by the
gene is IKZF3. American College of Allergy, Asthma and Immunology
• rs36095411 is located at position 38031865 in (ACAAI), American Academy of Allergy, Asthma and
Chromosomal region 17q21 with an effect allele “G,” and Immunology (AAAAI), and the Joint Council on Allergy,
the nearest gene is ZPBP2. Asthma and Immunology, AR can be classified as seasonal,
• rs35569035 is located at position 38035624 in perennial, and episodic. Alternatively, Allergic rhinitis and
Chromosomal region 17q21 with an effect allele “T,” and its impact on asthma (ARIA)’s guideline divide AR into
the nearest gene is ZPBP2-GSDMB. “intermittent allergic rhinitis” and “persistent allergic rhini-
• rs9303279 is located at position 38073968 in tis” according to the duration of symptoms, the former
Chromosomal region 17q21 with an effect allele “C,” and occurring less than 4 days/week, or less than 4 consecutive
the nearest gene is GSDMB. weeks, and the latter occurring more than 4 days/week and
• rs8076131 is located at position 38080912 in more than 4 consecutive weeks (AR3). American Academy
Chromosomal region 17q21 with an effect allele “A,” and of Otolaryngology—Head and Neck Surgery Foundation
the nearest gene is ORMDL3. (AAO-HNSF) has published the “Clinical Practice Guideline:
• rs7221814 is located at position 38089717 in Allergic Rhinitis (AGAR)” in 2015. According to AGAR,
Chromosomal region 17q21 with an effect allele “G,” and AR may be classified by (1) the duration of exposure to aller-
the nearest gene is ORMDL3-LRRC3C. gens, such as seasonal (e.g., pollens), perennial/year-round
• rs62067034 is located at position 38063738 in (e.g., dust mites), or episodic (occasional encounters in the
Chromosomal region 17q21 with an effect allele “T,” and patient’s living environment, e.g., visiting a home with pets);
the nearest gene is GSDMB. (2) frequency of symptoms; and (3) severity of symptoms
• rs9303277 is located at position 37976469 in (AGAR).
Chromosomal region 17q21 with an effect allele “T,” and
the nearest gene is IKZF3. 32.1.5.2 Clinical Appearance
• rs11557467 is located at position 38028634 in AR can be initially diagnosed as having allergic signs and
Chromosomal region 17q21 with an effect allele “T,” and one of the symptoms of nasal obstruction, rhinorrhea, itch-
the nearest gene is ZPBP2. ing, and sneezing. Allergic signs mainly include clear water-
• rs2290400 is located at position 38066240 in like discharge, pale nasal mucosa, edema, red eyes, and tears,
Chromosomal region 17q21 with an effect allele “C,” and but not limited to this. Because allergy is usually a systemic
the nearest gene is GSDMB. inflammatory response, AR complications such as conjuncti-
• rs4795405 is located at position 38088417 in vitis, otitis media, sinusitis, laryngopharyngitis, adenoid
Chromosomal region 17q21 with an effect allele “T,” and hypertrophy, and bronchial asthma are also common [22].
the nearest gene is ORMDL3. The AGAR 2015 expert group believed that although it
was difficult to diagnose AR conclusively without the results
of allergen detection, the preliminary diagnosis of AR based
32.1.5 Allergic Rhinitis on patient’s medical history and physical examination was
also entirely reasonable.
32.1.5.1 Overview
Allergic rhinitis (AR) is an IgE-mediated inflammatory dis- 32.1.5.3 Laboratory Diagnosis
ease characterized by nasal congestion, rhinorrhea (nasal
drainage), sneezing, and/or nasal itching (AGAR). The main Supportive Methods for the Diagnosis of AR Include
clinical symptoms were nasal itching, sneezing, and runny Skin Prick Test (SPT) and Serum Specific IgE
nose. AR is one of the most common diseases in the outpa- Dust mites and pollen are the most common allergens caus-
tient department of otolaryngology and is also a public that ing AR and are the main targets of laboratory testing. AGAR
seriously affects the health and quality of life of the whole 2015 no longer uses allergen detection as a supportive or
society. In recent years, the global incidence of AR has been exclusive diagnostic basis but recommends that patients with
clinical diagnosis of AR and empirical treatment be ineffec- 32.1.6.2 Clinical Appearance

tive, or that the diagnosis is not clear, or that patients who Food allergy can involve multiple organ systems. The clini-
need specific allergens for immunotherapy should undergo cal manifestations of food allergy vary according to the
allergen detection again. In fact, the diagnosis of AR through organs accumulated by allergens. The severity of clinical
diagnostic examination and the identification of allergen manifestations is related to the degree of allergenicity in
types and severity are of great significance for the prevention food and the susceptibility of the host.
(avoidance of contact with related allergens) and treatment
(allergen-specific immunotherapy) of AR [23]. IgE-Mediated Food Allergy
IgE-mediated food allergy in digestive system is a rapid-
Inhalation Allergen Screening Test (Phadiatop) onset reaction. The clinical symptoms are rapid and can
Phadiatop is a screening test for respiratory allergic diseases occur from minutes to 1–2 h after eating. The main manifes-
launched in recent years. Phadiatop contains 95% of the tations were oral allergic syndrome, infantile colic, and gas-
common allergens in the air. Phadiatop test results will show troesophageal reflux in children. Oral allergic syndrome:
positive when the patient is allergic to some allergens and itchy mouth and throat, swollen tongue, and/or swollen
there is specific IgE in serum [24]. throat appear within minutes after eating certain foods, but
symptoms usually disappear quickly. Infantile colic often
ECP Detection manifested as paroxysmal irritability without cause, persis-
As one of the main markers of eosinophils (EOS) activation, tent crying at night, curled legs, abdominal distension, and
ECP is widely used in the diagnosis and treatment of allergic excessive exhaust. Patients who eat allergic food can some-
diseases. For suspected AR patients with obvious symptoms, times quickly develop skin symptoms or exacerbate skin
serum ECP is a better and convenient assistant screening symptoms in chronic allergic patients. Common are urti-
tool, and it can also be used as an objective index to judge the caria, vascular edema, atopic dermatitis, etc. And sometimes
severity of the patient’s condition [25]. the manifestation of food allergy is allergic asthma and aller-
gic rhinitis [27, 29].
32.1.6 Food Allergy Non-lgE-Mediated Food Allergy

Gluten-sensitized celiac disease is a small intestinal disease
32.1.6.1 Overview that occurs in children or adults with genetic susceptibility. It
Food allergy is an adverse health effect arising from a spe- is induced by ingestion of gluten-containing food. The main
cific immune response that occurs reproducibly on exposure manifestations of clinical CD in adults are chronic diarrhea,
to a given food. Food allergies are common (up to 10% weight loss, anemia, and so on. In addition to the gastrointes-
affected), and there are a lot of data showing that the preva- tinal symptoms such as vomiting and diarrhea, children also
lence has been rising over the past 20–30 years. Food aller- show stagnation of development and short stature. Food
gies seem to be more severe in western developed countries protein-associated enterocolitis syndrome is a severe vomit-
and are more common in children than in adults. About 8% ing and diarrhea syndrome in infants and young children
of children have food allergy in USA, among which 2.4% caused by non-IgE-mediated allergic reactions induced by
have multiple food allergies, and about 3% experience severe milk and/or soybeans. It is common in infants and young
reactions [26]. In Europe, it was found that an overall life- children aged 1–3 months. Its clinical features are repeated
time self-reported prevalence is of. There are several major vomiting, diarrhea, or dehydration 1–4 h after ingestion of
food allergens include peanuts, tree nuts, fish, shellfish, eggs, suspicious food. The symptoms are sometimes very serious
milk, wheat, soybeans, and seeds [27, 28]. and manifest obvious symptoms of infection and poisoning.
Food Allergy is deferent from food intolerance, which has There is no etiological evidence of infection. Pulmonary
nothing to do with the immune system. “Allergies” and hemosiderosis with cow’s milk hypersensitivity also named
“allergic diseases” are diseases involving the immune sys- as Heiner’s syndrome: symptoms include anemia, cough,
tem changes. These immune system changes fall into two even hemoptysis, recurrent pneumonia, dysplasia. It is gen-
categories: IgE-mediated and Non-IgE-mediated. The symp- erally believed that avoiding milk-related diet can alleviate
toms of the former one are the result of interaction between the disease [27, 29].
the allergen and a type of antibody known as IgE, which is
thought to play a major role in allergic reactions, and the Mixed IgE and Non-IgE-Related Food Allergies
later one has the symptoms which are the result of interac- The disease is characterized by eosinophilic inflammation in
tion of the allergen with the immune system, but the interac- the gastrointestinal tract. It can manifest dysphagia, vomit-
tion does not involve an IgE antibody [28]. ing, diarrhea, intestinal obstruction, and malabsorption.
446 H. Mu et al.
Fig. 32.1 Diagnostic approach of food allergy. OFC Oral food challenge
Laboratory Diagnosis foods (Fig. 32.1). Diagnostic tests include physical exami-

The diagnosis of food allergy composes the history, epide- nation, elimination diets, skin prick test (SPT), sIgE tests,
miology, pathophysiology, and test results leading to a and oral food challenge (OFC), allergen-specific IgG4
diagnosis, including identification of the trigger food or measurement, etc. [27].
Component Resolution Diagnosis (CRD) 4 impairs Th2 responses and IgE class switching, which
The basic principle of CRD is to determine allergens by induces the cow’s milk allergy.
detecting IgE combined with specific allergen molecules. In • IL-5: IL-5 regulates the eosinophilic inflammation.
clinical laboratories, there are two types: singleplex and Demethylation of IL-5 increases the risk of developing
multiplex assays, which are used to detect single allergen food allergy
and multiple allergens, respectively. The analytical method • IL-10: IL-10 suppresses allergic inflammation. Increased
of CRD is classical immunofluorescence or immunolumi- methylation of IL-10 induces oral tolerance.
nescence analysis. CRD detects specific antigen compo- • IFN-γ: IFN-γ suppresses Th2 responses of allergic dis-
nents, and CDR can provide more accurate diagnostic eases. Increased methylation of IFN-γ is associated with
information than the assay for IgE binding to extracts com- food allergy, such as Hen’s egg, cow’s milk, peanut.
prised of mixtures of proteins. CRD test is considered as an • IL-17A: IL-17A increases allergic inflammation.
important test for the diagnosis of food allergy, and more and Methylation of IL-17A is associated with food allergy.
more studies have confirmed its value. Common allergen • HLA-DR and HLA-DQ: HLA-DR and HLA-DQ present
components are shown below: [27] antigen-derived peptides, mostly of exogenous origin, to
CD4+ helper T cells. Recognize allergenic proteins,
• Common allergen components in peanut include Ara h 1, including peanut proteins, which then leads to specific
Ara h 2, Ara h 3, Ara h 6, and Ara h 9 (especially southern IgE responses. Methylation of HLA-DR and HLA-DQ is
Europe). associated with peanut allergy.
• Common allergens in hazelnut are Cor a 9, Cor a 11, Cor • FOXP3: FOXP3 maintains immune tolerance to aller-
a 14. gens. FOXP3 DNA methylation level is associated with
• Jug r 1 and Jug r 3 are the allergens in walnut. food allergy.
• Cashew contains the common allergen Ana o 3. • DHX58: DHX58 is responsible for antigen recognition in
• Brazil contains the allergen Ber e 1. the innate immune response to various RNA viruses and
• Egg contains the allergen Ovomucoid. some DNA viruses. Demethylation of DHX58 is associ-
• Milk contains the allergen Casein. ated with allergic inflammation.
• Common allergen components in Soy include Gly m 5, • ZNF281: Represses GAST, which stimulates the stomach
Gly m 6, Gly m 8. mucosa, produces digestive enzymes, ensures smooth
• Tri a 19 is the common allergen in wheat. muscle contraction, and releases histamines. Increased
methylation ofZNF281 is associated with the gastrointes-
Gene Detection tinal reactions.
Major histocompatibility complex (HLA) gene family has • EIF4E2: Stimulates postnatal growth. Demethylation of
been identified associate with the susceptibility of food allergy. EIF4E2 is associated with allergic inflammation.
HLADRB1*08 and DQB1*04 alleles were increased in fre- • HTRA2: Regulates the perception of abdominal pain and
quency among peanut-allergic subjects. GWAS study identi- smooth muscle contraction in the gastrointestinal tract.
fied peanut allergy-specific loci in the HLA-DR and HLA-DQ Increased methylation of HTR2A is associated with gas-
gene region on chromosome 6p21.32 which were tagged to trointestinal symptoms.
the 2 single nucleotide polymorphisms (SNPs) rs7192 and
rs9275596 in subjects of European ancestry [26, 30].
Moreover, HLA-DQB1 (rs9273440) is another peanut allergy- 32.1.7 Drug Allergy
specific gene. Filaggrin (flg) dysfunction mutation has been
confirmed to be associated with atopic dermatitis (AD) and 32.1.7.1 Overview
other allergic diseases, such as peanut allergy. FLG variants (rs
Drug allergy is a series of immune-mediated hypersensitivity
1933064 and rs12123821) are associate with a common and reactions caused by drugs, with varying mechanisms and
food sensitization [26, 30]. The clade B serpin (SERPINB) clinical presentations [33]. Adverse drug reactions (ADRs)
gene cluster at 18q21.3 and the cytokine gene cluster at 5q31.1
are a major problem during the drug therapy. ADRs are com-
can increase the risk for any food allergy [30]. mon in everyday clinical practice, affecting 15–25% of
DNA methylation is an important mediator of gene- patients; serious reactions occur in 7–13% of patients. ADRs
environment interactions in food allergy, because host DNA are classified as predictable reactions and unpredictable
methylation status can be affected by environmental factors reactions, and unpredictable reactions, including drug
and subsequently predisposes people to food allergy [31, allergy, occur in approximately 20–25% of patients who
32]. Details are shown as follows: experience ADRs [33, 34].
The basis of drug allergy is the immunological mecha-
• IL-4: IL-4 enhances the differentiation of naïve CD4+ T nism of four allergies proposed by Gell and Coombs [35].
cells into IL-4-producing Th2 cells. Demethylation of IL- Type I, also known as immediate reactions, is mediated by
448 H. Mu et al.
drug-specific IgE antibodies. Type II and Type III are lesions on mucous membranes, particularly of the mouth
cytotoxic reactions mediated by IgG or IgM antibodies and and lips, as well as on truncal area.
immune-complex reactions, respectively. Type IV, known as • TEN: causative drugs of TEN are same as SJS, and clini-
delayed-type hypersensitivity reactions, is mediated primar- cal features are similar to SJS, but usually involve signifi-
ily by T cells and has been recently classified into 4 sub- cant epidermal detachment and potentially
types: IVa, IVb, IVc, and IVd, with the immune response of life-threatening.
Th1, Th2, cytotoxic T cells, and T cells (IL8/CXC8), respec-
tively [36, 37]. Other organs can be affected by drug-induced allergic
reactions. Hemolytic anemia, leucopenia, and thrombocyto-
32.1.7.2 Clinical Appearance penia may be caused by penicillin, sulfonamides, anticonvul-
Immediate reactions (IRs): IRs present as isolated symptoms sants, cephalosporins, quinine, heparin, thiazides, and gold
(angioedema, urticaria, conjunctivitis, bronchospasm, and salts. Hepatitis and cholestatic jaundice may be induced by
rhinitis,) or as a severe reaction such as anaphylaxis. Urticaria/ sulfonamides, phenothiazines, carbamazepine, erythromy-
angioedema and anaphylaxis are the most common [38]. cin, anti-tuberculosis agents, allopurinol, and gold. Penicillin,
Nonimmediate reactions (NIRs): The skin is the organ most sulfonamides, allopurinol, proton pump inhibitors (PPIs),
frequently and prominently affected by drug-induced allergic ACE inhibitors, and NSAIDs can cause interstitial nephritis
reactions. Maculopapular exanthema (MPE) and delayed and glomerulonephritis [33].
urticaria are the most common. Acute generalized exanthem- Drug allergy can induce multi-organ reactions such as
atous pustulosis (AGEP), fixed drug eruption (FDE), eczema, [33]:
and erythema multiforme (EM) are other presentations. Both
skin and other organs can be affected in drug allergy, as in • Anaphylaxis, induced by antibiotics, neuromuscular
drug rash with eosinophilia and systemic symptoms (DRESS)/ blocking agents, anesthetics, radiocontrast media, and
drug-induced hypersensitivity syndrome (DiHS), vasculitis, recombinant proteins (e.g., omalizumab), with the clini-
and Stevens-Johnson syndrome (SJS)/toxic epidermal necrol- cal features of urticaria/angioedema, bronchospasm, gas-
ysis (TEN) [34]. The clinical manifestations of drug allergy trointestinal symptoms, and hypotension.
on skin are involved in exanthemata, urticaria, angioedema, • DRESS (drug rash, with eosinophilia and systemic symp-
fixed drug eruption, SJS, TEN [33]. toms), caused by Anticonvulsants, sulfonamides, minocy-
cline, allopurinol, strontium ranelate, and mAbs, with the
• Exanthemata can be induced by Allopurinol, penicillins, clinical features of cutaneous eruption, fever, eosino-
cephalosporins, anticonvulsants, sulfonamides, and philia, hepatic dysfunction, and lymphadenopathy.
mAbs. The clinical features include as follows: (1) dif- • Serum sickness, caused by heterologous antibodies, inf-
fuse, fine macules, and papules, (2) Evolve over days post liximab, allopurinol, thiazides, antibiotics (e.g., cefaclor),
drug initiation. bupropion, and mAbs. The clinical features are urticaria,
• Urticaria and angioedema are caused by Antibiotics, arthralgias, and fever.
angiotensin-converting enzyme (ACE) inhibitors, anti- • DILE (drug-induced lupus erythematosus), induced by
convulsants, neuromuscular blocking agents, platinums, hydralazine, procainamide, isoniazid, quinidine, minocy-
radiocontrast media, non-steroid anti-inflammatory drugs cline, antibiotics, and anti-TNF-α agents, with the clinical
(NSAIDs), narcotics, and mAbs. The clinical features are features of arthralgias, myalgias, fever, and malaise.
included as follows: (1) onset within minutes to hours of • Vasculitis, caused by sulfonamide antibiotics and diuret-
drug administration, (2) potential for anaphylaxis, and (3) ics, hydralazine, penicillamine, propylthiouracil, and
often gE-mediated. mAbs. The clinical feature is cutaneous or visceral
• Fixed drug eruption is induced by Sulfonamide and tetra- vasculitis.
cycline antibiotics, NSAIDs, acetylsalicylic acid (ASA),
sedatives, chemotherapeutic agents, and anticonvulsants. 32.1.7.3 Laboratory Diagnosis
The clinical features are (1) hyper-pigmented plaques
occurring at the same site upon, and (2) re-exposuring to General Laboratory Examination
the culprit drug. The diagnosis of drug hypersensitivity reactions (DHRS)
• SJS can be caused by sulfonamides, nevirapine, cortico- relies on a comprehensive review of detailed clinical history
steroids, anticonvulsants, NSAIDs (oxicams), allopuri- and complemented with skin tests (STs), such as skin prick
nol, phenytoin, carbamazepine, lamotrigine, barbiturates, testing (SPT), when indicated. Additional tests, such as con-
psychotropic agents, pantoprazole, tramadol, and mAbs. trolled drug provocation tests (DPTs), may be needed
The clinical features include as follows: (1) fever, sore because STs might not always be predictive of the clinical
throat, fatigue, ocular involvement, (2) ulcers, and other outcome [39, 40]. However, DPTs should not be performed
in high-risk patients. In nowadays, more and more tests are screening before dapsone treatment for patient safety. HLA-
being added to the diagnostic panel and are becoming avail- B*58:01 allele is associated with allopurinol-induced Dress
able for clinical use. For clinicians, it is important to under- and SJS/TEN, and its predictive value is being evaluated in
stand the value of in vitro testing in diagnosing or estimating different populations [35, 36, 42]. Other genetic predispos-
drug allergies and to recognize the advantages and limita- ing factors are displayed below [35, 42]:
tions of current in vitro testing [39–41].
Tryptase and histamine determination are used for esti- • Dapsone can induce reactions of fever, rash, and systemic
mating immediate drug hypersensitivity reactions (IDHRs) symptoms in Han Chinese who carry the HLA alleles
at the acute phase. Tryptase determination can be used for HLA-B*13:01 and HLA-C*03:04.
assessment of mast cell involvement, but there are difficul- • Flucloxacillin can induce reactions of drug-induced liver
ties in performing the test in the right time kinetic of peak injury (DILI) in Caucasian who carry the HLA alleles
tryptase caused by short half-life (30–120 min). And the HLA-B*57:01 and HLA-DRB1*07:01.
comparison with basal levels is needed. Histamine determi- • Lamotrigine can induce reactions of SCAR in Caucasian
nation can be used for assessment of mast cell and/or baso- (predominantly) who carry the HLA alleles HLA-
phil involvement, but the half-life of histamine is very short. A*68:01, HLA-B*58:01, and HLA-DQB1*06:09; it can
There is no commercial test available. And the comparison induce SJS/TEN in Han Chinese who carry HLA-
with basal levels is also needed [39]. B*13:01, HLA-B*15:02, HLA-C*08:01, and HLA-
sIgE and BAT are used for identifying the relevant drug or DRB1*16:02; it also induces SJS/TEN and MPE in Han
drugs of IDHRs at resolution phase. The advantage of sIgE Chinese who carry HLA-B*15:19, HLA-B*44:08, HLA-
assay is the serum sample can be easily stored and trans- B*55:01, and HLA-B*81:01.
ported, the limitations include the available for a limited • Lapatinib induces reactions of DILI in Multiethnic (pre-
number of drugs, and the time interval from reaction is criti- dominantly Caucasian) with the association of HLA-
cal for sensitivity. For BAT, a wide panel of drugs is avail- DQA1*02:01, HLA-DQB1*02:02, and
able, but blood samples cannot be stored. HLA-DRB1*07:01.
Lymphocyte transformation test (LTT), cytokine determi- • Lumiracoxib induces reactions of DILI in Multiethnic
nation, and LTT combined with cytokines and cytotoxicity (predominantly Caucasian) with the association of HLA-
assays (Cyto-LTT) are used for identifying the relevant drug DQA1*01:02, HLA-DQB1*06:02, HLA-DRB1*15:01,
or drugs of NIDHRs (nonimmediate drug hypersensitivity and HLA-DRB5*01:01.
reactions) at the resolution phase. The limitations of LTT and
• Methazolamide can induce SJS/TEN in Han Chinese and
cytokine determination are (1) highly dependent on clinical Korean who carry the alleles HLA-B*59:01 and
entities and (2) low sensitivity in severe bullous skin reac- HLA-C*01:02.
tions. Cyto-LTT identifies the culprit drug based on cytokine• Nevirapine causes SJS/TEN in Black who carry HLA-
production and cytotoxicity, and it might even help in diag- C*04:01; it induces Skin rash and systemic or organ-
nosis of subset of patients with SJS/TEN [39]. specific symptoms in Caucasian with the association of
HLA-B*14, HLA-C*08, and HLA-DRB1*01:02; it also
Gene Detection causes reactions of Skin rash and hepatitis in Caucasian
Recent studies have shown that HLA alleles are associated with the association of HLA-DRB1*01:01; it also induces
with increased severe cutaneous allergic reaction (SCAR) DILI in Caucasian with the association of HLA-
risk, especially in reactions involving abacavir, carbamaze- DRB1*01:01; it can induce Skin rash and systemic or
pine, dapsone, and allopurinol. HLA-B*57:01 was associ- organ-specific symptoms in Han Chinese who carry the
ated with severe DHR induced by abacavir, with sensitivity HLA allele HLA-C*04; it can induce Skin rash in Indian
ranging from 46 to 80% and specificity from 98 to 99%. who carry the HLA-B*35allele; it can induce Skin rash
Therefore, gene screening can reduce the incidence of and hepatitis reaction in Multiethnic who carry the HLA-
abacavir-induced hypersensitivity. In the case of C*04allele; it also causes DILI in Multiethnic (predomi-
carbamazepine-induced SJS/TEN, HLA-B*15:02 is strongly nantly Black African) with the association of
correlated with the Asian population, and in patients with HLA-B*58:01, HLA-C*02:10, HLA-C*03:02, HLA-
other SCARs, such as Drug rash with eosinophilia and sys- C*12:03, and HLA-DRB1*01:02; it also causes Skin rash
temic symptoms (DRESS), HLA-A*31:01 has proved to be in Thai who carry the alleles of HLA-B*35:05 and
a predisposing factor [39]. The European Drug Administration HLA-C*04.
and the Food and Drug Administration of the United States • NSAIDs and “cold medication” may induce SJS/TEN in
have recommended testing in high-risk groups. In patients Brazilian and Indian people who carry the allele of HLA-
with SCAR induced by dapsone, a correlation with HLA- B*44:03; it also induces SJS/TEN in Japanese with the
B*1301 was found, suggesting the importance of gene association of HLA-A*02:06, HLA-A*24:02, and
450 H. Mu et al.
LA-B*44:03; it also induces SJS/TEN in Korean who respiratory rate of 18 breaths per minute. There is large ery-
H
carry HLA-A*02:06. thema on facial, trunk, and limbs, with non-depressed facial
• Oxcarbazepine can cause an MPEH in Chinese who carry swelling, difficulty to open eyes (edema), and itching, scal-
the alleles of HLA-B*13:02, HLA-B*15:02, HLA- ing, without pus.
B*15:19, HLA-B*15:27, HLA-B*15:58, HLA-B*27:04,
HLA-B*27:09, HLA-B*38:02, HLA-B*48:04, and Routine Laboratory Diagnosis Laboratory examination
HLA-B*56:01. showed that WBC was 2.1×109/L, N% was 57.7%, RBC was
• Phenytoin may induce SJS/TEN in Han Chinese with the 4.33×1012/L, PLT was 155×109/L, alanine aminotransfer-
association of HLA-B*13:01, HLA-B*15:02, HLA- ase (ALT) was 41 IU/L, aspartate aminotransferase (AST)
C*08:01, and HLA-DRB1*16:02 alleles; it also causes was 45 IU/L, creatinine clearance rate (Cr) was 64 μmol/L,
SJS/TEN in Thai who carry HLA-B*15:02. γ-glutamyl transpeptidase (γ-GGT ) was 62 IU/L, Na was
• Sulfamethoxazole can induce SJS/TEN in Thai who carry 130 mmol/L, K was 4.5 mmol/L, CL was 95 mmol/L, and Ca
the alleles of HLA-B*15:02, HLA-C*06:02, and was 2.7 mmol/L.
HLA-C*08:01.
• Sulfasalazine causes DRESS in Han Chinese with the Gene Detection HLA-B∗15:02 allele was positive.
association of HLA-B*07:02, HLA-B*13:01, HLA-
B*15:05, HLA-B*39:01, and HLA-B*58:01. Final Diagnosis Drug allergy induced by Carbamazepine.
• Ticlopidine can induce DILI in Japanese who carry HLA-
A*33:03, HLA-B*44:03, HLA-C*14:03, HLA-Management Carbamazepine treatment was stopped. The
DRB1*13:02, and HLA-DQB1*06:04 alleles. patient was administered Vitamin C, Dexamethasone, and
• Aromatic anticonvulsants can cause SJS/TEN in Han Cimetidine intravenously and was given loratadine orally.
Chinese, Thai, Indian, Malaysian, Vietnamese, After 1 week, the symptom was relieved.
Singaporean, and Hong Kongese, who carry the allele of
HLA-B∗15:02; it also induces DRESS in Han Chinese, This case was from Tianjin First Central Hospital.
European, and Spanish with the association of
HLA-A∗31:01.
• Carbamazepine may induce DRESS/SJS/TEN in Northern 32.2 Autoimmune Diseases
European, Japanese, and Korean who carry the HLA-
A∗31:01 allele; it also causes SJS/TEN in Han Chinese who Ling Fang and Feng Xie
carry the HLA-B∗15:11 and HLA-B∗15:02, in Japanese
who carry HLA-B∗15:11, HLA-B∗59:01, HLA-B∗15:02,
HLA- B∗13:01, and HLA-B∗51:01, in Spanish who carry 32.2.1 Overview
HLA-B∗38:01, in Thai who carry HLA-B∗15:02, and it
also causes SJS/TEN in Malaysian with the association of Normally, the body’s immune system identifies its own tis-
HLA-B∗15:02, HLA-B∗13:01, and HLA-B∗51:01. sue components as itself. Generally, it does not produce
immune response or only produces weak immune response,
which is called self-tolerance. Autoimmunity refers to the
32.1.8 Typical Medical Case phenomenon that the body’s immune system responds to its
own substances and produces autoantibodies or autoreactive
Clinical Background A female patient, 37 years old, was T lymphocytes when its tolerance is destroyed. There are
diagnosed with type II diabetes in local hospital due to thirst, many kinds of autoantibodies or sensitized lymphocytes in
excessive drinking, and hydrouria for more than 3 months. normal human serum, which can eliminate senescent cells
The patient had been treated with insulin for 1 month, and in vivo and exert the effect of immune stabilization.
then the treatment changed to take Glipizide sustained release Autoimmune disease (AID) is defined as autoimmune
tablets orally. Because of poor curative effect, the patient was disease only when the autoimmune response exceeds the
treated with insulin and Novolin. One day, she had been given physiological limit or lasts too long, then destroys the normal
Carbamazepine because of lower limb pain. Three days later, tissue structure and causes the corresponding clinical symp-
there were many rashes and itching all over the body. toms [43].
Physical Examination Physical examination showed 32.2.1.1 Classification of Autoimmune Diseases

patient’s detailed information as follows: temperature (T) of According to the scope of pathological changes, it can be
37.1 °C, blood pressure (BP) of 95/65 mmHg divided into organ-specific and non-organ-specific autoim-
(1 mmHg = 0.133 kPa), heart rate 100 beats per minute, and mune diseases. The former is limited to some specific organs
or tissues, and can detect autoantibodies or autoreactive T 32.2.1.3 Pathogenesis of Autoimmune

lymphocytes, such as chronic thyroiditis, Addison’s disease, Diseases (Fig. 32.2)
autoimmune hemolytic anemia, etc. The latter involves a
variety of tissues, organs, and connective tissue, such as RA 32.2.1.4 The Appearance of Autoantigens
and SLE. Concealed antigen release: Concealed antigen refers to some
antigen components isolated from the immune system in
32.2.1.2 Common Characteristics anatomical position, such as sperm, eye contents, etc.
of Autoimmune Diseases Changes in the properties of autoantigens: Biological,
There are autoantibodies (or) and sensitized lymphocytes physical, chemical, and other factors can change the proper-
targeting autoantigens in the peripheral blood of patients. ties of autoantigens, stimulate the immune response of the
Pathological features are immune inflammation, and the body, and cause AID. For example, denatured IgG stimulates
extent of injury corresponds to the distribution of antigens the body to produce anti-denatured IgG antibodies (RF),
targeted by autoantibodies or sensitized lymphocytes. causing RA.
Similar disease models can be reproduced in animals, and Common antigen induction: Some foreign antigens have
diseases can be metastasized in homologous animals through the same or similar antigenic determinants as specific human
serum or lymphocyte. Often accompanied by the following tissue antigens, and immune responses to these antigenic
characteristics: Unknown etiology, mostly spontaneous or determinants can cause autoimmune diseases. For example,
idiopathic; the course of disease is generally longer, mostly group A B hemolytic streptococcus infection is prone to
recurrence, and remission alternate; there is a genetic ten- rheumatic heart disease or glomerulonephritis.
dency, but mostly not the result of a single gene; more women
than men, older than adolescents; most patients can be 32.2.1.5 Abnormal Immune Regulation
detected in the serum of anti-nuclear antibodies or other Lymphocyte bypass activation, polyclonal stimulant bypass
autoantibodies; easily associated with immunodeficiency activation, abnormal expression of costimulatory factors,
disease or malignant tumors. and imbalance of TH1 and TH2 cells.
Fig. 32.2 Pathogenesis of A

autoimmune diseases “Resting”
T cell
tissue APC
self-tolerance
Self Self-tolerance
antigen
B
APC expresses
Microbe costimulatory Self-
molecules reactive
Activation T cell
of APC
Activation of
APCs
Self Self
antigen B7 CD28 tissue
Presentation of Autoimmunity
antigen by APC
Activation
of T cell
Molecular
mimicry
Self
Self-reactive T cell tissue
Microbial that recognizes
antigen microbial peptide Autoimmunity
452 H. Mu et al.
32.2.1.6 Abnormal Expression of Fas/FasL glomeruli, joints, and small vessel walls of various organs,
Fas belongs to the TNFR/NGFR family, also known as CD95, activating complements and causing local injury.
and is expressed on various cell surfaces. FasL is expressed
on the surface of activated T cells. Fas/FasL pathway can 32.2.2.3 Type IV Hypersensitivity
induce apoptosis. In Fas/FasL gene-deficient patients, because Firstly, when CD4+ Th1 cells encounter the same target anti-
of the impaired apoptotic mechanism, the clonal proliferation gen again, they produce inflammatory injury mainly caused
of T and B cells is out of control, and AID is prone to occur. by the infiltration of monocytes and lymphocytes by releas-
IL-1B and NO produced in type I diabetes induce Fas expres- ing cytokines and chemokines. Secondly, CD8+ CTL cells
sion in islet B cells and FasL expression in activated CTL. B recognize target cells, release perforin, granules, and other
cells were damaged by Fas/FasL. mediators, leading to target cell lysis, or induce target cell
apoptosis through Fas/FasL pathway.
32.2.1.7 Genetic Factors
AID has a familial genetic tendency and is positively corre-
lated with the detection rate of some HLA genotypes, mani- 32.2.3 Common Autoimmune Diseases
fested in HLA-B, DR antigens. Such as HLA-DR3, Fas/FasL
gene defect; Compulsory spondylitis HLA-B27; Type I dia- 32.2.3.1 A utoimmune Hemolytic Anemia
betes chromosome 6q27 [44]. (AIHA)
Erythrocyte shows changes in antigenicity and produces
autoantibodies against surface antigens of erythrocyte mem-
32.2.2 The Mechanism of Immune Injury branes after abnormal immune function or taking certain
in Autoimmune Diseases (Fig. 32.3) drugs. Autoantibodies bind to autoantigens, activate comple-
ments, destroy red blood cells, and lead to anemia.
32.2.2.1 Type II Hypersensitivity Clinical manifestation: Anti-erythrocyte autoantibodies;
The combination of autoantibodies (IgG, IgM) and autoanti- Coombs test positive; shortened erythrocyte life span; AIHA
bodies destroys cells through three ways: first, activating is more common in middle-aged women; secondary cases
complements, forming membrane attack complexes, and dis- are mainly secondary to lymphatic malignancies, connective
solving cells; second, promoting macrophages to phagocy- tissue diseases, infections and drug use; the drugs causing
tose and destroy target cells through Fc and C3b conditioning; AIHA are penicillin, quinidine, isoniazid, sulfonamides,
third, destroying target cells through ADCC, such as autoim- methyldopa, etc.
mune hemolytic anemia [45]. Anti-erythrocyte antibodies can be divided into three cat-
egories: WAIHA, IgG type, 37 °C binding to RBC, no aggre-
32.2.2.2 Type III Hypersensitivity gation of RBC. CAD, the agglutinin is IgM type, which
After autoantibodies bind to antigens, they form medium- binds to RBC at low temperature to agglutinate and induces
sized complexes, activate complements, and cause inflam- agglutinin syndrome. Donath Landsteiner antibody is IgG
mation and injury. For example, in SLE, IC deposits on type. It binds to two complement components at low tem-
Fig. 32.3 The mechanism of immune injury in autoimmune diseases

perature. When the temperature rises to 37 °C, it activates the 2. Anti-DNA antibodies: natural (double-stranded) DNA

complement chain, leading to hemolysis and paroxysmal (dsDNA) and variable (single-stranded) DNA (ssDNA)
cold hemoglobinuria (PCH). antibodies. There are many methods to detect dsDNA
antibody, such as indirect immunofluorescence, indirect
Case 1, Primary WAIHA enzyme-labeled antibody, complement-binding antibody,
A 68-year-old woman was treated for fatigue, Hb 93 g/L, enzyme-linked immunosorbent assay, and radioimmuno-
MCV 89.7 fL, RET 119×109/L, Haptoglobin < 14 mg/dL, assay (Farr method). Farr method is the most specific and
LDH 267 U/L, TBIL 0.8 mg/dL, erythrocyte polychroma- reliable method to detect dsDNA antibody. It is the inter-
tism in blood smears, DAT anti-IgG++, anti-C3 weak nationally recognized standard method to detect anti-
positive. dsDNA antibody.
This case was from China-Japan Union Hospital of Jilin 3. Anti-extractable nuclear antigen (ENA) antibodies: two-
University. dimensional immunodiffusion, convective immunoelec-
trophoresis, Western blotting, and ELISA.
32.2.3.2 Immune Thrombocytopenic Purpura
(ITP) Case 2, SLE
The main manifestations are skin and mucosal purpura, Female patient, 32 years old. He was admitted to the hospital
thrombocytopenia, increase of megakaryocytes in bone mar- on December 23, 2004, because of joint pain for nearly 2
row, multiple females, patients with anti-platelet antibodies, years, eyelid edema for 16 months, dry cough for 1 month,
which shorten the life of platelets [46]. and lack of consciousness for 20 days. There was pain in the
Clinical manifestations: Acute type: (1) Purpura may proximal interphalangeal joint of both hands. Fever appeared
have a history of upper respiratory tract infection or viral 14 months ago, the highest temperature was 40 °C, urinary
infection before it appears. (2) Acute onset, often with fever, protein (+++).
skin bruises, and even massive hematoma. Hemorrhage is Laboratory examination: Hemoglobin 78 g/L, WBC
common in nasal, gingival, and oral mucosa. (3) Severe vis- 5.2 × 109/L, platelet 120 × 109/L. Urine routine: Protein
ceral bleeding can occur, such as gastrointestinal tract, uri- 5 g/L. Serum albumin 18 g/L, liver and kidney function was
nary tract, reproductive tract, and even intracranial normal. The erythrocyte sedimentation rate was
hemorrhage. (4) Most platelets are lower than 20 × 109/L. 98 mm/H. Complement C3 409 mg/L. Anti-nuclear antibody
Chronic type: (1) The onset of the disease is slow, with (ANA) 1:640 (+), homogeneous type, anti-double-stranded
long-term subcutaneous hemorrhage, gingival and oral DNA (dsDNA) antibody (+), Anti-Sm (+), RNP, rRNP and
mucosal hemorrhage, women often with menorrhea as the rheumatoid factor (RF), anti-neutrophil cytoplasmic anti-
main manifestation. (2) About 10% of recurrent cases had body (ANCA), and anticardiolipin antibody (ACL) were all
mild splenomegaly. (3) The moderate reduction of platelet negative.
was 30–80 × 109/L. This case was from China-Japan Union Hospital of Jilin
University.
32.2.3.3 Systemic Lupus Erythematosus (SLE)
It is an inflammatory connective tissue disease involving 32.2.3.4 Rheumatoid Arthritis (RA)
multiple organs and systems, which occurs in young A systemic connective tissue inflammation characterized by
women. Its clinical symptoms are complex, including fever, joint lesions occurs mostly in young adults, with more
rash, arthralgia, kidney injury, cardiovascular disease females than males.
(including pericarditis, myocarditis, and vasculitis), pleu- The main clinical manifestations are characterized by
risy, neurological symptoms, gastrointestinal symptoms, symmetry of the joints and surrounding tissues, multiple
anemia, etc. The disease is often progressive and difficult to lesions, some cases may have heart, lung, and vascular
alleviate. Immunological examination showed that IgG, involvement. Immunological examination showed that rheu-
IgA, and IgM increased significantly, especially IgG. There matoid factors appeared in serum and synovial fluid, and
were many kinds of autoantibodies (mainly anti-nuclear serum IgG, IgA, and IgM levels increased. Other
antibody series) and immune complexes in serum, and the autoantibodies can also appear, such as anti-keratin antibody,
level of complement decreased in active phase. Anti- anti-RA33 antibody, anti-cyclic citrulline antibody (anti-CCP
dsDNA and anti-Sm antibodies are characteristic markers antibody), anti-perinuclear factor, and so on, which are very
of the disease [47]. helpful for the early diagnosis of rheumatoid arthritis [48].
Experimental Diagnosis of SLE Case 3, RA

1.
Detection of anti-nuclear antibodies by The patient was a woman with multiple joint pain and swell-
immunofluorescence ing for 3 years. The affected joints included proximal and
454 H. Mu et al.
metacarpophalangeal joints of both hands, wrists, left ankle, usually lateral ophthalmoplegia, such as blepharoptosis, stra-
and knees. The patient complained of pain in the finger and bismus, and diplopia. In severe cases, eye movement is obvi-
wrist joints at the beginning, often migrating between these ously limited, and even the eyeball is fixed. Facial and
joints, accompanied by morning stiffness. pharyngolaryngeal muscles were involved in the expression
Examination: WBC 7.9 × 109, Hemoglobin 96 g/L, of indifference, bitter smile; continuous chewing weakness,
Platelet 369 × 109, ESR 48 mm/h, RF189 IU/mL, Anti-CCP drinking cough, dysphagia; speech with nasal sound, dyspho-
antibody 136.4 U/mL. Positive radiographs of both hands nia, etc. When sternocleidomastoid and trapezius muscles
showed that the space between proximal hands and metacar- were involved, it was difficult to raise the head, turn the neck,
pophalangeal joints was narrow, bone destruction, wrist joint and shrug the shoulders. Muscle involvement of extremities is
space narrow, bone destruction. The knee color Doppler mainly due to proximal weakness, which is manifested by
ultrasound showed that the bone surface of both knees was difficulty in lifting arms, combing hair, and climbing stairs.
rough, the synovium of both knees was thickened, and the Respiratory muscle involvement often leads to dyspnea.
lumen of both knees was hydrocele.
This case was from China-Japan Union Hospital of Jilin 32.2.3.7 P ulmonary Hemorrhagic Nephritis
University. Syndrome
Anti-glomerular basement membrane IV collagen antibody
32.2.3.5 Sjögren’s Syndrome (SS) can be detected in patients with good pasture syndrome.
Sjögren’s syndrome is a chronic autoimmune disease that Because alveolar basement membrane and glomerular base-
invades exocrine glands, especially salivary glands, and lac- ment membrane have common antigens, both lungs and kid-
rimal glands. It can involve multiple organs at the same time. neys develop simultaneously.
Sjogren’s disease and dry keratitis are the main clinical man- The main clinical manifestations: Before the onset of the
ifestations of SS. Anti-SSA and anti-SSB positive. disease, some patients had respiratory tract infection, and
The main clinical manifestations: (1) xerostomia: Most then repeated hemoptysis. Most of the patients appeared
patients complain of xerostomia, and the serious cases are before the kidney lesions. The elderly were years, the short
due to mucous membranes, teeth and tongue stickiness; ram- ones were months, and a few occurred after nephritis. When
pant dental caries is one of the characteristics of this disease; hemoptysis occurs, pulmonary diffusive function decreases,
Adult mumps; the tongue is characterized by tongue pain, hypoxemia occurs, and anemia is common. Kidney manifes-
dry and cracked tongue surface, atrophy and smooth tongue tations: proteinuria, erythrocyte, and tubular type were pres-
papilla; infection of ulcer or oral mucosa. (2) Dry keratocon- ent in each case, and gross hematuria was present. Renal
junctivitis: The mucin secreted by the lacrimal gland dysfunction, however, progresses at different rates. Some
decreases, and symptom such as dry eyes, foreign body sen- patients may present with acute renal failure within 1–2
sation, and less tears occur. Serious cases cry bitterly without days. Most of them develop to uremia within a few weeks to
tears. Some patients have recurrent suppurative infection of months. A few develop slowly and recur after stabilization or
eyelid margin, conjunctivitis, keratitis, etc. (3) The exocrine remission.
glands of superficial parts such as nose, hard palate, trachea Serological examination: Preliminary tests may include
and its branches, digestive tract mucosa, and vaginal mucosa anti-nuclear antibody (ANA) spectra, anti-double-stranded
may be involved, making them secrete less and showing cor- (ds) DNA, anti-neutrophil cytoplasmic antibody (ANCA),
responding symptoms. anti-basement membrane (GBM) antibody, and anti-
phospholipid antibody. Circulating anti-GBM antibodies
32.2.3.6 Myasthenia Gravis (MG) were positive in Goodpasture syndrome. Under the micro-
Acetylcholine receptor autoantibodies exist in patients, scope, P-ANCA was positive in polyarteritis, Churg-Strauss
which bind to the acetylcholine receptor, internalize and vasculitis, and oligoimmune glomerulonephritis (PIGN).
degrade it, reduce the reactivity of muscle cells to acetylcho-
line released by motor neurons, and cause skeletal muscle
motor weakness. 32.2.4 Major Immunological Detection
The main clinical manifestations: The prominent feature of Autoimmune Diseases
of myasthenia in MG patients is daily fluctuation. Myasthenia
aggravates after fatigue in the afternoon or evening and 32.2.4.1 D
etection of Autoantibodies and Its
decreases after morning rise or rest. All skeletal muscles can Clinical Significance
be involved in the whole body. Extraocular muscles are the The autoantibodies commonly detected in clinic include
most common, followed by facial and pharyngolaryngeal rheumatoid factor, anti-nuclear antibody, and anti-ENA
muscles and proximal limbs muscles. The first symptoms are antibody.
Systemic Lupus Erythematosus (SLE) 32.2.4.2 Clinical Significance

There are many kinds of autoantibodies (mainly anti-nuclear of Immunoglobulin and Complement
antibody series) in the serum of SLE patients, including anti- Examination
nuclear antibody, anti-DNA antibody, anti-extractable Significance of immunoglobulin detection: autoantibody
nuclear antigen (ENA) antibody, anti-Sm antibody, and so production, therefore, serum IgG increased significantly,
on. Anti-DNA antibody is divided into anti-double-stranded IgM, IgA also increased, the fluctuation of Ig content in dis-
DNA antibody and anti-single-double-stranded DNA anti- ease activity and stability is related, such as SLE, RA Ig
body. Anti-double-stranded DNA antibody and anti-Sm anti- increased.
body are characteristic markers of SLE [49]. Significance of complement detection: In AID induced by
SLE autoantibody detection methods: (1) The detection type II and III allergic mechanism, the consumption of com-
method of anti-nuclear antibodies is immunofluorescence plement increases and the content decreases during active
assay. (2) There are many methods for the determination of phase, and the disease alleviating complement can return to
dsDNA antibody, such as indirect immunofluorescence, normal. AID induced by autoreactive T lymphocyte had no
indirect enzyme-labeled antibody, complement-binding anti- significant change in complement.
body, enzyme-linked immunosorbent assay, and radioimmu-
noassay (Farr method). Farr method is the most specific and 32.2.4.3 Lymphocyte Detection
reliable method for the detection of dsDNA antibody. It is the AID is mostly related to autoantibodies, but the main patho-
internationally recognized standard method for the detection genesis of AID is the disorder of immune regulation and
of anti-dsDNA antibody. (3) Anti-extractable nuclear antigen response. Therefore, the change of lymphocyte number and
(ENA) antibody detection methods are including two- function is an important factor in mediating immunopatho-
dimensional immunodiffusion, convective immunoelectro- logical injury. If CD4/CD8 T cells were inverted during SLE
phoresis, Western blotting, and ELISA. activity, the level of cytotoxic T cells (CTL) increased, and
B1 cells in B cell subsets increased significantly.
Rheumatoid Arthritis (RA): Detection
of Autoantibodies to RA 32.2.4.4 Detection of Cytokines
Anti-keratin antibody, anti-RA33 antibody, anti-cyclic citrul- Immunoregulation disorder of AID is characterized by
line antibody (anti-CCP antibody), anti-perinuclear factor, imbalance of Th1 and Th2. Th1 cells activate and secrete a
and so on, these tests are very helpful for the early diagnosis large number of cytokines such as IFN-, IL-2, and TNF-
of rheumatoid. which can promote the production of TDTH, CD8+ CTL and
inhibit Th2 cells. Th2 cells can secrete a large number of
Autoimmune Hemolytic Anemia IL-4, IL-5, IL-10, IL-13 when activated, which promotes B
Anti-erythrocyte anti-autoantibody can appear, and its detec- cell activation to produce antibodies and inhibits Th1 cells.
tion is helpful to the diagnosis of the disease. Anti-erythrocyte
anti-autoantibodies fall into three categories. (1) Temperature 32.2.4.5 Application Principles
antibody: IgG type, binding to RBC at 37 °C, not aggregat- of Immunoassay for Autoimmune
ing RBC. (2) Condensin: IgG type. It can bind with RBC to Diseases
agglutinate at low temperature, causing agglutinin syndrome. Firstly, ANA, IgG, IgA, IgM, complement (CH50 or C3),
(3) Donath Landsteiner antibody: IgG type. Low tempera- RF, and so on were detected. Then ENA, dsDNA, anti-keratin
ture binds to two complement components. When the tem- antibody, and anti-CCP antibody were detected. The final
perature rises to 37°, it activates complement, leading to decision can be made after combined with clinical manifes-
hemolysis and paroxysmal cold hemoglobinuria (PCH) [50]. tations, dynamic observation of laboratory indicators, and
selection of appropriate examination items.
Sjögren’s Syndrome (SS)
1. The detection methods of anti-SS-A and anti-SS-B anti-
bodies include two-way diffusion (DID), convective 32.3 A
cquired Immune Deficiency
immunoelectrophoresis (CIE), and immunoblotting (IBT). Syndrome
2. Other immunological abnormalities are including hyper-
gammaglobulinemia and polyclonal, causing increased Yan Zhang and Huanhuan Chen
erythrocyte sedimentation rate. RF is mostly IgM type,
IgG and IgA type are few, CIC can be positive. Acquired immune deficiency syndrome, which is known
3. Other autoantibodies in SS patients are anti-nuclear anti- as AIDS, is a mixed immunodeficiency disease that caused
bodies (ANA). by human immunodeficiency virus (HIV) infection and char-
4. Tear secretion test and salivary secretion test [51]. acterized by T cell immunodeficiency. It targets the most
456 H. Mu et al.
important T4 lymphocytes in the body’s immune system,

env gp120
devours and destroys the T4 lymphocytes in large quantities, env gp41
which lead to the entire destruction of the body’s immune
system and ultimately the loss of human resistance to dis-
gap p17
eases and death.
RNA
gap p24
32.3.1 Overview proteins P7,P9
AIDS is a highly harmful infectious disease caused by HIV

infection. HIV is a virus that attacks the body’s immune sys-
tem. It takes the most important CD4 T lymphocytes in the
human immune system as the main target, destroying the
cells in large quantities and depriving immune function. As a
Fig. 32.4 HIV gene structure
result, the human body is prone to various diseases, malig-
nant tumors may occur, and mortality rate is high [52].
Human immunodeficiency virus (HIV), which is known been shown to neutralize antigenic epitopes. On the gp120
as AIDS (Acquired Immunodeficiency Syndrome) virus, is a V3 loop, the V3 loop is an important functional region of
virus that can cause defects in the human immune system. In envelope proteins and plays an important role in the fusion of
1981, the human immunodeficiency virus was first discov- viral and cell. Gp120 is linked to the transmembrane protein
ered in the United States. It is a lentivirus that infects human
gp41 by a non-covalent bond. Gp41 fuses with target cells,
immune system cells and is a type of retrovirus. causing the virus to enter cells. Experiments have shown that
gp41 also has strong antigenicity and can induce antibody
32.3.1.1 HIV Morphology responses.
HIV virus particles are spherical, icosahedral symmetrical, The TaT gene-encoded protein binds to LTRs to increase
90~130 nm in diameter, with high density tapered core and the transcription rate of all genes in the virus, and it also
glycoprotein spike mosaic envelope. The core contains two promotes translation of viral mRNA after transcription.
copies of RNA molecules, viruses, reverse transcriptase The Rev gene product is a cis-acting factor that inhibits
(p66, p51), protease (p15), integrase (p31), etc. the expression of cis-acting repression sequence (Crs) in env
and gag, which enhances the expression of gag and env
32.3.1.2 HIV Gene Structure genes, and synthesizes the corresponding viral structural
The viral genome consists of two identical positive strand proteins.
RNAs, each of which is about 9.2–9.8 kb in length. Both The Nef-encoded protein P27 has a negative regulatory
ends are long terminal repeats (LTRs) containing cis- effect on the expression of HIV gene to delay viral replica-
regulatory sequences that control the expression of pro- tion. This protein acts on the LTRs of the HIV cDNA, inhib-
viruses. Promoters and enhancers have a negative regulatory its integrated viral transcription, which may be necessary for
region in the LTRs. The sequence between LTRs encodes at HIV to maintain a sense of continuity in the body.
least nine proteins and can be divided into three categories: The Vif gene is not essential for HIV, but may affect the
structural proteins, regulatory proteins, and accessory pro- free HIV infectivity, virion production, and in vivo
teins (Fig. 32.4). transmission.
The gag gene encodes a polymeric precursor protein of The VPU gene is unique to HIV-1, and it is also essential
about 500 amino acids, which is hydrolyzed by protease to for efficient replication of HIV, assembly, and maturation of
form P17, P24 nuclear protein, thus RNA will not be virions.
destroyed by external nucleases. The Vpr gene-encoded protein is a weak transcriptional
The Pol gene encodes a polymerase precursor protein that activator that plays a role in the in vivo reproductive cycle.
be cleaved to form protease, integrase, reverse transcriptase, The HIV-2 gene structure differs from HIV-1 in that it does
and ribonuclease H, all of which are required for viral not contain the VPU gene but has an unidentified VPX gene.
proliferation. The nucleotide sequence of HIV-1 and HIV-2 were exam-
The env gene encodes a precursor protein of approxi- ined by nucleic acid hybridization, which were only 40%
mately 863 amino acids and is glycosylated to gp160, gp120, identical. The env gene expression product stimulates the
and gp41. Gp120 contains a neutralizing epitope, which has antibody produced by the body without cross-reactivity.
32.3.1.3 Virus Characteristics Generally, the initial symptoms are like the cold and flu.
The main helper of the human T lymphocyte system, once They may have general fatigue, loss of appetite, fever, etc.
invaded into the body cells, the virus will be integrated with As the condition worsens, the symptoms increase day by
the cells, which will be difficult to eliminate for life; day. For example, candida infection occurs in the skin and
It has been widely found in the blood, semen, vaginal mucous membranes, herpes simplex, herpes zoster, purple
secretions, milk, cerebrospinal fluid, and brain tissue fluid spots, blood blisters, blood spots, etc., may occur; gradually
with neurological symptoms in infected person. Among invade internal organs, persistent fever of unknown cause,
them, blood, semen, and vaginal secretions have a higher up to 3–4 months; cough, shortness of breath, difficulty
concentration; breathing, persistent diarrhea, blood in the stool, hepato-
The resistance to the external environment is weak. The splenomegaly, complicated with malignant tumors. The
effective disinfection method for hepatitis B virus is also clinical symptoms are complex and variable, but not all of
effective for disinfecting HIV; the above symptoms appear in every patient. Invasion of the
The infected person has a long incubation period and a lungs often causes difficulty in breathing, chest pain, cough,
high mortality rate; etc.; invading the gastrointestinal tract can cause persistent
The genome of HIV is more complex than any known diarrhea, abdominal pain, breathing, weight loss, etc.; it can
viral gene. also invade the cardiovascular system and the nervous
system.
32.3.1.4 Route of Transmission of AIDS
AIDS is transmitted through blood, sexual contact, and 32.3.2.1 General Symptoms
mother-to-child. Sustained fever, weakness, night sweats, and extensive gen-
AIDS is transmitted through sexual intercourse (vaginal eralized lymphadenopathy are the general symptoms.
pay, oral sex, anal sex) between men and women. The greater Especially in the neck, armpits, and inguinal lymph nodes
risk of AIDS infection is more sexual partners. are more obvious. Lymph nodes are more than 1 cm in diam-
HIV can spread AIDS through contaminated blood or eter, with a solid texture, active, painless. Weight loss can
blood products. They can enter human body through non- reach more than 10% within 3 months; up to 40%, the weight
strict sterilization surgery, injections, acupuncture, dental, loss of patients is particularly noticeable.
beauty, and other equipment.
Women infected with AIDS are likely to transmit HIV to 32.3.2.2 Respiratory Symptoms
their fetus or baby. Without precautions, about a third of the Long-term cough, chest pain, difficulty breathing, and blood
fetus and infant will be infected. in the sputum are the typical respiratory symptoms.
People who shaking hands, hugging, kissing a courtesy,
eating common meals, sharing work tools, office supplies, 32.3.2.3 Digestive Symptoms
such as coins in their daily lives and work with AIDS patients Loss of appetite, anorexia, nausea, vomiting, diarrhea, and
or people living with HIV will not be infected. blood in the stool are obviously when severe. Drugs that
AIDS will not spread through public facilities such as toi- commonly used to treat digestive tract infections are ineffec-
let, telephone, tableware, bedding, bath, or pool. Mosquito tive for this type of diarrhea.
bites do not infect AIDS. Coughing and sneezing do not
spread AIDS [53]. 32.3.2.4 Nervous System Symptoms
Nervous system symptoms are dizziness, headache, unre-
sponsiveness, mental retardation, mental disorders, convul-
32.3.2 Clinical Appearance sions, hemiplegia, and dementia, etc. [54].
The incidence of AIDS is higher in young adults, and 80% of 32.3.2.5 Skin and Mucous Membrane Damage
the cases were between 18 and 45 years old, which is the age Skin and mucous membrane damage are herpes simplex,
of active sexual activity. Later stage of AIDS is often accom- herpes zoster, inflammation of the mouth and throat mucosa,
panied by some rare diseases such as pneumocystis pneumo- and ulceration.
nia, toxoplasmosis, atypical mycobacteria, and fungal
infections. 32.3.2.6 Tumor
After HIV infection, there is no clinical manifestation in A variety of malignant tumors may occur, red or purple-red
the first few years to more than 10 years. Once developed rashes, papules, and infiltrative lumps can be seen on the sur-
into AIDS, patients can have many clinical manifestations. face of the Kaposi sarcoma [55].
458 H. Mu et al.
32.3.3 Detection Method as a confirmatory experiment because it has a relatively long

window period, is slightly less sensitive and costly. With the
The method for detecting viral antigens and antibodies in increased sensitivity of third- and fourth-generation HIV
body fluids of HIV-infected person is convenient to operate diagnostic reagents, WB has become increasingly unable to
and easy to popularize. At present, antibody detection is par- meet its requirements as a confirmatory experiment.
ticularly common; however, the determination of HIV P24 Another type of screening confirmation reagent approved
antigen and viral genes has become an increasingly impor- by the FDA is the Immunofluorescence-Test (IFA). IFA is
tant place in the detection and importance of HIV infection. less expensive than WB and is relatively simple to operate,
and the entire process ends in 1–1.5 h. The main disadvan-
32.3.3.1 Antibody Detection tage of this method is that it needs expensive fluorescence
HIV antibodies in serum are an indirect indicator of HIV detectors, experienced professionals to observe and evaluate
infection. According to its main scope of application, exist- the results, and the results cannot be preserved for a long
ing HIV antibody detection methods can be divided into time. The FDA now recommends that negative or positive
screening tests and confirmatory tests. IFA results be used when the final result of a blood donor
cannot be determined by WB, but not as a criterion for blood
Screening Test eligibility.
Screening tests are mainly used to screen blood donors, so
they require easy operation, low cost, and are sensitive and 32.3.3.2 Antigen Detection
specific. In 2012, the world’s main screening method was Pathogen detection mainly refers to direct detection of virus
still ELISA, with a few particle agglutination reagents and or viral genes from host samples by virus isolation and cul-
rapid ELISA reagents. The ELISA has high sensitivity and ture, electron microscopic observation, viral antigen detec-
specificity and is easy to operate. It only needs to be equipped tion, and gene assay. As the first two methods are difficult,
with a microplate reader and a plate washer in the laboratory. and special equipment and professional technicians are
It is especially suitable for large-scale screening in the needed, only antigen detection and RT-PCR (reverse tran-
laboratory. scription PCR) can be used for clinical diagnosis. The HIV-1
Particle agglutination test is another simple and conve- P24 antigen test can be used for the uncertainty or window
nient operation method with low cost. The method can be diagnosis of HIV-1 antibody; the early differential diagnosis
judged by the naked eye and has high sensitivity. It is espe- of infants born to HIV-1 antibody-positive mothers; positive
cially suitable to use in developing countries or a large num- result of fourth-generation HIV-1 antigen/antibody ELISA
ber of blood donors. The disadvantage is that the samples reagent while the HIV-1 antibody is confirmed to be nega-
must be fresh. tive. P24 antigen detection generally uses ELISA double
The Dot-blot assay developed in the late 1980s is an antibody sandwich reagent, the reagent must be approved by
extremely simple, short-lived, rapid ELISA method. Most of SDA for registration, and positive result must be confirmed
the process can be done within 5–10 min or even 3 min. But by the neutralization test according to the reagent specifica-
the method is much more expensive than ELISA and particle tion. The sensitivity of HIV-1 P24 antigen detection is
agglutination reagents. 30–90%. This result is only used as a supplementary diagno-
The human immunodeficiency virus antibody oral muco- sis for HIV infection and cannot be diagnosed accordingly.
sal exudate detection kit (colloidal gold method) belongs to Negative result of HIV-1 P24 antigen test only indicates that
the category of lateral immunochromatography (gold immu- there is no response in this test, and HIV infection cannot be
noassay). Based on immunochromatography technology, excluded. Generally, it is not used as a routine diagnostic
with manual operation, visual reading results, 20 min rapid item in the clinic.
diagnostic reagent, qualitatively yields test results, this
method can be used for the detection of HIV-1 and HIV-2 32.3.3.3 Nucleic Acid Detection
antibodies in oral mucosal exudate samples. It can be used HIV nucleic acid testing can be used for the auxiliary diag-
for initial screening of patients who are willing to consult nosis of HIV infection, disease monitoring, guiding treat-
and test, who do not want to take blood or fainting. This ment programs and efficacy judgment, predicting disease
method is suitable for primary screening test. Anyone who is progression, etc. Commonly used methods for detecting HIV
positive to the reagent needs further screening and viral load include RT-PCR (reverse transcription PCR),
confirmation. NASBA (nucleic acid sequence amplification), branched
DNA (bDNA) hybridization, and real-time PCR. It is worth
Confirmatory Reagent noting that each HIV RNA quantification system has its low-
The most commonly used method to confirm screening test est detection limit, which is the lowest copy number or inter-
positive serum is Western blot (WB), which is only suitable national unit that can be measured. When RNA is not
detected in the RNA quantitative test, it does not mean that amplification of RNA by nucleotide sequences. The prin-
the sample does not contain viral RNA, thus the HIV nucleic ciple of NASBA is to extract viral RNA and amplify it by
acid is qualitative. Negative test can only report the negative adding AMV reverse transcriptase, nuclease H (Rnase H),
results of this experiment, but cannot exclude HIV infection; T7 RNA polymerase and primers.
positive HIV nucleic acid test can be used as an auxiliary 3. LCR (Ligase Enzymatic Chain Reaction): A probe ampli-
indicator for the diagnosis of HIV infection, while it cannot fication technique based on the interconnection of target
be used alone in the diagnosis of HIV infection. When molecule-dependent oligonucleotide probes, which is a
reporting the results of HIV nucleic acid quantitative test, the promising new in vitro development after PCR.
instrument reading should be reported, which indicating the 4. Gene chip detection technology: It is a combination of
experimental method, sample type and sample size used. PCR technology and nucleic acid molecule hybridization.
When the measurement result is less than the minimum By using HIV genome analysis, the highly conserved
detection limit, the minimum detection limit level should be sequence of the virus is used as an identification index to
indicated. directly detect viral pathogens, which greatly improves
HIV nucleic acid qualitative testing can also be used for the accuracy of diagnosis.
the auxiliary diagnosis of HIV infection and is applied in
basic research such as analysis of HIV gene subtypes and 32.3.3.4 C D4+ and CD8+ T Lymphocyte
mutations. PCR or RT-PCR is commonly used technique, Detection
and amplification reagents are commonly used in molecular T lymphocyte is the most important cell group in the body’s
biology laboratories. Primers can be designed from the lit- immune system. They maintain the body’s immune function
erature or by themselves. All or common strains should be of normal organs. HIV mainly invades human CD4+ T lym-
covered as much as possible, or composite primers can be phocytes, causing defects in their number and function,
used. The qualitative test results should be reported with the destroying the body’s immune function. HIV infection patient
reaction conditions and the primer sequences used. In addi- with CD4 count below 200 or an AIDS-defining illness is the
tion, by using the high sensitivity of nucleic acid detection criteria for a diagnosis of AIDS. It eventually leads to a vari-
method, using integrated nucleic acid amplification detec- ety of opportunistic infections and tumors. CD4+ and CD8+
tion technology and method, collection nucleic acid detec- T lymphocytes can be used to understand the immune status
tion can be performed on the highly suspected infected of HIV-infected patients in disease stage, to understand the
population, antibody negative sample and infected person in patients’ condition based on the disease stage, and to deter-
the window period. This method is more cost-effective than mine the routine treatment plan and drug efficacy.
single sample nucleic acid detection.
32.3.3.5 Virus Isolation and Culture
Qualitative Detection When the human body is infected with the virus, it will be
PCR technology is commonly used to detect HIV RNA from carried for life. HIV is widely distributed in human lympho-
the plasma of patients 1–14 days after HIV infection. It can cytes, monocytes, macrophages, tissues, blood, and other
be used for the diagnosis of patients with acute infection, body fluids. Isolation and cultivating HIV from these materi-
uncertainty of antibody detection and blood screening. als can study the distribution, variation, and drug resistance
of HIV in humans.
Quantitative Detection
1. RT-PCR (Reverse transcription PCR) technique: Viral
32.3.3.6 HIV-1 Genotype Resistance Detection
RNA is retrieved into cDNA by reverse transcriptase, fol- With the development of antiviral therapy and clinical appli-
lowed by polymerase chain reaction, exponential amplification of novel target antiviral drugs, more than 200 gene
cation of DNA fragments, denaturation of the amplified mutations that related to various types of HIV-1 have been
product, and binding to multiwell plates, which is detected identified, which have different impaction of drug resistance
by enzyme-linked system. Since RT-PCR technology can and viral biological characteristics. At present, there are two
amplify products to the level of gel electrophoresis or methods for detecting drug resistance, one is phenotype
real-time fluorescence within 2 h, a variety of improved resistance drug analysis, the virus needs to be cultured
rapid RT-PCR assays can achieve rapid clinical diagnosis in vitro, which is complicated and expensive. Therefore, the
of HIV. rapid and accurate detection of genes is becoming more and
2. NASBA (Nucleic acid sequence-dependent amplifica-
more important for clinical drug guidance.
tion): A new technique that based on RT-PCR while there AS-PCR (Allele specific PCR), RFMP (restriction frag-
is no need to divide reverse transcription and PCR ampli- ment mass polymorphism), Gene chip technology, Sanger
fication into two steps, consisting of a pair of primer- sequencing, and Deep sequencing can be used for detection
mediated, continuous, in vitro specific isothermal of HIV-1 genotype resistance.
460 H. Mu et al.
32.3.4 Therapy develops, HIV is likely to develop cross-resistance to other

PIs. Since the development of combination therapies, drug
There is still a lack of effective drugs to eradicate HIV resistance and cross-resistance have become the worst set-
infection worldwide. The current treatment goals are as fol- backs in the fight against HIV/AIDS [58].
lows: maximizing and lasting reduction of viral load; obtain-
ing immune function reconstruction and maintaining
immune function; improving quality of life; reducing HIV- References
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Liver Diseases
33
Qishui Ou, Hong Mu, Chunlei Zhou, Zhaojing Zheng,
and Juan Geng
The liver, with four lobes of unequal size and shape, is a eases, etc. In this chapter, the application and the latest
reddish-brown, wedge-shaped organ with dual blood supply. progress of molecular diagnosis in some common liver dis-
As the heaviest parenchymatous organ in the human body, it eases are presented.
mainly lies in the right upper abdomen and a small part in the
left rib region, that is, it is below the diaphragm, nearby the
stomach, and covers the gallbladder (Fig. 33.1). Lobules, the 33.1 Viral Hepatitis
functional units of the liver, are composed of millions of
hepatocytes which are radially arranged in the center of cen- Qishui Ou
tral veins.
The liver is a huge “chemical plant” of the human body
and up to thousands of biochemical reactions are finished in 33.1.1 Overview
the hepatocytes. Firstly, the liver acts as a vital role in quanti-
ties of nutritional substance metabolism such as carbohy- Viral hepatitis, a severe international public health problem,
drate, fat, protein, etc., meanwhile it is involved in the is caused by hepatitis viruses which are a group of viruses
synthesis and storage of vitamins and the inactivation of hor- that mainly infect the hepatocytes. At present, there are five
mones. Secondly, as the largest digestive gland, the liver can recognized human viral hepatitis: hepatitis A, B, C, D, and
secret bile which can promote the metabolism of lipid. E. Prior to the treatment of viral infection, we need to detect
Thirdly, toxins, metabolites of drugs, and some wastes gen- virus, viral components, and the related antibodies, which can
erated by metabolism can be detoxified in the liver. Fourthly, provide reliable basis for identification of the pathogeny, ill-
the liver has a great importance in regulating the dynamic ness assessment, and treatment plan. The detection is usually
balance of the body’s coagulation and anticoagulation sys- performed using immunological methods in the clinic which
tems, because it produces almost all coagulation factors. could not satisfy the demand for the early diagnosis and treat-
Finally, the liver can exert immunity by phagocytizing, iso- ment of viral infection due to the unsatisfactory sensitivity or
lating, and eliminating exogenous and endogenous antigens. specificity. And molecular biological methods have been
Though it is an indispensable organ in sustaining life, the widely used in early diagnosis, genotyping, drug resistance
liver has the tendency toward many diseases. According to monitoring, and dynamic monitoring of viral load in recent
the etiology, liver diseases can be divided into infectious years. The virus-specific genes and the whole genome can be
diseases, tumor diseases, vascular diseases, metabolic dis- directly sequenced by molecular biological methods with the
eases, toxic diseases, autoimmune diseases, genetic disadvantages of high efficiency, sensitivity, and specificity.
Q. Ou (*) 33.1.2 Hepatitis A

The First Affiliated Hospital of Fujian Medical University,
33.1.2.1 Overview
H. Mu (*) · C. Zhou Hepatitis A results from hepatitis A virus (HAV) infection
Tianjin First Central Hospital, Tianjin, People’s Republic of China
which is classified into the Picornaviridae family. The trans-
Z. Zheng (*) · J. Geng mission of HAV is mainly through the fecal-oral route, that is,
Shanghai Children’s Medical Center, Shanghai Jiao Tong
University School of Medicine, Shanghai, people ingest the food or water that is contaminated by feces
People’s Republic of China occasionally. The patients and recessive infected people are

464 Q. Ou et al.
Fig. 33.1 Anatomical
structure of the human liver
the sources of transition who excrete HAV with their feces. Nucleic Acid Molecular Hybridization
Fortunately, with the use of inactivated vaccines worldwide, RNA is extracted from the patient’s blood sample and hybrid-
the epidemic of hepatitis A has been effectively controlled. ized with HAV gene probes labeled with radionuclide or
non-radionuclide. Detection of hybridization signals can
33.1.2.2 Clinical Appearance determine whether HAV RNA exists in samples. Nucleic
The clinical manifestation of HAV infection is related to the acid molecular hybridization is more sensitive than RIA or
age of the patient and the virus load, which mainly includes ELISA.
the digestive tract symptoms, fever, fatigue, anorexia, and so
on. During the infection, the body would produce the corre- RT-PCR
sponding antibodies which have neutralizing activity, and HAV RNA is extracted and reverse transcribed into
then the body will gain resistant to HAV once recovery. cDNA. The amplified products by PCR are separated by
SDS-PAGE and identified by staining dyes (e.g., ethidium
33.1.2.3 Laboratory Diagnosis bromide).
Immunological Detection 33.1.2.4 Management

Commonly, Enzyme-Linked Immunosorbent Assay (ELISA) Hepatitis A is a self-limited disease. Currently, it is mainly
is mainly applied to detect viral antigen; antibodies includ- given supportive therapy and symptomatic treatment.
ing IgM and IgG are usually detected at the same time: the Patients with acute jaundice hepatitis should be treated in
former increases 1–4 weeks after acute infection and lasts for isolation ward about 3 weeks after onset until the clinical
a short time (3–6 months) which could be indicators of acute symptoms disappear. It is greatly emphasized that the
infection; the latter usually appears 4 weeks after infection patients should have early bed rest; they can gradually
and peaks at week 24, which last for many years or even for increase activity until symptoms decrease.
a lifetime. The titer of antibodies could be dynamically mon-
itored to evaluate the vaccination effect and the epidemio- 33.1.2.5 Typical Medical Case
logical investigation. Clinical Background A 27-year-old male smoker was
admitted for complaining of fatigue and poor appetite for 8
Molecular Biological Detection days.
There are two main types of HAV RNA detection methods:
nucleic acid molecular hybridization and reverse transcription Initial Laboratory Values Anti-HAV IgM antibodies 10.42
polymerase chain reaction (RT-PCR). However, neither of COI, TBIL 47.4 μmol/L, DBIL 39.2 μmol/L, IBIL
them is recommended for routine detection of suspected acute 8.2 μmol/L, ALB 37.6 g/L, ALT 540 U/L, AST 72 U/L, GGT
hepatitis A. 158 U/L.
33 Liver Diseases 465
Hepatic Magnetic Resonance Imaging (MRI) (1) 33.1.3.3 Laboratory Diagnosis

Heterogeneous fatty liver (2) Hilar lymph node
enlargement. Immunological Detection
Laboratory Test Performed Anti-HAV antibody is used HBsAg

for distinguishing hepatitis A from other confusing diseases. HBsAg is a glycoprotein presenting on the surface of Dane
The other tests were all for auxiliary diagnosis. Antibody granules, which can be detected in the 4–7 weeks after infec-
detection is commonly used screening method. tion. It can stimulate the body to produce protective antibod-
ies—anti-HBs. As one of the indicators of infectivity, HBsAg
Final Diagnosis Several tests were performed for screen- indicates that the liver has been infected by HBV, thus it can
ing this disease, such as anti-HAV antibodies, TBIL, ALT, be found in (1) latent and acute phase of hepatitis B; (2)
AST, and that sort of things. The diagnosis can be con- chronic HBsAg carriers.
firmed by positive anti-HAV IgM antibodies test. In addi-
tion, since the IgM antibodies could be detected several Anti-HBs Antibodies
days after infection, anti-HAV IgM antibodies should be They are one kind of protective antibodies found in (1)
tested dynamically even though the initial result is negative recovery period of acute hepatitis B; (2) vaccination of hepa-
if the patient is suspected of hepatitis A. Therefore, accord- titis B vaccine; (3) passive acquisition of antibodies.
ing to the above examination results, this patient was diag- Therefore, they are often used as an indicator of rehabilita-
nosed as hepatitis A. tion after acute infection or the effectiveness evaluation.
This case was from the First Affiliated Hospital of Fujian HBeAg
Medical University. As an indicator of HBV replication activity and high infec-
tivity, it appears at the same time with or later than HBsAg.
The positive rate is related to the titer of HBsAg, viral DNA
33.1.3 Hepatitis B replication, and liver damage. Continuously positive HBeAg
for more than 10 weeks suggests a transition to chronic liver
33.1.3.1 Overview disease. At the same time, it can be used to evaluate antiviral
The pathogen of hepatitis B is hepatitis B virus (HBV). treatment. Quantitative detection of HBeAg is of great
Virtually, there are probably 2 billion people afflicted with importance to predict persistent virological response.
HBV, of which approximately 257 million are chronic hepa-
titis B patients, especially in Africa and Asia countries [1, 2]. Anti-HBe Antibodies
Thanks to improvement in the socioeconomic status, avail- Compared with anti-HBs antibodies, anti-HBe antibodies
able antiviral treatments, and universal vaccination pro- have no neutralization activity. Anti-HBe antibodies are
grams, the prevalence has been significantly controlled. common in (1) patients with negative HBeAg in the conva-
However, what inevitable is that the epidemic situation is lescent stage of hepatitis B. They indicate that: (a) part of
still a fluctuation in Europe (e.g., Italy, Germany) due to the HBV is inhibited or cleared; (b) replication is weakened; (c)
flowing population. It was reported that the death toll associ- infectivity is reduced. (2) Some chronic hepatitis, cirrhosis,
ated HBV had increased by 33% from 1990 to 2013, which and hepatocellular carcinoma (HCC) result from HBV.
was 686,000 cases in 2013 [3].
HBcAg
33.1.3.2 Clinical Appearance The existence of HBcAg reveals the replication of HBV and
Generally, asymptomatic acute infection may occur to peo- is helpful to evaluate the curative effect and prognosis of
ple at any age, including infants, children, and the immuno- hepatitis B. However, it is difficult to be detected due to its
suppressed, such as the HIV infected, are susceptible to existence inside of the hepatocytes.
HBV. When infected, most patients may be asymptomatic,
while some of them may have jaundice and dark urine along Anti-HBc Antibodies
with hepatomegaly and tenderness. As for the chronic Anti-HBc-IgM antibodies are the markers of early infection
patients, they may have fatigue or loss of appetite. The con- of HBV and are of great significance for acute HBsAg-
dition of the patients would get worsen as their disease pro- negative hepatitis B: (1) they are indicators of viral replica-
gresses over time if not well control [4]. tion activity; (2) they can be used to identify chronic active
466 Q. Ou et al.
hepatitis. Anti-HBc-IgG antibodies are the earliest nontarget probe 1-sequence-target probe 2-bDNA complex is
neutralizing antibodies found in convalescence from acute formed, and the content of nucleic acids could be quantified
infection and chronic persistent infection. High titer of by the catalytic reaction of the enzymes. The most important
anti-HBc-IgG indicates current HBV, while low titer is an feature of bDNA technology is that it is a direct detection of
indicator of the previous infection, which is of epidemiologi- target nucleic acid with fixed magnification. In addition, it is
cal significance. a detection of high stability and high repeatability, with accu-
rate results and less pollution during operation. The disad-
Molecular Biological Detection vantage of the bDNA technique is that it has few amplification
Quantitative Analysis of HBV DNA multiples, low sensitivity, and narrow detection range, there-
HBV DNA serves as a symbol of viral replication and trans- fore it is not suitable for low-level detection. Compared with
mission. Quantitative detection of HBV DNA exerts an enor- 3.5 × 105–5.7 × 107 copies/mL of the detection range of the
mous function in evaluating viral replication, infectivity, and first generation of bDNA technique, the second generation of
the efficacy of antiviral drugs. bDNA technology could reach 2 × 105 copies/mL, but it is
still not sensitive enough, which limits the application of this
Run-In PCR Technology A large number of specific cop- method.
ies of nucleic acid fragments can be obtained quickly and
conveniently by the conventional PCR method. However, it Run-In Nucleic Acid Hybridization The specimens are
has been replaced by other methods due to its incompetence dotted on the cellulose nitrate film and hybridized with
of gene quantification, poor repeatability, high false-positive labeled HBV DNA oligonucleotide probes to detect HBV
rate, and the use of ethidium bromide which is a strong DNA in the specimens, which could be qualitatively or semi-
carcinogen. quantitatively without special equipment. Nucleic acid
probes labeled with I125 are used in liquid phase hybridiza-
Run-In Fluorescence Quantitative Polymerase Chain tion, whose lower detection limit is 1 × 106 copies/mL or
Reaction HBV DNA could be quantified by fluorescence 1–2 pg/mL.
quantitative PCR, which could accurately reflect the replica-
tion of HBV DNA, the patient’s condition, and the treatment Quantitative Analysis of HBV RNA
effect. The fluorescence quantitative PCR kit of high speci- HBV RNA is a 3.5 kb pre-genomic RNA (pgRNA) tran-
ficity and high sensitivity has been developed whose detec- scribed by HBV DNA, which is released to the extracellular
tion linear range is 2.5 × 102–2.5 × 109 copies/mL, so space binding to its corresponding capsid protein in the cyto-
fluorescence quantitative PCR has been widely used in the plasm without reverse transcription. Some studies have
clinic. found that HBV RNA in the serum is significantly correlated
with HBV DNA, suggesting that HBV RNA is likely to
Run-In Hypersensitive HBV DNA Technology HBV become a marker in the diagnosis of HBV infection [5]. At
DNA in samples is captured and purified by magnetic bead present, HBV RNA is mainly detected by fluorescence-
adsorption. The internal standard sample is added to the labeled PCR technology. In addition, pgRNA could be
extracted DNA and then the mixture is amplified. There is detected by rapid amplification of the end of the cDNA and
not only a high recovery rate and good repeatability, but also micro-drop digital PCR respectively.
an accurate result because the difference among samples due
to nucleic acid extraction and amplification is eliminated. HBV Typing
There is also a wide detecting range compared with the tech- The pathogenic HBV can be described by different serotypes
nology of Fluorescence Quantitative Polymerase Chain or genotypes. Based on the antigenicity of HBsAg, HBV is
Reaction, Hypersensitive HBV DNA Technology is more classified into ten serotypes, among which adr, adw, ayw,
accurate and reliable in the quantification of low viral load and ayr are the predominant ones. The distribution of sero-
(<104 IU/mL), whose lowest detectable level could reach types varies between regions and races. Adr is the dominant
10 IU/mL. serotype in Chinese Han nationality, followed by adw.
Based on the principle that a genotype could be identified
Run-In Branched-Chain DNA (bDNA) Technique It is a if the total nucleotide sequence of HBV differs by 8% or
hybridization signal amplification technique labeled with more, or if the S gene sequence by 4% or more, nine geno-
nucleic acid probes, which is different from PCR. Branched types have been identified. The sequence length varies in dif-
DNA is a synthetic DNA fragment with many side chains, ferent genotypes especially in the pre-S1 region. At present,
which could be labeled and activated. In the system, the tar- HBV genotyping could be done by Sanger sequencing,
get nucleic acid is not amplified, and the signal of the labeled reverse hybridization, gene chip, and so on. Ou and col-
nucleic acid probes is amplified step by step. After complet- leagues have established a melting curve typing method,
ing the hybridization reaction, a solid-phase-capture probe- which could distinguish B and C that are prevalent in China;
it is simple, rapid, practical, and low cost [6]. In addition, Ou into HCC. Hepatic failure deserved to be seriously noticed in
and colleagues have also established a double molecular bea- acute hepatitis B. Otherwise, antiviral therapy should be
con real-time PCR method for HBV DNA typing and quan- adopted to block maternal-neonatal transmission and to pre-
titative dimerization, whose results are accurate and helpful vent HBV-associated extrahepatic manifestations [10].
in clinical practice [7]. HBV serotype and genotype have
certain correspondence, but in some cases, different sero- 33.1.3.5 Typical Medical Case
types can be of the same genotype, while the same serotype Clinical Background A 47-year-old male smoker was
can be of different genotypes. admitted for discomfort in the right upper abdomen and
fatigue for at least 2 months.
Analysis of Drug Resistance of HBV
In the process of HBV replication, reverse transcription is Initial Laboratory Values HBsAg 380.52 IU/mL, anti-
essential in which HBV DNA polymerase works as a cata- HBs antibodies (−), HBeAg 1466.92 S/CO, anti-HBe anti-
lyst. HBV DNA polymerase is a reverse transcriptase that bodies (−), anti-HBc antibodies 9.09 S/CO, HBV DNA
contains both DNA polymerase functionality and RNase H 7.23 × 106 IU/mL, TBIL 20.2 μmol/L, DBIL 9.4 μmol/L,
functionality. And the defective repair function of HBV ALB 37.6 g/L, ALT 141 U/L, AST 121 U/L, GGT 144 U/L.
DNA polymerase results in the spontaneous mutation of
HBV DNA which could reach 105 times. Long-term antiviral Hepatic MRI (1) Cirrhosis, splenomegaly, multiple
treatment could also induce HBV mutation. In addition, enlarged lymph nodes in the hilar region; (2) atypical cavern-
HBV mutation occurs during immune response or ous hemangioma in segment IV of the liver; (3) small cysts
vaccination. in segment VII of the liver.
Nucleoside (acid) analogs are nucleoside drugs that can
inhibit HBV replication. The mechanism is that nucleoside Results with Interpretation Guideline The diagnosis of
(acid) analogs competitively bind to HBV DNA polymerase hepatitis B should combine signs, symptoms with laboratory
whose natural substrate is dNTP, and the synthesis of HBV results., For laboratory test, the assessment of serum markers
DNA stops which leads to inhibiting the replication. and hepatic fibrosis which can provide guidance of treat-
Therefore, the binding ability of nucleoside (acid) analogs ment. In addition, the use of non-invasive tests such as
with HBV DNA polymerase determines the efficacy of these abdominal ultrasound and elastography also are crucial in
drugs. When the amino acid sequence of HBV DNA poly- the evaluation of diagnosis, treatment, and prognosis.
merase and its spatial conformation change, the binding abil-
ity of nucleoside (acid) analogs to HBV DNA polymerase Final Diagnosis The positive results of HBsAg, HBeAg
decreases significantly which can result in the resistance to suggested that HBV infection and the ALT and AST level
nucleotide drugs. HBV mutations are more common in lami- indicated hepatitis. The level of HBV DNA directly reflected
vudine treatment, and the mutations occur in the gene of the capacity of viral replication and transmission. Combined
HBV DNA polymerase which is the target of lamivudine. At with the hepatic MRI results, this patient was diagnosed with
present, some HBV patients have been found to be resistant chronic hepatitis B (CHB).
to certain nucleoside (acid) analogs. And the incidence of
resistance to these drugs is different. This case was from the First Affiliated Hospital of Fujian
There are many detection methods with different sensitiv- Medical University.
ity for HBV DNA resistance mutation sites, including Sanger
sequencing, gene chip, reverse hybridization, pyrophosphate
sequencing, and next generation sequencing. Ou and col- 33.1.4 Hepatitis C
leagues reported that ARMS-LNA-qPCR method [8] was
used to detect well-known HBV mutation sites of drug resis- 33.1.4.1 Overview
tance and Cold-PCR method [9] was used to detect unknown/ Hepatitis C originates from the Hepatitis C virus (HCV)
known mutation sites. The sensitivity of these two methods which leads to about 350,000 deaths annually. Hence, it was
is high enough to give us a deep understanding of the rela- regarded as one of the serious infectious diseases in China
tionship between genotype resistance and phenotype after hepatitis B, tuberculosis, and dysentery [11, 12]. HCV
resistance. is a single-stranded, positive-sense RNA virus with approxi-
mately 9.5 kb genome. There is only one ORF in the whole
33.1.3.4 Management genome. Polyprotein precursors composed of 3011–3010
It is quite difficult to get rid of HBV once infected, but we amino acids are cleaved into viral structural proteins and
can have a good command of it by means of regular treat- nonstructural proteins by viral protease and host signal pep-
ments. Measures should be taken to postpone the progres- tidase. There are two untranslated regions (UTRs) called the
sion of fibrosis or cirrhosis and to avoid patients with CHB 5′ UTR and the 3′ UTR. 5′ UTR consists of 341 nucleotides
468 Q. Ou et al.
and forms four secondary domains that are necessary for Run-In HCV RNA in Serum PCR, nucleic acid hybrid-
virus replication and translation. It is so highly conserved ization, bDNA, gene chip, and transcription-mediated
that it is used as the basis for HCV genotyping. 3′ UTR can amplification system can be used to detect HCV RNA in
be divided into three domains: A genotype-specific variable serum.
region which is next to the coding sequence; a poly U region
in the middle which contains 50–62 nucleotides (varied by Genotyping of HCV
genotypes) and is essential for viral RNA replication; and the Region E1 and E2 among different HCV genotypes are more
highly conserved hairpin structure called X-tail at the 3′ end diverse than the C gene and some nonstructural protein-
which is of great importance to RNA virus replication. encoding genes. The sequence of 5′ UTR is the most con-
served. The variation degree and evolution rate of 5′ UTR is
33.1.4.2 Clinical Appearance very low, so 5′ UTR can be used to distinguish the main
Most patients with acute hepatitis C are acute non-jaundice genotypes. High variation in region NS5b makes it easy to
hepatitis with elevated ALT. About 80% of the acute HCV- distinguish different viral strains and is often selected as the
infected patients would progress to chronic hepatitis C, and basis for subtype differentiation. Based on the genetic differ-
the other patients would progress to hepatic fibrosis, HCC, or ences, we can distinguish HCV into 6 genotypes and about
hepatic decompensation if their illnesses were not under 100 subtypes.
effective control [13–15]. The manifestation of chronic hep- At present, the main methods used for HCV genotyping
atitis C is similar with chronic hepatitis B. The condition are as follows: sequencing analysis, PCR-RFLP, genotype-
becomes more serious if it is co-infection or superinfection specific nucleic acid probe hybridization, genotype-specific
with other hepatitis viruses, which often leads to chronic primer amplification, gene chip, and so on.
hepatitis, long infection process, different degrees of liver
pathological changes, and progressive aggravation. Run-In Sequencing Analysis It is the “golden standard”
for HCV genotyping, by which region E1, NS5b, and core
33.1.4.3 Laboratory Diagnosis regions of HCV genome can be directly sequenced and phy-
logenetic analysis can be done. However, the operation is
Immunological Detection tedious, time-consuming, and laborious, and the cost-
effectiveness is relatively low, which limits its clinical
Screening Test application.
The ELISA test is used to screen patients with anti-HCV
antibodies. Protein encoded by C22 (C), C33, NS3, and NS5 Run-In PCR-RFLP 5′ UTR, core region and NS5b all can
region are used antigens, therefore the false positive rate has be amplified, among which choosing 5′ UTR is the best in
decreased. detecting the genotypes of HCV, but the operation process is
relatively complex, and the detection types are limited.
Confirmatory
Recombinant Immunoblot Assay (RIBA) is of high specific- Run-In Genotype-Specific Nucleic Acid Probe
ity and is used as the confirmatory test once the sample is Hybridization It is the most commonly used method for
positive in preliminary ELISA screening. HCV genotyping. The results are accurate and the method
can be used for the detection of multiple-genotype HCV
Molecular Biological Detection infection. However, the detection result may be unsatisfac-
Detection of HCV Nucleic Acid tory if there is relatively low amount of HCV RNA in the
sample.
Run-In HCV RNA in Liver Tissues HCV RNA in liver tis-
sues can be detected by utilizing the technology of spot 33.1.4.4 Management
hybridization. The nucleic acid in tissue samples is extracted, Currently, what cheers us most is that hepatitis C is likely
denatured by deionized formamide, and then dotted on cel- to be cured. For patients who were suspicious of acute
lulose nitrate membrane. Specific nucleic acid probes are hepatitis C, the HCV RNA should be quantified dynami-
transformed into single strands by thermal denaturation, they cally for at least 4 weeks. They should maintain the thera-
bind to single strands of nucleic acid on cellulose nitrate pies until the HCV RNA test is negative. As for chronic
membrane based on the principle of base complementary infection, the treatment paradigm of DAAs is rapidly
pairing. Finally, the formed double strands are displayed by evolved and proved to be effective, while the clinical phy-
special coloration reaction. If the sample contains HCV RNA, sicians need to negotiate with patients and take their
a hybrid double-stranded nucleic acid would be formed, and actual condition into account to decide whether to adopt
the result is positive; otherwise, the result is negative. the therapy regimens [4].
33.1.4.5 Typical Medical Case Run-In Fulminant Hepatitis Due to the high pathogenic
Clinical Background A 34-year-old male smoker was potential of HDAg, clinical symptoms and liver damage are
admitted for persistent fatigue that lasted for more than half serious, and the mortality rate is high.
a year.
Superinfection
Initial Laboratory Values TBIL 14.5 μmol/L, ALT Run-In Self-Limited Hepatitis The symptoms and liver
196 U/L, AST 171 U/L, GGT 99 U/L; anti-HCV antibodies damage are limited, so patients can recover from it quickly.
50.51 COI, HCV RNA 1.35 × 107 IU/mL, HBsAg (−), Only a small number of patients with superinfections are
HBsAb (+), AFP 4.85 ng/mL. self-limited, most patients develop chronic hepatitis.
Results with Interpretation Guideline Run-In Chronic Active Hepatitis HDV has a directly effect
Anti-HCV IgM and anti-HCV IgG are not protective anti- on hepatocytes and accelerates the dissolution of hepato-
bodies, but are the markers of HCV infection. The positive cytes, 10–15% of patients developed cirrhosis or hepatocel-
of the former indicates the presence of HCV infection, and lular carcinoma (HCC) within 2 years. The patients may
the positive of the latter indicates symptomatic infection or have many clinical symptoms and serious liver histological
previous infection. HCV RNA is a direct marker that indi- damage. And their biological markers are often out of their
cates virus infection and replication. The results of HCV normal range.
genotyping are useful to judge the stage of the disease and
to formulate the individualized scheme of antitoxic 33.1.5.3 Laboratory Diagnosis
therapy.
Immunological Detection
Final Diagnosis Antigen Detection HDVAg can be detected by fluorescence
In this case, the positive results of anti-HCV antibodies and immunoassay or ELISA after the removal of HBsAg.
HCV RNA supported the diagnosis of HCV infection. HDVAg is transitorily seen in the early stage of acute hepati-
Meanwhile, the sign and other tests could be helpful in the tis D. However, the positive results may fluctuant in chronic
diagnosis of chronic hepatitis C. hepatitis D.
This case was from the First Affiliated Hospital of Fujian
Medical University. Antibodies Detection ELISA is usually used for antibody
detection. Anti-HDV-IgM, the only detection index of co-
infection antibody, is a diagnostic criterion for acute or early
33.1.5 Hepatitis D infection or persistent infection during the active period.
Anti-HDV-IgG helps to distinguish co-infection from super-
33.1.5.1 Overview infection. During co-infection, anti-HDV-IgM antibodies are
HDV, a unique defective virus, belongs to the Delta virus transient positive, but anti-HDV-IgG antibodies may appear
family. Its assembly depends on HBsAg, which means that it or disappear. The situation is exactly on the contrary when
completes its own replication and expression only in the overlapping infection occurs.
presence of HBsAg. The sources of infection are acute or
chronic hepatitis D patients, and the routes of HDV transmis- Molecular Biological Detection
sion are analogous to HBV. Based on the relationship HDV RNA detection can directly reflect the presence of the
between HDV and HBV infection, HDV infection can be virus, which is a useful indicator for HDV replication.
classified into two types: co-infection and superinfection, RT-PCR is used to detect HDV RNA in patients in the acute
and the latter mostly occur in chronic HBV infection and phase by designing the primer based on the conservative
HBV carriers. Superinfection can aggravate liver cell dam- region of HDV RNA, which is of high sensitivity and
age and cause severe or fulminant hepatitis [16]. specificity.
33.1.5.2 Clinical Appearance 33.1.5.4 Management

Hepatitis D is only prevalent in people infected with HBV, Currently, Peg-IFN-α is widely recommended in patients
therefore patients infected with HDV may manifest as co- suffering from hepatitis D, but the therapeutic effect varies
infection of HBV and HDV or superinfection. from person to person. Based on the treatment of Peg-IFN-α,
there are several combinations that add some NA(s) includ-
Co-Infection ing HBV/HDV entry inhibitors (Myrcludex-B), drugs inhib-
Run-In Acute Hepatitis The symptoms and liver damage iting the release of HBsAg (nucleic acid polymers), and
are limited, so most patients can recover from it quickly. inhibitors of the prenylation of the large HDV antigen to
470 Q. Ou et al.
compensate the defection of Peg-IFN-α. However, these This case was from the First Affiliated Hospital of Fujian
combinations are in clinical trials and require comprehensive Medical University.
evaluation [10].
33.2 Autoimmune Hepatitis

33.1.6 Hepatitis E
Hong Mu and Chunlei Zhou
33.1.6.1 Overview
Hepatitis E caused by the hepatitis E virus (HEV) is primar-
ily diffused through the gastrointestinal tract. Evidence
showed that there were 20 million individuals infected with 33.2.1 Overview
HEV, approximately a quarter of them were acute, and
resulted in 70,000 deaths in the world annually [17]. In recent years, viral liver diseases have been well controlled,
and we should be thankful for the widespread use of hepatitis
33.1.6.2 Clinical Appearance vaccination, standardized management of blood products,
The latent period of HEV varies from 2 to 9 weeks, averag- and development of antiviral drugs. However, the diagnosis
ing 40 days. Particularly, it predisposes young adults to hep- and treatment of other non-viral liver diseases, i.e., autoim-
atitis E. Jaundice is common. The patients often have mune liver disease (AILD) have become one of the major
symptoms in the digestive system, such as nausea, vomiting, problems in the field of liver diseases.
and so on. The proportion of fulminating hepatitis is high, Autoimmune hepatitis (AIH) is a rare form of AILD and
and the mortality rate of pregnant women suffering from is associated with parenchymal inflammation. This is caused
hepatitis E reaches about 31% [18]. However, HEV infec- by autoimmune attack of the liver cells and is characterized
tions are mostly self-limited, and once patients recover from by positive serum autoantibodies, high levels of aminotrans-
HEV infection, they will get a lifelong immunity against ferase and IgG. It is considered as the histological hallmark
HEV. of moderate to severe interface hepatitis [20]. AIH is widely
distributed and occurs in individuals of all ages, but women
33.1.6.3 Laboratory Diagnosis are more likely to be affected, with a ratio of 1:4 in men and
RT-PCR is used to detect RNA in the samples, and the sero- women [21]. According to the statistics, the incidence of
prevalence of HEV RNA can reach 70% during acute infec- AIH has remained the highest in Alaska, North America
tion. In clinical settings, the detection of HEV antibodies is (42.9/100,000), followed by European countries with a high
more carried out than HEV RNA. climbing rate of the disease (10.7–23.9/100,000) [22]
(Table 33.1).
33.1.6.4 Management Based on the autoantibody profile, AIH can be classified
Timely and effective control of the source of infection and into three subtypes [20, 23]: (1) type 1 AIH is characterized
blocking the transmission are important means to prevent by antinuclear antibodies (ANA) or anti-smooth muscle anti-
hepatitis E. f Hepatitis E may be prevented by some sanitary bodies (ASMA) and is the most common AIH with an inci-
measures. Uncontaminated food and water are of great dence rate of 60–80% in patients; (2) type 2 AIH is
importance. And the HEV vaccine could be used to prevent characterized by anti-liver/kidney microsomal antibody type
the infection. For hepatitis E patients, symptomatic treat- 1 (ALKM-1), or anti-liver/kidney microsomal antibody type
ments combined with plenty of rest are often adopted [19].
33.1.6.5 Typical Medical Case Table 33.1 Simplified criteria for the diagnosis of AIH
Clinical Background A 54-year-old male smoker was Variable Cut-off Points
admitted for icteric sclera and abnormal urine for at least 2 ANA or SMA ≥1:40 1
months. ANA or SMA ≥1:80 2
Or LKM ≥1:40
Or SLA Positive
Initial Laboratory Values TBIL 117.6 μmol/L, DBIL
IgG >upper normal limit 1
92.0 μmol/L, ALT 301 U/L, AST 148 U/L, GGT 168 U/L,
>1.10 times the upper 2
LDH 278 U/L, ALP 131 U/L, TBA 27.6 μmol/L, anti-HEV normal limit
IgM (+), anti-HEV IgG (+). Liver histology (evidence of Compatible with AIH 1
hepatitis is a necessary condition) Typical AIH 2
Final Diagnosis Absence of viral hepatitis Yes 2
The positive result of anti-HEV IgM is important for the ≥6:Proable AIH
diagnosis. Combined with the symptoms and other examina- ≥7:Definite AIH
tion results, this patient was diagnosed with hepatitis E. Addition of points achieved for all autoantibodies (maximum, 2 points)
3 (ALKM-3) and/or antibodies against liver cytosol type 1 response driven by the recognition between T cells and anti-
(LC1) and is observed in very few cases of 3–4%; and (3) gens or autoantigens expressed by genetic susceptibility
type 3 AIH is characterized by antibodies against soluble [30]. The genetic predisposition to AIH is linked to the
liver antigens/antibodies against liver pancreas (SLA/LP). human leukocyte antigen (HLA) gene that is located on the
By now, the etiology of AIH still remained unclear, but is short arm of chromosome 6 and is particularly located in
believed to be associated with genetic predisposition, auto- HLA class II DRB1 alleles [31]. In North America and
antibodies, immune disorder, environment, and other factors. Europe, HLA-DR3 (HLA-DRB1*0301) and HLA-DR4
The presence of autoimmune responses might be associated (HLA-DRB1*0401) encode HLA-DR3 and DR4 antigens,
with the genetic susceptibility of individuals, molecular sim- respectively, contributing mainly to the type I AIH suscepti-
ulation of viruses, and various physical and chemical factors bility [32]; whereas HLA-DR4 is more common than
[24]. Since AIH is caused by the loss of immune tolerance, HLA-DR3 in Asia. It should be noted that the diversity of
previous studies hypothesized that the mechanism of genetic susceptibility not only develops across the oceans,
immune-mediated injury plays an important role in this pro- but also occurs within the same continent. For example, the
cess [25, 26]. The development of liver injury involves a major susceptible gene in China and Japan is DRB1*04059
complex network of many immune cells and molecules [27– [33], but it is B27 and cw411 in western India [34], and
29]. Previous studies showed that various stimulants activate DR6 in Pakistan [35]. Type 1 AIH has a strong association
innate immune cells to secrete inflammatory mediators, within the HLA loci, Floreani and colleagues summarized
which further triggers adaptive immune cells forming an the HLA alleles associated with type 1 AIH in 2018 [36].
inflammatory network in the liver. This network includes Age of presentation is variable, and the disease may present
multiple immune response mechanisms such as autoantigen- at any age from childhood to late life; such variation suggests
antibody reaction, cytokine release, and oxidative stress, that the disease shares multiple factors contributing to etio-
together resulting in liver tissue damage. The main idea of pathogenesis in common with other autoimmune conditions
AIH is that the disease involves hepatic inflammatory (Fig. 33.2).
Fig. 33.2 Factors proposed

to alter the risk of developing
autoimmune hepatitis,
presented by category. Many
of these factors are
speculative or unconfirmed
(including AIRE, FAS/FASL,
CTLA4, and GATA2
mutations outside of specific
syndromes), but the HLA-D
allele, SH2B3 variants,
female sex, age, pregnancy,
and exposure to several drugs
have been confirmed to be
associated with the
development of autoimmune
hepatitis. Abbreviations: AIRE
autoimmune regulator, CMV
cytomegalovirus, CTLA4
cytotoxic T lymphocyte
antigen 4, EBV Epstein-Barr
virus, FAS/FASL CD95 and
CD95 ligand, GATA2
GATA-binding factor type 2,
HAV hepatitis A virus, HCV
hepatitis C virus, HEV
hepatitis E virus, HIV human
immunodeficiency virus,
HLA-D human leukocyte
antigen D allele, NAFLD
nonalcoholic fatty liver
disease, PD-1/PD-L1
programmed death receptor 1/
programmed death receptor
ligand 1, SH2B3 gene
encoding adaptor protein also
known as Lnk, Tr1 type 1
regulatory T cell, Treg
regulatory T cell
472 Q. Ou et al.
33.2.2 Clinical Appearance 33.2.3.2 Autoantibody
Despite varied clinical manifestations of AIH, most of the ANA

patients had an insidious onset, showing chronic liver dis- ANAs are also known as anti-nucleic acid antibodies. These
ease in general. However, it is also accompanied by atypical are a group of antibodies produced by DNA, RNA, protein,
symptoms such as sleepiness, fatigue, anorexia, nausea, or molecular complexes of the substances present in the
weight loss, and general discomfort [22]. Physical examina- nucleus. ANA is the first autoantibody that is found in the
tion may reveal the signs of hepatomegaly, splenomegaly, serum of AIH patients and is still considered to be the most
ascites, and occasional peripheral edema. About 20% of sensitive antibody for AIH diagnosis. The mechanism of
patients do not develop any significant symptoms and can be ANA formation in AIH is still unknown but is currently
established only with elevated serum aminotransferase levels believed to be associated with the release of nuclear compo-
in physical examination [37]. Cirrhosis is another problem nents after liver cell damage, and/or the loss of tolerance of
that needs to be concerned. The percentage of AIH patients B cells to these substances. Though ANAs are the serum
progressing to cirrhosis varies from 5% to 45% in different markers for AIH, they are not specific to the disease. This is
countries [22]. By the time AIH is diagnosed, about 1/3 of because positive ANA expression can be detected not only in
patients have already developed cirrhosis, and some might patients with chronic viral hepatitis or other autoimmune
present hematemesis and black stool due to esophageal- diseases, but also in healthy elderly people. Indirect immu-
gastric variceal bleeding as the initial symptoms. Few nofluorescence is the most preferred method of detection for
patients might be accompanied by fever. Apart from the ANAs. Normally, Hep-2 cells are used as substrate for the
above, about 30% of patients would show symptoms of acute detection of total ANA [40]. ANA shows multiple fluores-
hepatitis with the rapid increase of transaminase (≥10 times cence patterns including homogenous, speckled, and granu-
of normal upper limit). They are often accompanied by jaun- lar. Most of the AIH patients displayed homogeneous
dice, and severe cases might progress to acute liver failure if fluorescence in the liver. Patients with positive ANA on indi-
untreated timely [38]. There are still some patients that might rect fluorescence or negative ANA, but are highly suspected
show intermittent AIH where clinical or biochemical abnor- of AILD, ELISA or immunoblotting assist in detecting spe-
malities could be alleviated spontaneously, even recovered cific ANA antigens (dsDNA, SSA, gp210 for example),
completely, and reoccurred after a period of time. Also, AIH thereby facilitating the diagnosis and differential diagnosis.
is complicated with other organic or systemic autoimmune
diseases, such as Hashimoto’s thyroiditis, diabetes, rheuma- Anti-Smooth Muscle Antibodies (ASMA)
toid arthritis, etc. AIH presents a variety of clinical manifes- ASMAs were first found by Johnson and colleagues in
tations, most of which are chronic liver disease, but also patients with chronic hepatitis (lupus-like hepatitis) in the
acute onset, acute and chronic liver failure. Special groups year 1965 [41]. A year later, Whittingham and colleagues
such as children, the elderly, pregnant women have different related ASMA to AIH, as ASMA was disappeared when AIH
clinical characteristics. We need to fully understand the con- was improved [42]. ASMA targets the backbone proteins of
sistency and specificity of AIH and adopt appropriate treat- smooth muscle, including actin, vimentin, desmin, and tubu-
ment strategies. lin. Purified actin, which is also called G-actin, is a polar
molecule with a molecular weight of 46 kD. Globular G-actin
forms a microfilament called F-actin, which is an AIH-
33.2.3 Laboratory Diagnosis specific autoantigen, and is targeted by F-actin-specific
ASMA, an antibody belonging to the IgG class. Actin-
33.2.3.1 Serum Biochemical Indicators specific ASMAs are specific for type 1 AIH. In particular, the
The AIH-accompanied serum biochemical abnormalities specificity of ASMA in AIH diagnosis could be nearly 100%
mainly include liver cell damage, increased AST and ALT when purified actin was used as the coating antigen; and
activities, and normal or slightly elevated ALP and GGT lev- similarly in indirect fluorescence when the titer is over 1:40.
els. However, these indicators are not specific to AIH [39]. It As for non-actin-specific ASMAs, they demonstrated no
is noteworthy that the change of serum aminotransferase organ specificity and can be detected in patients with AIH,
cannot reflect intrahepatic inflammation accurately, and chronic active hepatitis, primary biliary cirrhosis, infection,
minor increase or decrease of the indicator do not necessarily malignant tumors, and hematological diseases. Couto and
point to mild or inactive hepatic diseases, meaning that the colleagues [43] reported that persistent ASMA (>1:80 in
diagnosis of AIH could not be excluded completely. In severe indirect fluorescence) and anti-F-actin antibody (>1:40 in
or acute cases of AIH, the level of total bilirubin (TBiL) ELISA) are significantly associated with serological indica-
might also be raised significantly. tors and histological characteristics of type 1 AIH patients.
ASMA (1:80) and F-actin (1:40) are associated with disease ducted by Chen and colleagues [48] in a total of 1297 AIH
activities both biochemically and histologically, and they are cases from 8 centers of America, Germany, and Japan. The
also used in the prediction of the high rate of treatment fail- results revealed that patients positive for anti-SLA/LP anti-
ures in patients with type I AIH. bodies had a threefold higher death risk than those negative
patients and a recurrence risk of twofold than their counter-
Anti-Liver/Kidney Microsomal Antibodies (ALKM) parts. The mechanism by which anti-SLA/LP antibodies
ALKMs were first identified in the serum of a patient with affected the disease is yet to be clarified, but considering the
chronic hepatitis in 1973. ALKM is an autoantibody that coexistence of anti-Ro52 and anti-SLA/LP antibodies in
reacts with proximal convoluted tubules and liver cell plasma type I AIH patients (about 96%) [49], it is speculated that the
[44]. ALKM is classified into three subtypes according to poor prognosis of anti-SLA/LP-antibody-positive patients
fluorescence staining, namely ALKM-1, ALKM-2, and might be associated with the concurrence of anti-Ro52 anti-
ALKM-3. Both ALKM-1 and ALKM-2 exhibit no species bodies. Since immunofluorescence failed to identify anti-
specificity, whereas ALKM-3 cannot be identified in fluores- SLA-LP antibodies with high sensitivity or specificity, the
cence staining when rat cells are used as substrate, and it is use of recombinant antigen-based methods (ELISA and
specific for humans as it only reacts to human liver cells. immunoblotting for example) for the detection of anti-SLA/
ALKM-1 mainly targets on human CYPD450IID6, and LP antibodies has been recommended by the guidelines of
ALKM-2 on UDP-glucuronosyltransferase (UGT). ALKM-1 EASL-AIH [20].
acts as the serological marker for type 2 AIH, but it can also
be detected in about 10% of patients with chronic hepatitis 33.2.3.3 Serum Immunoglobulin
C, and the titer is dependent on disease activity [45]. The elevation of serum IgG and/or γ-globulin is one of the
According to the previous studies, ALKM-2 is more com- characteristic serum immunological changes of AIH. Serum
monly observed in patients with drug-induced liver injury, IgG reflects the activity of liver inflammation, and immuno-
and ALKM-3 in hepatitis D patients. suppressive therapy can restore the level of it. Since this indi-
cator facilitates not only the diagnosis of AIH, but also the
Anti-Liver Cytosol Type 1 (LC-1) Antibodies evaluation of patient response to treatment, it should be
Anti-LC-1 antibodies were initially found in the serum of included as a routine test during the initial diagnosis, treat-
ALKM-1 positive AIH patients in the year 1988 by Martini ment, as well as follow-up. The clinical diagnosis and treat-
and colleagues [46]. Anti-LC-1 antibody is another marker ment of IgG-associated AIH can still be confused by
for type 2 AIH, where the high titers of the antibody indicate increased IgG or positive autoantibodies in patients with
type 2 AIH. Anti-LC-1 antibodies often coexist with ALKM- drug-induced liver damage or chronic hepatitis B. Hence, the
1, and the former could be the only autoantibody that can be IgG-associated AIH has drawn increasing attention in recent
detected in about 10% of type 2 AIH patients. Therefore, years and is investigated by several other researchers [50].
anti-LC-1 antibodies demonstrated a higher diagnostic spec- For example, IgG4-associated AIH showed common symp-
ificity when compared to ALKM-1. When coexisting, the toms of AIH, but is characterized by increased serum IgG4,
fluorescence of anti-LC-1 antibodies might be covered with infiltration of IgG4-positive plasma cells, high response to
that of ALKM, and so ELISA and immunoblotting are more hormone therapy, and low recurrence rate after treatment
frequently used for the detection of anti-LC-1 antibodies. [51]. Also, AIH patients often showed normal serum IgM,
According to a previous study, anti-LC-1 antibodies are but occasionally have increased serum IgA levels.
associated with the activity and progression of AIH [20].
33.2.3.4 Histopathology
Antibodies Against Soluble Liver Antigens/ AIH has diverse histopathological manifestations. Though
Antibodies Against Liver Pancreas (SLA/LP) the onset could be acute or chronic, and the degree of fibrosis
SLAs and LPs were initially believed to be different from could be low or high, the root of AIH is still the damage of
each other until Wies [46] has successfully cloned the full- the liver cell. The main pathological features of AIH include
length sequence of SLA from human liver tissue. After that, [20]: (1) interface hepatitis or piecemeal necrosis, which
SLA was recognized as an unknown soluble protein in the refers to the necrosis and loss of single or clustered interface
cytoplasm of hepatocytes and is actually the same as cells, thereby producing an insect-eaten appearance at the
LP. Nowadays, both of these are known as anti-SLA/LP anti- lobular-portal-interface. Inflammatory cells would then
bodies and are reckoned as serological markers for migrate along the necrotic interface and surround the dead
AIH. Positive expression of anti-SLA/LP antibodies indi- liver cells. (2) Lymphocyte-plasma cell infiltration is
cates higher severity of the disease, more serious histological observed at the portal area and is peripheral. Plasma cells are
changes, and greater possibilities of recurrence, liver trans- often seen in the portal area, but also within the lobules. In
plantation, and even death [47]. A meta-analysis was con- AIH, most of the plasma cells are IgG-positive, while only a
474 Q. Ou et al.
few are IgM-positive. (3) Hepatic rosette formation is a pseu- considered the influence of virus, drug, alcohol, and other
doglandular structure formed by 2–3 liver cells that show factors. Considering the complexity of this system, it
hydropic degeneration, and dilated bile capillaries can be remained to be suitable only for clinical studies, or the diag-
seen in the center. The structure is named so due to its resem- nosis of patients with complex symptoms. In 2008, the
blance of a rose, and it is often found around the limiting IAIGH proposed a simplified AIH diagnostic system that
plate. (4) Emperipolesis is considered as the histological mainly focused on four aspects, namely autoantibody, IgG
manifestation of lymphocyte entry into the hepatocytes. It is level, histopathological manifestations, and excluded viral
often seen in interface hepatitis and is another typical mani- hepatitis [54]. One point is added to each item that meets the
festation of AIH. The histologic features of autoimmune following criteria: (1) ANA or SMA ≥ 1:40. (2) IgG > upper
hepatitis are shown in Fig. 33.3 [52]. AIH also presents some normal limit. (3) Liver histology (evidence of hepatitis is a
rare histologic findings, including central lobular necrosis, necessary condition) compatible with AIH. Two points are
the presence of multicellular or megakaryocyte hepatocytes, added to each item that meets the following criteria: (1) ANA
and bile duct injury. or SMA ≥ 1:80 or LKM ≥ 1:40 or SLA positive. (2)
IgG > 1.10 times the upper normal limit. (3) Typical AIH
33.2.3.5 Conclusion liver histology. (4) Absence of viral hepatitis. Judgment cri-
The International Autoimmune Hepatitis Group (IAIHG) teria: Total score ≥ 6 Proable AIH, ≥7 definite AIH. By com-
developed a scoring system for AIH diagnosis in 1993 and paring the two scoring systems, Czaja [55] reported that the
updated it in 1999 [53]. Apart from the scores based on ami- comprehensive system has a higher sensitivity (100 vs.
notransferase, IgG, ANA, and liver histology, the system also 95%), whereas the simplified system has a higher specificity
a b
c d
Fig. 33.3 Histological features of autoimmune hepatitis (Haematoxylin and Eosin). (a) Moderate interface hepatitis (magnification 200); (b)
Lymphocyte and plasma cell infiltration (magnification 400); (c) Hepatocyte rosette (magnification 400); (d) Emperipolesis (magnification 400)
(90 vs. 73%) as well as accuracy (73 vs. 82%). Therefore, Table 33.2 Geographical distribution of major risk factors for primary
Czaja concluded that the comprehensive scoring system is liver cancer all over the world
more suitable for the diagnosis of atypical cases. As for Alcohol HBV HCV Others
(%) (%) (%) (%)
patients suspected with AIH could not be diagnosed by the
Europe
simplified scoring system, it is suggested to use the compre-
Western 32 13 44 10
hensive scoring system to avoid misdiagnosis. For unex- Central 46 15 29 10
plained liver dysfunction or liver disease, the clinical practice Eastern 53 15 24 8
guidelines for AIH published by the European Society for North America 37 9 31 22
the study of the liver (EASL) in 2015 put forward the diag- Andean Latin America 23 45 12 20
nostic process of AIH [20]. For the liver disease of unknown Asia
origin, indirect immunofluorescence (IFL) is the preferable East Asia 32 41 9 18
and main technique for routine autoantibody test for all auto- Asia-Pacific 18 22 55 6
antibodies except SLA/LP (ELISA or blot). If ANA, SMA, South-East Asia 31 26 22 21
Africa
LKM1/LC1 or SLA/LP positive, consider AIH (Test also for
North Africa, Middle 13 27 44 16
elevated IgG levels) and do the liver biopsy for further con- East
firmation. If the autoantibodies are negative and clinical sus- Southern 40 29 20 11
picion remains, repeat testing in specialty lab (including (sub-Saharan)
pANCA and specific immunoassays for LKM1, LKM3, Western (sub-Saharan) 29 45 11 15
LC1, SLA/LP, F-actin, Ro52, gp210, sp100). The procedures Notes: HBV hepatitis B virus, HCV hepatitis C virus
in the guidelines for the diagnosis of AIH not only combine
AIH-related autoantibodies and liver pathology, but also diagnosed at advanced stages. The 5-year survival rate of
exclude other AILDs. During the occurrence and develop- patients with HCC at early stages reaches 50–70% and drops
ment of AIH, the titer and specificity of autoantibodies can to 5% at advanced stages.
be changed, and individuals who are serum-negative at the As reported, primary liver cancers own relationships with
time of diagnosis can also express common autoantibodies in various potential etiologies (Table 33.2). The incidence of
the later course of the disease. Autoantibodies can be clini- HCC is associated with both age and gender. The progres-
cally tested repeatedly to correct the diagnosis and classifica- sively increment associating with the advance of age reaches
tion of disease. The overall goal of AIH treatment is to obtain a peak at 70-year-old [57]. The male/female ratio for HCC is
liver histological remission, prevent the development of liver estimated as high as 2–2.5:1. Of all the globally HCC cases,
fibrosis and the occurrence of liver failure, extend the sur- those cases attributed to hepatitis B virus (HBV) infection,
vival period of patients and improve the quality of life of hepatitis C virus (HCV) infection, and other medical reasons
patients. All patients with active AIH should be treated with accounted for 54%, 31%, and 15%, respectively [56]. Serum
immunosuppressive therapies, and the treatment regimens E antigen positivity, high viral load, or genotype of HBV act
and drug dosages can be adjusted according to disease activ- as independent predictors of HCC development [58–60].
ity. Currently, the main treatment regimen for AIH is predni- Furthermore, metabolic syndrome, as well as HIV infection,
sone monotherapy or combined azathioprine therapy. is taken as supplementary factor into account to contribute to
the occurrence of HCC in patients with chronic viral hepati-
tis [61, 62]. Higher frequency of HCC appears among
33.3 Hepatocellular Carcinoma patients with alcoholic liver cirrhosis, acute hepatic por-
phyria, and porphyria cutanea tarda [63, 64]. Growing evi-
Hong Mu and Chunlei Zhou dences indicate that HCC tends to develop in patients with
non-alcoholic fatty liver disease (NAFLD) associated with
metabolic syndrome, diabetes, and obesity [65–67].
33.3.1 Overview Prognostic assessment is a vital step in the management
of HCC cases following the diagnosis of the disease. The
Liver cancer has been reported as the fifth most common classification of cancer is necessary prior to the established
cancer and identified as the second cause of death world- prognosis and the selection of the optimal treatment for the
wide. In addition, 854,000 new cases and 810,000 deaths best candidates. Several staging systems have been employed
were annually reported, which accounted for 7% of all can- to offer a clinical classification of HCC. Six out of these
cer cases [56], and approximately 90% of new cases are clas- comprehensive staging systems have been tested widely
sified into hepatocellular carcinoma (HCC), causing a including the French classification, the Cancer of the Liver
serious health problem worldwide. Diagnosis of HCC mainly Italian Program (CLIP) classification, the Barcelona-Clinic
relies on imaging techniques and biopsy, but most cases are Liver Cancer (BCLC) staging system, the Chinese University
476 Q. Ou et al.
Prognostic Index (CUPI) score, the Hong-Kong Liver Cancer echotexture on the US, making the identification of small
(HKLC) staging system and the Japan Integrated Staging tumors to be highly difficult. Serum markers, including
(JIS). BCLC staging system is common to be used by the alpha-fetoprotein (AFP), Protein induced by vitamin K
doctor to make decisions about treatment referring to both absence/antagonist-II (PIVKA-II), alpha-fucosidase, and
the number and size of tumors in the liver and the status per- glypican have been assessed for early diagnosis of HCC,
formed by patients. Following the guidelines modified from however, their performance found disappointing and unsatis-
the BCLC classification, patients with HCC are divided into factory in serological tests for surveillance [71–73].
five stages ranging from 0 to D based on the analysis of the
pre-established prognostic variables. Stage 0, A, and B 33.3.2.2 Diagnosis
sequentially corresponding to very early, early and interme- Regarding the classification of liver cancer into distinct types
diate stage presents the patient with a single nodule (<2 cm), based on morphological parameters, pathohistological diag-
a single nodule of any size or 2–3 nodules (<3 cm), and many nosis is taken as the gold standard into account to define
nodules respectively when the patient feels well and his liver HCC and also distinguish it from others, that presents similar
works well. The ablation and resection are alternative meth- clinical features. Pathological diagnosis of HCC is made fol-
ods employed to treat the patient at stage 0; The resection, lowing the criteria issued by the World Health Organization
transplant as well as ablation can be chosen to deal with the (WHO) classification and the International Consensus Group
patient at stage A; Chemoembolization can be used in the for Hepatocellular Neoplasia. Specificity of HCC diagnosis
treatment of the patient at stage B. Stage C known as on the basis of liver biopsy has been reported to reach 100%
advanced stage indicates the patient with cancer spreading [74]. Although the sensitivity of the HCC diagnosis based on
into organs other than liver such as blood vessels and lymph liver biopsy may be affected by all the effective factors, such
nodes. A patient at stage C feels uncomfortable but his liver as lesion’s size, location, differentiation, and even surgeon’s
still works well and needs systemic therapy. Stage D as ter- and pathologist experience, it was as high as 90% for differ-
minal stage indicates a patient with severe liver damage ent tumor sizes as previously reported [75]. In general,
which leads to loss of liver function. The one at stage D feels pathologically defining HCC for nodules being less than
uncomfortable. He is not transplantable but requires the best 2 cm in size is challengeable. A prospective study reported a
supportive care. The determination of prognosis relied on the 60% positive result for the first liver biopsy on a tumor of
variables associated with tumor status involving size, num- size 2 cm or smaller [76]. Additionally, immunohistological
ber as well as the existence of vascular invasion, the liver markers mainly contribute to the identification of HCCs with
function described by serum level of bilirubin, presence of poor prognosis [77]. It also was revealed that medical imag-
portal hypertension and preservation of liver function and ing plays a pivotal role in the diagnosis of HCC and facili-
health status defined by the Eastern Cooperation Oncology tates the detecting primary liver tumor and HCC staging by
Group (ECOG) classification as well as the presence of presenting the peculiar vascular derangement, often occur-
symptoms [68]. ring in the process of hepatic carcinogenesis [78]. Computed
tomography (CT) and magnetic resonance imaging (MRI)
are regarded as recommended contrast-enhanced imaging
33.3.2 Surveillance and Diagnosis techniques in the diagnosis of HCC. As to the patients with
cirrhosis, HCC cases are usually determined based on the
33.3.2.1 Surveillance contrast-enhanced imaging, and the corresponding proce-
Imaging and serological examinations are typically con- dures presenting in HCC diagnosis is briefly provided as fol-
ducted for HCC surveillance. Relying on their advantages, lows: multiphasic contrast-enhanced CT, multiphasic
such as non-invasive, the absence of risks, reputability, a contrast-enhanced MRI or gadoxetic-enhanced MRI was an
relatively moderate cost, and the possibility of observing the alternative method used to detect small HCC nodules(<1 cm)
onset of other complications of cirrhosis early, ultrasonic which shows features of growth or changes in pattern in
(US) technology is taken as the most widely used imaging ultrasound in the pre-contrast phase or large nodules(>1 cm).
test for surveillance into account. The sensitivity of the US A positive signal which is identified in any one of these tech-
when is exploited in a surveillance test ranges from 58 to niques can serve as an HCC imaging hallmark. A tech such
89% and its specificity is higher than 90% [69]. However, a as other modality multiphasic contrast-enhanced CT, multi-
meta-analysis showed that the majority of HCC tumors could phasic contrast-enhanced MRI, gadoxetic-enhanced MRI, or
be detected with US surveillance with a sensitivity of 94%, contrast-enhanced ultrasound is recommended to be utilized
while in the case of a sensitivity of 63%, US technology was to confirm the negative imaging signal which appears in the
effective for detection of HCC at early stage [70]. In addi- previous step. The HCC diagnosis can be also determined if
tion, HCC surveillance in cirrhotic patients by the US is a there is a positive signal at this step. A biopsy is further sug-
main challenge, especially in the case of very coarse liver gested to be used for exclusion of HCC if there is no HCC
imaging hallmarks. A nodule of non-HCC malignancy or combination with AFP, may be a clinically useful adjunct
benign can be determined in a biopsy assay. Either a re- marker for diagnosis of HCC [86].
biopsy or a recheck using imaging tools is necessary or if a
consequence obtained in biopsy assay is unclear. As reported 33.3.3.2 Glypican-3 (GPC3)
previously, it can be stated that MRI with specificity ranging Glypican-3 (GPC3) is a heparan sulfate proteoglycan
from 85 to 100% owns higher sensitivity compared with CT (HSPG) that is attached to the cell surface via a glycosyl-
scanning [79]. For tumors being smaller than 20 mm in size, phosphatidylinositol (GPI) anchor [87, 88]. GPC3 is known
the sensitivity of MRI and CT is 62% and 48%, respectively, as a member of the family, containing six cell-surface pro-
while those figures may increase up to 95% and 92% corre- teins who have similar sizes, ranging from 60 to 70 kDa and
spondingly when tumor size would be equal or larger than structures [89]. The finding that the ignorable expression
20 mm [80]. level of GPC3 mRNA in noncancerous liver, referring to nor-
mal, cirrhotic, and focal nodular hyperplasia livers would be
detectable in 75% of HCC cases, suggesting GPC3 as a
33.3.3 Biomarkers potential marker for HCC diagnosis [90]. In recent years, the
cut-off values of serum GPC3 levels used in the detection of
The biological molecules that served as markers to identify HCC changed from 3.9 to 300 ng/mL with sensitivity and
HCC can be classified into four categories, including bearing specificity ranging from 36–65% to 65–100%, respectively
embryonic and glycoprotein antigens, enzymes and iso- [71, 91, 92]. The expression level of GPC3 did not show any
zymes, genes, and cytokines. relationship with tumor size [91]. In the diagnosis of HCC at
the early stage, the sensitivity of 55.1% and the specificity of
33.3.3.1 Alpha-Fetoprotein (AFP) 97% were observed. Moreover, the sensitivity increased to
AFP is mainly used as the most frequent tumor biomarker 76% in identical cases when GPC3 was employed along with
for HCC. It is a glycoprotein that has been reported to consist AFP [93].
of 591 amino acids and one asparagine-linked bi-antennary
complex-type sugar chain [81]. The normal range for serum 33.3.3.3 Golgi Protein-73 (GP73)
AFP levels is 10–20 ng/mL and a level > 400 ng/mL is usu- GP73, a resident Golgi membrane protein, is a potential
ally regarded as diagnostic. AFP has been extensively applied serum biomarker for the diagnosis of liver diseases and
to the diagnosis of HCC except for surveillance because HCC. In the normal human liver, GP73 is expressed in bili-
abnormal serum AFP level may cause flares related to HBV ary epithelial cells, but detection is negligible in hepatocytes
or HCV infection, exacerbation of underlying liver disease [94]. However, upregulated expression of GP73 has been
or the development of HCC in only a small percentage of identified in hepatic cells in liver disease [95]. A meta-
tumors at early stage may eventually invalid the performance analysis showed that the sensitivity of serum GP73 level
of AFP for surveillance [82, 83]. A pathological threshold for ranged from 69 to 95%, while the specificity varied from 35
serum AFP concentration in the diagnosis of HCC is gener- to 97% in the diagnosis of HCC [96]. The sensitivity and
ally desired to be 20 μg/L with a sensitivity and a specificity specificity of GP73 accompanied with AFP-L3 for HCC
of 60.0% and 90.6%, respectively. In concentration of diagnosis reached 94.0% and 93.1%, respectively [97].
400 μg/L, serum AFP concentration is taken as a reliable fac-
tor into account for HCC diagnosis with a specificity of more 33.3.3.4 Alpha-l-Fucosidase (AFU)
than 99.4% [84]. AFP owns distinct isoforms, which have AFU, a lysosomal enzyme present in all mammalian cells,
non-identical affinities to certain lectins or different patterns has been proposed as a tumor marker for the diagnosis of
after electrophoresis. According to their diverse reactions to HCC in many studies [98]. By measuring the level of cleaved
Lens culinaris agglutinin (LCA), AFPs can be classified into colorimetric substrate p-nitrophenyl-α-l-fucopyranoside
three variants: AFP-L1, AFP-L2, and AFP-L3. As AFP-L3 (PNFP) in several cases, the serum AFU level can be accord-
produced by malignant liver cells binds to LCA with high ingly determined. It is noteworthy that serum AFU levels
affinity, the ratio of L3 fraction to total AFP likely acts as an were provided in accordance with the catalytic activity of
effective marker to discriminate HCC from other chronic enzymes using enzymatic units. AFU serves as a serum
liver lesions. There is a sensitivity of 96.9% and a specificity marker based on the fact that the AFU concentrations are
of 92% in the diagnosis of HCC using AFP-L3 at the cut-off significantly higher in patients with HCC compared with the
value of 15% [85]. AFP-L3 accompanied with total serum corresponding concentrations in healthy adults and patients
AFP levels to detect HCC provides the most promising con- with benign liver tumors. The sensitivity of AFU to detect
sequence, in which AFP-L3% > 35% has 100% specificity HCC varies from 60 to 90%, while the specificity ranges
for HCC, and also it was revealed that AFP-L3%, used in from 55 to 98% [99, 100]. Furthermore, AFU rises
478 Q. Ou et al.
6–9 months ahead of HCC patients as described by ultraso- able supplementary marker in the diagnosis of HCC [106].
nographic imaging, indicating that AFU can be employed as The usage of SCCA-IgM in accompany with AFP resulted in
an earlier marker to detect HCC. a sensitivity of 70% and a specificity of 100% in the diagno-
sis of HCC [107].
33.3.3.5 P rotein Induced by Vitamin K Absence/
Antagonist-II (PIVKA-II) 33.3.3.7 MicroRNAs
Protein induced by vitamin K absence/antagonist-II MicroRNAs (miRNAs) refer to a class of small non-coding
(PIVKA-II), also known as Des-γ-Carboxyprothrombin RNA ranging from 17 to 25 nucleotides and perform their
(DCP), is an abnormal prothrombin that has been widely functions, namely RNA silencing, by directly binding to the
used as a tumor marker for HCC and could be predictive of complementary sequences within 3′ untranslated region of a
worse tumor behavior and prognosis. The defect in carboxyl- target mRNA [108–111]. They are conserved among various
ation results in the frustration of the binding of Ca cations to species and involved in distinct cellular processes, such as
PIVKA-II, impairing its action on prothrombin in the pro- proliferation, differentiation, and apoptosis by regulating the
cess of coagulation [101]. It was revealed that serum expression level of their target genes. MiRNAs act as
PIVKA-II levels in 63% of patients with HCC were more oncogenes as well as tumor suppressor genes in the develop-
than 0.1 U/mL and the corresponding levels in 48% of ment of cancer. The dysregulations of miRNAs have been
patients were above 0.3 U/mL. In a test performed to distin- verified to be associated with several types of cancer. The
guish HCC from the cases of cirrhosis and chronic, PIVKA-II striking advantages of miRNAs as potential markers in the
showed a sensitivity of 72.7% along with a specificity of detection of HCC are detectable and stable in clinical sam-
90.0% [102]. The combination of PIVKA-II with AFP used ples, including frozen or formalin-fixed paraffin-embedded
for the detection of HCC yielded a sensitivity of 74.2% and tissues, blood, serum, as well as blood plasma. In order to
a specificity of 87.2% [103]. In order to the diagnosis of distinguish HCC cases from patients with chronic hepatitis,
HCC at early stage, a multicenter case-control research miR-21, whose level increases in serum, yielded a sensitivity
showed that PIVKA-II alone yielded a sensitivity of 56%, of 61.1% and a specificity of 83.3% [109]. Both miR-15b
while the combination of PIVKA-II with AFP exhibited an and miR-130b levels were significantly elevated in the serum
expected sensitivity of 87%, in addition to a disappointing of patients with HCC. When they were employed for the
specificity of 69% [72]. Although PIVKA-II has an accept- detection of HCC, the achieved sensitivity was 98.3% and
able performance, it was uncovered that the serum levels of 87.7%, while the specificity was 81.4% and 15.3%, respec-
PIVKA-II not only increased in the HCC cases, but also in tively. Regarding their high sensitivity, the analyses on the
patients who were treated with coumarin anticoagulants led serum concentrations of miR-15b and miR-130b could con-
to decrease in the effectiveness of PIVKA-II utilized as a tribute to the detection of HCC at early stage [110].
biomarker to detect HCC.
33.3.3.8 Other Biomarkers
33.3.3.6 S quamous Cell Carcinoma Antigen
(SCCA) Tumor Specific Protein 70 (SP70)
SCCA is a useful tumor marker for the diagnosis and man- Owing to the lack of accuracy, most current tumor markers
agement of squamous cell carcinoma. It has two isoforms, are limited in clinical. Some candidate markers have been
SCCA1 and SCCA2, both of which are expressed in a layer reported in recent years. SP70 was reported to be a new pro-
of the squamous epithelium. The presence of SCCA is asso- tein mainly expressed in non-small cell lung cancer (NSCLC)
ciated with various types of cancer, including HCC [104]. with the relative molecular mass of 70 kDa. It could be a sen-
The highly expressed SCCA appears to be associated with sitive biomarker for monitoring timely response to chemo-
tumorigenesis. It not only protects tumor cells from apopto- therapy in patients with advanced NSCLC [111, 112]. Not
sis, but also promotes their growth in vivo. Additionally, only that, it was found to be highly expressed in HCC tissues,
SCCA has shown to have a potential to determine HCC. When and significantly associated with the tumor stage, metastasis,
SCCA is applied to differentiate patients with HCC from early recurrence, and viral load. Compared with AFP and
those with cirrhosis, SCCA has a sensitivity of 84.2% and a DCP alone or combined detection of two markers, the combi-
specificity of 48.9% [105]. SCCA-IgM is recognized as the nation of AFP and SP70 presented the best diagnostic effi-
variant IgM immune complex formed by the combination of cacy in differentiating HCC from LC (AUC = 0.772, 95% CI
SCCA with IgM, and its expression level increases in the 0.720–0.820), which proved that SP70 could serve as a com-
primary-phase of hepatocarcinogenesis, however, it cannot plementary tool for liver cirrhosis surveillance. Moreover,
be detected in healthy adult. SCCA-IgM outperforms the serum SP70 levels in patients with early recurrence increased
free form of SCCA in the detection of HCC with a sensitivity significantly after hepatectomy and showed an independent
of 89% and a specificity of 50%. Regarding its high sensitiv- correlation with RFS (Figs. 33.4, 33.5, 33.6, 33.7 and 33.8).
ity and low specificity, SCCA is typically utilized as a valu- The relevant data of SP70 in this chapter are from the First
a b
c d
Fig. 33.4 The expression of SP70 in liver tissue is determined by immunohistochemistry. (a) HCC; (b) corresponding paracancerous tissues; (c)
intrahepatic cholangiocarcinoma (ICC); (d) hepatic hemangioma
a P<0.001 b P<0.001 c P<0.001
120 120 120

80 80 80
40
40 40
SP70(ng/ml)
SP70(ng/ml)
SP70(ng/ml)
30 30 30
20 20 20
15.9
12.0 14.3 14.8
10 8.7 9.1 10 9.0 10.7 10 10.1
13.2
5.7
0 0 0
C
C
0/
H
BL
IC
P
e
P
H
ag
ag
ag
C
e
ag
St
St
St
St
Fig. 33.5 (a) Serum SP70 levels in different groups and (b, c) associations with BCLC and CP stage. (ICC intrahepatic cholangiocarcinoma, BLD
benign liver disease, HC healthy control; *P < 0.001)
480 Q. Ou et al.
a HCC vs BLD + HC
b HCC vs BLD
c HCC vs LC
1.0 1.0 1.0
0.8 0.8 0.8
Sensitivity
0.6 0.6 0.6
Sensitivity
Sensitivity
0.4 0.4 0.4
SP70 SP70 SP70

0.2 AFP 0.2 AFP 0.2 AFP
PIVKA PIVKA-II PIVKA-II
Reference Line Reference Line Reference Line
0.0 0.0 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
d HCC vs BLD + HC
e HCC vs BLD
f HCC vs LC
1.0 1.0 1.0
0.8 0.8 0.8
0.6
Sensitivity
Sensitivity
Sensitivity
0.6 0.6
0.4 0.4 0.4
SP70+AFP SP70+AFP SP70+AFP

0.2 SP70+PIVKA-II 0.2 SP70+PIVKA-II 0.2
SP70+PIVKA-II
AFP+PIVKA-II AFP+PIVKA-II AFP+PIVKA-II
Reference Line Reference Line Reference Line
0.0 0.0 0.0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0
Fig. 33.6 (a–c) Comparison of receiver operating characteristics curves of SP70, AFP, PIVKA-II, and (d–f) three different multi-marker
combinations
a P<0.001 b
P=0.136 c P=0.052
80 40000 80000
60
40
40 30000 60000
PIVKA-II(mAU/m)
SP70(ng/ml)
AFP(ng/ml)
30 20000 40000
20
10000 20000
10
0 0 0
Before resection After resection Before resection After resection Before resection After resection
Recurrence (n=45) Recurrence (n=45) Recurrence (n=45)
d P=0.051 e P=0.059 f P=0.009

40 2000 40000
30 1500 30000
PIVKA-II(mAU/m)
AFP(ng/ml)
SP70(ng/ml)
20 1000 20000
10 500 10000
0 0 0
Before resection After resection Before resection After resection Before resection After resection
Control (n=40) Control (n=40) Control (n=40)
Fig. 33.7 Changes of SP70, AFP, and PIVKA-II levels before and after hepatectomy in recurrence group and control group
a Low SP70 b Low AFP c Low PIVKA-II

High PIVKA-II
Recurrence-free survival
High SP70
1.0 1.0 High AFP 1.0
P<0.001 P=0.057 P=0.163
0.5 0.5 0.5
0.0 0.0 0.0

0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
Time (months) Time (months) Time (months)
Fig. 33.8 Kaplan-Meier analysis of recurrence-free survival (RFS) stratified according to the median of tumor marker. (a) SP70; (b) AFP; (c)
PIVKA-II
Affiliated Hospital of Nanjing Medical University, from trols. Exosomal hnRNPH1 mRNA showed the sensitivity of
October 2017 to December 2018. The reference interval of 85.2% and the specificity of 76.5% in the discrimination
SP70 (ELISA) is less than or equal to 7.5 ng/mL. between patients with HCC and ones with chronic hepatitis
B. Moreover, the sensitivity and specificity increased up to
Long Non-coding RNAs (LncRNAs) 87.5% and 84.8% when hnRNPH1 mRNA in combination
Long non-coding RNAs (lncRNAs) belong to a kind of with AFP was subjected to the same study [115].
non-coding RNA molecule that exceeds 200 nucleotides in
length. Recent report showed that the levels of plasma
ZFAS1 in the HCC patients were upregulated as compared 33.3.4 Genetic Variations
with the patients with cirrhosis, hepatitis B as well as
healthy controls and had positive relationships with the One of the most important mechanisms underlying the devel-
concentrations of AFP in serum. The sensitivity and speci- opment of HCC is the accumulation of genetic alterations.
ficity of ZFAS1 were 55.7 and 90.0% in the assays per- The mutation that may induce cancer progression can be
formed to differentiate HCC patients from healthy controls detected through sequencing of whole genome, whole
[113]. In addition, the upregulated levels of other cancer- exome, and transcriptome, which are deciphered by various
related lncRNAs including MALAT1 and HOTAIR in the kinds of sequencing approaches (e.g., next-generation
clinical tissue samples were documented to be associated sequencing (NGS) and clonal sequencing). The most impor-
with poor prognosis and recurrence of HCC in the previous tant alterations located at the TERT gene promoter mutations
reports. were found in 60% of patients suffering from HCC. HBV
can promote HCC in many ways. There is a large amount of
Circular RNAs (CircRNAs) data describing the multiple pathways involved in this pro-
Circular RNAs (circRNAs) refer to a class of RNAs pre- cess, including the accumulation of genetic damage due to
sented as loops as the results of back-splicing events. The immune-mediated hepatic inflammation, the induction of
expression level of hsa_circ_0001649 was found to be oxidative stress, virus-specific mechanisms involving the
reduced obviously in HCC tissues when compared with insertional mutagenesis with the integration of HBV DNA
adjacent liver tissues and exhibited the sensitivity of 0.81 into the host genome. The typical site integrated by HBV in
and the specificity of 0.69 in the experiment aiming to dis- the genome of the host is the TERT gene [116]. Another criti-
tinguish cancerous tissue from adjacent non-cancerous liver cal hotspot genetic change related to HCC is G-T
tissue [114]. single-nucleotide transversion in the DNA sequence encod-
ing P53 protein, resulting in a variety of amino acids. These
Contents in Exosome findings indicate that there may be a tight relationship
The experimental data documented in recent literatures indi- between G–T transversion and HCC cases. Additionally, the
cated that heterogeneous contents in exosome have the recurrent varieties are also identified in other genes related to
potentials to act as biomarkers in the diagnosis of cancers. the occurrence of HCC cases, such as CTNNB1, ARID1,
The levels of nuclear ribonucleoprotein H1 (hnRNPH1) RPS6KA3, NFE2L2, and IRF2 [117]. The performance of
mRNA was significantly upregulated in the serum exosome genetic variations exploited as biomarkers aiming to detect
derived from HCC patients as compared with patients with HCC is currently in progress, hopefully leading to great
liver cirrhosis, chronic hepatitis B as well as healthy con- achievements.
482 Q. Ou et al.
33.3.5 Treatment MRI An HCC lesion with 3.9 cm in diameter at segment 8

of the liver was seen in Fig. 33.9.
Treatment options for HCC include hepatectomy, liver trans-
plantation, local ablative therapy, hepatic artery transcatheter Laboratory Data ALT 29.5 U/L (7.0–40.0 U/L), AST
treatment, radiotherapy, chemotherapy, targeted therapy, and 27.4 U/L (13.0–35.0 U/L), GGT 59.4 U/L (7.0–45.0 U/L),
supportive care. Molecular targeted therapies have changed the ALP 116.0 U/L (30.0–120.0 U/L), HBV-DNA 4.12E+03 IU/
landscape of cancer management. Sorafenib, lenvatinib, and mL (<500 IU/mL), HBsAg positive (negative), HbsAb nega-
regorafenib are currently the only drugs approved for advanced tive, HBeAg negative (negative), HBeAb positive (negative),
HCC management. Some large-scale international multicenter HbcAb positive (negative), AFP 2.03 ng/mL (<20 ng/mL),
phase III clinical trials have proved that these drugs have cer- PIVKA-II 97 mAU/mL (<40 mAU/mL), SP70 14.9 ng/mL
tain survival benefits for advanced HCC in different countries, (≤7.5 ng/mL).
regions, and backgrounds [118–120], but more drugs have
failed to meet clinical end points in phase III trials. Histopathological Examination HCC, Edmondson-
Steiner grade I (Fig. 33.10).
33.3.6 Typical Medical Case Treatment The patient underwent hepatectomy.
Clinical Background A 58-year-old male patient came to Results with Interpretation Guideline Guidelines from
the hospital for pain in the right upper quadrant for 2 months. the American Association for the Study of Liver Diseases
He had a history of chronic hepatitis B for more than 10 years. (AASLD) and the European Association for the Study of the
a b
Fig. 33.9 MRI imaging. (a) arterial phase; (b) portal phase
a b
Fig. 33.10 Pathological results. (a) hematoxylin-eosin staining; (b) immunohistochemical staining
Liver (EASL) recommend surveillance being currently pri- Table 33.3 Clinical manifestations of Wilson’s disease
marily imaging-based (CT or MRI). HCC can be diagnosed Age at onset of
radiologically, without the need for biopsy if the typical Clinical symptoms symptoms
imaging features are present. If imaging features are not typ- Hepatic >2 years
ical, other imaging models or lesion biopsy can be used. For Incidental finding of increased serum
transaminases
non-cirrhosis patients, the diagnosis should be based on
Acute hepatitis
pathology. Tissues that are not clearly defined for HCC
Hepatomegaly
should be stained with special markers, such as glypican 3 Fatty liver
and glutamine synthetase to improve diagnostic accuracy. Acute liver failure with hemolysis
However, tumor markers still play an important role in the Portal hypertension: Esophageal varices,
diagnosis and monitoring of HCC because they are conve- splenomegaly, low platelet count
nient, cost-effective, available, and repeatable, such as AFP, Decompensated cirrhosis with ascites
PIVKA-II, AFP-L3, and new marker SP70. Neurological and psychiatric Usually >15 years
Dysarthria Case reports 7–9
years
This case was from the First Affiliated Hospital of Nanjing Dysphagia, excessive salivation
Medical University (also named Jiangsu Province Hospital). Mood/behavior changes including
depression, irritability
Incoordination (e.g., handwriting
deterioration)
33.4 Wilson Disease
Declining performance at school
Resting and intention tremors
Zhaojing Zheng and Juan Geng Gait disturbance, dystonia, rigidity
Mask-like face, risus sardonicus
Stroke-like symptoms
Overview Ophthalmic >10 years
Wilson disease (WD), also known as hepatolenticular degen- KF rings at slit-lamp examination
eration, is a rare autosomal recessive disease, with a quoted Hematological >7 years
prevalence of 1 in 30,000, and incidence ranging from 15 to Acute/chronic hemolytic anemia
30 per million [121, 122]. It is characterized by abnormal Other Case reports, age
cannot be defined
accumulations of copper in several tissues, particularly in
Renal
liver, brain, and cornea. Clinical features associated with Renal tubular dysfunction(Fanconi
WD are protean even within families, it can mainly manifest syndrome, tubular acidosis, aminoaciduria)
as hepatic, neurologic, hematologic, or psychiatric distur- Nephrolithiasis
bances, or a combination of them. The diagnosis of WD is Nephrocalcinosis
not always straightforward considering the diverse clinical Cardiac
presentations and variable biochemical testing results. Cardiomyopathy, subclinical dysfunction
Genetic information gained from genetic testing is always Arrhythmia
Endocrine
indispensable for diagnosis, prognosis, management, and
Hypoparathyroidism
genetic counseling for the patient as well as his/her family. Other
Pancreatitis
Skin lipomas
33.4.1 Clinical Appearance Skeletal
Rickets/osteopenia/osteoporosis
Although most patients develop symptoms in adolescence to Arthropathy
early adulthood, the age of first symptoms varies widely KF Kayser–Fleischer
from the first decade to the fourth and fifth decades of life.
Common symptoms and clinical features of WD are involvement is variable, ranging from asymptomatic hepa-
described in Table 33.3 [123]. tosplenomegaly with mild elevations of certain liver
enzymes, to complete liver failure. Associated symptoms
33.4.1.1 Clinical Manifestations include nonspecific general symptoms, ascites, and jaun-
Patients with hepatic WD usually present features of acute dice, as well as hematemesis and melena caused by portal
hepatitis, fulminant hepatic failure, or progressive chronic hypertension. In general, the younger the patient is at the
liver disease in the form of either chronic active hepatitis or onset of symptoms, the greater the degree of liver involve-
cirrhosis of the macronodular type. The extent of liver ment will be.
484 Q. Ou et al.
33.4.1.2 Neurological and Psychiatric naked eye, a slit-lamp examination is necessary to confirm

Manifestations the presence of K–F rings. Sunflower cataracts are brilliantly
Neurological manifestations can be categorized as: (1) an multicoloured and are visible only by slit-lamp examination,
akinetic-rigid syndrome similar to Parkinson’s disease; (2) and they do not impair vision. Although K–F rings are very
pseudosclerosis dominated by tremor; (3) ataxia; and (4) a common in WD, especially in patients with neurological dis-
dystonic syndrome, which often leads to severe contractures. eases, sunflower cataracts are rarely observed. Both of them
Neuropsychiatric symptoms and signs, including decreased are reversible after medical therapies or liver transplantation.
scholastic performance, hand-eye discoordination, and
behavioral changes that may foretell more florid neurologi-
cal manifestations. Other findings include drooling, spastic- 33.4.2 Diagnosis
ity, chorea, athetosis, myoclonus, micrographia, dyslalia,
hypomimia, and dysarthria. Achieving the diagnosis of WD depends on maintaining a
high suspicion index. According to a recent case report, all
33.4.1.3 Ophthalmic Manifestations subjects with symptomatic or asymptomatic liver disease of
Ocular manifestations include the Kayser–Fleischer ring unknown cause, or extrapyramidal features along with a past
(K–F ring, the most important sign in WD) and sunflower or family history of similar hepatic or neurological illnesses
cataracts. K–F rings are most apparent at the periphery of the in other siblings, should be screened for WD.
cornea. They are caused by the granular deposition of copper The diagnosis of WD is still based on laboratory and sug-
on the inner surface of the cornea in the Descemet’s mem- gestive clinical phenotypes, and cannot be made by a single
brane. The upper pole is affected first. The rings have a test alone: a combination of tests is always required
golden-brown appearance. Although sometimes visible to the (Fig. 33.11) [123, 124]. Two sets of diagnostic algorithms
Fig. 33.11 Diagnostic
approach to Wilson’s disease.
ALT alanine aminotransferase,
AP alkaline phosphatase, AST
aspartate aminotransferase
Table 33.4 Scoring system for Wilson’s disease diagnosis Ceruloplasmin

Diagnosis score Ceruloplasmin is the major carrier of copper in the blood.
Kayser–Fleischer rings Measurement of the serum ceruloplasmin level is important
Present 2 for the diagnosis of WD. The normal concentration of ceru-
Absent 0 loplasmin measured by the enzymatic assay varies among
Neurological symptomsa laboratories (with a lower limit of 0.15 ~ 0.2 g/L). In Wilson’s
Severe 2
disease, the serum ceruloplasmin level significantly
Mild 1
Absent 0
decreased and usually lower than 0.1 g/L [125]. Because
Serum ceruloplasmin ceruloplasmin acts as an acute phase protein as well as a cop-
Normal (>200 mg/L) 0 per transporter, false high levels could be observed when
100–200 mg/L 1 patients have active inflammation, pregnancy, or taking
<100 mg/L 2 estrogen. Any value below 0.2 g/L is abnormal, and a reduced
Haemolytic anaemia level can be seen in up to 95% of cases of WD [126]. Low
Present 1 levels of ceruloplasmin can also be found in cases of Menkes
Absent 0 disease, hereditary aceruloplasminemia, protein-losing
Liver copper (in absence of cholestasis)
enteropathy including celiac disease and severe hepatic
>5 × ULN (>250 μg/g) 2
insufficiency, and heterozygous carriers of WD.
50–250 μg/g 1
Normal (<50 μg/g) −1
Total Serum Copper
Rhodanine positive granulesb 1
Urinary copper (in absence of acute hepatitis) Although it is a copper overdose condition, the total serum
Normal 0 copper (which includes copper incorporated in ceruloplas-
1–2 × ULN 1 min) in WD generally decreases in proportion to the decrease
>2 × ULN 2 in circulating ceruloplasmin. However, in patients with WD
Normal, but >5 × ULN after penicillamine 2 who have a severe liver injury, serum copper may be within
Mutation analysis the normal range or markedly elevated in the setting of acute
Homozygous 4 liver failure (ALF) due to the release of copper in the liver
Heterozygous 1 tissue and the increase in free copper in the blood. The serum
No mutations detected 0
non-ceruloplasmin-bound copper concentration can be esti-
ULN upper limit of normal
mated from the serum copper and serum ceruloplasmin lev-
a
Typical abnormalities on brain MRI
b
If no quantitative liver copper available els but is dependent on the accuracy of the methods for
4 or more: diagnosis established; 3: diagnosis possible, more tests measuring both serum copper and ceruloplasmin. Total
needed; 2 or less: diagnosis very unlikely serum copper has a poor performance for diagnostic pur-
poses but could be more valuable for the prediction of ther-
have been published and are widely accepted, including the apy response and treatment monitoring. Extremely low
scoring system for diagnosis (Table 33.4) [124]. The scoring values may indicate that systemic copper depletion may
system collects biochemical, clinical, and genetic data from occur in some patients with prolonged treatment.
individual patients to provide a quantitative score.
Urinary Copper Excretion
33.4.2.1 Liver Function Tests Urinary copper is derived from the so-called free (non-
In the acute manifestations of WD with liver failure, typical caeruloplasmin- bound) copper circulating in plasma. In
findings are high total bilirubin levels (>300 mmol/L, Wilson’s disease, the 24 h urinary copper excretion is
>17.5 mg/dL), relatively low serum transaminases levels increased, and the concentration taken as suggestive of dis-
(100–500 IU/L), low serum alkaline phosphatase level that ease is greater than 100 μg per 24 h (>1.6 μmol/24 h) [122].
was associated with zinc deficiency, and a low alkaline phos- The reference limits for normal 24 h excretion of copper vary
phatase (IU/L)-to-total bilirubin (mg/dL) ratio < 1 [123]. between laboratories, with many taking 40 μg per 24 h
However, these findings are not specific features of WD. (0.6 μmol/24 h) as the upper limit of normal. This limitation
seems to be a better threshold for diagnosis because the test
33.4.2.2 Biochemical Investigations sensitivity is increased. However, basal 24-h urinary copper
WD is biochemically characterized by low ceruloplasmin excretion may be less than 1.6 μmol/24 h at presentation in
and total serum copper levels increased 24-h urinary copper 16–23% of patients, especially in children and asymptomatic
excretion, and abnormally high hepatic copper content. siblings [127]. Since urinary copper excretion is negligible
486 Q. Ou et al.
in healthy individuals, a urinary copper excretion above sequenced [124]. Promoter mutations or gene dosage prob-
0.64 μmol/24 h may suggest Wilson’s disease in asymptom- lems, such as complete exon deletions, are rare and therefore
atic children. Interpreting of 24-h urinary copper excretion do not need to be included in a routine genetic testing of
may be difficult due to the overlapping findings with other ATP7B. Predominant mutations have been reported in spe-
types of liver disease (e.g., autoimmune hepatitis, chronic cific populations, such as Eastern Europe (H1069Q), Spain
active liver disease, or cholestasis, particularly during acute (Met645Arg), Sardinia (c-441427del15), Japan (229insC,
hepatic failure of any origin). Heterozygotes may also have Arg778Leu), Costa Rica (Asp1279Ser), and China, Korea,
higher copper excretion than the control, rarely exceeding and Taiwan (Arg778Leu), facilitating the molecular diagno-
the normal range levels. sis [129–132]. Therefore, in a given population, a stepwise
approach initially focuses on sequence analysis of ATP7B
33.4.2.3 Liver Biopsy and Liver Copper Content mutation hotspots, which is likely to be more cost-effective
The measurement of copper in the liver is the most important in a clinical setting. The genetic detection algorithm of
diagnostic test for patients who are prompted by other data Wilson’s disease is shown in Fig. 33.12.
but cannot make the diagnosis of WD. Copper lev-
els<250 mg/g dry weight (normal <50 mg/g dry weight) in
non-cholestatic patients are considered to be diagnostic of 33.4.3 Correlation Between Phenotype
WD in adults. It has been reported that sampling error might and Genotype
occur in up to 20% of WD patients due to the uneven distri-
bution of copper in the liver. The accuracy of liver copper Despite the allelic heterogeneity of the ATP7B gene, several
measurements is improved by a sample of sufficient size research groups attempted to analyze phenotype–genotype
(preferably >1 cm long, min. 0.5 cm) which should be placed correlation data. Truncation mutations are associated with
on a small piece of paper for drying and subjected to atomic fulminant hepatic failure and early onset, suggesting that
absorption analysis in a dry plastic copper-free container. It complete loss of function leading to early copper overload
has also been suggested to perform twice liver biopsy and [133–135]. In contrast, patients with H714Q mutation tend
use the entire sample core for copper determination [123]. to have late, neurologic manifestations [136]. This mutation,
found in ~20% of patients, affects the phosphorylation
33.4.2.4 Imaging domain and has some residual functional activities, which
Imaging plays an important role in the diagnosis of WD and may account for a slower accumulation of copper and hence,
patient monitoring during therapy. Brain CT scan is rela- a later onset of the disease. The most common mutation in
tively insensitive but can reveal hypodensity and atrophy of Caucasian patients from Europe and North America is
the bilateral basal ganglia, brainstem, cerebellum, and cere- H1069Q and is located in the loop motif with the disruption
bral cortex. In comparison, MRI is more sensitive in the of ATP binding. This mutation accounts for ~40% of all
detection of brain abnormalities in WD. Other abnormalities, mutations, especially from individuals in Eastern Europe and
such as tectal plate hyperintensity, central pontine Germany [129]. The homozygosity of this mutation is rela-
myelinolysis-like abnormalities, and concurrent signal of the tively common to allow a meaningful phenotypic-genotypic
basal ganglia, thalamus, and brainstem are much more com- correlation. However, homozygous patients have an almost
mon. Rarely, Wilson’s disease can result in diffuse white equal chance for either neurologic (51.7%) or hepatic
matter abnormality which should be regarded as a possible (48.3%) manifestations. Overall, the presence of this mutant
cause of diffuse leukoencephalopathy. Other imaging tech- allele was seen in 39.8% of patients with hepatic and 51.9%
niques, such as 7 T MRI, T2-weighted imaging, MR spec- with neurologic manifestations [137]. N1270S homozygote
troscopy, transcranial brain parenchyma sonography (TCS), predisposes to liver disease with frequent fulminant liver
or SPECT are currently only used in research [124, 126]. failure as a feature, c.2299insC predisposes to hepatic and
A1003T to neurologic symptoms [128, 138].
33.4.2.5 Genetic Testing
The use of mutation screening to identify defects in the
ATP7B gene can provide etiological confirmation of WD in 33.4.4 Genetic Counselling
the affected symptomatic or pre-symptomatic individuals.
More than 500 mutations within the ATP7B gene (locus Genetic counseling is essential for families of patients with
13q14.3) have been identified, and most of the affected WD, and screening first-degree relatives is recommended by
individuals are compound heterozygotes [123, 128]. Two both European and American guidelines. Family members of
pathogenic ATP7B mutations can be detected in 98% of affected individuals are similarly affected (with two mutant
patients with clinically confirmed Wilson’s disease if the genes) with a probability of 25% for siblings and 0.5% for
entire ATP7B coding region and adjacent splice sites are offspring. The assessment should include physical examina-
Fig. 33.12 Genetic testing algorithm of Wilson’s disease
tions, measurement of serum ceruloplasmin, liver function retrieved by either chorionic villus sampling or amniocente-
tests, and molecular testing for ATP7B mutations or haplo- sis. In diagnostic laboratories, the causative mutations estab-
type studies if not available. Screening for newborns is not lished by family DNA studies or analysis of affected
warranted and screening may be delayed until 1 to 2 years of probands are directly targeted by PCR and Sanger sequenc-
age. Moreover, the risk of WD in offspring increases in con- ing, and if one or both mutations are unknown, a linkage
sanguineous families and specific populations with high car- analysis with short tandem repeats can be used as an alterna-
rier frequencies. Finally, considering the possibility of late tive strategy [139, 140]. These approaches are highly reliable
onset of WD, parents of children newly diagnosed with WD and accurate for defining the genotype of the fetus, and if the
should also be screened by performing liver tests, explora- fetus is affected, the couple is allowed to make a decision
tions of copper metabolism, and suitable genetic testing. The regarding termination of pregnancy. In addition, denaturing
pre-symptomatic individual and placement on the treatment high performance liquid chromatography (DHPLC) tech-
regimen can then be monitored as appropriate prior to the nique and Circulating Single-Molecule Amplification and
onset of clinical symptoms. Resequencing Technology (cSMART) were used and proved
to be a powerful tool for WD prenatal diagnosis [141, 142].
Recently, with the advent of whole-exome sequencing,
33.4.5 Prenatal Diagnosis pathogenic mutations can be readily identified for almost any
and Preimplantation Genetic Diagnosis monogenic disease, and this approach has been proven to be
particularly useful to identify novel or rare mutations in fam-
The diagnosis of monogenic diseases during pregnancy is ilies and to aid in establishing a more definitive diagnosis of
currently performed by molecular analysis of the fetal cells individuals with unclear clinical phenotypes.
488 Q. Ou et al.
a c
d
b
Fig. 33.13 The genetic testing results of the classic case with Wilson’s ray analysis (CMA) result depicting exonic deletion encompassing
disease. (a) the chromatograph of ATP7B Sanger sequencing by which ATB7B gene exon2 and exon 3 in the proband. (c) Microdeletion of
a pathogenic variant c.2668G>A (p.V890M) was detected both in the ATB7B gene exon2 and (d) exon was validated in the proband by qPCR,
proband and his mother. (b) The screenshot of chromosomal microar- and also detected in the proband’s father as well as his brother
33.4.6 Typical Clinical Case sible for the missing pathogenic variations. A heterozygous
microdeletion of ATP7B exon 2 and exon 3 was detected by
An 8-year-old girl was referred due to hepatosplenomegaly. CMA in the patient. This exonic deletion of ATP7B was
On admission, she was found to have abdominal distension further validated by qPCR in the patient as well as in her
and yellow sclera for duration of 4 days, fever of 3 days. father and brother. Taken together, the proband had a com-
Laboratory testing showed that the serum levels of cerulo- pound heterozygous mutation consisted of maternal
plasmin decreased to 0.08 g/L, in addition to mildly elevated c.2668G>A and paternal Del Exon 2–3 (Fig. 33.13) that
serum concentrations of liver enzymes with glutamic-pyruvic contributed to the development of Wilson disease in this
transaminase (GPT) of 69.0 IU/L and glutamic oxaloacetic patient.
transaminase (GOT) of 279.0 IU/L. The total bilirubin (TBil) This case was from Shanghai Children’s Medical Center.
in serum also increased to 195.4 μmol/L. Kayser–Fleischer
rings were observed in this girl. There is no abnormality
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136. Houwen RH, Juyn J, Hoogenraad TU, et al. H714Q mutation in
Wilson disease is associated with late, neurological presentation. J
Med Genet. 1995;32:480–2.
Pancreatic Diseases
34
Lu Yang, Huanyu Ju, Yuan Mu, and Chunrong Gu
This chapter stocks up information about pancreatic disor- 34.1.1 Overview

ders, methods of detection, and use of laboratory test results
in recognizing and characterizing patterns of pancreatic The most common type of pancreatic cancer (PC) is pan-
diseases. creatic ductal adenocarcinoma (PDAC). PDAC accounts for
The pancreas is a coarsely lobed gland lying at the back 90% of all pancreatic malignancies, and the dismal 5-year
of the abdomen, extending from the duodenum to the splenic survival rate is about 8% [1, 2]. It is the fourth leading cause
hilum. The organ is divided into the head, neck, body, and of cancer death in men and women in the United States.
tail. The anterior part of the pancreas is in direct contact In 2019, the estimated number of new pancreatic cancer
with the posterior wall of the stomach. The pancreas extends cases in the United States is 56,770, and the death caused
backwards into the inferior vena cava, superior mesenteric by pancreatic cancer is 45,750 [3]. PDACs are solid, poorly
vein, splenic vein, and left kidney. The uncinate process is demarcated tumors, hard and yellowish-white to gray, and
a part of the head of the pancreas that extends posteriorly usually between 2 and 5 cm in diameter. In spite of all the
to the superior mesenteric vein and artery. Anatomy of the research it has launched over the past 30 years, the 5-year
pancreas is described in Fig. 34.1. survival rate is still low. We are far from having an effec-
The pancreas is really two glands that are intimately tive strategy for early diagnosis and treatment of this deadly
mixed together into one organ. The pancreas consists of an disease. Most risk factors, such as diabetes, obesity, male
exocrine and endocrine chamber. Exocrine tissue is made gender, and Helicobacter pylori infection, all cause mild
up of acinoles, which are involved in the secretion of diges- (<twofold). Smoking is the greatest environmental risk fac-
tive enzymes that are transported to the gut and secreted in tor (about threefold risk). Genetic factors are the greatest risk
large quantities of alkaline fluid, and endocrine Chambers, factors for PDAC development. Mutation carriers of known
which contain islets involved in the secretion of pancreatic genetic syndromes such as familial atypical multiple mole
hormones such as insulin, glucagon, and somatostatin. melanoma (FAMMM), Peutz-Jeghers’s syndrome, or famil-
Disorders of pancreas include pancreatitis, cystic neo- ial pancreatic cancer (which means individuals with two or
plastic lesions, pancreatic cancer, and other diseases. The more cases of PDAC in their family without a known muta-
detail information is summarized in Fig. 34.2. tion) are all the known genetic factors.
34.1 Pancreatic Ductal Adenocarcinoma 34.1.2 Clinical Appearance
Lu Yang Most pancreatic cancers are diagnosed with nonspecific

abdominal pain or jaundice or both. If the tumor grows in
the head of the pancreas near the bile duct, the only clinical
symptom is jaundice. Many patients present with advanced
secondary symptoms associated with large malignant tumors
and/or metastatic spread with back pain (direct invasion of the
L. Yang (*) · H. Ju (*) · Y. Mu (*) · C. Gu (*) abdominal plexus) or malignant ascites. Unexplained weight
Department of Laboratory Medicine, The First Affiliated Hospital loss is sometimes the only sign. About 80% of patients have
an unresectable disease due to metastatic transmission or
e-mail: y.mu@njmu.edu.cn locally advanced disease at the time of diagnosis.

494 L. Yang et al.
splenic artery
pancreatic
hormones
bile duct
pancreas
digestive
interlobular duct enzymes
islet of
intralobular duct duodenum
langerhans
intercalated duct
α-Cells
β-Cells
lobule
acinar cells
Fig. 34.1 Anatomy of the pancreas, the pancreas is divided into the head, neck, and body
Organ Disease type Clinical appearance Laboratory diagnosis
Nonspecific abdominal
Pancreatic ductal
pain Tumor markers
adenocarcinoma
Jaundice
Pain Exocrine pancreatic

Chronic pancreatitis Exorcine enzymes or hormones
pancreatic insufficiency Pancreatic function tests
Pancreas
Intravenous
Insulinoma Whipple Triad
secretin tests
Intraductal papillary Upper abdomen

Tumor markers
mucinous neoplasm discomfort or pain
Fig. 34.2 Description of pancreatic diseases

34 Pancreatic Diseases 495
The development of diabetes should be a strong reminder CA19-9, CA242, and CEA for the diagnosis and prognosis
to doctors of the potential for pancreatic cancer. Compared of pancreatic cancer [7].
with the general population, people over 50 with late-onset
diabetes have an eightfold increased risk of developing pan- CA242
creatic cancer within 3 years of diagnosis. Most malignant CA242 is a carbohydrate antigen containing sialic acid that
tumors occur in the head of the pancreas due to the low attaches to core proteins/lipids detected on the cell surface or
resectability and poor prognosis of the tumor in the tail of in serum. Elevated serum CA242 levels have been clinically
the pancreas. used as biomarkers for the diagnosis of pancreatic, colorec-
tal, and other cancers.
Kuusela and colleagues [8] first reported the elevation of
34.1.3 Laboratory Diagnosis serum CA242 in pancreatic cancer patients in 1991. Further
studies have shown that CA242, CA19-9, and CA50 have
34.1.3.1 Mucin Tumor Markers similar sensitivity and specificity in the diagnosis of pancre-
atic cancer. In general, the sensitivity of CA242 is lower than
CA19-9 that of CA50 and CEA, but CA242 has a higher specificity in
CA19-9 is the most widely used pancreatic tumor marker and the diagnosis of pancreatic cancer. Combined with CEA or
has become the standard for comparison of other markers. It CA50 can increase the sensitivity of CA242. Another study
was developed for the colorectal cell line SW1116 in 1979 confirmed this view, showing that the sensitivity of CA242,
and was originally known as the gastrointestinal carcinoma CA19-9, and CA50 to the diagnosis of pancreatic cancer was
antigen (GICA) [4]. CA19-9 is the preferred tumor marker 66.2%, 70.6%, and 70.6%, respectively. The sensitivity of
in clinical practice today, but the main problem with CA19-9 CA242 and CA50 combined as diagnostic biomarkers was
is its specificity, which ranges from 60 to 90%. In addition, 75.0%. Combined application of CA242 with CA19-9 and
abnormal levels may be observed in some benign diseases, CA50 can significantly improve the sensitivity and specific-
especially in jaundice, where CA19-9 may reach 1000 U/ ity of the diagnosis of pancreatic cancer. CA19-9, CA50, and
mL. The European tumor marker group (EGTM) considers CEA are common biomarkers of pancreatic cancer [9].
CA19-9 to be of little diagnostic value, especially in the early
stages of the disease, but this biomarker may be of interest as DU-PAN-2
an adjunct to radiological methods, mainly in cases without In 1984, Metzgaretal [10] described this monoclonal anti-
jaundice. The comprehensive national cancer network showed body after immunizing it with pancreatic cancer cell line
that the results of CA19-9 could be used as an aid to distin- HPAF. Epitopes are mucin oligosaccharides acidified by
guish between patients with inflammatory pancreatic disease saliva, but different from Lewis antigen. The sensitivity of
and patients with pancreatic adenoma, although negative does serum DU-PAN-2 was 38–94% by competitive immuno-
not exclude malignancies (mainly leusi-ageno type) and false assay. Data from the Japanese pancreatic cancer registry
positives in jaundice need to be taken into account [5]. showed a weak or no correlation between du-pan-2 and
CA19-9, while a relatively high correlation between span-1
CA50 and CA19-9 [11].
Carbohydrate antigen 50 (CA50) was initially reported as a
tumor-specific antigen expressed on the surface of human CAM17.1/Wheat-Germ Agglutinin (WGA)
colon-205 cancer cells [6]. Subsequently, elevated serum CAM17.1 is a monoclonal antibody developed using a mix-
CA50 levels were observed not only in patients with colorec- ture of Coll 2–23 colorectal cancer cells, PC/a a multiple
tal cancer, but also in patients with other types of cancer. Escherichia coli cells and meconium. It binds to intestinal
Finally, serum CA50 was clinically detected as a biomarker mucins [12], is sialic acid dependent, and agglutinates all of
for cancer. an extensive donor panel of normal adult red blood cells but
Elevated serum CA50 levels are most effectively in diag- not cord blood, suggesting that it recognizes the sialylated I
nosing pancreatic cancers with a sensitivity of more than antigen. The sensitivity and specificity of CAM17.1 in the
84%. Although CA50 is also expressed in normal pancre- diagnosis of pancreatic cancer were 78–83% and 76–82%,
atic tissue, elevated CA50 is mainly found in patients with respectively. It is negative in cystic fibrosis sera, unlike
pancreatitis and pancreatic adenocarcinoma. Among them, CA19-9, and false-positive rates in acute and chronic pan-
the expression of CA50 in malignant pancreatic tissues is creatitis are no higher than for other disease control groups.
higher than that in benign pancreatic tissues. As a serum bio- An avidin-biotin-amplified version of the assay has since
marker, CA50 can distinguish pancreatic carcinomas from been marketed, and a prospective assessment of this assay
chronic pancreatitis with a sensitivity of 46% and a specific- has produced even better results, with sensitivity of 91% and
ity of 91%, respectively. It is often used in combination with specificity of 92%.
496 L. Yang et al.
34.1.3.2 Other Markers It is quite possible that some of the anti-mucin antibodies
that are used in pancreatic cancer diagnosis cross-react with
Tumor-Specific Protein (SP70) carbohydrate structures expressed by CEA; however, the
SP70, the corresponding antigen of NJ001, has been dem- antibodies used in CEA assay bind to the protein portion of
onstrated to be located on cytoplasm and NSCLC cell mem- the molecule. Although occasional studies have shown good
brane. The relative molecular mass (Mr) of SP70 protein is results in pancreatic cancer diagnosis, the sensitivity and
70 kDa. NJ001, a monoclonal antibody that is responsive to specificity have generally proved too low for it to be used as
NSCLC cells, was screened from the self-made tumor mono- a single test. Published sensitivities range from 30 to 92%
clonal antibody library. By blocking SP70 cell membrane, and specificities from 58 to 95%.
NJ001 can effectively inhibit spc-a1 cell proliferation in vivo
and in vitro, and induce spc-a1 cell apoptosis [13, 14]. In Membrane Antigens
addition, our recent study showed that high levels of SP70 Common epithelial cell surface antigen (EPM-1) is present
can be detected in the serum of patients with pancreatic can- in the serum of normal persons, but is reduced to low levels
cer (PC). or undetectable in patients with gastrointestinal malignancy,
A double antibody sandwich ELISA method was used to which is different from all other markers [16]. Pa-25 and
detect SP70 in serum of 162 patients with PC, 61 patients Pa-42, two monoclonal antibodies against pancreatic can-
with benign pancreatic disease (BPD), and 122 healthy cer cell lines (Capan-1), were detected. These antibodies are
control (HC) from the First Affiliated Hospital of Nanjing consistent with a wide range of normal epithelial tissue and
Medical University from May 2016 to October 2017 in our with most epithelial neoplasms. This antigen can be detected
study. The reference range of SP70 (ELISA) in this study as 400 kDa glycoprotein in serum.
was ≤7.5 ng/mL. The serum SP70 concentration of PC group RCAS1 (receptor-binding cancer antigen expressed on
was significantly higher than that of HC group (P < 0.001), SiSo cells) is a novel tumor-associated antigen, which is
as well as that in patients with BPD (P < 0.05). There was expressed in a variety of malignancies. Monoclonal antibody
no significant difference between BPD group and HC group 22-1-1 was isolated from mice immune to SiSo cells [17].
(Fig. 34.3). Immunohistochemical studies have reported the presence of
RCAS1 in liver, gallbladder, stomach, esophagus, and pan-
Carcinoembryonic Antigen creas. Using the recently developed ELISA system for the
Carcinoembryonic antigen (CEA) was originally thought detection of soluble RCAS1, it has been demonstrated to
to be an oncofetal protein specific for colon cancer, but has have a sensitivity of 55% and specificity of 92%.
subsequently been shown to be present in both normal and
diseased pancreatic tissues, as well as tissues of other organs. PAM4
It was first defined by Gold and Freedman in 1965 [15]. It Recently, Gold and colleagues [18] developed the monoclo-
has a molecular mass of 180 kDa. Carbohydrate content is nal antibody PAM4, which is highly specific to the muc1
50–60%, including the expression of blood group antigens. produced by pancreatic cancer. This antigen has been identi-
fied in more than 90% of pancreatic cancers and their precur-
100 *** sors, but not in normal pancreas. The results showed that the
sensitivity of immunoassay to pancreatic cancer was 77%,
*
the specificity was 95%, and the positive likelihood ratio was
80 16.8. In immunohistochemical and enzyme-linked immuno-
assays, the total sensitivity and specificity of pam4 mucin
SP70(ng/mL)
60 in the tissues and serum of healthy individuals and cancer

patients with known cancer stages were 82% and 85%,
respectively, consistent with previous results. One exciting
40
finding in their study was that 92% of stage I cancer cases
were above the threshold for a positive result. Serum mean
20 antigen concentration is closely related to disease stage, sug-
gesting that PAM4 has potential application value in early
0 diagnosis of pancreatic cancer.
C
PC
H
BP
MicroRNAs
MiRNAs are 19–24 bases of non-coding RNA and play an
Fig. 34.3 Serum SP70 levels were increased in PC patients (PC pan-
creatic cancer, BPD benign pancreatic disease, HC healthy control; important regulatory role in post-transcriptional gene expres-
***
P < 0.001; *P < 0.05) sion. MiRNA was first discovered by Victor Ambros et al.
in 1993 while working with Caenorhabditis elegans [19]. mon mutated genes were found, including MLL3, SMAD3,
In recent years, microRNAs (miRNAs) have been reported FBXW7, and ARID1A. Germline mutations in BRCA2 and
as potential biomarkers for a variety of cancers. It is worth CDKN2A, as well as germline mutations that are less com-
noting that each of the previous studies proposed a different mon in rca1, PALB2, and ATM, have been found in a small
marker for the PC cycle. For example, plasma samples from subset of patients with familial PDAC [25].
50 cancer patients and 10 healthy subjects were used, as
well as anti-chemotherapy pancreatic cell lines, Ali and col-
leagues. It was suggested that serum miR-21 and other miR- 34.1.4 Management
NAs could predict the aggressiveness of PC [20]. Similarly,
Ganepola and colleagues examined plasma in both PC and In summary, many new markers based on proteins, DNA, and
non-pc patients and concluded that the sensitivity and speci- RNA are being investigated as markers for invasive pancre-
ficity of the three miRNAs, namely miR-642b, miR-885-5p, atic cancer. While some of the newly described markers are
and miR-22, as diagnostic tools were all greater than 90% promising, many require further validation using best labora-
[21]. It is suggested that the evaluation of eight miRNA tory methods and appropriate patient populations before they
co-markers (miR-6075, miR-4294, miR-6880-5p, miR- can be considered for use in a clinical setting. Because the
6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR- best time to cure pancreatic cancer is in its early stages, clini-
4476) is of clinical value in the identification of patients with cal studies involving pancreatic cancer markers must focus
cholangiocarcinoma who may benefit from surgery [22]. on patients with early pancreatic cancer. Currently, biomark-
Such tests would provide additional information to those at ers and imaging techniques are not recommended as routine
risk and, if necessary, prompt the use of more expensive and screening tools for screening asymptomatic patients in the
sometimes invasive imaging studies. Although the biological general population. Screening of high-risk patients with EUS
role of miRNAs is still under discussion, these unique mark- is widely accepted, but evidence of effectiveness and cost-
ers have the potential to lead to early detection and improved effectiveness is still needed. Continued efforts are needed to
survival. find effective tests to identify patients with non-genetic risk
factors who would benefit from screening and to develop less
34.1.3.3 Proteomics invasive and more cost-effective screening models aimed at
The current genomics-based techniques cannot reflect controlling pancreatic cancer.
the quantitative dynamic signal changes within the tumor. The tumor marker assays, particularly some of the well-
Therefore, “functional” diagnosis based on proteomics can characterized mucin assays such as CA19-9, DU-PAN-2,
serve as a useful complementary tool to provide the most and CAM17.1/WGA, are robust and reproducible and have
direct link to the cancer cell phenotype [23]. Since most tar- similar predictive value to current scanning techniques.
geted drugs are signal modulators, protein-based diagnostics Furthermore, they provide a biochemical test that, if used
will be extremely useful in assessing therapeutic response, appropriately, complements the diagnostic accuracy of “ana-
identifying potential mechanisms of drug resistance, and tomic” scanning. They deserve to have an established role in
guiding further therapeutic decisions, all of which are impos- (a) the diagnosis of no jaundiced patients with unexplained
sible to use with tumor tissue. Lim and colleagues reported upper abdominal pain or weight loss; (b) the investigation of
the use of a multiplex proteomic-based assay, Collaborative patients with a known pancreatic mass or cyst; and (c) moni-
Enzyme Enhanced Reactive-immunoassay (CEER™). The toring following treatment for pancreatic cancer. They are
CE™ platform can simultaneously detect the abundance and not currently sufficiently specific for the screening of asymp-
activation of multiple key signaling molecules in biologi- tomatic patients. However, in the future, novel technologies
cal tissues that are uniquely unregulated, demonstrating the including protein profiling may provide markers sufficient
clinical feasibility, high sensitivity, specificity, and reliabil- for early detection of pancreatic cancer.
ity of the technology. In the current era of precise oncology,
this technique can provide important proteomic information
within the tumor and is critical in assessing therapeutic effi- 34.1.5 Treatment
cacy and providing secondary signaling changes to identify
resistance mechanisms [24]. Surgical resection is the only potentially effective treatment
for pancreatic cancer. However, less than 20% of patients
34.1.3.4 Genetic Testing met the surgical criteria at the time of diagnosis. The pri-
Pancreatic tumor targeting gene may be a future biomarker mary goal of surgery is to achieve a negative (R0) resection
for PDAC gene diagnosis. The PDAC gene is very com- margin. After radiologic evaluation, only patients with a high
plex. The most commonly targeted PDAC genes are KRAS, R0 resection rate were considered to be a good early surgical
CDKN2A, TP53, and SMAD4. In addition, some less com- option.
498 L. Yang et al.
Considering the poor efficacy of surgery alone in pan- Results with Interpretation Guideline The diagnosis of
creatic cancer, numerous efforts have been made to improve pancreatic cancer is difficult and requires multiple investi-
5-year survival in pancreatic cancer patients with chemother-gative modalities. Imaging techniques will always be
apy, radiation, or both. required and have the advantage of assessing tumor size
and spread and coincident intraabdominal disease.
However, problems with these techniques include the
34.1.6 Typical Medical Case insensitivity of ultrasound due to poor pancreatic visualiza-
tion, the expense and invasive nature of CT and endoscopic
Clinical Background A 66-year-old male smoker pre- retrograde cholangiopancreatography, and the fact that dif-
sented to his primary care physician complaining of upset of ficulty can arise in distinguishing benign from malignant
upper abdomen, reclusion, epigastralgia distention, and pancreatic masses. Tumor markers have five potential
anorexia for at least 1 month. USES in patient care: they can be used to screen, diagnose,
determine prognosis, monitor treatment, and detect recur-
Abdominal CT Scan Abdominal CT revealed a low- rence. In this case, serum SP70 and CA19-9 levels were
density mass in the body and tail of the pancreas. CT scan both elevated. Our previous research shows that SP70 has
results were showed in Fig. 34.4. higher sensitivity in distinguishing pancreatic cancer from
benign pancreatic disease compared with other commonly
Initial Laboratory Values WBC 5.34 × 109/L, Lymphocyte used tumor biomarkers. Serum SP70 could be used as a
1.30 × 109/L, RBC 3.90 × 1012/L, RDW 15.6%, CRP marker for better assessment of space-occupying lesion in
36.80 mg/L, Lp-PLA 2462 ng/mL. pancreas.
Tumor Biomarker CA19-9 158.5 U/mL (<39.00 U/mL), Final Diagnosis According to the results of elevated tumor
SP70 30.9 ng/mL (≤7.5 ng/mL), CEA, CA72-4, and AFP markers and abnormal CT findings, the patient underwent
were normal. pancreatectomy. Histological examination showed that pan-
creatic adenocarcinoma.
Histopathological Examination Pancreatic adenocarci-
noma, see in Fig. 34.5. SP70 expression in pancreatic cancer This case was from the First Affiliated Hospital of Nanjing
and benign pancreatic disease were described in Fig. 34.6. Medical University (also named Jiangsu Province Hospital).
a b
Fig. 34.4 CT scan results. Abdominal CT revealed a low-density mass in the body and tail of the pancreas. (a) Arterial phase; (b) Portal phase
34.2 Chronic Pancreatitis exocrine compartment. Functional consequences include

recurrent or persistent abdominal pain, diabetes mellitus
Huanyu Ju (endocrine insufficiency), and indigestion (exocrine insuf-
ficiency). The incidence rate in European countries is 5–10
per 100,000 inhabitants, and the annual incidence reported
34.2.1 Overview worldwide is roughly similar in all countries, 5–14 per
100,000 inhabitants, with an incidence rate of about 30–50
Chronic pancreatitis (CP) is a syndrome of endocrine and per 100,000 [27]. Common causes of chronic pancreatitis
exocrine gland dysfunction secondary to progressive inflam- include toxic factors (such as alcohol or smoking), metabolic
mation and chronic fibrosis of the pancreatic acinar, with abnormalities, idiopathic mechanisms, genetics, autoimmune
permanent structural damage [26] due to atrophy and/or responses, and obstruction mechanisms. The TIGAR-O risk
replacement of fibrous tissue caused by the endocrine and factor classification system is described in Fig. 34.7.
34.2.2 Clinical Appearance
Pain is the most common symptom of chronic pancreatitis

(80–95% of patients) [28]. The pain usually occurs in the
upper abdomen and may radiate to the back. The pain may
be related to nausea and vomiting. Pain usually occurs after
a meal, although it can be taken independently of oral admin-
istration. Most patients reported numbness and pain lasting
more than 24 h, and 68% reported pain in the upper abdo-
men. Thirty-nine percent of patients reported back pain, 50%
reported pain in the upper left quadrant, and 32% reported
pain in the upper right quadrant. About 6% of patients feel
pain in the shoulder. Although abdominal pain is the most
common symptom in CP patients, some patients still have
no symptoms.
A few patients developed other symptoms related to
Fig. 34.5 The histopathology of pancreatic adenocarcinoma (Hema- pancreatic exocrine dysfunction. Exocrine pancreatic insuf-
toxylin-eosin staining, ×100) ficiency means that the pancreas does not secrete enough
a b
Fig. 34.6 SP70 expression in pancreatic cancer and benign pancreatic disease (a) Benign pancreatic disease; (b) Pancreatic cancer
(Immunohistochemistry, ×200)
500 L. Yang et al.
Fig. 34.7 TIGAR-O risk Toxic-metabolic

factor classification system
• Alcoholic • Tobacco Smoking
• Hypercalcemia • Hyperlipidemia oxidative
• Chronic Renal Failure stress
• Medications • Toxins
Idiopathic
• Early Onset • Late Onset

• Tropical
- Tropical Calcific Pancreatitis
toxic
- Fibrocalculous Pancreatic Diabetes
metabolic
Genetic
• Autosomal Dominant
- Hereditary Pancreatitis
• Autosomal Recessive
- CFTR Mutations
- SPINK1 Mutations necrosis
- Cationic Trypsinogen fibrosis
- α-1-antitrypsin Deficiency
chronic
Autoimmune
pancreatitis
• Isolated Autoimmune Chronic
Pancreatitis
• Syndromic Autoimmune Chronic
sape
Pancreatitis
hypothesis
• Sjogren Syndrome-associated Chronic
Pancreatitis
• IBD-associated Chronic Pancreatitis
• PBC-associated CP
Recurrent And Severe Acute Pancreatitis

stone
• Post-necrotic(Severe Acute Pancreatitis)
and duct
• Recurrent AP obstruction
• Vascular Disease/ischemic
• Post-radiation
Obstructive
• Pancreatic Divisum
• Sphincter of Oddi disorders(Controversial)
• Duct Obstruction(Tumor) large duct
• Pre-ampullary Duodenal Wall Cysts
• Post-traumatic Pancreatic Duct Scars
enzymes to maintain proper digestion of nutrients. Chronic with chronic pancreatitis, and its incidence increases with
pancreatitis, pancreatic cancer, and prepancreatic resection the development of the disease. Overall, 45% of patients with
are the main causes of adult exudate pancreatic dysfunc- chronic pancreatitis had overt diabetes. People with pancre-
tion. Typical symptoms of exome pancreatic insufficiency atic diabetes have an increased risk of hypoglycemia, lead-
include abdominal cramps, bloating, flatulence, fat spillage, ing to increased mortality. Episodic hypoglycemia occurs in
and malnutrition. Generally, weight loss is a major symp- up to 79% of patients with pancreatic diabetes, and severe
tom of pancreatic exocrine dysfunction with fat fever, while hypoglycemia occurs in up to 41% of patients. Glucose
hypoproteinemia or fat-soluble vitamin malabsorption is intolerance and diabetes may occur later in the course of the
uncommon. disease.
More rarely, patients seek treatment because they develop In general, the first symptoms that prompted patients to
diabetes, loss of endocrine function, or cachexia as the first seek medical attention were abdominal pain, which origi-
signs of chronic pancreatitis. Diabetes is common in patients nated in the upper abdomen and radiated in a band to the
back, weight loss (80% of patients), and fatty acid poison- below the normal value will the clinical symptoms of exo-
ing (50% of patients), followed by diabetes in about 26–80% crine insufficiency appear [32]. About one-third of patients
of patients. Some patients have severe radiologic pathologic with chronic pancreatitis have clinically related indigestion.
changes but do not experience pain for unknown reasons. The decrease of exocrine function usually precedes obvious
morphological changes, so the sensitivity of the detection of
exocrine pancreatic function to early changes is higher than
34.2.3 Laboratory Diagnosis that of imaging studies.
34.2.3.1 E xocrine Pancreatic Enzymes or Indirect Pancreatic Function Tests

Hormones Fecal Elastase-1 Currently, the determination of fecal elas-
Some people speculate that low serum amylase can be tase is considered to be the most appropriate detection
used to diagnose chronic pancreatitis due to the decrease method [33]. The FE-1 test measured fecal levels of elastin-
of intestinal amylase secretion during chronic pancreatitis. ase-1, a proteolytic enzyme produced by pancreatic acinar
Unfortunately, pancreatic isoamylase is completely normal cells that binds to bile salts and passes through the intestine
in many patients with mild to moderate chronic pancreati- with minimal degradation. Trypsin accounts for 6% of the
tis, with a reported sensitivity of only 60%, with variations protein in pancreatic juice. Compared with other serine pro-
ranging from 12 to 100% depending on the severity of the teases, this enzyme is highly stable as it passes through the
disease. The same question applies to the determination of intestinal tract and can be detected 5–6 times in excess stool
lipase and trypsin. Although serum amylase and lipase may (median concentration 1200 g/g). Fecal elastase is deter-
be elevated in CP, their levels are usually normal. In addi- mined by enzyme-linked immunoassay (ELISA), and there
tion, changes in the pancreatic enzyme profile can also be are polyclonal and monoclonal detection kits on the market.
caused by pancreatic cancer, diabetes, or other malabsorp- To measure fecal elastase, only small amounts of stool
tion. Therefore, the detection of pancreatic serum enzymes are required (100 mg) and it is not necessary to test multiple
lacks diagnostic accuracy and specificity. samples because inter-assay variability is low (8–15%). The
total sensitivity of fecal elastase test to mild exocrine dys-
34.2.3.2 Human Pancreatic Polypeptide (PP) function was 63%, and that of moderate to severe exocrine
The only hormone in the serum that may serve as a diag- dysfunction was 100%, compared with gold standard secre-
nostic tool for pancreatitis is human pancreatic polypeptide tion CCK test. The FE-1 test is easy to perform and samples
(PP). PP contains 36 amino acids and is found in the islets can be obtained by the patient or their chief physician com-
and exocrine parts of the pancreas. Patients with chronic pared to many other pancreatic function tests, which can be
pancreatitis had lower PP levels after meal or after exoge- expensive and require specialized testing equipment.
nous stimulation. Sensitivity to PP can be as high as 90% in
patients with severe chronic pancreatitis and fat fever, and Fecal Chymotrypsin In the chymotrypsin secreted into the
as low as 50% in patients with mild to moderate disease. duodenum, 5% of chymotrypsin maintained the enzyme
The sensitivity of fasting plasma levels below 125 pg/mL activity in feces and was determined by the colorimetric
to chronic pancreatitis was 70%. If normal, this test would enzyme reaction of the substrate n-glutaryl-l-phenylalanine-
exclude chronic pancreatitis with an accuracy of 90%, but p-nitroanilide (GNPNA). Chymotrypsin prevents degrada-
35% of healthy volunteers would still show a level below tion in feces by binding to insoluble detritus in faces and is
125 MCG/mL [29, 30]. As a diagnostic test, PP test has been stable at room temperature for several days, enabling the
abandoned by clinical routine and used for direct detection sample to be transported to a reference laboratory.
of exocrine pancreatic function or more sensitive indirect Chymotrypsin below 3 U/g in feces indicates late CP. Fecal
detection of pancreatic function. chymotrypsin assay has no clinical value for early detection
of CP, but its sensitivity to advanced disease is between 50
34.2.3.3 Pancreatic Function Tests and 80%, sensitivity to cystic fibrosis is between 80 and 90%
Pancreatic function test (PFTs) is usually divided into indi- [34], and specificity is between 50 and 100% [35, 36]. As
rect (non-invasive) or direct (invasive) [31]. Pancreatic exo- with all fecal protease tests, watery diarrhea, such as short
crine and endocrine function test is the second diagnostic bowel syndrome, can be diluted to generate false-positive
tool for chronic pancreatitis. Exocrine deficiency refers to the results (low fecal chymotrypsin).
total or partial reduction of the secretion of amylase, lipase,
protease, or bicarbonate in the pancreas. The most common Pancreolauryl Test Among the indirect oral pancreatic func-
cause of loss of exocrine function in adults is chronic pancre- tion tests, the serum trypsin test is the most widely accepted
atitis. The human pancreas has a large exocrine reserve. Only method for detecting and grading damage to glandular func-
when the secretion of pancreatic lipase is reduced to 10% tion. The analysis was based on the intake of fluorescein
502 L. Yang et al.
dilaurate (0.25 mmol) at a standard breakfast (20 g bread, placing a nasoduodenal tube with two Chambers: one that
20 g butter, and 200 mL). The pancreatic dodecyl test can also collects gastric juice from the stomach to prevent it from
quantify severe exocrine dysfunction by reducing the increase stimulating pancreatic secretion; the second was placed
of serum luciferin in the hemofiltration and renal replacement behind the Treitz ligament; and duodenal fluid was collected
therapy in patients with renal insufficiency. The sensitivity every 15 min. The CCK test of pancreatic juice is the gold
and specificity of severe exocrine insufficiency were 82% and standard for the detection of pancreatic function, with a total
91%, respectively. The detection sensitivity of mild exocrine sensitivity and specificity of 90% [41]. Even though the pan-
insufficiency was 51%. The trypsin test is considered to be an creatic juice CCK test is the most accurate test for pancreatic
indirect non-invasive pancreatic function test with a high clin- function, only a few professional centers use the technique in
ical relevance [37, 38]. In summary, PLT is a simple, inexpen- clinical practice or in clinical trials. Some authors used the
sive, non-invasive screening method with a specificity of Lund test instead of hormones to stimulate the extracellular
83–91% for the diagnosis of pancreatic insufficiency. pancreas, but this more “physiological” approach ended up
being less sensitive to detecting early functional changes, and
13
C-Mixed Triglyceride Breath Test Another noninvasive bicarbonate could not be measured in the collected chylous.
approach for evaluating pancreatic exocrine insufficiency is
assessment of CO2 exhalation after digestion of 13C-labeled Endoscopic Secretin Test Stevens and colleagues [42]
synthetic substrates such as mixed triglyceride, triolein, showed that duodenal fluid collected endoscopically after
and hiolein, which are enzymatically cleaved by pancreatic secretin stimulation showed the same yin-yang secretion
enzymes in the duodenum but which are not resorbed and curve as found in the standard “test tube.” However, this col-
can therefore be detected in exhaled breath over time [39]. lection requires an endoscope to last at least 30 min, placing
After an overnight fast, a standard solid test diet containing a huge burden on the endoscope physician and the patient.
13C triglyceride (250 mg) and 16 g fat (40 g bread, 20 g One possibility for overcoming the limitations of invasive
butter, and 200 mL water) was administered orally [40]. function testing might be secretin-stimulated magnetic reso-
Spread the base on the butter for the best mixture. nance cholangiopancreatography (MRCP).
Metoclopramide was administered orally at 10 mg 30 min Intravenous secretin causes a rapid outflow of bicarbon-
before breath test to avoid potential problems related to ate-rich fluid from the pancreatic exudate, which can be
gastric emptying. A 6-h respiratory sample was collected quantified semi-quantitatively, and this is markedly reduced
every 15 min prior to ingestion of the test meal. The quo- in patients with impaired exocrine function. The sensitivity
tient of 13CO2/12CO2 in each respiratory sample was and specificity of secretin-stimulated MRCP were 69% and
determined by mass spectrometry, and the global 6-h cumu- 90%, respectively. As magnetic resonance imaging gradually
lative recovery rate (CRR) of 13CO2 was considered as the replaces computed tomography in the diagnosis of chronic
test result (cut-off point: 29%). pancreatitis, secretin-stimulated MRCP may become a valu-
able diagnostic tool.
Fecal Fat Quantitation Quantitative determination of fecal
fat using the classic van DE Kamer (alcohol extraction) tech- Evaluation of Endocrine Function The incidence of exo-
nique is the standard test for determining fat efflux as a char- crine and endocrine functions increases over time. About
acteristic symptom of reduced exocrine function. The trial 20% of patients with alcoholic chronic pancreatitis develop
lasted for 3 days and included 80–100 g of fat per day. Feces overt diabetes 6 years after onset. After 10 years of onset,
were collected 24 h a day during the same period. After los- about 50% of patients with alcoholic pancreatitis showed
ing 90% of exocrine function, the fat excretion in excrement signs of impaired glucose metabolism and decreased insulin
increases significantly, this is the mark of fat dyspepsia. Mild secretion [43]. According to the who guidelines for the diag-
to moderate exocrine impairment is usually compensated for nosis of diabetes, pancreatic endocrine function should be
clinically. The Van DE Kamer trial has become unpopular assessed by fasting and 1 h postprandial-glucose levels, oral
with patients, nurses, and technicians because of the amount glucose tolerance tests, and glycosylated hemoglobin
of smelly poop it requires. levels.
Direct Pancreatic Function Tests 34.2.3.4 Genetic Testing

Secretin-Cholecystokinin Test Pancreatic enzyme activity In addition to evaluation of exocrine and endocrine function,
as well as bicarbonate concentration are measured in the duo- considerable attention is currently paid to the etiology of the
denal juice after stimulation with the entero hormones secre- disease. Recent results from molecular and genetic studies
tin (1unit/kg i.v.) and CCK (25−100ng/kg). This involves suggest that a significant number of patients with chronic
pancreatitis suffer from a genetically determined or inherited 34.2.5 Treatment

disease. This is mainly true for patients who were formerly
classified as suffering from idiopathic pancreatitis, for those Although the guidelines summarize current approaches
with onset of the disease before age 25 years, or those with a to diagnosing and managing chronic pancreatitis, the dis-
positive family history for chronic pancreatitis or pancreatic ease needs to be redefined to focus on basic pathways and
cancer. Patients who suffer from chronic pancreatitis due to informational biomarkers rather than end-stage pathol-
mutations in the cationic trypsinogen gene are burdened with ogy. The aim is to establish a reasonable framework and
a 70–140-fold increased risk of developing pancreatic can- method for early diagnosis, classification, and progno-
cer, particularly if they smoke. Whether this is also true for sis. A new definition of the mechanism should include
patients who carry SPINK1 or CFTR mutations needs to be two components, because the combination of these char-
determined. Genetic testing for the most common and clini- acteristics defines the syndrome: the nature of chronic
cally relevant trypsinogen gene mutations (N29I and R122H pancreatitis and the established characteristics of chronic
or R122C) can be recommended for patients with chronic pancreatitis.
pancreatitis who have first-degree relatives suffering from
pancreatitis or pancreatic cancer, and for those with chronic
pancreatitis or recurrent bouts of acute pancreatitis before the 34.2.6 Typical Medical Case
age of 25 years and no identifiable risk factor [44]. Genetic
testing for clinically unaffected relatives is not indicated and Clinical Background A 41-year-old female nonsmoker
should only be performed within ethics committee-approved presented to her primary care physician complaining of
research protocols. The simplest restriction enzyme screening recurrent abdominal pain or discomfort for at least 1 month
test is now the BstU digest, which will discover more than and aggravate 1 week.
50% of hereditary pancreatitis patients in most of the world.
Abdominal CT scan Atrophy of the pancreas and mild
dilation in pancreatic duct. CT scan results were showed in
34.2.4 Management Fig. 34.8.
The aim of treatment is to reduce symptoms (pain is the most Endoscopic Ultrasonography All sizes of duct were
common, followed by exocrine and endocrine insufficiency) involved and showed irregular dilation, loss of lining epithe-
and to prevent further disease progression and disease- lium, encroachment of periductal connective tissue to form
related complications such as bile duct obstruction, gastric stenosis, pancreatic cysts or complete disappearance of acini,
outlet obstruction, and portal vein thrombosis. and ducts in the territory drained by a blocked duct.
a b
Fig. 34.8 CT scan results. Abdominal CT revealed atrophy of the pancreas and mild dilation in pancreatic duct. (a) Arterial phase; (b) Portal
phase
504 L. Yang et al.
patients suspected as having CP, those requiring differential

diagnosis from other pancreatic diseases
follow-up observation for 2-3 months
Characteristic imaging findings

history taking/physical examination (or characteristic histological findings)
Definite Probable Early finding No

finding finding (EUS, ERCP) finding
biochemical tests/functional tests(BT-PABA)
More than More than More than

2 of 1 - 3 2 of 1 - 4 2 of 1 - 4
Evaluation items yes no yes no yes no

1. Repeated upper abdominal pain
2. Abnormal serum/urine enzyme levels
yes
3. Abnormal exocrine function Rule out
4. Continuous heavy drinking(more than 80g/day) other disease
no
Definite CP Probale CP Early CP
Consider
other disease
Fig. 34.9 Diagnostic path for chronic pancreatitis. The figure shows a schematic flow diagram for the diagnosis of chronic pancreatitis (CP)
Initial Laboratory Values WBC 3.79 × 109/L, Lymphocyte ing (MRI), endoscopic ultrasound, and pancreatography in
1.46 × 109/L, RBC 4.57 × 1012/L, RDW 12.5%, PT 0.020 ng/ the diagnosis of chronic pancreatitis exocrine pancreatic
mL, Lp-PLA2 331 ng/mL. function testing, pathological diagnosis and genetic testing.
Tumor Markers SP70 6.2 ng/mL, CA19-9, CEA, CA72-4, Final Diagnosis Several tests were performed for screening
AFP were normal. this disease, such as WBC, RDW, PT, Lp-PLA2, and SP70.
All these tests were for screening. If inflammatory biomark-
Results with Interpretation Guideline The definitive diag- ers were higher, suggesting that a high risk for CP.SP70 was
nosis of chronic pancreatitis is sometimes difficult, especially used for differential diagnosis of benign and malignant dis-
if the disease is not considered by the physicians treating the
eases. The other tests were all for auxiliary diagnosis.
patient. How chronic pancreatitis is suspected, based on
Endoscopic ultrasonography and the pathological examina-
signs, symptoms, and laboratory results, and how the diagno-
tion were the diagnostical golden standard. So according to
sis is developed. The diagnosis is very detailed and consists of
the pathological examination results, this disease was diag-
all these items, including history taking, physical examina-
tion, determination methods for pancreatic enzymes in the nosed as chronic pancreatitis. Diagnostic path for chronic
blood and urine, significance of various imaging methods pancreatitis is showed in Fig. 34.9.
chest and abdominal radiography, abdominal ultrasonogra- This case was from the First Affiliated Hospital of Nanjing
phy, computed tomography (CT), magnetic resonance imag- Medical University (also named Jiangsu Province Hospital).
34.3 Insulinoma Insulinoma is difficult to diagnose due to a variety of tempo-

rary symptoms and can lead to patients being misdiagnosed
Yuan Mu as a mental or neurological disease. Up to 20% of patients
with a final diagnosis of insulinoma have a prior diagnosis
that has previously been assigned to explain their symp-
34.3.1 Overview toms, usually an undefined seizure, hypotension, or other
diagnosis [49].
Pancreatic neuroendocrine neoplasm (PNETs) is a tumor
caused by cells (also known as islet cells) in the pancreas
that produce hormones [45]. These tumors are classified as 34.3.3 Laboratory Diagnosis
“functional” or “nonfunctional,” depending on whether they
release symptomatic peptide hormones, usually based on 34.3.3.1 C -Peptide Inhibition Test with Hog
established patterns of tumor subtypes. Insulin
Insulinoma is a functional neuroendocrine tumor that In patients without insulinoma, 1-h infusion of pig insulin
originates from the pancreatic neuroendocrine islet cells or resulted in a decrease in plasma C-peptide levels, but not in
pluripotent stem cells. It is usually benign and can stimulate patients with insulinoma. This is because the insulin release
the production of insulin independently of glucose levels, of insulinoma cells is not inhibited by exogenous insulin,
causing clinical syndromes. Insulinoma is the most common while the insulin secretion of normal cells is inhibited by the
functional endocrine tumor of the pancreas, with an esti- increase of plasma insulin [50].
mated incidence of one to three per million per year. There is
an age-specific peak in the fifth decade of life, with a slightly 34.3.3.2 Intravenous Secretin Test
higher incidence in women than in men. About 10% are mul- for Insulinoma
tiple, less than 10% are malignant, and 5–10% are associated Four minutes after intravenous injection of trypsin (2 units/
with male 1 syndrome. The latter tumor is usually multiple kg body weight), plasma insulin in patients without insuli-
and may be malignant in 25% of cases. A preliminary under- noma increased by more than 200%, while plasma insulin
standing of the main symptoms is followed in most cases in patients with insulinoma did not. The principle of this
by a careful laboratory examination, complex imaging, and experiment is based on two characteristics: (a) normal cells,
ultimately delicate surgery. Clearly, a multidisciplinary team not insulinoma cells, release insulin under the stimulation of
approach is needed [46, 47]. secretin; (b) in patients with insulinoma, normal pancreatic
cells produce significantly less insulin. Therefore, in patients
with insulinoma, the secretin stimulation test does not lead
34.3.2 Clinical Appearance to increased plasma insulin, because insulinoma cells do not
respond to secretin and normal cell insulin production is
The insulin-producing cells are the most common islet cells. reduced [51].
Insulinoma is the most common functional PNET (35–40%
of lesions) and is characterized by intermittent hyperinsu- 34.3.3.3 The 72-h Fast Test
linemia leading to symptomatic hypoglycemia. The clinical Further controlled trials included the 72-h rapid, the gold
manifestations of these tumors are the classic Whipple triad: standard for determining the diagnosis of insulinoma.
intermittent hypoglycemia, central nervous system (CNS) Patients and controls fasted for 72 h under strict medical
dysfunction associated with hypoglycemia, and reversal of supervision. In the 72 h fast test, blood glucose, insulin,
CNS dysfunction by glucose therapy [48]. C-peptide, and proinsulin levels were measured every 4 h
Symptoms of central nervous system dysfunction asso- after overnight fasting. Additional samples were taken when
ciated with hypoglycemia, also known as neuroglycogen blood glucose <2.5 mmol/L or clinical symptoms of hypo-
reduction, can include delirium, anxiety, syncope, convul- glycemia were present. When patients developed symptom-
sions, vision changes, amnesia, and coma. Other symptoms atic hypoglycemia with blood glucose below 2.5 mmol/L,
are caused by sympathetic nervous system stimulation, the rapid trial was discontinued 72 h before. C-peptides, pro-
including tremors, palpitations and sweating. Most com- insulin, and insulin are also extracted from the blood when
monly, hypoglycemia occurs on an empty stomach, but the patient develops symptoms and blood glucose levels are
up to 10% of patients report only postprandial symptoms. below 2.2 mmol/L (or 40 mg/dL). The failure of proper insu-
506 L. Yang et al.
lin suppression in the presence of hypoglycemia confirms the [52]. Intraductal papillary myxoma of the pancreas (IPMN)
spontaneous secretion of insulinoma. is a group of heterogeneous lesions growing in the pancre-
atic duct system, which may involve the main pancreatic
34.3.3.4 Genetic Testing duct (MD-IPMN), branching pancreatic duct (BD-IPMN), or
Germline DNA testing for hereditary tumor syndromes is both (mixed IPMN) [53], and is the most common imageally
recommended only in certain cases: family history or clinical identifiable precursor of pancreatic cancer.
presentation suggests men-1 or von Hippel-Lindau disease Intraductal papillary myxoma (IPMN) is one of the most
(VHL); the presence of multiple tumors; or a precancerous common cystic tumors of the pancreas. Intraductal papillary
lesion of the pancreas. Mutation analysis should be per- mucinous tumor is pathologically defined as a lump-forming
formed to detect male-1 or VHL mutations (after informed pre-invasive tumor (neoplastic intraepithelial tumor) grow-
consent). ing in the pancreatic duct [54]. IPMNs include a range of
lesions, from those that look very harmless to those that are
completely intramucosal cancerous. This spectrum is now
34.3.4 Management recategorized as “low-grade dysplasia – high-grade dyspla-
sia – invasive carcinoma.” IPMN is a precancerous lesion
Treatment decisions for PNETs are based on whether the that invades adenocarcinoma and accounts for 5% of pancre-
tumor causes hormone overdose, the possibility of surgical atic tumors. A 2013 case-control study concluded that a prior
treatment, and the treatment of metastatic disease. According history of diabetes (especially insulin use), chronic pancre-
to the clinical situation, different PNET requires different atitis, and a family history of PDAC were all associated risk
treatment methods. factors. Smoking does not appear to be a risk factor but may
Insulinoma is the most common functional PNET and accelerate the malignant progression of IPMN. Some genetic
must be recognized because its symptoms are insidious. diseases, such as McCune-Albright syndrome, involve muta-
Drug therapy for metastatic or inoperable diseases is to con- tions in the GNAS gene that predispose to IPMN. Mutations
trol symptoms or tumor growth, and the only treatment is still in GNA and KRA are frequently present in IPMN and are
surgery. Recognizing the symptoms and thinking about this found even in early carcinogenesis prior to the development
rare network is the basis for proper diagnosis and treatment. of invasive diseases [55, 56]. IPMN has a progressive pro-
gression from benign to malignant (adenoma-carcinoma
sequence). The incidence of IPMN is increasing, mainly
34.3.5 Treatment due to advances in imaging and an increase in the elderly
population. IPMNs account for 21~33% of pancreatic cystic
PNET is a clinical entity with high morbidity and mortal- lesions. There was a slight male-to-female predominance,
ity due to the occurrence of advanced disease at the time of with 60% of the lesions occurring in males. They usually
diagnosis. Clinical suspicion must exist when evaluating a occurred in the sixth to seventh centuries [57].
patient with a cluster of related symptoms, and ideally a pro-
vider capable of following the patient’s clinical progress for
weeks to months can initiate appropriate work. For patients 34.4.2 Clinical Appearance
with suspected PNET, it is recommended to carefully docu-
ment the history (including, where appropriate, to assess the The clinical manifestations of IPMN are varied and related
family history of an inherited disease). to tumor size and survival. Even if there are no typical clini-
cal symptoms of IPMN, the patient’s medical history plays
an important role in the diagnosis of IPMN. About 80% of
34.4 I ntraductal Papillary Mucinous patients with IPMN have only nonspecific clinical symptoms,
Neoplasms such as discomfort, nausea and vomiting, abdominal or back
pain, and weight loss. Some patients have pancreatic symp-
Chunrong Gu toms. They suffer from attacks of abdominal or back pain
and slow development of exocrine and endocrine pancreatic
insufficiency. This phenomenon is associated with obstruc-
34.4.1 Overview tion of the pancreatic duct. In contrast to chronic pancreatitis
(CP), patients with IPMN are mostly female, older, and less
Intraductal papillary myxoma (IPMN) is a cystic tumor likely to have a history of alcohol consumption. Patients with
originating from the epithelial cells of the pancreatic ductal symptoms appear to be at higher risk for malignancy than
system. It is characterized by the proliferation of cells that other patients. A history of pancreatitis can cause IPMN to
form papillae and secrete mucin, leading to cystic dilatation be misdiagnosed as CP [58].
34.4.3 Laboratory Diagnosis S100 Protein

Recent studies have investigated the activity of S100 fam-
34.4.3.1 M
ucin Tumor Markers and Oncofetal ily members in IPMNs [61]. The S100 family is a group of
Antigens proteins involved in important signaling pathways, such as
Ca2+ signaling networks. In IPMNs, several members of
CEA this family, such as S100P, have higher levels of expression
CEA is the most commonly used biomarker for diagnosis in large pancreatic tissues than in non-tumor pancreatic
and follow-up of IPMN. CEA, a cell surface glycoprotein, is tissues. In addition, in microisolated cells, the expression
elevated in more than 60% of patients with pancreatic ductal of S100P in pancreatic ductal adenocarcinoma was asso-
adenocarcinoma. It is released from the outer membrane of ciated with IPMN cells. Interestingly, S100P was highly
the cancer cell and then freely circulates throughout the body. expressed in IPMNs associated with chronic pancreatitis
As for the diagnosis of IPMN, the study shows that its cut-off in pancreatic juice. Therefore, the determination of S100P
value is 192 ng/mL. Unfortunately, CEA cannot distinguish level in pancreatic juice can be used to identify neoplasia
between benign and malignant cysts. In addition, it was also and chronic pancreatitis. In addition, S100 calcium-binding
reported to be elevated in mucinous cystic tumors and to be protein A4 (S100A4) is another member of this family, and
low in 30% of the samples. Therefore, the evaluation of serum its expression is related to tumor metastasis. S100A4 was
CEA level is the preferred method. Serum CEA has a sensi- only expressed in 7.4% of adenomas and borderline atypi-
tivity of 18% to malignant and invasive IPMNs, too low to be cal hyperplasia, and in 42.9% of IPMN-derived carcinomas.
used as a screening method, especially in high-risk patients. We know that S100A4 is a promising malignancy marker
On the other hand, the specificity of serum CEA is about because we can use it to diagnose and study IPMN. The
95%, which can be used for the diagnosis of IPMN malig- activity of S100 family members needs to be further stud-
nancy. Serum CEA level of >5 ng/mL is considered an effec- ied in order to use some of these compounds as biomarkers
tive predictor of malignancy and aggressiveness. However, it for pancreatic cancer.
is clear that elevated serum CEA levels are not a clear marker
for determining a treatment regimen that includes treatment mAb Das-1
with increased severity, such as surgery [59]. Recently, Das and colleagues developed a monoclonal anti-
body, called mAb Das-1, against a colonic epithelial pheno-
CA19-9 type [62]. Both the EUS-FNA cyst fluid analysis (specificity
Carbohydrate antigen 19-9 (CA19-9) is another biomarker and sensitivity of 100% and 89%, respectively) and histolog-
commonly used to diagnose IPMNs. It is a tumor-associ- ical specimens from resected high-grade IPMNs (specificity
ated glycoprotein. CA19-9 and CEA have a good predic- and sensitivity of 95% and 85%, respectively) showed that
tive effect on the diagnosis and prognosis of breast ductal Das-1 has potent reactivity.
adenocarcinoma, but CA19-9 has a good predictive effect on
the malignancy of breast ductal adenocarcinoma. CA19-9 MicroRNAs
level is increased in 85% of patients with pancreatic ductal Finally, we should mention that microRNAs are a very
adenocarcinoma. The sensitivity and specificity of CA19-9 promising field in the field of biomarkers. MicroRNAs (miR-
were 44% and 85%, respectively. In addition, increased NAs) are small single-stranded RNA molecules involved in
CA19-9 levels were associated with the presence of invasive the regulation of gene expression. MiRNAs are abnormally
IPMN. Therefore, for patients with positive tumor markers, expressed in pancreatic cancer and its precursor lesions such
we should not attempt limited excision when deciding or as IPMN. In particular, miRNA-21 (miR21) and miRNA-155
instructing surgery. (miR155) were significantly higher in invasive IPMN than in
non-invasive IPMN and normal pancreatic tissue. In addi-
34.4.3.2 Other Tumor Markers tion, the up-regulated expression of miR21 and mir155 in
IPMN of carcinoma in situ was greater than that of IPMN
CF protein adenomas [63].
The ability of laboratory, radiology, and endoscopy to dis-
tinguish between low risk (low and moderate dysplasia) and 34.4.3.3 Genetic Testing
high-risk (high-grade dysplasia and invasive) IPMN is cur- IPMNs usually contain activation mutations of KRAS and
rently limited. Because of the limitations of imaging and GNAS, inactivation mutations of RNF43, CDKN2A/p16,
cytology in defining high-risk IPMN, the diagnostic value of and TP53, and few mutations of BRAF, PIK3CA, stk11, and
CF analysis has been widely evaluated [60]. SMAD4.
508 L. Yang et al.
34.4.4 Management This case was from the First Affiliated Hospital of Nanjing
Medical University (also named Jiangsu Province Hospital).
A recent meta-analysis identified the relationship between
IPMN specific genetic changes in the pancreas and malig-
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endoscopic pancreatic function testing is optimized using duode-
Digestive Tract Disease
35
Genyan Liu, Yuqiao Xu, Shiyang Pan, Weijuan Song,
Jia Wang, Fei Jin, Zhenzhen Cai, Yi Zhang, and Xiang Qian
35.1 Gastritis
Genyan Liu and Yuqiao Xu
Stomach, one of the body’s digestive system, is the

expanding part of esophagus. The shape of the stomach can
be affected by the state of fullness. When the stomach is
completely empty, it is slightly tubular with a capacity of
about 50 mL. It consists of five regions cardia, fundus, cor-
pus, antrum, and pylorus (Fig. 35.1).
The fundus and corpus harbor acid-secreting glands,
whereas the antrum harbors alkaline-secreting surface epi-
thelium and endocrine, gastrin-secreting G-cells [1]. The
Fig. 35.1 Anatomy of the stomach
most important function of the stomach is fixed as an aid
to digestion. The stomach is also an important endocrine
organ producing an array of peptide hormones important for the gastric mucosa that displays changes related to etiology
both enteric and non-enteric physiology including ghrelin and the host response, involves damage, inflammation, and
and leptin [2]. Meanwhile, gastric juice represents a barrier regeneration. Assessing gastritis involves a clinical examina-
to microbes in saliva and ingested food. If this bactericidal tion, serology (pepsinogens and antibodies against infectious
activity is weakened by an elevation of gastric pH, microbes agents and/or auto-antigens), endoscopy (applying standard-
will be allowed to survive in the stomach. ized biopsy protocols), and histology [3].
Gastric diseases include functional gastropathy and
organic gastropathy. Functional gastropathy includes gas-
tric dysfunction, gastric neurosis, rapid gastric movement, 35.1.1 Classification
slow gastric movement, gastric motility weakness, gastro-
paresis, gastroptosis, gastric mucosal prolapse, and so on. 35.1.1.1 Based on the Course of Disease
Organic gastropathy includes gastritis, gastric ulcer, gastric Acute Gastritis Acute gastritis is a transient type of gastri-
polyp, benign, and malignant tumors of the stomach, and tis that has an acute beginning, and it causes gastrointestinal
so on. Gastritis is considered an inflammatory condition of pain and hemorrhage. It can develop in a hemorrhagic or
nonhemorrhagic, ulcero-erosive, or nonerosive manner, and
it develops as a result of the stress that the mucosa is exposed
G. Liu (*) · Y. Xu · S. Pan (*) · W. Song · J. Wang · F. Jin · Z. Cai to [4].
X. Qian
of Nanjing Medical University, Nanjing, Jiangsu, Chronic Gastritis Chronic gastritis is a multistep, pro-
People’s Republic of China gressive, and lifelong inflammation in human beings. It
e-mail: liugenyan@njmu.edu.cn; sypan@njmu.edu.cn refers to chronic inflammation or atrophic lesions of the gas-
Y. Zhang (*) tric mucosa. The essence of the gastric mucosa is that after
Department of Clinical Laboratory, Qilu Hospital of Shandong repeated damage to the gastric mucosa, the mucosa changes
University, Jinan, Shandong, People’s Republic of China
e-mail: yizhang@sdu.edu.cn
due to the specific regeneration ability of the mucosa,

512 G. Liu et al.
characterized by a loss of normal mucosal glands either in Autoimmune Gastritis

antrum or corpus or in both [5]. Autoimmune gastritis (AIG) is one of the chronic inflamma-
tory diseases of the stomach, finally presenting with atrophy
35.1.1.2 Based on Etiology of the mucosa. Its inflammatory and continuous atrophy is
The main forms of gastritis include Helicobacter pylori gas- restricted to corpus and fundus, because the autoimmune
tritis, chemical gastritis, and autoimmune gastritis. reaction only targets parietal cells. Parietal cells are epithe-
lial cells situated in the glands of the corpus and fundus,
Helicobacter pylori Gastritis producing hydrochloric acid and intrinsic factors. The acid-
Helicobacter pylori (H. pylori) is the most common etio- ification of the stomach is primarily managed by the gas-
logical agent in gastritis, with a prevalence of approximately tric H+/K+ATPase, the proton pump, which is the causative
50%, and the majority of infected individuals are asymp- autoantigen and which is recognized by CD4+ T cells [14].
tomatic [6]. Genetic sequence analysis has indicated that Chronic inflammation leads to an atrophy of the mucosa,
human being has coevolved with H. pylori for more than with a decrease and final total loss of parietal cells during
58,000 years [7]. Since its discovery in 1982, H. pylori has the progression of the disease. As a result, pH increased and
been closely linked to a diverse spectrum of gastrointestinal intrinsic factor lost, which is an intrinsic factor for uptake of
diseases [8]. Although the majority of individuals infected vitamin B12.
with H. pylori remain asymptomatic throughout their life-
time, essentially almost all develop chronic inflammation
[9]. The person-to-person transmission of this infection is 35.1.2 Clinical Manifestation
believed to occur via multiple routes: fecal-oral, oral-oral as
well as environmental transmission through a contaminated Acute gastritis, with acute onset, often show the performance
water supply [10]. H. pylori has urease, an enzyme that of excruciating abdominal distension pain, nausea, vomit-
hydrolyzes urea and releases ammonia, which can neutralize ing, and diarrhea symptoms. The clinical manifestations of
gastric acid, allowing H. pylori to survive and colonize the chemical gastritis are chronic hemorrhage, gastric mucosa
gastric mucosa [11]. Posselt G et al. have reported a novel edema and erosion. Acute erosive hemorrhagic gastritis is
mechanism and identified strong c-Abl threonine 735 phos- caused by a certain acute or chronic disease, including pri-
phorylation (pAblT735) mediated by the type-IV secretion mary ulceration and hemorrhagic focus formation.
system (T4SS) effector d-glycero-β-d-manno-heptose-1, Chronic gastritis is also caused by a variety of causes
7-bisphosphate (βHBP) and protein kinase C (PKC) as a of chronic gastric mucosal inflammatory disease. General
new c-Abl kinase. pAblT735 interacted with 14-3-3 pro- clinical manifestations are abdominal discomfort, recurrent
teins, which caused cytoplasmic retention of c-Abl, where attacks, anorexia, emesis, and gastric mucosa hemorrhage.
it potentiated Hp-mediated cell elongation and migration. Abdominal distension, burning pain, and pressing pain are
Further, the nuclear exclusion of pAblT735 attenuated cas- the characteristics of chronic gastritis. Chronic superficial
pase-8 and caspase-9-dependent apoptosis. Importantly, in gastritis occurs mostly in young men and women over the
human patients suffering from Hp-mediated gastritis c-Abl age of 20, with the manifestation of mild epigastrium symp-
expression and pAblT735 phosphorylation were drastically toms. Chronic atrophic gastritis mostly occurs in patients
enhanced as compared to type C gastritis patients or healthy over 40 years old. This type of gastritis is caused by gas-
individuals [12]. tric mucosa atrophy and gastric acid deficiency. With weak
appetite, emaciation, anemia, fatigue, cleft lip, and tongue
Chemical Gastritis collapse are the main manifestations of this gastritis. Chronic
Bile reflux, nonsteroidal anti-inflammatory drugs, and other hypertrophic gastritis is rare in clinical practice. Gastric
chemical injuries (possibly alcohol, etc.) may be expected to mucosa hyperplasia can be seen by gastroscopy and histol-
result in a broad spectrum of histological mucosal lesions, ogy. The main clinical manifestations are hyperacidity, gas-
associated with low-grade inflammation of the gastric tric bleeding, vomiting, and recurrent pain, which is also
mucosa. These conditions are currently defined as chemical known as one of the early symptoms of stomach cancer.
gastritis or gastropathies. A concomitant histamine-mediated
vascular response and the release of other pro-inflamma-
tory cytokines produce vascular ectasia, edema, muscularis 35.1.3 Laboratory Diagnosis
mucosa hyperplasia, and variable mucosal fibrosis. It usually
features a puzzle of low-grade lesions such as inter-foveolar 35.1.3.1 Endoscopy
edema, foveolar hyperplasia, muscularis mucosa hyperpla- Endoscopy is highly accurate for the diagnosis of upper gas-
sia, and vascular ectasia in histology [13]. trointestinal diseases. Endoscopic atrophic appearance may
35 Digestive Tract Disease 513
predict pathologic atrophy, however, some patients with

pathologic atrophy, even intestinal metaplasia, do not have
typical endoscopic appearance. We found that endoscopic
appearance, such as the absence of rugae, presence of visible
vessels, was not sensitive to diagnose pathologic atrophy, but
it was very specific for predicting pathologic atrophy.
35.1.3.2 Histology
Staining is the critical part of the histological exam, like
HE staining, Giemsa, HP silver stain, and so on. The crite-
ria of histopathological grading are as follows, for grading
of activity, in mild cases, the lamina propria of the mucosa
is infiltrated with few neutrophils; in moderate cases, more
neutrophils are seen in the mucosal layer and can also be
seen in between superficial epithelial cells, pit epithelial
cells, and glandular epithelial cells. In severe cases, more
dense infiltration of neutrophils or abscess on pits can be Fig. 35.2 Detection of Helicobacter pylori
seen in addition to what is seen in moderate activity. While
for grading of chronic inflammation, chronic gastritis can
be graded according to the density of chronic inflammatory fluorescent in situ hybridization (PNA-FISH) is a highly sen-
cells. Normal: number of mononuclear cells less than 5 high sitive (97% sensitivity) and specific (100% specificity) tech-
power field; mild: some chronic inflammatory cells are local- nique for the diagnosis of H. pylori infection. PNA-FISH can
ized in the superficial layer of the mucosa, but not more than identify the coccoid form of H. pylori which is usually unde-
1/3 of the depth of the mucosa; moderate: a more dense accu- tectable by routine histological exam because this method
mulation of chronic inflammatory cells, but not exceeding could avoid individual biasness from morphological identifi-
2/3 of the depth of the mucosa; severe: a dense accumulation cation [17]. Despite the advantages of PNA-FISH in detect-
of chronic inflammatory cells occupying the whole depth of ing H. pylori, its disadvantages such as laborious preparation,
the mucosa [15]. requiring fluorescent microscope, and particular expertise to
read the slides, may limit the broad use of this method.
35.1.3.3 Helicobacter pylori
H. pylori is a Gram-negative microaerophilic spiral-shaped Rapid Urease Tests Based on the activity of the H. pylori
bacterium with flagella, which enables it to colonize the urease enzyme, the presence of H. pylori in gastric biopsy
human gastrointestinal tract. The prevalence of H. pylori is specimen converts the urea test reagent to ammonia, result-
differed in the geographic regions, age, socioeconomic sta- ing in an increase in the pH and a color change on the pH
tus, living environment, and occupation. H. pylori could per- monitor. Because of its highly specific, rapid, inexpensive,
sist for decades in the harsh stomach environment, damaging and widely available, rapid urease test is the most useful
the gastric mucosa and changing the pattern of gastric hor- invasive test for the diagnosis of H. pylori infection.
mone release, which can affect gastric physiology.
The tests detecting H. pylori include invasive diagnostic Culture Culturing of H. pylori requires a particular trans-
tests and non-invasive diagnostic tests (Fig. 35.2). port medium, growth medium, and incubation environment.
The commonly used media include Pylori agar, Skirrow
Endoscopy Conventional endoscopic exam is usually per- agar, Columbia blood agar, Brucella agar, Brain heart infu-
formed to diagnose H. pylori-associated diseases and is also sion, or Trypticase soy agar, supplemented with sheep or
an instrument used to obtain specimens, usually gastric horse blood. The agar plates are usually incubated in a
mucosa from biopsy, for further studies on other invasive microaerobic environment (80–90% N2, 5–10% CO2, 5–10%
tests [16]. Conventional endoscopy can increase diagnostic O2) at 35–37 °C for at least 5–7 days because H. pylori has
accuracy, but it is time-consuming and cannot provide better been considered a microaerophile. Culturing has almost
results than other invasive tests. 100% specificity.
Histology Histology is usually considered to be the gold Polymerase Chain Reaction Polymerase chain reaction
standard in the direct detection of H. pylori infection. HE (PCR) has been applied extensively for the diagnosis of H.
staining is usually sufficient for the diagnosis of H. pylori pylori in gastric biopsy specimens, gastric juice, saliva, stool,
infection in routine clinical practice. Peptide nucleic acid and variable specimens. Compared with other tests, PCR
514 G. Liu et al.
provides excellent sensitivity and specificity. Several target acidification and neutralization. Fractions 1–5 are nominated
genes including 16S rRNA, 23S rRNA, UreA, glmM, UreC, group 1 pepsinogens(PG I) and fractions 6–7 are the group
HSP60, and VacA genes, had been used for the detection of II pepsinogens(PG II). PG I originate from the fundus gland,
H. pylori. Furthermore, the benefits of faster results, fewer and PGII is secreted by the cardia gland, fundus gland,
bacteria required in the sample enable clinicians to make a pyloric gland, duodenum, and pancreas. Most of the synthe-
quicker and more accurate decision on patient’s treatment. sized PG enter the gastric cavity and are activated by pepsin
under the action of acidic gastric juice to exert the function
Urea Breath Test By the urease activity of H. pylori, the of decomposing the protein. Only a small amount of PG
13
C- or 14C-labeled urea ingested by the patient is hydrolyzed (about 1%) penetrates into the blood circulation through the
to label CO2 in the stomach, then labeled CO2 is absorbed in capillaries of the gastric mucosa. The change of serum PG
the blood and exhaled by breathing in which labeled CO2 can content can directly reflect the state of gastric mucosa, and it
be measured. With nearly 95% sensitivity and specificity is a specific biomarker reflecting the severity of lesions and
under standardized procedures, the urea breath test is a lesions in different parts of the gastric mucosa (Table 35.1).
highly accurate and reproducible test. The advantage of serum detection PG: high detection rate,
low cost of detection, simple and easy to operate, simple
Stool Antigen Test Stool antigen test (SAT) is another non- operation, no adverse reactions after the examination.
invasive method, which detects the presence of H. pylori
antigen in stool samples, with good sensitivity and specific- 35.1.4.2 Gastrin 17
ity, 94% and 97% respectively in the global meta-analysis, in Gastric 17 (G-17) is a protein that is specifically secreted
the diagnosis of H. pylori infection [18]. Enzyme immunoas- from antral G-cells, and it has been suggested that its serum
say and immunochromatography assay are two types of level may reflect the severity of antral atrophy more accu-
SATs used for H. pylori detection, using either polyclonal rately than serum total gastrin. G-17 in serum, used as a test
antibodies or monoclonal antibodies. for the early detection of chronic atrophic gastritis, yielded
an overall sensitivity of 48% and an overall specificity of
Antibody-Based Tests Serological tests have also fre- 73% [20]. The majority of recent studies have used one
quently been used in screening for epidemiological studies of these immunoassays to determine the level of G-17 in
due to their inexpensive, rapid, and acceptability to patients. serum.
Numerous serological tests are based on the detection of
anti-H. pylori IgG antibody are widely available for H. pylori 35.1.4.3 HOX Transcript Antisense RNA
diagnosis and the EIA test is the most common and accurate Zhang Z and et al. detected the expression of HOX transcript
technique among them. antisense RNA (HOTAIR) collecting from patients with
superficial gastritis, atrophic gastritis, atypical hyperplasia,
Molecular Examinations Utilizing PCR to detect H. pylori and gastric cancer as well as normal controls. The results
in the stool is a reliable and rapid technique, which is espe- showed that the expression of HOTAIR was higher in gastric
cially appealing for children as a non-invasive test. Stool cancer than in normal tissues, but reached the highest level in
PCR also provides the advantages of identifying specific atrophic gastritis, suggesting that HOTAIR may be involved
genotypes and antibiotic-resistance of the microorganism in the molecular process of nonresolving inflammation [21].
[19]. Oral cavity has been implicated as an extra-gastric res-
ervoir of H. pylori. Saliva and dental plaque were the speci-
mens commonly used to detect H. pylori in the oral cavity Table 35.1 Clinical significance of serum pepsinogens results
and PCR was the most common and reliable test used in Project Normal Clinical significance
recent studies. PG I 67– <67 μg/L: Atrophy or damage of the
200 μg/L gastric body and fundus mucosa
>200 μg/L: It may be related to diet, drug
stimulation, or H. pylori infection, as well
35.1.4 Biomarker as gastric and duodenal ulcer
PG II 0–15 μg/L >15 μg/L: It may be related to H. pylori
35.1.4.1 Pepsinogens infection, gastric ulcer
The proteolytic fractions of human gastric mucosal extracts duodenal ulcer and gastric antrum
diseases
were analyzed by agar electrophoresis, numbered 1–8 in PGR(PG >7.5 <7.5: It may be related to superficial
decreasing order of electrophoretic mobility. Fractions 1–7 I/PG II) gastritis, atrophic gastritis, H. pylori
have characteristics of pepsinogens, with proteolytic activ- infection, gastric ulcer, duodenal ulcer,
ity resistant to alkalinization, but damaged by sequential and gastric antrum diseases
35.1.5 Typical Medical Case Final Diagnosis According to the results of tumor markers,
laboratory examinations, and pathology, the patient was
Clinical Background A 72-year-old male, teacher, had diagnosed with moderate chronic superficial gastritis.
unexplained upper abdominal discomfort for 1 year. With
early satiety, abdominal distension after the meal, and anxi- This case was from the First Affiliated Hospital of Nanjing
ety, he came to the hospital for physical examination. Medical University (also named Jiangsu Province Hospital).
Endoscopy The gastric antrum mucosa and the gastric fun-

dus mucosa can see edema, erythema, and apophysis. There 35.2 Diarrhea
is a bulge in the bottom of the stomach.
Shiyang Pan and Weijuan Song
Pathology Gastric Antrum: The gastric antrum mucosa can
see the mucosal layer, chronic superficial gastritis, mild.
Gastric fundus: superficial mucosa, chronic superficial gas- 35.2.1 Overview
tritis, mild. The pathological results show chronic superficial
gastritis (Fig. 35.3). The result of SP70 immunohistochemi- Diarrhea is a kind of common symptom, it is to show defecat-
cal staining is negative. ing frequency exceeds the frequency of routine habit apparently,
fecal material is rarefied and moisture increases, the daily def-
Tumor Markers SP70: 6.97 ng/mL (<7.5 ng/mL); CY21- ecating amount exceeds 200 g, or contains undigested food or
1:3.61 ng/mL (<3.3 ng/mL); CA19-9, CEA, CA72-4, AFP pus/blood stool, and mucus. Diarrhea is often accompanied by
were normal. Plasma circulating DNA:131.20 ng/mL. defecation urgency, anal discomfort, incontinence, and other
symptoms. Healthy people have about 9 L of liquid entering the
Laboratory Examination H. pylori antibody including gastrointestinal tract every day, through which water is absorbed,
Ure A, Ure B, Cag A and Vac A were negative. PGI: 161.9 μg/ and the final water in feces is only about 100–200 mL. Clinically,
mL (70–165 μg/mL); PGII:8.1 μg/mL (3–15 μg/mL); PGI/ diarrhea can be classified into acute and chronic diseases accord-
PGII:19.9 (7–20); G-17:1.4 μg/mL (1–15 μg/mL). ing to the length of the disease course. Acute diarrhea occurs rap-
idly within 2–3 weeks, mostly due to infection. Chronic diarrhea
Results with Interpretation Guideline The clinical symp- refers to a course of more than 2 months or recurrent diarrhea
toms of chronic gastritis mainly include dyspepsia, abdomi- with an intermittent period of 2–4 weeks. The etiology is more
nal distension, stomachache, and so on. The results of SP70, complex and may be due to infectious or non-infectious factors.
tumor markers were negative. The result of plasma circulat-
ing DNA was higher than healthy adult. The result of pathol-
ogy also suggested gastritis. The results of H. pylori 35.2.2 Acute Diarrhea
antibody including Ure A, Ure B, Cag A, and Vac A were
negative, which means this patient did not have H. pylori Onset is urgent and the course of the disease is in 2–3 weeks.
infection. It can be divided for watery diarrhea and dysentery kind
a b
Fig. 35.3 Pathological results. (a) hematoxylin-eosin staining (×100); (b) immunohistochemical staining (×100)
516 G. Liu et al.
diarrhea, former feces do not contain blood or pus, can- Vibrio cholerae, and Campylobacter species. If the food is
not accompany inside tenesmus, abdominal pain is lighter; contaminated with bacteria and kept at room temperature
The latter has pus and blood stool, often accompanied by for several hours, the bacteria will multiply increasing the
tenesmus and abdominal cramps. Infectious diarrhea is often risk of infection for people who eat the food. Parasites, par-
accompanied by abdominal pain, nausea, vomiting, and ticularly protozoa (such as Cryptosporidium spp., Giardia
fever. Small intestinal infections are often watery diarrhea, spp., Entamoeba histolytica, Blastocystis spp., Cyclospora
and large intestinal infections often contain bloody stools. cayetanensis), are often the cause of diarrhea in chronic
Causes of acute diarrhea can be seen in Fig. 35.4. infection. Under healthy living conditions, an otherwise
healthy person usually recovers from viral infections within
35.2.2.1 Infections a few days. However, for people who are sick or malnour-
Acute infectious diarrhea is due to the destruction of the upper ished, diarrhea can cause severe dehydration and can be
intestinal cortex by intestinal pathogens, pathogens, or toxins, life-threatening.
resulting in the destruction of normal functions and inflamma-
tion. The causes of infectious diarrhea include viruses, bacteria, 35.2.2.2 Poisoning
and parasites. Infectious diarrhea is often called gastroenteritis, Food poisoning such as eating undercooked lentils, poison
gastroenteritis is one of the main causes of death in children poisoning, pufferfish poisoning, heavy metal poisoning, pes-
under 5 years old [22]. Rotavirus, Norovirus, Adenovirus, and ticide poisoning, etc. can lead to acute diarrhea.
Astrovirus are known to cause viral gastroenteritis. Noroviruses
are considered to be the most common cause of non-bacterial 35.2.2.3 Medications
diarrheal in children and adults in developed countries [23]. More than 700 drugs are known to cause diarrhea. Drugs
Although rotaviruses are considered to be the main cause of are known to cause diarrhea include laxatives, antacids,
diarrhea in children in developing countries, noroviruses are heartburn drugs, antibiotics, antineoplastic drugs, anti-
rapidly becoming the most prevalent etiological agent of severe inflammatory drugs, and various dietary supplements [25].
gastroenteritis in children in countries where rotavirus vaccines
have been implemented in immunization programs [24]. 35.2.2.4 Other Diseases
The most common types of bacterial gastroenteritis are Acute attack of ulcerative colitis, Acute hemorrhagic necrotic
Escherichia coli, Salmonella, Shigella, Dysentery bacillus, enteritis, and Food allergy, etc.
Fig. 35.4 Causes of acute diarrhea

35.2.3 Chronic Diarrhea [29]. The pathogenesis of IBS has not been elucidated yet.
It is a multifactor syndrome with multiple mechanisms, such
A recent report suggests that both increased frequency and as micro inflammation, intestinal flora changes, high visceral
altered consistency of change are indications of organic sensitivity, immune dysfunction, and so on.
etiology [26]. And thus a pragmatic definition incorporates
these elements: Chronic diarrhea is more complex, mainly
refers to defecating frequency to increase, daily defecate is 35.2.4 Laboratory Examination
more than three times, loose stool or not formed, fecal water
content is more than 85%, accompany mucus, pus blood 35.2.4.1 Stool Tests
sometimes, lasts 2 months above, or recurrent diarrhea with
the intermittent period of 2–4 weeks. Chronic diarrhea may Fecal Bacterial Culture
be caused by intestinal mucosal lesions, excessive bacterial Fecal bacterial culture is still the “gold standard.” Patients
reproduction in the small intestine, intestinal transport func- with fever or abscess and blood stool should undergo fecal
tion defects, insufficient digestive capacity, intestinal move- specimen culture and drug sensitivity to facilitate the adjust-
ment disorders, certain endocrine diseases, and intestinal ment of treatment regimen after the experience of treatment.
tumors. Because pathogeny is different, can be companied In recent decades, to prevent and control cholera, the CDC
with the symptom such as abdominal pain, calorific, angular, has required diarrhea patients to get 100% cholera culture
abdominal mass. Causes of Chronic diarrhea can be seen in rate during the intestinal outpatient consultation. At present,
Fig. 35.5. clinically common mild cholera is often diagnosed through
Irritable bowel syndrome (IBS) is another possible cause fecal culture.
of diarrhea, usually manifested as abdominal discomfort,
with relief from bowel movements and unusual bowel move- Stool Routine
ments (diarrhea or constipation) at least 3 days a week for Observation with naked eyes, such as mucus and blood in
the past 3 months [27]. IBS is a functional disease character- stool specimens, suggests an inflammatory process. Under
ized by frequent abdominal pain and changes in defecation the microscope, red and white corpuscles may indicate
habits. No pathological changes were found in gastrointes- inflammatory diarrhea; fish-like movement under dark vision
tinal examination [28]. Possible risk factors for IBS include can indicate Vibrio cholerae, and braking test is needed to
youth, women, stress, and depression; however, the role of identify Vibrio cholerae; If diarrhea takes blood, and without
smoking as a risk factor is controversial in various studies leucocyte suggesting that it may be hemorrhagic Escherichia
Fig. 35.5 Causes of chronic diarrhea

518 G. Liu et al.
coli EHEC (O157:H7/O104:H4), or infection of amoeba and patients with inflammatory diarrhea increased. Hemolytic
Clostridium difficile (the latter two pathogens can destroy uremic syndrome (HUS) patients may have anemia and
white blood cells in feces. If suspected parasitic infection thrombocytopenia, hematuria, hemoglobinuria, and tubular
may choose to smear the eggs or oocysts. urine can be observed.
Detection of Virus, Virus Antigen, and Virus Nucleic 35.2.4.3 Serological Tests

Acid in Feces Commonly used serological methods include IF, CF, EIA, HI,
For enteroviruses that are difficult to cultivate, can exten- and NT tests. Double serum samples from patients in acute
sively be extracted from fecal specimens and observed by and convalescent stages are used for detection. If the titer
electron microscope or immune electron microscope. of serum antibodies in the convalescent stage increases four
Antigen detection commonly used to directly detect virus times or more than that in the acute stage, it is of diagnostic
infection in the gastrointestinal tract, which is faster and significance. Rapid detection of serum can be performed by
more sensitive. Immunofluorescence (especially for respi- ELISA or latex agglutination test. Finally, if clinicians still
ratory tract specimens, pharyngeal swabs, and living tissue suspect, duodenal biopsies should be carried out, and anti-
specimens) and enzyme immunoassay (especially for fecal body-negative celiac diseases account for 6.4–7% of celiac
specimens) are widely used methods. Compared with cell diseases [33].
culture, the sensitivity of virus detected by immunofluo-
rescence is increased by 40–60%. Other methods for direct 35.2.4.4 Others Tests
determination of antigen include immunochromatography X-ray barium agent examination and abdominal plain film
and latex agglutination. can show gastrointestinal lesions, intestinal dynamic state;
DNA was identified or isolated by DNA hybridization or Endoscopy plays an important role in the diagnosis of intes-
endonuclease. PCR can be used for the diagnosis of virus or tinal tumors and inflammatory lesions. Mucosal biopsies can
bacterial infection. In brief, bacterial multiplex PCR was used help detect early malignancies, precancerous lesions, and
to detect all kinds of bacteria, such as Salmonella, Shigella, certain parasites; The absorption function of the small intes-
Campylobacter jejuni, Campylobacter, Clostridium difficile, tine can be studied by fecal lipid assay, bile salt absorption
and Yersinia enterocolitica. Rotavirus group A, norovirus test, vitamin B12 absorption test, and dextroxylitol absorp-
genotype, adenovirus serotype 40/41, and astrovirus were tion test; Determination of gastrointestinal hormones and
detected by viral multiplex PCR [30]. chemicals in serum and urine; It has important diagnostic
Gastrointestinal Panel Assays (AGPA) is a new multiplex value for various gastrointestinal neuroendocrine tumors;
real-time PCR method, which can detect 13 kinds of bacte- Selective angiography and CT examination; It is especially
ria, 6 kinds of viruses, and 6 kinds of parasites in 4 kinds of valuable for the diagnosis of digestive system tumors such as
multiplex PCR reactions (two kinds of bacterial, one kind liver cancer and pancreatic cancer.
of viral, and one kind of parasitic). Gastrointestinal viruses,
especially norovirus genogroup II virus, were detected by
the AGPA in a high number of fecal electron microscope 35.2.5 Biomarkers
negative specimens. For bacterial pathogens, AGPA can
detect organisms growing in culture with high sensitivity 35.2.5.1 Calprotectin
and find several other types of E. coli, such as enteropatho- When the inflammatory process of neutrophil degranula-
genic E. coli (EPEC), enteroaggregative E. coli (EAEC), tion occurs, calprotectin is released. When inflammation
and non- O157 Shiga toxin-producing E. coli (STEC), occurs in the gut, calprotectin is released into the lumen of
which cannot be detected by traditional culture. In general, the gut and is stable enough to be detected in feces. There
the detection rate of AGPA is twice that of traditional meth- is evidence that calprotectin levels >250 μg/g feces are well
ods [31]. related to endoscopic inflammatory inflammation [34].
35.2.4.2 Blood Tests 35.2.5.2 Lactoferrin

High erythrocyte sedimentation rate(ESR), anemia, or low Lactoferrin is an iron-binding glycoprotein expressed by
albumin have a high specificity for organic disease. Iron activated neutrophils. During inflammation, lactoferrin
deficiency is a sensitive indicator of enteropathy, especially is released from the damaged tissues, which can regulate
coeliac disease [32], but not a specific test. The basic screen- inflammation and play a role in preventing infection as part
ing for malabsorption should include full blood count, urea, of the innate immune system. It can resist degradation and
and electrolyte, liver function test, vitamin B12, folate, cal- proteolysis and is not affected by the freeze-thaw cycle,
cium, ferritin, ESR, and CRP. The total number of leuko- making it a useful biomarker and an ideal marker of intesti-
cytes and neutrophils and neutrophils in peripheral blood of nal inflammation.
35.2.5.3 A nti-Saccharomyces cerevisiae population of onset, the number of diarrhea and the nature
Antibodies (ASCA) of feces, accompanying symptoms and signs, routine tests,
ASCA is an antibody with affinity to yeast Saccharomyces especially fecal examination. Etiological treatment and
cerevisiae wall antigen. CD patients tend to be positive for symptomatic treatment of diarrhea are important. In the
ASCA, which may be a useful biomarker to distinguish absence of a clear cause, the use of pain medication and
between UC and CD. ASCA positive children with CD ant diarrheal drugs must be careful, so as not to cover up
showed more extensive (endoscopic) and clinically severe the symptoms of misdiagnosis, then avoid any delay of the
disease. ASCA IgG is a useful prognostic marker in children illness.
with CD who are treated with biological products [35].
35.2.5.4 A ntineutrophil Cytoplasmic Antibody 35.2.7 Typical Medical Case

(ANCAs)
ANCAs are antibodies with affinity for neutrophils. The Clinical Background The patient, male, 16 years old, had
antibodies have been found under a variety of immune con- a 5-month history of intermittent diarrhea and had diarrhea
ditions. In ANCA staining, immunofluorescence micros- about six times a day when he visited the doctor.
copy observed that UC and CD patients have different He was with Bloody stools and intermittent pain in his left
patterns, in which UC patients mainly show perinuclear lower abdomen.
ANCA (pANCA) staining compared with CD patients [36].
However, as with ASCA, a significant number of healthy Endoscopy and Biopsy Endoscopy showed that the whole
controls were positive for pANCA. colon mucosa was hyperemic, highly edematous, the colonic
pouch became shallow, some of the colons showed lead
35.2.5.5 C-Reactive Protein (CRP) tube-like changes, the whole colon mucosa showed bleeding
CRP is one of the increased proteins in serum during acute spots and erosion, some of the mucosa showed ulcer forma-
diarrhea. CRP is produced in the liver and stimulated by tion, and the surface was covered with pus.
interleukin(IL)-6, tumor necrosis factor (TNF), and IL-1 in Biopsy pathology showed: (1) Ascending colon and trans-
the inflammatory site. Therefore, high CRP level is a sign of verse colon had acute and chronic inflammation of mucous
inflammation, but it lacks specificity. membrane, inflammatory exudation, crypt inflammation,
and crypt abscess in moderate active stage (2) Rectum had
35.2.5.6 Anti-Vinculin and Anti-Cytolethal acute, chronic inflammation of mucous membrane, severe,
Distending Toxin B Antibodies (CdtB) active period with the decrease of the crypt, and formation of
CdtB and vinculin are new biomarkers that rule-in and crypt inflammation.
distinguish IBS-D from other diarrhea causes and healthy
controls. The titer and positive rate of Anti-CdtB and anti- Blood Tests Electrolyte test (dry chemical method): sodium,
vinculin were different in IBS subtypes, In IBS-D and IBS- 130.6 mmol/L (normal range, 136–145 mmol/L), chlorine,
M, the antibody level and positive rate were higher, while in 93.8 mmol/L (normal range, 98–106 mmol/L); blood routine
IBS-C, the antibody level and positive rate similar to that of test: leukocyte, 10.59 × 109/L (normal range, 3.5–
the healthy control group were lower. These antibodies are 9.5 × 109/L), neutrophil, 6.79 × 109/L (normal range, 1.80–
helpful in the diagnosis of IBS-M and IBS-D, but not for 6.30 × 109/L), eosinophil, 0.65 × 109/L (normal range,
IBS-C [37]. 0.02–0.52 × 109/L), lymphocyte percentage, 15.39% (normal
range, 20–50%), monocyte percentage, 14.15% (normal
35.2.5.7 A ngiotensin-Converting Enzyme 2 range, 3–10%), platelet, 444 × 109/L (normal range, 125–
(ACE 2) 35 × 109/L), CRP, 10.9 mg/L (normal range, 3–8 mg/L); Anti
ACE 2 is expressed in the epithelium, submucosa, and mus- neutrophil cytoplasmic antibody group: PANCA, positive,
cularis of human and mouse colon [38], and its expression A-MPO-PANCA, weak positive.
levels in the plasma of IBD patients increased [39].
Stool Tests Fecal occult blood test was positive; bacterial
smear showed dysbacteriosis, decreased bacterial count, and
35.2.6 Conclusion more purulent cells; fecal calprotectin test set: calprotectin
1201.95 μg/g (normal range, 0–50 μg/g).
The key to the diagnosis of diarrhea is the diagnosis of the
primary disease or etiology, which needs to be based on CT Examination Whole abdominal CT showed the wall of
the starting condition and course of disease, age of onset, the rectum and sigmoid colon is obviously thickened.
520 G. Liu et al.
Final Diagnosis The most common early symptom of festations, as well as joints, skin, eyes, oral mucosa, and other
ulcerative colitis is bloody diarrhea. The results of colonos- parenteral damage. Approximately 80% of patients have
copy also showed acute and chronic inflammation of the small bowel involvement. It is usually in the distal ileum,
colon with crypt formation, and the wall of the rectum and with one-third of patients having exclusively ileitis. 50% of
sigmoid colon is obviously thickened, considering chronic patients have ileocolitis and from 20 to 25% of patients have
enteritis. disease confined to the colon. Involvement of the esopha-
gus, stomach, or duodenum is rare and is often associated
This case was from the First Affiliated Hospital of Nanjing with disease of the more distal small bowel or large bowel.
Medical University (also named Jiangsu Province Hospital). Almost one-third of patients have the perianal disease [44].
Patients are often insidious and slow, sometimes months
to years from early symptoms to diagnosis. The course of the
35.3 Crohn’s Disease disease is chronic, with varying lengths of the active period
and the remission period alternating, and the delay does not
Shiyang Pan and Jia Wang heal. A few acute onset, can be realized as acute abdomi-
nal disease, some patients can be misdiagnosed as acute
appendicitis. Abdominal pain, diarrhea, and weight loss are
the main clinical manifestations of the disease. However,
35.3.1 Overview the clinical manifestations of this disease are complex and
changeable, which are related to clinical type, lesion loca-
Crohn’s disease (CD), a subtype of inflammatory bowel tion, disease stage and complications.
disease (IBD), has been increasing incidence over the last The international classification of IBD was proposed by
few decades [40]. Though preferring in most cases the distal the Vienna classification proposed by the World Congress of
small bowel and the proximal large bowel, it may involve the Gastroenterology in 1998. It considered the age of onset (A),
entire gastrointestinal tract from the mouth to the perianal disease location (L), and disease behavior (B) (Table 35.2).
area. There is big variation in the incidence and prevalence In 2005, the current Montreal Classification was proposed
of CD based on environment, geographic region, and eth- at the Montreal World Congress of Gastroenterology. The
nic groups. The annual incidence of CD in North America Montreal classification was essentially a revision of the
is reported to be 3.1–20.2/100,000. Rates are higher in peo- Vienna classification, which kept the three predominant
ple of Jewish descent [41]. Epidemiological data in China parameters (Table 35.2). Montreal classification is widely
show that the age-standardized incidence of IBD in Daqing used in both research and clinical practice, there is very
City, Heilongjiang Province is 1.77/100,000, and that in excellent inter-observer agreement in CD [45]. Studies have
Zhongshan City, Guangdong Province is 3.14/100,000, and shown that the patients with the predominantly inflammatory
the number of patients has been increasing rapidly over the disease are very likely to develop either fistulizing or stric-
past 20 years [42]. The age of Crohn’s disease onset has a turing complications within the next 20 years [46]. Lémann
bimodal distribution. The first peak occurs between the ages Index (LI) is a newly scoring system to quantify the global
of 15 and 30, and the second peak occurs mainly among digestive tract damage mediated by CD over a period of time
women between the ages of 60 and 70 years [43]. Based on [47]. It is considered equivalent to the sharp index used to
the epidemiological, genetic, and immunological data, the quantity joint space narrowing in Rheumatoid arthritis [48].
CD is considered to be a heterogeneous disorder with the
characteristics of lifelong disease, no special curative drugs, Table 35.2 Vienna and Montreal classification
high surgical rate, and disability rate, seriously affecting the Vienna classification Montreal classification
life and work of patients. Age at A1: <40 A1: <16
diagnosis A2: >40 A2: b 17–40
(years) A3: >40
Location L1: Ileal L1: Ileal
35.3.2 Clinical Manifestation L2: Colonic L2: Colonic
L3: Ileocolonic L3: Ileocolonic
CD is typically characterized by parenchymal inflammation L4: Upper L4: Upper disease modifier
of the intestine, which is more common in the terminal ileum or isolated upper disease
and the adjacent colon. The digestive tract, from the mouth Behavior B1: Non-stricturing, B1: Non-stricturing,
non-penetrating non-penetrating
to the anus, is involved in a segmental distribution. It has B2: Stricturing B2: Stricturing
abdominal pain, diarrhea, weight loss as the main clinical B3: Penetrating B3: Penetratin
manifestations, often fever, fatigue, and other systemic man- P: Perianal disease
ifestations, perianal abscess or fistula and other local mani- modifier
35.3.3 Laboratory Diagnosis an anemia of chronic disease (usually normal mean corpus-
cular volume [MCV]) or an iron deficiency anemia (MCV is
The pathogenesis of CD is not clear. It is closely related to bac- often low), which occurs due to acute or chronic blood loss
terial or viral infection, abnormal immune function, environ- and malabsorption (iron, folic acid, and vitamin B12), and
mental, and genetic factors. The diagnostic of CD is usually may also reflect reduced intake or a chronic inflammatory
based on the presenting symptoms, signs, and basic laboratory burden. In addition, erythropoietin is often involved in eryth-
abnormalities. The main features of diagnosis of CD include ropoietin production. Erythropoietin, which is produced by
a combination of radiographic, endoscopic, pathological find- the kidney, promotes the differentiation and survival of ery-
ings, and serologic examination. Serologic studies are helpful throid progenitor cells in the bone marrow. In the chronic
in the diagnosis of CD including CRP (C reactive protein), course of CD, high levels of tumor necrosis factor(TNF)
ESR (erythrocyte sedimentation rate), hematologic tests, fecal inhibit the secretion of erythropoietin, which is an important
calprotectin, perinuclear antineutrophil cytoplasmic antibod- mechanism of CD anemia caused by the cytokine network in
ies (pANCA), anti-Saccharomyces cerevisiae antibodies the course of chronic inflammation.
(ASCA), anti-flagellin (antiCbir1), and anti-Escherichia coli The occurrence of anemia can make the patient’s qual-
outer membrane protein C (OmpC) antibody, etc. ity of life, cognitive ability, ability to work decline, and
the chance of hospitalization greatly increased. It has been
35.3.3.1 CRP and ESR shown that decreased hemoglobin levels are associated with
CRP is the most common clinical indicator of acute phase increased clinical disease activity scores. Due to the high
response. It is a pentameric protein composed of five mono- prevalence of malnutrition in CD, vitamin B12, and folic
mers. Under normal circumstances, CRP is produced by acid levels often need to be evaluated. Vitamin B12 defi-
hepatocytes at low levels (0.1 mg/L) and has the function of ciency can occur in patients with CD who have significant
activating complement and promoting phagocytosis of gran- terminal ileum disease or in patients who have had terminal
ulocytes and macrophages. Interleukin (IL)-6, tumor necrosis ileum resection.
factor-α (TNF-α), and IL-1beta may peak at 350–400 mg/L
under the influence of CRP, which stimulates rapid produc- 35.3.3.3 F ecal Calprotectin and Other Fecal
tion of liver cells, such as acute inflammation. Compared Marker
with other acute phase proteins, CRP has a shorter half-life Calprotectin is a 36-kDa calcium and Zn-binding protein
(19 h), so it rises early after inflammation and falls rapidly found in neutrophils, which is released into the gut when
after inflammation subside. white blood cells are damaged, resists enzyme breakdown,
CRP levels and ESR are associated with disease activ- is not affected by diet, and is stable at room temperature for
ity. ESR is the rate at which red blood cells migrate through 7 days. Fecal calprotectin (FC) is derived from calprotectin
the plasma. ESR will depend on plasma concentration and in colonic mucosa and can reflect the histological severity
the number and size of red blood cells. Anemia, polycythe- of inflammation. FC can be used as an evaluation method
mia, and thalassemia all affect ESR. ESR peaks much more for mucosal healing, reduce the need for colonoscopy dur-
slowly than CRP, and even if a patient’s clinical symptoms ing IBD follow-up, evaluate the response to treatment and
or inflammation improve, it can take days to decrease. And predict postoperative recurrence. Stool samples should be
ESR increases with age. tested for the presence of white blood cells (WBCs), occult
At present, basic and clinical studies have shown that blood, routine pathogens, ova, parasites, and Clostridium
the imbalance between preinflammatory cytokines and anti- difficile toxin. An obvious reason to search for fecal mark-
inflammatory cytokines is the most important pathogenesis ers is that FC has the advantages of non-invasive, easy
leading to CD. Therefore, it is important to detect CRP lev- access to specimens, simple and quick operation, can be
els and ESR, which are well correlated with inflammatory checked repeatedly in a short period of time, cheap price,
disease activity. They are closely related to clinical activity, and so on.
endoscopic activity, and histological activity of inflamma- A number of neutrophil-derived proteins present in stools
tory bowel disease, and can be used to monitor treatment have been studied, including fecal lactoferrin, lysozyme,
response, predict disease progression, and classify sub- elastase, myeloperoxidase, and calprotectin [49]. The pres-
groups of patients. ence of fecal neutrophil-derived biomarkers (calprotec-
tin and lactoferrin) is an excellent way to detect intestinal
35.3.3.2 Hematologic Tests information. They correlate closely with endoscopic activity
CD is characterized by diarrhea, abdominal pain, and loss of in CD and allow serial monitoring of disease activity and
body mass, and many complications may occur during the treatment success and can even serve in predicting clinical
course of the disease. Anemia is common and may be either relapse or sustained remission.
522 G. Liu et al.
35.3.3.4 Other Serological Markers been identified through large-scale genome-wide association
Perinuclear antineutrophil cytoplasmic antibodies (pANCA) studies [54]. The NOD2 gene, the interleukin (IL)-23 recep-
and anti-Saccharomyces cerevisiae antibodies (ASCA) have tor gene, and the autophagy-related 16-like 1 (ATG16L1)
been found in patients with Crohn’s disease. The combination gene associated with examples of single-nucleotide poly-
of positive pANCA and negative ASCA has high specificity for morphisms that confer susceptibility to CD [55]. These genes
ulcerative colitis, while the inverse pattern—positive ASCA, play a role in innate immunity and regulation of the epithelial
negative pANCA—is more specific for Crohn’s disease barrier. NOD2 variants are predictors of a more complicated
[50]. According to the World Gastroenterology Organization disease behavior including ileal involvement, stenosis, and
(WGO), ulcerative colitis is more likely to occur when the penetrating disease behaviors, and the need for surgery [56].
test results are pANCA positive and ASCA antigen nega- These laboratory tests have not been routinely used clinically
tive. However, the pANCA test may be positive in Crohn’s and remain a research tool, although variants may identify
disease, and this may complicate obtaining a diagnosis in more aggressive CD patients. Ultimately, we may be able to
otherwise uncomplicated colitis [51]. ASCA positive patients use genetic testing to characterize patient’s disease behavior
with Crohn’s disease have a higher rate of surgery and require and guide early therapy [57].
earlier surgery in the course of the disease, regardless of the
site of involvement. Other serological markers, such as anti- 35.3.3.7 Endoscopy
flagellin (antiCbir1) and Pseudomonas fluorescens (anti-12), Upper or lower GI endoscopy, which can assess disease loca-
are found in more than 50% of Crohn’s disease cases. Anti- tion and obtain tissue for pathological evaluation, is routinely
flagellin (antiCbir1) is independently associated with diseases used to confirm the diagnosis of CD. The endoscopic appear-
of the small intestine, penetrating, and fibrous stenosis. In fact, ance of CD is highly variable and changes with disease activ-
the serum response to cbir1, an antibody associated with IBD, ity and duration, but it is invasive, painful, and cannot be
has been shown to distinguish pANCA-positive ulcerative observed in patients with intestinal stenosis.
colitis from ulcerative colitis, such as Crohn’s disease [52].
Escherichia coli outer membrane protein C (OmpC) is 35.3.3.8 Imaging
the main outer membrane protein of Escherichia coli, which CTE or MRE is the standard imaging examination for the
maintains the basic morphology of the membrane and has evaluation of intestinal inflammatory lesions so far, and it
strong immunogenicity. It can enhance the role of macro- should be included as a routine examination for the diagnosis
phages in presenting antigens, stimulate the body to produce of CD. The examination may reflect inflammatory changes
body fluids and cellular immunity, and play an important in the intestinal wall, the location and range of lesion dis-
role in maintaining host immunity. The production of anti- tribution, the presence of stenosis and its possible nature
OmpC antibodies in the body can lead to the host’s immune (inflammatory activity or fibrous stenosis), and extraluminal
regulation disorder against the antigens of its own intesti- complications, such as fistula formation, abdominal abscess,
nal flora. Anti-OmpC antibodies combined with ASCA can or cellulitis [58].
improve the diagnostic sensitivity to CD. In addition, the WHO once proposed six diagnostic criteria for CD for diag-
positive expression of anti-OmpC antibody in CD patients nosis (Table 35.3), which was recently again recommended by
may indicate the combination of intestinal perforation or fis- World Gastroenterology Organization (WGO) [59].
tula formation, which is conducive to the determination of
the prognosis of the disease.
35.3.4 Conclusions
35.3.3.5 Microbiology
Stool culture, ova and parasite studies, bacterial pathogen In recent years, CD-specific serological markers have
culture, and evaluation for Clostridium difficile infection are aroused wide concern and made remarkable progress. With
required before making a definitive diagnosis of idiopathic the rapid development of proteomics, metabolomics, and
inflammatory bowel disease [53]. Clostridium difficile toxin other technologies exhibition, more and more new serologi-
assay should be performed on any patient hospitalized with cal indicators have been found. The diagnosis of CD should
a flare of colitis, because pseudomembranous colitis is com- be combined with clinical features, characteristics of the
monly superimposed on IBD colitis. endoscopic view, and histopathological findings were com-
bined. But these are mostly expensive invasive methods and
35.3.3.6 Genetic Testing the operation is complex. Currently, the serological exami-
CD is a heterogeneous disease with complex interactions nation is convenient, non-invasive, cheap, and fast. Various
between genetics, environmental exposures, and the intesti- CD-related serological markers have provided help for the
nal microbiome. To date, more than 200 genetic loci associ- diagnosis, differential diagnosis, efficacy evaluation, and
ated with IBD and greater than 71 CD susceptibility loci have prognosis of the disease.
Table 35.3 Diagnostic criteria for CD

Clinical manifestations Radiographic examination Endoscopy Biopsy Surgical specimens
1. Discontinuous or segmental changes + + +
2. Pebbly appearance or longitudinal ulcer + + +
3. Changes in the whole wall inflammatory + + + +
response
4. Noncaseating granuloma + +
5. Fissure, fistula + + +
6. Perianal lesions +
Those with (1), (2), (3) are suspected; plus (4), (5), (6) one of the three can be diagnosed; have the (4), as long as plus, (1), (2), (3) three of the two
can also be diagnosed. “+” represents the clinical manifestation of this condition
35.3.5 Typical Medical Case
Clinical Background A 42-year-old male patient was

admitted to the hospital due to “diarrhea, bloody stool for a
month, abdominal pain and abdominal distension l day.” The
patient had abscess and blood stool a month ago, five times
per day, fever appeared 7 days ago with a body temperature
of 38.5 °C, oral “cefaclor.” There were no similar cases in the
family. Physical examination: temperature 37.7 °C, heart
rate 120 times/min, breathing 22 times/min, the blood pres-
sure 110/54 mmHg (1 mmHg = 0.133 kPa), normal develop-
ment, clear consciousness, poor spirit. No enlargement was
found in superficial lymph nodes. Abdominal muscle ten-
sion, abdominal tenderness, rebound pain, liver, spleen, and
ribs did not touch, suspicious abdominal mobility dullness,
intestinal sound disappeared. Abdominal CT shows free gas Fig. 35.6 The histopathology of the diseased intestinal wall
in the abdominal cavity.
Laboratory Test Several tests were performed for screen-

ing this disease. WBC 13.79 × 109/L, Neutrophil 9.46 × 109/L,
RBC 3.37 × 1012/L, HGB 100 g/L, PLT 284 × 109/L, ESR
30 mm/h, CRP 80.5 mg/L, HGB 100 g/L, MCV 75 fl, fecal
calprotectin 150 ng/mL, ASCA and ANCA are positive, anti-
Cbir1 130 ng/mL. The increase of these examination indexes
strongly suggests the possibility of CD.
Histopathological Examination It is a lacunar ulcer that

extends deep into the muscular layer, thickening, and edema
of the submucosa. This is accompanied by inflammatory cell
infiltration, see in Fig. 35.6 (hematoxylin-eosin staining
×100).
Colonoscopy The colon is seen as a cobblestone (Fig. 35.7).
Results with Interpretation Guideline The patient under-

went exploratory laparotomy during which multiple ruptures
Fig. 35.7 The colonoscopy of the patient
of the colon were found, and the partial resection was patho-
logically diagnosed as Crohn’s disease. The definitive diag-
nosis of CD is sometimes difficult, especially if the disease is ical manifestations, laboratory examination, endoscopic
not considered by the physicians treating the patient. The examination, imaging examination and histopathological
diagnosis requires a comprehensive analysis combining clin- examination, and close follow-up. With high sensitivity and
524 G. Liu et al.
specificity, serological markers can detect most patients in the level of the eleventh thoracic vertebra [61]. The esopha-
the early stage of the disease, which is helpful for the deter- gus is usually about 25 cm (10 in) in length [62] The upper
mination of clinical stage, efficacy, prognosis, and recur- esophagus lies at the back of the mediastinum behind the
rence of the disease. trachea, adjoining along the tracheoesophageal stripe, and in
front of the erector spinae muscles and the vertebral column.
This case was from the First Affiliated Hospital of Nanjing The lower esophagus lies behind the heart and curves in front
Medical University (also named Jiangsu Province Hospital). of the thoracic aorta. The esophagus passes down the right
pulmonary artery, the left main bronchus, and the left atrium
from the bifurcation of the trachea. At this point, it passes
35.4 Esophageal Cancer through the diaphragm [61].
SCC tends to occur in the upper- and middle-third of the
Shiyang Pan, Fei Jin, and Zhenzhen Cai esophagus. AC usually occurs in the distal third of the esopha-
gus, so do other rare esophageal cancers such as small cell carci-
noma, choriocarcinoma, melanoma, sarcoma, and lymphoma.
35.4.1 Overview
The esophagus is a part of the digestive tract whose main 35.4.2 Epidemiology
function is to push food into the stomach. Esophageal-
related diseases include esophageal cancer, benign esopha- Esophageal cancer is a common malignant disease world-
geal tumor, the erosive burn of esophagus, achalasia of the wide, with more than 400,000 new cases per year. Two major
cardia, diverticulum of the esophagus, and so on. types, squamous cell carcinoma (SCC) and adenocarcinoma,
Esophageal cancer (EC) is the eighth most common can- account for over 95% of esophageal cancers. SCC is the
cer in the world and the sixth most common cause of can- most common histology worldwide, but adenocarcinoma is
cer death. Esophageal squamous cell carcinoma (ESCC) and more frequent in the United States. SCC commonly occurs
adenocarcinoma (EAC) are two major histopathological types in developing countries and is typically associated with the
of esophageal cancer. In the past 30 years, the incidence of EC consumption of tobacco and alcohol. The disease is four
has been increasing. The 5-year survival rate for advanced can- times more prevalent in men, and race also appears to be a
cer is still very poor, even with improved surgical techniques factor. The incidence of SCC is significantly higher among
and adjuvant chemoradiation therapy. It is extremely impor- African Americans compared with their white counterparts,
tant to understand esophageal cancer biology. SCC commonly even after adjusting for socioeconomic status and tobacco
occurs in developing countries and is typically associated with and alcohol use. Worldwide, parts of the Middle East, Central
the consumption of tobacco and alcohol. Adenocarcinoma Asia, and China have the highest rates of SCC, after adjust-
typically occurs in white men in developed countries, and the ing for tobacco and alcohol use, indicating that there may be
important etiologic factors are obesity, chronic gastroesopha- some genetic predisposition or other environmental factors.
geal reflux, and Barrett’s esophagus. Adenocarcinoma typically occurs in white men in
The esophagus, commonly known as the food pipe or gul- developed countries, and the important etiologic factors
let, is an organ in vertebrates through which food passes. With are obesity, chronic gastroesophageal reflux, and Barrett’s
the aid of peristalsis and contraction, food passes through the esophagus. Although endoscopic screening is useful for
esophagus from the pharynx to the stomach. The esophagus early cancer detection, 50% of superficial esophageal can-
is a fibromuscular tube, which travels behind the trachea and cers (those confined to the submucosa) have nodal metasta-
heart, passes through the diaphragm, and empties into the sis. Most esophageal cancers are diagnosed at an advanced
uppermost region of the stomach. The anatomical structure stage, and the prognosis after surgical resection with neoad-
of the esophagus is shown in Fig. 35.8. When swallowing, juvant chemoradiotherapy remains unsatisfactory; the 5-year
the epiglottis tilts backwards to prevent food from entering survival rate is <50% after curative surgery.
the larynx and lungs.
The esophagus is one of the upper parts of the digestive
system. There are taste buds on its upper part [60]. It starts 35.4.3 Etiology
at the back of the mouth, goes down through the rear part of
the mediastinum, through the diaphragm, and into the stom- The two main types have different risk factors (see in
ach. In humans, the esophagus usually begins at the level of Table 35.4). Squamous cell carcinoma is related to lifestyle
the sixth cervical vertebra behind the cricoid cartilage of the factors such as smoking and alcohol, while Adenocarcinoma
trachea, enters the diaphragm at about the level of the tenth is related to long-term acid reflux [63]. Tobacco is a risk fac-
thoracic vertebra, and ends at the cardia of the stomach, at tor for both types.
Fig. 35.8 Anatomy of the

esophagus
Table 35.4 Risk factors for squamous cell carcinoma and adenocarci- 35.4.3.1 Squamous Cell Carcinoma
noma of the esophagus Tobacco and alcohol are strong risk factors for SCC, and
Squamous cell they have a synergistic effect on risk. The disease is four
Risk factor carcinoma Adenocarcinoma times more prevalent in men, and race also appears to be a
Geography Southeastern Africa, Western Europe, North factor. The incidence of SCC is much higher among African
Asia, Iran, South America (United States),
America Australia Americans compared with their white counterparts, even
Race Black, White White men after adjusting for socioeconomic status and tobacco and
women alcohol use. Worldwide, parts of the Middle East, Central
Gender Male > female Male > female Asia, and China have the highest rates of SCC, indicating
Alcohol ++++ / that there may be some genetic predisposition or other envi-
Tobacco ++++ ++ ronmental factors. SCC is associated with certain intrinsic
Obesity / +++
disorders of the esophagus, such as Plummer-Vinson syn-
GERD / ++++
drome and achalasia. Other hereditary cancer syndromes
Diet and ++ +
nutrients: associated with esophageal SCC include tylosis and Fanconi
Genetic aspects ++ + anemia. Patients with a history of caustic ingestion are at
Socioeconomic ++ / significantly increased risk for SCC.
conditions
526 G. Liu et al.
35.4.3.2 Adenocarcinoma to cancer [65, 66]. EGFR overexpression, partly due to gene
Adenocarcinoma typically arises in the setting of Barrett’s amplification, is correlated with tumor progression, mini-
esophagus. In addition to GERD, smoking and obesity are mal response to chemotherapy, and poor prognosis. Gene
risk factors for adenocarcinoma. As with SCC, there is a amplification and overexpression of cyclin D1 are detected
male predominance. There are also familial forms of Barrett in 25–50% of esophageal SCC and cyclin D1 is an indepen-
esophagus that increase the risk of adenocarcinoma. dent prognostic marker confirmed by multivariate analysis
[65, 66]. Homozygous deletion and hypermethylation of the
p16INK4a gene are found in 50–60% of esophageal SCC and
35.4.4 Screening cause dysregulation of the G1/S checkpoint and abnormal
proliferation, resulting in metastasis and poor prognosis [67].
Squamous dysplasia is a precancerous lesion of squamous Loss of FHIT expression occurs even in normal-appearing
cell carcinoma. It is difficult to detect in asymptomatic indi- squamous epithelium, which has been heavily exposed to
viduals and there is no standardized screening procedure to environmental carcinogens such as tobacco and alcohol.
detect this condition. Patients with Barrett’s esophagus are In esophageal adenocarcinoma, a p53 mutation is also
at higher risk and may receive regular endoscopic screening an early event in carcinogenesis, as it is detected in Barrett
for the early signs of cancer [64]. Because the benefits of esophagus and dysplasia [65, 68] Alterations in the tran-
screening for adenocarcinoma in asymptomatic people are scription of FHIT and p16INK4a also occur in adenocarcinoma
unclear, it is not recommended in the United States. There at an early stage. HER2 amplification and overexpres-
are screening programs in some areas with high incidence of sion are found in 20–30% of esophagogastric and Barrett’s
squamous cell carcinoma in the world. esophagus-related adenocarcinomas. In Barrett esophagus
and esophageal adenocarcinoma, the loss of p27 expression
is associated with malignant transformation and poor prog-
35.4.5 Clinical Diagnosis nosis [68].
In regard to epigenetic alterations in esophageal SCC,
The diagnosis of esophageal cancer is almost always made genes including APC, CDH1, p16INK4a, RARβ, and Ras
by endoscopic biopsy. Endoscopy should be performed in association domain family protein 1 (RASSF1A) are highly
any patient with dysphagia, even if the barium esophagram methylated [67].CDH1 methylation is observed in 70% of
is suggestive of a motility disorder. Classically, esophageal esophageal SCC and is associated with invasion, metasta-
cancers appear as friable, ulcerated masses, but the endo- sis, and poor prognosis. Hypermethylation of RARβ and
scopic appearance can be varied. Early-stage tumors may RASSF1A that causes cell cycle deregulation is found in
appear as ulcerations or small nodules. More advanced 50–60% of esophageal SCC. As in SCC, esophageal ade-
tumors are more likely to be friable masses but may also nocarcinoma is also characterized by frequent methylation
appear as strictures or ulcerations. In many cases, the initial of APC, CDH1, and p16INK4a [67]. CDH1 methylation and
endoscopist may not recognize the presence of cancer and reduced expression are associated with metastatic ability.
a single biopsy may not be diagnostic. Therefore, multiple Hypermethylation of p14ARF and p15INK4b is uncommon in
biopsies should be performed for any suspicious lesions. Barrett’s esophagus-associated carcinogenesis. The lower
During endoscopy, the location of the tumor relative to the frequency (10%) of hMLH1 methylation is consistent with
incisors and GEJ should be noted, as well as the length of the lower prevalence of MSI in esophageal adenocarcinoma
the tumor and degree of obstruction. The most proximal compared with gastric and colorectal adenocarcinomas.
extent and circumferential extent of any Barrett’s esophagus MGMT inactivation by DNA methylation is frequently
should also be noted according to the Prague criteria. For found in Barrett esophagus (40%) and esophageal adenocar-
small tumors or nodules, an experienced endoscopist should cinoma (60%), whereas 20% of normal squamous epithelia
perform endoscopic mucosal resection (EMR) to provide a show MGMT methylation.
specimen that accurately assesses the depth of invasion. These aberrant methylations mentioned above can be used
as biomarkers for molecular diagnosis of esophageal can-
cer. Methylation profiles of multiple genes including APC,
35.4.6 Molecular Diagnosis CDH1, MGMT, p16, and RUNX3 serve as indicators of the
neoplastic progression of Barrett’s esophagus and are inde-
35.4.6.1 G enetic and Epigenetic Alterations pendent prognostic factors for esophageal adenocarcinoma
Detection and Its Implications [69, 70]. DNA methylation detected in serum is useful for
In esophageal SCC, mutation of the p53 gene occurs at an screening and monitoring of esophageal cancers. P16 meth-
early stage of carcinogenesis and is found in 40–60% of ylation is found in the sera of 10–20% of esophageal SCC
SCC and less commonly in non-cancerous mucosa adjacent patients and correlates with poor prognosis [71–73]. APC
methylation is found in the sera of 25% of esophageal AC the other hand, the expression of miR-21, miR-192, miR-
patients and in the sera of <10% of esophageal SCC patients, 195, and miR-223 increased in esophageal adenocarcinoma
and high serum levels of APC methylation are significantly while the expression of miR-203 is decreased [81]. The
associated with reduced patient survival [74]. levels of miR-30e and miR-200a correlate with survival
of esophageal adenocarcinoma patients, while decreased-
35.4.6.2 Molecular Diagnosis miR-375 expression is associated with shorter survival [83].
of Micrometastasis and CTCs Overexpression of miR-148 enhances the effect of cispla-
Compared with gastric cancer, the sentinel node mapping for tin and 5-FU, which provided a basis for the potential use
esophageal cancer is relatively complicated, but it provides of miRNAs to predict or improve the response to chemo-
useful information on individualized selective lymphadenec- therapy [84]. During the development from normal mucosa
tomy, which reduces the incidence rate of esophageal cancer to adenocarcinoma via Barrett’s esophagus, upregulation in
and maintains the quality of life for patients [75]. Detection turn of miR-21, miR-93, miR-192, and miR-194 is observed
rates of sentinel nodes by the 99mTc–tin colloid method or [85]. Upregulation of miR-192 and miR-215 and downregu-
fluorescent dye imaging have been reported to be satisfactory; lation of miR-203, miR-205, and let7c are a “progression
the sensitivity is 90–100% for clinical stage T1–T3 patients signature” for the progression from Barrett’s esophagus to
and 45% for patients who received neoadjuvant chemora- adenocarcinoma which may serve as molecular markers for
diation therapy, respectively [76, 77]. A sensitive real-time neoplastic progression [86].
RT-PCR system, using CK19, CK20, SCC antigen, and CEA
as marker mRNAs, efficiently detects micrometastasis in 35.4.6.4 Genetic Polymorphism
sentinel nodes. CTCs in the blood can also be detected in Many studies have assessed the risk of genetic polymor-
esophageal cancer patients by the RT-PCR–based molecular phism and esophageal cancer with a majority of these stud-
method. CEA mRNA is detected in the sera of 60% of esoph- ies conducted in Asian countries [87–89]. Meta-analyses of
ageal SCC patients and is related to tumor invasion, vessel ALDH2, MTHFR, CYP1A1, CYP2E1, GSTP1, GSTM1,
involvement, nodal metastasis, and advanced stage [78, 79]. and GSTT1 have found significant correlations between
The presence of CTCs detected by CEA expression is an ALDH2 × 1 × 2 and CYP1A1 Val allele and increased risk
independent factor for shortening hematogenous disease- of esophageal cancer. ALDH2 is a polymorphic gene whose
free interval. CTC positivity is correlated to the decrease of individual genotypes determine concentrations of acet-
E-cadherin expression in the primary tumor. Detection of aldehyde in the blood after drinking, whereas CYP1A1 is
CTC by the RT-PCR–based method is useful to predict the involved in the activation of major types of tobacco procar-
recurrence in patients with esophageal SCC. cinogens such as polyaromatic hydrocarbons and aromatic
amines. The increased risk of esophageal SCC is associated
35.4.6.3 miRNA-Based Molecular Diagnosis with ADH2 × 1 × 2 and p53 codon 72 Pro/Pro genotypes.
Changes in the expression pattern of miRNAs have poten- GSTP1 (Ile105Val) is a risk factor for Barrett esophagus
tial clinical applications in the development of biomark- and adenocarcinoma in Caucasian males [90]. GSTP1 is
ers to identify the presence and progression of esophageal the major isoform expressed in the esophagus and elimi-
cancer and to assess chemosensitivity and radiosensitivity nates DNA oxidative products. In addition to protein-coding
of tumors [80, 81]. The expressions of miR-10b, miR-92a, genes, pre-miRNAs also possess polymorphisms that affect
miR-93, miR-192, miR-194, and miR-205 in ESCC are esophageal cancer risk [91]. For example, C-T SNP in pre-
increased in tumor tissues compared with normal esopha- miR-196a (rs11614913) and G>C variant in premiR-146a
geal mucosa, while the expressions of miR-100, miR-125b, raise esophageal SCC in the Chinese population [91] These
miR-133a, miR-133b, miR-143, miR-145, miR-203, miR- findings are applicable to molecular diagnosis in identifying
205, and miR-375 are reduced. Overexpression of miR-21, individuals at high risk for developing esophageal cancer.
miR-23a, miR-26a, miR-96, miR-103, miR-107, miR-128b,
and miR-129 detected in ESCC is associated with progno- 35.4.6.5 Novel Molecular Markers
sis. Increased expression of miR-200c correlates not only
with poor prognosis but also with decreased sensitivity to ADAMTS16
chemotherapy in patients with ESCC. Increased expres- Although traditional serum tumor markers just like SCC
sion of miR-296 is also associated with chemoresistance. antigen and CYFRA21-1 (fragment of CK19) have been
Detection of circulating miRNAs provides a new comple- used clinically as biomarkers, but with low sensitivity and
mentary tumor marker for ESCC. The plasma level of miR- low specificity. To search for novel biomarkers for esopha-
21 is higher and that of miR-375 is lower in SCC patients geal cancer, a SAGE library was generated from esophageal
than in control groups [82]. High plasma concentrations SCC and compared with the library from normal esophageal
of miR-21 are significantly correlated with recurrence. On mucosa [91]. Many upregulated and downregulated genes
528 G. Liu et al.
were identified that might be candidate diagnostic mark-

ers and therapeutic targets. ADAM metalloproteinase with
thrombospondin type 1 motif 16 (ADAMTS16) was the
most upregulated gene in esophageal SCC. ADAMTS16
is expressed in 40% of esophageal SCC at high levels, as
shown by quantitative RT-PCR, whereas SCCA1-encoding
SCC antigen is expressed in only 20% of esophageal
SCC. ADAMTS protein is secreted from cancer cells, and
knockdown of ADAMTS16 inhibits cell growth and invasion
ability. Therefore, ADAMTS16 could be a new diagnostic
and therapeutic target in patients with ESCC. SAGE data
provide a list of genes associated with the development and
progression of esophageal cancer.
Tumor Specific Protein (SP70) Fig. 35.9 Levels of SP70 in esophagus group
SP70, a specific antigen recognized by monoclonal antibody
NJ001, is a marker of non-small cell lung cancer. Previous
researches have indicated that the expression of SP70 is
closely related to the clinical pathological features and prog-
nosis of lung cancer. It is found that SP70 is also abnormal
in esophageal cancer patients. The double antibody sand-
wich ELISA method was used to detect SP70 in the serum
of 66 patients with esophageal cancer, 18 patients with the
benign esophageal disease, and 53 healthy persons from
the First Affiliated Hospital of Nanjing Medical University
from September 2017 to June 2018. At the same time, SP70
was compared with CEA, CY211, and NSE, which were
Fig. 35.10 ROC curve of SP70, CEA, CY211 and NSE in esophagus
detected by electrochemiluminescence. The distribution of group
SP70 in the serum of patients with esophageal cancer was
significantly higher than that of patients with esophageal
benign lesion and healthy physical examination group [9.1 removal, while more locally advanced cancers are treated
(7.1, 11.3) and 7.1 (5.9, 11.0), P = 0.016;9.1 (7.1, 11.3) with chemotherapy, chemoradiotherapy, surgical resection,
and 7.0 (6.0, 7.8), P < 0.001], but there was no significant or combinations of these (see in Fig. 35.11). Patients with
difference between patients with esophageal benign lesion esophageal cancers which are not suitable for operation are
and healthy physical examination group [7.1 (5.9, 11.0) and treated with systemic chemotherapy.
7.0 (6.0, 7.8), (P > 0.05)] (see in Fig. 35.9). The AUC of
SP70, CEA, CY211, and NSE in diagnosing esophageal can- 35.4.7.1 Ablation
cer was 0.718, 0.572, 0.702, and 0.556 respectively (see in Various endoscopic ablative and resection techniques have
Fig. 35.10). Compared with AUC = 0.5, SP70 and CY211 been developed that have largely supplanted the role of
have diagnostic efficacy (P < 0.05). The sensitivity and spec- esophagectomy for high-grade dysplasia. The most com-
ificity of SP70 were 54.8% and 76.9% respectively, while monly used technology today is radiofrequency ablation
the sensitivity and specificity of CY211 are 8.24% and 100% (RFA). RFA is much more effective than photodynamic
respectively. The patients with high SP70 before the opera- therapy with a lower stricture (and overall complication)
tion were followed up. It was found that the serum SP70 rate. RFA may be delivered. With a circumferential balloon
decreased about 7 days after operation [10.3 (9.1, 11.5) and or an electrical plate using a bipolar electrode that transmits
8.8 (7.3, 10.3), P < 0.001]. radiofrequency energy, which generates heat and destroys
superficial tissue. The treated mucosa is replaced by neo
squamous mucosa. The standard ablation program uses two
35.4.7 Therapy double pulses of 12 J/cm2. The balloon is then repositioned
distally, and the procedure is repeated until the entire seg-
Treatment of esophageal cancer dependents on the charac- ment of Barrett’s esophagus is treated. If there are areas of
teristics of patient (including fitness) and tumor, mainly the residual Barrett esophagus on follow-up endoscopy, those
TNM stage; early tumors may be suitable for endoscopic segments may be treated with more focal ablation.
Staging:
Endoscopy, EUS
CT, PET
Early stage Locally advanced

OSCC and OAC Non-metastaticc
T1-T2 NO MO T3-T4 or N1 -N3 MO
Resection
Endoscopic Squamous cell
Adenocarcinoma
surgery cancer
Neoadjuvant
Definitive Neoadjuvant Neoadjuvant
or perioperative
chemoradiotherapy chemoradiotherapy chemoradiotherapy
chemotherapy
EXCLUDE M1
Surgery only if
local
recurrence or
tumour persistence Surgery Surgery Surgery
Fig. 35.11 Suggested algorithm for management of localized esophageal cancer
Multiple studies have demonstrated the effectiveness of suppression after ablation. A repeated endoscopy should
RFA for eradicating Barrett esophagus and dysplasia. In be performed 3 months after ablative therapy, prefer-
a multicenter European trial,136 patients were randomly ably with high-resolution endoscopy and some form of
assigned to RFA versus surveillance, 1.5% of patients treated chromoendoscopy. Many patients will require more than
with ablation progressed to cancer versus 8.8% in the sur- one ablation session to eradicate all Barrett’s esophagus.
veillance arm [92]. RFA was able to wipe out dysplasia in There is also a small risk that areas of Barrett epithelium
93% of patients. could be hidden beneath areas of the new squamous epi-
thelium, known as buried glands. Malignancy can arise
35.4.7.2 Cryotherapy within these buried glands, and these cancers may be more
Cryotherapy is an alternative ablative technique that uses difficult to identify during endoscopy. The clinical signifi-
extreme cold instead of heat to destroy tissue. There have been cance of this phenomenon is unknown, and the incidence
no head-to-head comparisons between cryotherapy and RFA, of malignancy developing within these areas of buried
but reports indicate similar efficacy to RFA [93]. Cryotherapy glands appears to be very low [93]. Nevertheless, eradica-
is generally well tolerated with little pain and low stricture tion justifies future surveillance of ablated patients. After
rates. One advantage of cryotherapy compared with RFA is eradication of Barrett’s esophagus, fundoplication may
that cryotherapy does not require a probe to be in contact with also be considered for the treatment of reflux, although
the tissue. However, a decompression tube is required to pre- studies have not conclusively demonstrated the effective-
vent overdistention of the stomach and intestine with gas. ness of antireflux surgery for the prevention of esopha-
Regardless of what ablation technology is used, geal cancer the potential implications of unrecognized
patients should have close surveillance and long-term acid incomplete.
530 G. Liu et al.
35.4.7.3 Endoscopic Mucosal Resection administered doses of 20–40 Gy before resection aim to
One limitation of ablative therapies is the limited depth of reduce local recurrence and to raise survival rates. With one
penetration. Another disadvantage is the lack of definitive exception, all of these trials included patients with SCC only,
pathologic analysis. Therefore, patients with nodular or and none of the trials demonstrated significant benefits of
raised Barrett esophagus or other abnormalities suggestive of adding radiation therapy to resection.
superficial invasive cancer should undergo EMR rather than Although the lower radiation doses (20–40 Gy) may have
ablation. EMR provides larger samples to accurately deter- been inadequate, clinicians were wary of combining higher
mine the depth of invasion. EMR resects the full thickness dose radiation before surgery, given the toxicity risks (Note
of the mucosa, down into the submucosa. Therefore, it is an that radiation delivery and particles used in therapy were
excellent therapeutic option for superficial lesions with a low very different in the past compared with current therapy)
risk of nodal metastases. However, high rates of locoregional recurrence after sur-
gery led to the consideration of adjuvant radiation therapy
35.4.7.4 Esophagectomy for esophageal cancer. The rationale for this approach was
The role of esophagectomy as a single modality treatment the ability to deliver higher doses (40–60 Gy) of radiation
for esophageal cancer is diminishing. Most tumors are found postoperatively without worsening perioperative complica-
after symptoms develop, at which point they are usually tions. Postoperative radiation therapy for esophageal can-
locally advanced or metastatic. Locally advanced tumors cer appeared to be potentially beneficial in several trials,
should be treated with multimodality therapy. Asymptomatic although the data are conflicting and subject to selection bias.
tumors are usually found during surveillance for the Barrett’s
esophagus. These are typically superficial and can be treated 35.4.8.2 Chemotherapy
with EMR with lower complication rates than with esopha- The cause of death from esophageal cancer is mainly due
gectomy. This leaves a relatively narrow subset of tumors to metastatic disease. Intuitively, systemic chemotherapy has
that are treated appropriately with surgery only. As discussed the potential to target micrometastatic deposits. Even in the
earlier, T1b tumors have a significant risk for nodal metas- setting of a seemingly localized disease, it usually down-
tasis and in general should be treated with esophagectomy. stages marginally resectable tumors, allowing improved
High-risk T1a lesions (larger tumors or lesions with lym- complete (R0) resection rates, and decreases the incidence
phovascular invasion) could also be considered for esopha- of locoregional recurrence [95]. The synergistic effect of
gectomy. Extensive, multifocal lesions and ulcerated tumors chemotherapy with radiation strengthens the argument for
may also be difficult to eradicate endoscopically and would its use. Importantly, when it is administered preoperatively,
be appropriate candidates for esophagectomy. the biologic response can be evaluated and quantified patho-
An area of controversy is the best treatment for clinical logically in terms of pathologic tumor histoviability, and the
T2N0 tumors. Esophagectomy with an adequate lymphad- degree of this response has been correlated as an indicator of
enectomy would be expected to lead to an overall 5-year outcome. Current chemotherapeutic regimens are based on
survival of anywhere between 40 and 65% for a pathologic platinum compounds (cisplatin and carboplatin) in combina-
T2N0 cancer, depending on the histology, grade, and location with 5-fluorouracil or taxanes as a doublet.
tion of tumor [94]. Unfortunately, a clinical stage of T2N0 is
not accurate in the majority of cases, and many patients are 35.4.8.3 Chemoradiation Alone
found to have node-positive disease on final pathology after Chemoradiation may be administered in a preoperative or
esophagectomy. postoperative setting, as definitive bimodality therapy, or
as part of trimodality therapy when combined with surgery.
Concomitant administration of chemotherapy and radiation
35.4.8 T
reatment Modalities Used in Locally has a synergistic effect with increased tumor cytotoxicity at
Advanced Esophageal Cancer low doses. The validity of chemoradiation use for all loca-
tions of esophageal cancer is based on encouraging results
35.4.8.1 Radiation Therapy of definitive chemoradiation for cervical esophageal SCC.
Radiation was employed as the first treatment modality for
esophageal cancer. Early experiences with radium bougies 35.4.8.4 Chemoradiation and Surgery
and external beam radiation demonstrated esophageal tumor When used alone, each cancer treatment modality has its
regression with occasional complete tumor responses. With limitations, ranging from inadequate therapeutic effect to
the evolution of surgical care, radiation became a part of a excessive toxicity. The synergistic effect of chemoradiation
multidisciplinary approach to esophageal cancer therapy combined with surgical resection maximizes the chances of
with the goal of sterilizing areas within or around the opera- effectively treating both locoregional disease and potential
tive field. Early randomized trials of neoadjuvant radiation undetectable metastases.
35.4.8.5 Surveillance 35.4.9.2 Targeting the HER2 Signaling Pathway

Patients who have received definitive chemoradiation ther- Amplification and overexpression of HER-2/neu (c-erbB-2)
apy (bimodality therapy) for esophageal cancer continue to in esophageal cancer have also been considered to be fac-
worry that the disease may happen again either as locore- tors of poor prognosis. But, in fact, there is few relevant data
gional or distant metastatic recurrence. The purpose behind about HER-2/neu amplification in esophageal cancer and its
the periodic surveillance of patients who completed defini- implications for clinical treatment.
tive bimodality therapy is to potentially implement salvage
therapy for locoregional failure. Evidence-based surveil- 35.4.9.3 Angiogenesis Inhibitors
lance algorithms are not available; however, most provid- Agents that inhibit vascular endothelial cell growth factor
ers conduct clinical examination and a variety of imaging (VEGF) and the angiogenesis process are also associated
or endoscopy studies on patients every 3–6 months. This with the treatment of many types of cancers. VEGF is over-
method is often expensive and anxiety provoking for expressed in 30–60% of patients with esophageal cancers.
patients, and it may not change the final outcome for the Bevacizumab is the most widely studied anti-angiogenesis
patient. Considering the fact that more than 98% of local agent, which is still in the clinical evaluation stage for esoph-
recurrences occur in the first 36 months, most authors sug- ageal cancer and this approach could be an important supple-
gest vigilant surveillance during this time after bimodality ment to the treatment of this cancer.
therapy to potentially catch recurrences as soon as possible
to render salvage surgery a feasible strategy. 35.4.9.4 Others
In the past decade, more and more attention has been paid
35.4.8.6 P alliative Options for Esophageal to the metabolic pathway of cancer as the target of cancer
Carcinoma treatment. Inhibitors of fatty acid synthase are probably to be
Patients with poor performance or distant metastatic disease investigated at a clinical level soon. But so far studies have
at the time of diagnosis are not suitable for aggressive locore- shown that this metabolic pathway could provide a new tar-
gional therapy. The goal of treatment in these circumstances get for the treatment of esophageal cancer.
is either to palliate existing symptoms or potentially to avoid
future complications related to the disease extent. Metastatic
esophageal carcinoma may be manifested with a variety of 35.4.10 Typical Medical Case
symptoms, depending on the disease spread; however, dys-
phagia, odynophagia, chest pain, fatigue, and weight loss are Clinical Background A 51-year-old male patient was
likely to be among the most common symptoms. Palliative admitted to the hospital due to a recent exacerbation of dull
treatment is always individualized according to the patient’s pain in the sternum after eating for more than half a year.
physiologic status, symptoms, disease degree, and wishes.
Options for palliation range from best supportive care for EUS Esophageal lesions.
symptom control to the use of chemotherapy or radiation,
esophageal stent placement, and enteral nutrition support. Tumor Marker CY21-1 1.09 ng/mL (<3.3 ng/mL), CEA
With advancements in image-guided percutaneous and 2.28 ng/mL (<4.7 ng/mL), NSE 12.34 ng/mL (<16.3 ng/
endoscopic procedures, surgical procedures for palliation of mL), SP70 9.1 ng/mL (≤7.5 ng/mL).
esophageal carcinoma have become exceedingly rare.
Histopathological Examination High-grade intraepithe-
lial carcinogenesis of esophageal squamous epithelium with
35.4.9 Molecules of Companion Diagnosis squamous cell carcinoma, grade II, see in Fig. 35.12. SP70
expression in tissues of esophageal cancer and benign esoph-
35.4.9.1 Targeting the EGFR Signaling Pathway ageal tumor was described in Fig. 35.13.
Epidermal growth factor receptor (EGFR) is one of the
most commonly altered genes in human cancer, and its Treatment Radical resection of esophageal cancer.
changes include overexpression, amplification, and muta-
tion. Targeted inhibition of EGFR activity inhibits signal Results with Interpretation Guideline
transduction pathways, consequently affecting tumor cell Esophageal cancer has a high degree of malignancy, and its
proliferation and resistance to apoptosis. The most common early symptoms are not typical, which leads to the difficulty
EGFR-targeting agents include small-molecule tyrosine of early diagnosis. Most of the patients in clinical treatment
kinase inhibitors and monoclonal antibodies, which have are in the middle and late stages, with a very poor prognosis.
been used clinically for treating various malignancies with With the improvement of esophageal surgery technology and
EGFR mutations or abnormal expression of the receptor. the innovation of radiotherapy and chemotherapy technol-
532 G. Liu et al.
ogy, the resection rate of esophageal cancer and the survival 1,000,000 new cases and an estimated 783,000 deaths [96].
rate of patients have been improved. Although the incidence rate has decreased in recent years, the
This case was from the First Affiliated Hospital of Nanjing survival rate of GC patients is still not optimistic. The devel-
Medical University (also named Jiangsu Province Hospital). opment of GC involves a multistep process with highly het-
erogeneous and it has been shown a higher risk of recurrence
and metastasis in advanced GC patients. In recent years, multi-
35.5 Gastric Cancer modal strategies including the neoadjuvant and/or adjuvant
treatments with surgery have been recommended to improve
Yi Zhang the prognosis of GC. The researches focus on the molecu-
lar markers for early diagnosis and individualized treatment
might be infancy and effective way to improve the survival
35.5.1 Overview rate of patients. This section will discuss the recent progress on
GC-related molecular markers based on the risk factors for GC,
Gastric cancer (GC) is the fifth most frequently diagnosed which might provide a theoretical basis for the clinical applica-
cancer and third in terms of mortality worldwide, with over tion and individualized treatment according to GC guidelines.
Most patients with early GC have no noticeable symptoms.

A few people may feel only nonspecific symptoms such
as nausea, vomiting, or some upper gastrointestinal symp-
toms. The more obvious symptoms (including pain, weight
loss, abdominal discomfort, anemia, malnutrition, and even
cachexia) usually occur with the advanced stage of GC
patients. There are also special manifestations depending on
the location of the tumor. Cardiac cancer may present pro-
gressively dysphagia and reflux symptoms, while antrum
cancer may cause pyloric obstruction. When the tumor
destroys the blood vessels, there may be symptoms of gastro-
intestinal bleeding such as hematemesis and melena. Some
patients may present symptoms such as back pain or malig-
Fig. 35.12 The histopathology of esophageal cancer nant ascites because of metastatic spread.
a b
Fig. 35.13 SP70 expression in tissues of Esophageal cancer and benign esophageal tumor. (a) SP70 positive expression (b) SP70 negative expres-
sion (Immunohistochemistry staining, ×200)
35.5.3 Risk Factors for Gastric Cancer Table 35.5 The molecular classification of GC proposed by TCGA
Organization subtype Gene mutation
GC is a multifactorial disease associated with complex envi- TCGA Tumors positive PI3KCA mutations, DNA
ronmental and genetic factors. Approximately 90% of GC for Epstein-Barr hypermethylation, JAK2,
virus PD-L1, PD-L2,
are adenocarcinomas, which arise from the glands of the EBV-CIMP;CDKN2A
mucosa or the most superficial layer of the stomach. Several Microsatellite Mutations of genes encoding
risk factors for GC have been identified including older age, unstable tumors targetable oncogenic signaling
male gender, smoking history, Helicobacter pylori infec- proteins
tion, atrophic gastritis, and family history. However, it is Genomically RHOA, CDH1,CLDN18-
stable tumors ARHGAP, RHO-family
supposed to have some different epidemiologic patterns and GTPase-activating proteins
causes between cardia (in the area adjoining the esophageal- Tumors with TP53, RTK-RAS, receptor
gastric junction) and noncardia (arising from more distal chromosomal tyrosine kinases and marked
regions) regions of the stomach. For example, on one hand, instability aneuploidy
the risk factors associated with cardia GC might include
obesity and patients with a history of gastroesophageal
reflux disease or Barrett’s esophagus. On the other hand, 35.5.5 Laboratory Diagnosis
noncardia GC usually occurs on a background of H. pylori
infection, and perhaps of dietary factors such as high intake 35.5.5.1 P rinciples of Biomarker Testing by
of salty and smoked food. Besides, the high risk of genetic NCCN Guidelines for GC
factors includes the history of hereditary tumor syndromes The identification and evaluation of biomarkers for GC have
such as Lynch Syndrome, hereditary diffuse-type gastric been developed quickly in recent years. Nowadays, molecu-
cancer syndrome, Peutz–Jeghers Syndrome, and Juvenile lar biomarkers are emerging research hotspots in the field
Polyposis Syndrome, which might be prevented by detect- of malignant tumor research, and have important clinical
ing germline mutation. However, only 1–3% of GC cases significance for early diagnosis, prognosis monitoring, and
arise as a result of inherited syndromes mentioned above. especially for the evaluation of treatment responses. NCCN
Nowadays, more investigations of genetic risk factors are guidelines for GC have already been recommended to assess
still working on aiming to discover more valuable high- HER2 detection to guide trastuzumab therapy. In 2018, the
penetrance genes. section of “Principles of Pathologic Review and Biomarker
Testing” was extensively revised and updated new recommen-
dations with MSI and PD-L1 testing for advanced GC [97].
35.5.4 Historical Classification of GC Following the diagnosis of GC relying on histopathol-
ogy, it is necessary to carry out related molecular tests and
Currently, the historical classification and clinical staging guide the treatment according to molecular typing. The
are the most valuable predictive and prognostic factors for positive rate of HER2 in Chinese patients with GC ranging
GC. Several classification systems have been developed. from 12 to 13%. For early GC, the studies showed that the
The first one is Lauren’s classification system (1965), which outcome in survival rates of HER2-negative intestinal-type
divides GC into two major groups based on the intestinal GC patients was the best, but that the rates of HER2-positive
type and diffuse or poorly differentiated type. Another sys- diffuse GC patients were the worst. The patients with HER2-
tem is proposed by the World Health Organization (WHO) positive advanced GC can benefit from trastuzumab treat-
and divides GC into four categories including tubular, pap- ment. High microsatellite instability (MSI-H) ratio varies
illary, mucinous, and discohesive subtypes. Recently, the from 11 to 33%. In MSI-H GC, patients with preoperative
rapid development of molecular pathology also promotes chemotherapy plus surgery had a poor prognosis compared
novel classifiers that can be used as the guidelines for the with patients with surgery alone, suggesting that MSI/MMR
therapy. The Cancer Genome Atlas (TCGA) project pro- status testing may be helpful to identify GC patients who
posed a molecular classification of GC into four subtypes: might require preoperative chemotherapy. In addition, the
tumors positive for Epstein-Barr virus, microsatellite unsta- updated guideline for hereditary diffuse gastric cancer sug-
ble tumors, genomically stable tumors, and tumors with gests that the CDH1 mutation of patients whose ages were
chromosomal instability. Importantly, the novel molecular between 18 and 40 should be tested before prophylactic total
classification could be employed as the guidelines for clini- gastrectomy.
cal targeted therapy and be evaluated in the clinic by exist-
ing testing including MSI and emerging genomic assays for 35.5.5.2 Traditional Biomarkers of GC
predictive gene mutations and amplifications. The detailed Nowadays, the biomarkers for GC which are most frequently
classification is shown in Table 35.5. used in clinic include CEA, CA72-4, CA19-9, pepsinogen
534 G. Liu et al.
I/II (PG I/II), and Gastrin. However, the low sensitivity for 60
P<0.05
early gastric cancer detection made these markers not be
used for screening and early diagnosis of gastric cancer. In
recent years, studies have suggested that pepsinogen (PG)I/II P<0.05
40
can be used as an indicator for gastric cancer. Pepsinogen is
SP70(ng/ml)
a pepsin precursor secreted by the gastric mucosa. It belongs
to the aspartic protease family and is mainly synthesized by
gastric main cells and cervical mucus cells. It can be divided 20
into two subgroups: PGI (secreted by the main cells of the
fundus gland) and PG II (secreted by the fundic glands, car-
diac glands, pyloric glands, and Brunner glands). The model
for the intestinal type of GC is preceded by several precan- 0
cerous stages including superficial/atrophic gastritis, small
C
D
H
G
BG
intestinal metaplasia colonic metaplasia, and dysplasia. The
ratio between PG I and PG II can reflect the functional sta- Fig. 35.14 Serum levels of SP70 (ng/mL)
tus of gastric mucosa and is significantly correlated with the
extent and severity of gastric mucosal atrophy. When gastric
1.0
mucosa occurs, the serum pepsinogen content also changes. 7
Studies have shown that PG I/PG II ratio is significantly
reduced in GC patients, and a lower PGI/II ratio in patients
might predict a higher risk of GC, which can be used as a
Sensitivity
continuous marker of GC. SP70 AUC=0.722

0.5
CEA AUC=0.649
35.5.5.3 Molecular Biomarkers of GC CA724 AUC=0.499
CA199 AUC=0.539
Tumor Specific Protein 70 (SP70)
0.0
SP70, a specific antigen recognized by monoclonal antibody 0.0 0.5 1.0
NJ001, is a marker of non-small cell lung cancer. Previous 1-specificity
studies have shown that the expression of SP70 is closely
related to the clinical pathological features and prognosis of Fig. 35.15 ROC curve analyses of SP70, CEA, CA72-4, CA19-9
lung cancer. It is found that SP70 is also abnormal in gas-
tric cancer patients. The double antibody sandwich ELISA HER2/neu
method was used to detect SP70 in the serum of 70 patients Human epidermal growth factor receptor 2 (HER2 or HER2/
with gastric cancer, 50 patients with benign gastric disease, neu), a member of the human epidermal growth factor recep-
and 66 healthy persons from the First Affiliated Hospital tor (EGFR) family, is a proto-oncogene encoded by ERBB2
of Nanjing Medical University from October 2017 to July gene locus on chromosome 17. It has been demonstrated that
2018. The distribution of serum SP70 in gastric cancer(GC) HER2 was predominantly overexpressed in more than 30%
group was significantly higher than that of patients in gas- intestinal-type GC. Therefore, HER2 testing is of great sig-
tric benign disease (GBD) and healthy control (HC) group nificance in intestinal-type adenocarcinoma treatment. It is
[8.0 (6.8, 10.6) and 6.5 (5.5, 7.5), P < 0.001; 8.0 (6.8, 10, 6) now approved to be used to monitor and determine the GC
and 7.0 (6.0, 7.8), P < 0.001], but there was no significant patients with recurrence or metastasis and gastroesophageal
difference between patients with gastric benign lesion and junctional adenocarcinoma to evaluate HER overexpression
healthy physical examination group [6.5 (5.5, 7.5) and 7.0 levels. The expression level of HER2 was detected using
(6.0, 7.8), (P > 0.05)] (Fig. 35.14).At the same time, SP70 immunohistochemistry (IHC), fluorescence in situ hybrid-
was compared with CEA, CA72-4, and CA19-9, which were ization (FISH), and other methods. The evaluation criteria
detected by electrochemiluminescence. The AUC of SP70, of HER2 expression is recommended to use Trastuzumab
CEA, CA72-4, and CA19-9 in diagnosing gastric cancer was for Gastric Cancer (ToGA) standard. The NCCN guide-
0.722, 0.649, 0.499, and 0.539, respectively. The diagnostic lines recommend FISH for IHC detection with HER2 (score
efficiency of SP70 was better than that of CEA, CA72-4, and 2+). The HER2 overexpression detected by IHC (score 3+)
CA19-9 (P < 0.05) (Fig. 35.15). is positive. Recent studies have shown that serum levels of
HER2 extracellular domain are correlated with the tissue Circulating Nucleic Acids
levels of HER2 in metastatic GC, which might be a poten- During recent years, circulating nucleic acids, such as long
tial alternative assay in future. The NCCN guidelines also noncoding RNAs (lncRNAs) which consisted of more than
suggest that trastuzumab can be added to the chemotherapy 200 nucleotides, have shown potential applications in the
regimen for metastatic adenocarcinoma patients with HER2 molecular diagnosis of GC. Exosomes can participate in
overexpression. cellular communication and have been indicated to become
potential candidates for non-invasive diagnosis. Our recent
MSI study investigated the existence of the HOXA transcript at
Microsatellite instability (MSI)usually refers to DNA meth- the distal tip (HOTTIP) in the circulating exosomes and the
ylation or gene mutation mismatch repair (MMR) gene dele- potential roles in GC [100].
tion, resulting in changes in DNA sequences with the length Serum exosomal HOTTIP were detected from 246
from 1 to 6 or even more base pairs, which occur at thou- subjects (126 GC patients and 120 healthy people)
sands of locations within the genome. MMR consists of a by RT-qPCR. The results demonstrated that exosomal
series of enzymes that specifically repair DNA mismatches, HOTTIP was significantly upregulated in GC patients
by removing errors generated during DNA replication, main- compared with normal control (Fig. 35.16 and showed a
taining genome stability, avoiding mutations, and indirectly demonstrated a higher diagnostic capability with the AUC
inhibiting tumorigenesis. In most intestinal-type GC patients, for exosomal HOTTIP (0.827) than CEA, CA 19-9 and
it is frequently seen a high-level of microsatellite instabil- CA72-4 (AUC = 0.653, 0.685 and 0.639, respectively)
ity (MSI-H) and less frequent local lymph node metastasis (Fig. 35.17). The Kaplan–Meier analysis showed that there
which might due to the hypermethylation of the promoter is a correlation between upregulated levels of exosomal
regions of mismatch repair genes (MLH1 and MSH2). Some HOTTIP and poorer overall survival (log rank P < 0.001).
studies have shown that the patients might have a better And the univariate and multivariate COX analysis indi-
prognosis with MSI-high tumors in comparison to MSI-low cated that the overexpression of exosomal HOTTIP could
tumors with different capacity in tumor invasion and lymph be an independent prognostic factor for GC (Fig. 35.18).
node metastases. The MAGIC trial study has shown that The above findings revealed that exosomal HOTTIP might
patients with operable GC cancer with MSI-H have superior be a potential biomarker for the diagnosis and prognosis
survival rates compared with patients with MSI-L or micro- of GC.
satellite stable tumors when treated with surgery alone. It has
been recommended that MSI phenotype should be system-
atically defined because of its potential role in perioperative 8
treatment [98]. In 2017, the FDA approved MSI-H/dMMR
as the biomarker for all solid tumors using Pembrolizumab.
Relative levels of exosomal HOTTIP
This epoch-making approval also demonstrates the effective-

ness of MSI-H/dMMR as a biomarker for immunotherapy. 6
PD-L1
Recently, with the development of immunology, more stud- 4
ies have focused on anti-tumor immunity therapy and the
immune checkpoint inhibitor with programmed cell death 1
(PD-1) is also adapted generally in GC patients [99]. PD-1 is
a CD28 family member receptor, while PD-L1(B7-H1) is a 2
B7-family member which regulates T cell functions through
PD-1. It has been reported that GC patients with MSI or
EB-positive subtype and positive lymph node metastasis are
0
more likely to express PD-L1. In addition, recent clinical tri-
Gastric Cancer Healthy Control
als have indicated that targeting PD-L1/PD-1 pathway, such
as Pembrolizumab, has proved to be a novel target for the Fig. 35.16 The levels of exosomal HOTTIP were upregulated in GC
treatment of GC patients. serum
536 G. Liu et al.
a 100
b 100
c 100
80 80 80
Sensitivity
Sensitivity
Sensitivity
60 60 60
40 40 40
HOTTIP CEA CA 19-9

20 20 20
0 0 0
0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
100-Specificity 100-Specificity 100-Specificity
d e f
100 100 100
80 80 80
Sensitivity
Sensitivity
Sensitivity
60 60 60
40 40 40
CA 72-4 CEA+CA 19-9+CA 72-4 HOTTIP+CEA+

20 20 20 CA 19-9+CA 72-4
0 0 0
0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100
100-Specificity 100-Specificity 100-Specificity
Fig. 35.17 The ROC curves of biomarkers including (a) HOTTIP, (b) CEA, (c) CA19-9, (d) CA72-4 and (e, f) the combination of them
The Other Genetic Susceptibility Genes the ERBB2 gene, mutations, and all target mutations related
The development of GC is a multistep process with genetic to the efficacy of Herceptin in the upstream and downstream,
alterations including p53, E-cadherin gene (CDH1), caudal- and comprehensively guide Herceptin targeted therapy for
type homeobox transcription factor 2 (CDX2), MET, SMAD, GC. The potential molecular biomarkers for gastric cancer
ERBB2, and so on. The inactivation mutation of the p53 were shown in Fig. 35.19.
occurs in early GC patients ranging from 38 to 71%, and
most has been identified to occur in the intestinal type of
GC. The pathological expression of p53 may be associated 35.5.6 Typical Medical Case
with lymph node metastasis and chemosensitivity, which
can also predict a decreased cumulative survival. However, Clinical Background A 88-year-old male patient came to
CDH1 mutations are usually present in diffuse-type GC and the hospital for pain in the left upper abdominal for 2 weeks,
the detection of CDH1 mutation may be helpful in the identi- especially after eating hard food. One month ago, the patient
fication of asymptomatic mutations carriers with GC family began to have black stool without obvious inducement.
syndrome. The combination of CDH1 and CDX2 detection
might be used as prognostic factors for GC patients. The Gastroscopy Irregular ulcers were found on the small
frequency of MET amplification in gastric cancer is about curved side, with white moss on the surface, congestion and
5% and dynamic monitoring of ctDNA suggests therapeu- edema of the surrounding mucosa, 44 cm from the upper
tic efficacy and explores relevant resistance mechanisms. In edge of the lesion to the incisor teeth, the lesion extended to
addition, approximately 21% of the SMAD gene mutation the gastric angle, and the biopsy was fragile and easy to
carriers of JPS patients occur in GC patients. The NCCN bleed.
guidelines clearly state that GC patients need to be tested for
ERBB2 gene amplification to guide the use of Herceptin for Enhanced CT The mucosa of the lesser curvature of the
targeted therapy. NGS detection can detect the mutations of stomach was thickened with enhancement (Fig. 35.20).
a b c
1.0 1.0 1.0
0.8 0.8 0.8

Overall survival rate

0.6 0.6 0.6
0.4 0.4 0.4
0.2 0.2 0.2

Low CEA in serum Low CA 19-9 in serum Low CA 72-4 in serum
0.0 High CEA in serum 0.0 High CA 19-9 in serum 0.0 High CA 72-4 in serum
0 20 40 60 0 20 40 60 0 20 40 60
Survival time (months) Survival time (months) Survival time (months)
d e
1.0 1.0
0.8 0.8
0.6 0.6
0.4 *** 0.4
0.2 0.2 ***

Low exosomal HOTTIP in tissue Low HOTTIP in tissue
0.0 High exosomal HOTTIP in tissue 0.0 High HOTTIP in tissue
0 20 40 60 0 50 100 150
Survival time (months) Survival time (months)
Fig. 35.18 Association between tumor markers including (a) CEA, (b) CA19-9, (c) CA72-4, (d, e) HOTTIP and overall survival in GC
Fig. 35.19 Overview of Biomarkers for grastic cancer

molecular biomarkers for GC
DOWNREGULATION UPREGULATION
Protein coding genes Protein coding genes
RBMS3, S100A12, CDK5RAP3 FAM46C, Apelin, AGR2, CISD2, Trop2, PTTG3P,

GPR155, Tryptase, PROX1, FOXP3, SASH1, FPX050, SYT8, PRMT5, CCL2, MAGED2,
SAMSN1, Barx2, BUB1, SPRY2, BAK, PRMT1, NRAGE, KLHL6, SCIN, MET4, TREM2,
MGMT ANOS1, HOXA1, NRP-1, PCDHB9,
ADAR1, FGF9, CYR61, MMP16, REG4,
Methylated genes KIAA1199, TPX2, STMN 1, ATM, GGT
FAT4, RNF180, MZB1, MFSD4, GFRA3,
KCNMA1, NDRG4, NR4A3, MAP1LC3Av1 microRNAs
microRNAs MiR-23b, MiR-125b, MiR-196a/b, MiR-215,

MiR-BART20-5p
MiR-101, MiR-148a, MiR-195, MiR-203,
MiR-204, MiR-206, MiR-383, MiR-490-3p, Long noncording RNAs
MiR-503, MiR-647,MiR-933
PVTl, UCA1, CARLo-S, XIST,
Long noncording RNAs
KRT18P55, MALATl, LINC00673,
LINC00675, LOC100130476 DANCR, NEAT2
538 G. Liu et al.
Histopathological Examination Grade II, ulcerative type,

TMN stage: T3N1M0.
Recover The serum SP70 of patients dropped to 6.2 ng/mL

7 days after the operation.
This case was from the First Affiliated Hospital of Nanjing

Medical University (also named Jiangsu Province Hospital)
35.5.7 Conclusion
Molecular markers are emerging research hotspots in the field

of malignant tumor research, and have played important roles
in early diagnosis, prognosis monitoring, and targeted therapy
of tumors in the clinic. With the development of NGS tech-
niques, more cancer-related genes including oncogenes, tumor
suppressor genes, miRNAs, and DNA methylation have been
identified in recent years. However, the qualification standards
for the testing should be well-established in the future.
Fig. 35.20 CT scanning
35.6 Colorectal Cancer
Shiyang Pan and Xiang Qian
35.6.1 Overview
The colon and rectum form part of the digestive system and
are often called the large bowel. The large bowel is about
150 cm in length and is defined as a portion of the intestine
from the ileocecal valve to the anus. According to its vas-
cular supply and extraperitoneal or retroperitoneal location,
it is divided into five segments: cecum (with appendix) and
ascending colon, transverse colon, descending colon, sig-
moid colon, and rectum (Fig. 35.24). The colon, arranged
like the letter M, surrounds the small intestine.
Fig. 35.21 Tissue biopsy (H&E staining)
The rectum is the last part of the intestine and is con-
nected to the sigmoid colon above and the anus below. Rectal
Tissue Biopsy There is a small focus of atypical hyperpla- anatomy is usually divided into three parts (Fig. 35.25). The
sia of glandular epithelium in the lesser curvature of the lower rectum is an area of about 3–6 cm around the edge of
stomach. (H&E staining) (Fig. 35.21), (SP 70 3+) the anus. The middle rectum extends from 5–6, to 8–10 cm,
(Fig. 35.22), (SP 70−) (Fig. 35.23). and the upper rectum goes approximately from 8–10, to
12–15 cm from the anal verge, although the retroperitoneal
Laboratory Test WBC 3.1 × 109/L (3.50–9.50 × 109/L), portion of the large bowel often reaches the upper limit about
RBC 2.87 × 1012/L (4.30–5.80 × 1012/L), HGB 72 g/L (130– 12 cm from the anal verge. The determination of the location
175 g/L), AFP 4.00 ng/mL (<20 ng/mL), CEA 1.15 ng/mL of the boundary between the rectum and sigmoid colon is of
(<4.7 ng/mL), CA199 0.84 U/mL (<39.00 U/mL), SP70 great significance in defining adjuvant therapy.
9.8 ng/mL (<7.5 ng/mL), Fecal occult blood: Weak positive. The main function of the large bowel is to absorb water,
electrolytes, and other substances from feces, and to form,
Treatment Radical distal gastrectomy + Roux-en-Y store, and excrete feces. The large bowel also has secret-
gastrojejunostomy. ing functions, such as secreting mucus to protect mucous
Fig. 35.22 Tissue biopsy (SP70 immunobiological staining)
Fig. 35.23 Tissue biopsy (SP70 immunobiological staining)
membranes and lubricate feces, making it easier for feces to 35.6.2 Epidemiology
descend and protecting the intestinal wall from mechanical
damage and bacteria. CRC is one of the most prevalent gastrointestinal malig-
Disorders of the large bowel include acute or chronic nant types in the world. In 2018, it was estimated that over
inflammatory bowel disease, colorectal polyps, colorectal 1.8 million people were diagnosed with CRC and 881,000
adenoma, colorectal cancer, colorectal carcinoid (neuroen- people died from this disease worldwide [101]. According to
docrine tumor), and other diseases. the date of the International Agency for Research on Cancer
540 G. Liu et al.
Fig. 35.24 The anatomy of

the colon
Fig. 35.25 The anatomy of Portion of rectum (cm from angle verge) left upper valve of Houston
the rectum
peritoneum
15
upper third
Ampulla of right middle valve of Houston

rectum
11
middle third
left lower valve of Houston
7
lower third
in 2012, CRC is the third most common type of cancer and entiated carcinoma, other carcinoma, and indeterminate car-
the fourth leading cause of cancer-related death in the world. cinoma. Over 98% of CRC are adenocarcinomas.
For both incidence and mortality, the rates of CRC were CRC has the highest recurrence rate within the first 5 years
much higher in males than in females, and in rural areas after surgical treatment. While for stage 1 tumor, 5 years of
than in urban areas. The rate of CRC increased greatly with survival rate is more than 90%, is the best cure rate type.
age, especially after 40 or 45 years old [102]. Most of the Therefore, the key to CRC treatment lies in early detection
patients were in the advanced stage. The histologic types and early diagnosis. At present, in addition to the endoscopic
of CRC include adenocarcinoma, mucinous adenocarci- examination, the application of tumor markers has become
noma, signet ring cell carcinoma, squamous cell carcinoma, an important means of early screening of tumors, among
adenosquamous carcinoma, medullary carcinoma, undiffer- which CEA and CA199 are serum markers of digestive tract
tumors that are widely used in clinical practice, but their low nonpolyposis CRC (HNPCC). The latter is mainly a genetic
specificity cannot meet the needs of early clinical diagnosis mutation caused by environmental factors.
of CRC.
35.6.3.3 Other Risk Factors
35.6.3 Aetiology Colorectal Polyps (Adenomatous Polyps)

It is generally believed that most CRC originates from ade-
The etiology of CRC has not been fully understood. At pres- noma, so adenomatous polyps are regarded as precancer-
ent, it is mainly due to the combination of environmental fac- ous lesions. The bigger the adenoma, the more irregular the
tors and genetic factors [103]. morphology, the higher the content of villi, the heavier the
epithelial dysplasia, and the greater the chance of cancer.
35.6.3.1 Environmental Factors The sequence evolution process of adenoma carcinoma has
Although the incidence of CRC in China and Japan is sig- been well understood. The occurrence of CRC is the evo-
nificantly lower than that in the United States, the incidence lution process of normal intestinal epithelial-hyperplasia
of CRC in the first generation that immigrated to the United change/small adenoma-early adenoma-middle adenoma-
States has increased, and the incidence of CRC in the second late adenoma-carcinoma-metastasis. The changes of onco-
generation is close to that in the United States. This epide- genes and tumor suppressors that accompany the different
miological characteristic of migration suggests that the inci- stages of this evolution process have been relatively clear,
dence of CRC is closely related to environmental factors, and the accumulation of multiple mutations of oncogenes
especially dietary factors. High-fat diets and insufficient and tumor suppressors is seen as the molecular biological
dietary fiber are generally considered to be the main factors, basis for the development of CRC. Gene mutation is the
which have been demonstrated in a number of epidemiologi- result of the combination of environmental and genetic
cal and animal studies (Table 35.6). factors.
35.6.3.2 Genetic Factors Inflammatory Bowel Disease

From a genetic standpoint, CRC can be classified as either Ulcerative colitis can produce cancerous change, more at
hereditary (familial) or non-genetic (sporadic). Examples of the young onset, the pathological range is wide and disease
the former include hereditary polyp syndrome and hereditary course elder.
Table 35.6 Influence factors for colorectal cancer Cholecystectomy

There are reports of increased incidence of CRC after chole-
Increased or decreased
Influence factors incidence cystectomy, which is believed to be related to the increase of
Sociodemographic factors secondary cholic acid entering the large intestine.
Older age ↑↑↑
Male sex ↑↑
Medical factors 35.6.4 Screening
Family history ↑↑
Inflammatory bowel disease ↑↑ Screening contributes to the early detection, diagnosis, and
Diabetes ↑ treatment of CRC, and is key to preventing CRC and reduc-
Large bowel endoscopy ↓↓ ing its cumulative mortality. In recent years, the mortality
Hormone replacement therapy ↓ of CRC in western developed countries has been declining,
Aspirin ↓ which is due to the early detection and treatment of early
Lifestyle factors
CRC and its precancerous lesions through screening [104].
Smoking ↑
Today, fecal occult blood testing (FOBT), fecal DNA test-
Excessive alcohol consumption ↑
ing, flexible sigmoidoscopy, colonoscopy, and CT colonos-
Obesity ↑
copy, and occasionally barium enema, are all used for CRC
Physical activity ↓
Diet factors
screening.
High consumption of red and ↑
processed meat 35.6.4.1 Screening Object
Fruit and vegetables ↓ It is recommended to establish the general risk group and
Cereal fiber and whole grain ↓ high-risk group on the basis of preliminary screening, and
↑↑↑ = very strong risk increase. ↑↑ = strong risk increase. ↑ = moderate give different screening schemes to improve the cost-benefit
risk increase. ↓↓ = strong risk reduction. ↓ = moderate risk reduction ratio and save a lot of manpower and material resources. The
542 G. Liu et al.
target population of screening includes all people with CRC than 1 cm, and timely detect early genetic lesions of CRC. It
warning symptoms such as blood stool, black stool, anemia, provides a simple and feasible method for early detection,
and weight loss, as well as those aged 50–74 without CRC diagnosis, and treatment of CRC.
warning symptoms.
Endoscopy
35.6.4.2 Screening Test Rectal colonoscopy and sigmoid colonoscopy are suitable
for colorectal lesions with a lower position. Changes in the
Questionnaire Survey Based on High-Risk Factors wall and lumen of the whole large intestine can be directly
It is a simple and economical screening method, and it is observed through colonoscopy, and the location and size of
recommended to help identify high-risk population of CRC the tumor can be determined [105]. And by colonoscopy, the
through questionnaire screening, so as to detect early CRC extent of infiltration can be preliminarily determined too.
and precancerous lesions (Fig. 35.26). Then diagnosis is made upon confirmation by histology.
Sigmoid colonoscopy and fibro colonoscopy can directly
Fecal Occult Blood Test (FOBT) observe the morphology of the whole colon and rectum
It is recommended to screen for early cancer and precancer- mucosa and can take biopsy under direct vision for suspi-
ous lesions with 3 consecutive fecal occult blood tests. Fecal cious lesions, which is of great value to improve the accu-
occult blood test is one of the most widely used screening racy of diagnosis, especially for the early diagnosis of small
methods for CRC and precancerous lesions, with a sensi- lesions. Endoscopy is recommended for all patients with
tivity of 47–87%. The detection methods include chemical suspected CRC, except the following: the general condition
method and immune method, the latter does not need to of the patient is not good enough to tolerate the examina-
restrict the diet, the impact of upper gastrointestinal bleeding tion; acute peritonitis, intestinal perforation, and extensive
is minimal, can quantitatively determine the low concentra- abdominal adhesion; perianal or severe intestinal infections.
tion of hemoglobin in feces, and then significantly improve
the detection rate of early CRC.
35.6.5 Clinical Diagnosis
Fecal DNA Testing
Screening colorectal cancer through fecal DNA testing 35.6.5.1 Clinical Manifestation
has the advantages of non-invasive, good patient compli- The predominant symptoms include abdominal pain, change
ance, high sensitivity, and high specificity. By analyzing the in bowel habits, and hematochezia or melena. Weakness,
genetic material in feces, we can detect progressive adeno- anemia, and weight loss are only seen in a minority of cases.
mas and colorectal cancer lesions with a diameter of more The age of onset of CRC in China is mostly 40–60 years old,
Fig. 35.26 Flow chart for

Medical history and
early CRC screening
physical signs
Physical check ( emphasize

digital rectal examination)
Laboratory examination: Endoscopic: proctoscopy,

Imaging examination: x-ray,
blood routine examination, sigmoidoscopy, fiber/electronic
ultrasound, CT/MRI, PET/CT
FOBT, CEA, CA19-9 colonoscopy + biopsy pathology
Laparotomy may be performed

in suspected cases that cannot
be pathologically confirmed or in
various emergency situations
Definite diagnosis and

staging, different staging with
different treatment options
and the peak of incidence is around 50 years old, but young tion is to clarify the depth of the intestinal wall, the extent
CRC under 30 years old is not uncommon. The median age of the spread to the intestinal wall, and the location of dis-
of onset of CRC in China is about 10 years ahead of that in tant metastasis [107]. Currently, CT examination of CRC is
Europe and the United States, and young CRC is more com- recommended for the following aspects. Firstly, to provide
mon than that in Europe and the United States, which is a staging of colorectal malignant tumors. Secondly, to find
characteristic of CRC in China. the recurrent tumor. Thirdly, to evaluate the response of the
CRC begins with occult disease, often with positive fecal tumor to various treatments. Fourthly, to clarify the internal
occult blood in its early stage, followed by the following structure and properties of barium enema or endoscopic find-
clinical manifestations. ings of the intestinal wall and extrinsic compression lesions.
Fifthly, to evaluate the intraperitoneal mass found by barium
Changes in Bowel Habits and Fecal Traits enema examination, and to clarify the origin of the mass and
Changes in bowel habits and fecal traits are often the earliest its relationship with surrounding organs. Finally, tumor loca-
symptoms of CRC. Take blood more as outstanding perfor- tion can be determined.
mance, or have dysentery kind of purulent blood. Sometimes The indication of MRI is the same as that of CT. MRI
performance is constipation, and defecate shape becomes is recommended as a routine examination for rectal cancer.
thinner. Also can show diarrhea and mushy defecate, or diar- MRI can provide preoperative staging of rectal cancer and
rhea and constipation alternant, fecal quality does not have evaluation of liver metastases of CRC. MRI may be used for
apparent mucous pus blood, see more at right side large suspected peritoneum and subcapsular liver lesions [108].
bowel cancer. Abdominal b-ultrasonography can be used to find out whether
patients have recurrence or metastasis, which is convenient
Abdominal Pain and rapid. Rectal endoscopic ultrasonography is recom-
Abdominal pain is also an early symptom of CRC, most mended for the diagnosis and staging of middle and lower
commonly seen in the right CRC. Presents as a blunt right rectal cancer. PET-CT is not recommended for routine use,
abdominal pain, or involves the right upper abdomen, mid- but can serve as an effective auxiliary examination for patients
dle upper abdomen simultaneously. Pathological change with the complex condition and whose diagnosis cannot be
can make gastric colonic reflex strengthens, so patients can confirmed by routine examination. Preoperative examination
appear abdominal pain after meal. Abdominal pain is aggra- prompt tumors are III period above, to understand the pres-
vated or spastic colic when CRC complicated with intestinal ence of distant metastasis, it is recommended to use.
obstruction.
35.6.5.4 Histopathology
Abdominal Mass Pathological biopsy is the basis of treatment for CRC. Patients
The location of the mass depends on the site of cancer, indi- diagnosed with invasive carcinoma by biopsy were treated
cating that the tumor has reached the middle and late stage. with standardized CRC. For example, due to the limitations
of biopsy sampling, pathological biopsy cannot determine
35.6.5.2 Digital Rectal Examination the depth of infiltration, and a case of high-grade intraepithe-
Digital rectal examination is the simplest and most important lial neoplasia is diagnosed, it is recommended that clinicians
examination method for the diagnosis of rectal cancer. It can integrate other clinical conditions, including the presence
not only find the tumor, but also determine the location, size, of vascularized cancer thrombus and lymphocyte response
shape, surgical method, and prognosis of the mass. Many around cancer, to determine the treatment plan. When con-
patients with rectal cancer often do not check in time and firmed as recurrent or metastatic CRC, it is recommended
be misdiagnosed as hemorrhoid, enteritis, and so on, which to test the Ras gene and other related gene states of tumor
delay treatment for a long time. tissue for further treatment. In conclusion, histopathology is
the gold standard for the diagnosis of CRC.
35.6.5.3 Imaging Examination
X-ray barium enema, CT, and MRI are three imaging diag-
nostic methods commonly used for CRC [106]. They each 35.6.6 Molecular Diagnosis
have advantages. Colon barium enema examination, espe-
cially barium double-contrast examination, is an important 35.6.6.1 Broad-Spectrum Tumor Serum
method for the diagnosis of CRC. Barium double-contrast Markers
examination can reveal filling defects, intestinal stenosis,
mucosal fold damage, and other signs, showing the site and CEA
range of carcinomas. However, patients suspected of hav- CEA is mainly used for colorectal cancer screening, and
ing ileus should choose carefully. The role of CT examina- about 70% of colorectal cancer patients have elevated serum
544 G. Liu et al.
CEA levels. Monitoring CEA level is helpful to observe them are used for the diagnosis of gastrointestinal malignan-
the growth and decline of cancer in tumor patients, and the cies, especially pancreatic cancer and CRC. Recent studies
increase of CEA level can predict the recurrence and liver have shown that preoperative serum CA242 can predict the
metastasis of colorectal cancer, which is 6–8 months ear- staging, lymph node metastasis, and tumor invasion depth
lier than imaging and clinical examination. Elevated CEA of colorectal cancer, which can be used as an independent
is commonly seen in colorectal, pancreatic, gastric, breast, predictor of 5-year survival rate of colorectal cancer [110].
and medullary thyroid cancers. However, smoking, preg-
nancy, cardiovascular disease, diabetes, nonspecific colitis, 35.6.6.2 Tumor Specific Protein (SP70)
and other diseases, some patients will also increase serum SP70, a specific antigen recognized by monoclonal anti-
CEA [109]. So CEA is not a specific sign of malignancy and body NJ001, is a marker of non-small cell lung cancer
has only auxiliary value in diagnosis. It has long been pro- [111]. Previous studies have shown that the expression of
posed that the determination of CEA can be used to monitor SP70 is closely related to the clinicopathological features
the treatment effect and recurrence of colorectal cancer after and prognosis of lung cancer. It is found that SP70 is also
surgical resection. In addition, serum CEA level is clearly abnormal in CRC patients. The distribution of SP70 in the
related to the stage of CRC. The more advanced the tumor serum of patients with CRC was significantly higher than
lesion, the higher the CEA concentration. However, its speci- that of patients with benign colorectal disease and healthy
ficity is not strong and sensitivity is not high, the early diag- control (P < 0.05), and there was significant difference
nosis of the tumor is not obvious. A persistent increase in between patients with benign colorectal disease and healthy
CEA after the drug or surgical treatment is often a sign of control (P < 0.05) (Fig. 35.27). The double antibody sand-
residual or recurrent tumors, and a drop to the normal range wich ELISA method was used to detect SP70 in the serum
is a sign of successful treatment. of 100 patients with CRC, 60 patients with benign colorec-
tal disease, and 60 healthy controls from the First Affiliated
CA19-9 Hospital of Nanjing Medical University from October 2017
CA19-9 can be used as an auxiliary diagnostic indicator to March 2018 in our study. The reference range of SP70
for malignant tumors such as colorectal, stomach, pancre- (ELISA) in this study was ≤7.5 ng/mL.
atic, and gallbladder cancers. In the serum of patients with
gastrointestinal malignancies, the content of CA19-9 is sig- 35.6.6.3 Liquid Biopsy
nificantly increased, but the value of early diagnosis is not Liquid biopsy is a new precision medical technique used to
significant, which is mainly used as the indicator for disease analyze biomarkers in blood, urine, and cerebrospinal fluid
monitoring and recurrence prediction. In addition, it is valu- [112]. There is evidence that liquid biopsy has prognostic
able for the differential diagnosis of digestive tract diseases. and predictive values in the treatment of CRC [113]. Here,
CA19-9 is increased in pancreatic cancer, gallbladder cancer, circulating tumor cells (CTCs) and cell-free DNA (cfDNA)
gastric cancer, colon cancer, and liver cancer. Acute pancre- are used as markers for CRC patients.
atitis, cholecystitis, and hepatitis also have different degrees
of increase. The combined detection of CA19-9 and CEA
can be used as a marker for recurrence detection of CRC
after operation.
CA242 and CA50
CA242 is a mucin-type carbohydrate antigen, which can
be used as a good tumor marker for pancreatic and colon
cancer. The sensitivity of CA242 to the diagnosis of CRC
is 60–72%. Its sensitivity is similar to that of CA19-9, but
its specificity and diagnostic efficiency are better than that
of CA19-9. CA50 is a nonspecific broad-spectrum tumor
marker. CA50 can be increased in the blood of many patients
with malignant tumors, such as lung cancer, liver cancer,
stomach cancer, ovary or cervical cancer, pancreatic or bile
duct cancer. Other tumors, such as rectal cancer and blad-
der cancer, showed an increase of more than 70% in CA50.
CA242 is a salivary acidifying carbohydrate antigen that is
almost always expressed with CA50, but the two are recog- Fig. 35.27 Serum SP70 levels were increased in CRC patients, BCD
nized by different monoclonal antibodies. Clinically, all of (benign colorectal disease), HC (healthy control)
CTCs 35.6.6.4 Genetic Biomarkers

In the early stage of CRC, tumor cells shed from the tumor
may be detected in the peripheral blood circulation. By Microsatellite Instability (MSI)
detecting circulating tumor cells, early tumor metastasis Microsatellites are small pieces of DNA in tandem repeat
can be detected and targeted therapy can be conducted. sequences that normally remain relatively stable. In the repli-
Studies have shown that tumor cells already exist in the cation process, these small replication units are prone to slip,
peripheral blood of patients before metastasis is detected. insert, deletion, and other replication errors. These replica-
Most of these tumor cells are eliminated by immune cells, tion errors are normally fixed. When the mismatch repairs
and the quantity of survival is very small. However, under functional defects, multiple alleles of different sizes will be
proper conditions, these tumor cells can further develop formed in the next round of replication, that is, microsatel-
into new tumor sites. The amount of CTC is closely related lite instability. MSI first observed in colon cancer, one after
to distant metastasis and prognosis of CRC patients and can another after gastric cancer, pancreatic cancer, lung cancer,
be used to determine the risk of postoperative recurrence bladder cancer, breast cancer, prostate cancer, and other
[114]. In CRC patients, the 5-year survival rate is inversely tumors found microsatellite instability phenomenon, prompt
proportional to the number of CTC in peripheral blood, and microsatellite instability may be another important mol-
CTC is about a year earlier than CEA in warning of distant ecules of tumor cells, according to the results of microsat-
metastasis in CRC patients. Tumor progression is propor- ellite instability is associated with tumor and development.
tional to CTC positive rate. Surgical operation can cause Microsatellite instability is only found in tumor cells, never
tumor cells to fall off and enter peripheral blood. CTC detected in normal tissues. In primary and metastatic tumors,
detection after surgically assisted chemotherapy has clini- microsatellite instability is distributed throughout the tumor.
cal application value of indicating potential metastasis risk, BAT-26 is considered to be a typical representative of CRC
providing a novel diagnostic tool for postoperative treat- microsatellite instability. The rate of mutation of the BAT-
ment monitoring of patients with CRC. 26 in HNPCC is significantly higher than that in sporadic
Tumor recurrence and metastasis are the main causes of CRCs. The specificity and sensitivity are very satisfied and
tumor death. CTC is a key step in the process of tumor blood valuable. It is so easy and economical that it can be used in
metastasis, providing a link between primary and metastatic clinical practice.
tumors. Early and accurate detection of CTC is crucial for
comprehensive treatment of patients with CRC and may be KRAS and BRAF Mutations
used as an evaluation indicator for monitoring treatment, KRAS gene is a proto-oncogene, about 35 kb in length,
evaluation of prognosis, and prediction of recurrence and located on chromosome 12. It is related to tumor forma-
metastasis risk of CRC, which is helpful for selecting indi- tion, proliferation, migration, diffusion, and angiogenesis.
vidualized treatment plans, thereby reducing the recurrence Studies have shown that about 30% of human malignant
rate and prolonging the survival period of patients. In addi- tumors are related to RAS gene mutation, and the products
tion, in some patients, the amount of tumor tissue obtained after RAS mutation can always be in an active state. KRAS
by biopsy is small, and it is demanding to get tissue, which mutations are common in leukemia, lung, rectal, and pan-
makes it very difficult to detect gene mutation, and also creatic cancers, with 35–45% of patients with colorectal
becomes a dilemma of individualized treatment monitoring. cancer having mutations [116]. Detection of KRAS muta-
The circulating tumor cells, which are easy to collect and tion is an important indicator for in-depth understanding of
have little trauma and good patient compliance, are the ideal oncogenes, development prognosis of various cancers, and
specimen source in the clinic. curative effect of radiotherapy and chemotherapy. Studies
also have demonstrated that a large proportion of patients
CfDNA with CRC have KRAS mutations, and these patients will not
CfDNA, which may come from normal or tumor cells, can benefit from targeted therapy for EGFR. Therefore, patients
be detected and quantified in the blood circulation of cancer with CRC should be routinely tested for KRAS mutations
patients. Several studies investigating the prognostic value before applying targeted drug therapy, thus providing a reli-
of the cfDNA for various cancer types have shown that high able basis for targeted therapy. Studies have confirmed that
levels of cfDNA are associated with a more adverse prog- the BRAF mutation rate is 10–15% in CRC, which is inde-
nosis than that of lower levels. The levels of cfDNA were pendent of KRAS mutation. In the sporadic MSI-H CRC,
indeed associated with survival in metastatic colorectal the mutation rate of BRAF is 40–50%, all of which leads to
cancer [115]. a significant decrease in survival rate.
546 G. Liu et al.
SMAD4 Mutation The decrease of its expression is closely related to the proxi-
SMAD family member 4 (SMAD4), also known as moth- mal tumor location, infiltrative growth, and high TNM stage
ers against decapentaplegic homolog 4, is a 552-amino acid of CRC. Decreased expression of CDX2 is associated with
involving in the transforming growth factor TGF-β signal- lymph node metastasis, distant metastasis, and poor progno-
ing pathway. SMAD plays a critical role in the progression sis in CRC patients. In conclusion, CDX2 may be an inde-
of CRC patients. Sporadic SMAD4 gene mutations were pendent prognostic factor in CRCs.
observed in some CRC patients. SMAD4 has been reported
as a potential prognostic indicator of patient survival. MutT-Related Proteins
MutT-related proteins, including MutT homolog 1 (MTH1),
35.6.6.5 Proteomics MTH2, MTH3, and Nudix hydrolase 5 (NUDT5), can effec-
tively degrade 8-oxoGua-containing nucleotides. It was
P53 found that in CRC cells and tissues which were closely
P53 gene is a tumor suppressor gene. In all malignancies, related to lymph node metastasis and AJCC staging, the
mutations in the gene appear in more than 50% of cases. expression of MutT-related proteins increased, while the
The protein encoded by this gene is a transcriptional factor, downregulation of MutT-related proteins inhibited the pro-
which controls the initiation of cell cycles. liferation of CRC cells [117].
The occurrence of CRC is the same as that of other dis-
eases, which is affected by heredity, environment, society, C-Met
behavior, and other aspects. However, numerous clinical The c-Met oncogene encodes a receptor tyrosine kinase,
trials have confirmed that the mutation of the p53 gene is called Tyrosine-protein kinase Met, which was found to be
closely related to the occurrence of some tumors, especially upregulated in different solid tumors and is significantly
colon cancer. Moreover, p53 gene mutation and overexpres- associated with poor prognosis. This is also associated with
sion of p53 protein have been confirmed to be correlated tumor progression and poor prognosis of CRC.
well. The p53 gene mutates and expresses in CRC, while
the p53 gene in normal gastrointestinal mucosa is negative. 35.6.6.6 Epigenetics
Studies have shown that the expression of the p53 gene in
well-differentiated adenomas, including familial multiple MicroRNA (miRNA)
colon polyps, an autosomal dominant genetic disease, and MiRNA is the noncoding single small RNA that is made
colorectal polyps is significantly lower than that in CRC, up of about 22 nucleotides and widely exists in eukaryotic
which supports the argument that p53 gene mutation plays cells. It regulates gene expression at the post-transcriptional
an important role in the development of CRC. In addition, level by pairing with the complete or incomplete bases of its
studies have shown that wild-type p53 gene can induce p21 target mRNA molecule, inducing degradation of mRNA or
gene expression and cause growth inhibition of various inhibiting its protein expression. MiRNA has the function
tumor cells, while the mutant p53 gene has no such effect. of oncogenes or tumor suppressor genes, and the abnormal
Usually in the process of CRC, the following stages can be expression of miRNA may cause changes of multiple target
experienced: normal mucosa, adenoma, CRC, metastatic genes, which may affect tumor biological processes such as
lesions. Numerous experiments have shown that the expres- cell proliferation, apoptosis, invasion, and metastasis.
sion of the p53 gene in the normal mucosa is negative, while As the research progresses, miRNAs that affect the prolif-
some studies have shown that the p53 gene in the adenoma eration, invasion, and metastasis of CRC cells are increasingly
stage is likely to be mutated and not further increased with found. MiRNA affects the proliferation, invasion, and metas-
the development of the tumor. The mutation rate of the p53 tasis of CRC mainly through proteins that regulate the occur-
gene in the process of CRC is relatively high, about 50–80%, rence and development of CRC, including Wnt/β-catenin,
and mainly occurs in exons 5–8, and studies have shown that epidermal growth factor receptor (EGFR), phosphatidylino-
p53 gene mutation is independent of the histological type sitol 3 kinase/serine-threonine kinase (PI3K/AKT), trans-
of adenoma and adenocarcinoma. Therefore, the detection forming growth factor (TGF-β), matrix m etalloproteinases
of p53 gene expression is conducive to the prediction of (MMPs) and so on. MiRNA expression levels are different
adenoma cancerization tendency and the detection of p53 between different diseases and at the precancerous lesion
gene protein products has become an important means for stage of CRC, which provide a new idea for early screening
the early diagnosis of CRC. and diagnosis of CRC. Some people compared samples of
colon cancer, colorectal adenomatous polyps, inflammatory
CDX2 bowel disease, and normal colon tissues, and found that miR-
Caudal-type homeobox protein 2 (CDX2) is a homologous 145 was significantly downregulated in cancer tissues [118].
protein responsible for maintaining the intestinal phenotype. In addition, there are specific differences in the expression
of other miRNAs in cancer tissues of CRC patients. MiRNA Table 35.7 Main biomarkers of CRC
can be secreted outside cells and become an important regu- Clinical
latory molecule in the cell microenvironment and body flu- Markers application Effect on prognosis
ids. MiRNA can be found in human milk, saliva, urine, and CTCs Diagnostic and High CTC count predicts poor
prognostic prognosis
peripheral blood. Detection of miRNA in easily obtained
CfDNA Diagnostic and Low levels of cfDNA predicts
specimens such as peripheral blood and feces is a better prognostic better survival
choice for early CRC screening. MSI Prognostic and MSI-H predicts better survival
therapeutic
Methylation KRAS Prognostic KRAS mutations predict poor
CpG island Methylator Phenotype (CIMP) mutations prognosis
BRAF Prognostic BRAF mutations predict poor
CpG island is mainly located in the promoter and first exon
mutations prognosis
region of the gene and is rich in non-methylated CpG dinu- SMAD4 Prognostic and SMAD4 gene mutation predicts
cleotide with a length greater than 197 bp. CpG island meth- mutations therapeutic poor prognosis
ylation abnormalities occurred in the early stage of CRC CIMP Prognostic CIMP positive predicts poor
lesions, with an incidence of about 10–20%. Under normal prognosis
conditions, special proteins with protective effects in the pro- ZNF331 Prognostic Methylation of ZNF331
predicts poor prognosis
moter region bind to the non-methylated state of CpG island.
LINE-1 Prognostic and LINE-1 hypomethylation
After methylation of CpG island, chromatin conformation therapeutic predicts poor prognosis
changes, and protein binding activity decreases, thereby
reducing the promoter activity, inhibiting gene transcription
and inactivation of tumor suppressor genes, thus inducing
tumor. The detection of CpG island methylation abnormality 35.6.7 Therapy
is helpful for the early diagnosis of tumors, the assessment
of tumor process and prognosis, and the guidance of clini- The radical treatment of CRC is still the surgical treatment.
cal diagnosis and treatment. P15 and p16 genes are located In addition to surgical treatment, radiotherapy, and chemo-
at 9p21. Through studying the methylation of p15 in CRC therapy are also commonly used to treat CRC, especially in
tissues, it is believed that methylation inactivation of CpG the late stage of the disease. Many researchers have found
island in the promoter of p15 promotes the occurrence and that miRNA is an important biological molecule that regu-
development of CRC [119]. Another study finds that CRC lates radiochemotherapy sensitivity. It was found that miR-
patients with p16CpG methylation have short survival. All 195 can increase the sensitivity of CRC cells to radiotherapy
above suggest that 9p could be used as a prognostic indicatorby inhibiting CARM1 [121]. Numerous studies have shown
of CRC. that miRNA has the clinical application value of increasing
drug sensitivity of CRC cells and reducing drug resistance.
ZNF331 Advanced solid tumors with MSI-H phenotype are often of
Classical zinc finger protein (ZNF)is one of sequence- significant efficacy for immune checkpoint inhibitors, such
specific DNA-binding proteins families and is encoded by as PD-1/PD-L1 antibodies. As early as 2016, the consensus
2% of the human genes. Studies have shown that promoter guidelines of the European Society of Medical Oncology
region methylation can regulate the expression of ZNF331, (ESMO) for metastatic CRC had proposed that MSI testing
and ZNF331 can inhibit the proliferation and colony forma- had a strong predictive value for immune checkpoint inhibi-
tion of CRC cells. Kaplan–Meier plots showed that CRC tor therapy in patients with metastatic CRC.MSI-H was a
patients with abnormal methylation of ZNF331 had poor key factor in the benefit of the checkpoint inhibitors. Based
5-year DFS and 5-year OS. Cox proportional hazards model on this, the Food and Drug Administration (FDA) approved
analysis showed that methylation of ZNF331 was an inde- pembrolizumab in 2017 for use in patients with advanced/
pendent prognostic value of CRC patients. metastatic solid tumors in dMMR/MSI-H who have no
satisfactory alternative therapy after previous treatment.
LINE-1 The absence of SMAD4 affects the chemotherapy effect of
Long interspersed nucleotide element-1 (LINE-1) is one of CRC. A study found a new mechanism for SMAD4 to regu-
the retrotransposons involved in the regulation of genomic late the chemotherapy sensitivity of 5-FU, namely, blocking
structure and function. Kaplan–Meier analysis showed that the cell cycle by inhibiting the cascade of PI3K/Akt/CDC2/
CRC patients with LINE-1 hypomethylation had higher apoptosis-inhibiting protein. Compared to CRC patients with
CRC-specific mortality (Table 35.7). In addition, multivari- hypermethylation, patients with LINE-1 hypomethylation
ate analysis showed that LINE-1 hypomethylation may be had significantly worse PFS and OS after chemotherapy.
an independent prognostic factor for advanced CRC [120]. Tumor LINE-1 hypomethylation was also associated with
548 G. Liu et al.
adverse effects of 5-FU combined with oxaliplatin and poor

prognosis in patients with advanced CRC.
Clinical Background A 69-year-old female patient pre-

sented to her primary care physician complaining of the
upset of abdominal pain and vomiting for a day.
B-Ultrasound Right intraperitoneal mixed echogenic mass.
Abdominal CT Scan The right wall of the transverse colon

was significantly thickened with enhancement, blurring of
the surrounding fat space, and multiple lymph nodes. The
CT scan photos were shown in Fig. 35.28. Fig. 35.29 The histopathology of transverse colon cancer (hematoxylin-
eosin staining, ×100)
Initial Laboratory Values WBC 6.92 × 109/L, Neutrophil
granulocyte 5.09 × 109/L, Hemoglobin 135 g/L, Platelet
257 × 109/L. expression in tissues of colorectal cancer and benign colorec-
tal disease was described in Fig. 35.30.
Tumor Marker CA19-9 8.46 U/mL (<39.00 U/mL), CEA
2.37 ng/mL (<4.70 ng/mL), SP70 12.9 ng/mL (≤7.5 ng/mL). Treatment Right hemicolectomy.
Histopathological Examination Poorly differentiated car- Treatment Outcome Good postoperative recovery.
cinoma of the transverse colon, see in Fig. 35.29. SP70
Results with Interpretation Guideline Early diagnosis
and treatment are crucial for CRC patients. Serum levels of
CA19-9 and CEA were normal in this patient, while SP70
was increased. CEA and CA19-9 are serum markers of
digestive tract tumors that are widely used in clinical prac-
tice, but their low specificity cannot meet the needs of early
clinical diagnosis of CRC. Imaging examinations will
always be necessary and have the advantage of assessing
tumor size. However, it is difficult to differentiate between
benign and malignant colorectal masses. Histopathology is
the gold standard for the diagnosis of CRC, but it is invasive
and there is a risk of failure. SP70, as a newly developed
tumor marker, can be used for differential diagnosis of
benign and malignant diseases.
This case was from the First Affiliated Hospital of

Nanjing Medical University (also named Jiangsu Province
Fig. 35.28 The CT scan in CRC Hospital).
a b
Fig. 35.30 SP70 expression in tissues of colorectal cancer and benign colorectal disease. (a) CRC (SP70 positive expression) (b) BCD (SP70
negative expression) (Immunohistochemistry staining, ×200)
15. Fang JY, Liu WZ, Shi Y, et al. Consensus on chronic gastri-
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Kidney Diseases
36
Zhaojing Zheng, Juan Geng, Ye Jiang, Meijuan Zhang,
Ruixia Yang, Gaoxia Ge, Huaguo Xu, and Xiaojie Zhang
36.1 Polycystic Kidney Disease 36.1.1 Overview
Zhaojing Zheng and Juan Geng Polycystic kidney disease (PKD), as one of the most com-
mon heritable kidney diseases, is clinically and genetically
The kidneys are two bean-shaped organs in the renal sys- heterogeneous [1], and is characterized by bilateral renal
tem. They are located on the left and right in the retroperi- cysts, and usually affects adult patients. Frequent compli-
toneal space and are about 11 cm in length in adult humans. cations of PKD include dangerously high blood pressure
The functional substance, or parenchyma, of the kidney can (hypertension), pain in the back or sides, blood in the urine
be classified into the outer renal cortex and the inner renal (hematuria), recurrent urinary tract infection, kidney stone,
medulla. These structures take the shape of renal lobes, each and heart valve abnormality. Additionally, people with PKD
of which contains a renal cortex surrounding a portion of the have an increased risk of an abnormal bulging (aneurysms)
medulla called a renal pyramid. Between the renal pyramids in the aorta or in blood vessels at the base of the brain.
are called renal columns. Nephrons, the urine-producing func- Aneurysms can be life threatening if they tear or rupture.
tional structures of the kidney, span the cortex and medulla. PKD can be inherited in a dominant (ADPKD) or recessive
The initial filtering portion of a nephron is the renal corpuscle (ARPKD) pattern. Genetic information gained from genetic
located in the cortex. This is followed by a renal tubule. Part testing is pivotal for disease diagnosis, prognosis, manage-
of the renal cortex, a medullary ray is a collection of renal ment, and genetic counseling.
tubules that drain into a single collecting duct (Fig. 36.1). ADPKD is the most common inherited renal disease and
The kidneys perform many crucial functions, including one of the commonest Mendelian human disorders with an
maintaining overall fluid balance, regulating and filtering overall prevalence of 1/500–1000 [2, 3]. This approximates
minerals from the blood, filtering waste materials from food, to about 12.5 million affected individuals worldwide [1]. In
medications, and toxic substances, creating hormones that general, ADPKD is a late-onset multisystem disorder char-
help produce red blood cells, promote bone health, and regu- acterized by bilateral renal cysts, liver cysts, and an increased
late blood pressure and so on. risk of intracranial aneurysms. Other manifestations include:
Due to all the vital functions the kidneys perform and the cysts in the pancreas, seminal vesicles, and arachnoid mem-
toxins they encounter, the kidneys are susceptible to vari- brane; dilatation of the aortic root and dissection of the
ous problems. Some of these conditions including polycystic thoracic aorta; mitral valve prolapse; and abdominal wall
kidney disease, nephropathy, kidney cancer, and Hantavirus hernias. Renal manifestations include hypertension, renal
hemorrhagic fever with renal syndrome are described below. pain, and renal insufficiency. Approximately 50% of individ-
uals with ADPKD have end-stage renal disease (ESRD) by
age 60 years. The prevalence of liver cysts increases with age
Z. Zheng (*) · J. Geng
and occasionally results in clinically significant severe poly-
University School of Medicine, Shanghai, cystic liver disease (PLD). The overall risk of intracranial
People’s Republic of China aneurysms is fivefold higher than in the general population
Y. Jiang (*) · M. Zhang · R. Yang (*) · G. Ge · H. Xu (*) and further increased in those with a positive family history
X. Zhang of aneurysms or subarachnoid hemorrhage. There is substan-
Department of Laboratory Medicine, The First Affiliated Hospital tial variability in the severity of renal disease and other extra-
renal manifestations even within the same family.
e-mail: huaguoxu@njmu.edu.cn

554 Z. Zheng et al.
structure of kidney
In contrast, ARPKD is a severe, typically early-onset Kidney Disease: Improving Global Outcomes (KDIGO)
form of renal cystic disease. It is rare and typically of child- Controversies Conference. ADPKD should be suspected in
hood onset and occurs in 1: 6000 to 1: 50,000 live births individuals with the following:
[4–7]. A “Potter” oligohydramnios phenotype, along with
massively enlarged kidneys, pulmonary hypoplasia, char- 1. Multiple bilateral renal cysts and the absence of manifes-
acteristic facies, and contracted limbs with club feet, is tations suggestive of a different renal cystic disease
always observed in affected fetuses. Approximately 30–50% 2. Cysts in other organs, especially the liver, but also semi-
of affected neonates die shortly after birth from respiratory nal vesicles, pancreas, and arachnoid membrane
insufficiency due to pulmonary hypoplasia and thoracic 3. Enlargement of the kidneys or liver on physical

compression by the excessively enlarged kidneys. ARPKD examination
patients may present with clinically significant congenital 4. Hypertension in an individual younger than age 35 years
hepatic fibrosis, with portal hypertension, requiring close 5. An intracranial aneurysm
monitoring, surgical shunting procedures, and kidney and/or 6. A family history of ADPKD
liver transplantation.
The diagnosis of ADPKD is established in a proband with
ANY of the following: Age-specific ultrasound criteria and an
36.1.2 Diagnosis affected first-degree relative with ADPKD; Age-specific MRI
criteria and an affected first-degree relative with ADPKD;
PKD and cysts on the liver or other organs may be found on Identification of a heterozygous pathogenic variant in PKD1,
CT scan, MRI scan, ultrasound B, and Intravenous pyelo- PKD2, GANAB, DNAJB11 and other unknown genes.
gram (IVP). If several members of the patient family have ARPKD should be suspected in individuals with bilater-
PKD, genetic tests should be undertaken to determine the ally enlarged, diffusely echogenic kidneys. Diagnosis is typi-
mutation status of PKD genes. cally made based on clinical presentation and radiographic
For ADPKD, MRI or contrast-enhanced CT examina- findings [8–10]. Ultrasonography (US) is the diagnostic
tion, which has much higher sensitivity than ultrasound to method of choice for assessing fetal and pediatric ARPKD
detect cysts and is routinely performed in most transplanta- because it is cost-effective, painless, widely available, and
tion centers to define the donor kidney anatomy, provides does not require radiation. It is predominantly useful in
further evidence for the diagnosis of the disease. Practically, identifying renal abnormalities, but abdominal US may also
sequence analysis and deletion/duplication analysis of PKD1 indicate biliary ductal involvement or splenic enlargement
and PKD2 are usually performed at first. A multi-gene panel in those with ARPKD. However, renal US alone is never
that includes PKD1, PKD2, GANAB, DNAJB11, and other diagnostic. The mutational spectrum of PKHD1 is charac-
genes of interest is most likely to identify the genetic cause terized by significant allelic heterogeneity with many vari-
of the condition at the most reasonable cost while limiting ants unique to single families in “non-isolate” populations.
identification of variants of uncertain significance and patho- Given the size ofPKHD1 gene and the absence of mutational
genic variants in genes whose correlation with the underly- hotspots, conventional PKHD1 molecular testing by Sanger
ing phenotype not fully established yet. Diagnostic criteria sequencing is labor-consuming and cumbersome [11, 12].
for ADPKD are discussed in the executive summary of the NGS streamlines this process and makes molecular diagno-
36 Kidney Diseases 555
sis of ADPKD more time- and cost-effective, esp., in clinical egy is chosen, it is important to be aware of the strengths and
settings. In addition, as the number of genes associated with pitfalls of each method [13]. For PKD, the testing approach
PKD increases rapidly, high-throughput technologies such should be able to detect copy-number variations (CNVs) such
as a multigene panel or whole-exome sequencing represent as heterozygous deletions (e.g., in HNF1B) and to cover even
powerful approaches and allow simultaneous analysis of complex genomic regions such as the PKD1 gene [1].
many genes in a single test at a relatively low cost. Specific The diagnosis of ADPKD is mostly based on imaging
diagnostic criteria of ARPKD are typical findings on renal studies using well-established criteria in the presence of a
imaging and one or more of the following: positive family history [14]. However, genetic testing may
be indicated in patients without a positive family history,
1. Imaging findings consistent with biliary ductal ectasia with equivocal imaging findings, with atypical clinical fea-
2. Clinical/laboratory signs of congenital hepatic fibrosis tures (e.g., extrarenal findings suggestive of other syndromic
(CHF) that leads to portal hypertension and may be indi- renal cystic disease), in young at-risk subjects (<25 years
cated by hepatosplenomegaly and/or esophageal varices old) being evaluated as a living-related kidney donor, and
3. Hepatobiliary pathology demonstrating a characteristic for pre-implantation genetic diagnosis/screening [15]. For
developmental biliary ductal plate abnormality and resul- patients suspected to have ADPKD without a positive fam-
tant CHF ily history, the differential diagnosis should be undertaken
4. Absence of renal enlargement and/or characteristic imag- to include several other genetic (e.g., isolated polycystic
ing findings in both parents, as demonstrated by high- liver disease, autosomal recessive polycystic kidney disease,
resolution ultrasonography (HRUS) examination autosomal dominant tubulointerstitial disease, tuberous scle-
5. Pathologic (biopsy or autopsy) or genetic diagnosis of rosis complex, and Von Hippel–Lindau syndrome) and non-
ARPKD in an affected sibling genetic (e.g., simple renal cysts, and acquired renal cystic
kidney disease) causes and genetic testing of multiple cystic
disease genes is often required [16, 17]. By contrast, patients
36.1.3 Genetic Testing Algorithm with bilaterally enlarged kidneys filled with numerous cysts
are highly likely affected with ADPKD; nonetheless, in
In the past, strategies of genetic testing for PKD were mainly the absence of validated diagnostic criteria, genetic testing
based on time- and cost-intensive single-gene testing. Along is often performed to establish the diagnosis of ADPKD
with the rapid development of genomics and sequencing of de novo onset. Additionally, genetic testing is useful in
chemistry, NGS-based genetic testing has transformed deeply patients or families with suspected complex genetic etiol-
the molecular diagnostic arena of PKD. Target sequencing, ogy (i.e., bilineal disease resulting in early-onset and severe
whole-exome sequencing, and even whole-genome sequenc- PKD or discordant kidney disease severity between affected
ing have emerged as powerful diagnostic tools for PKD in relatives) [18]. The approach for diagnosis of ADPKD using
research as well as in clinical settings. Whatever primary strat- imaging- and genetics-based approach is shown in Fig. 36.2.
Fig. 36.2 Diagnostic
algorithm for autosomal Renal cysts
dominant polycystic kidney
disease (ADPKD)
Positive family history of
ADPKD?
Yes No
Unusal clinical features? Unusal clinical features?
No No
Fulfill ultrasound
Bilateral small kidneys?
diagnostic criteria?
Yes Yes Yes Yes

No Consider genetic No Acquired renal cysts Consider other renal
Dx: ADPKD
testing disease cysts disease
556 Z. Zheng et al.
The large size of PKHD1 poses significant challenges to PKHD1 is the main gene for ARPKD and one of the larg-
current DNA sequencing methods. Other diagnostic chal- est disease genes in the human genome, extending over a
lenges relate to the high frequency of missense mutations genomic segment of at least 470 kb and including a mini-
and private mutations in “non-isolate” populations [19]. In mum of 86 exons. To establish the genotype–phenotype
cases with strong clinical and/or histopathological evidence correlations for PKHD1 is hampered by multiple allelism
for ARPKD, mutation detection rates of about 80–85% and the high rate of different compound heterozygotes. No
have been demonstrated for patients across the entire clini- significant clinical differences have been observed between
cal spectrum [11]. Several other cilia-related disease genes patients with missense and those patients with truncat-
may mimic (“phenocopy”) ARPKD. For instance, 2% of all ing mutation in trans; thus, the milder mutation obviously
ADPKD patients express an early onset, severe phenotype defines the phenotype [29]. The phenomenon of discordant
that is clinically indistinguishable from ARPKD [20]. Expert phenotypes observed in siblings suggests that the pheno-
opinions are as follows [21]: types cannot be simply explained on the basis of the PKHD1
genotype alone. Modifying genes, epigenetic changes, and
1. Given the high number of phenocopy disorders, muta- variations in other non-coding regions of the genome are
tional analysis of PKHD1 using current single-gene test- thought to be associated with the wide interfamilial clini-
ing methodologies should not be considered as a first-line cal variability [8]. Recently, mutations in DZIP1L have
diagnostic approach for infants and children presenting been described in patients with a moderate clinical course of
with an ARPKD-like phenotype. ARPKD [30]. So far, the data suggest that the type or loca-
2. Pathogenicity predictions for missense variants represent tion of DZIP1L mutations does not determine the severity
another diagnostic challenge; caution is required when of the clinical course. Patients harboring missense mutations
only novel or rare missense changes are detected. on both parental alleles displayed a comparable phenotype
3. More robust next-generation sequencing methods that
to patients with two truncating DZIP1L alleles [1] No geno-
allow simultaneous investigation of multiple cystic kid- type–phenotype correlations have been established to date
ney disease genes will increasingly become available. for either PKHD1 or DZIP1L. Most PKHD1 pathogenic
variants are “private” or unique to single families [11].
36.1.4 Correlation Between Phenotype

and Genotype 36.1.5 Genetic Counselling
The majority of ADPKD patients carries a pathogenic germ- It is only possible to discuss in detail the clinical course,
line variation in PKD1 and about 15–20% harbor a patho- disease spectrum, and recurrence risk if the nature of the
genic variation in PKD2 [22]. PKD2 is usually significantly patient’s genotype is known. Understandably, it makes a
milder than PKD1 with the development of end-stage renal huge difference for parents who already have an affected
disease (ESRD) at a later age and a lower prevalence of arte- child and who plan to have further pregnancies, if they carry
rial hypertension and urinary tract infections. Patients with a 50% (as for ADPKD), 25% (as for ARPKD), or practically
PKD1 develop ESRD approximately 20 years earlier than no recurrence risk. The latter holds true when the genetic
patients with PKD2, with a median age of onset of ESRD variant in the patient arose de novo.
at 58.1 years and 79.9 years, respectively [1]. The greater Most individuals diagnosed with ADPKD have an affected
severity of PKD1 is due to the development of more cysts at parent, while some individuals diagnosed with ADPKD have
an early age, not to faster cyst growth [23]. A recent study the disorder as a result of a de novo pathogenic variant. If the
including more than 500 pedigrees with ADPKD further pathogenic variant found in the proband cannot be detected
demonstrated that the type of PKD1 mutation significantly in either of the parent, possible explanations include a de
correlated with renal survival [24]. The average age at onset novo pathogenic variant in the proband or germline mosa-
of ESRD in affected individuals with truncating PKD1 vari- icism in a parent. If the parent is the individual in whom the
ants is 55.6 years compared to 67.9 years for those with pathogenic variant first occurred, she or he may have somatic
non-truncating PKD1 variants, suggesting that a significant mosaicism for the variant and may be mildly/minimally
proportion of in-frame variations are likely hypomorphic affected. The risk to siblings of the proband depends on the
[25, 26]. Family studies have identified incompletely pen- genetic status of the proband’s parents. If a parent of the pro-
etrant non-truncating PKD1 variants that are associated with band is affected or has a mosaic pathogenic variant, the risk
less severe disease [27]. Recently, truncating PKD2 patho- to siblings of inheriting the variant is 50%. Meanwhile, his
genic variants were found to be associated with more severe or her family members may also be at risk.
disease with lower EGFR expression than non-truncating ARPKD is caused by biallelic pathogenic variants in
pathogenic variants [28]. PKHD1 and DZIP1L. The parents of an affected child are
obligate heterozygotes for the pathogenic variants in PKHD1 While those who have not been informed with knowledge
or DZIP1L. They are usually asymptomatic and are not at of being at increased risk but in which routine prenatal ultra-
risk of developing ARPKD. At conception, each sibling of sound examination reveals enlarged cystic kidneys have the
a proband has a 25% chance of inheriting both pathogenic possibility to develop low-risk pregnancies. For those, there
variants and being affected, a 50% chance of inheriting a are some suggestions as follows: (1) karyotyping analysis or
single pathogenic variant and being a carrier, and a 25% risk chromosomal microarray analysis (CMA) and detailed fetal
of inheriting neither pathogenic variant nor not being a car- ultrasonography should be performed to evaluate for the
rier. The offspring of an individual with ARPKD are obligate presence of a chromosomal abnormality and/or other con-
carriers of PKHD1 pathogenic variants. Each sibling of the genital anomalies in a fetus not known to be at increased risk
proband’s parents is at a 50% risk of being a carrier of a for ARPKD; (2) genetic testing of PKHD1 may be appropri-
PKHD1 pathogenic variant. ate. Failure to detect two pathogenic variants, however, does
With improved clinical management now even patients not completely exclude the diagnosis of polycystic kidney
with early disease manifestation often reach adulthood and disease. A second PKHD1 variant may have been missed by
may wish to have their own children. For these patients, it is current testing techniques or the fetus may have ADPKD;
of major importance to have knowledge about the underlying (3) renal ultrasound examinations of both parents should
genetic defects for their disease in order to assess the genetic be considered in all fetuses with suspected ARPKD, i.e., an
risk of recurrence in the family. While dominant will mean ARPKD diagnosis not confirmed by PKHD1 genetic testing
50% recurrence risk, recessive inheritance will usually result to evaluate for the possibility of ADPKD.
in a <1% risk for offspring unless consanguineous marriage.
36.1.7 Typical Clinical Case

36.1.6 Prenatal Diagnosis
and Preimplantation Genetic Diagnosis A 46-year-old male patient was referred due to a suspected
diagnosis of PKD. He was found to have increased serum
Due to a recurrence risk of 25% and oftentimes devastating BUN and Cr on routine health check-ups at the age of 41. His
and early manifestations, many parents of ARPKD children father was reported to be a patient of PKD but without more
seek early and reliable prenatal diagnosis to guide future information about disease subtypes.
family planning. Frequently, ARPKD patients are identified To confirm his diagnosis of PKD, whole-exome sequenc-
by ultrasound only late in pregnancy or at birth. However, ing (WES) was performed with Agilent SureSelect Human
fetal sonography at the time when termination of pregnancy All Exon V6 kit and Illumina sequencing platform. After
(TOP) is usually performed may fail to detect enlargement standard bioinformatic pipeline processing and proband-
and increased echogenicity of kidneys or oligohydramnios only variant filtering strategy, the patient was found to harbor
secondary to poor fetal urine excretion. Therefore, an early a heterozygous c.5778del variation in the PKD1 gene, which
and reliable PND for ARPKD in “at risk” families is only was categorized as “likely pathogenic” according to ACMG
feasible by molecular genetic analysis. PND should only standard and validated by Sanger sequencing (Fig. 36.3).
be offered to those families in which the diagnosis has been This case was from Shanghai Children’s Medical Center.
previously unequivocally confirmed in an affected fam-
ily member. Still, molecular prenatal diagnostics is usually
done after chorionic villus sampling (CVS), an invasive pro- 36.2 Nephropathy
cedure already quite late in the pregnancy (no earlier than
10–12th week of gestation). Interested families should also Ye Jiang and Meijuan Zhang
be informed on the possibility of pre-implantation genetic
diagnosis (PGD) at some single diagnostic centers and that
this procedure always requires a high degree of coordination 36.2.1 Overview
and work-up in advance. A future scenario might be noninva-
sive prenatal testing (NIPT) of the fetal genotype by screen- Kidney is composed of more than one million neph-
ing a maternal blood sample. rons. Each nephron consists of three parts: glomerulus,
For ARPKD, there exist high-risk pregnancies and low- Bowman’s capsule and tubules (Fig. 36.4). Damage to any
risk pregnancies. Patient with a 25% risk based on fam- part can cause nephropathy. Nephropathy is a global health
ily history increases the risk of high-risk pregnancy. Once problem. Acute kidney injury (AKI) and chronic kidney
the PKHD1 pathogenic variants have been identified in an disease (CKD) are increasing in incidence. It is clear that
affected family member, prenatal testing for a pregnancy at the incidence of AKI has been steadily rising at a rate that
increased risk for ARPKD and PGD are possible options. is disturbingly high, and it is increasingly recognized that
558 Z. Zheng et al.
Fig. 36.3 Classic case of polycystic kidney disease due to PKD1 ing with integrated genome visualization (IGV) browser; the lower row
c.5778del. The upper row shows the reads harboring the likely patho- shows the chromatograph of PKD1 c.5778del by Sanger sequencing
genic variant c.5778del in PKD1 gene through whole-exome sequenc-
AKI predisposes to the progression of CKD toward end- paramount importance. Biomarkers that will help diagnose
stage kidney disease (ESKD), which ultimately requires kidney injury, predict the progression of kidney disease, and
dialysis or kidney transplantation [31]. According to the provide information on the effectiveness of therapeutic inter-
World Health Organization, approximately 850,000 patients vention will be important adjuncts to our standard manage-
develop ESKD every year [32]. Across the globe, the treatment strategies.
ment of ESKD poses a major challenge for health care sys-
tems and the global economy. The burden of kidney disease
is most significant in developing countries and is adversely 36.2.2 Classification
influenced by inadequate socioeconomic and health care
infrastructures. Glomerular disease is a group of diseases with different eti-
Importantly, kidney disease progression may be curtailed ology, pathogenesis, clinical manifestations, pathological
if the disease is diagnosed early. Hence, detection and man- changes, course of disease, and prognosis. It can be divided
agement of kidney diseases, whether acute or chronic, in into various types including glomerular diseases, renal tubu-
the early, reversible, and potentially treatable stages are of lar diseases, interstitial nephritis, etc.
to normal. Severe hypertension and even hypertensive

encephalopathy may occur in a few patients.
Renal Dysfunction The early onset of the disease may be

due to decreased glomerular filtration rate, water and sodium
retention, and a small number of patients even oliguria
(<400 mL/day). Renal function can be temporarily impaired,
manifested as mild azotemia. After more than 1–2 weeks,
urine volume increased gradually, and renal function returned
to normal gradually in a few days after diuresis. Only a few
patients can manifest acute renal failure and need to be dif-
ferentiated from acute nephritis.
Congestive Heart Failure Frequent occurrence in the acute

phase, severe retention of water and sodium, and hyperten-
sion are important incentives, and urgent treatment is needed.
Fig. 36.4 The structure of the nephron
Abnormal Immunological Examination Transient decrease
of serum complement C3: more than 2 weeks after onset, and
36.2.2.1 Glomerular Diseases gradually returned to normal within 8 weeks, which is of great
significance in the diagnosis of this disease. The serum anti-
Acute Glomerulonephritis streptococcal hemolysin “O” titer could be increased.
Acute glomerulonephritis is common in children, espe-
cially boys. Usually, it starts 1–3 weeks after the precursor Causes and Pathogenesis
infection. The latency is equal to the time needed to induce This disease is usually caused by the infection of beta-
immune complexes after the first immunization of patho- hemolytic streptococcus “nephrogenic bacteria” (group A,
genic antigen. The latency of respiratory tract infections is type 12, etc.). It is common after the infection of streptococ-
shorter than that of skin infections. The onset of the disease cus such as upper respiratory tract infection, scarlet fever,
is more acute, the severity of the illness is different, the mild skin infection, and so on. The severity of infection is not
is subclinical (only abnormal urine routine); the typical is entirely consistent with the occurrence and severity of acute
acute nephritis syndrome, severe can occur acute renal fail- nephritis. The disease is mainly caused by immune response
ure. Most of the cases have a good prognosis and can be self- induced by infection.
healed in a few months. It is endocapillary proliferative glomerulonephritis. It is
characterized by diffuse proliferation of endothelial cells
Signs and Symptoms and mesangial cells under light microscopy and infiltration
Hematuria and Proteinuria Almost all patients had glo- of neutrophils. Immunofluorescence showed that IgG and C3
merulogenic hematuria, and about 30% of them had naked were deposited along the capillary wall and mesangial area
hematuria, which was the first symptom of the disease and in coarse granules. Electron microscopy showed hump-like
the reason the patients go to a doctor. It can be accompanied deposits of large electronic dense substances under glomeru-
by mild to moderate proteinuria, and about 20% of the lar epithelial cells.
patient presents proteinuria in the range of nephrotic syn-
drome. In addition to red blood cells, early urinary sediment Molecular Markers
also showed increased white blood cells and epithelial cells, Serum Glomerular Filtration Markers
and granular tube and red blood cell tube.
Blood Urea Nitrogen Blood urea is a low-molecular-
Edema Edema is often the initial manifestation of the dis- weight waste product derived from dietary protein catabo-
ease. Typical manifestations are eyelid edema in the morning lism and tissue protein turnover, and its levels are inversely
or mildly depressed edema in the lower limbs. A few severe correlated with a decline in the GFR. The normal range of
cases can affect the whole body. urea nitrogen in blood or serum is 5–20 mg/dL (1.8–7.2 mmol
urea/L) [33]. BUN is a not sensitive and specific marker for
Hypertension Most patients have transient mild hyperten- acute or chronic kidney disease. However, for patients with
sion, often associated with retention of water and sodium. advanced CKD (e.g., CKD 4–5), some authorities have sug-
After diuretic treatment, blood pressure can gradually return gested averaging urea clearance and creatinine clearance to
560 Z. Zheng et al.
serve as a more accurate estimate of the true GFR. This cell debris, and the latter may not necessarily reflect disease
approach is suggested in part because at these lower levels of status. An improved and standardized laboratory method
renal function, creatinine clearance overestimates the (secre- is urgently needed to facilitate the measurement of urinary
tion) GFR and urea clearance underestimates the GFR. BUN podocyte numbers. Alternative methods that indirectly assess
is measured by spectrophotometry. Because of these undesir- the number of podocytes in urine include detection of mes-
able limitations of creatinine and BUN as markers, there has senger RNA (mRNA) and protein levels of podocyte-specific
been a great deal of interest in the identification of improved proteins by polymerase chain reaction (PCR) and enzyme-
biomarkers for kidney injury. linked immunosorbent assay (ELISA), respectively.
Cystatin C For the last 10–15 years, there has been a tre- Podocalyxin Podocalyxin is the most commonly used
mendous amount of research investigating serum cystatin C marker protein for detecting podocytes in urine. A highly
as a marker of GFR, and urinary cystatin C excretion has O-glycosylated and sialylated type I transmembrane protein
been proposed as a tubular injury marker. Cystatin C is a of approximately 140 kDa, podocalyxin is expressed in
low-molecular weight protein produced at a constant rate by podocytes, hematopoietic progenitor cells, vascular endothe-
all nucleated cells and eliminated exclusively by glomerular lial cells, and a subset of neurons. Urinary podocalyxin has
filtration. It is small (13 kDa) and has a positive charge at been reported as a marker of activity in a number of diseases,
physiologic pH [34]. It is neither secreted nor reabsorbed by including IgA nephropathy, diabetic nephropathy, lupus
renal tubules but undergoes almost complete catabolism by nephritis, post-streptococcal glomerulonephritis, focal seg-
proximal tubular cells, and thus, little, if any, appears in the mental glomerulosclerosis, and preeclampsia [37, 38].
urine under normal circumstances. Any impairment of reab- Unfortunately, because podocalyxin is expressed on a num-
sorption in proximal tubules can lead to marked increases in ber of cell types, the presence of podocalyxin in the urine is
urinary levels of cystatin C in humans and animals. not always reflective of urinary podocytes.
β-Trace Protein β-Trace protein (BTP), also referred to as Nephrin Nephrin, a transmembrane protein of the immuno-
prostaglandin D synthase, has emerged as another promising globulin superfamily, is a component of the filtration slit dia-
biomarker for GFR. BTP is a small protein with a molecular phragm between neighboring podocytes [39, 40].
weight of 23–29 kDa, depending on the size of the glycosyl Immunohistochemical analysis and in situ hybridization
moiety. BTP is primarily eliminated by glomerular filtration have shown that nephrin is primarily expressed in glomerular
and its concentrations in urine range from 600 to 1200 μg/L podocytes. On the basis of these observations, it has been
[35]. Concentrations of BTP are not affected by commonly proposed that nephrin is a key component of the glomerular
used immunosuppressive medications such as prednisone, filtration barrier, which plays a pivotal role in preventing pro-
mycophenolate mofetil, and cyclosporine. This feature is tein leakage.
especially useful in the evaluation of kidney function in kid-
ney transplant recipients. Unlike serum creatinine values, Treatment
age and race were not associated with BTP concentrations. The treatment of this disease is mainly rest and symptomatic
Several new GFR estimation equations based on BTP have treatment. Patients with acute renal failure should be dialyzed
been developed for use in kidney transplant recipients. until they recover naturally. This disease is a self-limiting
However, these equations require external validation in disease. Glucocorticoids and cytotoxic drugs should not be
larger and more diverse patient groups. In contrast to creati- used.
nine, one limitation of using BTP is the lack of widespread
availability and standardization of the assay [36]. General Treatment In the acute stage, we should rest in
bed and gradually increase the activity after the disappear-
Urinary Glomerular Cell Injury Markers ance of naked hematuria, edema, and blood pressure return
Podocyte Count Numerous studies have reported that the to normal. In the acute stage, we should eat a low salt diet
number of podocytes shed is significantly higher in patients (less than 3 g/day). It is not necessary to limit protein intake
with active glomerular disease than in healthy controls and in in patients with normal renal function. Limit fluid intake in
patients with inactive disease. Importantly, podocyte number patients with acute renal failure with significant oliguria.
in urine correlates with disease activity (assessed by renal
biopsy) and has been shown to decline with treatment. Treatment of Infectious Focus If the infection exists, it
The methods used to count urinary podocytes, however, should be treated accordingly.
are limited by several factors: cytologists are needed to
perform the counting; the process is very time consuming; Symptomatic Treatment Including diuretic detumescence,
urine sediments contain whole viable podocytes as well as hypotension, prevention of cardio-cerebral complications.
Antihypertensive drugs can be added when hypertension con- Causes and Pathogenesis

trol is still unsatisfactory after rest, low salt, and diuresis. Only a few cases of chronic nephritis are caused by the
development of acute nephritis. The pathogenesis of most
Dialysis Treatment When a small number of patients with chronic nephritis is immune-mediated inflammation. In
acute renal failure have dialysis indications, dialysis treat- addition, nonimmune and noninflammatory mechanisms
ment should be timely to help patients through the acute play an important role in the development of disease, such
phase. as the long-term compensation of surviving nephron in the
“three high” state of high blood flow perfusion, high filtra-
Chronic Glomerulonephritis tion, and high transmembrane pressure, which leads to glo-
Chronic glomerulonephritis is referred to proteinuria, hema- merulosclerosis in the long run.
turia, hypertension, and edema as the basic clinical manifes- Pathological types are diverse, mesangial proliferative
tations. The onset of the disease is different, the condition glomerulonephritis including IgA and non-IgA mesangial
of the disease is prolonged, the disease progresses slowly, proliferative glomerulonephritis, mesangial capillary glomer-
and the renal function can be reduced to varying degrees. It ulonephritis, membranous nephropathy, and focal segmental
has the tendency of deterioration of renal function and will glomerulosclerosis are common pathological changes. If the
eventually develop into a group of glomerular diseases with lesions progressed to a later stage, all the above mentioned
chronic renal failure. Because of the different pathological different types of pathological changes can be transformed
types and stages of this group of diseases, the main clinical into glomerular sclerosis with varying degrees of tubular
manifestations may be diverse. atrophy and renal interstitial fibrosis. In the late stage of the
disease, the size of the kidney shrinks and the cortex of the
Signs and Symptoms kidney becomes thinner, the pathological type can be trans-
Chronic glomerulonephritis can occur at any age, but formed into sclerosing glomerulonephritis.
mainly in young and middle-aged men. Most of them have
a slow and insidious onset. The clinical manifestations are Molecular Markers
diverse. Proteinuria, hematuria, hypertension, and edema are Including the serum glomerular filtration markers and uri-
the basic clinical manifestations. They may have different nary glomerular cell injury markers as mentioned in acute
degrees of renal dysfunction. The severity and duration of glomerulonephritis.
the disease can be delayed, and progressively develop into
chronic renal failure. Laboratory examinations were mostly Treatment
mild urinary abnormalities. Urinary protein was often 1–3 g/ The main purpose is to prevent or delay the deterioration of
day. Urinary sediment microscopy showed an increase in red renal function and prevent serious complications. The fol-
blood cells and a tubular pattern. Blood pressure may be nor- lowing comprehensive treatment measures can be adopted.
mal or slightly elevated. Renal function is normal or slightly
impaired (creatinine clearance decreased or mild azotemia), Actively Control Hypertension and Reduce Urinary
which can last for several years or even decades. Renal func- Protein Hypertension and urinary protein are important fac-
tion deteriorates gradually and the corresponding clinical tors to accelerate glomerulosclerosis and promote deteriora-
manifestations (such as anemia, high blood pressure) appear, tion of renal function. Active control of hypertension and
and enter the uremic stage. reduction of urinary protein are two important links. Patients
If blood pressure is not well controlled, renal function with chronic nephritis often have volume-dependent hyper-
deteriorates rapidly and the prognosis is poor. In addition, tension due to sodium retention. Therefore, hypertension
some patients suffer from acute attacks of infection and patients should be restricted to salt (NaCl <6 g/day) and using
fatigue, or acute deterioration after using nephrotoxic drugs. thiazide diuretics such as hydrochlorothiazide. When Ccr is
After prompt removal of incentives and appropriate treat- less than 30 mL/min, loop diuretics should be used instead of
ment, the condition can be alleviated to a certain extent, but thiazides, but they should not be used too much and for a long
it may also lead to irreversible chronic renal failure. Most time. ACEI or ARB not only has the function of lowering
patients with chronic glomerulonephritis have chronic pro- blood pressure but also has the function of reducing urinary
gressive impairment of renal function. Pathological type is protein and delaying the deterioration of renal function. Thus,
an important factor determining the progress of renal func- ACEI or ARB is the preferred drug for treating hypertension
tion (such as mesangial capillary glomerulonephritis and and/or reducing urinary protein in chronic nephritis. And in
membranous nephropathy), but it is also related to reason- order to achieve the goal of reducing urinary protein, the dos-
able treatment. age used is usually higher than the conventional hypotensive
562 Z. Zheng et al.
dose. Hyperkalemia should be prevented in patients with Massive Proteinuria Nephrotic-range proteinuria is typi-
renal insufficiency when ACEI or ARB is used, and when cally defined as greater than 3–3.5 g of protein in a 24-h
serum creatinine is greater than 264 μmol/L, it is necessary to urine collection.
closely monitor serum creatinine and potassium to prevent
side effects. In addition, beta-blockers and calcium channel Hypoalbuminemia Patients with hypoproteinemia lose a
blockers can be combined or selected. large amount of albumin in the body, the plasma total protein
is less than 50 g/L, and albumin is less than 30 g/L.
Limit Protein and Phosphorus Intake in Food Patients
with renal insufficiency and nitrogen deficiency should limit Hyperlipidemia When the symptoms of hyperlipidemia
the intake of protein and phosphorus, use high-quality low are lighter, there are occasional dizziness, numbness, palpi-
protein diet or add essential amino acids or alpha-keto acids. tations, etc., often accompanied by physical obesity; When
heavier, it appears as a headache, palpitation, fatigue, and
Glucocorticoids and Cytotoxic Drugs In view of the fact squint; Long-term can cause coronary heart disease, stroke,
that chronic nephritis includes many diseases, it is advisable angina and so on.
to differentiate the use of such drugs. However, patients with
normal renal function or only slightly impaired, normal renal Causes
volume, mild pathological type (such as mild mesangial pro- Primary nephrotic syndrome has no background diseases,
liferative nephritis, early membranous nephropathy, etc.), whereas secondary nephrotic syndrome has many back-
higher urinary protein, and have no contraindication, it can ground diseases. Secondary NS may result from diverse
be tried and gradually withdrawn if invalid. underlying causes, including renal diseases such as minimal
change nephropathy, focal and segmental glomerulosclero-
Anticoagulant, Fibrinolytic, and Antiplatelet Depolymer- sis, membranous nephropathy, IgA nephropathy, or a con-
ization Drugs These drugs can inhibit fibrinogenesis, genital predisposition. Alternatively, nephrotic syndrome
platelet aggregation and reduce complement activity, but the may develop due to diabetes, amyloidosis, viral infections,
efficacy is uncertain. malaria, pre-eclampsia, systemic lupus erythematosus, or
other systemic disorders that affect the kidneys. Immune
Avoid Factors that Aggravate Kidney Damage Avoid complex injury of the glomerulus by cancer antigens may
infections, fatigue, pregnancy, and nephrotoxic drugs (such cause membranous nephropathy. It has also been associated
as aminoglycoside antibiotics, aristolochic acid containing with nonsteroidal anti-inflammatory drugs, gold, lithium,
traditional Chinese medicine, etc.) which may lead to dete- mercury, interferon-b-1a, pamidronate, penicillamine, or
rioration of renal function. heroin use [41].
Nephrotic Syndrome Pathogenesis

Nephrotic syndrome (NS) refers to excessive proteinuria, Minimal Change Nephrotic Syndrome Most common
with associated hypoalbuminemia, edema, and hyperlip- pathology found in childhood (77–85%). Usually idiopathic.
idemia. It is caused by increased permeability through the Light microscopy of renal biopsy samples shows no change,
damaged basement membrane in the renal glomerulus espe- on electron microscopy, effacement of the foot processes can
cially infectious or thrombo-embolic. It is the result of an be seen. Immunofluorescent staining for immune complexes
abnormality of glomerular permeability that may be primary is negative.
with a disease specific to the kidneys or secondary to con-
genital infections, diabetes, systemic lupus erythematosus, Focal Segmental Glomerulosclerosis 10–15% of cases.
neoplasia, or certain drug use. Light microscopy of renal biopsy sample shows scarring, or
sclerosis, of portions of selected glomeruli which can prog-
Signs and Symptoms ress into global glomerular sclerosis and tubular atrophy. In
Edema The predominant symptom of NS is edema. In the most cases, negative immunofluorescence.
early phase, edema appears in local parts such as the eyelids;
in the advanced phase, patients may develop periorbital or Membranoproliferative Glomerulonephritis More com-
genital edema, ascites, or pleural or pericardial effusion. monly presents as nephrotic syndrome. Involves immune
Persons who present with new edema or ascites, without complex deposition. Immunofluorescence staining shows
typical dyspnea of congestive heart failure or stigmata of cir- the granular pattern. On light microscopy, can see thickened
rhosis, should be assessed for nephrotic syndrome. basement membrane.
Membranous Glomerulonephritis Just 2–4% of cases in may be low). Tests are best done as indicated by clinical
children, but the most common type in adults. Thickened context. Consider: Serum glucose or glycosylated Hb
basement membrane and granular pattern on immunofluo- (HbA), antinuclear antibodies, hepatitis B and C serologic
rescence. On electron microscopy, characteristic “spike and tests, serum or urine protein electrophoresis, cryoglobu-
dome” appearance seen, with membrane deposition growing lins, rheumatoid factor, serologic test for syphilis (e.g.,
around subepithelial immune complex deposition [42]. rapid plasma reagin), HIV antibody test, complement lev-
els (CH50, C3, C4).
Complications
Venous Thrombosis Venous thrombosis is one of the most Ultrasonographic Individuals with a single kidney may be
important complications of NS. The most common sites of prone to developing focal glomerulosclerosis, having only one
venous thrombosis in adults are in the deep veins of the kidney is also a relative contraindication to kidney biopsy.
lower limbs, although thrombosis can also occur in the renal Ultrasonography also demonstrates renal echogenicity.
veins and can cause pulmonary embolism. Arterial thrombo- Increased renal echogenicity is consistent with intrarenal
sis is rare in patients with NS. fibrosis.
Infection Loss of immunoglobulins in the urine may Renal Biopsy Indicated for the following: congenital
increase the risk of infection, with susceptibility to bacteria nephrotic syndrome, children older than 8 years at onset, ste-
or viral infections such as varicella. roid resistance, frequent relapses or steroid dependency, sig-
nificant nephritic manifestations.
Renal Failure Acute kidney injury is considered a rare
spontaneous complication of NS. It can coexist with NS Treatment
when it is caused by the same factors that lead to edema and General Treatment Measures Creating a negative sodium
proteinuria, such as lupus nephritis and drug-induced inter- balance will help reduce edema, presumably as the underly-
stitial nephritis. ing illness is treated or as renal inflammation slowly
resolves. Patients should limit their sodium intake to 3 g/day
Hyperlipidemia Elevated lipid levels are a common feature and may need to restrict fluid intake (to less than approxi-
of NS. Proteinuria also causes hypoproteinemia that stimu- mately 1.5 L/day.
lates lipoprotein synthesis by the liver and decreased lipid
catabolism because of reduced lipoprotein lipase. Serum Diuretics Diuretics are the mainstay of medical manage-
total cholesterol, very low-density lipoprotein, intermediate- ment; however, there is no evidence to guide drug selection
density lipoprotein, low-density lipoprotein, and lipoprotein or dosage. Patients with nephrosis are resistant to diuretics,
(a) may all be increased in nephrotic syndrome. Clearance of even if the glomerular filtration rate is normal. Loop diuret-
these atherogenic lipoproteins is also impaired by dysfunc- ics act in the renal tubule and must be protein-bound to be
tion of receptor-mediated mechanisms. effective. Serum proteins are reduced in NS, limiting the
effectiveness of loop diuretics, and patients may require
Laboratory Diagnosis higher-than-normal doses. Other mechanisms for diuretic
Urine Tests Nephrotic-range proteinuria will be apparent resistance are also possible. Oral loop diuretics with twice-
by 3+ or 4+ readings on the dipstick, or by semiquantitative daily administration are usually preferred because of the lon-
testing by sulfosalicylic acid. A 3+ reading represents ger duration of action. However, with severe NS and edema,
300 mg/dL of urinary protein or more, which correlates with gastrointestinal absorption of the diuretic may be uncertain
a daily loss of 3 g or more and thus is in the nephrotic range. because of intestinal wall edema, and intravenous diuretics
Urine samples over 24 h (for an accurate measurement), pro- may be necessary. If there is still an inadequate clinical
teinuria (3 g protein) is diagnostic [43]. response, patients may be treated by changing to intravenous
loop diuretics, adding oral thiazide diuretics, or giving an
Blood Tests The serum albumin level is classically low in intravenous bolus of 20% human albumin prior to an intrave-
nephrotic syndrome, serum albumin often is <25 g/L. Creatinine nous diuretic bolus [44].
concentrations vary by degree of renal impairment. Total cho- Diuresis should be relatively gradual and guided by daily
lesterol and triglyceride levels are typically increased. weight assessment, with a target of 1–2 kg/day [45]. This
may be clinically important if over-rapid diuresis leads to
Serologic Studies Role of testing for secondary causes acute renal failure from reduced glomerular blood flow,
of nephrotic syndrome (is controversial because yield despite persistent edema [46].
564 Z. Zheng et al.
ACE Inhibitors Angiotensin-converting enzyme (ACE) Persistent Hematuria Without Edema, Proteinuria Usually,
inhibitors have been shown to reduce proteinuria and reduce gross hematuria appears after the upper respiratory tract
the risk of progression to renal disease in persons with infection and lasts several hours to a few days, maybe associated
nephrotic syndrome. The recommended dosage is unclear. with abdominal pain, low back pain, muscle pain, or low fever.
Most persons with nephrotic syndrome should be started on
ACE inhibitor treatment to reduce proteinuria, regardless of Asymptomatic Proteinuria with or Without Hematuria
blood pressure. Most patients with IgAN show mild proteinuria and 24-h urine
protein quantitation <1 g.
Corticosteroids Treatment with corticosteroids remains
controversial in the management of nephrotic syndrome in Acute Nephritis Syndrome Some patients of IgAN show
adults. It has no proven benefit but is recommended in some hematuria, proteinuria, edema, hypertension in the clinic.
persons who do not respond to conservative treatment. But it
is more clearly established that children respond well to cor- Nephrotic Syndrome A small number of patients can be
ticosteroid treatment [47]. Classically, minimal change dis- characterized by massive proteinuria, Hypoalbuminemia,
ease responds better to corticosteroids than FSGS; however, high edema, hyperlipidemia syndrome.
this difference is found primarily in children with nephrotic
syndrome. Causes
IgAN is an autoimmune disease, causing antibody-medi-
Lipid-Lowering Therapy Some evidence suggests an ated destruction of the glomerular basement membrane. It
increased risk of atherogenesis or myocardial infarction in is hypothesized that mucosal infection is the culprit of the
persons with nephrotic syndrome, possibly related to disease as most patients develop gross hematuria from IgA
increased lipid levels. However, the role of treatment for nephropathy after upper respiratory tract infections, specifi-
increased lipids is unknown and, at present, the decision to cally pharyngitis. However, most cases of IgA nephropathy
start lipid-lowering therapy in persons with nephrotic syn- are idiopathic.
drome should be made on the same basis as in other patients.
Pathogenesis
Antibiotics Therapy Antibiotics should be used in cases of The main processes leading to the pathogenesis of IgAN are
infection. increased production of IgA1, defective galactosylation of
IgA1, binding of antibodies against the galactose-deficient
Anticoagulation Therapy The decision to treat with anti- IgA1 with an accumulation of immune complexes in the
coagulants should be made individually. Anticoagulation is mesangium, and activation of the mesangial cells. Patients
not routinely used for the primary prevention of thrombotic with IgA nephropathy have excess deposits of galactose-
events in patients with NS. Adult patients with NS should be deficient IgA1 in their serum and glomerular mesangium.
assessed individually for underlying disease. Additional con- The galactose-deficient immunoglobulin A1 has less galac-
siderations are the severity of NS (i.e., serum albumin less tose in the hinge region of the heavy chains and, subse-
than 20–25 g/L may be more likely to prompt anticoagula- quently, is recognized as a neo-antigen, triggering the
tion prophylaxis), preexisting thrombophilic states, and the formation of autoantibodies and circulating immune com-
overall likelihood of serious bleeding events from the use of plexes that accumulate in the mesangial cells. This mesan-
oral anticoagulation. gial deposition causes the release of proinflammatory and
pro-fibrotic mediators and results in mesangial proliferation,
IgA Nephropathy extracellular matrix synthesis, and podocyte damage. These
IgA nephropathy (IgAN) also known as Berger disease, is mesangial-induced mediators are also filtered in the urine
considered an immune complex-mediated disease and typi- and activate proximal tubular epithelial cells, causing tubu-
cally affects young adults, but can also occur in children and lointerstitial scarring [48].
the elderly. The disease presents with gross hematuria and Histologically, IgAN is characterized by: diffuse prolifer-
upper respiratory infection, with slow progression to end- ation of mesangial cells and matrix; hypercellular or normal
stage renal disease in up to 50% of affected patients. It also glomeruli with diffuse necrotizing crescentic glomerulone-
can be accompanied by varying degrees of proteinuria. The phritis; mesangial involvement resembling focal and seg-
diagnostic hallmark of IgAN is the predominance of IgA mental glomerulosclerosis. The most common abnormality
deposits in the glomerular mesangium. is mesangial hypercellularity. Immunofluorescence showed
IgA deposition in the mesangial area, or with capillary wall
Signs and Symptoms distribution, often accompanied by C3 deposition. Under the
The clinical manifestations of IgAN are mainly of four types: electron microscope, dense matter is mainly deposited in the
mesangial area, sometimes with a large cluster shape, which component of the kidney and are vulnerable to a variety of
has important diagnostic value. injuries including hypoxia, proteinuria, toxins, metabolic
disorders, and senescence. It has long been believed that
Laboratory Diagnosis tubules are the victim of injury.
Urine Tests Generally, proteinuria is not heavy, but about
15% of cases can present a large amount of proteinuria. Signs and Symptoms
Urine sediment examination, hematuria accounted for almost The main function of the renal tubule is the proximal tubular
100%. reabsorption function, as well as the urine concentration and
dilution function of the distal renal tubule. The reabsorption
Serum Immunology IgA is elevated in about 40% of of glucose, amino acids, and phosphorus is in the proximal
patients; IgA rheumatoid factor (IgA-RF) is positive; IgA- tubules. Therefore, the renal tubular urinary tract can occur
fibronectin polymer (IgA-FN) is positive; Immune com- in renal tubular injury. Urine routine tests show that urine
plexes of IgA type can also be increased. sugar is positive and blood sugar is normal. Amino aciduria,
phosphate urine, hypokalemia, hyperuricemia, hypocalce-
Blood Tests The main manifestation is that the GFR is mia, hyperuricemia, metabolic alkalosis, and hypertension
reduced, and BUN and Scr are slowly increased. may also occur. Low specific gravity urine, nocturia, renal
tubular acidosis, etc. may also occur.
Renal Biopsy Diagnosis of IgAN depends on biopsy find-
ings, particularly by immunofluorescence microscopy. Causes and Pathogenesis
In response to injury, tubular epithelial cells undergo changes
Treatment and function as inflammatory and fibrogenic cells, with the
Treatment options are patients dependent based on clinical consequent production of various bioactive molecules that
findings and disease evolution. drive interstitial inflammation and fibrosis. Innate immune-
sensing receptors on the tubular epithelium also aggravate
General Treatment Prevent colds and overwork. immune responses. Necroinflammation, an autoamplification
Nephrotoxic drugs should be used carefully. Diet should loop between tubular cell death and interstitial inflammation,
consist of adequate protein. Salt intake is restricted to control leads to the exacerbation of renal injury. Furthermore, tubu-
blood pressure. The blood pressure target is 130/80 mmHg. lar cells also play an active role in progressive renal injury
via emerging mechanisms associated with a partial epithe-
Antibiotics Therapy Antibiotics should be used in cases of lial-mesenchymal transition, cell-cycle arrest at both G1/S
infection. and G2/M checkpoints, and metabolic disorder.
Renal tubular damage can be caused by direct factors,
Immunosuppressive Therapy Prolonged immunosuppres- secondary factors (glomerular or vascular).
sive therapy with cytotoxic agents and prednisone may ben-
efit a subgroup of patients with progressive IgAN [49]. 1. Renal tubular epithelial cells (TEC) have strong self-
renewal ability, with damage factors removed, TEC can
Blood Purification Therapy Patients with acute renal fail- be quickly repaired.
ure or who have formed chronic renal failure need hemodi- 2. Renal tubular cells are not only passive victims but also
alysis or peritoneal dialysis. can synthesis and release a variety of biological active
factors through TEC activation, then recruit inflammatory
Renal Transplantation For the few who progress to cells in the tubules, amplify the inflammatory cascade,
develop end-stage renal disease (ESRD), renal transplanta- leading to TEC apoptosis, necrosis, extracellular
tion is an option. But IgAN recurrence rate is high after Increased matrix production, eventually renal dysfunc-
transplantation. tion and renal fibrosis.
3. The TEC that cannot be repaired normally can cause cell
Others Conservative therapy with angiotensin-converting cycle arrest, and the blocked TEC secretes a large amount
enzyme inhibitor or angiotensin receptor blocker to slow of factors such as TGF-β and connective tissue growth
proteinuria is recommended in all patients [50]. factor (CTGF) to promote the formation of renal
fibrosis.
36.2.2.2 Tubular Injury
Kidney is composed of more than one million nephrons. As shown in Table 36.1 and Fig. 36.4, a variety of cyto-
Each nephron consists of three parts: glomerulus, Bowman’s kines have been shown to be produced by activated TECs,
capsule, and tubules (Fig. 36.4). Renal tubules are the major including IL-1β [51, 52], IL-18 [51, 53], IL-15 [54, 55],
566 Z. Zheng et al.
Table 36.1 List of pro-inflammatory cytokines produced by TECs performance characteristics of urine sediment examination
Cytokines and its inter- and intraobserver variability have not been well
effects Cytokines effects characterized. The presence of specific findings in urine sedi-
IL-1b Triggers pro-inflammatory cytokines and initiates ment can aid in the diagnosis of acute and chronic kidney
acute-phase responses disease prior to more invasive testing. Examination of urine
IL-18 Triggers pro-inflammatory cytokines
sediment can help discriminate proliferative glomerular dis-
IL-6 Pro-inflammation
ease from nonproliferative diseases. Hematuria and red blood
IL-15 CD103+ T cell recruitment
IL-16 CD4+ T cell recruitment cell casts are a sensitive early sign of relapse inpatients with
IL-34 Neutrophil and macrophage recruitment lupus nephritis. White blood cell casts may be seen in tubu-
TNF-a Triggers pro-inflammatory cytokines lointerstitial nephritis, but also may be seen in glomerulone-
Innate and adaptive immunity apoptosis phritis and pyelonephritis. Muddy brown casts, granular
CSF-1 Macrophage recruitment and adhesion casts, and kidney tubular epithelial cells and casts can indi-
Polarization into an M2 phenotype
cate acute tubular injury but can be seen in a variety of other
TWEAK Cell death in the presence of
TNF-α/IFN-β Pro-inflammation
acute and chronic kidney diseases [72].
Fas ligand Apoptosis However, the diagnostic utility of urine sediment analy-
CTGF Triggers pro-inflammatory cytokines sis results in clinical practice is uncertain due to a lack of
VEGF Macrophage recruitment standardization in reporting of results and the potential for
significant inter- and intraobserver variability. Most studies
of the performance of urine sediment involve clinicians with
IL-16 [56], TNF-α [57], TWEAK [58, 59], Fas ligand [60, many years of experience and an interest in examining urine
61], CTGF [62, 63], and vascular endothelial growth fac- sediment. This may contrast with the experience of the gen-
tor [64–66]. Recently, emerging evidence indicates that the eral population of practicing nephrologists, internists, and
expression of colony-stimulating factor 1 is upregulated in trainees. There is a need for a single standard for reporting
TECs and may be responsible for the polarization of renal urine sediment analysis results that correlates with results
macrophages and recovery from AKI [67–69]. TEC-derived of kidney biopsies and clinical outcomes. Future studies are
IL-34 also plays a key role in aggravating macrophage infil- needed to quantify the diagnostic characteristics of urine
tration and tubular cell injury, leading to persistent ischemic sediment examination in a variety of kidney diseases and
acute kidney injury (AKI) and subsequent chronic kidney assess the inter- and intraobserver reliability [73].
disease (CKD) [70]. These findings support the notion that
activated TECs gain the inflammatory phenotype that drives α1-Microglobulin α1-Microglobulin is a low-molecular-
the immune response by producing inflammatory cytokines weight glycoprotein of approximately 27–30 kDa and a
directly in an autocrine manner or indirectly through the member of the lipocalin superfamily [74]. It is primarily syn-
infiltrating leukocytes in a paracrine manner. thesized in the liver and is available both in free form and as
a complex with IgA. α1-Microglobulin has been detected in
Molecular Markers human serum, urine, and cerebrospinal fluid. Urine and
Microscopic examination of the urine has been used for serum values have been found to be elevated in patients with
many years to gain insight into the severity of glomerular renal tubular diseases [74]. α1-Microglobulin is freely fil-
and tubular injury. Other components of the urine have been tered at the glomerulus and completely reabsorbed and
used to quantitate tubular cell injury in a more specific and catabolized by the normal proximal tubule. Megalin medi-
sensitive fashion. These markers have been demonstrated ates the uptake of this protein in the proximal tubule.
to be extremely valuable in detecting kidney injury in the Therefore, an increase in the urinary concentration of α1-
setting of AKI. Moreover, some of these biomarkers, such microglobulin indicates proximal tubular injury or dysfunc-
as interleukin-18 (IL-18), kidney injury molecule-1 (KIM- tion. The urinary levels of α1-microglobulin are influenced
1), neutrophil gelatinase-associated lipocalin (NGAL), and by age. The normal range in populations younger than
liver-type fatty acid-binding protein (L-FABP), have been 50 years is less than 13 mg/g of creatinine and in those
shown to be potentially useful in a variety of contexts in both 50 years or older is less than 20 mg/g of creatinine. In com-
acute and chronic kidney injury. Here, the utility of urine parison with β2-microglobulin, α1-microglobulin is more
microscopy is described briefly and some of the emerging stable over a range of pH levels in the urine, making it a more
biomarkers of tubular injury are discussed. acceptable urinary biomarker.
Urine Microscopy Examination of urine sediment is a time- β2-Microglobulin β2-Microglobulin is a low-molecular-

honored test relied on by generations of nephrologists to aid weight polypeptide with a molecular weight of 11.8 kDa. It
in the diagnosis of kidney diseases [71]. The actual diagnostic is present on the cell surfaces of all nucleated cells and in
most biologic fluids, including serum, urine, and synovial injury both as a result of leakage from cells (preformed
fluid [75]. β2-Microglobulin is normally excreted by glomer- enzymes) or by upregulation of their production in response
ular filtration, reabsorbed almost completely (approximately to injury. Because they are localized to specific cells along the
99%), and catabolized by the normal proximal tubule in nephron, the presence of certain enzymes in urine can give a
humans. Megalin mediates the uptake of this protein in the clue to the specific location of the kidney injury. N-Acetyl-
proximal tubule. In healthy individuals, approximately 150– glucosaminidase (NAG) is a lysosomal enzyme primarily
200 mg of β2-microglobulin is synthesized daily with a nor- localized to the proximal tubule [78]. Glutathione- S-
mal serum concentration of 1.5–3 mg/L [76]. Any pathologic transferase (GST) is a family of enzymes with eight different
state that affects kidney function results in an increase in β2- classes found throughout the nephron [79]. However, the α
microglobulin levels in the urine because of the impeded isoform is present in proximal tubular cells alone, whereas
uptake of β2-microglobulin by renal tubular cells. For spot the π isoform is found in only distal tubular cells. Alkaline
urine collections, the concentration of β2-microglobulin in phosphatase, GGT, alanine aminopeptidase, and lactate
healthy individuals is typically 160 μg/L or less or 300 μg/g dehydrogenase are brush-border enzymes originating in
of creatinine or less. Unlike serum levels of urea, those of the proximal tubule that normally are present in urine in
β2-microglobulin are not influenced by food intake, making small quantities and increase significantly in the setting of
this polypeptide an attractive marker for malnourished AKI. Tubular enzymeuria has been noted in patients with
patients with low serum urea levels. In patients with CKD, a broad range of kidney diagnoses, including acute tubular
increases in serum β2-microglobulin levels reflect the necrosis, interstitial nephritis, nephrotoxicity, and acute
decrease in glomerular function. In patients with ESKD, transplant rejection. No individual enzyme has been shown
serum levels of β2-microglobulin are usually in the range of to be a consistent predictor of AKI or need for dialysis
20–50 mg/L. β2-Microglobulin accumulation is linked to across all studies, and although some have shown promise in
toxicity because the molecule precipitates and forms fibril- certain homogenous populations, these results have not been
lary structures and amyloid deposits, particularly in bone and replicated across heterogeneous groups. This may be due in
periarticular tissue, leading to the development of carpal tun- part to the fact that enzymuria is increased in many settings
nel syndrome and erosive arthritis. Elevations of β2- and is not specific for AKI. There also may be settings in
microglobulin have been reported in several AKI and CKD which activity of the enzymes is decreased by exogenous
clinical settings, including in cadmium toxicity and follow- factors, for example, although NAG has been shown to be
ing cardiac surgery, liver transplantation, and renal trans- a sensitive marker of AKI in certain situations, the presence
plantation. In idiopathic membranous nephropathy, of heavy metals and other nephrotoxins in urine inhibits
β2-microglobulin level was identified as a superior indepen- its activity, reducing the sensitivity of the assay. There is a
dent predictor of the development of renal insufficiency. suggestion that panels of enzymes may be more useful than
Other studies have reported that β2-microglobulin performs individual enzymes in diagnosing kidney disorders, although
as well as, if not better than, serum creatinine for the detec- their primary use may lie in the diagnosis of drug nephrotox-
tion of acute kidney injury in critically ill children or after icity, in which levels of these enzymes increase prior to any
cardiac surgery in adults. increase in creatinine level and may help guide drug dosing.
Serum concentrations of β2-microglobulin should be inter-
preted cautiously because they are altered significantly in Interleukin-18 IL-18 is an 18 kDa pro-inflammatory cyto-
various diseases, including rheumatoid disorders and several kine that is expressed primarily by macrophages, but also by
types of cancers. Initially, it was believed that the increase in monocytes, dendritic cells, and kidney epithelial cells. It has
β2-microglobulin levels in CKD is solely due to declines in a role in the innate and adaptive immune response and is
kidney function, but later studies have shown that other fac- upregulated in inflammatory states. IL-18 is thought to be
tors, including increased synthesis of β2-microglobulin, may one of the mediators of injury in ischemic AKI. Inhibition of
contribute in patients with ESKD. Another significant draw- IL-18 has been shown to be effective in treating inflamma-
back associated with the use of urinary β2-microglobulin as a tory disorders in mice, and interstitial IL-18 expression is
marker of kidney injury is its instability in urine at room tem- increased in mouse models of AKI. Mice deficient in cas-
perature, particularly when the pH is less than 5.5; for this pase, an IL-18-activating enzyme, develop less acute tubular
reason, the urine should be alkalinized and frozen at −80 °C necrosis than wild-type mice in models of ischemia. Urinary
immediately after collection [77]. IL-18 originates in the kidney tubular epithelium and thus
was proposed as a potential marker of AKI [80].
N-Acetyl-β-d-glucosaminidase and Glutathione-S-trans‑ IL-18 level has been shown to increase early in patients
ferase The kidneys contain large amounts of enzymes with sepsis in the intensive care unit and was a good predic-
that perform specialized functions in tubular cells. These tor of AKI, particularly when combined with NGAL level.
enzymes can be released into the urine in the event of kidney IL-18 levels increased in patients with acute tubular necrosis,
568 Z. Zheng et al.
but not those with prerenal AKI or CKD and healthy con- been reported: liver (L), intestinal (I), muscle and heart (H),
trols. Similarly, increasing IL-18 levels predicted AKI in epidermal (E), ileal (I1), myelin (M), adipocyte (A), brain
pediatric patients undergoing cardiac surgery. Higher post- (B), and testis (T). L-FABP is a cytoplasmic protein expressed
operative urinary IL-18 excretion also has been shown to in proximal tubules that binds free fatty acids and transports
identify AKI and predict the progression of AKI after cardiac them to the mitochondria for metabolism. Increased urinary
surgery in adults. Potential limitations of IL-18 derive from L-FABP excretion has been noted in patients with AKI (par-
the fact that it may be a more generalized marker of inflam- ticularly ischemic), nephrotoxicity, and severe sepsis in the
mation rather than a specific marker of AKI, particularly in absence of AKI [84, 85].
older age groups, for who there may be underlying baseline
decreased kidney function. Netrin-1 Netrin-1 is a 50–75 kDa, laminin-like protein, ini-
tially recognized as a chemotropic factor, that plays an essen-
Kidney Injury Molecule-1 Kidney injury molecule-1 tial role in guiding neurons and axons to their targets. Studies
(KIM-1 in humans, Kim-1 in rodents), which is also referred have now revealed diverse roles of netrin-1 beyond axonal
to as T cell immunoglobulin and mucin domains-containing guidance, including development of various organs, angio-
protein-1 (TIM-1) and hepatitis A virus cellular receptor-1 genesis, adhesion, tissue morphogenesis, inflammation, and
(HAVCR-1), is a type I transmembrane glycoprotein with an tumorigenic processes. Netrin-1 is expressed in several tis-
ectodomain containing a six-cysteine immunoglobulin-like sue types, including brain, lung, heart, liver, intestine, and
domain, two N-glycosylation sites, and a mucin domain. kidney. It is an axonal-guidance molecule that is not
KIM-1 initially was found to be highly upregulated in a rat expressed in normal tubular cells but is highly expressed
model of ischemic kidney injury [81]. It is a transmembrane after kidney injury, with levels increasing within 2 h of an
protein that is expressed at very low levels in normal kidney, acute insult. Levels correlate with the degree of injury and
but its production increases in dedifferentiated proximal return to normal as the injury resolves. Netrin-1 seems to be
tubular cells in the presence of ischemic or nephrotoxic a promising early biomarker for AKI, but additional studies
AKI. The extracellular domain of KIM-1 appears in urine need to be conducted in larger cohorts with AKI due to vari-
shortly after ischemic injury and can be detected readily by a ous causes to further evaluate its potential [86, 87].
new KIM-1 urinary dipstick, potentially making it a conve-
nient and readily measured marker of AKI [82]. Neutrophil Gelatinase-Associated Lipocalin Neutrophil
Early studies of humans found that, in common with the gelatinase-associated lipocalin (also known as lipocalin 2 or
rat, KIM-1 expression was increased in kidney biopsy speci- lcn2) is one of the biomarkers of AKI that has been studied
mens from patients with acute tubular necrosis. Elevated extensively [88]. It is involved in innate immunity and is
urinary KIM-1 level predicted increased risk of mortality or expressed primarily by immune cells, but also by hepato-
need for dialysis in hospitalized patients with AKI and was cytes and kidney tubular cells. It originally was identified
a sensitive predictor of AKI in children undergoing cardiac through transcriptome analysis in a mouse model of
surgery. Its performance as a biomarker of AKI may prove to ischemia-reperfusion injury in which its production in kid-
be better in patients with normal baseline kidney function, as ney tubules was noted to increase rapidly after kidney injury.
is the case for many novel biomarkers. This may be because It is readily detectable in urine and resistant to degradation
although expression is very limited in normal kidney tissue, by proteases, making it a potentially ideal biomarker of
there is upregulation of expression in various CKDs, with the AKI. NGAL appears in urine early after injury, and studies
degree of expression correlating with tubulointerstitial fibro- of cultured human tubular cells confirmed that it is produced
sis. Its role in CKD currently is under investigation [81, 82]. in response to hypoxic injury. Urinary and plasma NGAL
levels have been shown to correlate with the degree of kidney
Liver-Type Fatty Acid-Binding Protein Urinary fatty injury, and levels return to baseline on resolution of
acid-binding protein 1 (FABP1) has been proposed to be a AKI. Interestingly, the source of plasma and urinary NGAL
useful biomarker for early detection of AKI and monitoring appears to be different. Most urinary NGAL is produced in
of CKD. Also known as L-type or liver-type fatty acid- tubules in response to injury, whereas most plasma NGAL
binding protein (L-FABP), which will be used in this book, originates in distant organs, where its production is upregu-
FABP1 was first isolated in the liver as a binding protein for lated in the setting of AKI. Commercial assays are available
oleic acid and bilirubin. FABP1 binds selectively to free fatty for NGAL measurement [89].
acids and transports them to mitochondria or peroxisomes, Urinary NGAL has been shown to be a possible early bio-
where free fatty acids are β-oxidized and participate in marker of AKI in adults and children after cardiac surgery
intracellular fatty acid homeostasis [83]. There are several and after administration of radiocontrast. Similarly, eleva-
different types of FABP, which are ubiquitously expressed in tions of NGAL levels predict AKI in patients presenting to
a variety of tissues. At this time, nine different FABPs have the emergency department. A large meta-analysis of stud-
ies of NGAL in AKI found that there was no advantage in IGFBP-7 remained the top two performing biomarkers for
using urinary NGAL as opposed to serum NGAL. NGAL the prediction of RIFLE Injury or Failure stage within the
performed better in children, although it remained useful in first 12–36 h of study enrollment, providing AUCs of 0.77
adults, suggesting that there may be other factors that influ- and 0.75, respectively. When these two biomarker values
ence NGAL levels in this population. A wide range of cutoff were multiplied together, they demonstrated an improved
values for urinary NGAL was used in the various studies, but ability to detect this same endpoint (AUC = 0.80). A com-
a level 150 ng/mL appeared to be the most appropriate, par- mercial assay for these biomarkers is currently available in
ticularly when commercial assays were used. Interestingly, Europe, but the TIMP-2 and IGFBP-7 test has not been
normalization of results to urinary creatinine level did not approved for clinical use in the United States.
appear to affect the test accuracy. Elevated NGAL level in
the absence of elevated serum creatinine level may be prog-
nostically significant and suggests that creatinine level may 36.3 Kidney Cancer
misclassify individuals with currently subclinical kidney dis-
ease. This was borne out by a meta-analysis that found that Ruixia Yang and Gaoxia Ge
elevations in NGAL level even in the absence of an elevated
creatinine level predicted worse outcomes. This finding sug- This section covers the common malignant tumors of the
gests that there may need to be further changes in the way kidney.
that we define AKI to incorporate these biomarkers.
Albuminuria Proteinuria has long been recognized as a 36.3.1 Overview

consequence of kidney damage. Measurement of urinary
protein and urinary albumin is a central component of screen- 36.3.1.1 Summary of Renal Tumors
ing for and monitoring CKD. The glomerulus acts as a bar- Renal tumors are one of the most common tumors in the
rier to filtration due to pore size and charge selectivity, while urinary system. European and American countries have a
most filtered albumin is reabsorbed in the proximal tubule. higher incidence than Asian countries. The morbidity in
Thus, the presence of albuminuria usually indicates damage urban areas is higher than that in rural areas. Danish and
to the filtration barrier, but also may be the result of proximal Swede had the highest incidence. Japanese and Yugoslavs
tubular dysfunction. have the lowest incidence. Renal tumors can occur at any
Multiple studies have demonstrated that albuminuria is an age. In recent years, the incidence of renal tumors has been
independent risk factor for mortality in the general popula- on the rise. Because the early symptoms of renal tumors
tion. Albuminuria also is an independent risk factor for the are not obvious, nearly 30% of the patients had reached the
progression of diabetic and nondiabetic CKD. As a result, it late stage when they found out and missed the best time for
has been proposed that stage 3 CKD be stratified further by treatment.
the presence or absence of albuminuria. Reduction in albu- The etiology of renal tumors is unknown. According to
minuria is a commonly accepted goal of treatment in patients statistics, smokers have a higher incidence than non-smokers,
with CKD, although it is not as yet an acceptable surrogate as well as a higher incidence of cadmium exposure in occu-
endpoint in clinical trials. However, the US Food and Drug pations and family genetic predisposition. Renal tumors are
Administration (FDA) recently qualified its use as a bio- more common in patients with retinal vascular disease or
marker of proximal tubular injury from nephrotoxin expo- polycystic kidney disease.
sure in animal studies.
36.3.1.2 T he Classification of Malignant Kidney
TIMP-2 and IGFBP-7 Tissue inhibitor metalloproteinase- Diseases
2 (TIMP-2) and urine insulin-like growth factor-binding
About 95% of renal tumors are malignant, and benign ones
protein-7 (IGFBP-7) have been shown to serve as biomark- are rare. Malignant renal tumors can be classified into two
ers of AKI in the setting of critical illness [90, 91]. They were types according to the characteristics of pathological anat-
originally discovered as part of a three-center discovery omy: renal cell carcinoma and renal pelvic carcinoma.
cohort of 522 subjects. These patients had AKI stemming
from sepsis, shock, major surgery, and trauma. More than
300 potential markers were evaluated, with TIMP-2 and 36.3.2 Renal Cell Carcinoma
IGFBP-7 being the two that best predicted the development
of KDIGO stage 2 or 3 AKI. This finding was then validated Renal cell carcinoma (RCC) is one of the most common
in a prospective international multicenter observational study malignant tumors in the urinary system. It originates from
of 728 subjects. In this validation study, TIMP-2 and the renal parenchymal urotubular epithelial system, thus also
570 Z. Zheng et al.
known as renal adenocarcinoma. It accounts for about 2–3% Traditional Biomarkers for Renal Cell Carcinoma
of adult malignancies and 80–90% of adult renal malignan- Carcinoembryonic antigen (CEA) is a tumor-associated anti-
cies [92]. gen first extracted from colon cancer and embryonic tissues
The etiology of renal cancer remains unclear. by Gold and Freedman in 1965. It is an acidic glycoprotein
Epidemiological studies have shown that smoking, obesity, with the characteristics of a human embryonic antigen. It is a
occupational exposure to carcinogens, hypertension, dia- broad-spectrum tumor marker and can be detected in patients
betes, blood transfusion, radiation, related drugs, food, and with renal cancer.
so on may be related to the pathogenesis of renal cancer.
In addition, chronic kidney disease patients with long-term Novel Molecular Markers for Renal Cell Carcinoma
dialysis are also found to have a high incidence of kidney Transforming Growth Factor β (TGF-β)
cancer. TGF-β is a multifunctional cytokine, which plays an impor-
The typical triad of renal cell carcinoma is hematuria, tant role in embryonic development, wound healing, innate
lumbago, and mass. When renal cancer invades the renal immunity, and tumor progression through the mediation of
pelvis, there is hematuria; pain is mainly caused by enlarge- its receptor. The expression of TGF-β1 was low in the periph-
ment of renal cancer mass and swelling of renal capsule, eral blood of normal people and high in the serum of patients
often blunt pain. The pain caused by renal cancer invading with renal cell carcinoma. The level of TGF-β1 in serum of
surrounding organs and lumbar muscles is relatively severe patients with renal cell carcinoma post-operation was signifi-
and persistent, such as blood clot obstruction of the ure- cantly lower than that pre-operation, suggesting that it may
ter, it is colic. About one-third of the patients had systemic be related to the occurrence of renal cell carcinoma. TGF-β1
symptoms, such as fever, hypertension, rapid erythrocyte can be used as a serological marker of renal cell carcinoma.
sedimentation rate, anemia, abnormal liver function, immu- Measurement of serum TGF-β1 could be used as a valuable
nological abnormalities, abnormal hormone levels, elevated assessment of the disease [93].
urinary polyamines, elevated blood carcinoembryonic anti-
gen (CEA), varicocele, etc. Carbonic Anhydrase IX
The clinical diagnosis of renal cell carcinoma depends Carbonic anhydrase IX is a transmembrane glycoprotein
mainly on imaging examination. Pathological examination is composed of acidic amino acids. It is a member of the
needed for confirmatory diagnosis. With the development of carbonic anhydrase family. Carbonic anhydrase IX is not
medical laboratory technology, more and more biochemical, expressed or underexpressed in most normal tissues but
immunological and molecular markers are used in laboratory highly upregulated in renal clear cell carcinoma [94].
diagnosis of renal cell carcinoma.
PTEN
36.3.2.1 S erological Markers of Renal Cell PTEN is a kind of protein with phosphatase activity encoded
Carcinoma by the PTEN gene [95]. It can antagonize the activity of
Biomarkers of renal cell carcinoma include traditional bio- phosphorylase such as tyrosine kinase and inhibit the occur-
markers and novel molecular markers (Table 36.2). rence and development of tumors. The detection of PTEN is
helpful for clinical assessment of clinical stage and patho-
Table 36.2 Serological markers of renal cell carcinoma logical grade of renal clear cell carcinoma. It is of great sig-
nificance to the diagnosis, treatment, and prognosis of renal
Markers Functions of markers
Traditional CEA Broad spectrum tumor markers,
clear cell carcinoma.
markers increased in serum of patients with
renal cancer B7-H1
New TGF-β High expression of peripheral B7-H1, also known as PD-L1 or CD274, is a cell surface
molecular serum in patients with renal cancer, protein, belonging to the B7 family T cell costimulatory
markers used in the evaluation of the
condition molecules. The positive expression of B7-H1 was positively
Carbonic High expression in renal transparent correlated with the mortality and recurrence rate of patients
anhydrase cell carcinoma with renal clear cell carcinoma. B7-H1 was an independent
IX prognostic factor of renal clear cell carcinoma [96].
PTEN Clinical staging and pathological
classification of renal transparent
cell carcinoma Insulin-Like Growth Factor II mRNA Binding Protein 3
B7-H1 An independent prognostic factor (IMP3)
of renal transparent cell carcinoma Insulin-like growth factor II RNA binding protein 3 (IMP3)
IMP3 Strong positive expression in renal is a member of the insulin-like growth factor RNA binding
transparent cell carcinoma protein family [97]. It is mainly expressed in embryonic and
tumor tissues. IMP3 is considered as a carcinoembryonic pro-

tein. Its expression in renal cell carcinoma is significant, and
its strong positive expression in renal clear cell carcinoma.
Other Laboratory Diagnostic Testings

Other laboratory testings commonly used in diagnosis, man-
agement, and prognosis include measurement of BUN, Cr,
glucose, alkaline phosphatase, lactate dehydrogenase, hemo-
globin, erythrocyte sedimentation rate, and whole blood cell
counting.
36.3.2.2 Typical Case

Clinical Background A patient, female, 79 years old.
Hypoechoic right kidney hilus was found during B-ultrasonic
examination. Then the patient had an enhanced CT examina-
tion of the middle and lower abdomen, and results show that Fig. 36.5 Pathology of renal cell carcinoma (×200)
the right kidney is occupied. There is a high risk of kidney
cancer. The patient feels sore in the waist after tiredness. No
frequent urination, urgent urination, and pain; no hematuria; in patients with interstitial nephritis are often the predispos-
no lumbago, no fever, no cough and expectoration; no chest ing factors for renal pelvic cancer. Renal pelvis squamous
tightness and palpitation. The patient came to Jiangsu cell carcinoma is rare. It is associated with long-term urinary
Province Hospital for further diagnosis and treatment. stones, infection, and other risk factors.
The early symptoms of renal pelvic cancer are pain-
Physical Examination T 36.7 °C, P 68 times/min, R 18 less gross hematuria. A few patients may suffer from
times/min, BP 127/60 mmHg. No percussion pain of double lumbar discomfort, dull pain, and distension after obstruc-
kidney area. No tenderness in double ureteral region. tion of the junction of the pelvis and ureter by tumors.
Occasionally, renal colic may be caused by coagula-
Diagnosis and Treatment Process After admission, rele- tion or tumor abscission, hydrocele due to enlargement
vant examinations were carried out. The GFR of the left or obstruction of tumors, lumbar mass is rare, and a few
kidney is 19.8 mL/min. The GFR of the right kidney is patients have urine road irritation, anemia, and cachexia
28.5 mL/min. No contraindications were found in other in late-stage patients.
examinations. Because the function of the left kidney is The clinical diagnosis of renal pelvic carcinoma mainly
very poor, a partial right nephrectomy is recommended. depends on imaging examination. Pathological examination
Postoperative pathological results showed clear cell carci- is needed for confirmation. With the rapid progress of labora-
noma of the right kidney, the size of the tumor is tory diagnostics, more and more biomarkers are used in the
3 cm × 2 cm × 1.1 cm, local hemorrhage, and cystic change laboratory diagnosis of renal pelvic cancer.
of tumor. See in Fig. 36.5.
36.3.3.1 Biomarkers of Renal Pelvic Carcinoma
This case was from the First Affiliated Hospital of Nanjing Biomarkers of renal pelvic carcinoma include traditional
Medical University (also named Jiangsu Province Hospital). markers and novel molecular biomarkers (Table 36.3).
Traditional Biomarkers for Renal Pelvic Carcinoma

36.3.3 Renal Pelvis Carcinoma β2 Microglobulin
β2 microglobulin (β2-MG) is a small molecule globulin pro-
Pyelonephrosis is a urothelial malignancy occurring in the duced by lymphocyte, platelet, and neutrophil. Serum β2MG
renal pelvis or calyx epithelium. Transitional cell carcinoma in patients with renal pelvic cancer increases because of the
(TCC) is the most common type of renal pelvic cancer, increased secretion of serum β2-MG by inflammatory cells
accounting for about 10% of renal tumors. It can be accom- or tumor cells. Serum β2-MG can be used to evaluate renal
panied by ureteral, bladder, or contralateral pelvic tumors. function.
Mostly at 40–70 years of age, the ratio of men to women is
2:1. It can be single or multiple. Neuro-Specific Enolase
Long-term use of analgesics, chronic inflammation or Neuro-specific enolase exists in nerve cells, neuroendocrine
stone stimulation, and exposure to carcinogenic chemicals cells, and tumor cells. Some studies have found that the con-
572 Z. Zheng et al.
Table 36.3 Serological markers of renal pelvis carcinoma on April 10, 2013. the urine routine test was performed
Markers Functions of markers again On September 14, 2013, the results show that urine
Traditional β2 globulin For the evaluation of kidney red blood cell (+++). Then the patient had a CT scan, the
markers function and the diagnosis of results show that the left renal pelvis is occupied. There
renal pelvic carcinoma was a high risk of renal pelvic carcinoma, right renal cyst.
Neuro-specific High expression of neuro-
enolase specific enolase in patients
The patient came to Jiangsu Province Hospital for further
with renal pelvic carcinoma diagnosis and treatment.
New Hipper Lindau Early gene mutations in renal
molecular Cancer suppressor pelvic carcinoma are Physical Examination T 36.5 °C, P 80 times/min, R 20
markers gene manifested in abnormalities of times/min, BP 120/75 mmHg. There is no mass in the renal
the VHL gene
area, no tenderness in kidney areas, no tenderness in the ure-
G250 antigen More than 98% of renal pelvic
carcinoma has the expression teral region, no swelling, and obvious tenderness in the blad-
of G250 antigen der area.
TH protein Elevated the protein antibody
antibody in patients with renal pelvic Laboratory Examination Hemoglobin: 117 g/L (130–
carcinoma
175 g/L), Urine routine: occult blood (+), Serum total protein
59.1 g/L (65–85 g/L).
tent of neuro-specific enolase in patients with renal pelvic
cancer is 34 times higher than that in normal people. The Imaging Examination The CT of the urinary system shows
content of neuro-specific enolase returns to normal after that the left renal pelvis is occupied, right renal cyst.
resection and increases again when the tumors recur.
Diagnosis and Treatment Process There were no chills
Novel Molecular Biomarkers for Renal Pelvic and fever, no obvious progressive emaciation, no nausea and
Carcinoma vomiting, no waist and abdomen pain, no palpitation and
Oncogene Marker chest tightness. No contraindications were found in other
Hippel Lindau tumor suppressor gene (VHL) is located on examinations. Radical operation of left renal pelvis cancer is
the short arm of chromosome 3, and its mutation rate is as recommended, Postoperative pathological results showed
high as 50%. More than 98% of patients with renal pelvic that urothelial carcinoma of left renal pelvis, IILevel, Cancer
cancer have VHL gene mutation, most of them are heterozy- cells infiltrate renal medulla. The size of the tumor is
gote mutation, and more than 80% of patients had del 3p or 2.5 cm × 2 cm × 2 cm. No residual cancer cells were found
other chromosome 3 abnormality. at the ureter cutting edge. See in Fig. 36.6.
G250 antigen was identified and cloned from a variety of
renal cell carcinomas, and its sequence was identical to that This case was from the First Affiliated Hospital of Nanjing
of carbonic anhydrase. More than 98% of patients have G250 Medical University (also named Jiangsu Province Hospital).
antigen expression, which can be used as a tumor marker for
renal pelvic carcinoma [98].
Tamm–Horsfall Protein Antibody

Tamm–Horsfall protein antibody is a glycoprotein with anti-
genicity. The majority of patients with renal pelvic cancer
have higher TH protein antibodies than normal people [99].
36.3.3.2 Typical Case

Clinical Background A patient, male, 75 years old, the
patient found that the color of morning urine became darker
without obvious inducement 4 months ago. If drinking
more water, the color of urine becomes lighter. No obvious
waist and abdomen pain, no nausea and vomiting, no palpi-
tation and chest tightness, no urgency of urination, fre-
quency of urination, urination pain, no abdominal distention
and pain. The patient did not pay attention to this phenom-
enon. The patient found that urine occult blood positive,
urine red blood cell negative during physical examination Fig. 36.6 Pathology of renal cell carcinoma (×40)
36.4 H
antavirus Hemorrhagic Fever tion, Hantavirus infection has become a global public health
with Renal Syndrome problem, with at least 30,000–50,000 cases occurring every
year. The reports are mainly concentrated in Asia, and the
Huaguo Xu and Xiaojie Zhang epidemic situation in China is more severe [102]. Vascular
endothelial cells are the main target cells for the action of
Hantavirus, and the vascular endothelial barrier becomes
36.4.1 Overview disordered after virus infection, especially in capillaries and
small vessel endothelial cells [103]. The main features of the
According to different serotypes or genotypes, Hantavirus disease caused by Hantavirus are increased vascular perme-
can be roughly divided into at least 40 kinds. There are dif- ability and damage to small blood vessels throughout the
ferences in clinical symptoms and disease severity caused body.
by different kinds of viruses, with which Human beings are There are two diseases caused by Hantavirus:
easy to be infected when they contact with the rodents car- Hantavirus lung syndrome (HPS) and Hantavirus Nephrotic
rying the virus (Fig. 36.7) [100]. The dominant in the field Hemorrhagic Fever (HFRS). Renal syndrome hemorrhagic
was Apodemus agrarius followed by Dalin agrarius, and fever, also known as epidemic hemorrhagic fever, is a seri-
dominant in the house was Rattus norvegicus followed by ous natural epidemic disease caused by Hantavirus, of which
Apodemus agrarius (Fig. 36.8) [101]. With the development the prevalence rate is the worst in China [104]. Hantavirus
of globalization and the increase of people’s communica- belongs to the Hantaviridae of Bunyavirales in the arbovirus,
which is a zoonotic pathogen [105]. Pathogens that cause
HFRS include a variety of Hantaviruses, such as Seoul virus
and Tula virus, which have been reported mostly in Asia
[106]. The HFRS syndrome caused by the Hantavirus sub-
type virus and Dobrava–Belgrade virus is the most severe
and has the highest incidence [107]. The spread of rodent
feces and secretions aerosols is one of the known methods
of virus transmission to humans across species, and infected
animals can also be transmitted through biting.
36.4.2 Pathogenesis
36.4.2.1 Pathogenesis
Different types of Hantavirus correspond to different disease
profiles and different epidemiological characteristics. The
pathogenesis of the seven types of Hantavirus infections cur-
Fig. 36.7 Hantavirus cell membrane particles (red arrow) seen by rently identified in China is unclear, and the mechanism of
electron microscopy
Hantavirus-induced diseases has achieved results. In HFRS,
the main part of the virus replication in the human body is in
blood vessels. There are great changes in vascular permeability
and decreased blood pressure due to endothelial dysfunction,
of which kidney damage is the most obvious. In HPS, most of
the sites of action are in the lungs, and the spleen and gallblad-
der may also be damaged. The early symptoms of HPS are
often similar to the flu, often appearing about 2 weeks after
the infection, and the respiratory symptoms worsening about
1 week after the onset of symptoms, such as dyspnea most
studies suggest that Hantavirus is the initiator of the disease.
On the one hand, viral infection can cause damage to the func-
tion and structure of infected cells; on the other hand, viral
infection induces the immune response of the human body and
the release of various cytokines, which not only removes the
Fig. 36.8 Rattus norvegicus virus, protects the body, but also causes damage to the body tis-
sues. It is generally believed that Hantavirus enters the human
574 Z. Zheng et al.
body and then reaches the whole body with blood flow. The tion to granular IgG deposition, there is linear IgG depo-
virus first combines with the receptor p3 integrin expressed on sition in the basement membrane of renal tubules,
the surface of platelets, endothelial cells, and monocytes, and suggesting that clinical platelet reduction and tubular
then enters the cells as well as liver, spleen, lung, kidney, etc. damage are related to type II allergic reaction.
The tissue, after further replication, is released into the blood- C. Electron microscopy revealed that lymphocytes attacked
stream, causing viremia, resulting in cell degeneration, necro- renal tubular epithelial cells, and it is believed that the
sis, or apoptosis due to viral infection and immune response virus can damage the body cells through cytotoxic T
induced by infection, and release of various cytokines, thereby cells. It is suggested that there is a type IV allergy. As for
impairing organ function. Because Hantavirus is a pan-tropic the status of the above-mentioned I, II, and IV allergic
infection in humans, it can cause multiple organ damage. reactions in the pathogenesis of this disease, further
Mechanisms of cell and organ damage include: research is needed.
Direct Action of the Virus The Role of Various Cytokines and Mediators

The main basis is: (1) Clinically, the patient has a viremia Hantavirus can stimulate the body to produce a large num-
period and has corresponding symptoms of poisoning. (2) ber of cytokines and interleukins from macrophages and
Different serotypes of the virus cause the severity of clini- lymphocytes of the body. IFN-r, IL-2, TH2 cytokine IL-10
cal symptoms diversely. (3) It also has different virulence and etc. cause clinical symptoms and tissue damage. For
to suckling mice. It is indicated that the severity of clinical example, IL-1 and TNF can cause fever. A certain amount of
symptoms after EHF patients is closely related to the differ- TNF can cause shock and organ failure. In addition, elevated
ence of viral antigens and virulence. EHF patients can detect levels of endotheliolysin, thromboxane B2, angiotensin II,
Hantavirus antigens in almost all organ tissues, especially in etc. can Significantly causes renal parenchymal ischemia and
vascular endothelial cells of EHF basic lesions. Moreover, reduced glomerular perfusion, and promote the occurrence
cells with antigenic distribution often develop lesions. (4) of renal failure.
Normal human bone marrow cells and vascular endothelial
cells cultured in vitro, in the absence of cellular immunity 36.4.2.2 Pathology and Physiology
and humoral immunity, cell membrane and organelle dam- The pathological changes of this disease are most obvious in
age after infection with EHF virus, indicating that cell dam- small blood vessels and kidney diseases, followed by organs
age is the direct effect of Hantavirus. such as the heart, liver, and brain. The basic lesion of EHF
is swelling, degeneration, and necrosis of endothelial cells
Immunity in small blood vessels including small arteries, venules, and
Injury caused by immune complex (type III allergy): In this capillaries. The wall of the tube is irregularly contracted and
patient, serum complement is decreased in the early stage, expanded, and finally fibrillar necrosis and disintegration.
and specific immune complexes are present in the blood There may be microthrombus formation in the lumen, result-
circulation. In recent years, it has also been found that the ing in edema and hemorrhage of surrounding tissue due to
patient’s small blood vessel wall, glomerular basement extensive small vessel disease and extravasation of plasma.
membrane, renal tubule and renal interstitial blood vessels Kidney fat edema and hemorrhage can be seen in the kid-
have immune complex deposition. The immunohistochemi- neys. Microscopic examination showed glomerular conges-
cal method proves that the antigen is EHF virus antigen, and tion, basement membrane thickened, renal tubular epithelial
there is a complement cleavage fragment, so it is considered cells degeneration and necrosis and tubular compression and
that the immune complex is the cause of blood vessel and narrowing or occlusion, and renal interstitial cell infiltration.
kidney damage in this disease. Other immune reactions: Electron microscopy showed that the glomerular capillary
After the EHF virus invades the human body; it can cause an endothelial cells had different degrees of swelling. In the oli-
innate immune response in the body. It is found that: guria, individual glomeruli showed capillary endothelial cell
necrosis, neutrophils and platelets, and low between endo-
A. The specific IgG antibody was elevated at the time of no thelial cells and basement membrane. Cardiac changes in the
clinical symptoms of the disease, and its rising level was cavity are mainly extensive bleeding under the right atrial
positively correlated with the positive rate of mast cell subendocardium. Myocardial fibers have different degrees
degranulation, suggesting the presence of type I allergic of degeneration, necrosis, and some can be broken. The
reaction. pituitary gland is markedly hyperemic, hemorrhagic, and
B. There are immune complexes in platelets of EHF patients. coagulative necrosis. There was no significant change in the
Electron microscopic observation of renal tissue in addi- pituitary gland.
Shock platelets. Thrombocytopenia in patients with EHF is associ-

The third to seventh days of the disease course often appear ated with the maturation of bone marrow megakaryocytes,
hypotension shock called primary shock. Shock that occurs increased platelet consumption, and increased destruction.
after the oliguric period is called secondary shock. The cause (3) Abnormal blood coagulation mechanism: Because DIC
of primary shock is mainly due to extensive damage to the consumes a large number of clotting factors, in addition,
small blood vessels of the whole body, increased vascular DIC causes secondary fibrinolysis, increased fibrinogen
permeability, and a large amount of extravasation of plasma degradation products, and increased heparin substances can
into loose tissues, such as retroperitoneal and soft tissues of cause coagulation abnormalities. Causes of A. DIC: The
organs, resulting in decreased blood volume. In addition, incidence of DIC in EHF patients can reach 35–70%, except
due to plasma extravasation, the blood is concentrated, and in the recovery period, which can occur in other periods,
the increased blood viscosity can promote the occurrence of especially in the hypotensive shock phase and the oliguria
disseminated intravascular coagulation (DIC), resulting in phase. This is an EHF virus or immune complex that dam-
stagnant blood circulation and blocked blood flow, thereby ages endothelial cells in the capillaries or small vascular,
further reducing the effective blood volume. The main causes resulting in the vascular basement membrane collagen expo-
of secondary shock are major bleeding, secondary infection, sure, thereby activating factor XII, causing a chain reac-
and insufficient supplementation of water and electrolytes tion that causes endogenous coagulation. In addition, EHF
during the polyuria, resulting in insufficient blood volume. patients with plasma extravasation, increased blood concen-
tration and viscosity, as well as shock and oliguria acidosis,
Bleeding have promoted DIC. B. Increased heparin content: About
The factors of bleeding in EHF patients are more compli- 80% of patients with EHF have elevated heparin levels in
cated, sometimes involving multiple factors. It is gener- the blood from the onset of fever. In addition to increased
ally believed that the small bleeding points of the skin and release of mast cells in the body, reduced heparin inactiva-
mucous membrane during the fever period are caused by tion due to liver damage, decreased heparin excretion due to
capillary damage, thrombocytopenia, and abnormal plate- renal failure, and decreased extravasation of plasma proteins
let function. Hypotension shock period to pre-urinary and reduced heparin binding, all contribute to an increase in
period, mainly DIC cause is abnormal blood coagulation free heparin.
mechanism, in addition to thrombocytopenia and dysfunc-
tion, heparin substances and uremia which can also cause Acute Renal Failure
bleeding. (1) Small blood vessel injury: small blood vessel The causes include: (1) Renal blood flow disorder: due to
lesions in EH patients mainly manifested as swelling and plasma extravasation, blood volume reduction and blood
degeneration of endothelial cells, severe fibrin-like necrosis, concentration, blood flow is insufficient, resulting in a sharp
and even disintegration of the vessel wall, which can cause a decline in glomerular filtration rate. (2) Immunological dam-
lot of blood oozing and bleeding. There are three reasons for age of the kidney: The deposition of immune complexes in
the damage of small blood vessels: (A) Hantavirus directly the glomerular basement membrane and the renal tubular
acts on vascular endothelial cells and damages them. (B) basement membrane has been confirmed, and the glomer-
Hantavirus antigen and antibody complexes are deposited in ular basement membrane and renal tubular epithelial cells
small blood vessels attract neutrophils to phagocytose anti- can be damaged after activation of complement. Cytotoxic
gen–antibody complexes with the participation of comple- T cells can also cause tubular damage. (3) Interstitial edema
ment, and release proteolytic enzymes in lysosomes, thereby and hemorrhage: renal interstitial edema caused by plasma
damaging endothelial cells. (C) Microcirculatory disorders extravasation, as well as renal medullary congestion, blood
due to shock and other causes, causing hypoxia and vascu- pressure to force the renal tubules, can reduce urine output.
lar endothelial cells to cause degeneration and necrosis. (2) (4) Renal ischemic necrosis hypotension shock and DIC lead
Thrombocytopenia and dysfunction: under normal condi- to renal vascular microthrombus formation, which can cause
tions, platelets are arranged along the wall of the blood ves- ischemic necrosis of renal parenchymal cells. (5) Activation
sel, which has the function of maintaining capillary integrity of renin and angiotensin II: The renal artery is contracted,
and reducing capillary fragility and permeability. The and renal cortical blood flow is reduced, and glomerular fil-
decrease in platelets leads to an increase in capillary fragil- tration rate is decreased. (6) Renal tubular obstruction: renal
ity and permeability, and in addition to blood plate coagula- tubule lumen can be blocked by protein, tube type, etc., urine
tion due to barriers to adhesion, aggregation, and release of discharge blocked.
576 Z. Zheng et al.
36.4.3 Course of Disease is drunk. Mucosal congestion is seen in the conjunctiva of

the eye, the soft palate in the mouth, and the pharynx. Skin
Symptoms of HFRS usually occur within 2 weeks after bleeding is more common in the armpits and chest and back.
infection, and a few patients take 2 months to show symp- It is often scratched and sputum-like. The mucosal hemor-
toms. The onset of the disease is acute and symptoms are rhage is common in soft-sputum-like hemorrhage, and the
typical, including severe headaches, body pain, blushing, conjunctiva is flaky. A small number of patients have nose-
redness, etc., followed by severe symptoms of effective cir- bleeds, hemoptysis, melena, or hematuria. For example,
culating blood volume and insufficient tissue perfusion, such on the 4th to 6th day of the disease, large ecchymoses may
as acute shock and acute renal failure [108]. The clinical appear in the waist, buttocks, or injection site, which may be
symptoms of different subtypes of Hantavirus showed dif- caused by DIC. This is a severe manifestation. The oozing
ferent severity. Hantan virus can cause severe HFRS, Seoul edema sign is mainly manifested in the conjunctival edema.
virus mainly causes medium HFRS, and Pumara virus only When the light eyeball rotates, the bulbar conjunctiva appears
causes nephritis as light HFRS. In the typical case, there are to be paralyzed. In severe cases, the bulbar conjunctiva is
five periods of fever, hypotension shock, oliguria, polyuria, blister-like and even protrudes. Some patients have eyelids
and recovery. Over-stage phenomena can occur in atypical and facial edema, and ascites can also occur. Generally, the
and light cases, while in severe cases there is an overlap heavier the edema is, the heavier the condition is. (4) Kidney
between fever, shock, and oliguria. damage: mainly showing urinary protein-positive, tube type
can be found by microscopy.
36.4.3.1 Fever Period
In addition to fever, it mainly manifests as systemic poison- 36.4.3.2 Hypotension Shock Period
ing, capillary damage, and kidney damage. (1) Fever: a small It generally occurs on the 4th to 6th disease days, and later
number of patients start with low fever, gastrointestinal dis- may appear on the 9th disease day. Most patients have a drop
comfort, and respiratory infection-like prodromal symptoms. in blood pressure at the end of the fever or heat withdrawal.
Most patients have a sudden onset of chills and fever, body A small number of shocks occur after heat retreat, which is
temperature between 39 ~ 40 °C, with frequent heat and different from bacterial infections. The duration of hypoten-
relaxation heat, the majority of the heat cycle is 3–7 days, sion or shock, in a few hours, the elderly can reach more
a few for more than 10 days. The higher the general body than 6 days, usually 1–3 days. The duration of the disease is
temperature and the longer the heat history are, the heavier related to the severity of the condition and whether the treat-
the symptom is. Mild patients with symptoms after heat ment is timely and correct. When most patients begin to have
retreat, the severity of patients with severe retreat is worse. insufficient blood volume, they can regulate the blood ves-
(2) Systemic poisoning: most patients have body aches, sels of the skin and viscera by regulating the neurohumoral
headaches, and low back pain. A small number of patients fluid, and maintain normal blood pressure. At this time, the
have eyelid pain and rotate with the eyeball. Headache, heartbeat can be increased by the increase of catecholamine
back pain, and eyelid pain are generally referred to as “three secretion. When blood volume continues to drop, hypoten-
pains.” Headache is caused by cerebral vasodilatation and sion occurs, even shock. At this time, his face was pale, his
congestion; low back pain is related to congestion, edema, limbs were cold, his pulse was weak or could not be touched,
and retroperitoneal edema around the kidney; eyelid pain is and his urine volume was reduced. When the blood supply
caused by edema around the eyeball, and severe cases may to the brain is insufficient, irritability, paralysis, and ambi-
be accompanied by elevated intraocular pressure and blurred guity may occur. Mild patients may not have hypotension
vision. Most patients may have symptoms of gastrointestinal or shock. A small number of patients with refractory shock
poisoning, such as loss of appetite, nausea, vomiting, hic- have cyanosis due to long-term poor perfusion of tissue and
cups, abdominal pain, and diarrhea. If the abdominal pain is promote DIC, cerebral edema, acute respiratory distress syn-
severe, the abdomen has tenderness and rebound pain, and drome, and acute renal failure. At this time, the patient devel-
it is easy to be misdiagnosed as acute abdomen and undergo oped shortness of breath, coma, convulsions, and extensive
surgery. Such patients are caused by extreme hyperemia and bleeding.
edema in the mesenteric area. Diarrhea can carry mucus and
blood and is easily misdiagnosed as enteritis or dysentery. 36.4.3.3 Oliguria
Some patients may have neuropsychiatric symptoms such Oliguria is followed by hypotensive shock; some patients have
as drowsiness, irritability, convulsions, or convulsions, and no clinical hypotension shock period, from the fever period
most of these patients develop severe. (3) Capillary damage directly into the oliguria. There are also oliguria and hypoten-
signs: mainly manifested as congestion, bleeding, and exu- sion shock overlap, this time should be differentiated from
dation edema. Skin congestion is mainly seen in the facial, prerenal oliguria. It is generally considered that the urine vol-
neck, chest, and other parts of the flushing and the heavy one ume is less than 500 mL/24 h for oliguria and <50 mL/24 h
for anuria. A small number of patients without significant the elderly can be several months. According to the amount
oliguria and azotemia, called no oliguric renal insufficiency, of urine and azotemia can be divided into three phases.
which is glomerular damage and renal tubular damage is not (1) Transition period: urine volume increased from 500 to
serious, only affect the glomerular creatinine and urea nitro- 2000 mL/day, although the urine volume increased, but urea
gen excretion. The oliguria period usually occurs on the 5th nitrogen and creatinine increased, the symptoms worsened,
to 8th disease days, and the duration is shorter than 1 day, many patients died of complications due to complications,
and the elderly are more than 10 days, usually 2–5 days. The should be special Pay attention to the condition. (2) Early
discharge of membranes in the urine is severe. The clinical urine: urine volume more than 2000 mL/day, no improve-
manifestations of oliguria are uremia, acidosis, and water and ment in azotemia, symptoms are still heavy. (3) Polyuria
electrolyte disorders. Severe patients may have high blood later: urine volume exceeded 3000 mL/day, and increased
volume syndrome and pulmonary edema. (1) Uremia: gas- day by day, azotemia gradually decreased, and mental appe-
trointestinal symptoms such as anorexia, nausea, vomiting, tite improved day by day. The amount of urine in this period
abdominal distension, diarrhea, and oral ulcers may occur due can reach 4000 ~ 8000 mL/day, a few can reach more than
to the stimulation of urea nitrogen and ammonia. Often there 15,000 mL/day. In this period, if the water and electrolyte
are intractable hiccups, which can cause dizziness, headache, supplements are insufficient or secondary infection, second-
irritability, lethargy, paralysis, and even neurological symp- ary shock may occur, and symptoms such as hyponatremia
toms such as coma and convulsions. In most patients, hem- and hypokalemia may occur.
orrhage is aggravated by thrombocytopenia and dysfunction,
increased heparin levels, or DIC. It is manifested in increased 36.4.3.5 Recovery Period
skin freckle, nosebleeds, blood in the stool, hematemesis, After the period of polyuria, the urine volume is restored to
hemoptysis, hematuria, and vaginal bleeding. Intracranial about 2000 mL/day, and the spirit and appetite are basically
hemorrhage or other visceral hemorrhage can still occur in restored. It usually takes 1–3 months for physical strength to
a small number of patients. (2) Acidosis: Metabolic acidosis fully recover. A small number of patients may have symp-
occurs due to the accumulation of acidic metabolites, mani- toms such as hypertension, renal dysfunction, myocardial
fested by increased breathing or Kussmaul breathing. (3) strain, and hypopituitarism.
Water and electrolyte disorders: due to water, sodium reten- The course of the disease can be divided into five stages:
tion, tissue edema is aggravated; patients may appear facial, According to the difference of fever, the severity of poisoning
limb edema, and even ascites. The electrolyte disorder in this symptoms, and the severity of hemorrhage, shock and renal
period is mainly hyperkalemia, dilute hyponatremia, and function damage, it can be divided into five types clinically.
hypocalcemia. Hypokalemia and hypermagnesemia can also
occur in a small number of patients. Because hypokalemia Light Type The body temperature is below 39 °C, the
and hyperkalemia can cause arrhythmia, it is advisable to symptoms of poisoning are light, and there is no other bleed-
periodically check serum potassium and ECG for identifica- ing phenomenon except the bleeding point. Kidney damage
tion. Hypoxic sodium is mainly characterized by dizziness is light, no shock and oliguria.
and burnout, and severe symptoms such as blurred vision
and cerebral edema. Low blood calcium can cause hand and Medium Body temperature 39 ~ 40 ° C, the symptoms of
foot spasms. (4) High blood volume syndrome: manifested poisoning are heavier, there is obvious conjunctival edema,
as body surface venous filling, increased systolic blood pres- the systolic blood pressure is less than 12 kPa (90 mmHg) or
sure, increased pulse pressure, and thus large pulse. The face the pulse pressure is less than 3.5 kPa (26 mmHg). Have
is full and the heart rate increases. The severity of this disease obvious bleeding and oliguria, urine protein +++.
is parallel to the duration of oliguria and the level of azotemia.
If the urea nitrogen rises by 21 mg/dL or more, it is a highly Heavy Body temperature ≥ 40 °C, poisoning and exudation
decomposing renal failure, and the prognosis is poor. signs are serious, may have toxic mental symptoms, and
shock, skin ecchymosis and channel bleeding. Oliguria per-
36.4.3.4 Polyuria sists within 5 days or within 2 days without urine.
In this period, the reabsorption function of the new renal
tubule is not perfect, and the hypertonic diuretic effect caused Criticality One of the following conditions occurs on a
by the retention of urea nitrogen and other substances causes heavy basis: (1) refractory shock; (2) major organ bleeding;
a significant increase in urine output. Most patients enter this (3) oliguria more than 5 days or no urine for more than
period after oliguria, and a small number of patients can be 2 days, BUN > 42.84 mmol/L; (4) heart failure, pulmonary
transferred to this period from the fever period or hypoten- edema; (5) cerebral edema, cerebral hemorrhage or brain and
sive period. The polyuria usually occurs on the 9th to 14th other central nervous system complications; (6) severe sec-
day of the disease, and the duration is shorter than 1 day, and ondary infection.
578 Z. Zheng et al.
Atypical Fever below 38 °C, skin mucosa may have scat- 36.4.4.3 Biochemical Tests
tered bleeding points, urine protein ±, blood, urine specific Blood Urea Nitrogen and Creatinine Most patients in the
antigen, or antibody positive. hypotensive shock phase, a small number of patients in the
late fever, urea nitrogen and creatinine began to rise, peaked
at the end of the transition period, declined in the late
36.4.4 Diagnosis polyuria.
The definite diagnosis of HFRS cannot be based on clini- Acidity and Alkalinity Blood gas analysis during fever is
cal symptoms alone, but also requires serological evidence more common with respiratory alkalosis, which is related to
to assist diagnosis. IgG antibody titer was measured twice fever and hyperventilation. Metabolic acidosis is the main
within 1 week. The serum antibody titer in the recovery stage of shock and oliguria.
phase was four times or higher than that in the acute phase.
At the same time, the serum Hantavirus IgM antibody was Electrolytes During the course of the disease, sodium,
positive, which was seemed as strong evidence of infection. chlorine, and calcium in the blood can always decrease,
For patients with acute fever-like flu-like symptoms, patients while phosphorus and magnesium are increased. Blood
with renal failure of unknown origin should think of HFRS potassium is at a low level during the fever period and
early to prevent missed diagnosis. shock period, and the oliguria period is elevated. However,
a small number of patients still have hypokalemia during
36.4.4.1 Blood Routine oliguria.
White Blood Cell Count The first 1–2 days of disease are
normal, and gradually increase after the third disease day, up Coagulation Function Thrombocytopenia begins during
to (15 ~ 30) × 109/L. A small number of critically ill patients the fever phase, and its adhesion, aggregation, and release
can reach (50 ~ 100) × 109/L. function are reduced. Platelets are often reduced to below
50 × 109/L. The clotting time is shortened during the high-
Classification of White Blood Cells Neutrophils increase coagulation period of DIC. During the consumptive low
at the beginning of the disease, the left side of the nucleus coagulation phase, fibrinogen is reduced, prothrombin is
moves, and there are poisonous particles. In severe cases, prolonged, and thrombin time is prolonged. Fibrin degrada-
immature cells can be seen to be leukemia-like. After the tion (FDP) increases as the fibrinolytic phase enters.
fourth to fifth disease days, lymphocytes increased and more
atypical lymphocytes appeared. Because atypical lympho- 36.4.4.4 Special Inspections
cytes can also occur in other viral diseases, they cannot be Virus Isolation The serum, blood cells, and urine of the
used as the main basis for disease diagnosis. patients in the febrile phase are inoculated with Vero-E6 cells
or A549 cells to isolate Hantavirus.
Hemoglobin and Red Blood Cells As blood extravasation
leads to blood concentration, the number of hemoglobin and Antigen Examination Serum of early patients, peripheral
red blood cells increases from the late stage of fever to the blood neutrophils, lymphocytes, and monocytes, as well as
hypotensive shock period, reaching 150 g/L and 5.0 × 1012/L urine and urine sediment cells, using Hantavirus polyclonal
or more. (4) Platelets are reduced from the second disease or monoclonal antibodies, can detect Hantan Viral antigen.
day, generally around (50 ~ 80) × 109/L, and visible The colloidal gold rule is more sensitive with commonly
platelets. used immunofluorescence or ELISA.
36.4.4.2 Urine Routine Specific Antibody Detection Including detection of spe-

Urine Protein It can appear on the second disease day, and cific IgM or IgG antibodies in serum. The IgM antibody
the urine protein on the 4th to 6th day is often +++ or ++++. was positive at 1:20 and was detected on the second day of
The sudden appearance of large amounts of urinary protein is onset. IgG1:40 is positive, and a fourfold increase in titer
helpful for diagnosis. In some cases, there is a membrane in after 1 week has diagnostic value. It is currently believed
the urine, which is a mixture of a large amount of urine protein that the detection of nucleoprotein antibodies is conducive
mixed with red blood cells and exfoliated epithelial cells. to early diagnosis, while the detection of G2 antibodies is
beneficial to prognosis. Recent foreign studies on immuno-
Microscopic Examination Visible red blood cells, white chromatographic rapid test using recombinant nucleopro-
blood cells casts, and large fused cells can be found in the tein (NP) as antigen to detect IgM antibody in patients for
urine sediment, which is the fusion of the urinary exfoliated 5 min can produce results with sensitivity and specificity of
cells of the EHF virus under acidic conditions. 100%.
PCR Technology RT-PCR method for detection of Anti-virus According to our experience with Xi’an Medical
Hantavirus RNA is highly sensitive for early diagnosis [108]. University, the ribavirin-treated group was significantly bet-
ter than the control group in terms of fever, disappearance of
36.4.4.5 Other Auxiliary Inspections urinary protein, rise in platelets, and overdue. In addition, we
Liver function serum alanine aminotransferase (ALT) is also carried out detection of Hantavirus antigen in granulo-
elevated in about 50% of patients, and a small number of cytes and found that Hantavirus antigen in granulocytes was
patients have elevated serum bilirubin. obviously lower than that in the control group after 3 days of
An electrocardiogram can have sinus bradycardia, con- application of ribavirin. This indicates that early treatment
duction block, and other arrhythmia and myocardial dam- with ribavirin can inhibit the virus, reduce the disease and
age [109]. In addition, when the blood is high, the T wave is shorten the course of the disease.
high, and when the blood is low, the U wave appears.
Patients, who have problems with elevated intraocular Improving the Symptoms of Poisoning High heat is
pressure, are often indicated as severe. Patients with cerebral mainly caused by physical cooling. Avoid strong sweating
edema can be seen with optic disc edema and venous conges- and antipyretics to prevent further loss of blood volume due
tion and dilatation. to sweating. Prevention of DIC: If the symptoms of hyper-
About 30% of patients with chest X-rays have pulmonary thermia, poisoning, and exudation are severe, the clotting
edema and congestion, and about 20% of patients have pleu- time should be checked regularly. Intravenous method within
ral effusion and pleural reaction. 3 min or activated partial thromboplastin time (APTT) within
34 s for hypercoagulable state, can be given a small dose of
36.4.4.6 Differential Diagnosis heparin anticoagulation.
The fever period must be distinguished fever from upper
respiratory tract infections, sepsis, acute gastroenteritis, and The Principle of Treatment for Hypotension Shock
bacillary dysentery. The shock phase should be differentiated Period Positively fill the volume, pay attention to correct-
from other septic shocks. The oliguria phase is differentiated ing acid.
from acute nephritis and other causes of acute renal failure.
Patients with obvious bleeding need to be differentiated from The Principle of Treatment During Oliguria Stable, promot-
peptic ulcer bleeding, thrombocytopenic purpura, and other ing, guiding, and translucent. That is to stabilize the body envi-
causes of DIC. The main manifestation of ARDS should be ronment, promote diuretic, catharsis, and dialysis treatment.
distinguished from those caused by other causes. Patients
with abdominal pain as the main sign should be identified Multi-urinary Treatment Principles The transitional
with surgical acute abdomen. period and early treatment of polyuria are the same as oligu-
ria. The late polyuria is mainly to maintain the balance of
water and electrolytes and prevent secondary infections.
36.4.5 Treatment
Recovery Period Treatment Principle To supplement
No specific vaccine or treatment is currently available for nutrition, gradually resume work. After discharge, you
HFRS, which mainly relies on supportive and symptomatic should take a rest for 1–2 months. Renal function, blood
treatment. It has been reported in China that treatment with pressure, and pituitary function are checked regularly. If
ribavirin within 7 days after fever can reduce mortality and there is any abnormality, it should be treated promptly.
shorten the course of the disease. The treatment of this dis-
ease is based on comprehensive therapy, early application of
antiviral therapy, and symptomatic physiology for symptom- 36.4.6 Typical Medical Case
atic treatment in the advanced stage. “Early and nearby” is
still the principle of treatment of this disease including Early Clinical Background Patient, female, 49 years old, high
detection, early rest, early treatment, and nearby treatment. fever for 6 days, headache, eyelid pain, low back pain for
Pay attention to the prevention and treatment of shock, kid- 3 days. Three days after returning from Maoming Tourism in
ney failure, and bleeding during treatment. Guangdong on April 28, he suddenly became chilly at home
and continued to have fever (above 39 °C), followed by
The Principle of Treatment During Fever Anti-virus, headache, back pain, body aches, fatigue, anorexia, nausea,
reducing extravasation, improving symptoms of poisoning, vomiting, and other symptoms. During the tourism period,
and preventing DIC. the sanitary conditions in the hotel were poor. It was often
580 Z. Zheng et al.
found that the rats were infested, but they denied that they 11. Bergmann C, Kupper F, Dornia C, et al. Algorithm for efficient
were bitten by the rats. The food and the outer packaging PKHD1 mutation screening in autosomal recessive polycystic
kidney disease (ARPKD). Hum Mutat. 2005;25:225–31.
carried by them were bitten. The family members of the 12. Krall P, Pineda C, Ruiz P, et al. Cost-effective PKHD1 genetic
patients stated that the food in the home was contaminated testing for autosomal recessive polycystic kidney disease. Pediatr
by rats, the family members of the patients, and other joint Nephrol. 2014;29:223–34.
tourism. There is no disease, and there is no history of wild 13. Eisenberger T, Decker C, Hiersche M, et al. An efficient and com-
prehensive strategy for genetic diagnostics of polycystic kidney
accommodation. Physical examination: bulbar conjunctival disease. PLoS One. 2015;10:e0116680.
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15. Pei Y, Watnick T. Diagnosis and screening of autosomal domi-
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Cardiovascular Disease
37
Zhou Zhou and Yahui Lin
The cardiovascular system is the conveyor system of the and other factors. In this chapter, we will introduce acute
body which consists of the heart, blood vessels, and the myocardial infarction, heart failure, myocarditis, congenital
blood itself. The heart is a cone-shaped muscular organ that heart disease, and arrhythmia.
normally weighs between 230 to 340 g. A healthy adult heart
is the size of your fist: about 12 cm long, 9 cm wide, and
6 cm thick. A fibrous pericardium, a double-walled sac, 37.1 Acute Myocardial Infarction
encloses the heart. The wall of the heart is made up of a spe-
cial type of muscle called cardiac muscle, also called myo- Zhou Zhou and Yahui Lin
cardium. The heart is divided into the right and left sides of
the heart by cardiac septum. The left and right side each
separated into two chambers, called atrium and ventricle, by 37.1.1 Overview
bicuspid valve and tricuspid valve, respectively. Therefore,
the four chambers of the heart contain the right atrium, left Acute myocardial injury (AMI), also called a heart attack,
atrium, right ventricle, and left ventricle. Blood is received refers to myocardial necrosis due to lack of blood supply.
with the right atrium and left atrium from the rest of the body The Fourth Universal Definition of Myocardial Infarction
and the lungs, respectively. The right ventricle pumps blood denotes that AMI is the presence of acute myocardial injury
to the lungs while the left ventricle pumps out blood to the with abnormal levels of myocardial biomarkers and clinical
rest of the body (Fig. 37.1). evidence of acute myocardial ischemia [1]. According to the
The cardiovascular system circulates blood from the heart presence or absence of ST-segment elevation on the ECG,
to the lungs and around the body, as well as pumps blood AMI is classified into ST-elevation MI (STEMI) and non-ST-
through itself. The left and right coronary artery from the elevation MI (NSTEMI). STEMI, NSTEMI, or UA (unstable
root of the aorta is two major coronary arteries, which branch angina) are considered acute coronary syndrome (ACS).
into a network of arteries surrounding the heart’s surface. According to pathological, clinical, and prognostic differ-
These blood vessels surrounding the top of the heart which ences, AMI is further classified into seven types: (1) Type 1
supply cardiac muscle, septum, and sinoatrial node with MI, caused by coronary atherothrombosis after atheroscle-
nutrition and oxygen are called coronary arteries due to look- rotic plaque disruption. (2) Type 2 MI, caused by an imbal-
ing like a “crown.” When blood streams in these coronary ance between oxygen supply and demand. (3) Type 3 MI,
arteries become less or completely disappear because of nar- defined by a sudden cardiac death with symptoms suggestive
rowing or blockage of the blood vessels, myocardial injury of myocardial ischemia without the opportunity for bio-
or damage is caused by a region of myocardial ischemia. marker, or a fresh or recent thrombus in the infarct-related
Cardiovascular diseases, which include diseases of the artery by autopsy examination. (4) Type 4a MI, defined by an
heart, are mainly noncommunicable and related to lifestyle AMI associated with a percutaneous coronary intervention
(PCI). (5) Type 4b MI, defined by an AMI related with a stent
Z. Zhou (*) · Y. Lin thrombosis after PCI procedure. (6) Type 4c MI, defined by
Center of Laboratory Medicine, Beijing Key Laboratory for an AMI associated with restenosis after percutaneous coro-
Molecular Diagnostics of Cardiovascular Diseases, State Key
Laboratory of Cardiovascular Disease, National Center for nary intervention. (7) Type 5 MI, defined by an AMI associ-
Cardiovascular Diseases & Fuwai Hospital, Chinese Academy ated with coronary artery bypass grafting (CABG).
of Medical Sciences & Peking Union Medical College,
e-mail: zhouzhou@fuwaihospital.org

584 Z. Zhou and Y. Lin
Fig. 37.1 The anatomical structure of the heart
37.1.2 Clinical Appearance Medicine (IFCC TF-CB), hs-cTn assay’s characteristics need
meet two points: first, the CV% at 99th percentile should be
Prolonged (20 min or more) angina, characterized by pres- less than 10%; second, cTn concentration in more than 50%
sure, heaviness, squeezing, burning, or choking sensation, is apparently healthy men and women can be detected, and the
the most common symptom. Some patients with AMI feel cTn concentration is above the limit of detection (LoD). If
the pain radiate to the left arm (seldom to the right arm or assays detect cTn concentration in less than 50% apparently
both arms), jaw or neck. Many atypical presentations, such healthy population, the assay is defined as con-cTn assay,
as sweating, nausea, vomiting, fatigue, palpitations, syn- which CV% at 99th percentile should be less than 10%, too [2].
cope, abdominal pain, can be observed in about 30% of Myocardial injury is defined if cTn concentration is
patients, especially in elder women with diabetes or renin higher than the 99th percentile upper reference limit (URL).
dysfunction. Even some patients present a syndrome. Acute myocardial injury is defined that if cTn concentration
presents a rise and/or fall of more than 20%. Chronic myo-
cardial injury presents a persistently elevated cTn level [1].
37.1.3 Laboratory Diagnosis Type 1, 2, and 3 acute myocardial infarction is defined
when there is elevated and reduced values of cTn with at least
Cardiac troponin (cTn), including I and T, are the primary bio- one value exceeding the 99th percentile URL and at least one
markers in diagnosis of AMI, preferably high-sensitivity cTn of ischemia evidence, including symptoms of myocardial
(hs-cTn) is more sensitive and specific than contemporary cTn ischemia, new ischemic ECG changes, the appearance of
assay (con-cTn). According to clinical laboratory practice rec- pathological Q waves, imaging evidence of new loss of viable
ommendations published by the Academy of the American myocardium or new regional wall motion abnormality
Association for Clinical Chemistry (AACC) and the Task Force (RWMA) in a pattern consistent with an ischemic etiology
on Clinical Applications of Cardiac Bio- Markers of the and identification of an angiography-based or autopsy-based
International Federation of Clinical Chemistry and Laboratory thrombus in coronary (not for types 2 or 3 MIs).
37 Cardiovascular Disease 585
A diagnosis of STEMI can be made depending on an shown in Fig. 37.2. Lots of data from large multicenter
ST-segment elevation of 12-lead ECG results when patients clinical trials demonstrate that 0 h/1 h algorithm is realiz-
with chest pain arrived at an emergency department. able when hs-cTn assay is employed. But different hs-cTn
Although blood sampling for cardiac troponin is recom- assays need to use different cutoff levels within the 0 h/1 h
mended routinely in the acute phase, waiting for the cTn algorithm (Fig. 37.3).
results is no necessary to initiate reperfusion or revascular- Types 4a acute myocardial infarction are defined by an
ization treatment. elevation of cTn values >5 times of 99th percentile URL
A diagnosis of NSTEMI is complexed and depends on after percutaneous coronary intervention (PCI) procedure
cTn level and ischemia evidence. If cTn level detected ≤48 h. Type 5 acute myocardial infarction is defined by an
using con-cTn assay is lower than 99th percentile URL and elevation of cTn values >10 times of 99th percentile URL
chest pain onset presented before at least 6 h, suspected within 48 h after coronary artery bypass grafting (CABG)
NSTEMI patients with GRACE score less than 140 can be operation. The cTn level of patients is stable or falling before
ruled out. Hs-cTn assay can rapidly rule-out non-ACS the procedure and with at least one of ischemia evidence
according to the baseline of cTn level (below LoD) if chest including new ischemic ECG changes (only for type 4a MI),
pain happens more than 3 h. However, a negative cTn level appearance of new pathological Q waves, imaging evidence
employing point-of-care testing (POCT) for cTn cannot of new loss of myocardial viability with an ischemic etiol-
make a decision of rule-out due to lower sensitivity and ogy, angiographic results consistent with a procedural com-
diagnostic accuracy, although POCT is often shorter turn- plication due to flow-limiting such as coronary dissection,
around time. Serial measurement of cTn concentration is blockage of a major epicardial artery, occlusion of side-
recommended when a rule-out diagnosis cannot be made. branch by thrombus, disruption of collateral or distal
0 h/3 h algorithm is usually employed, which detail is circulation.
Fig. 37.2 0 h/3 h algorithms Acute Chest Pain with Suspected NSTEMI
using cTn assays in NSTEMI
cTn < 99th URL cTn > 99th URL
Pain>6h Pain<6h cTn< 5 x 99th URL cTn> 5 x 99th URL
Serial testing cTn after 3h
∇
0-3h cTn < 50%
∇ ∇
0-3h cTn < 50% 0-3h cTn > 50%
Pain Free, GRACE<140,
differential diagnosis
Rule-out or Stress testing Differential diagnosis Rule-in and management

2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation;
European Heart Journal (2016) 37,267-315
Fig. 37.3 0 h/1 h algorithms

using cTn assays in NSTEMI Acute Chest Pain with Suspected NSTEMI
hs-cTn: 0h < A (only pain > 3 h) hs-cTn: 0h >

_D
or ∇ Others ∇ or
0h < B & 0-1h < C 0-1h >
_E
0h/3h
Rule-out Rule-in
algorithm
2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation;
European Heart Journal (2016) 37,267-315
Type 4b and 4c acute myocardial infarction follows the transport blood to peripheral tissues or organs with enough
same criteria of type 1 MI. Type 4b MI is caused by stent/ force or/and fill with enough blood.
scaffold thrombosis according to angiography or autopsy evi- The prevalence of HF in China and the United States is
dence. It is classified as acute (0–24 h), subacute (>24 h), late approximately 0.9–2% among the adult population, increas-
(1–30 day), and very late (1–12 month) type 4b MI depending ing significantly after age 65 [5–7]. Both cardiovascular and
on the timing of the PCI procedure. Type 4c acute myocardial non-cardiovascular can cause HF. Ischemic heart disease,
infarction is restenosis associated with PCI procedure, but hypertension, and chronic kidney disease are the most com-
there is no culprit lesion or thrombus in the infarct area. mon causes of HF. Other pathologies, such as metabolic dis-
eases, congenital heart disease, immune-mediated disease,
and arrhythmias, can also lead to HF [4].
37.1.4 Management Impaired left ventricular ejection fraction (LVEF) is fre-
quently observed in a wide range of patients with HF; how-
Relief of ischemic pain is very important since the pain could ever, LVEF of some patients with HF is mid-reduced or even
promote vasoconstriction and increase the workload of the normal. According to ESC criteria, HF can be classified into
heart. Oxygen therapy should be applied in the presence of a three groups: (1) HFrEF-HF with a reduced ejection fraction
blood oxygen saturation of less than 90% or respiratory (<40%); (2) HFmrEF-HF with a mid-range ejection fraction
distress. (40–49%); (3) HFpEF-HF with normal ejection fraction
Once STEMI diagnosis is made a decision, patients (≥50%). The terminology is a practical benefit to differing
should be treated immediately with primary PCI if the antici- aetiologies and co-morbidities and responses to treatment.
pated absolute time from STEMI diagnosis to PCI-mediated To describe the time course of HF, New York Heart
reperfusion is less than 2 h; otherwise, patients should be Association (NYHA) functional classification defines four
treated with thrombolytic therapy as soon as possible. CABG functional classes based on symptoms of heart failure and
is an alternative treatment, especially in multivessel or com- exercise tolerance: (1) Class I: physical activity is unlimited,
plex STEMI [3]. and ordinary physical activity does not result in shortness of
NSTEMI patients should be treated with anticoagulation, breath, fatigue or heart palpitations; (2) Class II: physical
which regimen should be depended on conservative or inva- activity is faintly limited. Ordinary physical activity causes
sive management strategy. Coronary angiography and revas- undue breathlessness, fatigue, or palpitations but patients
cularization are recommended. The optimal timing of feel comfortable at rest; (3) Class III: physical activity is sig-
angiography should be classified into four categories based nificantly limited. Ordinary physical activity results in short-
on the risk profile of the individual patient [4]. ness of breath, fatigue, or heart palpitations but patients still
feel comfortable at rest; (4) Class IV: physical activity is
completely limited. Any physical activity can cause signifi-
cant symptoms, even at rest [8].
37.1.5 Conclusion
Acute myocardial infarction is acute myocardial injury,

which diagnosis depends on troponin level and clinical char-
acters specially NSTEMI. In clinical application of troponin,
The clinical appearance of HF is often multifarious. Typical
pain time is important to rule out NSTEMI when employing
symptoms include breathlessness, orthopnea, paroxysmal
the basic value of troponin. Serial measurement of troponin
nocturnal dyspnea, reduced physical activity levels, fatigue,
is very useful to rule in NSTEMI. Hs-cTn assay reduces
and swelling in the foot or ankle. Specific signs of HF may
obviously made decision time.
be observed, such as raised jugular venous pressure, hepato-
jugular reflux symptom, and additional heart sounds (gallop
rhythm). Atypical symptoms include dizziness, syncope,
37.2 Heart Failure palpitations, and nocturnal cough. Discrimination of HF is
often difficult due to nonspecific symptoms and signs,
Zhou Zhou and Yahui Lin especially in obese patients, elder patients, and patients with
chronic lung disease.
37.2.1 Overview
37.2.3 Laboratory Diagnosis
Heart failure (HF) is defined as a complex clinical syndrome
in which the heart is no longer able to pump enough blood to Natriuretic peptides (NPs) secreted from the heart are hor-
meet the physical requirements because the heart is unable to mones related to natriuretic and kaliuretic bioprocedure. NPs
consist of atrial natriuretic peptide (ANP), brain (B-type) >25% increase of NT-proBNP and >50% increase of cardiac
natriuretic peptide (BNP), and C-type natriuretic peptide troponin were significantly associated with a greater risk for
(CNP) [9]. Pre-pro ANP is a 151-amino-acid precursor con- systolic dysfunction, HF events, and cardiovascular death. A
tains a 25–amino-acid signal sequence, N-terminal ANP1–98 multi-biomarker strategy including NT-proBNP, mid-
(NT-ANP), and the biologically active ANP99–126. The plasma regional proANP, high-sensitivity cardiac troponin, cystatin-
concentrations of ANP1–98 are higher than ANP99–126, because C, and urinary albumin-to-creatinine ratio is predictive for
the half-life of ANP1–98 (40–60 min) is longer than ANP99–126 new-onset HF.
(2–5 min). Pre-pro BNP is a 134-amino-acid precursor con- ST2 (suppression of tumorigenicity 2) is an interleukin 1
tains a 26-amino-acid signal sequence, N-terminal BNP1–76 receptor-like 1 (IL1RL-1). Membrane-bound ST2 activates
(NT-proBNP), and the biologically active BNP77–108. The the transcription factor nuclear factor-κB (NF-κB) and the
half-life of BNP77–108 (20 min) is much shorter than that of mitogen-activated protein kinase (MAPK) pathway via
NT-proBNP (120 min). interaction with interleukin 33 (IL-33) that is secreted by
ANP, mainly produced by cardiomyocytes of the cardiac necrotic cells. Ultimately, IL-33/ST2L signaling leads to the
atria, reduces cardiac volume load through enhancing diure- production of inflammatory factors and a Th2 immune
sis, natriuresis, vasodilation, and inhibition of renin–angio- response. Truncated soluble ST2 (sST2), a decoy receptor
tensin–aldosterone system (RAAS) in response to increased of IL-33, is detectable in circulating blood and modulates
atrial wall tension during heart failure. BNP, primarily the IL-33/ST2L signaling pathway. sST2 levels correlate
secreted from cardiomyocytes of the cardiac ventricles, has with features of worse and prognosis in HF; meanwhile,
similar physiological action in response to increased left and/ sST2 is not affected by age, renal function, or body mass
or right ventricular volume and pressure loads. The majority index. sST2 levels <35 pg/mL indicate a low risk of adverse
of CNP is found in the central nervous system and vascular outcomes.
endothelial cells with very low plasma concentration.
Measurements of NPs are considered as primary bio-
markers to diagnose or exclude HF in acute dyspnea, prog- 37.2.4 Management
nosis and admission in acute and chronic HF and evaluation
of pre-discharge prognosis. Early diagnosis and treatment are very important in acute HF
For patients with suspected non-acute HF, NPs should be to make life longer. Initial evaluation and continued noninva-
measured if an individual has prior clinical history (such as sive monitoring of the patient’s pulse oximetry, blood pres-
myocardial infarction, revascularization, arterial hypertension, sure, respiratory rate, and a continuous ECG instituted within
diuretics use, orthopnea, and paroxysmal nocturnal dyspnea), minutes, are essential to evaluate the adequacy of ventilation,
or present abnormality of ECG and physical examination peripheral perfusion, oxygenation, heart rate, and blood
(such as rales, heart murmur, bilateral ankle edema, jugular pressure. According to the type and severity of HF, all
venous dilatation and laterally displaced). When the plasm patients with suspected acute HF should have a diagnostic
level of NT-pro BNP < 125 pg/mL or BNP < 35 pg/mL, other workup and appropriate pharmacological and non-
diagnoses should be considered. Echocardiography should be pharmacological treatment in parallel.
carried out to confirm HF when plasm level of NT-pro Aims of management of HF are treating aetiologies of
BNP ≥ 125 pg/mL or BNP ≥ 35 pg/mL. HF, reducing symptoms, increasing your lifespan, and
For patients with acute dyspnoea and suspected acute HF, improving your quality of life by medicines (e.g., ACE
NPs should be measured in all patients since NPs is sensitiv- inhibitors, angiotensin receptor blockers, beta-blockers,
ity to rule-out non-cardiac dyspnoea. When BNP < 100 pg/ diuretics, and digoxin) or medical procedures (e.g., implant-
mL, NT-proBNP <300 pg/mL, or MR-proANP <120 pg/mL, able cardioverter defibrillator or heart transplant).
cardiac dyspnoea is unlikely. When NT-proBNP >450 pg/mL
for <50 years, >900 pg/mL for 50–75 years, and > 1800 pg/
mL for >75 years, or BNP >400 pg/mL, positive predictive 37.2.5 Conclusion
value of acute HF will be improved. Other laboratory tests,
including cardiac troponin, D-dimer, procalcitonin, blood HF is a complex syndrome and signs involving many causes
creatinine and urea nitrogen, electrolytes, glucose, blood gas and pathological processes. NPs are key biomarkers in diag-
and liver function, should be performed to detect ACS, pul- nosis, prognosis, and admission of HF. Soluble ST2 is prom-
monary embolism, infection, kidney, and liver function [10]. ised a novel biomarker to predict the risk of HF. In the future,
Aggregate evidence supports that NPs are valuable risk the multi-biomarker strategy may be valuable to prognosti-
biomarkers to predict the long-term development of HF. Both cation and treatment strategies.
37.3 Myocarditis Table 37.2 Noninfectious aetiologies of myocarditis

Drugs Aminophylline, amphetamine, anthracyclin,
Zhou Zhou and Yahui Lin catecholamines, chloramphenicol, cocaine
cyclophosphamide, doxorubicin, 5-fluorouracil,
mesylate, methysergide, phenytoin,
trastuzumab, zidovudine, Interleukin-2
37.3.1 Overview Hypersensitivity Drugs azitromycin,
reactions benzodiazepines, clozapine,
Myocarditis is an inflammatory disease of the myocardium cephalosporins, dapsone,
dobutamine, lithium,
with an infiltration of inflammatory immune cells to the heart diuretics, thiazide,
muscle cells. Myocarditis is associated with dilated cardio- methyldopa, mexiletine,
myopathy (DCM), ventricular arrhythmias, heart failure, Streptomycin, sulfonamides,
cardiogenic shock, or cardiac death. Specifically, about 12% non-steroidal anti-
inflammation drugs, tetanus
of sudden cardiac death in young adults is linked to acute toxoid, tetracycline, tricyclic
myocarditis [11]. antidepressant
Myocarditis could be caused by several infectious and Venoms Bee, wasp, black widow
noninfectious agents. Infectious causes include viruses, spider, scorpion, snakes
bacteria, protozoa, fungi, spirochete (Table 37.1). Systemic Churg–Strauss syndrome, collagen diseases,
diseases sarcoidosis, Kawasaki disease, scleroderma
Noninfectious causes include direct toxic of drugs and
Autoimmune Dematomyositis, inflammatory bowel disease,
other diseases, such as systemic diseases or autoimmune diseases rheumatoid arthritis, sjögren syndrome,
diseases (Table 37.2) [12]. systemic lupus erythematodes, Wegener’s
granulomatosis, giant cell myocarditis
Others Heart stroke, hypothermia, transplant rejection,
Table 37.1 Infectious aetiologies of myocarditis radiation injury
Viruses RNA viruses Picornaviruses Echovirus
(Coxsackie A/B
virus) Virus-induced myocarditis and post-infectious inflam-
Poliovirus matory cardiomyopathy have been increasingly considered
Hepatitis virus as the most common cause. Approximately 70% of patients
Orthomyxovirus Influenza virus with myocarditis were infected with enteroviruses, adeno-
Paramyxoviruses Respiratory viruses, parvovirus B19, or human herpesvirus 6 using
syncytial virus nuclear detection and in situ hybridization technologies.
Mumps virus
Among all the virus agents, coxsackie B viruses, which
Togaviruses Rubella virus
Flaviviruses Dengue fever
belong to the family Picornaviridae and the genus
Yellow fever Enterovirus, account for one-quarter of viral myocarditis
DNA viruses Adenovirus A 1, 2, 3, and 5 cases. Coxsackievirus-mediated myocarditis includes three
Erythrovirus 1 (B19V) and 2 phases: Phase I, during hours to 7 days, involves viral entry
Herpesviruses Human through receptor-mediated endocytosis and activation of
herpesvirus 6 innate immunes response; Phase II, for 1–4 weeks, involves
A/B
viral replication, myocyte degradation, and activation of
Cytomegalovirus
adaptive immune response; Phases III, during months to
Epstein–Barr
virus years, involves virus clearance, inflammation disappear-
Varicella-zoster ance, and recovery, otherwise the development of dilated
virus cardiomyopathy.
Retrovirus HIV
Bacteria chlamydia (C. pneumonia/psittacosis) haemophilus
influence, legionella, pneumophilia, brucella
clostridium, francisella tularensis, neisseria meningitis,
mycobacterium (tuberculosis), salmonella,
staphylococcus, streptococcus A, S. pneumonia, The clinical appearance of both acute and chronic myocardi-
tularemia, tetanus, syphilis, Vibrio cholera tis is highly variable and complex, including fatigue,
Fungi actinomyces, aspergillus, candida, cryptococcus, decreased exercise tolerance, fever, dyspnoea, palpitations,
histoplasma, nocardia
Protozoa Entamoeba histolytica, leishmania, Plasmodium
chest pain, syncope, gastrointestinal symptoms, or cardio-
falciparum, Trypanosoma cruzi, Trypanosoma brucei, genic shock. Some patients with myocarditis are asymptom-
Toxoplasma gondii, Larva migrans, Schistosomiasis atic presentations. Cardiac symptoms are indistinguishable
Spirochete Borrelia recurrentis, leptospira, Treponema pallidum from acute coronary symptoms, arrhythmogenic right ven-
tricular cardiomyopathy, cardiac arrhythmias, amyloid heart in late diagnosis because CK-MB back to basal levels within
disease, heart failure, and sudden cardiac death. 36–48 h.
Viral myocarditis frequently has rash, fever, loss of appe- Brain natriuretic peptide (BNP) and N-terminal–pro-
tite, abdominal pain, and respiratory or gastrointestinal syn- brain natriuretic peptide (NT-proBNP) levels are commonly
drome. The cardiac damage and syndrome in male patients elevated as a prognosis biomarker. NT-proBNP concentra-
with viral myocarditis are more severe than in female tion (7962 ng/mL vs. 1771 ng/mL) was significantly higher
patients, since women might have natural hormones to regu- in the Chinese patients with fulminant myocarditis than
late inflammatory reactions. Young patients with acute myo- those with non-fulminant myocarditis. BNP mean level is
carditis often present more fulminant signs and symptoms, significantly higher in the non-survivor pediatric myocarditis
which may resemble angina with acute myocyte infarction or than in the survivor children (24 U/L vs. 8 U/L) [18]. A sig-
pericarditis. The suspected myocarditis had 75% dyspnea, nificantly higher BNP level is associated with poor outcomes
32% chest pain, and 18% arrhythmias in the European Study in adolescents with clinical myocarditis and normal left ven-
of the Epidemiology and Treatment of Inflammatory Heart tricular (LV) systolic function [19].
Disease [13].
37.3.3.3 Inflammation Biomarkers
Nonspecific inflammation biomarkers, such as leukocyte
37.3.3 Laboratory Diagnosis count, C-reactive protein (CRP), and erythrocyte sedimenta-
tion rate are commonly elevated in myocarditis although
37.3.3.1 Endomyocardial Biopsy they are not used as diagnostic markers. Laboratory tests
Endomyocardial biopsy (EMB) remains the “gold standard” should include erythrocyte sedimentation rate and CRP lev-
for the diagnosis of myocarditis. In accordance with the els according to a position statement of the European Society
Dallas criteria, lymphocytic infiltration in association with of Cardiology Working Group on Myocardial and Pericardial
myocyte necrosis is defined as acute myocarditis. EMB Diseases [17]. Inflammatory cytokines that cause myocar-
might differentiate between giant cell myocarditis and sar- dial injury, including tumor necrosis factor (TNF), IL-1α,
coidosis. Notably, the sensitivity of the EMB and the correla- IL-1β, IL-2, IL-6, IL-10, and IFN-γ, are also raised during
tion between cardiovascular magnetic resonance (CMR) and the autoimmune reactions phase. Meanwhile, the cytokines
EMB for detection of myocarditis is rather low due to sam- are nonspecific in the diagnosis of myocarditis. Serum levels
pling time, sampling position, and operator experience [14]. of IL-10 are significantly higher in fulminant myocarditis,
It is a necessity to evaluate risk and cost when EMB is particularly in non-survivor myocarditis (74.0 pg/mL vs.
employed to confirm myocarditis. In clinical practice, it is 16.4 pg/mL); therefore, IL-10 level may predict the mortality
common that myocarditis is diagnosed according to clinical of fulminant myocarditis patients [20].
evaluation, biomarkers, and noninvasive imaging.
37.3.3.4 Pathogenetic Diagnosis
37.3.3.2 Cardiac Biomarkers The diagnostic value of positive viral or bacterial antibodies
Cardiac troponin (cTn), a special marker of myocardial is poor due to their antibodies are highly prevalent in the
injury, significantly rose when myocyte is damaged directly general population without infective myocarditis. It was
by virus or indirectly by abnormal immune responses, there- pointed out that positive viral or bacterial antibodies were
fore both troponins I and T are significantly elevated in myo- not correlated with EMB results.
carditis. Contemporary troponin T assay has a sensitivity of With the rapid development of molecular diagnostic tech-
71% and specificity of 86% with a cut-off value of 0.052 ng/ nologies, molecular analysis of the genome of pathogenic
mL in children with acute myocarditis [15]. High sensitive agents using issue or blood may be a potential tool in the
troponin T (hs-cTnT) has a sensitivity of 83% and specificity diagnosis of infective myocarditis. It has been reported that
of 80% with a cut-off value of 50 pg/mL for predicting acute the correlation between CMR and viral PCR in the blood
myocarditis in adults [16]. Prolonged troponin level upon was 84%. The obtained gene sequence of pathogenic agents
99th percent reference upon level is associated with a worse may identify pathogenetic subtypes and assist the manage-
prognosis. Therefore, it is recommended that troponins ment of infection myocarditis.
should be assayed in all suspected myocarditis [17]. The
serial change value of cTn concentrations for 3–24 h may
help distinguish myocarditis from acute coronary 37.3.4 Management
syndrome.
Creatinine kinase MB (CK-MB) lacks sensitivity and Confirmation of the specific causes of the disease is effective
specificity in the diagnosis of acute myocarditis, especially to manage acute and chronic myocarditis. The antiviral
immune response or anti-autoimmune injury can reduce car- 37.4.2 Clinical Appearance
diomyocyte damage directly by the virus. If the pretreatment
damage is severe, treatment against etiology may help halt Different types of heart defects lead to a range of clinical
the rapid progression of the disease but not achieve signifi- phenotypes in different congenital heart diseases. The over-
cant improvement in cardiac ventricular function. On the all clinical characteristics of these patients include: diapho-
other hand, the benefits of antiviral therapy and immuno- resis, tiredness, feeding difficulties, breath hard, cyanosis,
modulatory drug treatment are still controversial during the clubbed fingernails, and abnormal cardiac auscultation [23].
subacute stages of myocarditis. Therefore, preventing HF is Even some patients with severe congenital heart disease had
still an attractive strategy to treat patients with myocarditis. lower motor ability and lung function parameters.
37.4.2.1 Excessive Sweating

37.3.5 Conclusion Patients with CHD usually have higher evaporation loss than
normal children. Increased sweating may be part of the
So far, inexpensive, sensitive, and specific diagnostic bio- explanation. Some researchers believe that sweating and
markers for the diagnosis of myocarditis are not available. congestive heart failure can be linked. People with heart dis-
Molecular inflammatory markers in peripheral blood com- ease may have more heat loss especially those with conges-
bined with imaging and newer immunohistological staining tive failure to some extent. One way to dissipate this heat is
techniques are able to improve the diagnosis and prognosis increasing sweating [24].
of acute myocarditis. There remains a need for better bio-
markers to detect the early stage of acute myocarditis. It is 37.4.2.2 Poor Feeding
also important to explore novel biomarkers which more In infants, difficulty eating can be the first sign of decline of
accurately assess myocarditis severity and prognosis. heart function that occurs in about 30% of the patients with
CHD [25].
37.4 Congenital Heart Disease (CHD) 37.4.2.3 A cyanotic and Cyanotic Congenital

Heart Diseases
Zhou Zhou and Yahui Lin Some patients with congenital heart disease have cyanosis, a
bluish-purple color in the skin or mucous membrane caused
by an increase in deoxygenated hemoglobin in the blood.
37.4.1 Overview Therefore, CHD could be categorized into cyanotic and acy-
anotic types. Blood flow in the heart of cyanotic CHD is
Congenital heart disease (CHD) is one of the most com- characterized by right-to-left shunt, leading to hypoxic blood
monly seen congenital defects in live birth [21], accounting into oxygenated circulation, e.g., severe tetralogy of Fallot,
for one-third of all kinds of congenital malformations. It total anomalous pulmonary venous drainage, and transposi-
refers to anatomical abnormalities caused by abnormal tion of great vessels. Moreover, cyanotic CHD is often
development of the heart and great vessels during embryonic accompanied by the occurrence of clubbing. Acyanotic CHD
development. Accounting for 0.4–1% of the newborn babies includes left-to-right shunt and no shunt, e.g., atrial and ven-
means that 1.4 million babies worldwide suffer from CHD tricular septal defects, patent ductus arteriosus [26].
every year. However, the incidence of CHD cannot be
underestimated. 37.4.2.4 Heart Murmurs
Many pathogenic factors of CHD are known till now, When abnormal blood flow flowed through chambers of the
including genetic factors, environmental factors, and/or heart, a heart murmur is heard in the heartbeat, which can
genetic-environment interactions. Understanding the cause be measured with a stethoscope to check for heart defects.
of CHD is important in estimating the recurrence rate, guide The relationship between murmurs and congenital heart
treatment options, and can be used to develop public health disease is complex and varies with the observed population.
recommendations to reduce the prevalence of CHD [22]. In A healthy person often has heart murmurs. These murmurs
recent years, genetic testing techniques such as chromo- are usually harmless as the result of normal blood flow in
somal microarray and next-generation sequencing (NGS) the chambers of the heart and vessels. However, heart mur-
were in wide use, making it accessible to understand the murs may be the only finding among those patients with
underlying genetic factors. CHD [27].
37.4.3 Laboratory Diagnosis or normal cells to compare the differences of DNA expres-
sion between them to detect copy number variants (CNVs).
CHD can be prompted by pulse oxygen saturation, but it CNVs range widely from one gene to the variation of mil-
only can be used to screen for critical congenital heart dis- lions of base pairs, and it is one of the pathogenesis of CHD
ease since it has many limitations for diseases with late [32]. In fact, deletions are more harmful than duplications
changes in Pulse Oximetry and only can be used as a screen- due to haploinsufficiency. Previous studies have shown that
ing test [28]. Diagnosis of CHD usually depends on imaging children of CHD with pathogenic CNVs have poorer prog-
detection, which has highly investigator dependent, and noses and outcomes than children without pathogenic CNVs
Doppler gradients sometimes may be misleading with some [33, 34]. Array CGH is the most common technology for the
diseases which are difficult to image [29]. Genetic detection detection of CNVS. There are syndromes like Williams syn-
is increasingly being applied to the detection of congenital drome and Noonan syndrome caused by CNVs that can be
heart disease. Genetic causes of CHD can be classified into detected by Array CGH. These syndromes are always con-
the following categories: chromosomal aneuploidies, large comitant with CHD [35]. Because of CNVs usually involves
chromosomal rearrangements, single-gene mutations, copy multiple genes, it is hard to distinguish the entire phenotype
number variations. And common detection methods for CHD are caused by the effect of single genes or multiple genes.
contain the following [30]: The studies of the association of CNVs with CHD are not
clear enough, it is still unclear that the other determinants of
37.4.3.1 Karyotyping CHDs are existing or not, but they may interact at a genetic
Aneuploidy is the lack or addition of one or several chromo- level.
somes in a euploid chromosome. Some syndromes like
Down syndrome and Turner syndrome, concomitant with 37.4.3.3 Whole-Exome Sequencing
congenital anomalies including CHD, are caused by chromo- Whole-Exome Sequencing (WES) is a genomic analysis that
somal aneuploidies. Down syndrome is the most common uses sequence capture technology to test the protein-coding
aneuploidy, usually caused by trisomy 21, which is also the region for high throughput sequencing followed by Next-
most common chromosomal cause of CHD. The same tri- Generation Sequencing (NGS). It can be used to discover
somy also occurs in 13 and 18 chromosomes. Turner small genetic variations such as SNPs or INDELs in the exon
Syndrome usually appears in females with loss of part or all region, which is associated with CHDs [30]. The majority of
of the X chromosome. Diseases caused by chromosome CHD occur as non-syndromic and isolated defects, about a
abnormality usually concomitant with CHD [30]. hundred genes have been already confirmed that possess
Karyotyping is the gold standard genomic test for aneu- definitive or presumptive association with CHDs such as
ploidy and large chromosomal deletions or duplications NKX2.5, GATA4, and NOTCH1. WES is necessary to apply
detection, usually performed on metaphase chromosomes. In to test those CHD-associated genes. Family studies is also
CHD patients, the frequency of chromosome abnormality important to perform since different laboratories may have
detected by karyotyping ranged from 3% to 23%. In addition different interpretations for the same variation and the same
to the chromosome aneuploidy mentioned above, karyotype variation between the family members can cause different
can also be used for the detection of chromosome deletions phenotype which means the ability of targeted gene sequenc-
or duplications like deletion of the short arm of chromosome ing based on phenotype becomes more complicated. The use
4, the cause of Wolf–Hirschhorn syndrome and of chromo- of WES in family studies can help the establishment of a
some 5, the cause of Cri-du-Chat syndrome. As is reported, genetic cause of CHD, which can provide more accurate
chromosomal deletions or duplications are heritable, and recurrent risk for future pregnancy to effectively decrease the
there is always an indication of parents’ karyotype [31]. It is morbidity of CHD. The limitation of WES is that it only can
necessary to detect the karyotype of the patient’s parents for detect the variations in exon regions as well as ignore the
evaluating whether the patient’s karyotype is heritable or a variations of other regions except for exons and some large
new variation. There is also necessary to do genetic counsel- genetic variations [36].
ing for the families to access the risk of recurrence.
Karyotyping is also can help the prevention of heritable 37.4.3.4 Whole-Genome Sequencing
chromosome abnormality but cannot discover small genetic Same as WES, Whole-Genome Sequencing (WGS) can
variation. detect small genetic variation in exon, but it also can reflect
whole information of the genome. For variations occurring
37.4.3.2 Array CGH outside the exon, it can be detected by WGS, which is bet-
Array comparative genomic hybridization (Array CGH), a ter than WES. It is obvious that the hereditary constitution
Molecular cytogenetic technology, is performed by hybrid- of CHD seems polygenic, and the risk of heritable variation
izing the DNA probes of both abnormal cells and reference is widely distributed throughout the genome, which cannot
be detected by WES. But the limitation of WGS is that the like drug or alcohol abuse and stress, can contribute to the
huge whole-genome drives the price of the test increased development of arrhythmia. Its occurrence can also be sec-
and the universal application of WGS may lead to waste of ondary to other heart diseases like hypertension, diabetes,
resources [37]. and congenital heart disease, as well as injuries like the heal-
ing process from surgery. Gender difference appears to exist
in several but not all types of arrhythmia and the male sex is
37.4.4 Management considered as a risk factor in some of them. The most promi-
nent one is found in Brugada Syndrome patients with 8- to
Patients with CHD could have better outcomes if they got 10-folds more male than female [38]. Furthermore, similar
proper care. Therapeutic schemes of CHD are depending on trend in gender is also seen in patients with CPVT and PCCD
the specific type of CHD and overall development. Different [39, 40].
CHD require different treatment methods according to the
clinical situation. Typical effective patient management is a
comprehensive, elaborative, and all-around corporate strat- 37.5.2 Clinical Appearance
egy with the collaboration of electro-physiologist, surgical
cardiologist, and anesthesiologists. Each type of arrhythmia has its own characteristic symp-
toms and phenotypes, including dizziness, palpitations,
abnormal heart beating, and the feeling of weakness [21].
37.4.5 Conclusion Those patients were combined with coronary heart disease
or severe left ventricular dysfunction may even suffer from
The availability of new molecular technologies has facili- severe consequences including heart failure or sudden car-
tated gene discovery and altered medical and cardiac care for diac death (SCD). Within over 600,000 annual death in the
many CHD patients. Many major medical centers in the USA due to HF or SCD, around half of them were due to
United States now have an accurate molecular diagnosis of arrhythmias.
the CHD technology, which can not only get an early diagno- At the early stage of the disease progress, patients are
sis but also can determine the risk of recurrence of the fam- normally asymptomatic, although some abnormal electro-
ily, providing reproductive choice. A carefully taken history cardiogram occasionally occurred during physical examina-
of familial CHD, and clinical symptoms (including other tion. At the late stage of the disease progress, serious
extra-cardiac defects) into consideration were recommended bradycardia or chronotropic incompetence (the movement
in patients in whom undergoing a clinical molecular cannot be increased heart rate) symptoms of the patient are
diagnostic. presented. Owing to the sinus intermittent or highly atrio-
ventricular block, patients with the former symptoms may
appear amaurosis or syncope. Cases without any symptoms
37.5 Arrhythmia are very rare.
37.5.1 Overview
Arrhythmia is a group of disorders involving irregular
rhythms of heartbeat (too fast, too slow, or erratically), which The most effective way to diagnose an arrhythmia and
can occur at any age [21]. There are many types of arrhyth- identification of subtypes was by an electrical recording of
mia including bradycardia, tachycardia, flutter or fibrillation, heart rhythm called an electrocardiogram (ECG). For
premature contraction, etc. Among them, those primary elec- example, the hallmark of long QT syndrome is the finding
trical disorders are also referred to as channelopathy, some of of QT prolongation on surface ECG, in association with a
which are caused by defective genes leading to the cardiac risk of ventricular arrhythmias, syncope, or sudden death;
channel dysfunction involved in the action potential. The short QT syndrome SQTS is characterized by an abbrevi-
common channelopathies included long QT Syndrome ated QT interval and a risk of both atrial and ventricular
(LQTS), short QT Syndrome (SQTS), Brugada Syndrome, arrhythmias. The diagnosis of arrhythmias is based on clin-
catecholaminergic polymorphic ventricular tachycardia ical grounds, but the incorporation of genetic testing in the
(CPVT), and progressive cardiac conduction disease evaluation of arrhythmias can be a powerful supportive
(PCCD). However, the most common type of arrhythmia is tool, particularly as the results may assist in risk stratifica-
atrial fibrillation (AF), which mainly affects the elderly. tion, guide therapy, and facilitate family screening. Genetic
Genetic factors including rare variants in ion channels and testing for arrhythmia should not preclude a careful clinical
SNPs in predisposing genes, and other environmental factors evaluation.
37.5.3.1 LQTS 1/10000 [41]. CPVT can be presented as autosomal domi-

At least 16 LQTS-related pathogenic genes are currently nant or recessive, and nowadays recognized CPVT patho-
reported, including 9 pathogenic genes, which encode genic genes include RYR2 and CASQ2. The RYR2 gene
voltage-gated potassium, sodium, calcium channel pro- encodes a ryanodine receptor and is autosomal dominant,
teins, and their associated regulatory proteins. Among with a detection rate of 65% [42]. CASQ2 gene encodes tro-
them, KCNQ1 (LQTS1), KCNH2 (LQTS2), and CN5A ponin, which is autosomal recessive, with a detection rate of
(LQTS3), those three pathogenic genes can explain about 3–5% [43].
75% of patients, and the remaining pathogenic genes can
explain 5–10% of patients [39]. LQTS is mainly in autoso-
mal dominant inheritance. In addition to autosomal domi- 37.5.4 Management
nant inheritance, KCNQ1 and KCNE1 can also cause
Jervell–Lange-Nielsen syndrome related to deafness by The purpose of arrhythmia patient management is to relieve
autosomal recessive inheritance mode. Owing to the lack of symptoms and prevent sudden cardiac death. There is a lack
specific clinical manifestations, most LQTS cannot be clas- of effective medical treatment. Patients with clinically symp-
sified only by clinical manifestations and traditional labora- tomatic bradycardia, complete atrioventricular block, and
tory tests. The precise classification depends on genetic high-risk of sudden cardiac death require a pacemaker. In the
diagnosis. view of the disease development, the evaluation of ECG
should be regularly. For symptomatic patients who do not
37.5.3.2 SQTS meet the pacemaker implant criteria, longer-term ECG
SQTS is usually in autosomal dominant inheritance. At least recordings, such as Holter monitoring and loop recording,
three genes have been identified to be associated with its should be considered to detect intermittent deterioration of
pathogenesis, and the pathogenicity of the KCNH2 gene is conduction abnormalities. Patients with movement intoler-
clear. This gene encodes a voltage-gated potassium channel ance should undergo an exercise test to detect insufficiency
protein that can explain about 20% of patients, suggesting or exercise-induced atrioventricular block in heart rate
that there are additional pathogenic genes to be discovered. changes. Echocardiography should also be reviewed regu-
SQTS has a certain explicit delay, the age of onset is differ- larly in specific patients, especially in patients with LMNA,
ent, and it can also bear the disease-causing gene for life DES, and SCN5A mutations, as well as patients with clinical
without any clinical appearance. In clinical gene diagnosis, symptoms suspected of having heart failure. The physician
detection should include the KCNH2 gene (gene ID: 3757, should develop an individualized plan for the frequency of
genetic pattern: autosomal dominant, gene percentage follow-up depending on the severity of the conduction abnor-
18–33%). mality, the speed of disease progression, the presence of
symptoms, and the age of the patient.
37.5.3.3 Brugada Syndrome
Brugada syndrome is autosomal dominant, and more than 20
related pathogenic genes have been reported, but currently, 37.5.5 Conclusion
only the pathogenicity of the SCN5A gene encoding the car-
diac sodium channel alpha subunit is clear. In clinical gene The establishment of a clinical diagnosis of arrhythmia
diagnosis, the detection should include the SCN5A gene requires an electrocardiogram to show a signal abnormality
(gene ID: 6331, genetic pattern: autosomal dominant, gene at any single or multiple levels. When evaluating young
percentage 10–15%). patients with unexplained cardiac conduction defects, it is
advised to consider the family history of the upper and
37.5.3.4 Catecholaminergic Polymorphic lower generations. For young (<50 years old) primary
Ventricular Tachycardia arrhythmia patients with or without cardiomyopathy or con-
Catecholaminergic polymorphic ventricular tachycardia genital heart disease, genetic testing should be considered,
(CPVT) is a rare but serious hereditary arrhythmia and ion especially in the presence of a positive family history. In
channel disease. It manifests as episodes of syncope when younger patients, arrhythmia incidence is lower and more
exercising or activating. The ventricular tachycardia can be likely to be heritable; in older patients, arrhythmia incidence
revoked by itself or converted to ventricular fibrillation. If is higher and more likely to cause deterioration of the con-
cardiopulmonary resuscitation is not performed in time, duction system. In summary, detailed clinical examinations
SCD can be caused. The prevalence of CPVT has still not of family members suspected of having an arrhythmia,
been seen in large-scale epidemiological surveys at home including their age of onset and commonalities, are impor-
and abroad, usually as case reports or single-center small tant for understanding hereditary presence, patterns, and
sample size data, reported in the West by approximately phenotypic identification.
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Lung Disease
38
Liang Ming, Ting Sun, Haitao Ding, Juan He, Wenjuan Wu,
Min Zhang, Simin Yang, Huaguo Xu, Fang Ni,
Shiyang Pan, Qun Zhang, and Yongping Lin
The lungs are the respiratory organs and the important hema-
topoietic organs of the human body. Lungs are located in the
chest on both sides of the heart. Each lung of human is
wrapped in a thin membranous capsule called the pleura, and
each is connected to the trachea through its large air passage
and to the heart through the pulmonary arteries. The lungs
are soft, light, spongy, and elastic organs that normally con-
tain some air after birth (Fig. 38.1).
The main function of the lungs is gas exchange, extract-
ing oxygen from the air and transferring it to the blood-
stream, and releasing carbon dioxide from the bloodstream
into the air. Respiration is driven by different muscular sys-
tems, primarily the diaphragm in human beings. Also, lungs
can provide airflow that makes vocal sounds. Moreover, the
Fig. 38.1 Diagram of the human lungs
L. Ming (*) · T. Sun

Department of Clinical Laboratory, The First Affiliated Hospital lungs are not only implicated in gas exchange, but also per-
of Zhengzhou University, Zhengzhou, Henan, form an important endocrine function.
People’s Republic of China Lung disease refers to disorders that affect the lungs.
e-mail: mingliang@zzu.edu.cn
The common lung diseases including lung cancer, cystic
H. Ding (*) · J. He fibrosis, pneumonia, and tuberculosis will be discussed
Inner Mongolia People’s Hospital,
Huhhot, Inner Mongolia Autonomous Region,
below.
W. Wu (*) · M. Zhang · S. Yang
Shanghai East Hospital South Branch Affiliated to Tongji 38.1 Lung Cancer
H. Xu · F. Ni · S. Pan (*) Liang Ming and Ting Sun
e-mail: huaguoxu@njmu.edu.cn; sypan@njmu.edu.cn
38.1.1 Overview
Q. Zhang
The First Affiliated Hospital of Nanjing Medical University,
Lung cancer is the leading cause of cancer death worldwide,
Nanjing, People’s Republic of China accounting for one-quarter of all cancer deaths [1]. The
Y. Lin
elderly population is the predominant sufferer of lung can-
Department of Clinical Laboratory, The First Affiliated Hospital of cer with a median age of 70 years at diagnosis. Nearly 70%
Guangzhou Medical University, Guangzhou Medical University, of lung cancer patients are over 65 years old, while less than
Guangzhou, People’s Republic of China

596 L. Ming et al.
Fig. 38.2 Histopathological
typing of lung cancer
3% of people under the age of 45 years. Lung cancer is usu- 38.1.2 Etiology and Pathogenesis
ally diagnosed at an advanced stage, leading to high mortal-
ity. The 5-year survival rate is poor, about 15% for all stages Smoking is the most common etiology of lung cancer, as pre-
combined. Even in patients with early excision or locally vious studies reported; tobacco usage is responsible for over
advanced chemoradiotherapy, up to 90% eventually relapse. 80% of lung cancer deaths. In addition, about 10–15% of
The World Health Organization (WHO) sorts lung cancer cases occur in nonsmoker. These cases are usually caused by
into two histological subtypes: non-small cell lung cancer a combination of genetic and environmental factors.
(NSCLC), which accounts for about 85%, as well as small- However, a significant fraction of nonsmoking lung cancer
cell lung cancer (SCLC), which accounts for the remaining patients is not clearly associated with environmental risk
15% [2, 3]. NSCLC includes lung adenocarcinoma, lung factors.
squamous carcinoma, and large cell carcinoma subtypes. Tumorigenesis is a multistage, gradual process, driven
Computed tomography (CT) has been recommended to be by a sequential accumulation of epigenetic and genetic
the best approach for lung cancer screening [4] (Fig. 38.2). abnormalities [6]. Consistent with most cancers, lung can-
The US National Lung Screening Trial (NLST) pointed out cer is initiated by either the activation of oncogenes or the
that individuals randomly screened with low-dose CT scans inactivation of tumor suppressor genes. Carcinogens cause
had 20% lower lung cancer mortality than those with tradi- driver mutations in lung cancer include KRAS, HER2,
tional chest X-rays [5]. Although low-dose CT screening has BRAF, EGFR, ROS1, ALK, RET, MEK1, and PIK3CA
been recognized as the foremost advance for lung cancer [7]. Moreover, DNA methylation, chromatin remodeling,
early detection over the past decades, the pros and cons of non-coding RNA expression, and epigenetic changes are
lung cancer screening remain controversial. The large num- key events involved in each step of lung cancer pathogen-
ber of false positives and potential overdiagnosis are causes esis [8].
for concern. Although most lung cancers are still diagnosed
at an advanced stage, significant advances have been made in
molecular diagnosis for selecting patients at the highest risk. 38.1.3 Clinical Appearance
The key to precision diagnosis of lung cancer is to develop
novel molecular biomarkers, which not only play a signifi- Lung cancer usually causes no signs or symptoms at the
cant role in diagnosis, but also provide valuable information early stage, which typically occurs at the advanced stage.
for therapeutic target selection. Signs and symptoms suggesting lung cancer include:
38 Lung Disease 597
Respiratory symptoms Coughing, wheezing, shortness of and should be available to patients at high risk of lung cancer
breath, or coughing up blood. [5]. CT screening should be performed at indicated time or
frequency, as the extended monitoring increases the risk of
Systemic symptoms Fever, weakness, or weight loss. radiation exposure and is costly.
Symptoms owing to cancer compression Chest pain, bone Magnetic Resonance Imaging
pain, superior vena cava syndrome (SVCS), or difficulty If lung cancer is confirmed by biopsy, Magnetic Resonance
swallowing. Imaging (MRI) is primarily used to assess the size, extent,
and degree of its spread to adjacent structures.
38.1.4 Routine Diagnosis (Fig. 38.3) 38.1.4.2 Endoscopic and Histopathological

Examination
38.1.4.1 Imaging Tests Laboratory diagnosis, including pathological and laboratory
tests, can improve the accuracy of the evaluation of the
Chest X-Ray benign or malignant tumors in patients with pulmonary nod-
The chest X-ray is usually the first radiological test per- ules judged by imaging examination. Laboratory diagnosis is
formed on the basis of a detailed history and physical exam- indispensable for the evaluation of lung cancer stage since
ination. It may show pulmonary mass or enlarged lymph imaging examination cannot reflect the biological character-
nodes. It should be emphasized that the chest X-ray alone is istics of tumor cells.
not enough to rule out lung cancer, and early lung cancers
are easily missed with these tests [9]. Sputum Cytology
Sputum cytology is one of the most convenient and nonin-
CT Scan vasive methods to diagnose lung cancer and is commonly
CT scan is usually the second step either to track abnormal used in lung cancer screening. But its application is lim-
chest X-ray findings or to assess complicated symptoms in ited to those cancers that extend into the airways. Sputum
those patients with a normal chest X-ray. In addition, low- cytology is inaccurate sometimes and can miss some can-
dose CT screening can reduce the risk of lung cancer deaths cer cells.
Fig. 38.3 Routine procedures for the diagnosis and treatment of lung cancer
598 L. Ming et al.
Bronchoscopy poor [10]. Molecular diagnosis provides a new opportunity

Bronchoscopy is the most common diagnosis of lung cancer. for the diagnosis and treatment of lung cancer [11].
During a bronchoscopy, a tube is inserted into the airways to Developing molecular diagnostics to identify more poten-
visualize and take tumor samples. This procedure is used tially clinical genetic variants in smaller samples obtained
when the tumor is found in the large airways and the tumor via minimally invasive techniques is a huge challenge.
biopsies can be taken from the airways.
38.1.5.1 Routine Biomarkers
Mediastinoscopy Although there is still no specific marker for lung cancer,
Mediastinoscopy is performed by incision at the base of the there are some markers that have a reference value for the
neck and insertion of surgical tools behind the sternum to diagnosis of lung cancer and for the distinction of tissue
extract tissue samples from the lymph nodes. It is the gold types, such as CEA, CYFRA21-1, SCC, NSE, and
standard for clinical evaluation of mediastinal lymph node ProGRP. Furthermore, the main value of these markers is to
status of lung cancer. reflect the changes in tumor load indirectly through dynamic
observation, to judge the development of the disease and the
Needle Biopsy therapeutic effect (Fig. 38.4).
Needle biopsy, also known as needle aspiration, is a less
invasive procedure that uses a hollow needle to remove some 38.1.5.2 C
ompanion Diagnostic Biomarker
cells from a suspicious area of the body and examine them (Fig. 38.5) (Table 38.1)
under a microscope to confirm the diagnosis. In a needle
biopsy of lung nodules, imaging techniques such as CT, EGFR
ultrasound, and sometimes MRI are frequently adopted to EGFR including wild-type and mutant is a common marker
lead the instruments to the abnormal growth sites. Biopsy for companion diagnosis. Lung cancer patients with wild-
samples of metastatic lung cancer can also be obtained from type EGFR gene do not respond to targeted drugs, whereas
lymph nodes or metastatic sites. those with the gene mutation are responders. Activating
mutations in EGFR, mainly in exon 19 (deletion) or in exon
21 (L858R), which increase the sensitivity of EGFR tyrosine
38.1.5 Molecular Diagnosis kinase inhibitors, are the first and most important discovery
of targeted therapy for lung cancer.
Although the diagnosis of lung cancer has made remarkable The prevalence of EGFR mutation is high in Asian non-
progress in previous decades, prognosis of the disease is still smoker women and adenocarcinoma patients, but very low
Fig. 38.4 The application of

routine tumor markers in lung
cancer diagnosis and
treatment
38 Lung Disease 599
in squamous cell carcinoma [12, 13]. Patients with these

mutations are more sensitive to tyrosine kinase inhibitors
(TKIs), Erlotinib and Gefitinib, two recommended TKIs for
the first-line treatment of EGFR mutation-positive
NSCLC. Unfortunately, TKIs are not effective for all patients
with EGFR-positive mutations. Moreover, many patients
sensitive to EGFR TKIs would develop resistance after
9–12 months. T790M in exon 20 is the most frequent sec-
ondary mutation for drug resistance, occurring in 40–60% of
patients. Osimertinib is a third-generation and irreversible
EGFR inhibitor, which targets the T790M mutation and the
primary activating EGFR mutations. But Osimertinib is still
unable to avoid drug resistance as the C797S mutation in
some patients causes new resistance [14].
Thus, the EGFR mutation detection is of great signifi-
cance for prognosis, therapeutic schedule design, and post-
Fig. 38.5 Single oncogenic driver paradigm of lung adenocarcinoma treatment monitoring [15]. The common methods for
molecular classification detecting EGFR mutations include sequencing, real-
time PCR, microfluidics digital PCR, dHPLC, and so on.
Table 38.1 Detection and the clinical significance of the companion diagnostic biomarkers in lung cancer
Gene Mutation style Detection methods Clinical significance Targeted drugs
EGFR Exon 19 (deletion) qRT-PCR microfluidics digital Histological typing and guideline for Erlotinib Gefitinib
PCR dHPLC sequencing drug therapy
Exon 21 (L858R) qRT-PCR microfluidics digital Histological typing and guideline for
PCR dHPLC sequencing drug therapy
Exon 20 (T790M) qRT-PCR microfluidics digital Implication for drug resistance to EGFR Osimertinib
PCR dHPLC sequencing TKIs
ALK Rearrangement qRT-PCR Molecular typing or staging of lung Crizotinib
FISH cancer staging; guideline for drug
IHC therapy
sequencing
C1156Y qRT-PCR microfluidics digital Implication for drug resistance to ALK Alectinib
G1269A PCR TKIs Ceritinib
L1196M dHPLC
sequencing
ROS1 Rearrangement qRT-PCR Molecular typing or staging of lung Crizotinib
FISH cancer staging; guideline for drug
IHC therapy
sequencing
KRAS Codon 12 (G → T) qRT-PCR Implication for drug resistance to EGFR
Codon 13 reverse-hybridization of TKIs
Codon 61 biotinylated PCR sequencing
BRAF Exon 15 (V600E)/(D594G) qRT-PCR and sequencing Guideline for drug therapy and Vemurafenib
exon 11 (G469A) implication for drug resistance to EGFR Dabrafenib
TKIs
MET Amplification exon 14 FISH Guideline for drug therapy Tivantinib
splice mutations SISH Onartuzumab
qRT-PCR sequencing Erlotinib
Crizotinib
HER2 Amplification dimerization FISH, IHC, SISH, and Guideline for drug therapy Afatinib
mutation sequencing Trastuzumab
PD-1/ Amplification IHC, ELISA Prognosis evaluation and guideline for Nivolumab
PD-L1 drug therapy Pembrolizumab
Atezolizumab
600 L. Ming et al.
ALK and ROS1 shorter overall survival (OS) and lower response to platinum-
The ALK gene is located on chromosome 2 and encodes a based chemotherapy than the outcome of patients with
transmembrane tyrosine kinase. ROS1, another receptor BRAF wild type. The tumor cells that present BRAF V600E
tyrosine kinase, can be constitutively activated when its tyro- mutation are extremely sensitive to selective BRAF and
sine kinase domain fuses with a partner gene such as CD74 MEK inhibitors. Vemurafenib and dabrafenib are at present
[16]. ALK and ROS1 rearrangements have been identified in the most studied BRAF V600E inhibitors. Then, the evalua-
2–7% and 1–2% of NSCLC patients, respectively [17, 18]; tion of the analysis of V600E mutation and the BRAF inhibi-
these rearrangements result in new fusion genes with trans- tion significantly improved the outcomes in the patients with
forming activity. Crizotinib, a tyrosine kinase inhibitor ini- NSCLC. BRAF mutations can be detected by real-time PCR,
tially used as a c-MET kinase inhibitor, has a significant NGS, and so on.
therapeutic effect in patients with ALK or ROS1 rearrange-
ments. Patients with ALK rearrangements are more suscep- MET
tible to drug resistance after a few months of treatment. MET gene encodes an isodimer of transmembrane receptor
Mutations in the TK domain of ALK, including C1156Y, tyrosine kinase (RTK), which is activated by binding hepato-
G1269A, and L1196M, interfere with Crizotinib binding to cyte growth factor (HGF) to regulate cell activity. MET/HGF
ALK, leading to the acquired resistance. After the failure of signaling pathway can be activated by MET amplification or
Crizotinib treatment, patients with ALK rearrangements exon 14 splicing mutations. MET amplification (defined as
showed increased response to second-generation ALK inhib- ≥6 copies) is present in 2–4% of untreated NSCLC and in up
itors, such as Alitinib or Ceritinib [19]. For patients with to 20% of EGFR/TKI-resistant NSCLC patients, while MET
ROS1 rearrangements, the clinical efficacy of Crizotinib is exon 14 splicing mutations are present in 3–4% of lung ade-
not very significant [20] and new ROS1 inhibitors are being nocarcinoma cases. MET amplification may be responsible
designed and planned for clinical trials. for cancer progression after treatment with EGFR and other
RT-PCR, FISH, IHC, and sequencing are common meth- TKIs. Combination of MET inhibitor (Tivantinib or
ods for detecting ALK and ROS1 rearrangement. Onartuzumab) with EGFR inhibitor (Erlotinib) has been
identified as a promising therapeutic option to overcome
KRAS acquired resistance caused by MET amplification [24, 25].
Oncogene Kras encodes a GTP-binding protein, whose Meaningful clinical response to MET inhibitors, as
mutations activate the oncogenic cellular processes activate Crizotinib, has been reported and longer survival in NSCLC
cancer-causing cellular processes. KRAS mutation is the patients with MET exon 14 skipping mutations is associated
most frequent driving factor for lung cancer, seen in 25% of with MET inhibitors. FISH, silver in situ hybridization
patients with adenocarcinoma. In NSCLC, KRAS and EGFR (SISH), and q-PCR are commonly used to detect MET
mutations are mutually exclusive [21]. KRAS mutations in amplification.
lung cancer usually occur in codon 12, characterized by G–T
conversion frequency, occasionally in codon 13, and rarely HER2
in codon 61. All clinical efforts targeting KRAS were disap- As a member of EGFR family, human epidermal growth fac-
pointing until AMG510 targeting KRAS G12C showed sig- tor receptor 2 (HER2) amplification or dimerization muta-
nificant efficacy in clinical trials. Various approaches to tion results in the same signal transduction effect as EGFR
inhibit KRAS-driven pathways are still under development. mutation. Overexpression of HER2 appears in 35% of lung
Nowadays, KRAS mutations can be detected by pyrose- cancers, and amplification appears in 10% of lung cancers.
quencing, real-time PCR, reverse-hybridization of biotinyl- HER2 mutations are found in about 2% of NSCLC, mainly
ated PCR, and next-generation sequencing (NGS). in adenocarcinoma tissues of women who are never smokers.
Most HER2 mutations are caused by inframe insertions of
BRAF exon 20. HER2 inhibitors (Afatinib and Trastuzumab) are
BRAF, an important member of RAF kinase family, regu- effective in HER2-positive NSCLC patients. FISH, IHC,
lates cell proliferation, apoptosis, and survival by activating SISH, and sequencing can be used to assess HER2 expres-
MAPK [22]. BRAF mutation was found in 2% of NSCLC sion and mutation.
patients, which was mainly composed of V600E mutation
[23]. Non-V600E BRAF mutations were very rare, less than PD-1/PD-L1
1% in a clinicopathological series. In the patients with BRAF The PD-1/PD-L1 signaling pathway is a strategy for cancer
V600E mutation, there is a significant correlation with cells, to escape from immunological surveillance, which
female gender, adenocarcinoma histology and never smok- inhibits immune response by preventing T cell proliferation,
ing while there is no correlation with staging. The outcome chemotaxis, and cytokine release. Currently, three
of patients with BRAF V600E mutations is featured by monoclonal anti-PD-1/PD-L1 antibodies have been approved
38 Lung Disease 601
for use in advanced NSCLC (Nivolumab, Pembrolizumab, Table 38.2 Results for measurement of tumor markers in the diagno-
and Atezolizumab) by FDA. Given the advances gained in sis of early-stage NSCLC and non-NSCLC
NSCLC individuals, the anti-PD-1/PD-L1 antibodies have Sensitivity Specificity
also been tried in SCLC patients and show considerable AUC (95%CI) (%) (%)
potential [26]. PD-L1 is expressed in various cancers, includ- SCLCC 0.808(0.715–0.901) 66.67 89.55
SP70 0.859(0.777–0.942) 69.23 92.54
ing 53–62% of NSCLC. At present, the expression of PD-L1
CEA 0.655(0.545–0.765) 25.64 91.04
is the best biomarker of NSCLC immunosuppressive
CYFRA21-1 0.529(0.413–0.646) 23.08 85.07
response. The benefits of checkpoint inhibitors in PD-L1 SCLCC+SP70 0.889(0.815–0.963) 87.18 82.09
positive patients are significantly better than in PD-L1 nega- SCLCC+CEA 0.819(0.735–0.904) 76.92 82.09
tive patients. Therefore, it is very important to accurately SCLCC+CYFRA21-1 0.811(0.717–0.905) 74.36 76.12
select patients who respond to anti-PD-1/PD-L1 monoclonal
antibodies. At present, IHC is most commonly used for
detecting PD-1/PD-L1. 19 cases of benign lung diseases, and 48 healthy controls
were enrolled. Peripheral blood circulating tumor cells
Others (CTCs, measured by flow cytometry assay), namely SCLCC,
Mutations in MAP 2 K1, RET, FGFRs, NRAS, and so on are serum SP70, CEA, and CYFRA21-1 levels were detected
also implicated in lung cancer progression [12]. before treatment. Analysis of ROC demonstrated that the
combination of SCLCC and SP70 could improve the diag-
38.1.5.3 Other Biomarkers nostic performance of NSCLC (AUC = 0.886), as well as in
early-stage NSCLC (AUC = 0.889). Furthermore, the com-
Tumor Specific Protein 70 bination showed superiority in detecting early-stage NSCLC
Tumor Specific Protein 70 (SP70), discovered by Prof. (Table 38.2). These findings highlight that peripheral blood
Shiyang Pan in 2012 [27], is a novel tumor marker of SCLCC and serum SP70 are sensitive indicators of early
NSCLC. Functional studies showed that SP70 could pro- diagnosis of NSCLC.
mote cell proliferation, metastasis, and resist cell apoptosis,
which is involved in diverse signal pathways. It is increased Therapy Efficacy Monitoring
in tissues, serum, and pleural effusions of patients with A total of 152 patients were enrolled to investigate the pre-
NSCLC. Several assays were developed for SP70 detection, dictive value of serum SP70 in response to chemotherapy in
such as immunohistochemistry, ELISA, flow cytometry patients with advanced NSCLC. After chemotherapy, serum
assay, and immunomagnetic beads capture-based liquid SP70 levels were significantly decreased in the partial remis-
biopsy. Recent researches suggest that SP70 plays an impor- sion (PR) group (P < 0.001) and increased in the progressive
tant role in auxiliary diagnosis, therapy efficacy monitoring, disease (PD) group (P < 0.001), but not significantly changed
and prognosis prediction in patients with NSCLC. in the stable disease (SD) group (P = 0.114). ROC analysis
showed that SP70 is better than CEA, CYFRA21-1, and
Auxiliary Diagnosis NSE. The median PFS of patients with decreased SP70 lev-
Serum SP70, CEA, NSE, and CYFRA21-1 from 150 NSCLC els after chemotherapy was longer than that of patients with
patients including 80 lung adenocarcinoma, 70 squamous stable or increased serum SP70 level (24 months vs
cell lung cancer, 25 SCLC, 25 benign lung disease (BLD) 12 months vs 2 months, P < 0.001), and the differences of all
patients, and 300 healthy controls (HC) were measured. other 3 tumor markers were not obvious (Fig. 38.6). Serum
Positive rates of SP70 in lung adenocarcinoma, squamous SP70 is a sensitive and real-time indicator of chemothera-
cell lung cancer, SCLC, and BLD were 68.8%, 51.4%, peutic efficacy in advanced NSCLC [28].
16.0%, and 12.0%, respectively, obviously higher than that
of HC (7.3%). NSCLC had a significantly higher positive Predict Prognosis
rate of SP70 than SCLC (60.7% vs 16.0%, P < 0.05) and SP70 is expressed in over 80% of NSCLC cancer tissues
BLD (60.7% vs 12.0%, P < 0.05). Meanwhile, positive rates (Fig. 38.7, Table 38.3). Immunohistochemical detection of
of CEA, NSE, and CYFRA21-1 (32.7%, 18.0%, and 37.3%) SP70 has high sensitivity and specificity for pathological
were significantly lower than SP70 in NSCLC (60.7% vs diagnosis of non-small cell lung cancer. The overall survival
32.7%, P < 0.05; 60.7% vs 18.0%, P < 0.05; 60.7% vs 37.3%, rate of patients with high (3+, n = 38) expression of the anti-
P < 0.05). SP70 is a valuable biomarker for the diagnosis of gen was significantly lower than that with low (2+, n = 47)
NSCLC. and negative (1+, n = 17) expression in 102 lung adenocarci-
In a prospective study by Pan et.al which was conducted noma. In addition, Kaplan-Meier survival curve analysis
at First Affiliated Hospital of Nanjing Medical University showed that the higher the expression of SP70, the shorter
from October 2018 to March 2019, 72 patients with NSCLC, the median survival time of patients (Fig. 38.8). Cox propor-
602 L. Ming et al.
a b
c d
Fig. 38.6 Kaplan-Meier curves for PFS in patients with different serum tumor markers. A, SP70. B, CEA. C, CYFRA21-1. D, NSE
Fig. 38.7 SP70 expression in a b

lung tissues (200×). A
staining score of “-“and “+”
are considered as negative
while a staining score of “++”
and “+++” are considered as
positive. (a) Benign lung
tumor, negative SP70
expression (−); (b) lung
adenocarcinoma, negative
SP70 expression (+); (c) lung
adenocarcinoma, low SP70 c d
expression (++); (d) lung
adenocarcinoma, high SP70
expression (+++)
38 Lung Disease 603
Table 38.3 SP70 expression in tissues (MS-PCR), bisulfite-sequence PCR, pyrosequencing, and
Number NJ001specific Positive NGS can be used to assess DNA methylation.
antigen rates (%)
Groups − + ++ +++ P value Potential Biomarkers
NSCLC ADC 183 0 28 81 74 84.1 0.288a Non-coding RNAs including microRNAs (miRNAs) and
SCC 72 0 15 37 20 79.2 long non-coding RNAs (lncRNAs), as well as exosomes are
Benign Lung 46 8 34 4 0 8.7 < 0.001b potential biomarkers for lung cancer diagnosis.
Disease
In lung cancer, miRNAs can be carcinogenic, cancer sup-
Adjacent 73 36 31 5 1 8.2 < 0.001c
normal tissue pressive, or double edged. So far, more than 2000 mature
NSCLC non-small cell lung cancer, ADC lung adenocarcinoma, SCC
small RNAs have been identified in humans, of which about
squamous cell lung cancer. −, +: negative, ++, +++: positive. 50 are related to the occurrence of lung cancer. For example,
a
ADC vs SCC; bNSCLC vs Benign Lung Disease; cADC vs Adjacent the sensitivity and specificity of microRNA-15b/microRNA-
normal tissue 27b to NSCLC were 100% and 84%, respectively [30], and
high levels of serum miR-21 were associated with poor prog-
100 nosis of lung cancer patients [31]. Real-time PCR, FISH,
+
80 ++ Northern blot, and miRNA microarray can be used to assess
Percent survival
++ + miRNAs.
60 Studies have shown that abnormal expression of lncRNAs
P=0.0007
40
may lead to the development of cancers, including lung can-
cer. Studies have confirmed that lncRNAs MALAT1,
20 HOTAIR, CDKN2B-AS1, CCAT2, PVT1, LUACT1,
0
SOX2-OT, and AFAP1-AS1 may be useful as new prognos-
0 20 40 60 80 100 tic biomarkers for lung cancer progression [32]. Real-time
Months after surgery PCR, lncRNA microarray, Northern blot, and sequencing are
common molecular methods for detecting of lncRNAs.
Fig. 38.8 Kaplan-Meier survival curve of lung adenocarcinoma
patients according to the SP70 expression The exosomes secreted by lung cancer cells could enrich
oncoproteins like EGFR and KRAS; promote the initiation
tional hazard model analysis suggested that the high expres- and progression of lung cancer, providing certain guidance
sion of SP70 was an independent risk factor affecting the for the early diagnosis and targeted therapy of lung cancer. In
overall survival time of NSCLC patients. Therefore, SP70 is a study of Huang and colleagues, through analysis of exo-
not only a molecular marker for assistant diagnosis, but also somes derived from NSCLC tissues and chronic pneumonia
a molecular marker for prognosis. tissues, they found that on the surface of the exosomes,
EGFR positive rate came up to 80% in the NSCLC speci-
DNA Methylation mens, while that in the chronic pneumonic tissues were only
DNA methylation refers to DNA methyltransferase-mediated 2%, suggesting that exosome-derived EGFR can serve as a
methylation of cytosine residues in CpG sequences. DNA biomarker for diagnosis and differential diagnosis of lung
methylation plays different roles in cell function according cancer [33]. Furthermore, exosome-derived RNA has been
to the gene region where methylation occurs. Global genomic reported to detect EGFR T790M with the sensitivity of 90%
instability induced by hypomethylation is considered to be and activating EGFR mutations with the sensitivity of 98%,
responsible for oncogene activation. Hypermethylation of making it possible to function as an indication of drug resis-
the promoter region of tumor suppressor genes leads to tance to targeted therapy [34]. Moreover, Zhao and col-
abnormal gene expression, which is common in cancer cells. leagues showed that the levels of plasma exosomal
DNA methylation has shown great potential in the screening hsa-miRNAs were related to EGFR mutation. These findings
and prognosis of lung cancer. Furthermore, DNA methyla- could guide the use of targeted therapy drugs and provide a
tion detection platform has been launched in recent years. new way for detecting gene mutations of NSCLC [35].
APC, RASSF1A and SHOX2 are common DNA methyla- Another two studies confirmed that the upregulation of exo-
tion markers in lung cancer. Professor Pan’s research showed somal miR-1246 and miR-208a was associated with radio-
that the methylation rates of APC and RASSF1A in NSCLC therapy resistance and overproliferation by targeting p21 and
patients were 59.0% and 66.7% in biopsy tissues, 42.5% and DR5, respectively, implying both exosomal miR-1246 and
52.5% in serum, and 24.1% and 43.1% in plasma, respec- miR-208a as prognostic biomarkers and new targets against
tively (Table 38.4) [29]. Methylation-specific PCR NSCLC [36].
604 L. Ming et al.
Table 38.4 Methylation frequency of RASSF1A and APC in different samples

Item Sample type Cases RASSF1A P APC P
Plasma 112 0.000 0.000
Lung cancer 58 43.1% (25/58) 24.1% (14/58)
Benign disease 31 6.5% (2/31) 3.2% (1/31)
Healthy 23 0% (0/23) 0% (0/23)
Serum 76 0.008 0.102
Lung cancer 40 52.5% (21/40) 42.5% (17/40)
Benign disease 13 7.7% (1/13) 15.4% (2/13)
Healthy 23 0% (0/23) 0% (0/23)
Tissue 54 0.009 0.052
Lung cancer 39 66.7% (26/39) 59.0% (23/39)
Benign disease 13 23.1% (3/13) 23.1% (3/13)
Normal tissue 2 0% (0/2) 0% (0/2)
38.1.5.4 Liquid Biopsy Table 38.5 Positive rates of peripheral blood SCLCC and serum
Liquid biopsy is noninvasive way alternative to surgical SP70, CEA, or CYFRA21-1, which were detected separately or jointly
in NSCLC patients without treatment
biopsies to discover cancer-related information through
blood or other body fluids. Assays of this nature using blood Items Positive/total (%)
SCLCC 45/72(62.50%)#*△
or other body fluids, but not tumor samples, are frequently
SP70 46/72(63.89%)#*△
referred to as liquid biopsies. Detection of exosomes, pro-
CEA 30/72(41.67%)○◇#*△
teins, platelet RNA, circulating tumor RNA (ctRNA), and
CYFRA21-1 31/72(43.06%)○◇#*△
circulating tumor cells (CTCs) and circulating DNA (ctDNA)
SCLCC + SP70 57/72(79.17%)
is involved in liquid biopsy. SCLCC + CEA 59/72(81.94%)
As mentioned above, lung cancer cells, especially the SCLCC + CYFRA21-1 58/72(80.56%)
NSCLC cells, express high levels of SP70 antigen. Thus, FCM Note: ○: compared to SP70, P < 0.05; ◇: compared to SCLCC in the
based SP70 detection in liquid biopsy can be achieved by uti- peripheral blood, P < 0.05; #: compared to SCLCC (peripheral
lizing fluorescence conjugated NJ001 antibody that targets blood) + SP70 (serum), P < 0.05; *: compared to SCLCC (periph-
SP70 antigen. The FCM data showed that the positive rate of eral) + CEA (serum), P < 0.05; △: compared to SCLCC (periph-
eral) + CYFRA21-1 (serum), P < 0.05
peripheral blood-specific circulating lung cancer cells
(SCLCC) of patients with NSCLC was 62.50%, which was
evidently higher than that of serum CEA or CYFRA21-1 Table 38.6 Diagnostic efficacy of tumor indicators in early-stage and
(Table 38.5). Besides, peripheral blood SCLCC level was CEA/CYFRA21-1-negative NSCLC
clearly higher in the early stage of NSCLC than that in patients Sensitivity Specificity
AUC (95%CI) (%) (%)
with benign lung diseases and healthy controls, with a positive
Early-NSCLC vs Non-NSCLC
rate of 66.67%. The sensitivity of combined detection of
SCLCC 0.808 66.67 89.55
peripheral blood SCLCC with serum SP70 in early NSCLC, (0.715–0.901)
as well as CEA and CYFRA21-1 double negative NSCLC, SP70 0.859 69.23 92.54
were 87.18% and 87.10%, respectively. Moreover, peripheral (0.777–0.942)
blood SCLCC was notably higher in the early stage of NSCLC CEA 0.655 25.64 91.04
than those in benign lung diseases and healthy controls (0.545–0.765)
CYFRA21-1 0.529 23.08 85.07
(Table 38.6), indicating a significant role of SCLCC detection (0.413–0.646)
by FCM in the early diagnosis of lung cancer. SCLCC+SP70 0.889 87.18 82.09
Recently, immuno-magnetic bead-based cell sorting is (0.815–0.963)
routinely used for the enrichment of cancer cells and can SCLCC+CEA 0.819 76.92 82.09
improve the sensitivity in liquid biopsy because of their sta- (0.735–0.904)
bility and large surface-to-volume ratios [37, 38]. For SCLCC+CYFRA21-1 0.811 74.36 76.12
(0.717–0.905)
instance, biochemically functionalized paramagnetic beads
CEA & CYFRA21-1-negative NSCLC vs Non-NSCLC
which are coated with NJ001 can recognize and capture SCLCC 0.845 70.97 89.55
SP70 positive malignant cells in a magnetic field. The cap- (0.755–0.936)
tured cells were utilized to perform NGS-based molecular SP70 0.867 74.19 92.54
analysis. The NGS analysis of SP70 positive cells in NSCLC (0.775–0.958)
patients upon immune-magnetic separation demonstrated SCLCC+ SP70 0.895 87.10 82.09
(0.814–0.976)
that 60% of the altered genes were found, together with 65
38 Lung Disease 605
Next Generation Sequencing Analysis of Altered Signaling Pathways

in SP70 Positive NSCLC Cells
APC PIK3CA
6.98% 2.33%
KRAS HRAS CTNNB1 PTEN TP53 RB

9.30% 2.33% 2.33% 9.30% 81.40% 9.30%
Cell proliferation, survival and cell cycle progression
Fig. 38.9 Next generation sequencing analysis of altered signaling pathways in SP70 positive NSCLC cells
loci. Among them, TP53 was the most common aberrations, ogy, results with better sensitivity and specificity could most
followed by KDR and EGFR. As shown in Fig. 38.9, genetic likely be obtained.
alterations affected signaling pathways in NSCLC. The most Currently, the technology of detecting EGFR alterations
common genetic alterations with high frequency in NSCLC in peripheral blood of treatment-naive NSCLC patients is
were involved in p53 signaling pathway, genetic alterations highly reliable and recommended in clinic. It is worth noting
involved in PI3K signaling pathway were identified in that a negative ctDNA result should be considered inconclu-
9.30%, which included PTEN mutation [39] and both PTEN sive, and other routine testing should be performed.
and PIK3CA mutations. The frequency of alterations in Wnt
pathway was also 9.30%, APC mutation was found in 6.98%,
while mutation of CTNNB1 was seen in 2.33% of NSCLC 38.1.6 Treatments and Therapies
patients. For the Ras signaling pathway, KRAS was the most
commonly altered gene, followed by HRAS. RB1 mutation Lung cancer therapy depends on the type of cancer, neo-
was found in 9.30% of all patients, which implied the impor- plasm staging, and the patient’s clinical presentation.
tant involvement of cell cycle regulators in NSCLC. Conventional treatments include surgery, chemotherapy,
Nowadays, CTCs analysis opens new possibilities for radiotherapy, targeted therapy, and palliative care. Before the
management of cancer patients. With ctDNA occurrence- targeted therapy was well developed, chemotherapy is the
based methods, tracking tumor evolution non-invasively now most commonly used treatment for NSCLC patients at stages
has the possibility. ctDNA could be detected in early stage of II and III.
cancers. However, in early stage of tumor, the amount of The combination of third-generation chemotherapy drugs
ctDNA may be under the detection limit for the tumor size. with platinum-based regimens showed a significant improve-
The histological subtype is also an important factor for the ment in lung cancer patients’ survival, with a 5 years survival
accuracy of liquid biopsy. Before surgery, ctDNA was rate improved by 11% [43]. Even so, the median survival is
detectable in 97% of lung squamous cell carcinoma cases, only 8–10 months, and the treatment-related adverse reac-
but only in 19% of adenocarcinoma. The relapses of 93% of tions are dramatically strong. In 2015, the FDA approved
NSCLC patients could be predicted by the consistent detec- nivolumab, an immunoassay checkpoint blocker (ICB), to
tion of postoperative ctDNA [40]. In comparison with rou- treat patients with disease progression during or after
tine tumor biomarkers, ctDNA showed up a greater positive platinum-based therapy, marking a new field of lung cancer
predictive value [41]. therapy, which is a major advance in the treatment of
Based on high throughput and deep sequencing, NGS advanced NSCLC [44]. The target therapy based on molecu-
could simultaneously analyze a large number of gene targets lar diagnosis extends the survival of advanced NSCLC
for different patients. The application of NGS to detect patients to several years [45]. Moreover, immunotherapy
ctDNA in lung cancer is feasible and valuable. The sequenc- often produces inspiring results and has received widespread
ing of ctDNA extracted from plasma specimens has a sensi- attention worldwide. The ICBs have been approved as the
tivity of 58% and a specificity of 87% by the gene panel standard treatment for advanced NSCLC patients with dis-
including hotspot regions of EGFR, ERBB2 (HER2), BRAF, ease progression after first-line chemotherapy [46–48].
PIK3CA, and KRAS [42]. With the development of technol- Although great advance has been made in target therapy and
606 L. Ming et al.
immunotherapy, not all patients can benefit from these thera- Results with interpretation guideline The pathological
pies, selecting the suitable patients through molecular diag- diagnosis was performed by CT-guided puncture of primary
nosis is critical. tumor and revealed lung adenocarcinoma (TTF1+, CK7+)
with the EGFR ex19del mutation. Several tumor markers
including SP70, CEA, and CA199, were increased.
38.1.7 Conclusion According to NCCN guidelines for non-small cell lung can-
cer, the patient was given zoledronic acid 4 mg every 28 days
Molecular diagnosis opens up a new field for the diagnosis and gefitinib 250 mg daily, obtaining a partial response.
and therapy of lung cancer patients. The development of After 9 months, the patient’s condition deteriorated, with an
novel molecular markers, especially those indicating a increased size of the primary tumor and liver metastases.
response to immunotherapy, will be the key to precision EGFR ex19del and T790M mutations were detected in
medicine for lung cancer. another biopsy of the primary tumor and confirmed in ctDNA
by digital PCR. The patient was treated with atezolizumab
and received stereotactic radiotherapy on the primary lung
38.1.8 Typical Medical Case tumor and liver metastases. Therefore, molecular diagnosis
plays an important role in the diagnosis and monitoring of
Clinical Background A 69-year-old female patient came to NSCLC.
the hospital for a cough, backache, and chest pain that per-
sisted for 4 months. The patient was non-smoking, without The case was from the First Affiliated Hospital of Nanjing
family history of cancer and other complications. Medical University (also named Jiangsu Province Hospital.
Chest CT Lung lesion (approximately 3.8 × 3.2 cm) of left

upper lobe. 38.2 Cystic Fibrosis
Laboratory Data SP70:7.53 ng/mL (↑), CEA: 76.79 ng/ Haitao Ding and Juan He
mL (↑), AFP: 3.05 ng/mL; CA199: 49.77 U/mL (↑); CA724:
1.56 U/mL; CYFRA21-1: 6.01 ng/mL; NSE: 13.09 ng/mL.
38.2.1 Overview
Histopathological Examination Left lung adenocarci-
noma, see Fig. 38.10. Cystic fibrosis (CF) is a manifestation of systemic diseases
specifically in the lungs, also known as cystic fibrosis of the
Treatment Chemotherapy. pancreas. This is a family congenital disease, an autosomal
recessive genetic disease, mainly caused by mutations in the
CFTR gene in the body. The main feature is the secretion of
systemic exocrine glands, mucus secretion, and viscous,
blocking the official cavity, causing the expansion of the offi-
cial cavity and fibrosis, resulting in the corresponding organ
dysfunction, rather than the secretion of sweat glands and
salivary glands in the mucous glands, the content of sodium
chloride is increased, so it is called systemic adenosis.
CF often involves multiple organs, including lung, pancreas,

gastrointestinal tract, liver, gallbladder, which is typically
characterized by chronic suppurative lung disease with bron-
chiectasis with bacterial infection, pancreatic insufficiency,
sweat high chloride and sodium. However, CF patients with
Fig. 38.10 Histopathological examination of left lung
adenocarcinoma
normal pancreatic function generally have only mild respira-
38 Lung Disease 607
tory symptoms, and the normal concentration of chlorine in creatic insufficiency, the correlation between the two is the
sweat does not exclude CF. highest, and for lung diseases, the correlation between the
two is lowest.
38.2.2.1 Respiratory System At present, most of the methods for detecting CFTR
The main clinical manifestations are pulmonary cystic fibro- mutant gene carriers are PCR-SSCP and denaturing gel gra-
sis caused by repeated airway obstruction and bronchial dient electrophoresis [51]. Short fluorescent fragment multi-
infection. The patient presented with cough, sputum viscous, ple PCR method is a semiquantitative method. Direct DNA
and difficulty to cough up. When combined with pulmonary sequencing results are reliable but expensive. Each of the
infection, the sputum was purulent. Repeated pulmonary above methods has advantages and disadvantages, and a
infection can cause bronchiectasis, lung abscess, and so on. combination of various methods can increase the detection
As the disease progresses, patients need long-term oxygen rate.
inhalation, eventually developing chronic pulmonary heart
disease, and 95% of patients are related to respiratory
failure. 38.2.4 Conclusion
38.2.2.2 Digestive System The current diagnostic criteria for CF are clinical manifesta-
Approximately 85% of CF patients have pancreatic insuffi- tions of one or more CFs, laboratory findings with abnormal
ciency and are predominantly children. Most patients CFTR (high concentrations of sweat chloride or abnormal
develop fat and protein malabsorption due to progressive nasal potential) or two pathogenic CFTR mutations point.
decline in pancreatic function, resulting in nutritional defi- Since the discovery of the CFTR gene in 1989, Western
ciencies. Patients with CF may have hepatomegaly and fatty scholars have done extensive screening and research on the
liver in the early stage, and advanced CF may have liver fail- CFTR gene in the Caucasian population. Although CF has a
ure. The most serious complications are portal hypertension very low incidence among Asians, Chinese may have a large
and esophageal varices bleeding. number of potential CF patients and carriers of CFTR muta-
tions. These mutations may differ from Caucasians in many
38.2.2.3 Other ways [52]. Therefore, the research of CFTR gene can help
Female women with CF often have low reproductive capac- improve medical level and promote human health.
ity, and male patients often have azoospermia. Some patients
may have arthritis and vasculitis.
38.3 Pneumonia
38.2.3 Laboratory Diagnosis Wenjuan Wu and Min Zhang
The diagnosis of CF is diagnosed by clinical symptoms, lab-

oratory results, and sweat tests [49]. Patients with one or 38.3.1 Overview
more CF manifestations or a family history of CF can be
diagnosed by identifying known CF mutant genes. Most CFs Common symptoms of pneumonia include productive cough,
are autosomal recessive diseases, mainly caused by muta- dry cough, chest pain, fever, and dyspnea. The site of the
tions in the CFTR gene in the body, and the genes are located primary inflammation is mainly located in the alveoli [53].
on the long arm of chromosome 7(7q.31.2). The most com- Severity is variable. About 450 million people (7% of the
mon CFTR lesion is a genetic mutation in ΔF508, which population) worldwide suffer from pneumonia, and about 4
results in the loss of phenylalanine at position 508 due to the million people die each year from pneumonia. Therefore, in
deletion of three nucleic acids [50]. Codons are deleted in the nineteenth century, pneumonia was called the “captain of
exons 9, 10, 11, 12, 19, 20, 21 of the NBFs of the coding the dead.” In the twentieth century, antibiotics and vaccines
region of CFTR and in exons 3, 4, 7, and 17b encoding the were developed, thus survival rates increased. However, for
transmembrane region, especially in the coding region NBFs. young people, the elderly, and people with chronic diseases
The 11th exon is surrounded by mutation hotspots, and only in developing countries, pneumonia remains the leading
11 of them are found in 5 adjacent codons. The mutation cause of death. In particular, pneumonia often reduces pain
form detected in patients with CBAVD in mainland China is for people who are dying, which is more common in older
△F508 mutation, which is related to pancreatic insuffi- people [54].
ciency and early manifestations. The CFTR genotype is According to location where it was acquired, people often
closely related to the CF phenotype. However, different CF divide infectious pneumonia into the following three catego-
phenotypes are not related to the CFTR genotype. For pan- ries—hospital-acquired pneumonia (HAP), community-
608 L. Ming et al.
acquired pneumonia (CAP), and healthcare-related may be accompanied by cervical lymphadenopathy, middle
pneumonia. The cause of infectious pneumonia is mainly ear infections, or joint pain. Compared with bacterial pneu-
bacterial or viral infection, and secondly, it also includes monia, viral pneumonia is more prone to wheezing. People
some other pathogenic bacteria, the use of drugs, and basic have classified pneumonia as “typical pneumonia” and
diseases. Impaired immune system, some basic diseases “atypical pneumonia” based on typical symptoms to predict
(such as diabetes, heart failure, asthma, COPD, and pulmo- the underlying cause. However, it is no longer emphasized
nary cystic fibrosis) and even smoking history are all risk due to the lack of evidence supporting this distinction.
factors for infectious pneumonia. Clinical symptoms, physi-
cal examination, chest X-ray, sputum culture, and blood tests
can be used as clues in the diagnosis of infectious 38.3.3 Laboratory Diagnosis
pneumonia.
In addition to infectious pneumonia, other clinically com- Pneumonia is commonly diagnosed based on a combination
mon pneumonias include organizing pneumonia, diffuse of physical signs and a chest X-ray [57]. However, it is dif-
alveolar injury, nonspecific interstitial pneumonia, lympho- ficult to distinguish bacterial origin from nonbacterial origin
cytic interstitial pneumonia, respiratory bronchiolitis, inter- by the common technologies, and them to confirm the under-
stitial lung disease, desquamation interstitial pneumonia, lying cause.
etc., which are also known as noninfectious pneumonia or Children’s pneumonia has been defined clinically by the
idiopathic interstitial pneumonia, belonging to a class of dif- World Health Organization (WHO) based on the physical
fuse lung disease [55]. Lipid pneumonia is another rare con- signs like cough or difficulty breathing, associated with chest
dition in which inhaled, or body fat enters the lungs. indrawing, a rapid breathing rate, or a decreased level of con-
In this chapter, we will focus on the value of clinical sciousness. The definition of the rapid breathing rate is as
molecular diagnostic techniques in infectious pneumonia. follows: >60 breaths/min in children under 2 months old,
>50 breaths/min in children 2 months to 1-year old, or >40
breaths/min in children 1 to 5 years old. The chest crackles
38.3.2 Clinical Appearance or increased breathing rate are less sensitive than lower chest
indrawing and low oxygen levels in children. Children under
Common symptoms after pneumonia infection include 5 years old may have other symptoms like grunting and nasal
cough, fatigue, fever, chills, shortness of breath, and sputum flaring. Lack of wheezing is an indicator of pneumonia
(approximately 60–90%). Other sub-common symptoms caused by Mycoplasma pneumoniae in children, but it is not
include severe or stinging chest pain during deep breathing accurate enough to decide whether or not macrolide treat-
(39–49%), and increased breathing frequency. Confusion is ment should be used based on this indicator. Chest pain is
found to be the most potentially prominent sign in elderly another indicator of pneumonia caused by Mycoplasma
patients. pneumoniae in children [58].
Children under five with infectious pneumonia usually Typically, vital signs and auscultation results are often
have fever, cough, and fast or difficult breath [56]. Fever also normal in patients with a lower probability of diagnosis of
occurs in many other diseases, but not occur in people with pneumonia. There seldom requires investigating the adults in
malnutrition, severe illness, or in the elderly, so that it is not mild cases. Recommended tests for inpatients include chest
a very specific symptom of pneumonia. Besides, children X-rays, pulse oximetry, and blood tests. In the whole blood
under 2 months old seldom cough. More severe symptoms, test, the whole blood cell count, serum electrolytes,
like unwillingness to drink, extremes of temperature, blue- C-reactive protein levels, and liver function tests have a cer-
tinged skin, ongoing vomiting, convulsions as well as a tain guiding significance for clinicians to use drugs [59].
decreased level of consciousness, may be found in children. Procalcitonin levels can help clinicians determine the cause
Bacterial pneumonia and viral pneumonia exhibit similar of pneumonia and whether to use antibiotics or not. Patients
clinical symptoms, including some classic clinical features. probably receive antibiotics when procalcitonin level
Pneumonia caused by Streptococcus pneumoniae may be ≥0.25 μg/L, more probably when it ≥0.5 μg/L, and scarcely
accompanied by rusty sputum. Klebsiella-induced pneumo- possible when it <0.10 μg/L. Merely CRP <20 mg/L is not a
nia may be accompanied by blood sputum (known as “cur- sufficient indication for antibiotic treatment [60].
rant jelly”). Abdominal pain, diarrhea, or insanity may be The above symptoms and signs can help clinicians make
seen in pneumonia caused by Legionella. Acute bronchitis, judgments about the disease, but some tests are still needed
tuberculosis, as well as pneumonia caused by Gram-negative to confirm the influenza infection. Whether or not the disease
bacteria are often accompanied by hemoptysis and lung is diagnosed as influenza has a major impact on treatment
abscesses. Pneumonia caused by Mycoplasma pneumoniae and management.
38 Lung Disease 609
38.3.3.1 Pathogenic Diagnosis detection methods, molecular diagnostic testing has

There are many advantages to identify pneumonia patho- developed into an important technology for the diagnosis of
gens. For example, clinicians can optimize the treatment of respiratory pathogens [61] (Tables 38.7 and 38.8).
patients with pneumonia based on the identification results.
However, clinical laboratories may have difficulty meeting 38.3.3.2 K ey Points for the Laboratory
these requirements by performing conventional methods. Diagnosis of Respiratory Infections
Determining the causative agent is cost-ineffective and sel- The first morning expectorated sputum is recommended for
dom changes management in community-managed patients. bacterial culture, and sputum culture combined with blood
Sputum culture is supposed to be carried out for patients culture is recommended for high-risk patients (such as CAP
with no responses to treatment. patients). When the pathogens are suspected to be fastidious
However, positive sputum culture results do not necessar- ones, for example, Bordetella pertussis, clinicians need to
ily indicate pathogenic bacteria; it may also due to coloniz- communicate with the laboratories to follow the correct and
ing bacteria. For patients with severe pneumonia, standardized procedures for sample collection and transpor-
immunosuppression, and HIV infection, it is often necessary tation. For pediatric patients, bronchoscopy with washings
to pay attention to their microbiological test results [60]. For culture is better than sputum culture [62]. In addition to cul-
patients with chronic progressive cough, additional culture ture and rapid antigen detection methods, nucleic acid ampli-
of Mycobacterium tuberculosis is required. For critically ill fication tests have also received increasing attention [63].
patients, sputum culture, blood culture, and urine antigen Polymerase Chain Reaction (PCR) is a rapid molecular
testing of Legionella and Streptococcus are recommended. diagnostic method by amplifying the DNA of pathogenic
Positive blood culture and pleural fluid culture results can bacteria remaining in a sample [62]. PCR tests can detect
decisively diagnose the type of microorganisms involved. It blood, nasopharyngeal swabs, and respiratory tract samples
is also recommended to test other specific organisms during to help diagnose pneumococcus, and are hardly affected by
the outbreak for public health. In addition to traditional antibacterial treatments because it does not require viable
Table 38.7 Laboratory diagnosis of common pathogens in community-acquired pneumonia

Transport issues and optimal
Etiologic agents Diagnostic procedures Optimum specimens Transport time
Bacteria
Streptococcus pneumoniae Gram stain Sputum, bronchoscopic Sterile container; RT, 2 h; 4 °C, >2–24 h
Culture specimens
Urine antigen Urine Sterile container, RT, 24 h; 2–8 °C,
>24 h-14 d
Haemophilus influenzae Gram stain Sputum, bronchoscopic Sterile container; RT, 2 h; 4 °C, >2–24 h
Culture specimens
Legionella spp. Urine antigen Urine Sterile container, RT, 24 h; 2–8°C,
L. pneumophila serogroup 1 >24 h-14 d
Selective culture on BCYE Induced sputum, bronchoscopic Sterile container; RT, 2 h; 4 °C, >2–24 h
NAATa specimens
Mycoplasma pneumoniae NAATa Throat swab, NP swab, sputum, Transport in M4 media or other
BAL fluid Mycoplasma-specific medium at RT or
4 °C up to 48 h; ≥48 h, −70 °C
Serology IgM, IgG antibody Serum Clot tube, RT, 24 h; 4 °C, >24 h
detection
Chlamydia pneumoniae NAATa NP swab, throat washings, Transport in M4 or other specialized
sputum, bronchial specimens transport medium at RT or 4 °C up to
48 h; ≥48 h, −70 °C
Serology (MIF) IgM antibody Serum Clot tube, RT, 24 h; >24 h, 4 °C
titer; IgG on paired serum 2–3
wk. apart
Mixed anaerobic bacteria Gram stain Bronchoscopy with protected Sterile tube with 1 mL of saline or
(aspiration pneumonia) Aerobic and anaerobic culture specimen brush thioglycolate; RT, 2 h;
>2–24 h
Pleural fluid (if available) Sterile container, RT, without transport
media ≤60 min;
Anaerobic transport vial, RT, 72 h
(continued)
610 L. Ming et al.
Viruses
Influenza viruses A, B Rapid antigen detection Nasal aspirates, nasal washes, NP swabs, throat washes, throat swabs,
DFA bronchoscopically obtained samples
Viral culture methods Transport in viral transport media, RT < 2 h; 5 d, 4 °C; >5 d, −70 °C
NAATb
Adenovirus DFA
Viral culture methods
NAATb
Parainfluenza viruses 1–4 DFA
NAATb
Respiratory syncytial virus Rapid antigen detection
DFA
NAATb
Human metapneumovirus DFA
NAATb
Coronaviruses NAATb
Rhinovirus Viral culture methods NAATb
Enteroviruses Viral culture methods NAAT
Abbreviations: BAL bronchoalveolar lavage, BCYE buffered charcoal yeast extract, DFA direct fluorescent antibody test, NAAT nucleic acid ampli-
fication test, NP nasopharyngeal, RT room temperature
a
In general, avoid calcium alginate swabs and mini-tipped swabs for NAATs
b
Several FDA-cleared NAAT platforms are currently available and vary in their approved specimen requirements and range of analytes detected.
Readers should check with their laboratory regarding availability and performance characteristics, including certain limitations
Table 38.8 Laboratory diagnosis of common pathogens in hospital-acquired pneumonia

Bacteria
Pseudomonas Blood culture Gram stain Blood Routine blood culture bottles, RT <24 h
aeruginosa aerobic and anaerobic culture Sputum Sterile cup or tube RT, 2 h; 4 °C, >2–24 h
Escherichia coli NAAT Endotracheal aspirates
Klebsiella pneumoniae BAL
Enterobacter spp. Protected specimen brush
Acinetobacter spp. samples
Staphylococcus aureus Lung tissue
Mixed anaerobes Gram stain Protected specimen brush Sterile tube with 1 mL of thioglycollate (for brush
(aspiration) Culture samples a lung tissue samples); Sterile container for tissue; RT, 2 h;
NAAT 4 °C, >2–24 h
Fungi
Aspergillus spp. Stain Endotracheal aspirates Sterile cup of tube RT, 2 h; 4 °C, >2–24 h
KOH with calcofluor; other BAL Clot tube 4 °C, ≤5 d; >5 d, −70 °C
fungal stains Protected specimen brush
Fungal culture samples
Histology Lung tissue Sterile cup; RT, 2 h; or formalin container, RT
2–14 d
Galactomann and (1–3) Serum, Sterile cup or tube RT, 2 h; 4 °C, >2–24 h
β-d-glucan BAL
Abbreviations: BAL bronchoalveolar lavage, NAAT nucleic acid amplification test, RT room temperature
bacteria. Studies have confirmed that PCR is more sensitive opment. First, in order to save amplification time and improve
than conventional methods such as culture when antibiotics efficiency, multiplex PCR technology has been developed.
have been received [61]. This technology is capable of qualitatively and quantitatively
The technology of rapid diagnosis of respiratory tract detecting multiple respiratory pathogens in a single proce-
infections is facing challenges and is under constant devel- dure, and is suitable for multiple types of samples. These
38 Lung Disease 611
Table 38.9 Rapid molecular microbiological tests for pneumonia diagnostics

Platform Pathogens Technology Clinical evidence
Gene Xpert MRSA/SA Multiplex-PC Rapid, accurate tool for blood and
Influenza viruses A. B respiratory samples
RSV
MALDI-TOF MS Microorganisms MS Identification directly Rapid identification of microorganisms;
from bacterial/fungal detects certain types of antibiotic
colonies resistance mechanisms
eSensor Respiratory Influenza A/B Multiplex-PCR Rapid identification of respiratory
Viral Panel Human metapneumovirus viruses
Rhinovirus Adenovirus B/E/C Co-infection detection
FilmArray Adenovirus Nucleic acid purification, Detection of several respiratory
Respiratory Panel Coronavirus 229E, OC43, NL63, HKU1 reverse transcription, pathogens in one test
Metapneumovirus Influenza A, H3, H1, high-order nested multiplex-
2009 H1 Parainfluenza virus 1, 2, 3,4, RSV PCR, and DNA melting curve
Rhinovirus/enterovirus analysis
Bordetella pertussis
M. pneumoniae
C. pneumoniae
pathogens include the most common multidrug-resistant Table 38.10 The antibiotic-resistant markers included in the multi-
bacteria of pneumonia, such as MRSA, Pseudomonas aeru- plex PCR technology
ginosa, Acinetobacter baumannii, and various Function Gene
Enterobacteriaceae (Table 38.9) [61, 63]. However, Gram β-lactam mecA, blaTEM, blaSHV, blaCTX-M,
blaDHA, blaEBC, blaOXA-51, blaKPC
staining and semiquantitative methods are still important as
Macrolides/ ermA, ermB, ermC, msrA, mefA
reference methods for identifying pathogens of respiratory lincosamides
infections. Fluoroquinolones GyrA, ParC
Second, detecting the antibiotic resistance of different bac-
teria is in need. The inconsistency between genotype and phe-
notype is challenging for detecting resistance of the bacteria the treatment of hospital-acquired pneumonia, there are a
by PCR, under the situation that there are more new resis- limited number of antibiotics available, including β-lactams,
tance mechanisms being discovered, so that PCR may cause aminoglycosides, uriquinolones, glycopeptides, and
false-negative results due to the potentially unknown mecha- oxazolidinones.
nisms. Also, some samples with positive genotype markers
results showed no phenotype resistance. Despite these draw-
backs, molecular techniques can hopefully improve the 38.3.4 Management
empirical treatment, by providing some early information on
the resistance profile. Nowadays, multiplex PCR being devel- There are many advantages of rapid molecular diagnostic
oped for the purpose of detecting some bacterial resistance tests, such as helping to distinguish between infections
markers in one process (Table 38.10) [61]. Recently, the next- caused by bacteria and viruses or identifying a specific
generation sequencing (NGS) has become a new molecular pathogen; providing information for disease surveillance
technique. Based on this, clinical metagenomics (CMg) has and antimicrobial stewardship; offering susceptibility
also been developed to diagnose hospital-acquired pneumo- results for antibiotics; monitoring the outcome of antibiotic
nia (HAP). This new technique can help optimize the antibi- therapy. Besides, outpatients as well as inpatients can both
otic regimen by identifying pathogens and antibiotic receive treatment according to the results of these molecular
resistance genes (ARGs). Therefore, provided that CMg technologies. And these techniques may be beneficial for
would be faster than conventional culture, the probabilistic critical patients to initiate an appropriate antimicrobial ther-
regimen used in HAP could be tailored faster, which should apy promptly, by doing which can probably reduce the risk
lead to an expected decrease of mortality and morbidity [64]. of mortality, by providing results in 1–2 h. Additionally,
At present, there are still many challenges in correlating rapid identification of antibiotic-resistant pathogens can
metagenomic data with antibiotic susceptibility results. In help isolate patients on time. Nevertheless, we come to real-
612 L. Ming et al.
ize that lungs are a dynamic microbiological ecosystem, and ing, especially for drug resistance Tuberculosis, simple cul-
an alteration of its microbiome may be involved in pneumo- ture or PCR is not accurate, effective, and quick for
nia. This seems to be challenging for microbiologists and diagnosing.
clinicians nowadays. Little evidence has shown that the new
technology can help clinicians to distinguish colonization
from infection. Besides, there need some cost-effective 38.4.2 Clinical Appearance
studies to learn the relative outcomes and costs of the new
technology [61]. The clinical appearances of TB largely rely on the locations
where tuberculosis bacillus is colonizing and proliferating.
The most commonly affected organ is the lung (pulmonary
38.3.5 Conclusion TB). Characterized symptoms of pulmonary TB include
chronic cough for more than three weeks, chest pain, or spu-
The development of molecular detection has improved the tum (sputum from deep in the lungs). TB disease in the
sensitivity and speed of detection, making it possible to lungs could also present with atypical symptoms such as
detect pneumonia pathogens quickly and accurately, and the weakness or fatigue, weight loss that could not be explained
use of broad-spectrum antibiotics will be reduced to a certain by other reasons, anorexia, chills, fever, and sweating dur-
extent. On this basis, antibiotic resistance can be reduced. ing sleep. Symptoms of TB in other organs are diverse and
Recently, it has been published that the multiple PCR tech- organ-specific. People who have latent TB infection are usu-
nology has many advantages in detecting community- ally asymptotic and their chance to spread TB to others is
acquired pneumonia. In the use of the new technologies, it is very low.
necessary to identify which pathogens to target and which Certain populations may be infected with TB and they
molecular tests to perform. Therefore, it is important to com- need to be tested for it. These populations include (1)
municate with the microbiology laboratories and clinicians Persons who have close contact with clinical diagnosed TB
in a timely manner. However, in order to determine whether patients; (2) Persons from a country where TB is prevailing,
the molecular detection technology can truly and effectively such as Asia, Eastern Europe, Africa, Russia, the Caribbean,
improve the diagnosis of respiratory pathogens and the treat- and Latin America; (3) Persons who stay at certain institutes
ment of patients, more research and evaluation should be or organizations where the infection risk of TB is high, such
conducted. In short, new technologies to optimize our knowl- as nursing homes, homeless shelters, and so on; (4)
edge, prevention, and treatment of pneumonia are just around Healthcare professionals whose patients are at high risk for
the corner [61]. TB disease; (5) Persons whose immune system is not well-
established, i.e., infants, children, and adolescents, and if
they have stayed with other people who are suspicious for
38.4 Tuberculosis latent tuberculosis infection or have clinically diagnosed TB
disease.
Wenjuan Wu and Simin Yang

38.4.1 Overview
In laboratory diagnosis, the “gold standard” of TB detection
Tuberculosis (TB) is a chronic infectious disease caused by is sputum microscopy and microbiological culture. Although
tuberculosis bacillus, which spreads among humans through direct microscopy shows the benefits of economical, quick,
the air. TB can cause serious health problems and even death and high specificity in clinical practice, the sensitivity of its
if not treated properly. Medications can prevent those who detection is less than 60% [65]. It takes over several weeks
are infected with TB but yet have no symptoms from disease for the samples to be cultured in liquid medium or 8–12 weeks
progressing in the future. Although an ancient disease, TB on solid medium, such as Löwenstein-Jensen medium, only
still poses a threat to everyone’s health. It is reported that if with the available setting and resources [66].
there were 9,600,000 cases and 1,500,000 deaths worldwide Mycobacterium Growth Indicator Tube (MGIT) is a kind of
in 2014. An increasing number of TB exhibits multidrug liquid culture platform, which can shorten the time to
resistance (MDR-TB, at least rifampicin and isoniazid resis- 6–12 days compared with 21 days of solid media [67, 68].
tance), and even extensively drug resistance (XDR-TB, NAAT has been playing a more and more significant part
MDR-TB with other fluoroquinolone and one second-line in diagnosing TB disease. The Xpert MTB/Rif runs on the
drug capreomycin/kanamycin/amikacin resistance). GeneXpert system. The system extracts nucleic acids, then
However, the treatment and management of TB are challeng- amplifies the rifampicin resistance determining region
38 Lung Disease 613
(RRDR) of the Mycobacterium tuberculosis complex Table 38.11 Characteristics of common sequence-based molecular
(MTBC), uses molecular beacons to automatically detect methods
mutations, and finally analyses the results with GeneXpert Assay Characteristics
system [69]. Through the method, the existence of MTBC Sanger • Can be used to sequence several hundred
and potential resistance to rifampicin can be detected in sequencing nucleotides and detect any mutations within
the range sequenced.
about 2 h. This test offers a sensitivity of 89% (85–92%) as • Can be optimized to test smear-positive
well as a specificity of 99% (98–99%) [70–73]. sediments or cultures.
As an alternative NAAT, Loop-mediated isothermal • If a gene and its association with drug
amplification (LAMP) is a novel thermostatic nucleic acid resistance is identified, an assay can be
developed to detect mutations.
amplification technique. Compared with the traditional poly- • TAT: 1–3 days.
merase chain reaction (PCR) technology, LAMP can amplify pyrosequencing • Short-segment real-time sequencing for
the target DNA at 65 °C by using 4 primers, 2 inner primers, mutations is concentrated within <100
2 outer primers, and DNA polymerase [74]. Compared with nucleotides.
• Can be optimized to test smear-positive
Xpert MTB/RIF test, the key advantage of LAMP is that a sediments or cultures.
thermal cycler is not required. Although a pooled sensitivity • If a gene and its association with drug
of 93% (92–95%) and specificity of 94% (92–95%) com- resistance is identified, an assay can be
pared to culture was suggested in a recent meta-analysis, the developed to detect mutations.
• TAT: <8 h.
heterogeneity test suggested that the heterogeneity was as
Targeted • Suitable for testing pure cultures.
high as 85% [75]. in-depth • More sensitive and can detect heteroresistance
Insertion sequence 6110 (IS6110) is a multi-copy con- massively with minor population as low as l%.
served fragment in the genome of Mycobacterium tuberculo- parallel • Longer TAT.
sis and belongs to the IS3 family. This sequence has a high (next-
generation)
copy number (approximately 10–25) in most Mycobacterium sequencing
tuberculosis isolates. Different strains can be distinguished Whole-genome • Suitable for testing pure cultures.
based on the number and position of IS6110 in the genome, sequencing • Open possibilities for identifying new targets
and it has been proven to have higher resolution and better for drug resistance detection.
• Longer TAT.
repeatability [76, 77]. Spoligotyping utilizes DNA polymor-
phisms at Direct Repeat locus (DRs). There are variable Abbreviations: TAT turnaround time
“spacer” regions interspersed between the DR locus which is
consisted of 36 bp DRs. Strains differ in the DR number and by NGS is associated with the accuracy and speed of diag-
the existence of intervals, which can distinguish certain spe- nosing MDR/XDR-TB, which is conducive to epidemiologi-
cies [78]. cal studies of TB. NGS can reassemble the produced reads of
VNTR, also known as Minisatellite DNA, is a small varying lengths into longer genome sequences (Table 38.11).
repeating DNA sequence. The designed PCR primers are While NGS is an advanced and practical technology, it
used to amplify, followed by gel electrophoresis to determine also has some related challenges to consider. Being capable
the size of the PCR product (amplicons), which in turn can of determining whether the differences between sequences
determine copy numbers [79]. The above methods are useful due to sequencing error or sequence variance is one of the
for epidemiological studies, but still have shortcomings, for challenges. There are various types of error, and the error
example, IS6110-RFLP needs a large amount of genomic rates also differ between each sequencing method. In order
DNA with high quality; spoligotyping is difficult to distin- to reduce the error, sufficient sequencing depth is needed to
guish similar species [80, 81]. generate consensus data for SNP calling, or it can be com-
In the past decade, the way genes have been sequenced pared with reference data sets (not possible if there are sys-
has revolutionized. Next-generation sequencing (NGS) tech- tematic errors). In addition, repeating elements consisting of
nology surpassed the original chain termination assay [82, a large number of base pairs within genomic DNA.
83]. The whole bacterial genome sequences can be detected The mechanisms mainly arise through point mutations in
by NGS in a relatively short time (hours–days). The cost is genes that encode target proteins or proteins required for
also much lower compared to the original method and cul- agent activation. The standard treatment of TB involves
ture is not necessary anymore [84]. The NGS technologies Isoniazid and Rifampicin. The Xpert MTB/Rif analysis of
on the market can be mainly divided into “short reads” and rifampicin resistance was performed in the rifampicin resis-
“long reads.” The former produces relatively short DNA tance determining region (RRDR), an 81 base pair mutation
sequences (base pairs <1000), while the latter produces lon- region of the rpoB gene. The target of rifampicin, b-subunit
ger DNA sequences (base pairs >5000). The depth measured of RNA polymerase, is coded by rpoB gene. 96% of pheno-
614 L. Ming et al.
typic rifampicin resistance is due to the mutations in this Antimicrobial Common

region. agent Gene mutations Comments
The resistance to rifampicin is also considered one of the PZA pncA No predominant pncA mutations are
symbols of MDR-TB [85, 86]. Isoniazid resistance is mainly mutations widely distributed
throughout the gene
caused by mutations in the gene katG or inhA. The former and its promoter. Some
may cause catalase activity to be lost or weakened, prevent- mutations, such as
ing it from activating INH, resulting in high levels of drug E37V, D110G, VI63A,
resistance. The latter is a direct target of activated INH and is A170V, and V180I, are
not associated with
related to low levels of resistance. The main cause of pyra- PZA resistance.
zinamide resistance is the reduction of pyrazinamide enzyme panD Mutations in this locus
activity caused by mutations of pncA gene. Ethambutol resis- may be associated with
tance is mostly caused by mutations in the embB gene or PZA resistance.
overexpression of Emb (Table 38.12). Quinolones gyrA D94G D94G is the mutation
A90V most frequently
associated with
quinolone resistance.
Table 38.12 Genes and associated drug resistance mutations in A90V is the second
MTBC most frequent
Antimicrobial Common mutation, usually
agent Gene mutations Comments associated with
lower-level quinolone
INH katG S315T(ACC) S315T(ACC) confers
resistance than
high-level INH
D94G. Moxifloxacin
resistance.
may still contribute to
inhA -15C/T Often associated with therapy.
promoter low-level INH
Amikacin rrs A1401G A1401G is the
resistance. May also be
mutation most
associated with
frequently associated
ethionamide resistance.
with amikacin
ahpC -48G/A, -52C/T, Associated with INH resistance.
promoter −54 C/T resistance.
Capreomycin rrs A1401G A1401G is the most
fabGl L(CTG)203 Associated with INH common mutation and
(or mabA) L(CTA) resistance. is usually associated
RIF rpoB* • Group 1: • S531L is the most with capreomycin
– S531L frequently detected resistance.
– H526Y mutation in Kanamycin rrs A1401G A1401G is the most
– H526D MDRTB, and it common mutation
• Group 2: confers RIF and associated with
– D516V RFB resistance. high-level kanamycin
• Group 3: • F514F(TTT) is the resistance.
most common silent
eis -10G/A Highly associated with
– Nonecommon mutation. It can be
promoter -12C/T kanamycin resistance.
• Group 4: detected by
-14C/T -12C/T may confer
– F514F probe-based assays.
-37G/T low-level kanamycin
(TTT) If the probe assay
resistance.
does not provide the
mutation identity, it
a
When the critical concentration is 1 μg /mL for RIF and 0.5 μg /mL for
can be RFB by cAST, the rpoB mutations can be divided into 4 groups: Group
misinterpreted as 1 mutations confer resistance to RIF and RFB. Group 2 mutations con-
RIF resistance. fer resistance to RIF but not to RFB. Group 3 mutations are also known
EMB embB M306V M306V is the most as “disputed mutations.” Isolates with these mutations often test suscep-
frequent mutation tible to RIF and RFB, but the presence of these mutations may increase
associated with EMB the risk of treatment failure or relapse, especially when INH is also
resistance. Not all resistant. Group 4 mutations are silent and do not confer resistance to
mutations in embB are RIF or RFB
associated with EMB
resistance.
38 Lung Disease 615
Fig. 38.11 Mycobacterium

tuberculosis sample Specimen collection
processing and detection Sputum smear for AFB
methods. Abbreviations: AFB within 24h
Acid Fast Bacilli, RIF Rapid molecular diagnostics
Rifampicin, INH Isoniazid RIF/INH resistance within 24h
Culture
Up to 8 weeks
Whole genome sequencing

Preliminary report with genotypic
susceptibilities within 9 days
Drug susceptibility
detection
About 3 weeks
Final report
The second-line anti-TB drug resistance can lead to

XDR-TB. The mutations associated with the resistance are
clear. Fluoroquinolones treat tuberculosis by inhibiting
MTB DNA gyrase. Changes among its subunits can result
in drug resistance. The 16s rRNA mutations result in other
second-line drugs kanamycin and amikacin resistance [87,
88]. These specific mutations make it easy to test by molec-
ular detections. However, the mechanism of agent resis-
tance is not completely clear. There are still 10–40%
mechanisms of resistant strains unestablished. In these
cases, the mechanism is unknown and the genomic basis of
agent resistance may be the presence of SNPs beyond the
reported resistance genes [89]. These SNPs can be accu-
rately detected by whole genome sequencing (WGS) [90].
WGS can be used to determine the mechanism of agent
resistance, and most importantly, as technology advances, Fig. 38.12 Chest CT Scan before therapy A
it can establish and refine a reference library of drug-resis-
tant mutations for comparison with the target strain. With Laboratory Data Sputum acid-fast smear was positive for
WGS, genotypic sensitivity tests can obtain results within a fluorescence staining (3+), Mycobacterium tuberculosis
few days of a positive culture, compared with traditional nucleic acid test was positive for molecular biology, and iso-
phenotypic tests (Fig. 38.11). niazid and rifampin gene tests were sensitive. Sputum cul-
ture was positive for Mycobacterium tuberculosis, and the
strain was identified as Mycobacterium tuberculosis com-
38.4.4 Typical Medical Case plex group. The phenotypic drug sensitivity results showed
that INH, RFP, SM, EMB, AK, K, CPM, and OFX were all
Clinical Background A 60-year-old female patient came to sensitive. After six months of treatment, sputum and acid-fast
hospital for cough, sputum, chest tightness for one month. smear staining were negative, and tuberculosis culture was
She lost weight in the last month. negative.
Chest CT scan Tuberculosis (Fig. 38.12). Treatment Antituberculosis therapy.

616 L. Ming et al.
operation of NGS to tuberculosis. However, competing

human and bacterial DNA and some paucibacillary samples
result in low genome coverage which hampered the applica-
tion. WGS is an economical, quick, and effective method for
resistance testing and molecular research, but more effort is
required to characterize resistance-associated mutations in
order to achieve a better relationship between resistance
genotypes and phenotypes. To improve the sensitivity of TB
diagnostics and treatment, it is necessary to implement tar-
geted methods, for example, capture MTBC cells or DNA
specifically.
38.5 Influenza
Fig. 38.13 Chest CT Scan after anti-tuberculosis therapy B
Huaguo Xu and Fang Ni
Results with Interpretation Guideline The diagnosis of
tuberculosis is based on etiology, including bacteriology,
molecular biology examination, chest imaging examination, 38.5.1 Overview
and so on. Acid-fast staining microscopy is rapid, simple,
and low-cost. Microscopic examination is still the main In recent years, the global highly pathogenic avian influenza
method for detecting Mycobacterium tuberculosis in clinical virus (H5N1) has killed more than 100 people in Asia,
specimens. It has been recommended by WHO and widely Europe, and Africa, and infected millions of poultry.
used, but its specificity is not high. If the clinical manifesta- Influenza, commonly known as “the flu,” is an infectious dis-
tions are not obvious and the imaging features are not typi- ease caused by frequent outbreaks of highly pathogenic
cal, mycobacterial culture is the gold standard, but the avian influenza virus. Influenza virus, a representative spe-
traditional Roche culture method takes a long time. WHO cies of Orthomyxoviridae, includes human influenza virus
recommended Mycobacterium tuberculosis molecular test as and animal influenza virus. Human influenza virus is divided
a method for detecting suspected drug-resistant tuberculosis into three types, which is the causative agent of several seri-
infection among adults and children, with high sensitivity ous influenza pandemics (Fig. 38.14). Typical clinical fea-
and specificity, and better diagnostic efficacy (Fig. 38.13). tures of influenza include high fever, runny nose, cough, and
muscle pain. The above symptoms usually appear within the
This case was from the Third People’s Hospital of Zhenjiang. first two days of infection with the virus. Except for a longer
duration of cough, the remaining symptoms usually do not
exceed one week. Influenza infections are often accompa-
38.4.5 Management nied by gastrointestinal symptoms, which are easily misdiag-
nosed as gastroenteritis and delay treatment.
1. Using molecular methods to diagnose TB can save time Influenza viruses are divided into type A (A), type B (B),
to a few hours in obtaining results (traditional methods type C (C), and type D (D), of which the first three viruses
take weeks). will infect humans, and type D is a newly discovered influ-
2. NGS helps distinguish between relapsed and re-infected enza virus that does not infect humans after recombination,
patients and facilitates the treatment of patients. but has the potential for infection (Table 38.13). The virus is
3. Convenience: Diagnosis, antibiotic resistance analysis, mainly transmitted in the air mainly through the respiratory
and epidemiological analysis may be merged into a single tract by coughing or sneezing droplets, especially when the
test through molecular methods, infected person and the susceptible person are in close con-
tact. It can also be transmitted through direct or indirect con-
tact with mucous membranes in the eyes, nasal cavity, mouth,
38.4.6 Conclusion etc. Before showing symptoms, recessive infection patients
may infect susceptible people who are not effectively pro-
Molecular methods are more promising in diagnosing and tected. The infection can be confirmed by detecting a virus in
managing TB, including drug-resistant TB. Shotgun the throat, sputum, or nose in the way of performing many
metagenomics sequencing can directly perform rapid TB rapid diagnostic tests. A polymerase chain reaction for
diagnosis and resistance test of sputum, which is an ultimate detecting viral RNA is more clinical value at the low viral
38 Lung Disease 617
Fig. 38.14 Schematic
diagram of influenza virus
structure
Table 38.13 Influenza virus classification of influenza. It cannot be effective for the same vaccine
Antigenic Representative injected for one year in the next year, because the mutation is
Type Subtype structure Popular time strain fast.
Influenza A A0 H0N1 1930–1946 A/PR/8/34
A1 H1N1 1946–1957 A/FM/1/4
A2 H2N2 1957–1968 A/Singapore/1/57
A3 H3N2 1968–1977 A/Hongkong/1/68
38.5.2 Pathogenesis
A1 and A3 H3N2 1977–Now A/BeiJing/32/92
H1N1 38.5.2.1 Transmission
Influenza B Victoria(BV) – Little Little variation There is a seasonal distribution of influenza virus infection,
Victoria(BY) outbreak
which mainly involves close unprotected contact with respi-
ratory droplets. When infected people sneeze and cough,
load concentrations. But it is worth noting that people can they need to cover their nose and mouth with paper to reduce
still be infected even if the pathogenic result is negative. the spread of virus particles into the surrounding air. The
Influenza spreads all around the world in an annual out- virus may also spread through contact with contaminants of
break, causing about three to five million cases of severe ill- the respiratory or gastrointestinal fluids. Viral RNA can
ness, which mainly infects unvaccinated people. An outbreak even be detected in the stool of diarrhea patients with influ-
of a new type of influenza H1N1 was declared to be a pan- enza, suggesting potential for fecal-oral transmission [91,
demic in June 2009, which might also affect other animals, 92]. The relative importance of the three modes of transmis-
including chickens and birds, causing huge economic losses sion that led to the outbreak of the virus is unknown. An
in the world. Frequent hand washing and wearing a surgical important factor associated with influenza virus transmis-
mask can reduce the risk of viral spread. Regular injection of sion and pathogenicity is the concentration of pathogen par-
vaccine is suggested by the World Health Organization to ticles that reach the site of host infection. Taking respiratory
those high-risk populations for preventing all kinds of types transmission as an example, most of the air source particles
618 L. Ming et al.
with a diameter of 5 μm to 10 μm can reach the trachea, and proteases in the throat and lungs is unable to infect other tis-
most of the particles with a diameter greater than 20 μm sues. However, the highly virulent influenza virus of which
cannot reach the lower respiratory tract. Infections caused HA can be cleaved by a variety of proteases, which can rap-
by influenza viruses in aerosols are also related to other fac- idly cause multi-tissue and multi-organ lesions to spread
tors: biological half-life in the air, types of influenza virus,throughout the body. The difference in the transmission of
and environmental factors. Impact of environmental factors these infections may be due to the severe inflammation and
on influenza virus transmission includes temperature, UV hypoxemia caused by multiple lesions in the lungs of the H5
radiation, and relative humidity, which significantly affect subtype.
the survival time of aerosols containing influenza virus. Influenza virus enters the body, and is mainly adsorbed on
Although influenza virus can persist in the human body, it the surface of upper respiratory epithelial cells. The innate
spreads on the surface of in vitro pollutants, such as door immune system recognizes and activates downstream signal-
handles and switches, which can only survive for several ing pathways, releases a series of pro-inflammatory cyto-
minutes on the surface of dry objects and can survive for kines and chemokines, immune cells chemotactically
several weeks or longer in the mucus. Avian flu is an infec- aggregate, and excessive immune responses cause histopa-
tious disease that causes acute, systemic septicemia and thology injuries, which are manifested by pathological
high lethality in birds. Among them, disinfection is particu- changes such as respiratory cilia epithelial cells metaplasia,
larly important in the prevention and control of avian influ- lamina propria hyperemia and edema [93–95]. After both
enza. At present, among the many disinfectant brands, it is TLR3 and RIG-I are knocked down, the interferon-induced
the only one that can be in line with international standards expression caused by influenza virus infection in human
and has been adopted by the World Health Organization alveolar epithelial cells will be suppressed, which indicates
(WHO). The only safe and effective fourth-generation disin- that TLR3 is involved in the regulation of the anti-influenza
fectant approved is chlorine dioxide. Time required for virus innate immune response and plays an important role in
influenza infection in normal adults appears: symptoms regulating the host’s inflammatory response [93]. In severe
peak on the second day after infection and last for an aver- cases, bronchial cells are extensively necrotic, hyaline mem-
age of 5 days. Children, immunocompromised and immu- branes are formed, alveolar epithelial cells and interstitial
nosuppressed patients are more infectious than adults and edema, and even tissues are extensively fibrotic. Unlike a
the virus can last for more than two weeks. common cold-associated rhinovirus, influenza often causes
local inflammation and systemic reactions in the body, but is
38.5.2.2 Pathology and physiology not entirely caused by inflammatory reactions. Influenza
The mechanisms by which influenza virus infection induces virus can be co-infected with bacteria: after lung infection,
body symptoms have been studied intensively. It is believed lung epithelial cells necrosis and fall off, and the physical
that one of the mechanisms is the inhibition of adrenocorti- barrier of the respiratory tract is damaged. Colonized bacte-
cotropic hormone (ACTH), which leads to elevated cortisol ria in the nasopharynx can enter the lungs and cause a double
levels. The timely detection of changes in serum cortisol in infection, which can spread to the whole body. The autopsy
patients with severe viral encephalitis has important clinical of influenza death cases found not only the lungs, but also
significance for treatment and assessment of prognosis. other organs and tissues, and the pathological changes were
Hemagglutinin (HA) can function mainly to bind to virus- also obvious [94]. More than 33% of autopsy results showed
specific receptors on the cell surface, mediate the fusion of diffuse congestion of brain tissue, interstitial hemorrhage of
the outer membrane of the virus with the intracellular body most myocardial cells, cytoplasmic porosity of hepatocytes,
membrane, release the viral nucleocapsid into the cytoplasm, and vacuole degeneration change.
and stimulate the body to produce protective antibodies. The
main factor determines the virulence of the influenza virus.
Frequent mutations in HA and changes in receptor binding 38.5.3 Epidemiology
sites may change the antigenic characteristics, host fitness,
and pathogenicity of influenza viruses. Influenza virus 38.5.3.1 Seasonal Variations
amplification and acquisition of infectivity in the host must Almost every winter, there will be regional outbreaks of flu
be activated by the host protease, called the cleavage of to varying degrees. Seasonal outbreaks may be caused by
HA. The distribution of host protease is closely related to the infrequent indoor ventilation and reduced outdoor activities
pathogenicity and tissue tropism of the virus. For example, in winter. Another explanation is that the winter air is dry, the
the weakly toxic influenza virus of which HA spreads suspended aerosol is dehydrated and lightened, the influenza
through the respiratory tract due to the cleavage of specific virus is better for survival at lower temperatures, and the
38 Lung Disease 619
virus particles are suspended in the air for a longer time. In 38.5.4 Course of the Disease
addition, bird flu viruses have similar seasonality. A number
of foreign studies have focused on the correlation between About one-third of the patients with influenza have no spe-
the seasonality of influenza infection and vitamin D levels: cific clinical symptoms at the initial stage. The emergence of
Simpson found that the easy outbreak of winter influenza influenza symptoms can occur suddenly one or two days
may be related to seasonal fluctuations in vitamin D levels, after infection. At first, the typical symptoms of influenza are
which are produced in the skin under sunlight. In winter, shivering, acute fever, general pain, etc. According to the
people usually stay indoors for a long time, with short sun- accompanying symptoms, influenza can be divided into sim-
shine hours, and their vitamin D levels decrease. Similar ple influenza, pneumonia influenza, poisoning influenza, and
studies have reached similar conclusions: Children with vita- gastrointestinal influenza. It is difficult to distinguish mild
min D levels below 10 nanograms per milliliter are 11 times influenza infection from common cold, and there is often no
more likely to develop respiratory disease than children with complication with self-limitation. Pneumonia type influenza
higher levels of vitamin D in the blood. includes primary viral pneumonia and secondary bacterial
pneumonia, accompanied by pulmonary inflammatory signs
38.5.3.2 Epidemic and Pandemic Spread and dyspnea symptoms. The pathogen culture is negative,
Almost every year, different types or subtypes of influenza and the influenza virus can be isolated positive. Secondary
virus will cause large-scale outbreaks, and the main reason is bacterial pneumonia will aggravate and delay the patient’s
that the influenza virus is constantly changing. The antigenic condition, which often occurs in the antiviral treatment with
variation of influenza virus includes two types: antigenic a trend of improvement, but the condition of high fever is
drift and antigenic transformation. Antigenicity drift is aggravated or repeated. Toxic influenza is characterized by
mainly due to a series of point mutations in the protein gene high fever, shock, and respiratory failure. Gastrointestinal
encoding HA and neuraminidase antigen (NA), leading to influenza often occurs in children, presenting with vomiting,
the replacement of amino acids in situ on the protein mole- abdominal pain, diarrhea, and fever. The incidence of viral
cule, which can cause medium and small-scale epidemics, encephalitis caused by influenza virus is relatively low. There
and the mutation size is often proportional to the scale of are only case reports at home and abroad. The clinical mani-
influenza outbreak. The antigenic drift of the virus is weak, festations are mainly meningitis and influenza symptoms,
and the antigenicity of some of the original strains is still and the brain parenchyma is not significantly affected, which
maintained. Human beings still have certain immune effects. is different from that of herpes virus encephalitis, which
“Antigenic transformation” refers to the redistribution of invades the central nervous system through olfactory nerve.
antigens to obtain completely new antigens, which is essen- Early identification and prophylactic treatment of influ-
tial to produce a new influenza virus causing global high enza virus is the key to prevent the severity of influenza,
pathogenicity. Human immunity will not be able to control because early treatment of influenza is easy to block virus
the transmission, and it is easy to appear global large-scale replication. In the above typical symptoms, the comprehen-
transmission. For example, avian influenza virus strain H9N2 sive judgment of fever and cough, pharyngeal pain, and nasal
and influenza virus strain H1N1. obstruction can improve the accuracy of early diagnosis. In
According to the 2017 “China influenza monitoring infor- addition, preventive treatment for the elderly and children
mation system” data, the three types of influenza viruses during the flu season is necessary to reduce mortality.
before virus isolation are H1N1, B, and H3N2 respectively.
Although influenza incidence rate and virus type vary con-
siderably each year, the seasonal outbreak of influenza virus 38.5.5 Diagnosis
causes the clinical degree of hospitalized patients, especially
the elderly and children, to increase significantly. The elderly It is not difficult to diagnose influenza in combination with
and children should be the focus of influenza prevention and clinical symptoms during the epidemic period, but labora-
control. It is reported that influenza causes millions of tory tests must be performed for diagnosis or epidemiologi-
diseases and up to one million deaths every year around the cal surveillance, including virus isolation, serological
world. Influenza spreads very quickly. It only takes two days diagnosis, and rapid diagnosis.
for a person to infect others. In addition, many people
become the peak of transmission when the symptoms are not 38.5.5.1 Virus Isolation and Identification
obvious. When the symptoms are typical, the ability to trans- Usually take the Nasal swab or throat swab of the patient
mit the virus has declined. At this time, isolation is not the within 3 days after the onset of the disease (Fig. 38.15). After
most effective public health intervention. antibiotic treatment, inoculate it in the amniotic cavity and
620 L. Ming et al.
Swab
Throat is
A sterile swab is swabbed in the
passed gently area of the
through the nostril tonsils
and into the Tonsil
nasopharynx
Fig. 38.15 Influenza virus sampling process
Fig. 38.16 Influenza
diagnosis flowchart
In the 7 days before the onset, there were close contact with
the confirmed cases of influenza in the infectious phase, and
Ask about medical history
influenza-like clinical manifestations appeared. Influenza-like
clinical manifesations have occured in areas where influenza
has been reported within 7 days before the onset of the
disease.
High fever, headache, body aches, cough or sore throat, acute

Influenza clinical manifestations onset
Influenza rapid antigen test positive
Laboratory test results Influenza virus nucleic acid test positive
Influenza virus-specific IgG antibody levels in the acute

and recovery phases were 4 or more times higher
allantoic cavity of chicken embryos of 9–11 days old. After by tissue culture cells (such as human embryonic kidney or
incubating for 3–4 days at 33–35 °C, amniotic fluid is col- monkey kidney), and the presence or absence of virus prolif-
lected. Hemagglutination test was performed with allantoic eration can be determined by the erythrocyte adsorption
fluid. If the hemagglutination test is positive, hemagglutina- method or the fluorescent antibody method (Fig. 38.16).
tion inhibition (HI) test is performed with known immune
serum to identify the type. If the hemagglutination test is 38.5.5.2 Serological Testing and Typing
negative, the chicken embryo is then blindly passaged for 3 Methods
times, and blood coagulation is still not observed to deter- Serological diagnosis was performed in the acute phase of
mine that the virus is negative. The virus can also be isolated the patient (within 5 days of onset) and during the recovery
38 Lung Disease 621
period (2–4 weeks of disease). The HI test was used to detect methods in that it detects live viruses and therefore in clinical
antibodies. A diagnosis can be made if the recovery period is sample testing and animal products.
more than four times higher than the serum antibody titer in The quarantine is accurate and practical. However, this
the acute phase. Normal human serum often contains non- method has certain requirements for specimens. Sputum,
specific inhibitors, so serum can be treated with trypsin or nasal extract, and nasal wash are the best specimens for
the like before the HI test, so as not to affect the HI test influenza virus culture and antigen detection. Relatively
results. The virus used in the HI test should be a virus strain speaking, the specificity and sensitivity of nasopharyngeal
closely related to the current epidemic, and the reaction swab specimens for influenza virus detection is lower.
results can be exact. Complement fixation (CF) can only
detect antibodies against NP and MP. These antibodies 38.5.5.3 Quick Diagnosis
appear early and disappear quickly. Therefore, the CF test Rapid diagnosis of patients mainly includes indirect or direct
can only be used as an indicator of whether it is a recent immunofluorescence detection of viral antigens. The
infection. patient’s turbinate mucosa or respiratory tract epithelial cell
The serological method is mainly to detect the level of smear is often taken, and the antigen is detected by immuno-
antibodies in the serum of patients. The most commonly fluorescence staining with fluorescein-labeled influenza
used method for the identification of influenza viruses is the virus immune serum, or the antigen in the pharyngeal spu-
hemagglutination inhibition assay, which is a serological tum is examined by ELISA. The monoclonal antibody can be
standard method for diagnosing human influenza virus infec- used to rapidly detect viral particles or virus-associated anti-
tion. HI is the collection of acute and recovery blood sam- gens of infected influenza A and B viruses in infected cells
ples, and the acute phase does not exceed the onset at the by immunoenzymatic labeling for only 24–72 h. At the
latest. It is diagnostically meaningful, and a single serum is National Influenza Prevention and Control Conference held
generally not used for diagnosis. The results of this type of on October 30, 2010, experts from the China CDC pointed
experiment are accurate and reliable, and do not require too out: in China, whether in the south or in the north, the epi-
much expensive equipment, can get a lot of virus materials demic is basically also an H3N2 virus (Table 38.13).
while completing the diagnosis. For scientific research and The rapid and effective detection technology of influenza
vaccine use, but the experiment belongs to the review sexual virus plays an extremely important role in the decision-
experiment, only valid for the discovered influenza virus, making of influenza prevention and early treatment, includ-
and the experimental period are longer, experimental tech- ing immunological methods and nucleic acid detection
niques are also more demanding. The current common methods, among which molecular biological detection tech-
method of virus antigen detection is mainly immunofluores- nology has attracted more and more attention due to its high
cence. First, the fluorescent substance is labeled on the anti- sensitivity. Rapid detection of samples from nasopharynx
gen (or antibody), and the antibody (or antigen) is detected swabs or sputum should be completed early after symptoms
by an immune reaction. If the two correspond to each other, appear, because virus shedding will decline after symptoms
an immune complex with a fluorescent substance is formed, appear, which is easy to cause false-negative results. Methods
and fluorescence is observed under a fluorescence micro- such as PCR, nucleic acid hybridization, or sequence analy-
scope. If the two do not correspond, they do not form immune sis are also used to detect influenza virus nucleic acids or to
complexes and fluorescence. The substance is washed away perform typing.
by water and does not show fluorescence under the micro-
scope. It is divided into direct Immunofluorescence and indi- Loop-Mediated Isothermal Amplification
rect immunofluorescence, the former is used by direct Loop-mediated Isothermal Amplification (LAMP) is a novel
detection of monoclonal antibodies against influenza virus in constant-temperature nucleic acid amplification method
clinical specimens. Viral antigen is a faster detection method, developed by Notomi and colleagues in 2000. The principle
but this method is for specimens. The requirement is higher, is to design four specific primers for six regions of the target
and there is a certain amount of call in the specimen. Suction gene, using a strand-replacement DNA polymerase in iso-
epithelial cells, otherwise it will result in false positives and thermal conditions (around 65 °C). The incubation of the
false negatives. The latter is an anti-immunoglobulin (anti- nucleic acid can be completed by holding for several tens of
antibody, generally IgG) that binds unlabeled specific minutes. There is no need for thermal denaturation of the
immune serum to antigens that may be present in the speci- template, long-term temperature cycling, cumbersome elec-
men, and then fluorescein-labeled, and binds to antigens that trophoresis, UV observation, and the like.
may be present on the specimen for the specific antibody
binding. The sensitivity of the indirect method is higher than Gene Chip Technology
that of the direct method due to the amplification of the inter- In recent years, the flu epidemic has been frequent outbreaks,
mediate antibody. This method differs from some other which requires High-throughput screening is also required
622 L. Ming et al.
for rapid diagnosis. Gene chip is high throughput, high sen- The high mutation rate of influenza virus determines that
sitivity, especially suitable for influenza virus genes and the specific influenza vaccine cannot prevent all influenza
sinks. The characteristics of the main polymorphism, various viruses. Each season, the influenza vaccine needs to be
frontier chip technologies are also used in the detection and remanufactured for the specific influenza virus strains that
typing of influenza viruses. may be prevalent, but it may not cover all active influenza
viruses. There is still a risk of infection when new or
Pyrosequencing Technology neglected strains of bacteria appear, or antibodies are not
Pyrosequencing technology, a new enzyme-linked cascade produced before or after vaccination. As a biological prepa-
sequencing technology for high-throughput, was developed ration, the incidence of adverse reactions of influenza vac-
by Nyren in 1987, which is referred to as high-accuracy cine itself is extremely rare; the most common is allergic
(99% accurate) and reproducible analytical techniques for reactions, mainly from different proteins. Caution or prohibi-
short to medium-length DNA sequence samples. tion of vaccines includes those whose autoimmunity is in
disorder half a month before vaccination, and those who are
Nuclear Acid Sequence-Based Amplification allergic to eggs and gentamicin.
Nuclear Acid Sequence-based Amplification (NASBA) is an
isothermal nucleic acid amplification method reported in Improve Your Own Immunity
1991 that focuses on RNA in samples (the experimental con- You can improve your body’s immunity and fight the virus
ditions can also be used to amplify DNA). NASBA is a by exercising your body. Normal work, life, learning should
sequence of primer-mediated, continuous, and uniform be combined with work and rest, excessive fatigue, resulting
in vitro specific amplification of single-stranded RNA. At in decreased resistance, highly susceptible to viral influenza.
about 40 °C, the template RNA can be amplified by about Strengthen nutrition, a balanced diet, diet should be light, eat
109 to 1012 times in about 2 h. more vegetables, and fruits rich in high vitamins, children
should not eat cold drinks.
38.5.6 Prevention and Treatment Other

Insist on washing your face with cold water to enhance the
38.5.6.1 Prevention ability of the nasal mucosa to adapt to the air. Keep abreast
of the weather changes and keep warm according to the
Influenza Vaccination weather. At the same time, strengthen physical exercise,
In recent years, influenza vaccine has achieved remarkable enhance the ability to adapt to the environment and the
results in reducing the incidence rate and mortality of influ- body’s immunity. In addition, during the cold epidemic, try
enza, and has become a major measure to prevent influenza. to go to places with dense population and wash your hands
Among the high-risk groups, the active vaccination rate of frequently. When the body is slightly uncomfortable, mild
influenza vaccine has increased significantly. The high-risk dry mouth, take the medicine immediately when the nose is
groups of influenza include infants, the elderly, medical staff, stuffed, drink plenty of water, pay attention to keep warm
and immunosuppressive patients. Attention should be paid to and rest, so that the condition will improve in time.
the influenza epidemic season. The immune response pro- Air conditioner should be cleaned before using air condi-
duced by the vaccination of influenza vaccine will not have tioner to avoid a large number of germs blowing out with the
adverse consequences for the high-risk population, but can wind; room temperature should be controlled above 24 °C,
significantly reduce the serious diseases and complications keep the indoor and outdoor temperature difference not more
caused by influenza [96]. Inactivated vaccines can even make than 7 °C, avoid the burden of body temperature adjustment
the immunosuppressive infected people produce certain pro- center; pay attention to air conditioning during sleep or do
tective antibodies. For the long-term gathering in the closed not blow the head directly on the fan.
environment, we should also pay attention to vaccination to In summer, sweating and consumption are large, and suf-
prevent the large-scale outbreak of influenza epidemic sea- ficient nutrition should be added to improve the body’s resis-
son and reduce the incidence of influenza-like symptoms in tance. Appropriate consumption of fish, meat, eggs, milk,
the population. The incidence of adverse reactions in elderly and beans to supplement protein, eat more fresh fruits and
patients with chronic obstructive pulmonary disease after vegetables to take vitamin C, and eat more hot and humid
influenza vaccination is low, but it can reduce the number of food, such as bitter gourd, peach, cucumber, mung bean, and
acute attacks and hospitalizations. However, the improve- so on.
ment of lung function, exercise tolerance and mortality still After taking preventive drugs, the incidence of colds can
need follow-up observation. generally be reduced by about 50%. In addition, people with
38 Lung Disease 623
weak constitutions can also be vaccinated in advance to pre- pneumonia. With the frequent and widespread use of neur-
vent colds. It is best to the flu vaccine in the fall, because the aminidase inhibitors, drug resistance has begun to appear,
temperature changes greatly in autumn and winter, and it is which makes researchers develop efficient and selective
the high incidence of flu. After the winter, when the spring inhibitors.
blooms, the flu will disappear. The flu vaccine can usually
take a shot every half a year. M2 Inhibitors
Pay attention to hygiene: Hygiene should be taken care of Amantadine derivatives, an antiviral drug, inhibit the replica-
to prevent illness from entering the mouth. Wash hands fre- tion of influenza A virus by inhibiting M2 protein. If these
quently, take a shower, change clothes, diligently, and be drugs are used in the early stage of infection, sometimes they
smashed, and the room is often ventilated. If you have a cold are effective for influenza A, but not for influenza B which
patient, pay attention to keep the distance! In the high season lacks M2 drug target. The drug resistance of H3N2 to aman-
of colds, try to be as small as possible. tadine has increased to 91%. The high level of resistance
may be due to the easy availability of amantadine as an over-
38.5.6.2 Treatment the-counter drug in China, and the widespread abuse of
Antiviral therapy: Patients with confirmed or suspected amantadine for the prevention of influenza outbreaks in
influenza virus infection should be treated with antiviral poultry.
therapy, and early treatment can achieve the effect. Two
types of antiviral drugs against influenza are neuraminidase
inhibitors (such as oseltamivir) and M2 protein inhibitors 38.5.7 Typical Medical Case
(such as adamantane derivatives) [97]. Unless bacterial sec-
ondary infection is identified, antibiotic therapy is ineffec- Clinical background The patient, male, 29-years old, had
tive. Early prevention of antiviral: it is recommended to take a hot day, cough, headache, sore throat for two days, went
neuraminidase inhibitor after contact with patients with con- to the clinic. Two days ago, after the patient suffered from
firmed influenza. When influenza outbreak season is used as cold, he developed cough, and coughed a small amount of
emergency prevention, children under 1-year old and preg- white sputum, accompanied by mild headache, salivation,
nant women can also use this as preventive medicine, but sore muscles, chest tightness, difficulty breathing, no nau-
there is no safety guidance for pregnant women. sea and vomiting, abdominal pain, and diarrhea. One day
Symptomatic and supportive treatment: To reduce the before the fever began, the body temperature was up to
temperature and relieve the pain, it is recommended to use 40 °C, with chills and chills. Physical examination: poor
such as paracetamol and non-steroidal anti-inflammatory spirit, breath sounds in both lungs, no obvious abnormali-
drugs to relieve fever and muscle ache related to influenza, ties in the ventral examination, no edema in both lower
and pay attention to rest and drink more water. Attention limbs.
should be paid to influenza symptoms (especially fever) in
adolescents and children when taking aspirin, which may
lead to Raynaud’s syndrome with a rare but potentially fatal Auxiliary Check
incidence. In addition, It is best to avoid close contact with Blood routine: WBC 6.3 × 109/L, NEUT% 42.5%, LYM%
other people and self-isolation to prevent the spread of 46.5%, peripheral blood smear visible 12% atypical
infection. lymphocytes.
Biochemical indicators: ALT 35 U / L, AST 41 U / L,
NA Inhibitors GGT 73 U/ L, Na 132 mmoL/L, normal myocardial enzymes,
NA Inhibitors have the advantages of broad spectrum, high PCT (−).
efficiency, and low resistance to all kinds of influenza T lymphocyte subset count: CD4+: 132 cells/μl, CD8+:
viruses. It is the most common antiviral drug to infect highly 54 cells/μl.
pathogenic avian influenza and new influenza viruses. NA, a Pathogen examination: Bacterial smear staining, fungal
glycoprotein on the surface of influenza A and B viruses, is smear staining, acid-fast smear staining, GM test, respiratory
the key enzyme to promote the replication of virus particles syncytial virus, Epstein-Barr virus, Mycoplasma, Chlamydia,
and release from infected cells. NA inhibitors can effec- and Legionella nucleic acid negative. Nasopharyngeal swab
tively prevent the process of virus replication and release. influenza A virus nucleic acid assay: (+); Influenza B virus
Compared with the adverse reactions, the prevention bene- nucleic acid assay: (−).
fits of healthy people are greater [98]. For those believed to Imaging examination: Chest X-ray: Interstitial infiltration
have the flu, they reduced symptoms by less than a day, but of both lungs (Fig. 38.17). Chest CT: Multiple lungs in the
there was no risk of complications such as hospitalization or lungs, the main pleural (Fig. 38.18).
624 L. Ming et al.
This case was from the Affiliated Jiangning Hospital of

Nanjing Medical University.
38.6 COVID-19
38.6.1 Overview
Qun Zhang, Shiyang Pan and Yongping Lin
Beginning in December 2019, a group of patients with pneu-

monia of unknown origin was first reported. By using molec-
ular techniques and unbiased DNA sequencing in samples
from patients with pneumonia, a previously unknown beta-
coronavirus was discovered, which was known as TARS-
CoV, SARS-CoV-2, or HCoV-19 (Fig. 38.19). The World
Health Organization (WHO) has named the new coronavirus
2019-nCoV [99]. Coronaviruses are enveloped RNA viruses
that, are distributed broadly among humans, other mammals,
and birds, and that cause respiratory, enteric, hepatic, and
Fig. 38.17 X-ray: Interstitial infiltration of both lungs
neurologic diseases. For the third time in as many decades, a
zoonotic coronavirus has crossed species to infect human
populations.
Although 2019-nCoV has typical coronavirus family
characteristics, it differs from coronaviruses associated
with human severe acute respiratory syndrome (SARS)
and the Middle East respiratory syndrome (MERS) [100].
However, novel coronaviruses use the same cell entry
receptor, ACE2, like SARS. Updates indicate that the
novel coronavirus possesses a strong capability to infect
humans, and coronavirus disease 2019 (COVID-19)
appears to be relatively mild as compared with SARS and
MERS. An outbreak of novel coronavirus has spread rap-
idly, with cases now confirmed in multiple countries [101].
The predisposing conditions for COVID 19 pneumonia
tended to be old age and medical comorbidities (such as
chronic pulmonary disease, diabetes, and other chronic
diseases), similar to previous viral infections such as influ-
enza H7N9. Fever, cough, and dyspnoea were the most
common symptoms in patients with COVID19 pneumonia,
consistent with the manifestation of lower respiratory tract
infections. By contrast, upper respiratory tract symptoms
were less common in these patients, indicating that the
cells targeted by the virus might be located in the lower
airway. Other nonspecific symptoms included dizziness,
diarrhea, vomiting, headache, and generalized weakness,
which occurred in around 2–9% of patients. In general, the
mortality rate of COVID19 so far is lower than that of
Fig. 38.18 Chest CT: Multiple ground-glass opacity in the lungs, the SARS or MERS coronavirus diseases. Severe disease
main pleural onset might result in death due to massive alveolar damage
and progressive respiratory failure [102]. Most patients
Treatment Symptomatic treatment, cooling, oseltamivir + have a good prognosis except for a few patients, most of
ganciclovir anti-infective treatment for a week, the symp- whom are the elderly and those with chronic underlying
toms were significantly relieved. disease.
38 Lung Disease 625
Fig. 38.19 Schematic
diagram of 2019 novel
coronavirus structure
Due to the lack of specific therapies and vaccines for 2019 sneezes or coughs, more than half a million virus particles
novel coronavirus diseases, early detection is crucial. The diag- can be spread to close people. The human-to-human trans-
nosis of COVID-19 is mainly confirmed by reverse mission of COVID-19 occurs primarily in families; the novel
transcription-polymerase chain reaction (RT-PCR) or genetic coronavirus is almost susceptible to all ages, and further
sequencing of breath or blood samples, and chest CT [103]. research is needed to determine whether it has immunity
However, with limitations of sample collection and transporta- after infection. Patients develop symptoms on average
tion, and kit performance, the sensitivity of RT-PCR was 5–6 days after infection, most of which can be cured by mild
reported low. In the current emergency department, chest CT, disease. Severe and high-risk populations are over 60 years
as a conventional imaging tool for the diagnosis of pneumonia, of age.
is relatively easy to operate, and can be quickly diagnosed. In COVID-19 can be spread in the following ways: through
this case, chest CT can help with the determination of COVID- direct transmission (when an infected person sneezes, mucus
19. The current sensitivity of RT-PCR detection is limited, and goes directly into another person’s eyes, nose or mouth); air-
chest CT may have some sensitivity but less specificity. borne routes (when someone inhales an aerosol produced by
an infected person coughing, sneezing, or spitting time), and
transmitted by hand–eye, hand–nose, or hand–mouth, or
38.6.2 Pathogenesis through a contaminated surface or direct personal contact,
such as a handshake. A small amount of literature has con-
38.6.2.1 Transmission firmed that the real-time quantitative PCR of 2019-nCoV in
The sources of infection seen so far are mainly patients with feces of patients with new coronavirus is positive, indicating
new coronavirus infections. Asymptomatic infections can that their feces may also be contagious. The relative impor-
also be the source of infection. When an infected person tance of these modes of transmission is unknown, and they
626 L. Ming et al.
may all contribute to the spread of the virus. During airborne vated after being heated to 56 °C (133 °F) for at least 30 min.
transmission, a single positive RN A genomic droplet with a Ethyl ether, 75% ethyl alcohol, chlorine-containing disinfec-
diameter of 50–200 nanometers, inhalation of only one drop- tant, peracetic acid, chloroform, and other lipid solvents can
let is sufficient to cause infection. Although a sneeze can effectively inactivate the virus, but chlorhexidine cannot
release as many as 40,000 drops of droplets, most droplets are effectively inactivate the virus.
large and will soon settle in the air. In a relatively closed envi-
ronment, prolonged exposure to high concentrations of aero- 38.6.2.2 Pathology and Physiology
sols may cause aerosol transmission. The time that 2019-nCoV Currently, the pathophysiology by which novel coronavirus
survives in the air appears to be affected by temperature lev- infections cause human symptoms is not fully elucidated,
els. Because the new coronavirus can survive in vitro, it can which is being studied. 2019-nCoV belongs to the subgenus
also spread through contaminated surfaces such as banknotes, Sarbecovirus. It is enveloped, round or elliptic in size, usu-
doorknobs, light switches, and other household items. The ally pleomorphic, and 60–140 nm in diameter. Its genetic
length of time that the virus survives on the surface varies, characteristics were significantly different from SARS-CoV
ranging from 1 to 2 days on hard, nonporous surfaces such as and MERS-CoV. Most of the proteins encoded by 2019-
plastic or metal, about 15 min on a dry paper tissue, and only nCoV, bat-SL-CoVZC45, and bat-SL-CoVZXC21 are simi-
5 min on the skin. However, if the virus is present in the lar in length, with only a few minor insertions or deletions.
mucus, this can protect it for longer. The understanding of the A significant difference is that the long-ear protein encoded
physical and chemical characteristics of coronavirus mainly by 2019-nCoV is compared to the bat SARS-like coronavi-
comes from the study of SARS-CoV and MERS-CoV. The ruses, SARS-CoV, and MERS-CoV (Fig. 38.20a). Through
virus is sensitive to ultraviolet light and heat. They are inacti- phylogenetic analysis of the receptor-binding domain of
a Non-structural polyprotein 1ab (7096 amino acids) Spike protein (1273 amino acids)
251 +C,13443 21541 21549 25730
TTTTTAAACCGGG
2019-nCoV E M 7 10b N
WH04 5’ 3’
29844 base pairs
3 8
9 1314
Non-structural polyprotein 1ab (7092 amino acids) Spike protein (1246 amino acids)
265 +C,13455 21543 21550 25290
MG772933 TTTTTAAACCGGG
E M 7 10b N
Bat_SL-CoVZC45
29802 base pairs 5’ 3’
3 8
3b 9 1314
265 +C,13389 21477 21484 25221
TTTTTAAACCGGG
MG772934 E M 7 10b N
Bat_SLCoVZXC21 5’ 3’
29732 base pairs
3a 8
3b 9 1314
265 +C,13398 21486 21493 25261
TTTTTAAACCGGG
E M 6 8b 9a
NC_004718.3
SARS-CoV 5’ 3’
29751 base pairs 3a 7a 9b
3b 7b 8a
279 +C,13398 21514 21705 25517
TTTTTAAACCGGG
NC_019843.3 4b 5 E N
MERS-CoV 5’ 3’
30119 base pairs 3 M
Fig. 38.20 Sequence comparison and genomic analysis of 2019-nCoV. (a) Coding regions of 2019-nCoV, bat-SL-CoVZC45, bat-SL-CoVZXC21,
SARS-CoV, and MERS-CoV. (b) Phylogenetic analysis of 2019-nCoV and other betacoronavirus genomes
38 Lung Disease 627
b 100 AY508724|SARS-CoV_NS-1
100 AY485277|SARS-CoV_Sino1-11
AY390556|SARS-CoV_G202|Guangzhou SARS-CoV Human
100 AY278489|SARS-CoV_GD01
KT444582|WIV16|Yunnan
100 KY417146|Rs4231|Yunnan
KY417151|Rs7327|Yunnan
100 KY417152|Rs9401|Yunnan
MK211376|BtRs-BetaCoV/YN2018B|Yunnan
KJ473816|BtRs-BetaCoV/YN2013|Yunnan
86 KY770859|Anlong-112|Guizhou
KY417145|Rf4092|Yunnan
100 MK211377|BtRs-BetaCoV/YN2018C|Yunnan
KY417142|As6526|Yunnan
100 KY417148|Rs424|Yunnan Bat-SL-CoV Bat
100 KJ473815|BtRs-BetaCoV/GX2013|Guangxi
JX993988|Yunnan2011
100 MK211374|BtRl-BetaCoV/SC2018|China
Clade 3 JX993987|Shaanxi2011
KJ473814|BtRs-BetaCoV/HuB2013|Hubei
DQ648857|Bat_CoV_279/2005|Hubei
100 GQ153547|HKU3-12|HK/2005
Q084200|HKU3-3|HK/2005
GQ153542|HKU3-7|HK/2007
Clade 2 KF294457|Longquan-140|China
WH04|2020-01-05
YS8011|2020-01-07
100 WH01|2019-12-26
WH03|2020-01-01
Clade 1 WH19002|2019-12-30
WH19008|2019-12-30 2019-nCoV Human
WH02|2019-12-31
Sarbecovirus
76 WH19001|2019-12-30
100 WH19004|2020-01-01
WH19005|2019-12-30
100 100 MG772933|bat-SL-CoVZC45|Zhejiang
100MG772934|bat-SL-CoVZXC21|Zhejiang Bat-SL-CoV Bat
KY352407|SARS-related_coronavirus|BtKY72|Kenya
100 100
Hibecovirus GU190215|Bat_coronavirus|BM48-31/BGR/2008|Bulgaria
KF636752|Bat_Hp-betacoronavirus/Zhejiang2013
Nobecovirus 100 KU762338|Rousettus_bat_coronavirus|GCCDC1_356|China
EF065513|Bat_coronavirus_HKU9-1|BF_0051|Guangdong
100 EF065509|Bat_coronavirus_HKU5-1|LMH03f|Guangdong
95 EF065505|Bat_coronavirus_HKU4-1|B04f|Guangdong
Merbecovirus 100
JX869059|Huntan_betacoronavirus_2c_EMC|Saudi Arabia MERS-CoV Human
KC545386|Beta_coronavirus_Erinaceas/VMC/DEU/2012|EricaceusCoV/2012-216/GER/2012|Germany
100 AY391777|Human_coronavirus_OC43|ATCC_VR-759|UK
Embecovirus 100 KM349744|Betacoronavirus_HKU24|HKU24-R05010|China Mouse
100 FJ647223|Murine_coronavirus_MHV-1|USA
MK167038|Human_coronavirus_HKU1|SC2521|USA Human
Fig. 38.20 (continued)
four different lineages of beta coronaviruses, although the cell serine protease TMPRSS2. The SARS-CoV S/ACE2
2019-nCoV was closer to bat-SL-CoVZC45 and bat-SL- interface has been clarified at the atomic level and it has been
CoVZXC21 at the whole-genome level, the receptor-bind- found that the use efficiency of ACE2 is a crucial determi-
ing domain of 2019-nCoV fell within lineage B and was nant of SARS-CoV’s ability to spread. The SARS-CoV S
closer to that of SARS-CoV (Fig. 38.20b). At present, the protein and 2019-nCoV S protein have approximately 76%
homology of SARS-coronavirus (bat-SL-CoVZC45) is sequence identity. However, it remains unclear whether the
more than 85% [100]. 2019-nCoV S protein uses ACE2 and TMPRSS2 to enter
Recently, scientists confirmed that the spike (S) protein of host cells, as the SARS-CoV S protein does.
coronavirus (referred to as S protein) helps the virus to enter In a new study, German researchers provide evidence that
the target cell. Cell entry depends on the binding of the sur- 2019-nCoV host cell entry is dependent on the SARS-CoV
face subunit S1 of the S protein to the cell receptor, which receptor ACE2 and can be blocked by clinically proven cell
helps the virus attach to the surface of the target cell. Also, serine protease TMPRSS2 inhibitors, and that TMPRSS2 is
cell entry requires activation of S protein priming by a cel- activated by 2019-nCoV for the S protein. In the lungs,
lular protease, which cleaved the protein at the S1/S2 and S2′ SARS-CoV mainly infects lung cells and macrophages.
sites of S and allowed the viral membrane to fuse with the However, the expression of ACE2 was not limited to the
membrane, a process driven by the S protein’s S2 subunit. lungs, and extrapulmonary spread of SARS-CoV in ACE2
SARS-CoV S protein enters the receptor with angiotensin- tissues was also observed. It has also been reported that
converting enzyme 2 (ACE2) and activates the S protein with 2019-nCoV can damage the central nervous system.
628 L. Ming et al.
It has been suggested that moderate expression of ACE2 in Wuhan (WHO). Chinese authorities made a preliminary
the upper respiratory tract may limit the spread of SARS- determination that the causative agent is a novel coronavirus
CoV. Given the increased potential transmissivity of (2019-nCoV). The origin of the zoonotic disease of the virus
COVID-19 relative to SARS, it may be speculated that this is unknown. Given its close similarity to bat coronaviruses, it
novel coronavirus may utilize cell attachment promotors is likely that bats are the primary reservoir for the virus [99].
more efficiently than SARS-CoV to ensure a robust infection It is essential to understand and will help define zoonotic
in ACE2 cells in the upper respiratory tract. This may include transmission patterns.
binding to cellular glycans, a function of the S1 subunit of According to recent reports, COVID-19 appears to be
some coronaviruses. Finally, it should be noted that ACE2 relatively mild compared to SARS and Middle East respira-
expression protects the lungs from damage and is downregu- tory syndrome. There is much to know about this infection.
lated by SARS-CoV S protein, which may contribute to the Most importantly, the extent of interpersonal transmission
respiratory disease SARS. and the extent of the clinical disease needs to be determined.
Convalescent SARS patients exhibit a neutralizing anti- Obviously, effective human-to-human transmission is a nec-
body response that can be detected even 24 months after essary condition for the large-scale transmission of this
infection, and this neutralizing antibody response focuses on emerging virus. However, the severity of the disease is a key
the SARS-CoV S protein. Also, experimental SARS vac- indirect factor in the ability of the virus to transmit and the
cines containing recombinant S proteins or inactivated ability to transmit the virus.
viruses induce neutralizing antibody responses. Although It appears that 2019-nCoV uses the same cellular receptor
verification experiments based on infectious viruses are as SARS-CoV (human angiotensin-converting enzyme 2
ongoing, the results of this new study suggest that the neu- [hACE2]) [104], so transmission is expected only after signs
tralizing antibody response to the SARS-CoV S protein may of lower respiratory tract disease develop. However, because
provide some protection against 2019-nCoV infection, infections do not cause serious illness, infected people often
which may have implications for outbreak control [104]. do not end up in the original health care center. Instead, they
The pathophysiology of unusually high pathogenicity for will go to work and travel, potentially spreading the virus to
SARS-CoV or MERS-CoV has also not been completely their contacts, resulting in a large-scale virus outbreak. Such
understood. Earlier studies have shown that increasing a as the following model: the change in receptor specificity
large amount of serum pro-inflammatory cytokines (such as necessary for effective human-to-human transmission of
IL1B, interleukin 6, IL12, IFNγ, IP10, and MCP1) is associ- avian influenza virus causes a downward movement to the
ated with lung inflammation and extensive lung injury in upper respiratory tract, thereby reducing the burden of dis-
patients with SARS. MERS-CoV infection has also been ease. Two major and recent examples are the H1N1 pan-
reported to induce increased concentrations of pro- demic virus and the H7N9 avian influenza virus. The H1N1
inflammatory cytokines (IFNγ, TNFα, IL15, IL17). We point pandemic influenza virus-caused by a relatively mild disease
out that patients infected with 2019-nCoV also have a large that binds to the upper respiratory tract receptors has become
amount of IL1B, IFNγ, IP10, MCP1, which may cause acti- epidemic in the population, and the H7N9 virus-bound to the
vation of T-helper-1 Th1 cell response. Besides, patients lower respiratory tract of the receptors, has a mortality rate
requiring ICU admission had higher GCSF concentrations, of about 40%. A few small clusters spread from person to
IP10, MCP1, MIP1A, and TNFα than those who did not person. Therefore, the severity of the disease is not necessar-
require ICU admission, suggesting that cytokine storms were ily related to the efficiency of transmission.
associated with disease severity. However, 2019-nCoV Even if a virus causes subclinical or mild disease in gen-
infection also initiated the secretion of T-helper-2 (Th2) eral, some people may be more susceptible and end up seek-
cytokines (such as IL4 and IL10) that inhibit inflammation, ing care. The majority of the novel coronavirus cases were
which is different from SARS-CoV infection. The associated with nosocomial transmission in hospitals, result-
characteristics of Th1 and Th2 responses in 2019-nCoV ing in at least in part from the use of aerosol-generating pro-

infection need to be further studied to elucidate its pathogen- cedures in patients with respiratory disease. In particular,
esis [105]. nosocomial super-spreader events appear to have driven
large outbreaks within and between health care settings. In
addition to the vulnerability of health care settings to out-
38.6.3 Epidemic and Pandemic Spread breaks of emerging coronaviruses, hospital populations are
at significantly increased risk for complications from the
Since mid-December 2019, several cases of a pneumonia- infection. Few Chinese medical staff has lost their lives as a
like disease (with symptoms including fever, difficulty in result. Age and coexisting conditions (such as diabetes or
breathing, cough, and invasive lesions on both lungs) of heart disease) are independent predictors of adverse out-
unknown causes have emerged in the central Chinese city of comes in COVID-19. Thus, emerging viruses that may go
38 Lung Disease 629
undetected because of a lack of severe disease in healthy lar standards will be crucial and, hopefully, successful in
people can pose a significant risk to vulnerable populations reducing the transmission of COVID-19 [107].
with underlying medical conditions. Currently, the number of people infected with 2019-nCoV
A lack of severe disease manifestations affects our ability continues to increase worldwide. As of August 19, 2020, the
to contain the spread of the virus. Identification of chains of cumulative number of patients was 23,717,157. The distribu-
transmission and subsequent contact tracing is much more tion of the infected population in the world is shown in the
complicated if many infected people remain asymptomatic following figure (Fig. 38.21).
or mildly symptomatic (assuming that these people can
transmit the virus). More pathogenic viruses that transmit
well between humans can generally be contained effectively 38.6.4 Course of the Disease
through syndromic (fever) surveillance and contact tracing,
as exemplified by SARS-CoV and, more recently, the Ebola Most patients presented with fever, dry cough, dyspnoea,
virus [106]. and bilateral ground glass opacities on chest CT scans. These
A distinctive feature of the SARS outbreak is that fear features of 2019-nCoV infection bear some resemblance to
plays an important role in economic and social consequences. SARS-CoV and MERS-CoV infections. However, few
Although specific anti-coronal virus therapies are still being patients with 2019-nCoV rhinorrhoea, sneezing, or sore
developed, we now know more about how to control such infection had prominent upper respiratory tract signs and
infections in communities and hospitals, which should alle- symptoms (e.g., throat), indicating that the target cells might
viate this fear. 2019-nCoV transmission may occur through be located in the lower airway. Furthermore, COVID-19
large droplets and contact, and based on our experience with patients rarely developed intestinal signs and symptoms
SARS-CoV and MERS-CoV, transmission through aerosols, (e.g., diarrhea), whereas about 20–25% of patients with
and pollutants may be less. Public health measures, includ- MERS-CoV or SARS-CoV infection had diarrhea. A few
ing community isolation, timely diagnosis, and strict adher- existing studies prove that the 2019-nCoV real-time quanti-
ence to universal preventive measures in health care facilities, tative PCR test results in feces of patients with new corona-
are essential to control SARS and MERS. Institution of simi- virus are positive, suggesting that their feces may also be
Fig. 38.21 People infected with 2019-nCoV worldwide as of August 19, 2020
630 L. Ming et al.
transfection. Fecal and urine samples should be tested to 4. Critical type

exclude a potential alternative route of transmission that is One of the following:
unknown at this stage [105]. (a) Respiratory failure occurs and requires mechanical
Based on the current epidemiological investigation, the ventilation.
incubation period is 1–14 days, mostly 3–7 days. The main (b) Shock occurs.
manifestations are fever, dry cough, and fatigue. A few (c) Combining other organ failures requires ICU moni-
patients have symptoms such as nasal congestion, runny toring and treatment.
nose, sore throat, myalgia, and diarrhea. Severe patients usu-
ally have dyspnea and/or hypoxemia one week after the
onset of symptoms, and severe patients can quickly progress 38.6.5 Diagnosis
to acute respiratory distress syndrome, septic shock, difficult
to correct metabolic acidosis, coagulation dysfunction, and In the absence of specific therapeutic drugs or vaccines for
multiple organ Functional failure, etc. It is worth noting that COVID-19, it is essential to detect the diseases at an early
in the course of severe and critically ill patients, there may be stage. The diagnosis of COVID-19 is primarily confirmed by
moderate to low fever, even without apparent fever. Mild the reverse transcription-polymerase chain reaction
patients showed only low fever, mild fatigue, and no pneu- (RT-PCR) or gene sequencing for respiratory or blood speci-
monia. Judging from the current cases, most patients have a mens, and Chest CT. However, with limitations of sample
good prognosis, and a few patients are critically ill. The fore- collection and transportation, and kit performance, the sensi-
cast for the elderly and those with the chronic underlying tivity of RT-PCR was reported low. In the current emergency,
disease is poor. Symptoms in children are relatively mild. Chest CT, as a routine imaging tool for pneumonia diagno-
sis, is relatively easy to perform and can produce a fast diag-
38.6.4.1 Clinical Classification nosis. In this context, the chest CT may provide benefits for
1. Mild type the determination of COVID-19. As recently reported, the
The clinical symptoms were mild, and no pneumonia current sensitivity of RT-PCR detection is limited, and chest
was manifested in imaging. CT may have some sensitivity.
2. Ordinary type
With fever, respiratory tract, and other symptoms, 38.6.5.1 B lood Routine and Biochemical
imaging show pneumonia. Indicators
3. Severe type In the early stage of the onset, the total number of white
Adults Meet any of the following criteria: blood cells in the peripheral blood was normal or decreased,
(a) Shortness of breath, RR ≥ 30 times/minute. and the lymphocyte count decreased. Some patients may
(b) In the resting state, the oxygen saturation is ≤93%. have increased liver enzymes, lactate dehydrogenase
(c) Arterial blood oxygen partial pressure (PaO2)/oxy- (LDH), muscle enzymes, and myoglobin; some critically ill
gen concentration (FiO2) ≤300 mmHg patients can see increased troponin. Most patients have
(1 mmHg = 0.133 kPa). elevated C-reactive protein (CRP) and erythrocyte sedi-
At high altitudes (above 1000 meters), PaO2 / FiO2 mentation rate and normal procalcitonin. In severe cases,
should be corrected according to the following formula: D-dimer increases, and peripheral blood lymphocytes pro-
PaO2/FiO2 x [Atmospheric pressure (mmHg) /760]. gressively decrease. Severe and critically ill patients often
Pulmonary imaging showed that the lesions pro- have elevated inflammatory factors. Lymphopenia is a
gressed significantly within 50–48 h. common feature in patients with COVID-19 and might be a
Children Meet any of the following criteria: critical factor associated with disease severity and
(a) Shortness of breath (<2 months old, RR ≥ 60 times/ mortality.
min;2–12 months old, RR ≥ 50 times/min; 1–5 years Furthermore, Plasma DNA is extracellular DNA present
old, RR ≥ 40 times/min; >5 years old, RR > 30 times/ in plasma. The concentration of plasma DNA in healthy peo-
min), excluding the influence of fever and crying. ple is less than 50 ng/mL. When trauma, infection, tumor,
(b) In the static state, the oxygen saturation is ≤92%. and other factors lead to tissue, and organ damage, DNA will
(c) Assisted breathing (groaning, wing flaps, triple con- be released from cells, and plasma DNA level will be signifi-
cave sign), cyanosis, intermittent apnea. cantly increased. Therefore, the plasma DNA level is an
(d) Refuse to feed or feed, with signs of dehydration. objective indicator of cell damage severity.
38 Lung Disease 631
38.6.5.2 Serological Detection of 2019-nCoV 2019-nCoV nucleic acid testing needs to be performed in
The serological test of 2019-nCoV used previously devel- a BSL-2 laboratory, Follow Standard Precautions when han-
oped nucleocapsid protein (N) from bats SARSr-CoV Rp3 as dling clinical specimens, all of which may contain poten-
antigen immunoglobulin and IgM enzyme-linked immuno- tially infectious materials. Standard Precautions include
sorbent assays (ELISAs), as this protein shared 92% amino hand hygiene and the use of personal protective equipment
acid identity to N protein of 2019-nCoV (Fig. 38.22) and (PPE), such as laboratory coats or gowns, gloves, eye protec-
showed no cross-reactivity against other human coronavi- tion, N95 masks, or protective screen.
ruses except SARSr-CoV7 [108]. Follow routine laboratory practices and procedures for
decontamination of work surfaces and management of labo-
38.6.5.3 Molecular Diagnostic Methods ratory waste.
Specimen Collection and Storage Detection of 2019-nCoV by Real-Time RT-PCR

According to the serial guidelines from NHC (National The specimens were first bathed in a water or metal bath at
Health Commission) and expert consensus from CSLM 56 °C for 45 min to achieve the effect of inactivating the
(Chinese Society of Laboratory Medicine), qualitative detec- virus. Then total RNA was extracted using the respiratory
tion of nucleic acid from the suspected patients in upper and sample RNA isolation kit.
lower respiratory specimens (such as nasopharyngeal or oro- The suspension was used for real-time reverse
pharyngeal swabs, sputum, lower respiratory tract aspirates, transcription-polymerase chain reaction (RT-PCR) assay of
bronchoalveolar lavage, and nasopharyngeal wash/aspirate 2019-nCoV RNA. Two target genes, including open reading
or nasal aspirate) collected from individuals who meet local frame 1ab (ORF1ab) and nucleocapsid protein (N), were
criteria for 2019-nCoV testing is carried out. In China, most simultaneously amplified and tested during the real-time
clinical labs collected oropharyngeal swabs as screening RT-PCR assay. The sequence is shown in Table 38.14.
tests specimen for virus RNA extracting following the proce- The real-time RT-PCR assay was performed using a
dure (Fig. 38.23). Only critical patients were suggested to 2019-nCoV nucleic acid detection kit according to the man-
collect lower respiratory specimens. ufacturer’s protocol (Fig. 38.24). The reaction mixture con-
Testing is limited to qualified laboratories designated by tains 19 μL of reaction buffer, 1 μL of enzyme solution, and
the local government. Biosafety-2 level and PCR certificate 5 μL of RNA template. RT-PCR assay was performed under
of compliance are the minimum requirements. Testing with the following conditions: incubation at 50 °C for 20 min and
the 2019-nCoV RT-PCR diagnostic panel is intended for use 95 °C for 10 min, 40 cycles of denaturation at 95 °C for
by trained laboratory personnel who are proficient in per- 15 s, and extending and collecting fluorescence signal at
forming PCR assays. 60 °C for 30 s.
Detection methods and sequence information of primers
Biosafety Considerations and probes for rRT-PCR panels are available on the CDC
All laboratories should perform a site- and activity-specific laboratory information website at COVID-19. WHO Corona-
risk assessment to identify and mitigate risks. virus disease (COVID-19) technical guidance also includes
Fig. 38.22 Amino acid sequence alignment of the nucleocapsid protein of 2019-nCoV to bat SARS-CoV Rp3 and SARS-CoV BJ01
632 L. Ming et al.
Inactivated virus
56°C, 45min
Educated worker with proper PPE: cap, goggles or face shield,
medical protective mask, gloves and coat
Extract sample Reaction buffer Enzyme solution

RNA (19 µL) (1 µL)
Prepare rRT-PCR Prepare master

Help the suspected patient to relax and open his mouth plate (5 µL RNA) mix (20 µL)
Run assay on ABI

7500 Fast Dx
Collect OP from the posterior pharyngeal wall with a
synthetic tip
Analyze data
Place swabs immediately into sterile tubes containig 2-3 ml of Report results
viral transport medium
Fig. 38.24 Summary of Testing Process
current molecular diagnostic methods in various countries.

(https://www.who.int/emergencies/diseases/novel-
coronavirus-2019/technical-guidance/laboratory-guidance).
Specimens should be packaged and transported to the lab
immediately
Assay Control Addition
All controls listed below must generate expected results for a
test to be considered valid:
1. Human Specimen Control (HSC): A human housekeep-

ing gene is used as reference genes in real-time PCR,
Specimens can be stored at 2-8°C for up to 72 hours or stored
at -70° C for a long period of time.
monitors for specimen preparation, and the entire process
of detection.
2. Positive Control: Run with each batch of specimens.

Monitors for failures of rRT-PCR reagents and reaction
Fig. 38.23 Flowchart of oropharyngeal swabs collection and conditions.
transport
3. Blank Control (BC): Nuclease-free water included in each
run. Monitors for reagent and system contamination.
Table 38.14 Primers and probes for rRT-PCR panels
Target Sequence Interpretation of Results
Target1 Forward primer: CCCTGTGGGTTTTACACTTAA 1. Threshold setting: Above the maximum level of Blank
(ORF1ab) Reverse primer: ACGATTGTGCATCAGCTGA Control.
Probe: 5′-VIC-CCGTCTGCGGTATGTGGAAAGG 2. Quality control: Before evaluating the specimen results,
TTATGG-BHQ1–3′
the Positive Control and Blank Control should be inter-
Target 2 Forward primer: GGGGAACTTCTCCTGCTAGAAT
(N) Reverse primer: CAGACATTTTGCTCTCAAGCTG preted. Negative control should be undetected. Positive
Probe: 5′-FAM- TTGCTGCTGCTTGACAGATT- control should be detected with Ct ≤ 32 and HSC should
TAMRA-3′ be detected.
38 Lung Disease 633
3. If the Positive Control, Blank Control, and HSC do not Detection of 2019-nCoV by Genetic Sequencing
meet the criteria, the entire run is invalid, and results On January 7, 2020, Chinese researchers shared the full
should not be reported. Repeat the entire process (speci- genetic sequence of 2019-nCoV through the National
men and control preparation, amplification, and Institutes of Health Gene Bank database and the Global
detection). Initiative on Sharing All Influenza Data (GISAID) database;
4. Specimen: A cycle threshold value (Ct-value) of 40 or a report about the isolation of 2019-nCoV was later pub-
less was defined as a positive test result, and a Ct-value of lished. As of 2020-03-23, there are a total of 1022 full-length
more than 40 was identified as a negative test [109]. virus sequences, of which 760 are of high quality. Moreover,
there are 101 full-length 2019-nCoV sequences in GenBank
The real-time RT-PCR test is intended for the qualitative and no SNP data are displayed (https://www.ncbi.nlm.nih.
detection of the SARS-CoV-2 RNA in nasopharyngeal and gov/genbank/sars-cov-2-seqs/). Based on the available high-
oropharyngeal swab specimens from individuals suspected quality new coronavirus genome sequence variations, the
of COVID-19 by their healthcare provider. world and China’s nucleotide haplotype maps were con-
Results are for the identification of SARS-CoV-2 RNA. The structed, respectively (Fig. 38.26). Circles represent haplo-
SARS-CoV-2 RNA is generally detectable in the upper respi- types, and the distance between circles represents the number
ratory during the acute phase of infection. Positive results are of sequence mutations. As of 2020-03-17, the 700 strains of
indicative of the presence of SARS-CoV-2 RNA; clinical cor- the virus have been sampled, with a total of 438 haplotypes.
relation with patient history and other diagnostic information Nucleic acid was extracted from rRT-PCR-positive speci-
is necessary to determine patient infection status. Positive mens (oropharyngeal and nasopharyngeal) and used for
results do not rule out bacterial infection or coinfection with whole-genome sequencing on both Sanger and next-
other viruses. The agent detected may not be the definite cause generation sequencing platforms (Illumina and MinIon).
of disease. Laboratories are required to report all positive Sequence assembly was completed with the use of
results to the appropriate public health authorities. Negative Sequencher software, version 5.4.6 (Sanger), minimap soft-
results do not preclude SARS-CoV-2 infection and should not ware, and version2.17 (Minion), and free Bayes software,
be used as the sole basis for patient management decisions. version 1.B.1 (MiSeq). Complete genomes were compared
Negative results must be combined with clinical observations, with the available 2019-nCoV reference sequence (GenBank
patient history, and epidemiological information. accession number NC045512.2) [110].
The CodeCheck™ SARS-CoV-2 RT-PCR Kit is intended
for use by qualified and trained clinical laboratory personnel 38.6.5.4 Chest CT for COVID-19
specifically instructed and trained in the techniques of real- Multiple small patchy shadows and interstitial changes
time PCR and in vitro diagnostic procedures (Fig. 38.25). appeared early, and the extrapulmonary bands were obvious.
Furthermore, it develops multiple ground glass infiltration
and infiltrates in both lungs. In severe cases, pulmonary con-
solidation and pleural effusion are rare. Interstibial mono-
nuclear inflammatory infiltrates, dominated by lymphocytes,
and were seen in both lungs (Fig. 38.27). Multinucleated
syncytial cells with atypical enlarged pneumocytes charac-
terized by large nuclei, amphophilic granular cytoplasm, and
prominent nucleoli were identified in the interalveolar
spaces, showing viral cytopathic-like changes [111]. Chest
CT imaging cannot confirm the diagnosis. Further diagnosis
requires immunohistochemical staining showing that some
alveolar epithelial cells and macrophages are positive for
new coronavirus antigens. And RT-PCR is positive for the
novel coronavirus nucleic acids.
38.6.5.5 Clinically Diagnosis

This designation is being used in Hubei Province only; in
these cases, no test was performed, but the diagnosis was
made based on symptoms, exposures, and presence of lung
imaging features consistent with coronavirus pneumonia.
Fig. 38.25 CodeCheck™ SARS-CoV-2 RT-PCR Kit

634 L. Ming et al.
Fig. 38.26 2019-nCoV
genome haplotype network
38.6.6 Prevention and Treatment fection and isolation work; take practical measures to ensure
the implementation of disinfection and isolation measures.
38.6.6.1 Prevention All surfaces and floors of the outpatient clinic and the ward
should be cleaned. When contaminated with pathogenic
Diagnosis and Isolation microorganisms, they should be cleaned before disinfection.
Supportive care for patients is typically the standard protocol The cleaning and disinfection work is divided into clean
because no specific effective antiviral therapies have been areas, semi-polluted areas, and contaminated areas. Suspect
identified. Therefore, early diagnosis of COVID-19 is crucial and confirmed patients of SARS should take isolation mea-
for disease treatment and control. However, due to inherent sures as soon as possible, and separate suspect and confirmed
difficulties in identifying and counting mild and asymptom- patients. When treating and caring for COVID-19 patients,
atic cases, many suspected and clinically diagnosed cases are medical personnel have to be in close contact and become a
still not isolated. These people are potential sources of infec- high-risk group of the disease. A poorly ventilated ward
tion. So the best way to control the novel coronavirus disease environment, critical condition of patients, and improper
is to isolate all individuals at home, which is what China is personal protection of medical personnel can increase the
doing now. risk of infection. Reduce occupational exposure of medical
COVID-19 leads to cross-infecting in hospitals. Ensuring personnel and strengthen personal protection.
the circulation of air is an important measure to control and COVID-19 patients often develop cytokine storms that
prevent COVID-19 infection in hospitals. At the same time, cause multiple organ failures. How to timely detect and
the air disinfection equipment with the sanitation license for intervene in the early stage is the key to improve prognosis.
disinfection products issued by the ministry of health shall As a sensitive indicator of body injury, the plasma DNA
be used for air disinfection. Attach importance to the disin- mentioned in the above diagnosis methods can sensitively
38 Lung Disease 635
Others
Always wash your face with cold water to enhance the ability
of the nasal mucosa to adapt to the air. Keep up with the
weather and stay warm according to the weather. At the same
time, strengthen physical exercise to enhance the ability to
adapt to the environment and the body’s immunity. In addi-
tion, try to avoid going to densely populated areas and
washing your hands frequently. When the body feels a little
discomfort, mild dry mouth, take medicine immediately
when nasal congestion, drink plenty of water, pay attention to
keep warm, and rest so that the condition improves in time.
The air conditioner should be cleaned before use to pre-
vent a large number of germs from blowing out with the
wind; the room temperature should be controlled above
24 °C, and the temperature difference between indoor and
outdoor should not exceed 7 °C to avoid burdening the body
temperature adjustment center; pay attention to the air condi-
tioner when sleeping, or do not blow your head on the fan.
Adequate nutrition should be added to increase the body’s
resistance. Eat fish, meat, eggs, milk, and beans appropri-
ately to supplement protein, eat more fresh fruits and vegeta-
bles to take in vitamin C, and eat more hot and humid foods,
such as bitter gourd, peach, cucumber, and mung bean.
Various places can use the appropriate amounts of Chinese
medicine to prevent according to the disease, local climate
characteristics, and different physical conditions.
Fig. 38.27 Transverse CT scans
Pay attention to hygiene: Hygiene should be taken care of
to prevent illness from entering the mouth. Wash your hands
reflect tissue injury and its severity. The plasma DNA quan- frequently, take a bath, change clothes, wash your hands fre-
tification can be used for the assessment of COVID-19 quently, and drop things frequently. The room is often venti-
patients’ condition, treatment efficacy monitoring, and prog- lated. Isolate individuals at home as much as possible if you
nosis judgment. go out and tend to keep a distance from other people, at least
Recently, the first affiliated hospital of Nanjing Medical above 1.5 m.
University and the First Affiliated Hospital of Guangzhou
Medical University cooperated to detect the plasma DNA 38.6.6.2 Treatment
concentration of 13 newly diagnosed COVID-19 cases (7 Unfortunately, no drug or vaccine has yet been approved to
cases of non-severe cases and 6 cases of severe cases). The treat human coronaviruses. The best thing we can do first is
results showed that the positive rate of plasma DNA in severe resting in bed, strengthening supportive treatment, ensuring
COVID-19 was 83.3% (5/6), while non-severe patients were adequate heat. Pay attention to the balance of water and elec-
negative. The prognosis of the severe patient with normal tricity to maintain the stability of the internal environment,
plasma DNA result was better than other severe patients. It etc. Several options can be envisaged to control or prevent
further suggests that the detection of plasma DNA can be emerging infections of COVID-19, including vaccines,
used for the diagnosis and prognosis of COVID-19. monoclonal antibodies, oligonucleotide-based therapies,
peptides, interferon therapies, and small-molecule drugs.
Improve Your Immunity However, new interventions are likely to require months to
You can improve your body’s immunity and resistance to years to develop. Given the urgency of the COVID-19 out-
viruses by exercising it. Normal work, life, and study should break, treatment programs mainly focus on the potential to
be combined with work and rest. Excessive fatigue leads to repurpose existing antiviral agents approved or in develop-
decreased resistance, which is extremely vulnerable to ment for treating infections caused by various viruses, based
COVID-19. Strengthen nutrition, a balanced diet, and the on therapeutic experience with two other diseases caused by
diet should be light, eat more vegetables and fruits rich in human coronaviruses: severe acute respiratory syndrome
vitamins; children should not eat cold drinks. (SARS) and the Middle East respiratory syndrome (MERS).
636 L. Ming et al.
2019-nCoV is an enveloped, positive-sense, single- structural proteins (such as spike glycoprotein), and acces-
stranded RNA beta-coronavirus. Similar to SARS and sory proteins (Fig. 38.28).
MERS, the 2019-nCoV genome encodes nonstructural pro- The four nonstructural proteins mentioned above are vital
teins (such as 3-chymotrypsin-like protease, papain-like pro- enzymes in the viral life cycle, and the spike glycoprotein is
tease, helicase, and RNA-dependent RNA polymerase), indispensable for virus–cell receptor interactions during
Fig. 38.28 Potential drug

targets for the novel a
coronavirus. (a) Gemonic
organization of 2019-nCoV;
(b) Drug binding pocket and
drugs
b
38 Lung Disease 637
viral entry. These five proteins were therefore recognized as Auxiliary check The physical examination revealed a body
attractive targets to develop antiviral agents against SARS temperature of 37.2 °C, blood pressure of 134/87 mm Hg, a
and MERS. pulse of 110 beats per minute, respiratory rate of 16 breaths
Initial analyses of genomic sequences from 2019-nCoV per minute, and oxygen saturation of 96% while the patient
indicate that the catalytic sites of the four 2019-nCoV was breathing ambient air. Lung auscultation revealed rhon-
enzymes that could represent antiviral targets are highly con- chi, and chest radiography was performed, which was
served, and share a high level of sequence similarity with the reported as showing no abnormalities. A rapid nucleic acid
corresponding SARS and MERS enzymes. Furthermore, amplification test (NAAT) for influenza A and B was nega-
protein structural analyses suggest that critical drug-binding tive. A nasopharyngeal swab specimen was obtained and
pockets in viral enzymes are probably conserved across sent for detection of viral respiratory pathogens by NAAT;
2019-nCoV, SARS, and MERS. It is, therefore, reasonable to this was reported back within 48 h as negative for all patho-
consider repurposing existing MERS and SARS inhibitors gens tested, including influenza A and B, parainfluenza,
for 2019-nCoV. respiratory syncytial virus, rhinovirus, adenovirus, and four
common coronavirus strains known to cause illness in
Virally targeted agents Nucleoside analogs that have been humans (HKU1, NL63, 229E, and OC43) [110].
approved and are under development may have the potential
to treat new coronaviruses. They include favipiravir, ribavi- Specimens were collected in accordance with CDC guid-
rin, remdesivir, and galidesivir. Nucleoside analogues are ance and included serum and nasopharyngeal and oropha-
usually derivatives of adenine or guanine. They can be used ryngeal swab specimens. After specimen collection, the
by RdRP to synthesize RNA strands, but their integration patient was discharged to home isolation with active moni-
into the RNA strand will prevent the further synthesis of toring by the local health department. One day later, the
RNA strands so that the synthesis of RNA strands will be CDC confirmed that the patient’s nasopharyngeal and oro-
terminated in advance. They can be used to treat broad- pharyngeal swabs tested positive for 2019-nCoV by real-
spectrum RNA viruses, including coronaviruses. time reverse-transcriptase-polymerase-chain-reaction
(rRT-PCR) assay.
Host-targeted agents Pegylated interferon-alpha-2a and After hospitalization, a sample of this stool was collected
alpha-2b have been approved for the treatment of hepatitis b for rRT-PCR testing, along with additional respiratory speci-
and hepatitis c virus infections. They may be used to stimu- mens (nasopharyngeal and oropharyngeal) and serum. The
late the innate antiviral response in patients with new corona- stool and both respiratory specimens later tested positive by
virus infection. Clinical trials have been initiated to examine rRT-PCR for 2019-nCoV, whereas the serum remained nega-
the efficacy and safety of a combination therapy of pegylated tive. A fourth chest radiograph showed streaky basilar opaci-
interferon and ribavirin approved for HCV in patients with ties in both lungs, a finding consistent with atypical
new coronavirus infection. However, it is not clear whether pneumonia.
pegylated interferon and nucleoside analogs have synergistic
effects in the treatment of new coronavirus. Since subcutane- Treatment Supportive treatment consists of 650 mg of
ous injections of interferon are associated with a variety of acetaminophen every 4 h and 600 mg of ibuprofen every 6 h.
side effects, this combination requires close monitoring of Given the concern about pneumonia, treatment with vanco-
the patient and may require a lower dose or discontinuation mycin (a 1750-mg loading dose followed by 1 g adminis-
of treatment depending on the patient’s response. tered intravenously every 8 h) and cefepime (administered
intravenously every 8 h) was initiated. Treatment with intra-
venous remdesivir (a novel nucleotide analog prodrug in
38.6.7 Typical Medical Case development) was initiated due to the radiographic findings.
The next day, the patient’s clinical condition improved [110].
Clinical background The patient, male, 35 years old, with
a 4-day history of cough and subjective fever, presented to an
urgent care clinic in Snohomish County, Washington. He dis- References
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Blood Disorders
39
Zhuang Zuo, Cheng Cameron Yin, Lixia Zhang, Lin Wang,
and Zhen Ren
The blood is a type of body fluid that delivers necessary sub- Blood cancers occur upon the initiation of several mecha-
stances such as nutrients and oxygen and excludes metabolic nisms. The common blood cancers involve myeloid neo-
waste products. In vertebrates including humans, blood is plasms and lymphoid neoplasms. This chapter stocks up
composed of blood cells suspended in plasma. Plasma, information about hematologic disorders, coagulation disor-
which constitutes 55% of blood fluid, is mostly water and ders and blood cancer, methods of detection, and use of labo-
contains proteins, glucose, mineral ions, hormones, carbon ratory test results in recognizing and characterizing patterns
dioxide, and blood cells themselves. The blood cells which of blood diseases.
are mainly derived from hematopoietic stem cells in bone
marrow are composed of red blood cells (RBCs/erythro-
cytes), white blood cells (WBCs/leukocytes), and platelets. 39.1 Blood Cancer
RBCs are the most abundant cells among them which con-
tain hemoglobin, an iron-containing protein, to facilitate Zhuang Zuo and Cheng Cameron Yin
oxygen transport. Vertebrate blood is bright red when its
hemoglobin is oxygenated and dark red when it is deoxygen-
ated. WBCs have nuclei, which distinguishes them from 39.1.1 Overview
RBCs and platelets. WBCs can be divided into neutrophils,
eosinophils, basophils, lymphocytes, and monocytes accord- Hematopoietic neoplasms, also called blood cancers, affect
ing to their physical and functional characteristics. Platelets the production and function of blood cells in blood, bone
are derived from the megakaryocytes of the bone marrow, marrow, lymph nodes, and other hematopoietic organs. The
and then enter the circulation. Circulating inactivated plate- main types of hematopoieitc neoplasms are leukemia, lym-
lets are biconvex discoid structures, whereas activated plate- phoma, and myeloma. According to the origin of the tumor
lets have cell membrane projections covering their surfaces. cells, hematopoietic neoplasms can generally be categorized
Platelets are only found in mammal. as myeloid neoplasms or lymphoid neoplasms.
Blood performs many important functions within the The current classification of hematopoietic neoplasms
body, including: supplying oxygen to tissues; supplying combines hematologic, morphologic, immunophenotypic,
nutrients such as glucose, amino acids, and fatty acids; and molecular genetic data. Common molecular tests of
removing waste such as carbon dioxide, urea, or lactic acid; hematopoietic neoplasms include gene mutation analysis,
immunological functions; messenger functions, such as detection of gene fusions caused by translocations and inver-
transporting hormones and delivering signaling of tissue sions, and B/T-cell clonality assays. These tests can be used
damage; as well as regulation of core body temperature. for cancer diagnosis, classification, prognosis, best course of
treatment, and measurable minimal residual disease (MRD)
monitoring. With the increasing application of allogeneic
stem cell transplant in treating hematopoietic neoplasms, rou-
Z. Zuo (*) · C. C. Yin
Department of Hematopathology, The University of Texas MD tine molecular analysis of chimerism is critical to monitor the
Anderson Cancer Center, Houston, TX, USA levels of donor and recipient cells in patients after transplant.
e-mail: zzuo@mdanderson.org; cyin@mdanderson.org Gene mutational analysis is performed by Sanger
L. Zhang (*) · L. Wang · Z. Ren sequencing (single gene sequencing), pyrosequencing (for
Department of Laboratory Medicine, The First Affiliated Hospital cases with well-defined “hot-spot” point mutation in a single
of Nanjing Medical University, Nanjing, Jiangsu, gene), or next-generation sequencing (NGS, multiple gene

642 Z. Zuo et al.
panels). Qualitative or quantitative polymerase chain reac- coexist in the same patient. This phenomenon suggests a
tion (PCR) is the function to detect the chromosomal translo- role of these mutations in MDS pathogenesis.
cations and inversions. Array-based Comparative Genomic Mutations in genes encoding splicing factors have been
Hybridization (aCGH) is used for genome-wide screening to reported in 50–80% of MDS cases. SF3B1 mutations have
detect copy number variations. Fluorescence in situ hybrid- been shown to be associated with a favorable prognosis and
ization (FISH) is a reliable method for crucial diagnosis of are correlated with the existence of ring sideroblasts (RS),
nearly all leukemias with characteristic chromosomal trans- found in 70–90% of MDS-RS and MDS/MPN with RS and
locations/inversions since false-negative results can occur by thrombocytosis (MDS/MPN-RS-T). In contrast, mutations
PCR because of the heterogeneity of chromosomal break- in SRSF2 and U2AF1 are more common in aggressive sub-
points. Conventional karyotypic analysis is still routinely types of MDS such as refractory anemia with excess blasts
detection for all new cases of myeloid neoplasms. (RAEB). DNMT3A mutations in approximately 10% of
MDS patients, with R882H being the most common, and
predict increased risk of leukemic transformation and infe-
39.1.2 Myeloid Neoplasms rior overall survival. IDH1 and IDH2 mutations are seen in
approximately 5% of MDS cases, mostly occur at R132 in
Myeloid neoplasms are clonal disorders arising in hemato- IDH1 and R140 or R172 in IDH2, and are correlated with
poietic stem cells and consist of Philadelphia chromosome unfavorable clinical outcome. ASXL1 (mostly in exon 12),
(Ph)-positive chronic myeloid leukemia, Ph-negative myelo- RUNX1, and EZH2 mutations occur in 10–20%, 10–15%,
proliferative neoplasms (MPN), acute myeloid leukemias and 5–10% of MDS cases, respectively, and all of these three
(AML), and myelodysplastic/myeloproliferative neoplasms mutations have demonstrated a positive correlation with
(MDS/MPN). In the past, the diagnosis of myeloid neo- poor overall survival. Patients with ASXL1 and RUNX1
plasms was mainly based on peripheral blood (PB) cell mutations also have an increased risk of leukemic transfor-
count, bone marrow (BM) morphologic findings, immuno- mation. TP53 mutations are present in approximately 5–10%
phenotypic features, and cytogenetic data. With the advance of MDS cases, and the majority occurs in the DNA binding
of molecular technologies, in particular the emergence of domain leading to impaired function. TP53 mutation in MDS
NGS, molecular testing now plays an essential role in the confers an aggressive clinical course, resistance to chemo-
diagnosis, classification, risk stratification, MRD monitor- therapy and dismal prognosis.
ing, and target therapy in myeloid neoplasms. We focus on The most cost-effective molecular work-up at the initial
the introduction of important somatic mutations in myeloid diagnosis of MDS is targeted NGS using a panel that covers
neoplasms that have been incorporated into the revised 2016 at least all the aforementioned genes. Cases with a single
edition of World Health Organization (WHO) classification mutation may be followed with a standalone molecular test,
and National Comprehensive Cancer Network (NCCN) such as pyrosequencing and mutation-specific PCR for “hot-
guidelines, and recent advance in the role of molecular test- spot” mutations, or Sanger sequencing (not ideal for MRD
ing in the clinical care of patients with myeloid neoplasms. monitoring due to low sensitivity) for genes without well-
defined “hot-spot” mutations. Cases with two or more muta-
39.1.2.1 Myelodysplastic Syndromes tions may be followed by the same NGS test.
Myelodysplastic syndromes are a crowd of hematopoietic
neoplasms characterized by cytopenia(s) and morphologic 39.1.2.2 Acute Myeloid Leukemia
dysplasia in at least one lineage of myeloid, erythroid, and Acute myeloid leukemias are a group of genetically heterog-
megakaryocytic cells, leading to ineffective BM hematopoi- enous diseases characterized by clonal proliferation of
esis and an increased risk of progression to AML. Recurrent immature blasts of myeloid lineage and disruption of normal
somatic mutations in over 50 genes have been identified in hematopoiesis. Patients can present either as de novo AML
MDS patients, and approximately 90% of the patients carry- or as AML arising from MDS.
ing at least one mutation, with a median of 2–3 mutations in The latter is usually related to complex karyotype, more
each patient. The most common mutations affect genes somatic mutations, and has a poor prognosis. As in MDS,
RNA splicing (SF3B1, SRSF2, U2AF1, ZRSR2), DNA cytogenetic abnormalities and gene mutation profile play
methylation (TET2, DNMT3A, IDH1, IDH2) and chromatin such important roles in risk stratification that recurrent
modification (ASXL1, EZH2). Other molecular aberrations molecular genetic data has been increasingly incorporated in
occur in genes involved in transcription regulation (RUNX1, AML classification. The 2016 revised WHO schemes recog-
TP53), signaling transduction (FLT3, NRAS, KRAS), and nize the following AML subtypes: AML with t(8;21)
cohesin complexes (STAG2, RAD21). Mutations within the (q22;q22.1)/RUNX1-RUNX1T1, AML with inv.(16)
same functional pathway are usually mutually exclusive, (p13.1q22) or t(16;16)(p13.1;q22)/CBFB-MYH11, acute
and mutations from different functional pathways whereas promyelocytic leukemia with t(15;17)(q22;q21)/PML-
39 Blood Disorders 643
RARA, AML with t(9;11)(p21.3;q23.3)/KMT2A-MLLT3, In addition to the above disease-defining mutations, lots
AML with t(6;9)(p23;q34.1)/DEK-NUP214, AML with inv. of other somatic mutations have been identified in AML with
(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2-MECOM, prognostic significance, such as genes in signal transduction
AML (megakaryoblastic) with t(1;22)(p13.3;q13.1)/RBM15- pathways. FLT3 mutations are the most common known
MKL1 and AML with BCR-ABL1. Most entities included in abnormality in AML with a diploid karyotype. There are two
this category involve balanced chromosomal translocations primary types of mutations: ITD in the juxtamembrane
or inversions activating transcription factors or signal trans- region (20–30% of AML) and point mutation in codon 835
duction pathways. The first three entities do not require a or 836 in the tyrosine kinase domain (TKD) (5–10% of
blast count of 20% in BM or PB for a diagnosis of AML. They AML). Both types of mutations lead to ligand-independent
are more common in young adults and are usually related to signaling. FLT3 ITD confers significantly inferior prognosis
a favorable prognosis. The gene fusions as a result of these in AML, and the outcome is proportional to the ratio of
translocations and inversions can be detected by karyotypic mutant to wild type allele. The prognostic significance of the
analysis, FISH or RT-PCR. It is of note that some of these point mutation in TKD remains controversial, although it is
abnormalities may be missed by conventional karyotyping, suggested by a few studies to be associated with poor clinical
especially inv.(16)(p13q22). Therefore, it is critical to screen outcome as well. FLT3 ITD can be detected by PCR/capil-
new patients with FISH and/or quantitative RT-PCR. In addi- lary electrophoresis or Sanger sequencing. The point muta-
tion, the RUNX1-RUNX1T1 transcript may persist following tion at codons 835 and 836 destroys an EcoRV restriction
therapy leading to the limited utility of qualitative RT-PCR site which can be used to detect these point mutations. KRAS
in MRD assessment. and NRAS mutations are seen in 5–10% of AML overall, and
Cytogenetic abnormalities can be detected in only are more common in AML with monocytic differentiation
50–60% of AML cases. Among AML patients with normal (30–40%). KRAS/NRAS mutations almost always occur at
karyotype, molecular screening has identified gene muta- codons 12, 13, and 61 that lead to constitutive activation of
tions in approximately 90% of cases. Three distinct entities RAS/RAF/MAPK signaling pathway, and they can best be
characterized by somatic gene mutations are recognized in detected by pyrosequencing. No significant prognostic
the current WHO classification: AML with NPM1 muta- impact of RAS mutation has been established, though some
tion, AML with biallelic mutation of CEBPA, and AML studies show that patients with RAS mutations may benefit
with mutated RUNX1. NPM1 mutations occur in 25–35% from high dose cytarabine consolidation. KIT mutations are
of AML overall and are associated with normal karyotype, seen more frequently in core binding factor in AML (20–
high frequency of FLT3 internal tandem duplication (ITD) 30%); and mainly consist of missense mutations in exon 17
and good response to induction chemotherapy. NPM1 (most commonly codon 816) in the kinase domain. KIT
mutations have been associated with a favorable clinical mutations confer KIT phosphorylation and constitutive acti-
outcome if the FLT3 ITD is absent. NPM1 mutations typi- vation of downstream effectors including STAT3, MAPK,
cally consist of insertions in exon 12 that lead to frameshift, and PI3K. KIT mutations override the good prognosis of core
with a duplication of TCTG at position 956–959 being the binding factor in AML. The D816V mutation can best be
most common, seen in 75–80% of cases. NPM1 mutations detected by mutation-specific PCR.
can be detected by PCR/capillary electrophoresis or Sanger Similar to MDS, genes involved in DNA methylation
sequencing. In addition, NPM1 mutations create a nuclear (TET2, DNMT3A, IDH1, IDH2), chromatin modification
export signal that mediates aberrant localization of NPM1 (ASXL1, EZH2), transcription regulation (TP53), and RNA
protein from the nucleus to the cytoplasm. As a result, splicing (SF3B1, SRSF2, U2AF1, ZRSR2) are frequently
immunohistochemistry to detect cytoplasmic expression of mutated in AML. No conclusive prognostic impact of TET2
NPM1 may be used as a surrogate for NPM1 mutations. mutation has been established; however, mutations in
CEBPA mutations are seen in 5–15% of AML and play a DNMT3A, IDH1, IDH2, ASXL1, EZH2, TP53, and U2AF1
role in blocking myeloid differentiation. N-terminal muta- have all been associated with inferior clinical outcome.
tions are usually nonsense mutations leading to expression The recommended algorithm for molecular testing in
of truncated CEBPA, whereas C-terminal mutations are AML include a screening quantitative multiplex RT-PCR for
usually in-frame mutations resulting in mutant CEBPA assessment of the more important recurrent translocations/
with decreased DNA binding capability; both are best inversions, such as t(8;21), inv(16)/t(16;16), t(15;17), t(9;22),
detected by Sanger sequencing. Biallelic, but not monoal- and targeted NGS using a panel of genes similar to that men-
lelic, somatic mutations in RUNX1 have been described in tioned for MDS. Any positive RT-PCR result should be fol-
10–20% of AML cases. RUNX1 mutations disrupt myeloid lowed by a quantitative real-time RT-PCR assay. Cases with
stem cell differentiation and are associated with unfavor- specific translocations/inversions can be best monitored with
able clinical outcome. RUNX1 mutations can be detected quantitative real-time PCR. Similarly, cases with a single
by Sanger sequencing. mutation can be followed by a standalone test, whereas cases
644 Z. Zuo et al.
with two or more mutations can be followed most cost- ment of CCyR and transplantation. qRT-PCR is recommended
effectively with NGS. NPM1 has been shown as the most at initial diagnosis, every 3 months till achievement of MMR
stable molecular marker for MRD monitoring. On the con- and every 6 months thereafter. At any time during the course
trary, FLT3 ITD is less stable and its role in MRD monitoring of therapy, an increased transcription of BCR-ABL1 fusion
has been in debate and needs to be used with caution. requires more frequent monitoring. Of note, neither FISH
Detection of certain mutations also allows for the selection nor qRT-PCR can detect cytogenetic abnormalities other
of AML patients for targeted therapy. FLT3 inhibitors have than t(9;22). Therefore, periodic karyotypic analysis is
been approved as a frontline therapy for FLT3-mutated AML essential for evidence of clonal evolution.
and have significantly improved the clinical outcome of this ABL1 kinase domain mutation is the most common
aggressive subtype of AML. Small molecular inhibitors for mechanism of resistance to TKIs, and has been reported in
IDH1 and IDH2 have been approved for relapsed/refractory 40–90% of cases with TKI resistance. More than 50 differ-
IDH1/IDH2-mutated AML and have demonstrated clear ent ABL1 point mutations have been identified, and different
clinical benefit. mutations vary in the resistance profile to TKIs, for exam-
ple, T315I confers resistance to both of the first and second
39.1.2.3 Chronic Myeloid Leukemia generation TKIs. Therefore, both the presence and type of
Chronic myeloid leukemia (CML) is a myeloproliferative point mutations may affect treatment decision. ABL1 muta-
neoplasm. It is molecularly defined by the appearance of the tions can be detected by several technologies. Sanger
Philadelphia chromosome (Ph) resulting from t (9;22) sequencing, though with a low sensitivity of 20%, is still the
(q34;q11.2)/BCR-ABL1 fusion. The chimeric BCR-ABL1 mainstay in the initial assessment of ABL1 mutation. More
fusion protein, which has constitutive tyrosine kinase activ- sensitive assays such as pyrosequencing (sensitivity 5%)
ity, plays an essential role in the pathogenesis of CML and and mutation-specific PCR (sensitivity 0.01–0.001%) may
the targeted therapy of tyrosine kinase inhibitors (TKIs). The be used after identifying a mutation by Sanger sequencing.
t (9;22) (q34;q11.2) can be detected by karyotypic analysis Targeted NGS can detect mutations in ABL1 as well as other
in ~95% of the cases at initial diagnosis. Rare cases carry genes.
cryptic translocation that can be recognized by FISH or
RT-PCR. Upon progression to accelerated or blast phase, the 39.1.2.4 Myeloproliferative Neoplasms
neoplastic cells usually acquire additional cytogenetic abnor- Myeloproliferative neoplasms (MPNs) are characterized by
malities, most often trisomy 8, a second copy of Ph, isochro- proliferation of at least one hematopoietic lineage with mini-
mosome 17q and trisomy 19, as well as mutations in ABL1 mal defects in maturation. Most MPNs are associated with
kinase domain leading to resistance to TKIs. Close monitor- well-defined molecular abnormalities involving genes that
ing the transcription level of BCR-ABL1 fusion and early encode protein tyrosine kinases that constitutively activate
detection of ABL1 mutation is crucial for timely therapeutic JAK-STAT signaling pathway. The three cardinal and mutu-
intervention. ally exclusive mutations, JAK2, CALR, and MPL, are referred
Cytogenetic response has been regarded as a major prog- to as “drive mutations” due to their role in driving the MPN
nostic factor for overall survival. Early achievement of com- phenotype. JAK2 V617F has been identified in 95–97% of
plete cytogenetic response (CCyR) within 12 months polycythemia vera (PV) and 50–60% of essential thrombo-
decreases the chance of disease progression. Although cythemia (ET) and primary myelofibrosis (PMF). High allele
karyotype analysis is less sensitive (sensitivity 5%), it is the burden of JAK2 V617F is related to an increased risk of
only method that can detect additional cytogenetic abnor- fibrosis and thrombosis. The remaining 3–5% of PV cases
malities (clonal evolution). It is recommended at initial diag- may carry mutations in JAK2 exon 12, or rarely, SH2B3 and
nosis, every 6 months till achievement of CCyR and then CBL. CALR mutations occur in the majority of JAK2-
once a year. FISH analysis with a quick turn-around time is negative ET and PMF. Two common types of CALR frame-
more sensitive (sensitivity 0.5%), which can be done on shift mutations in exon 9 have been reported: type 1 (52 bp
interphase cells and detects cryptic translocation. It is par- deletion) and type 2 (5 bp insertion); both resulting in mutant
ticularly useful when BM metaphase is unavailable. It is rec- protein which lose ER-retention motif (KDEL) at the
ommended at initial diagnosis, every 6–12 months until C-terminus. It has been shown that type 1 and type 1-like
CCyR is achieved. Timely achievement of major (MMR) CALR mutations predict favorable overall survival in
and complete (CMR) molecular response is related to more PMF. Missense mutations at codon 515 of MPL gene repre-
sustained cytogenetic responses. Quantitative real-time sent the least common driver mutation in MPNs, occurring in
RT-PCR has been regarded as the “gold standard” for the ~5% of ET and PMF. “Triple negative” accounting for ~10%
quantitative detection of BCR-ABL1 fusion. With a sensitiv- of MPNs cases, refer to patients with MPNs who do not har-
ity of 0.001%, it allows for monitoring MRD after achieve- bor any of the three aforementioned mutations.
Genome-wide analysis by NGS has identified co- 39.1.2.5 Myelodysplastic/Myeloproliferative

operating mutations which have benefits in the therapy and Neoplasms
prognosis of MPNs. Unlike driver mutations specific to Myelodysplastic/myeloproliferative neoplasms are charac-
MPNs, the co-operating mutations also occur in other hema- terized by both MDS and MPN, including the following enti-
topoietic and non-hematopoietic neoplasms, and include the ties: chronic myelomonocytic leukemia (CMML), chronic
same groups of mutations that are usually observed in other myelomonocytic leukemia (CMML), juvenile myelomono-
myeloid neoplasms, namely genes involved in DNA meth- cytic leukemia (JMML), MDS/MPN with ring sideroblasts
ylation (TET2, DNMT3A, IDH1, IDH2), histone modifica- and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN,
tion (ASXL1, EZH2), RNA splicing (SRSF2, U2AF1, ZRSR2, unclassifiable (MDS/MPN-U). The mutant profile of each
SF3B1), transcription regulation (RUNX1, TP53, CEBPA, entity is unique, and it is recommended to use a general tar-
ETV6, SETBP1), and signal transduction (KRAS, NRAS, KIT, geted NGS panel for molecular detection for initial evalua-
CBL, SH2B3, NF1). They contribute to disease progression, tion and/or subsequent monitoring.
clonal evolution, and the modification of disease phenotype. Somatic mutations have been found in ~90% of patients
A high-molecular risk (HMR) group defined by five of these with CMML. ASXL1 (30–40%), SRSF2 (40–50%), and TET2
genes (ASXL1, EZH2, SRSF2, IDH1, and IDH2), is a part of (50–60%) are the most common mutated gene, and the
prognostic scoring systems for patients with PMF, such as simultaneous mutations of TET2 and SRSF2 seems to be
MIPSS70. And a comprehensive mutation survey is recom- highly specific for CMML. Mutations affecting signaling
mended for this group of patients in search of a marker of pathways, such as KRAS, NRAS, JAK2, and CBL, are more
clonality. associated with the myeloproliferative group, whereas muta-
Another MPN with well-established molecular abnormal- tions affecting splicing factors, for example SF3B1 and
ity is chronic neutrophilic leukemia (CNL), characterized by U2AF1, are more familiar with group of myeloproliferative
persistent absolute neutrophilia. It has been shown that disorders. Other mutations with a relatively lower frequency
approximately 80–90% of CNL patients harbor mutations in involve RUNX1, SETBP1, EZH2, DNMT3A, IDH1, and
CSF3R. The majority of cases harbor a point mutation in the IDH2. The prognostic significance of TET2 mutation is
membrane proximal region (with T618 in exon 14 being uncertain, though it has been connected with favorable prog-
most common) which induces JAK-STAT pathway activa- nosis in the absence of ASXL1 mutation. Mutations in the
tion and is sensitive to ruxolitinib. On the contrast, a small rest of the aforementioned genes have all been identified as
subset of patients carry frameshift or nonsense mutations in independent poor prognostic factors of poor prognosis.
exon 17 which activate SRC pathway through mediating the Atypical chronic myeloid leukemia is a Ph-negative
truncation of the cytoplasmic tail and are sensitive to MDS/MPN. It is an exclusive diagnosis and the diagnosis of
dasatinib. it needs to rule out BCR-ABL1-rearranged CML, ET or PMF,
Screening for the presence of BCR-ABL1 fusion transcript JAK2/CALR/MPL-mutated PV and myeloid neoplasms with
and three driver mutations is recommended as the initial eosinophilia and PDGFRA/PDGFRB/FGFR1 rearrange-
work-up for all suspected MPNs. Depending on the clinical ment or PCM1-JAK2 fusion. In aCML (20–30%), SETBP1
settings, the algorithm for diagnosis of PV, ET and PMF can has been shown to be the most frequently mutated gene, and
be determined by sequential testing of JAK2 V617F and fol- its mutations usually occur together with ASXL1 mutations;
lowing testings of JAK2 exon 12 (for suspected PV or CALR) both predict inferior clinical outcome. CSF3R mutations
and MPL (for suspected ET and PMF), or by targeted NGS were initially reported in 45% of aCML; Nevertheless, in
using a panel of genes including the three drivers and com- following-up studies, it has been found that CSF3R muta-
mon co-operating mutations. For follow-up of cases with tions occured mainly in CNL (80–90%) but rarely in aCML
sole driver mutations, several methods can be used to detect (<10%). Due to the high existence of CSF3R mutations in
JAK2 V617F, including pyrosequencing (sensitivity 1%) or CNL, the diagnosis of aCML requires caution, in the exis-
mutation-specific qPCR or digital droplet (ddPCR) (sensitiv- tence of a CSF3R mutation. More recently, ETNK1 muta-
ity 0.01–0.001%). Sanger sequencing, with a sensitivity of tions have been reported in ~10% of aCML cases, as wells as
only 20%, is not recommended for JAK2 V617F follow-up. 3–14% of chronic myelomonocytic leukemia and 6% sys-
CALR mutation can be detected by PCR/capillary electro- temic mastocytosis. The clinical significance of these muta-
phoresis. MPL W515L/K can be detected using similar tions awaits further exploration.
approach as that used for JAK2 V617F. Ruxolitinib, a tyro- The feature of Juvenile myelomonocytic leukemia is the
sine kinase inhibitor for JAK1 and JAK2, has been shown to activation of Ras signaling pathway. Genetic mutations in
be beneficial in improving overall survival when applied to Ras pathway have been identified in nearly 90% of JMML
PMF patients. However, the modest reduction in mutant cases, including NRAS, PTPN11, KRAS, CBL, and NF1.
JAK2 allele burden restricts the role of JAK2 V617F in MRD These mutations can occur in the germline (NF1, CBL) or as
monitoring. somatic (PTPN11, NRAS, KRAS) lesions. JMML with
646 Z. Zuo et al.
PTPN11 mutation is rapidly fatal without allogeneic stem mias/lymphomas, mature B-cell leukemias/lymphomas and
cell transplantation. JMML with NRAS, KRAS or NF1 muta- acute lymphoblastic leukemias/lymphomas. According to
tion also has an aggressive clinical course. In the contrast, the distinctive immunophenotypes of B-cell differentiation
most children with CBL-mutated JMML have self-limiting stages, these tumors are typically diagnosed and classified.
disease. Furthermore, it has recently been discovered that T-cell tumors also contain acute lymphoblastic and mature
DNA methylation status is closely related to prognosis of lymphoid neoplasms. Mature T-cell malignant tumors have
JMML patients, with high methylation group more enriched much more sophisticated pathogenesis and clinical behavior.
in patients with PTPN11 mutations and worst prognosis, A subset of T-cell neoplasms are associated with viral infec-
whereas low methylation group comprising more children tions, for example, human T-cell leukemia virus (HTLV-1) in
with NRAS or CBL mutations and favorable outcome. adult T-cell leukemia/lymphoma and Epstein–Barr virus
Myelodysplastic/myeloproliferative neoplasms with ring (EBV) in NK-cell leukemias.
sideroblasts and thrombocytosis are characterized by the pres- The B-cell and T-cell clonality tests are the most common
ence of ≥15% ring sideroblasts and the count of platelets category of molecular testing in lymphoid malignancies. The
≥450 × 109/L. Over 80% of patients with MDS/MPN-RS-T process of recombination in B-cells and T-cells is the molec-
have mutations in SF3B1, and are often seen with concurrent ular basis for clonal detection, including the variable (V),
JAK2 V617F mutation (50-60%), or with CALR or MPL muta- diversity (D), and joining (J) segments of DNA in the immu-
tion (<5%) less frequently. Additional mutations include noglobulin heavy chain (IGH) and the T-cell receptor (TCR)
SETBP1 (10%), DNMT3A (15%), ASXL1 (20%), and TET2 genes, respectively, provides the molecular basis of clonality
(25%). SF3B1 mutation is associated with longer overall sur- testing. Currently, the most commonly used methods are
vival, and JAK2 V617F also predicts a more favorable progno- PCR based assays to evaluate IGH gene rearrangement in
sis. Poor prognosis is associated with the presence of ASXL1 B-cell tumors or T-cell receptor beta (TCRB) gene rearrange-
and SETBP1 mutations are associated with poor prognosis. ment and T-cell receptor gamma (TCRG) gene rearrange-
ment in T-cell tumors. The detection strategies and results of
oligoclonal, monoclonal, oligoclonal, and polyclonal B/T--
39.1.3 Lymphoid Neoplasms cell populations are shown in Fig. 39.1. In the IGH-VJ analy-
sis, all IGH J regions were identified by a consistent J primer,
Lymphoid neoplasms, including lymphoid leukemia, lym- and it was divided into three tubes to combine the forward
phoma, and myeloma, are produced by the proliferation and primers for the conservative frame region (FR)1, FR2, and
clonal expansion of B- and T-lymphocytes at various stages FR3. The polyclonal lymphocyte population is gaussian
of differentiation. In addition to morphologic evaluation, (normal) distribution because there are multiple PCR ampli-
immunophenotyping, cytogenetic profiling, gene mutation cons with different sizes that represent different recombina-
detection, and clonality analysis are usually used in the diag- tions of V(D)J fragments and the random nucleotide
nostic, prognostic analysis, and therapeutic stratifications of insertion/deletion at the ends of rearranging V, (D), and J
these patients. segments. By comparison, monoclonal B-cells produce
Most of the aggressive and indolent lymphoid neoplasms amplitons of the same size, and if there is a biallelic IGH
are from B-cell origin, including acute lymphoblastic leuke- rearrangement in the tumor clone, it appears as a single peak
Fig. 39.1 Lymphoid
clonality PCR assays for the IGH V D J TCRG V J TCRB V D J
CDRI/CDR2/CDR3
IGH, TCRG, and TCRB loci
Polyclonal
Oligoclonal
Monoclonal
with two different peaks. Considering the tendency of false become the standard of care for this disease. Genes com-
positive results and the differentiation of amplified oligoclo- monly found mutated in CLL are ATM, BIRC3, BTK,
nal and monoclonal patterns, especially in TCRG testing, it NOTCH1, XPO1, PLCG2, SF3B1, TP53. Interestingly, the
is more difficult to interpret TCR cloning by PCR. There are chromosomal location of the TP53 gene is at 17p13.3, which
two main reasons for these false positive results: false clones is deleted in about 10% CLL/SLL cases. In CLL/SLL, ATM
occur when only a small number of t cells are present in the gene alteration usually occurs as monoallelic loss in the form
sample, resulting in a PCR peak; and through typical TCRG of 11q23 deletion. The remaining ATM allele could be either
rearrangement to amplify non-cloned T-cells to produce a with or without mutation. ATM is a principal DNA damage
false positive clone pattern. Finally, it should be kept in mind response gene and biallelic ATM alterations lead to ATM
that due to lineage infidelity, about 40 percent of precursor B functional loss and chemoresistance.
leukemias has cloned TCRG or TCRB rearrangements. The immunoglobulin heavy chain (IGH) somatic hyper-
Therefore, where possible, TCRG and TCRB test results mutations (SH) status is another molecular marker with sig-
should be associated with histological findings prior to T-cell nificant prognostic value in CLL. Somatic hypermutation of
cloning. When inconsistencies are found, the term oligoclo- IGH locus occurs in B-cells that have undergone germinal
nal rearrangement should be used extensively. center (GC) affinity maturation and thus mark GC or post-
GC derivation in B-cell tumors. The post-GC subset has been
39.1.3.1 Lymphoid Leukemia shown to have a better prognosis and a different patholgen-
Acute lymphoblastic leukemia (ALL) occurs most frequently esis than the pre-GC subset. The same SH testing is also
in children. The tumor cells could derive from precursor indicated in hairy cell leukemia, a rare slow-growing of
B-cells or T-cells, though most of them are B-cell lineage. mature lymphoid B-cell disorder. Variable region of the IGH
IGH and TCR gene rearrangement studies are utilized as gene can be sequenced by conventional Sanger sequencing
markers of clonality as well as for MRD detection. or NGS technology. Compared to the germline sequence
Chromosome abnormalities are detected in approximately from a public database, the IGH sequence is thought to be
80 percent of ALL, which has been incorporated in the clas- evidence for somatic hypermutation with a homology of less
sification of the disease. A t(12;21)(p13.2;q22.1) proved to than 98% (i.e. >2% sequence variation). This cutoff is estab-
be the most common genetic lesion in childhood ALL, which lished since some few germline changes in the IGH locus can
fuses the ETV6 gene at 12p13.2 with the RUNX1/AML1 gene occur between different individuals due to inherited IGH
at 21q22.1. Other reciprocal translocations which could be gene polymorphism. SH testing is a complex assay to per-
commonly seen in ALL include t(8;14)(q24.2;q32.3), t(4;11) form. Under some circumstances in CLL, surrogate markers,
(q21.3;q23.3), t(1;19), and t(9;22)(q34.1;q11.2). These chro- such as detection of the expression of the ZAP70 kinase or
mosome abnormalities can be detected by FISH and surface CD38 (both associated with unmutated/pre-GC sta-
PCR. The t(9;22) occurs more often in adults than children. tus), are used to partially predict SH status.
Although this is usually a p190 isoform, different from the
p210 seen in CML, these patients respond to TKI treatment. 39.1.3.2 Lymphoma
In addition, conventional karyotyping has detected hyperdip- Lymphomas are a broad group of blood cancers of B and
loidy or hypodiploidy in a subset of ALL cases. Gene muta- T-lymphocytes. Every year, almost half of the blood cancers
tions often detected in B/T ALL include FBXWl, JAK2, are lymphomas. Immunophenotyping plays a central role in
KRAS, NOTCH1, NRAS, TP53. the diagnosis of these diseases. Common molecular markers
Chronic lymphocytic leukemia (CLL) is the most com- are of characteristic chromosomal translocations. They
mon form of leukemia in adults. CLL and small lymphocytic exploit the immunoglobulin (Ig) gene transcriptional regula-
lymphoma (SLL), its lymph node counterpart, usually pres- tory elements (promoters and enhancers) to drive a structur-
ent as low-grade and indolent disease process. Peripheral ally normal oncogene expression in a malignant B-cell or
lymphocytosis markers are CD5+, CD20+, CD23+ small B-cell precursor. For instance, IGH transcription elements
lymphocytes. They arise from monoclonal B-cells and char- on chromosome 14 are used in the t(14;18) translocation in
acterize in lacking reciprocal chromosomal translocations. follicular lymphoma to drive BCL-2 expression, and they are
Other types of chromosomal abnormalities, however, are still used in Burkitt’s lymphoma to overexpress c-MYC with a t
routinely detected in CLL patients. Among them, deletions (8;14) translocation. Less commonly, Burkitt’s lymphoma
of 11q23, 13q14, 17p13.3 (involving tumor suppressor gene can also use the light-chain κ and λ transcription elements on
TP53), and trisomy 12 have known diagnostic and prognos- chromosome 2 and 22, respectively, to overexpress c-MYC in
tic values. Sole abnormality such as13q14 deletion (involv- translocations t (2;8) and t (8;22). These light-chain tran-
ing microRNA-15a and microRNA-16) is related with good scription elements are also involved in other types of
prognosis, while a 17p deletion is often associated with poor lymphomas to drive the expression of BCL-6, PAX 5 or
survival. Testing of these features using FISH or aCGH has cyclin D1. These translocations are typically detected by
648 Z. Zuo et al.
breakapart or fusion FISH looking for rearrangement of the higher-grade lymphoma with DLBL morphology. Numerous
target gene loci. genetic changes have been associated with this transforma-
It is challenging to design PCR strategies to detect these tion, including trisomy 7, loss of p53, and c-MYC rearrange-
translocations because these Ig translocations do not gener- ments. Next-generation sequencing studies have identified
ate a unique fusion transcript. PCR must be done across the chromatin modifying gene (CMG) mutations as a hallmark
chromosomal breakpoint using genomic DNA and highly of follicular lymphoma. The most frequently mutated CMGs
sensitive reverse transcription quantitative PCR is not rec- include CREBBP, KMT2D, and EZH2, which may count in
ommended to us. Many of the breakpoints in these transloca- modifying normal B-cell differentiation programs and
tions can occur across a wide region, up to a million base impeding germinal center exit.
pairs, in some cases. Detection strategies by PCR rely on Mantle cell lymphoma is a rare but aggressive B-cell lym-
designing multiple PCR primer sets and focusing on the phoma that arises from the cells found in the mantle zone of
most common areas of translocation within the partner genes lymphoid follicles. It has characteristic cell surface markings
(Table 39.1). For follicular lymphoma breakpoint region of CD5+, CD10–, CD23–. In 90 percent of patients with
(MBR), a ~150 base pair region in the 3′ noncoding region of mantle cell lymphoma could find overproduction of cyclin
BCL2, is the site of chromosome 18 breakpoint in 65–70% of D1, in the form of a t (11;14) translocation that puts the
follicular lymphoma cases. For mantle cell lymphoma, the cyclin D1 gene under control of the Ig heavy chain transcrip-
most commonly tested area involves a 100–1000 base pair tion control elements. Cyclin D1 binds to and activates
major translocation cluster (MTC) upstream of the cyclin D1 cyclin-dependent kinases. An important target of this acti-
(CCND1) gene, which will detect 30–40% of cases. vated cyclin-dependent kinase complex is the retinoblastoma
Lymphomas with PCR-detectable breakpoints can be done (RB) gene product. In its hypophosphorylated state, RB
on follow-up samples with highly sensitive quantitative PCR inhibits entry into S-phase of the cell cycle by binding the
to monitor for residual lymphoma. Follicular lymphoma is transcription factor E2F. When RB is phosphorylated, E2F is
the most common type of the indolent lymphoma. Despite a freed to activate the transcription of genes which propel the
low cure rate, many patients can survive more than a decade. cell into S-phase. Therefore, overexpression of cyclin D1
The cell of origin of this disorder is thought to be the follicu- acts to overcome this late G1-phase checkpoint and maintain
lar center B-cell. Most cases of follicular lymphoma exhibit continuous proliferation of the tumor cells. In addition,
a t(14;18) translocation, resulting in deregulation of expres- mutations in NOTCH1 and TP53 are also seen in mantle cell
sion of the BCL2 proto-oncogene on chromosome 18. Gene lymphoma.
blocks programmed cell death (apoptosis), and overexpres- Lymphoplasmacytic lymphoma (LPL) is an uncommon
sion of this gene results in prolongation of the lifespan of type of mature B-cell lymphoma, usually with an indolent
involved cells, contributing to the pathogenesis of lym- clinical course. Wald Enstrom macroglobulinemia is a clini-
phoma. Of note, approximately 20% of diffuse large B-cell copathologic entity associated with an IgM monoclonal
lymphomas (DLBL) show the t (14;18) translocation, and gammopathy in the blood. Virtually, it is a manifestation of
therefore this translocation is not pathognomonic of follicu- LPL. Although there are no specific chromosomal abnor-
lar lymphoma. Follicular lymphoma can transform into a malities for LPL, many cases will demonstrate a t(9;14),
which juxtaposes the PAX-5 gene and the IgH locus. PAX-5
encodes the B-cell specific activator protein, which is a tran-
Table 39.1 Detection of lymphoma-associated translocations involv-
scription factor. Its expression is associated with increased
ing immunoglobulin gene enhancers
expression of genes important in early B-cell development
Gene
and decreased expression of the TP53 tumor suppressor. The
Translocation involved PCR strategy Alternate methods
t(14;18) BCL2 MBR breakpoint Dual-fusion FISH most frequent genetic aberration reported in LPL is the muta-
(q32;q21) primer detects G-banded tion of MYD88. MYD88 is a molecule that plays a part in
60–70% of cases karyotype Toll-like receptor and interleukin-1 receptor signaling lead-
t(11;14) CCND1 MTC breakpoint Dual-fusion FISH ing to enhanced B-cell survival. An activating point mutation
(q13;q32) primer detect G-banded
of MYD88 (MYD88 L265P) has been implicated in the
30–35% of cases karyotype
t(8;14) MYC None, given large Breakapart FISH pathogenesis of LPL. In LPL cells with the MYD88 L265P
(q24;q32) size of breakpoint to detect any MYC mutation, MYD88 complexes with Bruton tyrosine kinase
t(2;8) area at MYC partner (BTK) to promote tumor survival. Mutations in CXCR4 pres-
(p12;q24) ent less commonly in LPL patients but may identify a sub-
t(8;22)
(q24;q11) group with higher disease activity.
t(3;14) BCL6 None, given large Breakapart FISH The mucosa-associated lymphoid tissue (MALT) lym-
(q27;q32) size of breakpoint to detect any phomas are thought to arise from the extranodal counterpart
area at BCL6 BCL6 partner to post-follicular memory B-cells, which were found in the
marginal zone of lymph node follicles. These tumors are which confers a good prognosis. The t (2;5)(p23;q35) results
often localized, and their behavior is generally indolent. The in a chimeric gene encoding a fusion of the nucleophosmin
most common translocation in MALT lymphoma is t (11;18) (NPM) and anaplastic lymphoma kinase (ALK) proteins.
(q21;q21), the translocation represents the fusion of the NPM is a multifunctional protein which has been implicated
apoptosis inhibitor 2 gene, named BIRC2 (API2), on chro- in ribosome assembly, controlling of centrosome duplica-
mosome 11 and the MALT lymphoma-associated translocation, and acted as a shuttle protein in nuclear transport. It also
tion 1 (MALT1) gene on chromosome 18. The function of the possesses chaperonin and ribonuclease activities. ALK is a
fusion protein is thought to lead to inhibition of apoptosis. member of the insulin family of receptor tyrosine kinases. Its
The recurrent translocations in MALT lymphoma, constitu- natural ligand is unknown. The NPM-ALK fusion contains
tively activating the NF-κB pathway by associating BCL10 the oligomerization domain of NPM and the tyrosine kinase
and MALT1 in malignant lymphocytes, define this pathway domain of ALK. It results in a self-oligomerizing, constitu-
as an oncogenic event. tively active tyrosine kinase with transforming properties.
Burkitt’s lymphoma is a very high-grade B-cell malig- NPM–ALK can activate numerous downstream effectors,
nancy, nearly all of the African endemic Burkitt’s lymphoma including phospholipase C-γ, phophoinositol 3′-kinase, and
is found evidence for latent Epstein–Barr virus infection, but RAS.
in only 20% of the sporadic form found outside Africa. It has Large granular lymphocyte (LGL) leukemia is a rare
been suggested that Epstein–Barr virus plays a causative role clonal disease. It is characterized by a persistent increase in
by blocking apoptosis. Pathologically it is characterized by the number of cytotoxic T-cells or natural killer (NK) cells
small, non-cleaved cells with rapid cell division. Though fast with a clonal TCR gene rearrangement. It is associated with
growing, it is one of the most curable lymphomas, with long- recurrent infections, severe cytopenias and autoimmune dis-
term survival achieved in more than 90% of adult patients eases such as rheumatoid arthritis. JAK/STAT pathway acti-
when properly diagnosed and treated. Genetic testing plays a vation, deregulation of pro-apoptotic pathways (sphingolipid
key role in making the diagnosis of Burkitt’s lymphoma. The and FAS/FAS ligand), and activation of pro-survival signal-
genetic hallmark of Burkitt’s lymphoma is overexpression of ing pathways (PI3K/AKT and RAS) are known as hallmarks
the c-MYC oncogene, which is due to a translocation which of LGL leukemia. Activating somatic STAT3 mutations and
places c-MYC transcription under the control of elements at less commonly, STAT5B mutations are detected in LGL leu-
an immunoglobulin locus: t (8;14), t(2;8) or t(8;22). kemia cases.
Diffuse large B-cell lymphomas (DLBL) are a common, T-cell prolymphocytic leukemia (T-PLL) is a rare but
heterogeneous group of lymphomas with aggressive clini- highly aggressive T-cell neoplasm that mainly affects older
cal behavior. More than half of patients with this disease adults. The tumor cells in this disease are actually of post-
will be cured nowadays. There is no single characteristic thymic T-cell origin. The diagnosis of T-PLL is mostly by
genetic marker for these lymphomas. Of the numerous immunophenotyping. Genes suggested screening for muta-
abnormalities that have been identified, those involving the tions include IL2RG, JAK1, JAK3, STAT3, STAT5B.
BCL-6 gene at 3q27 are the most common, found in >80% Structure of the rearranged V-(D)-J and the locations of
of DLBL. BCL-6 is a transcriptional repressor and required the primers are shown on the left. Examples of polyclonal,
for germinal center formation. BCL-6 expression is now oligoclonal, and monoclonal amplification patterns detected
used clinically as a marker for germinal center origin, and using multicolor capillary electrophoresis detection.
also associated with relatively favorable prognosis.
Although no mutations have been specifically associated 39.1.3.3 Myeloma
with DLBL, screening for genes such as EZH2, TP53, Plasma cell myeloma is a genetically complex disease that
MYD88 are recommended. For example, mutations at arises from clonal plasma cell expansions and most com-
codon 646 of EZH2 are commonly recurrent in germinal monly occur in the bone marrow. Molecular studies have
center B-cell lymphomas, resulting in altered EZH2 enzy- revealed that intraclonal heterogeneity is a common feature
matic activity and enhanced tri-methylation of histone H3 in plasma cell myeloma. The primary genetic events contrib-
at lysine 27. This could be considered as a repressive chro- uting towards plasma cell immortalization can be broadly
matin modification. divided into a two group: a hyperdiploid group characterized
Anaplastic large cell lymphoma (ALCL) comprises a by trisomies of odd numbered chromosomes, and a nonhy-
group of T-cell lymphomas. The characteristic of ALCL is perdiploid group, characterized by IGH translocations to
the strong surface expression of the CD30 (Ki-1) antigen, a various partner chromosomes. It appears that overexpression
cytokine receptor in the tumor necrosis factor receptor fam- of the cyclin D family of genes is almost universal sequelae
ily. The majority of ALCL demonstrate T-cell surface mark- of primary events. Secondary genetic events are complex
ers and/or clonal rearrangements of the T-cell receptor locus. contributing to disease progression, involving secondary
Approximately 50% of the systemic ALCL carry the t (2;5), translocations, CNVs, acquired mutations, LOH, and epi-
650 Z. Zuo et al.
genetic modification. Significant mutations found in this dis-and platelet dense granules (ADP and serotonin, etc.) are
ease include BRAF, FGFR3, KRAS, TP53, and FGFR3. released, which activates additional platelets and supports
the plasma procoagulant system.
The plasma procoagulant system is characterized by the
39.1.4 Conclusion “cascade” of amplifying enzyme reactions resulting in the
activation of the final serine protease, thrombin. This cascade
The NGS introduced into clinical laboratory has greatly is caused by exposure to tissue factor (TF), which circulates
changed our methods for the diagnosis, risk assessment, after vascular injury. The exposed TF combines with FVIIa
monitoring, and treatment of hematopoietic neoplasms. to form a “Factor X-ase” activation complex, which either
However, it is known that some mutations at a low variant directly cleaves FX to FXa or activates FIX to FIXa. A sec-
allele frequency can also be detected in individuals without ond factor X-ase activation complex is formed by FIXa bind-
obvious hematopoietic neoplasms, especially in the elderly ing FVIIIa. FXa binds FVa to form a “prothrombinase
people, the so-called clonal hematopoiesis of indeterminate complex,” which activates prothrombin (FII) to thrombin
potential (CHIP). The most common “CHIP” genes include (FIIa). Factor X-ase and the prothrombinase complexes
DNMT3A, TET2, ASXL1, JAK2, TP53, SF3B1, SRSF2, and aggregate on negatively charged phospholipids in the acti-
CBL. The vast majority of people carrying “CHIP” never vated platelet envelope. Thrombin cuts fibrinogen to form
develop a hematologic malignancy, but they have a 10–15- fibrin monomers to produce a hemostatic thrombus, by acti-
fold increased risk. As a result, certain mutation data needs vating platelets and FXIII to FXIIIa, thereby cross-links
to be interpreted with caution and in consideration of the fibrin monomer chains to form insoluble hemostatic throm-
clinical context. While sequential testing of individual gene bus. Thrombin also can increase its own production through
mutation is still feasible in certain clinical settings, NGS activating FV, FVIII, and FXI in the feedback amplification
analysis to assess multiple mutations simultaneously is more loop [1].
cost-effective. The NGS will be widely applied to routine Although all three hemostatic mechanisms of hereditary
clinical care of patients with hematopoietic neoplasms in the or acquired disorders cause clinically abnormal bleeding,
next few years, which will aid in the development of more this chapter will focus on hemophilia A (HA) and B, von
refined prognostic models and identification of more targe- Willebrand disease (VWD).
table therapeutic markers.
39.2.1 Hemophilia
39.2 Coagulation Disorders
HA and HB are hereditary X-linked recessive hemorrhagic
Lixia Zhang and Lin Wang diseases caused by defects in factor VIII (FVIII) and factor
IX (FIX), which are encoded by the FVIII gene (F8) and FIX
Hemostasis is the coagulation in a physiological environ- gene (F9), respectively. All ethnic groups are affected
ment which leads to rupture of the sealed vascular sys- equally, with an estimated frequency of approximately
tem. There are three main mechanisms of normal 1/10,000. HA is more common than HB, accounting for
hemostasis: vasospasm, platelet thrombosis, and proco- 80–85% of the total number of patients with hemophilia.
agulant system [1]. Hemophilia generally influences men on the mother’s side.
Rupture of the vascular system exposes the extracellular About two-thirds of patients have a family history of bleed-
matrix to the blood and initiates the coagulation process. Von ing. However, both the F8 and F9 genes are apt to new muta-
Willebrand factor (VWF) mediates platelet adhesion to the tions, and as many as a third of cases are caused spontaneous
site of injury. VWF acts as a ligand for the platelet receptor mutations without a family history [2].
glycoprotein (GP)Ib/IX complex and fixes platelets to the
injury site by binding to exposed collagen. The binding of 39.2.1.1 Introduction
platelet GPIIb/IIIa, GPVI, and GPIa/IIa receptors to collagen
mediates the firm platelet adhesion. Subsequently, platelet- Hemophilia A
to-platelet binding is mediated by fibrinogen or VWF bind- HA is caused by a wide diversity of F8 gene events, and
ing to platelet GPIIb/IIIa. In platelets, the cleavage of PAR1 more than 2000 unique mutations have been recorded in the
results in signal transduction, leading to platelet activation. disease-specific databases (FVIII variant database; CHAMP
This process begins after platelets are exposed to very few Mutation List Database; accessed June 2017) [3].
thrombin, which is activated by binding to GP Ib. Platelet F8 contains 186 kilobases (kb) genomic DNA and 26
activation results in many significant changes. Finally, con- exons, located at the telomere end of the X chromosome
tents of alpha granules (fibrinogen, FV, FXI, and VWF, etc.) (Xq28) long arm. The messenger RNA (mRNA) is about
9 kb and the coding sequence is 7053 nucleotides. Recent Missense mutations influencing these cleavage sites were
studies have shown that the precursor protein is mainly syn- found in HA, leading to cross-reacting material positive HA,
thesized in sinusoidal and vascular endothelial cells with which has normal levels but low activity (1–7%) of FVIII
molecular weight about 293 kDa. FVIII protein has a mature antigen. The exact role of the A2 subunit remains unclear
sequence (2332 amino acids), after the secretion leader although the importance of the A2 domain for FVIII coagu-
sequence cleavage, with the domain of A1-a1-A2-a2-B-a3- lant activity has been verified by in vitro research. Domain B
A3-C1-C2. Most circulating FVIII contains heavy chains is cleaved during activation by proteolysis. Due to the expres-
(A1and A2 domains with variable B domain lengths) non- sion level of B-domain-deleted FVIII molecules is 5–10
covalently linked to light chains (A3, C1, and C2 domains), times higher than that of non-B-domain-deleted FVIII mol-
since FVIII protein is prong to proteolysis after secretion ecules, B domain may play a role in secretion or intracellular
(Fig. 39.2). FVIII procoagulant activity does not require a B processing of FVIII or both. It has been reported in HA for
domain. the mutations in this region. VWF-bound FVIII is protected
In the presence of phospholipid surfaces, proteolytic by activated protein C from inactivation. The VWF binding
cleavage by FIIa or FXa leads to the activation of FVIII to area of FVIII is assumed to be at the light chain N-terminus
FVIIIa. FVIII promotes FIXa-FVIIIa-Ca2+-PF3 complex to of FVIII and in the C2 domain. The FIXa binding site has
activate coagulation FX. FVIII circulates is combined with been located in the A2 domain and the light chain regions. In
VWF at a ratio of approximately 1:50 (FVIII:VWF) [4]. The addition, the FX binding site is located in the C-terminus of
cleavages at 372 by FIIa and FXa, or at 1689 by FIIa, are the A1 domain. Binding to phospholipids occurs in the C1
important for the procoagulant activity of FVIII. Cleavage at and C2 domains of the light chain FVIII, which is important
1689 releases FVIII from VWF, allowing FVIII to interact for FIXa and FVIII to activate FX activation. No harmful
with phospholipids and platelets. However, cleavages at 740 mutations in HA were found in the activation cleavage sites
or 1721 are without influence upon coagulant activity of FVIII (Fig. 39.3) [1].
(Fig. 39.3).
Fig. 39.2 FVIII peptide showing domains
Fig. 39.3 FIX peptide showing domains. Pre, pre-propeptide; pro, propeptide; GLA, GLA domain; EGF, EGF domain; act, activation peptide,
and catalytic domain
652 Z. Zuo et al.
Mutations in the F8 Gene the F9 gene can be found in hemophilia B mutation database
More than 2000 unique variants in the F8 gene were listed on (www.factorix.org).
the online database (http://www.factorviii-db.org). They can Missense mutations are the main mutations that typically
be divided into: gene rearrangement, gene sequence inser- cause mild disease, unless the mutations occur in residues
tion or deletion, and single base replacement. All types of that are essential for normal F9 function. Selected mutations
defects can cause serious disease, but the most important in the promoter region of the F9 gene have led to a unique
clinically manifestation is the inversion of F8 intron 22, phenotype, called hemophilia B Leyden, which is character-
which accounts for 40–45% of cases of severe hemophilia ized by severe disease at birth and gradually reduces severity
A. This inversion mutation origin always occurs in male during adolescence and puberty. However, some F9 pro-
germ cells during spermatogenesis. Intron 22 is inverted in moter mutations cause severe disease for life. Nonsense
more than 95% of hemophiliacs, and nearly all mothers are mutations in the signal peptide and propeptide regions cause
carriers. Another exon 1 inversion of F8 gene also occurs and severe HB. However, alterations in missenses leading to F9
affects up to 5% of severe HA patients [5]. Deletions are retention in hepatic cells have been described. The lack of
common, accounting for about 5% of characteristic muta- propeptide cleavage results in dysfunctional F9 molecules.
tions. Usually, these cause severe illness with FVIII activity Mutations in the GLA domain disrupt γ-carboxyglutamic
below 1%. Other patients typically have single base pair acid (post-translational modification), which is important for
changes (causing missense, frameshift, or splice junction collagen, activated platelets, and endothelial cells binding
mutations), duplications, or insertions. Single base pair site. Mutations in the EGF domain lead to disruption of cal-
changes that lead to missense mutations are spread through- cium binding site, which is essential for its procoagulant
out the F8 gene. activity and cofactor FVIII. Catalytic domain mutations
Some female carriers may have clinically significant often disrupt the catalytic triad (His 221, Asp 269, and Ser
bleeding. The molecular basis of symptomatic women 365) necessary to FIX protease function (Fig. 39.3). Due to
includes lyonization of the normal X chromosome and mutation at Ala10, an unusual FIX variant has normal base-
Turner syndrome, in which a dominant mutation in F8 gene line FIX activity. However, warfarin therapy leads to severe
is responsible for production abnormal FVIII. and disproportionate reductions in FIX activity (usually
Polymorphisms within the F8 gene (intragenic) or outside down to 1%) and bleeding in patients who were receiving
the F8 gene (extragenic) were used to assign haplotypes warfarin treatment and have a significant therapeutic interna-
(combinations of polymorphisms) for linkage analysis. tional normalized ratio (INR). A disproportionate prolonga-
Polymorphisms closely related to the gene can be used to tion of the activated partial thromboplastin time (APTT) is
track putative defective F8 genes. The frequency of carriers indicative of such a situation, which should be evaluated by
of these polymorphisms depends on the race of the study clotting factor assays and reveals the FIX deficiency.
group and needs to be considered when studying patients of Eight common polymorphisms are described in different
different races. ethnic groups of European and African descent; however,
they are less common in Asian and other ethnic groups. The
Hemophilia B most informative polymorphism in the Asian population is
Located on the long arm of the X chromosome (Xq27.1), the Hha I (allele frequency = 0.17). Another report, such as
F9 gene is much smaller than the F8, and FIX is mainly syn- Ala148Thr, occurs within the activation peptide. This has
thesized in liver cells. It spans 34 kb of DNA and containing nothing to do with F9 activity or antigen levels. The Thr
only 8 exons (a–h), which codes a 2.8 kb mRNA that is trans- allele is 0.3 in the white population, but is much less frequent
lated into a 415 amino acids protein (Fig. 39.3). The 18 in African Americans (0.053–0.15) and Asians (<0.01).
amino acid propeptide, encoded by the first exon is cleaved
after secretion. As a member of the serine protease family, 39.2.1.2 Clinical Manifestation
the domains of FIX protein are similar to FVII, X, and pro- Based on clotting factor level, the clinical phenotypes are
tein C. Like other serine proteases, FIX proteins require a classified as severe (≤1%), moderate (1–5%), or mild (>5%).
vitamin K-dependent post-translational γ-carboxyglutamic To the patient with hemophilia, the characteristic phenotype
acid (γ-Gla) residue. is the bleeding tendency, usually with lifelong history. The
severity of hemophilia bleeding is usually related to clotting
Mutations in the F9 Gene factor levels. Patients with mild hemophilia do not bleed
Most mutations in the FIX gene are point mutations, account- excessively before undergoing trauma or surgery, and spon-
ing for approximately 80%, and the rest are splice site, taneous bleeding is rare. For patients with moderate hemo-
frameshift, or gross deletions/rearrangements, each account- philia, occasional spontaneous bleeding and prolonged
ing for about 3–4%. More than 1000 different mutations in bleeding, accompanied by minor trauma or surgery, are
spontaneous bleeding into joints or muscles, mainly in identified, polymorphism linkage testing remains a useful
patients with severe hemophilia without an identifiable and inexpensive strategy for carrier diagnostics and prenatal
hemostatic challenge [1]. testing.
Traditionally, HA and HB are considered clinically indis-
tinguishable, repeated musculoskeletal bleeding, especially Hemophilia B
joint bleeding, is a sign of severe disease. However, this indi- The diagnosis of HB is determined by measuring FIX activ-
cates a clinical difference between HB and HA, with more ity rather than genetic testing. In consideration of FIX is a
severe degree of arthropathy in HA patients [6]. vitamin K-dependent protein, all patients with mild to mod-
erate reduced FIX activity should be evaluated to rule out
39.2.1.3 Laboratory Diagnosis vitamin K deficiency.
The following tests should be performed using the cord The molecular diagnostic strategies used for HB testing
blood for a known carrier of hemophilia: prothrombin time are similar to that discussed for HA, except that there is no
(PT), APTT, fibrinogen level, FVIII or FIX activity. All single predominant mutation in HB that is same as F8 inver-
patients with low FVIII activity should be excluded the diag- sions in HA. Compared with HA, where the large size of
nosis of Von Willebrand disease (VWD) due to FVIII nor- genes initially limited direct mutation analysis, direct muta-
mally relies on VWF for survival. The APTT of the affected tion detection in HB has been used for some time (186 kb/26
infant is prolonged, and the infant’s FVIII levels are compa- exons for F8 vs. 34 kb/8 exons for F9).
rable to that of adults, allowing accurate diagnosis. If the FIX Many F9 missense mutations provide knowledge about
level is less than 1%, severe hemophilia B can be diagnosed. the basic structure and function of the FIX protein. However,
Normal FVIII activity does not exclude carrier status, molec- from the perspective of molecular diagnosis, some clinically
ular genetic testing is the only option for diagnosis. important mutation types are worth highlighting.
Understanding what cause F8 and F9 mutation may help The first set of mutations worth noting are a variety of
predict the risk of acquiring factor inhibitors. Patients with gross deletions and rearrangements of F9 gene, which can
null genotypes (large deletions, nonsense mutations, and the lead to severe HB. It can be complicated by the development
F8 inversion mutations), gross F9 deletions or rearrange- of FIX inhibitors and anaphylactic reactions to FIX replace-
ments, have a significantly higher risk of developing inhibi- ment therapy. A second type of F9 mutation with important
tors. For carriers, prenatal diagnosis during pregnancy clinical consequences involves missense mutations in the
provides useful information for management of labor and propeptide-encoding sequence, leading to a significant
delivery and, sometimes, termination of pregnancy [7]. reduction in the affinity of the mutant protein for the vitamin
Molecular genetic testing of hemophilia has been possi- K-dependent carboxylase. The last set of F9 mutations worth
ble since the F8 and F9 genes were cloned in 1984 and 1982, identifying are those in the F9 promoter, 18 different point
respectively [7]. mutations have now been described in about 40 nucleotides
near the start of transcription. These mutations are associated
Hemophilia A with the HB Leyden phenotype, and due to androgen-
Two basic methods can be used for the mutation detection in dependent F9 gene expression, FIX deficiency undergoes at
hemophilia A: mutation screening strategy followed by least a partial spontaneous phenotypic breakdown after
sequencing abnormal region of the gene and direct sequenc- puberty.
ing of the F8 gene.
The detection of inversion mutations should be limited to 39.2.1.4 Management
patients with severe HA (FVIII activity <1%). For proband According to the hemophilia management guidelines of the
or high-risk female with a severe family history of HA, direct World Federation of Hemophilia (WFH), the main purpose
DNA analysis of the most common intron 22 inversion muta- of care is to prevent and treat bleeding with the insufficient
tion is usually performed by restriction digestion and clotting factor [2]. Best practice for the assessment and man-
Southern blot analysis. agement of acute joint bleeds and chronic synovitis in
An intron 22 F8 inversion mutation was found in approxi- patients with hemophilia are summarized in another guide-
mately 45% of patients with a severe HA phenotype. In line by the WFH [8].
molecular diagnostic laboratories, the detection of inversion HA and B, VWD, and FXI deficiency, can be treated with
mutations in any family affected by severe HA should be the single factor replacement therapies through the infusion of
first step of analysis. Detection of inversion use either a protein concentrates derived from either plasma or recombi-
Southern blot, or a long-range (>10 kb) PCR-based approach. nant DNA technology. The main recent development is the
Linkage analysis is rarely used for the genetic analysis of introduction of long acting products. By pegylation or fusion
hemophilia. However, if there is a family history of the dis- with neonatal Fc receptors or albumin, the half-life of FVIII
ease, and information-rich intragene polymorphisms can be and FIX products can be extended.
654 Z. Zuo et al.
The promise of genetic therapies for improved manage- tein for coagulation FVIII, stabilizing and protecting it from
ment of coagulation disorders is now beginning to be real- rapid removal from the circulation. It also has other func-
ized. The hemophilia is a perfect model genetic disease for tions and may be involved in processes such as inflammation
the application of gene therapy. They result from recessive and angiogenesis [12].
mutations in 2 well-characterized genes with minimal influ-
ence from other genetic modifiers. Furthermore, it was well Molecular Basis of Disease
recognized from the natural history of moderately severe The VWF gene is located near the end of the short arm of
and mild hemophilia that small increments in plasma clot- chromosome 12, with a length of 178 kb and consists of 52
ting factor levels would result in significant benefits in exons. The intron-exon boundary is roughly related to the
reducing the risk of bleeding. Aside from FVIII and FIX VWF domain. Part of unprocessed pseudogene on chromo-
gene therapy, the only other procoagulant targets that have some 22 was replicated to exons 23–34 of chromosome 12,
been explored to any extent in preclinical studies have been with a sequence variation of 3%.
VWF and FVIIa [9]. VWF is a 2813 amino acid peptide consisting of a 22
amino acid signal peptide, a 741 amino acid propeptide, and
39.2.1.5 Conclusions a mature 2050 amino acid peptides. After the signal peptide
Assessment of the patients with HA requires a careful his- is removed, the pro-VWF dimerizes at the C-terminal ends
tory and physical examination. Molecular genetic testing is via disulfide bond, and further polymerizes at the N-terminal
of great significance for the diagnosis and of HA and provid- ends to form a multimer with a size of 0.5 to more than 10
ing a genetic counseling. million daltons. The binding sites for various ligands have
been mapped to different domains of the VWF subunit
(Fig. 39.4). VWF gene mutations and polymorphisms can be
39.2.2 Von Willebrand Disease accessed on this website (www.vwf.group.shef.ac.uk).
In healthy people, VWF circulates as a high-molecular
39.2.2.1 Introduction weight (HMW) multimer carrying FVIII. Some people have
Von Willebrand disease (VWD) is an autosomal disease reduced VWF levels slightly, which may lead to a bleeding
characterized by VWF deficiency. It is the most common phenotype but not necessarily caused by defect in the VWF
inherited hemorrhagic disease known in human, with the gene. People with low VWF levels and bleeding tendency are
prevalence between 0.6% and 1.3% [6]. No racial or ethnic classified as low VWF, not VWD.
predisposition is identified. Both men and women are
affected, but women have a higher frequency of clinical Type 1 Von Willebrand Disease
manifestations. The VWD can be divided into three major Type 1 VWD is typically autosomal dominant. Although it is
categories: type 1 (reduced levels), accounting for 70–80%, the most prevalent form of the disease, accounting for
type 2 (abnormal function), accounting for approximately 65–75% of all VWD cases, little is known about its molecu-
20%, or type 3 (complete absence of VWF), accounting for lar pathogenesis. Diagnosis is established by detection of
<5% (Table 39.2) [10–12]. VWF, showing that plasma VWF antigen, ristocetin cofactor
Vascular endothelium and megakaryocyte are sources of activity, and FVIII activity decreased proportionally, and
plasma VWF synthesis and release. VWF is secreted into the VWF multimers were normal distributed. Several large-scale
plasma from the storage sites, platelet alpha granules, and population studies of the molecular genetic pathology of
Weibel–Palade bodies of endothelial cells. VWF combines type 1 VWD have shown that:
with collagen to form a bridge at vascular lesion, mediates In approximately 60% of families, the type 1 VWD phe-
platelet adhesion and aggregation, and acts as a carrier pro- notype is associated with the VWF gene. Candidate VWF
gene mutations are found in approximately 65% of patients
with type 1 VWD. More than 100 different candidate VWF
Table 39.2 Classification of von Willebrand disease [10]. gene mutations have been discovered. About 65% of the can-
Type Description didate VWF mutations are missense substitutions. Candidate
1 Partial quantitative deficiency of VWF VWF gene mutations occur from the 5′ flanking region to the
2 Qualitative VWF defect C-terminal domain of the protein. There are more than single
2A Decreased VWF-dependent platelet adhesion with selective
candidate VWF mutations in about 15% of patients [7].
deficiency of high-molecular-weight multimers
2B Increased affinity for platelet GPIb Although null alleles may be present in type 1 VWD,
2 M Decreased VWF-dependent platelet adhesion without most the mutations are actually missense mutations because
selective deficiency of high-molecular-weight multimers it is also has a quantitative deficiency of VWF. The missense
2 N Markedly decreased binding affinity for FVIII mutations in type 1 disease can result in VWF’s impaired
3 Virtually complete deficiency of VWF intracellular routing, secretion, and storage or faster
Fig. 39.4 Von Willebrand peptide domains
c learance. Studies showed that certain candidate mutations alleles. This situation poses a challenge in providing genetic
occur repeatedly, including Y1584C (8–25% of the type 1), counseling.
R924Q, R1205H, R1315C, R1374H, and R854Q [13–15].
Due to the extent of polymorphism exhibited by different Type 2 Von Willebrand Disease
ethnic groups, the pathogenicity distribution of missense Type 2 variants of VWD account for 25–35% of the total
changes is more complicated. It indicates that the genetics of VWD patients in most surveys. In type 2 VWD the pheno-
this complex trait is more complicated by allelic and locus type is determined based on specific functional defects or
heterogeneity, in addition to incomplete penetrance and vari- characteristics of the VWF protein, so the mutations are usu-
able expression. Although most patients with type 1 plasma ally dominant negative missense mutations confined to spe-
VWF levels <30% will show candidate VWF mutations, cific regions of VWF. The phenotype in type 2 disease is
patients with mild VWF deficiency (30–50%) are more pos- usually fully penetrant opposite to type 1. With the exception
sibly to have a phenotype that comes from several loci, of type 2 N disease, the genetic pattern of type 2 disease is
including the ABO blood group locus, are playing an impor- usually autosomal dominant [12].
tant pathogenic role. The identity of these additional genetic
modifiers is currently under investigation. Given the size and Type 2A Von Willebrand Disease
complexity of the VWF gene and the heterogeneity of allelic Type 2A VWD accounts for approximately 75% of all type 2
and locus, in most cases, the application of molecular genetic VWD and is autosomal dominant. High-molecular-weight
analysis to diagnose type 1 VWD is not warranted. However, (HMW) VWF multimers are reduced or missing, and lower-
sometimes this method may provide additional useful infor- molecular-weight (LMW) multimers are relatively increased
mation, and this situation may change as further technology or structurally abnormal. This phenotype can be caused by
advances and the potential identification of key genetic mod- mutations in at least three regions of the VWF subunit, A2
ifiers of VWF levels. domain, C-terminal CK domain, and D domain (Fig. 39.5).
Type 3 Von Willebrand Disease Type 2B Von Willebrand Disease

Type 3 VWD is autosomal recessive inheritance, which is Type 2B VWD accounts for about 20% of all type 2 VWD
characterized by a severe reduction in VWF antigen and ris- and is autosomal dominant. In patients with type 2B VWD,
tocetin cofactor activity, and a consistent decrease in F8 VWF antigens are variable reduced, and ristocetin cofactor
activity, leading to a more severe phenotype. The prevalence activity is inconsistently reduced. High and intermediate
of this disease is one in a million. Type 3 VWD mutations plasma VWF multimers are lost, but platelet VWF multimers
include deletions, frameshifts, and nonsense mutations. are normally distributed. Type 2B variant is different from
Homozygosity has been demonstrated in a few close rela- type 2A VWD in the presence of mild-to-moderate thrombo-
tives although most patients are typically compounded het- cytopenia. Causative missense mutations occur in the A
erozygous for these VWF mutations. Many parents of domain of the VWF gene, leading to a dominant gain in
patients with type 3 VWD are clinically unaffected although functional phenotype. Mutations in the A domain may dis-
most of these patients appear to have two defective VWF rupt regulatory sites that normally inhibits the binding of the
656 Z. Zuo et al.
Fig. 39.5 Type 2A VWD mutations regions of VWF subunit
A1 domain to platelet GPIb. Type 2B VWD must be different 60–80% of patients having bleeding after surgery or dental
from pseudo-VWD or platelet-type VWD, the latter being extractions [12]. Homozygous patients may experience
similar in performance. Patients with platelet-type VWD severe bleeding, such as hemarthrosis, or potentially fatal
have a primary platelet defects caused by the platelet GPIb/ gastrointestinal tract or central nervous system bleeding.
IX receptor mutations.
39.2.2.3 Laboratory Diagnosis
Type 2M Von Willebrand Disease VWD diagnosis due to personal or/and family bleeding his-
In patients with type 2M VWD, although the distribution of tory, in combination with laboratory tests showing VWF or/
VWF antigen and VWF multimers are normal, the activity of and FVIII abnormalities. The diagnosis of VWD is on the
ristocetin cofactor is reduced, reflecting the functional deficit basis of determination of VWF antigen, VWF-dependent
of VWF multimers. There may be ultra-high-molecular platelet adhesion level, VWF-ristocetin cofactor activity
weight multimers or uncleaved proVWF. Type 2M mutations assay, and the FVIII coagulant activity assay. When VWF
occur in the A1 region of the VWF gene, causing the binding antigen is undetectable or at level below 5 IU per deciliter,
affinity of VWF to platelet GPIb to decrease. type 3 VWD is diagnosed. Both VWF antigen and propep-
tide are at very low levels in type 3 disease or absent.
Type 2N (Normandy) Von Willebrand Disease However, antigen levels are low, but the propeptide levels
Poor binding of FVIII to VWF was caused by the mutations are minimal reduction or normal in severe type 1. A dispro-
in the FVIII binding domain of VWF. This binding defect portional reduced VWF-ristocetin cofactor activity as com-
results in a shorter half-life of plasma FVIII, and therefore pared with VWF antigen (≤0.6) indicates type 2 disease.
decreased in plasma FVIII activity. The VWF multimer dis- Reductions in FVIII activity which is greater than the reduc-
tribution, VWF antigen levels and ristocetin cofactor activity tion in VWF antigen (≤0.6) may indicate type 2N
are all normal. The 2N subtype mimics mild HA, but has an VWD. Determination of VWF and collagen binding can be
inherited autosomal recessive pattern rather than the X-linked used to identify cases of type 2M disease [12].
recessive pattern of HA. VWD was detected in 4.8% (58 of In the diagnosis of VWD, molecular testing is not rou-
1198) of patients previously diagnosed with mild HA in a tine. However, direct detection of specific mutations in
recent international survey. Three VWF gene mutations VWF gene can help distinguish between type 2N and mild
(Thr791Met, Arg816Trp, and Arg854Gln) account for 96% HA, and type 2A and type 2B. In type 3 disease family, the
of type 2N patients.16 Patients diagnosed with non-X-linked identification of specific mutations is helpful for prenatal
hereditary pattern “mild HA” should be considered type 2N diagnosis and genetic counseling. Techniques consist of
VWD. Generally, heterozygotes have normal F8 levels, and restriction fragment length polymorphism (RFLP) and
homozygotes have reduced F8 activity. Nevertheless, appar- direct sequencing. Indirect detection by linkage analysis can
ent heterozygotes with low FVIII levels usually have inher- be used for VWD, but with the exception of severe type 3
ited the second allele, forming a VWD type 1 (compound VWD [1], few have point out. Innovations in sequencing
heterozygotes). techniques, for example, next-generation sequencing, make
genotyping easier [12].
39.2.2.2 Clinical Manifestation Due to the large size of the VWF, the molecular genetic
The symptom severity varies great in patients with VWD, analysis of VWF genes is more challenging than the detec-
relating to the disease subtype, the level of VWF activity, tion of hemophilia. However, the availability of primers spe-
age, and sex. The most common symptoms are bruising and cifically designed to amplify VWF genes allows analysis of
epistaxis in children with VWD, but hematomas, menorrha- VWF gene sequences different from pseudogenes. The anal-
gia, and bleeding from minor wounds in adults. There are ysis of mRNA mutation is based on research, but considering
the instability of mRNA in transported specimens and the 39.2.2.5 Conclusions

complexity of the detection, it is not practical in clinical Improved diagnostic approaches and treatment options have
detection. emerged in the past decade. The introduction of more repeat-
able and rapid VWF functional assays will further improve
39.2.2.4 Management the diagnostic level. NGS will facilitate the routine identifi-
For most patients who have type 1 VWD or patients with cation of VWF mutations.
type 1 VWD classified as low levels of VWF and with type 2
VWD, desmopressin can increase plasma VWF concentra-
tions by stimulating endothelial cells to release endogenous 39.2.3 Hereditary Thrombocytopenia
VWF reserves, which VWF release is stimulated by an ago-
nist effect on the vasopressin V2 receptor. VWF containing Hereditary thrombocytopenia is a rare form of hereditary
concentrates, human plasma derived and viral inactivated are thrombocytopenic disorders characterized by thrombocyto-
the choice for treatment in type 3 and most type 2 VWD penia, often accompanied by abnormal platelet function.
patients. VWD patients with menorrhagia, gynecologic, and Patient has different degrees of bleeding tendency and a fam-
hormonal abnormalities should be excluded, and treatment ily history of thrombocytopenia. According to genetic meth-
with oral contraceptives containing estrogen and progestin ods, it is partitioned into autosomal recessive inheritance,
should be started. Tranexamic acid and aminocaproic acid, autosomal dominant inheritance, and sexual recessive
as fibrinolysis inhibitors, are important adjuvant treatment inheritance.
for bleeding from skin and mucous membranes. Gene ther- The molecular mechanisms of hereditary thrombocyto-
apy can be potentially used for severe type 3 VWD treat- penia are often associated with genetic mutations. At pres-
ment. Large VWF genes can be easily introduced into many ent, the molecular mechanism of an increasing number of
vectors, but gutless adenoviral vectors can easily accommo- hereditary thrombocytopenia is being elucidated
date a VWF (8.5 kb) gene [16]. (Table 39.3).
Table 39.3 Congenital thrombocytopenia characterized according to inheritance pattern, genetic mutations and associated findings
Inheritance Chromosomal
Syndrome (OMIM Entry) pattern Gene involved location Associated findings
MYH9-RD (multiple) Autosomal MYH9 22q12-13 Döhle like leukocyte inclusion bodies, nephritis,
dominant sensorineural hearing loss. and pre-senile
cataracts
Paris-Trousseau (Jacobsen) syndrome Autosomal FLI1 11q23.3-24 Cognitive and facial abnormalities
(188,025; 147,791) dominant
Platelet-type von Willebrand disease Autosomal GP1BA 17p13 Low VWF:RCo assay, decrease in HMWVW
(PT-VWD) (177820) dominant multimers, increased low-dose RIPA. Needs to
be differentiated from type IIb VWD
Radioulnar synostosis with Autosomal HOXA11 17p15.2 Radioulnar synostosis, clinodactyly, syndactyly,
amegakaryocytic Thrombocytopenia dominant hip dysplasia, sensorineural hearing loss and
(605432) progression to pancytopenia
Familial Platelet disorder with Autosomal RUNX1 22q22 Platelet aggregations shows impaired
propensity for myeloid malignancy dominant aggregation to collagen and epinephrine, 35%
(FPD/AML) (601399) of the cohort may go on to develop AML
ANKRD26-related thrombocytopenia Autosomal ANKRD26 10p2 Normal sized platelets with bone marrow shows
(610855) dominant dysmegakaryopoiesis. Possible increased risk of
leukemia
CYCS-related thrombocytopenia Autosomal CYCS 7p15.3 None
(612004) dominant
TUBB1-related Autosomal TUBB 16p21.3 None
macrothrombocytopenia (613112) dominant
Bernard-Soulier syndrome (231200) Autosomal GP1BA, 17p13, 22q11, Platelet aggregation studies show absent
recessive GP1BB, GP9 3q21 ristocetin induced response
Gray Platelet syndrome (139090) Autosomal NBEAL2 3p21 None
recessive
(mostly)
Congenital Amegakaryocytic Autosomal MPL 1p34 Progression to pancytopenia
Thrombocytopenia (CAMT) (604498) recessive
658 Z. Zuo et al.
Inheritance Chromosomal
Syndrome (OMIM Entry) pattern Gene involved location Associated findings
Thrombocytopenia with absent radii Autosomal RBM8A 1q21.1 Bilateral absent radii with thumb present. Lower
(TAR) syndrome (274000) recessive limb, cardiac, gastrointestinal, and renal
anomalies
Thrombocytopenia associated with Autosomal ABCG5, 2p21 Stomatocytosis, tendon xanthomas, and
sitosterolemia (210250) recessive ABCG8 premature atherosclerosis
Wiskott–Aldrich syndrome (X-linked X-linked WAS Xp11.22 Recurrent infections, eczema, with a risk of
thrombocytopenia) (301000) auto-immunity and lymphoid malignancy
X-linked thrombocytopenia with X-linked GATA1 Xp11.23 Dyserythropoietic anemia
dyserythropoiesis (300,367; 314,050)
FLNA-related thrombocytopenia (nd) X-linked FLNA Xq28 Possible association with periventricular
nodular heterotopia
Source: Kumar and Kahr 2013 [17]. OMIM: Online Mendelian Inheritance in Man
Fig. 39.6 Structure of the hemoglobin
39.2.4 Typical Medical Case able at the Globin Gene Server (http://globin.cse.psu.edu),
which provides data and tools for studying the function of
Clinical background A 11-year-old boy was admitted to DNA sequences, with a focus on those related to hemoglobin
hospital for surgical operation because of congenital heart production and the Human Gene Mutation Database (HGMB)
disease. The results of preoperative laboratory examination (http://www.uwcm.ac.uk/uwcm/mg/hgmd0.html) [18].
were as follows: PT 12.2 s, APTT 119.2 s, FIB 2.78 g/L, TT Hemoglobin (Hb) is a tetramer formed by connecting two
17.5 s, DD 1.98 mg/L. Then more tests were performed and alpha and two beta chains to four porphyrin rings consisting
the results were as follows: of one heme and one iron (Fe) ion, respectively (Fig. 39.6).
VIII 1.2%. The intron 22 F8 inversion mutation was found Here are six different types of globin chains in normal
by a long-range PCR. human Hb, which are designated by the Greek letters α, β, γ,
δ, ε, and ζ at different times during development. Separate
Final diagnosis hemophilia: A. genetic loci encode α and β chains. Three genes encoding
This case was from the First Affiliated Hospital of Nanjing α-like globins (5′-ζ-α2-α1-3′) clustered on chromosome 16
Medical University (also named Jiangsu Province Hospital). and five genes encoding β-like globins (5′-ε-Gγ-Aγ-δ-β-3′)
clustered on chromosome 11, are expressed in a develop-
mental order parallels to their structural arrangement
39.3 Hematologic Disorders (Fig. 39.7) [19]. The developmental regulation of these genes
can help to understand a lot of hemoglobin disorders. The
Lixia Zhang and Zhen Ren sequence of genes in each cluster is the same as the expres-
sion order during development. Several types of hemoglobin
are produced during the development of normal human
39.3.1 Hemoglobinopathies (Table 39.4).
Hemoglobin disorders are the most common genetic dis-
Hemoglobinopathies are the most common monogenetic diseases in the world, especially in areas where malaria has
orders globally. Online resource of the mutant alleles is avail- been endemic, such as Africa, all Mediterranean countries,
Fig. 39.7 The globin genetic

loci
β Chain 1
β Chain 2
Fe2+
Heme
α Chain 1
α Chain 2
Table 39.4 Comparative Chain Composition of Hemoglobin Types SCD is a leading cause of morbidity and mortality in sub-
Hemoglobin Polypeptide(Globin) Saharan Africa, followed by the Middle East and Indian sub-
Type Symbol Chains Stage continents [22]. In the presence of hypoxic tension in the
Hb Gower 1 ζ2ε2 2 zeta, 2 epsilon Embryonic microvessels, HbS polymerizes into fibers, causing erythro-
Hb Gower 2 α2ε2 2 alpha, 2 epsilon Embryonic cyte elongation and sickle stiffness. The sickle red cells have
Hb Portland ζ2γ2 2 zeta, 2 gamma Embryonic poor deformability and impede passage in the microcircula-
Hb F α2γ2 2 alpha, 2 gamma Fetal
tion that cause vascular occlusion, which is the characteristic
Hb A α2β2 2 alpha, 2 beta Normal
adults of this disease. These red blood cells have a shorter survival
Hb A2 α2δ2 2 alpha, 2 delta Normal time, leading to chronic hemolytic anemia.
adults SCD refers to any state in which the inheritance of HbS
can cause sickle of RBCs and therefore includes common
inheritance of HbS with another qualitative or quantitative β
the Middle East, the Indian subcontinent, and Southeast Asia globin chain defect, for example, in HbC(α2β26Glu → Lys), β0-
[20]. There are two main groups of hemoglobin diseases: thalassemia or β+-thalassemia, and other β globin. However,
thalassemia and structural hemoglobin disorders. The homozygous HbSS is the most common and severe form
decreased production or absence of α- and β-globin chains of caused by inheriting βS from both parents.
Hb are the characteristics of thalassemia. Sickle cell anemia, Sickle cell trait is not strictly a form of sickle cell disease.
HbE, and HbC diseases are common structural Hb disorders It is defined as inheriting HbS and a normal HbA gene from
that spread around the world [21]. In this chapter, we focus both parents, respectively. RBCs are not sickle shaped from
on the most common structural variations in sickle cell dis- people with sickle cell trait and these individuals are basi-
ease (SCD), as well as the disorders of globin expression, cally unaffected [23, 24].
α- and β-thalassemia.
Clinical Manifestation
39.3.1.1 Sickle Cell Disease Sickle cell can lead to lifelong weakness due to chronic ane-
mia, poor quality of life from organ damage, and early death.
Overview Signs of SCD usually begin in early childhood [25].
James B Herrick firstly described SCD in 1910, codon six of “Acute painful crisis” is the main clinical feature of SCD
the β-globin gene is changed by a single protein mutation which usually requires hospitalization. Several acute condi-
from a glutamic acid to a valine, characterized by the pres- tions such as the crisis of vascular occlusion, aplastic disor-
ence of HbS (α2β26Glu → Val, HBB: c.20A > T) (Fig. 39.8). ders, splenic sequestration, high hemolysis, liver disease,
This mutant homozygote leads to the autosomal recessive dactylitis, and acute chest syndrome can be described by
genetic disease SCD. SCD is the most common hemoglobin- sickle cell crisis [26].
opathy in humans. On the other hand, it is also the most com- Sickle cell trait has a better quality of life and the same
mon hereditary hematopathy. mortality rate as other general population. Because sickle
660 Z. Zuo et al.
ε Gγ Aγ δ β
5’ 3’ 11
ζ2Gγ2 ζ2Aγ2 α2 Gγ2 α2 Aγ2 α2 δ2

ζ2 ε2 α2 ε2 α2 β 2
Hb2 Gower 11 Hb Portland
Hb2 Gower 1
HbF HbA2 HbA
5’ 3’ 16
ζ α α
Fig. 39.8 Sickle hemoglobin molecules
cell trait is benign in nature, it generally does not have any

clinical significance. However, adverse conditions have been Abnormal red
reported due to the status of patient’s trait. A higher risk of cell morphology
exertional rhabdomyolysis was associated with it [27].
Therefore, sickle cell trait may not be entirely benign and the
patients should be treated aggressively once these complica-
tions occur.
Hb Electrophoresis
/HPLC
Laboratory Diagnosis
Abnormal red cell morphology, including sickled red cells
(in an acute crisis state), may be seen on peripheral smears
[28]. Hemoglobin electrophoresis is the most common tech-
nique distinguishing normal from abnormal hemoglobins, or PCR-RFLPM
high-performance liquid chromatography (HPLC). New /Real-time PCR
methods including DNA or antibody-based tests make accu-
rate point-of-care diagnostics possible [24]. Virtually all of
Fig. 39.9 Laboratory diagnosis of SC
the SCD cases can be diagnosed through direct DNA analy-
sis with polymerase chain reaction (PCR). PCR-RFLP is one
of the most commonly used methods. Restriction sites are diagnosis can be made by amniotic fluid puncture around
introduced by modified primers, and the βA, βS, and βC alleles 14th week of gestation. DNA diagnosis at 7th to 10th week
can be identified using Ava I and Sty I, respectively. It is of gestation is allowed to be performed by the extensive use
introduced that the βE allele in addition to the βA, βS, and βC of chorionic villus biopsy at present [28, 30].
alleles can be easily identified by a real-time PCR method
using hybridization probes [18] (Fig. 39.9). Management
Early childhood mortality caused by SCD have been sig- Streptococcus pneumoniae, neisseria meningitidis, and hae-
nificantly reduced by newborn screening programs which are mophilus influenzae type B, as well as hepatitis B virus and
widespread in most industrialized countries. Electrophoretic seasonal influenza vaccines should be given to all patients
or HPLC techniques become the first-line method for neona- with SCD besides all other standard vaccines due to the
tal screening. However, compared with current methods tan- functional asplenia. RBC transfusions may help improve
dem mass spectrometry seems to be a reliable technique quality and quantity of life. The severity of anemia should
[29]. Molecular testing is mainly used in prenatal diagnosis. be monitored and blood transfusions can be performed only
As DNA from the fetus is contained in the amniocytes, fetal when necessary. Iron chelation therapy should be consid-
ered to prevent the iron overload. Treatment of acute and Table 39.5 Genotypes of α-thalassemia
chronic pain should follow a rational guideline. Although Numbers of genes affected Genotypes Condition
RBC transfusions and hydroxyurea are important for the 1 αα/−α Silent carrier
treatment of SCD, their primary effect is to reduce morbid- 2 −α/−α or −−/αα α- thalassemia trait
ity and mortality [23]. 3 −α/−− HbH disease
Hematopoietic stem cell transplantation (HSCT) is the 4 −−/−− Hydrops fetalis
only possible treatment for SCD. HLA-matched sibling
transplantation procedures using marrow or cord blood stem hemoglobin synthesis. Patients with α-thalassemia trait have
cells have an overall survival and event-free survival rate of two deletions either on the same (−−/αα) chromosome (cis)
nearly 90 percent. Gene therapy which involves inserting a or on contralateral (−α/−α) chromosomes (trans).
normal gene into bone marrow of SCD patients are in a rapid α-thalassemia trait is only characterized by a mild hypochro-
development stage of scientific discovery and clinical mic microcytic anemia, but couples with these genotypes
research. New therapeutic agents including hydroxy- may give birth to a fetus with hydrops fetalis or children with
carbamide, short-chain fatty acid derivatives and histone HbH disease. HbH disease is caused by mutations of three of
deacetylase inhibitors, as well as immunomodulatory drugs the four alleles, and the α+ and αo mutations have a compound
are in development [24]. heterozygosity. During fetal development, the excess γ chains
form homotetramers (γ4), also known as Hb Bart’s, which
Conclusions can be detected instantly at birth. After that, excess β-globin
The worldwide burden of SCD is huge and still not fully chains form β4 tetramers (HbH), which are unstable and pre-
addressed. Newborn screening programs and prenatal diag- cipitate in developing red blood cells. Individuals with HbH
nosis can significantly reduce mortality and morbidity of the disease have moderate hemolytic anemia, while the no-copy
SCD. Renewed efforts should be worked hard on improved inheritance of the α gene (Hb Barts) is incompatible with life
diagnostics and treatments to reduce the devastating lethality [33]. The HbH Constant Spring α-globin mutation extends 3′
of sickle cell disease all over the world. mRNA sequences, abnormally lengthens α-globin chains,
and reduces globin production by unaffected allele.
Intracellular precipitation of oxidized chains of hemoglobin
39.3.2 Thalassemia Constant Spring damages membranes of red blood cells,
leading to hemolysis and a more severe anemia [33].
39.3.2.1 Overview −α3.7 and −α4.2 are the most commonly occurring single
Thalassemia is one of the common inherited monogenic dis- α-globin gene deletions, which are used to describe the
eases worldwide with about 1–5% of mutation carriers in the lengths of the potential deletions. The double α-globin gene
global crowd [31]. This is due to the reduced synthesis or deletions −SEA, −FIL, and −THAI are easily found in Southeast
stability of either α- or β-globin chain. The chain excess Asia while −MED and −(α)20.5 are more common in
caused by α:β imbalance precipitate within the red blood Mediterranean area. Non-deletion α-thalassemia alleles are
cells, resulting in membrane damage and red blood cell rare, but a mutation in the termination codon of the α2 gene
destruction [32]. The prevalence of thalassemia is highest in called αConstant Spring, is common in Southeast Asia.
geographic areas that most affected by malaria in history, The (−) genotype and the resulting risk of fetal edema is
Mediterranean, sub-Saharan Africa, the Middle East, the largely confined to Southeast Asia, however, the (−α) geno-
Asian-Indian subcontinent, and Southeast Asia are included. type is more common in other groups [18]. It is important to
However, the migration of these people has led to the occur- distinguish these two carrier states for accurate genetic
rence of the diseases worldwide [33]. counseling.
α-Thalassemia β-Thalassemia
About 95 percent cases of α-Thalassemia are caused by dele- β-thalassemia is caused by hundreds of β-globin gene muta-
tion, and the remaining few due to point mutations [34]. The tions, most mutations are point mutations, both coding and
α gene locus consists of paired alleles (αα /αα) on chromo- intervening (noncoding) DNA sequences are involved.
some 16 and that there are haplotypes with either one (-α) (α+ β-Thalassemia can be further subdivided into β0-thalassemia
thalassemia) or both (−) (αo thalassemia) copies of the α gene and β+-thalassemia where complete lack of β-globin chain
missing. Besides the normal genotype, four more genotypes synthesis and reduced β-globin chain production, respec-
are possible (Table 39.5). tively [34]. β-thalassemia minor (trait or carrier) represents
Patients with silent carriers have no clinical manifestation the heterozygotes for either type of allele, patients often have
of hemoglobinopathy, because the three remaining α genes clinically-asymptomatic-microcytic anemia while others can
directly synthesize a sufficient number of α chains for normal have no identified hematological abnormalities. β-thalassemia
662 Z. Zuo et al.
intermedia results from compound-heterozygous two On the contrary of α-thalassemia, 95% of β-thalassemia
β+-thalassemia alleles or one β+ and one β0 allele, including are caused by point mutations. A PCR method used to detect
anemia, hemolysis, iron loading, and occasional requirement the common specific mutation simultaneously is initially
for blood transfusion. Individuals with two β0-thalassemia used for any given ethnic region because a limited number of
alleles have the most severe β-thalassemia, leading to point mutations are common in different ethnic groups.
transfusion-dependent anemia, severe iron transfusion load Reverse dot-blot methods can be economical and screen for
requiring chelation therapy, and a reduction in life expec- multiple mutations. Amplification refractory mutation sys-
tancy [35]. HbE(α2β226Glu → Lys)/β-thalassemia is a tem (ARMS), also called allele specific PCR, is a diagnostic
β-thalassemia subtype that can result in a mild, moderate, technique that detects single base mutation including both
and severe thalassemia phenotype, the severe form is similar germline and somatic mutations. Detection of sickle cell
to β-thalassemia major, and the mild and moderate forms are anemia, non-deletional α-thalassemia and β-thalassemia are
similar to β-thalassemia intermedia. included in the applications of ARMS [37].
Sanger sequencing is currently the most practical method
39.3.2.2 Clinical Manifestation for detecting all possible mutations in the individual.
Thalassemia is clinically characterized by chronic hemoly- Denaturing high-performance liquid chromatography
sis, ineffective erythropoiesis (IE) and iron overload [31]. (DHPLC) has also been used to identify some unknown
Iron overload is either due to the increased iron absorp- mutations [34]. The next-generation sequencing (NGS) rep-
tion and release from the reticuloendothelial system or due to resents a new principle of sequencing technology after the
frequent blood transfusions. Excessive iron can cause dam- era of Sanger sequencing. However, there is no information
age to the heart, liver, and endocrine system which includes in the literature about sequencing of globin genes. Although
glands. The glands produce hormones which can regulate the not impossible, the high degree of homology between the α
entire body’s processes. Individuals with thalassemia are and β clusters genes makes the NGS methods very difficult,
more susceptible to infection with the spleen removed. The time-consuming, and expensive [36]. Laboratory diagnosis
task of removing destructed red blood cells in thalassemia of thalassemia is shown in Fig. 39.10.
leads to the enlargement of spleen. Splenomegaly worsens
anemia and reduces the life span of transfused red blood 39.3.2.4 Management
cells. Severe enlargement of the spleen may require resec- For different birth cohorts of patients with β-thalassemia,
tion. Increased proliferation of red blood cell precursors in blood transfusion and chelation therapy can prolong life
the bone marrow leads to bone marrow expansion followed expectancy, improve quality of life, comorbidities, and sur-
by bone deformities and low bone mass, especially in the vival. Allogenic hematopoietic stem cell transplantation
face and skull [35]. (AHSCT) techniques may provide a cure rate of 80 percent
for young people whose transplants come from HLA-
39.3.2.3 Laboratory Diagnosis matched-sibling donors. In recent years, gene therapy has
α- and β-thalassemia and their carrier states can be diagnosed
by standard red blood cell indexes such as hemoglobin, red
blood cell number, mean corpuscular volume (MCV), and Hb, RBC, MCV, RDW
red cell distribution (RDW) as well as Hb electrophoresis or
HPLC [36].
Molecular diagnostic techniques are focusing on demon-
strating the common deletions since most α-thalassemia are
caused by deletions of one or more HBA genes, which are
common in the affected populations. DNA blot analysis has Hb Electrophoresis/HPLC
been widely used before, but gap-PCR is currently the most
commonly used method [34]. The Southern blotting tech-
nique is still used in some laboratories for the unknown
α-deletion. However, multiplex ligation-dependent probe
amplification (MLPA) have been introduced recently. This α-thalassemia
method has high sensibility and reliability and replaced the gap-PCR/MLPA/Southern blotting
β-thalassemia
use of Southern blot. Although it is difficult for MLPA to
Reverse dot blot/ARMS/Sanger sequencing
consistently detect trans HBA deletions, MLPA has been
successfully used to describe the deletions of known and new
HBA locus that lead to α-thalassemia [34]. Fig. 39.10 Laboratory diagnosis of thalassemia
made significant progress in improving the treatment of thal- philia B may be less severe than hemophilia A. Haematologica.
assemia, and it is a rapidly developing treatment method in 2015;101:219–25.
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Endocrine and Metabolic Diseases
40
Hong Yuan, Jingyuan Zhao, Erfu Xie, Lujiang Yi,
Zhaojing Zheng, and Juan Geng
The endocrine system is the collection of glands that produce obesity. Endocrine disease is characterized by disordered
hormones that regulate metabolism, growth and develop- hormone release, aberrant response to signal, lack of a gland,
ment, tissue function, sexual function, reproduction, sleep, or structural enlargement in a critical site such as the thyroid.
and mood. In human beings, the major endocrine glands are Hypofunction of endocrine glands can occur as a result of
the thyroid gland and the adrenal glands. In all vertebrates, loss of reserve, hyposecretion, agenesis, atrophy, or active
the hypothalamus is the neural control center for all endo- destruction. Hyperfunction can occur as a consequence of
crine systems. Several glands that signal each other succes- hypersecretion, loss of suppression, hyperplastic or neoplas-
sively are usually referred to as axis. Besides, many other tic change, or hyperstimulation. In this chapter, diseases such
organs that are part of other body systems, including bone, as diabetes, thyroid diseases, dwarfism, adenopathy, and
pancreas, kidney, liver, heart, and gonads, have secondary gout are mainly described.
endocrine functions.
The endocrine system consists of a series of glands that
produce and secrete hormones that the body uses for a wide
40.1 Diabetes
range of functions. These control many different bodily
functions, including respiration, metabolism, reproduction,
Hong Yuan and Jingyuan Zhao
sensory perception, movement, sexual development, and
growth. Hormones are produced by glands and sent into the
bloodstream to the various tissues in the body. They send
40.1.1 Overview
signals to those tissues to tell them what they are supposed
to do. When the glands do not produce the right number of
Diabetes mellitus (DM) is one kind of metabolic diseases
hormones, diseases develop that can affect various aspects
which is characterized by hyperglycemia by virtue of insulin
of life.
deficiency and/or its biological dysfunction. Complications
Diseases of the endocrine system are common, involving
of diabetes affect nearly every tissue of the body [1]. Chronic
conditions such as diabetes mellitus, thyroid disease, and
hyperglycemia leads to various organs or systems dysfunc-
tion or failure including the eye, kidney, and nervous and/or
cardiovascular systems. Without appropriate therapy, many
H. Yuan (*)
Dalian Municipal Central Hospital, Dalian, People’s Republic serious complications may occur, including blurred vision,
of China headache, muscle weakness, poor wound healing, and pruri-
J. Zhao tus. Some acute complications include diabetic ketoacidemia
The First Affiliated Hospital of Dalian Medical University, and high osmolality and hyperglycemic nonketotic coma and
Dalian, Liaoning, People’s Republic of China severe long-term complications such as cardiovascular dis-
E. Xie (*) · L. Yi ease, chronic kidney disease, diabetic foot, stroke, and reti-
Department of Laboratory Medicine, The First Affiliated Hospital nopathy [2]. It is also a complex disease with high frequency
of Nanjing Medical University, Nanjing, Jiangsu, People’s associated with genetic factors. The number of patients is
Republic of China
e-mail: xieerfu@njmu.edu.cn increasing swiftly with the improvement of people’s quality
of life, population aging, lifestyle changes, and the progress
Shanghai Children’s Medical Center, Shanghai Jiao Tong of diagnostic techniques [1].
University School of Medicine, Shanghai, People’s Republic
of China

666 H. Yuan et al.
40.1.2 Classification and Clinical Appearance hemoglobin (A1C) of 6.0–6.4% other than a virtual diseased
status. However, meeting any of these criteria indicating
Most cases of diabetes can be broadly classified as type 1 individual is at high risk of developing diabetes. Literally, “at
diabetes, type 2 diabetes, Gestational diabetes, Monogenic high risk” does not mean “definitely.” Thus, not all people
diabetes syndromes, and other special categories of diabetes, with prediabetes will inevitably progress to diabetes. In fact,
which are summarized in Fig. 40.1. Other specific types of a large proportion of people diagnosed with IFG or IGT will
diabetes like genetic defects, hormones disturbance, drugs or return to normoglycemia. In general, IFG, FPG, and/or A1C
chemicals, infections, and immunity may inflict the normal is the primary tests for prediabetes definition. Of which, A1C
function of β-cells and insulin [3, 4]. Allocating a certain is an essential testing index to reflect a long-term and con-
type of diabetes diagnosis to an individual may depend on tinuous inner glucose level variation and is demonstrated a
the comprehensive clinical manifestation presenting at the tight connection with subsequent diabetes. Patients with pre-
time of diagnosis, whereas many patients cannot be exactly diabetes should be cautious about the high risk of obesity,
integrated into one single category. The clinical manifesta- hypertension, and dyslipidemia with disordered triglycerides
tions of typical types of diabetes are analyzed as follows [5]: and/or low HDL cholesterol, hence should be tested yearly.
40.1.2.1 C ategories of Increased Risk 40.1.2.2 Type 1 Diabetes Mellitus

for Diabetes (Prediabetes) T1DM is a kind of diabetes which is characterized by pan-
“Prediabetes” is a term referring to impaired fasting glucose creatic beta-cell destruction and resultantly definite insulin
(IFG), impaired glucose tolerance (IGT), or a glycated deficiency, which is inclined to ketoacidosis. Type 1 diabetes
Fig. 40.1 Diabetes classification

40 Endocrine and Metabolic Diseases 667
is more common in children and adolescents and is caused ume indicating the interrelationship between IFG and the
by autoimmune beta-cell destruction; insulin is absolutely development of T2DM [6].
deficient. The onset of the disease is more urgent, the symp- In fact, insulin resistance is not invisible apart from the
toms of polydipsia, polyuria, polyphagia, and weight loss are pathological condition when an individual bearing a sleeping
more obvious, often more serious, ketoacidosis tendency. disorder or stress. T2DM may occur when the pancreatic
Usually, sporadic, insulin autoantibodies can be detected, β-cells fail to compensate for the insulin resistance even after
such as GAD antibody, ICA antibody, and IA-2 antibody. upregulating insulin output. However, a genetic abnormality
Gene analyses HLA-DR3 and HLA-D4 are common. Long- is common to be detected in clinical in the T2DM sufferer.
term dependence on insulin therapy is required. Besides, There are existing two hypotheses of the development of
those diabetes cases with tight relationship with autoimmune T2DM. One is glucose toxicity, that is, persistent hypergly-
process yet whose exact etiology of beta-cell destruction is cemia may deplete the insulin secreting granules of β-cells.
still unknown also have been classified into this T1DM. The other one is lipotoxicity, during which free fatty acid
impinges the synthesis as well as the secretion of insulin.
Immune-Mediated Diabetes
This subtype accounts for 5–10% of type 1 diabetes and pre- 40.1.2.4 Gestational Diabetes Mellitus
viously called “insulin-dependent diabetes” or “youth onset In the second or third trimester of pregnancy, the increase in
diabetes of maturity” which results from autoimmune pan- insulin resistance of pregnant women, such as placental pro-
creatic β-cells destruction. There are characteristic pancre- lactin, estrogen, progesterone, and placental insulin, will
atic islet cell autoantibodies and antibodies against glutamate reduce their sensitivity to insulin. During which, the limita-
decarboxylase 65 (GAD65), insulin, ZnT8 and tyrosine tion of insulin secretion of the gravida fails to meet the
phosphatase IA-2/IA-2β. The manifestation of this type of increased requirement of insulin to maintain physical-level
diabetes varies dramatically among slightly nonspecific glucose which results in hyperglycemia, viz. the occurrence
symptom, classic “three polys and one little” symptom, or of gestational diabetes mellitus (GDM).
even diabetic coma. Onset may arise urgently in adolescents GDM usually emerges at the mid or third trimester of
with significant symptoms which tends to develop into dia- pregnancy and get recovery after delivery. Individuals with
betic ketoacidosis (DKA) if no appropriate therapy. For adult GDM may occur macrosomia, polyhydramnios, pruritus vul-
individuals, the onset symptoms at early stage may be vae, and recurrent candida yeast infection yet in absence of
implicit which is called “latent autoimmune diabetes in the classic “three polys and on little” diabetic symptoms. The
adults (LADA)”. Adverse Pregnancy Outcome (HAPO) and Hyperglycemia
study indicated that abnormal high maternal glycemia, which
Idiopathic Type 1 Diabetes can be observed by the level of fasting glucose concentra-
This type of diabetes has been used for diagnosis of some tion, postoral glucose tolerance test (OGTT) glucose concen-
cases of T1DM with no known etiology. Patients within this trations, and glycosylated hemoglobin, has a consistently
type show permanent insulinogenic and incline to DKA adverse influence on the pregnancy outcome.
without β-cell autoimmune antibodies detected. This form of
diabetes is not HLA associated but can be inherited. 40.1.2.5 Monogenic Diabetes Syndromes
Individuals in this category need absolute insulin replace- Monogenic diabetes syndromes are a type of diabetes caused
ment therapy. This type of diabetes is commonly seen in by the mutation of single but pivotal gene mediating islet
adults, especially after the fourth decades, yet can occur at β-cell development and functioning as well as insulin signal-
any age. Insidious onset accompanied by slight or even ing pathway. Monogenic diabetes syndromes normally can
absence of symptoms and seldom develops into DKA spon- be divided into two categories: neonatal diabetes and
taneously. Idiopathic diabetes is commonly defined with maturity-onset diabetes of the young (MODY).
comorbidities like obesity, dyslipidemia, or hypertension.
Neonatal Diabetes
40.1.2.3 Type 2 Diabetes Mellitus Diabetes occurring within 6 months of age can be defined as
This type of diabetes is characterized by insulin resistance neonatal diabetes, of which 80–85% cases have an underly-
and relative insulin deficiency. Commonly, type 2 diabetes ing monogenic defect [7]. Neonatal diabetes mellitus is now
mellitus (T2DM) has a tight connection with obesity and known to occur in approximately 1 in 90,000–160,000 live
metabolic syndrome. For some cases, no overt diabetes births, and more than 20 known genetic have been investigated
symptoms shown by virtue of whose β-cells still can func- for NDM [8]. Neonatal diabetes can be divided into perma-
tion adequately to maintain physical-level glucose via more nent or transient cases. Yet for the transient cases, half cases
insulin secretion to offset the increasing insulin resistance. may encounter recurrence but may be responsive to medica-
Yet individuals with IFG tend to have decreased β-cell vol- tion therapy except for insulin. Permanent cases are mostly
668 H. Yuan et al.
caused by autosomal dominant mutations in genes of β-cell Table 40.2 The criteria for diagnosis of diabetes
KATP channel (Kcnj11 and Abcc8, most frequent). The sec- Any one of the following is diagnostic:
ond cause is insulin gene mutations. Children manifested 1. HbA1c ≥ 6.5% (48 mmol/mol)
diabetes symptoms within 6 months of age may firstly be OR
considered as neonatal diabetes which needs immediate 2. FPG ≥ 7.0 mmol/L (126 mg/dL)
genetic testing. As the Kcnj11 gene also expresses in nerves OR
and muscles, about 20% cases may have nervous system 3. 2-h Plasma glucose ≥11.1 mmol/L (200 mg/dL) during an OGTT
comorbidity, like developmental delay, learning disorder, OR
epilepsy, or muscles weakness [9]. 4. Symptoms of hyperglycemia and casual plasma glucose
≥11.1 mmol/L (200 mg/dL)
Maturity-Onset Diabetes of the Young (MODY)

This type of diabetes has been defined by the appearance of
diabetes symptoms at an early age, usually before 25 years 40.1.3.2 D iagnostic Criteria for Diabetes
old yet clinical diagnosis sometimes maybe later. MODY is Mellitus
characterized by an autosomal dominant inheritance ten- It is sufficient to measure plasma glucose to diagnose diabe-
dency, and the pathologic foundation of MODY is insulin tes in patients with classic symptoms. Knowing the plasma
secretion defect other than insulin function defect. The glucose level is critical for confirming that symptoms are due
most common MODY types are HNF4A-MODY (MODY1), to diabetes. Sometimes, A1C may also be used to know how
GCK-MODY (MODY2), and HNF1A-MODY (MODY3), long a patient has had hyperglycemia.
wherein MODY3, which is caused by the mutation of The age of onset is a considerable implication for this
Hnf1a gene of hepatocyte nuclear factor (HNF), is the most diagnosis. Besides, diagnosis can be made for patients with
common seen monogenetic diabetes syndrome. Symptoms classic symptoms of hyperglycemia and a random blood glu-
may vary by the very type of MODY. For MODY3 cases, cose ≥200 mg/dL (11.1 mmol/L). The diagnostic criteria for
the main symptom is postprandial hyperglycemia, but sel- diabetes are summarized in Table 40.2.
dom DKA occurs, while for MODY2, the fasting hypergly-
cemia is always stable which do not need hypoglycemia 40.1.3.3 Gestational Diabetes Mellitus
therapy in general. Like MODY3, MODY1 cases often It is critical to identify pregnant women with GDM, as early
accompanied with macrosomia, hyperinsulinemia, and appropriate therapeutics can effectively decrease both mild
lower triglyceride level. and severe pregnancy-related complications. Of note, preg-
nant women diagnosed with diabetes during the first trimes-
ter of pregnancy should be considered as having preexisting
40.1.3 Diagnosis pregestational diabetes. GDM diagnosis can be made by the
following two strategies [10] (Table 40.3).
40.1.3.1 Prediabetes
IFG is defined as the FPG level within a range from 100 to
125 mg/dL while IGT as 2 h PG during 75 g OGTT levels 40.1.3.4 Monogenic Diabetes
within a range from 140 to 199 mg/dL, viz. 7.8– In general, monogenic diabetes manifests typically within
11.0 mmol/L. The threshold of IFG defined by the World the first 6 months of age and is difficult to distinguish from
Health Organization (WTO) is 110 mL/dL (6.1 mmol/L). type 1 diabetes only by the clinical manifestation. This type
The diagnosis can be made with the presence of IFG and/or of diabetes responses well to oral sulfonylurea to replace
IGT and/or A1C 5.7–6.4% (39–47 mmol/mol). Prediabetes insulin therapy. Therefore, children diagnosed as diabetes
is diagnosed by any of the following criteria (Table 40.1). within 6 months of age should accept neonatal diabetes
genetic testing. Besides, all people with a diagnosis of type 1
diabetes should be reviewed to determine if the diagnosis
Table 40.1 Categories of increased risk for diabetes (prediabetes)a occurred prior to 6 months of age, and if so, genetic testing
FPG 100 mg/dL (5.6 mmol/L) to 125 mg/dL (6.9 mmol/L) (IFG) should be performed. In addition, individuals diagnosed as
OR diabetes in children or early adulthood without classic fea-
2-h PG during 75-g OGTT 140 mg/dL (7.8 mmol/L) to 199 mg/dL tures of T1DM or T2DM, especially have a continuous fam-
(11.0 mmol/L) (IGT) ily history of early onset diabetes, should get MODY related
OR
genes test. Moreover, biomarkers screening, such as urinary
A1C 5.7–6.4% (39–47 mmol/mol)
C-peptide/creatinine ratio, and antibody screening, may be
a
For all three tests, risk is continuous, extending below the lower limit
of the range and becoming disproportionately greater at the higher end
also helpful in determining the potential candidates for
of the range MODY genetic testing [10].
Table 40.3 Diagnostic strategy for GDM Table 40.4 WHO criteria for interpreting 2-h OGTT
1. “One-step” 75-g OGTT or 2-h OGTT result, mmol/L (mg/dL)
2. “Two-step” approach with a 50-g (nonfasting) screen followed by 0h 2h
a 100-g OGTT for those who screen positive Impaired fasting 6.1 (110) to <7.0 <7.8 (140)
One-step strategy glucose (126)
Perform a 75-g OGTT, with plasma glucose measurement when the Impaired glucose <7.0 (126) <11.1 (200)
patient is fasting and at 1 and 2 h, at 24–28 weeks of gestation in tolerance
women not previously diagnosed with overt diabetes. Diabetes >7.0 (126) <11.1 (200)
The OGTT should be performed in the morning after an overnight
fast of at least 8 h.
The diagnosis of GDM is made when any of the following plasma
glucose values are met or exceeded: merular filtration rate reduced, and the renal sugar threshold
a) Fasting: 92 mg/dL (5.1 mmol/L) increased. At this time, although the blood sugar is elevated,
b) 1 h: 180 mg/dL (10.0 mmol/L)
the urinary sugar is a false negative. Conversely, if the renal
c) 2 h: 153 mg/dL (8.5 mmol/L)
Two-step strategy sugar threshold is decreased (such as pregnancy), urinary
Step 1: sugar can be positive, while the blood sugar is normal.
Perform a 50-g GLT (nonfasting), with plasma glucose
measurement at 1 h, at 24–28 weeks of gestation in women not 40.1.4.3 Glucose Tolerance Test
previously diagnosed with overt diabetes.
OGTT is required when blood glucose is above the normal
If the plasma glucose level measured 1 h after the load is >130 mg/
dL, 135 mg/dL, or 140 mg/dL (7.2 mmol/L, 7.5 mmol/L, or range and does not meet the cutoff for diabetes diagnosis.
7.8 mmol/L), proceed to a 100-g OGTT. OGTT should be carried out in the early morning. The WTO
Step 2: recommends that adults take 75 g glucose which dissolved in
The 100-g OGTT should be performed when the patient is fasting.
250–300 mL of water after 5 min, and venous blood glucose
The diagnosis of GDM is made if at least twoa of the following four
plasma glucose levels (measured fasting and 1 h, 2 h, 3 h during was measured after 2 h. WHO criteria for interpreting 2-h
OGTT) are met or exceeded. OGTT is shown in Table 40.4. Normal people can reach the
Carpenter- or NDDG (74) highest blood sugar level 1 h after taking the sugar and return
Coustan (73) to normal after 2 h. If the blood sugar still exceeds the nor-
a) Fasting 95 mg/dL 105 mg/dL mal level after 2 h, then it indicates that the glucose tolerance
b) 1 h (5.3 mmol/L) (5.8 mmol/L)
c) 2 h 180 mg/dL 190 mg/dL reduced. The intravenous glucose tolerance test is only suit-
d) 3 h (10.0 mmol/L) (10.6 mmol/L) able for postgastric resection, gastrojejunostomy, malabsorp-
155 mg/dL 165 mg/dL tion syndrome, or as a clinical research tool for evaluating
(8.6 mmol/L) (9.2 mmol/L)
glucose utilization. Dysglycaemia detected in an OGTT is
140 mg/dL 145 mg/dL
(7.8 mmol/L) (8.0 mmol/L) also a useful marker in the prediction of onset of type 1 dia-
NDDG National Diabetes Data Group betes in high-risk children [12].
a
ACOG recently noted that alternatively one elevated value can be used
for diagnosis 40.1.4.4 Glycated Hemoglobin (HbA1c)
Glycated hemoglobin is a slow, sustained, and irreversible
40.1.4 Laboratory Diagnosis nonenzymatic glycation product of hemoglobin and glucose
in red blood cells, which is difficult to separate in 2 weeks.
40.1.4.1 Blood Glucose When the blood glucose increases, the glycated hemoglobin
High blood sugar is the main basis for the diagnosis of dia- formed will be relatively high. Under normal physiological
betes. Glucose is considered as the predominant biomarker conditions, the amount of nonenzymatic glycation reaction
for hyperglycemia [11]. Plasma, serum, or whole blood can product is indirectly proportional to the concentration of the
be used. Plasma and serum blood glucose should be 15% reactants. Since the protein concentration remains relatively
higher than whole blood glucose. It is recommended to use stable, the level of saccharification is primarily determined
venous plasma for specific patients for diagnosis. The nor- by the glucose concentration and the time of the protein in
mal range is 3.9–5.6 mmol/L. Blood glucose measurement is contact with glucose. The lifetime of red blood cells in
also a major indicator of diabetes status and control. humans is generally 120 days. The glycated hemoglobin
content in the blood will remain relatively unchanged until
40.1.4.2 Urinary Glucose red blood cells die. Therefore, the glycated hemoglobin level
Urinary glucose positive is an important clue to the diagnosis reflects the average blood glucose level within 120 days
of diabetes, but urinary glucose negative cannot exclude the before the test, and it is a good indicator of long-term control
possibility of diabetes. When glomerulosclerosis, the glo- of diabetes [13].
670 H. Yuan et al.
40.1.4.5 Ketone Body insulin. Insulin cannot be converted to insulin in pancreatic

The ketone body is a general term for acetoacetic acid, β-cell tumors, rare familial hyperinsulinemia, leading to
β-hydroxybutyric acid, and acetone, which is a product of greatly increased in insulin and resulting in hypoglycemia.
the fat decomposition process. In the case of glucose metab- In patients with type 2 diabetes, both the proinsulin-to-
olism disorder, fat catabolism is strengthened, and ketone insulin ratio and insulin-converting intermediates are
body production is remarkably increased. When the produc- increased and are associated with cardiovascular risk factors.
tion of ketone bodies in the liver exceeds the utilization In addition, the increased proportion of proinsulin in insulin-
capacity of extrahepatic tissues, the blood ketone bodies can like substances can be used as an indicator for screening for
be increased, which is called ketosis. If ketosis occurs in the gestational diabetes, which is better than age, obesity, and
urine, it is called ketouria. The urine ketone body is negative hyperglycemia.
in normal urine, the presence of the urinary ketone body
indicated that type 1 diabetes in newly occurring patients and Glucagon
the curative effect is invalid, or important complications The secretion of glucagon is regulated by nutrients, auto-
occur in patients with type 2 diabetes or being treated. nomic nerve, islets, and gastrointestinal hormones. When
patients with alpha-cell carcinoma, diabetes, chronic pancre-
40.1.4.6 Lactic Acid and Pyruvic Acid atitis and obesity, etc., use adrenal hormones and growth hor-
Lactic acid is an intermediate in sugar metabolism; increased mone glucagon will reduce.
lactic acid in the blood refers to the enhanced lactic acid
action of the pyruvate anaerobic glycolysis pathway in the 40.1.4.8 Urinary Microalbumin
catabolism of glucose during severe hypoxia. Pyruvate is The urine protein is defined as positive when urinary albu-
derived from red blood cells, muscles, and various tissue min exceeds 300 mg/24 h. Detection of urinary microalbu-
cells. Normal lactate/pyruvate ratio was 10:1, in dynamic min is the earliest evidence of a risk of kidney and
equilibrium. cardiovascular disease, and large amounts of albuminuria
(>300 mg/24 h) suggest an increased risk of progression
40.1.4.7 Blood Glucose Regulator from kidney-related disease and kidney disease in the end
Insulin stage. To accept albuminuria test annually is recommended
Insulin is secreted by pancreatic β-cells, and plasma insulin by all major guidelines for patients with diabetes and/or kid-
can reflect the function of islet β-cells. Elevated plasma insu- ney disease, especially the postpubertal individuals in 5
lin can be seen in type 2 diabetes patients that are often years after diagnosis of type 1 diabetes and type 2 diabetes
obese, with hyperinsulinemia in the early and middle stages, patients at the time of diagnosis, regardless of treatment.
insulin β-cell tumor, insulin autoimmune syndrome, and
pituitary hypofunction. 40.1.4.9 Genetic Test
The clinical value of genetic markers in the current assess-
Insulin Release Assay ment and management of diabetic patients is very limited,
Insulin secretion includes basal secretion and stimulated but the mutational analysis is rapidly evolving in the classifi-
secretion. In the OGTT test, plasma insulin levels are mea- cation of the neonate and young patients with a dominant
sured at the same time to understand the function of pancre- family history of diabetes. The sequencing of the human
atic β-cells. Glucose-stimulated insulin release assay is an genome raises potent advantages to identify the genetic bases
important method to verify the function of islet β-cell secre- for both type 1 and type 2 diabetes.
tion, the number of β-cells, and the presence or absence of
insulin resistance. Type 1 Diabetes
Nearly 50% of the heritability of type 1 diabetes can be
C-Peptide ascribed to human leukocyte antigen (HLA) alleles [14].
The C-peptide is a polypeptide formed when the proinsulin Type 1 diabetes is closely related to HLADR12 (major histo-
is degraded, which is secreted by an identical molecule compatibility complex, class II, DR) and HLA-DQ (major
secretion as insulin, and the measurement also reflects the histocompatibility complex, class II, DQ) genes. HLA-
function of the islet β-cells. The determination of blood insu- DQA1 and HLA-DQB1 genotypes can be used as a predic-
lin and C-peptide is very critical for distinguishing type 1 tion index for diabetes development. HLA DQA1 * 0301,
and type 2 diabetes and guiding treatment. DQB1 * 0302, and DQA1 * 0501-DQB1 * 0201 haplotypes,
either alone or in combination, can be observed in almost
Proinsulin 90% of children with type 1 diabetes. Up to now, HLA-DQ
Proinsulin is a form of insulin stored in the body, and the and HLA-DR genetic factors are the foremost risk predic-
biological activity of proinsulin is quite low, about 10% of tions of type 1 diabetes. Although cannot be regarded as rou-
tine clinical diagnosis or classification, HLA-DR/DQ typing patients, still there is about 1–2% of healthy individuals are
still is useful to increase or decrease the presentation poten- associated with low risk of T1DM development yet have a
tial of type 1 diabetes in persons whose islet cell autoanti- certain autoantibody against insulin, GAD65, IA-2, or ZnT8.
bodies test is positive [15]. In addition, Non-HLA genetic The presence of multiple forementioned islet cell autoanti-
factors, including INS [protein tyrosine phosphatase, in bodies indicates extremely high risk (up to 90%) to develop
receptor type 22PTPN22(lymphoid)] and CTLA4 (cytotoxic T1DM.
T lymphocyte-associated protein 4) genes, also play a auxil-
iary role in diagnosis of those cases belonging to type 1 dia- Anti-Insulin Antibodies (IAA)
betes but with uncertain etiology. Notwithstanding screening There are two kinds of IAA; one is an autoimmune antibody
newborn children to identify their T1DM risk cannot be rou- that related to the onset of diabetes and existed before the
tine; if possible, it can really help determine the children onset of diabetes. The other is produced after the use of
with higher risk to develop into type 1 diabetes [16]. exogenous insulin during diabetes. When type 1 diabetes
begins, the positive rate of IAA can reach 30–40%. However,
Type 2 Diabetes the significance of IAA alone is very limited and combined
Less than 5% of patients with T2DM can be explained on with ICA and GADA can increase the predictive value of
the basis of molecular genetics, and most of these patients ICA for type 1 diabetes.
are accompanied by autosomal dominant or high insulin
resistance. Recently, GWAS have identified over 30 genetic Anti-Islet Cell Antibody (ICA)
factors that increase the risk of T2DM. However, the risk Anti-islet cell antibody is a general term for all antibodies
alleles in these loci predictive effects all are relatively too against pancreatic islet β-cells. It is currently believed that
small to significantly help predict the risk of T2DM devel- ICA positive predicts that autoimmune damage of β-cells,
opment [17]. which can only be used as a high-risk indicator of diabetes.
Sustained positive or high levels in children, it is highly pre-
Monogenic Diabetes dictive of type 1 diabetes.
The regimens of treatment and prognosis for monogenic
forms of neonatal diabetes mellitus deeply depend on which Antiglutamate Decarboxylase Antibody (GADA)
gene is affected. Advances in genetic testing make it avail- Type 1 diabetes is an autoimmune disease in which islet
able to take more efficient and comprehensive testing for β-cells are destroyed by genetically susceptible individuals
patients. It is technically feasible to detect mutations in through their own antigen-mediated immune responses.
maturity-onset diabetes mellitus of the young (MODY) GAD is the key target antigen for this immune response.
patients and their relatives. Eight different MODYs have Therefore, GAD-Ab is a relatively specific immune index for
been identified, including the mutations in the genes encod- prediabetes individuals. It can be used as a predictor of type
ing transcription factors, the GCK [glucokinase (hexokinase 1 diabetes and a census to identify high-risk groups and indi-
4)] gene, and the CEL [carboxyl ester lipase (bile salt- viduals with type 1 diabetes.
stimulated lipase)] gene. The emerging new technologies
and reduced costs of sequencing make it possible that the Islet Tumor-Associated Antigen-2 Autoantibody (IA-2)
detection of specific diabetes mutations will become Islet tumor-associated antigen (IA-2) is an important islet
routine. cell autoantigen discovered in recent years. IA-2 autoanti-
bodies are present in 60–80% of newly diagnosed patients
40.1.4.10 Autoimmune Markers with type 1 diabetes, while positive rates are approximately
Type 1 diabetes is mainly caused by insufficient insulin 1% in healthy controls and type 2 diabetes patients. IA-2 and
secretion mediated by an autoimmune attack of islet β-cells. its antibodies have important clinical application value for
Autoantibodies are present in most patients, either alone or pathogenesis, diagnostic typing, predictive screening, and
together, at some stage of the disease process, which is an early prevention and treatment of type 1 DM.
important marker of islet cell and function destruction. Islet
cell autoantibodies are composed of autoantibodies to native 40.1.4.11 Other
insulin, namely “insulin autoantibodies” (IAA), to islet cell The poor control of diabetes can accompany varying degrees
cytoplasm (ICA), to the 65-kDa isoform of glutamic acid of dyslipidemia, and blood fat should be detected.
decarboxylase (GAD65A), to two insulinoma antigen 2 pro- Electrocardiogram and echocardiography are used to under-
teins (IA-2A and IA-2βA), and to three variants of zinc stand cardiac function. Ophthalmologic examination is used
transporter 8 (ZnT8A). Although it can be helpful in recog- to determine if there is retinopathy caused by diabetes [18].
nizing individuals at high risk of T1DM development to Besides, researchers have tried to establish close relations
screen the islet cell autoantibodies of relatives of T1DM with alternative biomarkers such as glucose in sweat, tears,
672 H. Yuan et al.
saliva [19, 20], methylglyoxal (MG) [21], lipid metabolo- 40.1.6 Conclusion
mics, amino acids, and palmitoleate for the prediction of
types of DM [14]. Diabetes mellitus is a group of disorganized glycometabo-
lism caused by diverse etiologies. Till now, diagnosis and
categorization of certain type of diabetes mellitus are primar-
40.1.5 Management ily made based on the biomarkers, such as blood glucose,
urine glucose, and glycated hemoglobin. Besides, conven-
The development of evidence-based medicine has pro- tional tests like fasting plasma glucose, oral glucose toler-
moted the transformation of diabetes treatment concepts, ance, and postprandial plasma glucose criteria are still
which changed from traditional treatment to system man- indispensable for the diagnosis of diabetes mellitus. The
agement. The best management model is patient-centered diagnostic process of suspected diabetic patients is shown in
team management, including general and specialist physi- Fig. 40.2. Moreover, diet and lifestyle have a great influence
cians, diabetes instructors, dieticians, exercise rehabilita- on glucose levels, which may result in more complications in
tion, and patients and their families and establishing regular the assessment of diabetic conditions. Therefore, equal
follow-up and evaluation systems. Effective control of emphasis on keeping a healthy diet and physical exercise
blood glucose in early diabetic patients can delay the occur- should be put on the diabetic treatment as maintaining blood
rence and development of diabetic microangiopathy and glucose normally.
protect large blood vessel in a long-term and can keep the
β-cell function and improve insulin sensitivity.
Comprehensive control of risk factors for T2DM can sig- 40.1.7 Typical Medical Case
nificantly reduce the accident of macrovascular and micro-
vascular disease. Integrated diabetes management mainly Clinical Background A 51-year-old woman is 1.61 meters
includes diabetes education, medical nutrition therapy, tall and weight 63 kilograms. Symptoms including polydip-
exercise therapy, blood glucose monitoring, and medica- sia and fatigue lasted for more than 4 years. Four years ago,
tion treatment [22]. she was diagnosed as “diabetes” by a local hospital for treat-
Diabetes health education includes cultivation of diabe- ment; the condition control is relatively stable by taking the
tes prevention professionals, continuous education for medicine regularly. One month ago, the patient felt thirsty,
medical staff, and healthcare education for patients and the polydipsia, and fatigue was significantly aggravated. Fasting
relatives. In order to ensure that every diabetic patient blood glucose was significantly higher than before. After the
fully understands diabetes and master the self-manage- treatment with hypoglycemic agents, the symptoms did not
ment skills. Medical health treatment is an important part relieve significantly.
of comprehensive management, correcting metabolic dis-
orders to keep good metabolic control, reducing risk fac- Initial Laboratory Values FPG: 8.9 mmol/L, P1hPG:
tors for CVD, providing optimal nutrition to improve 16.3 mmol/L, P2hPG: 17.5 mmol/L, P3hPG: 16.2 mmol/L;
patient health, and slowing down the progression of beta- Urine routine: WBC 5–10/HP; Blood routine: WBC
cell dysfunction. The general principle is to determine a 1.5 × 109/L, neutrophils 0.8, lymphocytes 0.2.
reasonable total energy intake and rational and balanced
nutrients distribution and maintain the ideal constitution. Other Test Liver function, kidney function, blood lipids,
Exercise therapy also plays a pivotal role in diabetes man- hepatitis B virus, and urine microproteins are normal. chest
agement, especially in obese T2DM patients because exer- X-ray, electrocardiogram, liver and gallbladder spleen, and
cise is able to increase the endosomatic sensitivity to kidney B-ultrasound did not appear abnormal.
insulin and help maintain normal blood glucose level and
body weight. In addition, conditions test should be per- Physical Examination No abnormality in the physical
formed in patients with diabetes, including fasting blood examination.
glucose, postprandial blood glucose and HbA1c, and other
CVD risk factors and complications. Hypoglycemic agents Results with Interpretation Guideline Diabetes is a com-
should be applied promptly when diet and exercise cannot plex chronic and difficult disease. During treatment, it often
achieve glycemic control. Moreover, patients with a T1DM happens that the blood sugar suddenly increases significantly
with more than 5 years and all T2DM patients should be in the stable original hypoglycemic drugs, and the blood
screened for chronic complications every year after sugar fluctuations obviously enhanced. At this time, blood
diagnosis. sugar fluctuations should be considered. More common
Fig. 40.2 Diagnosis procedure of suspected diabetes
causes are infection, mood swings, fatigue, and insomnia. patient’s condition was good, the blood glucose was within
Infection is the most typical among them; women are par- the normal range. Glucose tolerance is normal after repeated
ticularly susceptible to urinary tract infections due to their review and no symptoms at all.
own physiological characteristics. In this patient, hypoglyce-
mic drugs are not always effective against increasing blood This case was from the First Affiliated Hospital of Dalian
sugar. Antibiotics are very crucial; blood sugar will naturally Medical University.
decline after the infection is eliminated. Therefore, an
increase in blood glucose fluctuations always indicates that
there is an incentive for increasing blood sugar fluctuations. 40.2 Thyroid Disease
At this point, the cause should be identified and treated,
instead of blindly adding hypoglycemic drugs. Erfu Xie and Lujiang Yi
Final Diagnosis (1) Type 2 diabetes, (2) Urinary infection. The thyroid gland is a butterfly-shaped gland located above
the trachea in the front of the neck.
Treatment “Xiaoke Pills” (traditional Chinese medicine Fully developed human thyroid gland consists of two
preparation containing glibenclamide as main ingredient) lobes composed of two lobes connected by a thin layer of
five capsules, 3 times/day, Metformin 250 mg, 3 times/day, tissue Gap, glands like a butterfly. The secretory units of the
ofloxacin 0.2, 2 times/day. Instruct patients to eat scientifi- thyroid gland are follicles that consist of an outer epithelial
cally and exercise moderately. cell layer. These cells rest on a basement membrane and
enclose amorphous materials call colloid. This section cov-
Treatment Outcome FPG: 5.3 mmol/L, P1hPG ers structure, function, and laboratory of thyroid and to be
7.8 mmol/L, P2hPG 7.6 mmol/L, P3hPG 5.2 mmol/L. The followed by different thyroid diseases including nontoxic
674 H. Yuan et al.
goiter, thyroid nodule, thyroiditis, hypothyroidism, and degradation of cholesterol and triglycerides, and an
hyperthyroidism, thyroid carcinoma, and the molecular diag- increasing demand for vitamin, increases calcium and phos-
nostic tests of these diseases. This section mainly introduces phorus metabolism, adrenal hormone receptors more sensi-
the conventional molecular biomarkers used to diagnose thy- tive to these substances. These effects are usually amplified
roid diseases. in patients with hyperthyroidism; it is reduced in patients
with hypothyroidism.
Thyroid function tests are the most common endocrine
40.2.1 Overview practice diagnostic evaluation [25]. Thyroid function tests are
used as screening tools to verify the clinical diagnosis of
Thyroid gland is located in the front lower part of the neck, hyperthyroidism and hypothyroidism, to assess the adequacy
at the fifth, sixth, and seventh vertebrae level. It is a bilobed of medications and to follow up on differentiated thyroid can-
organ joined by an isthmus and encased in a thin fibrous cap- cer. These tests include the hypothalamic–pituitary–thyroid
sule. The right and left lobes have a somewhat conical shape axis, assessment of radioactive iodine uptake tests, determi-
with convex anterior and lateral surfaces. Anatomy of the nation of urinary iodide and serum thyroglobulin, and studies
thyroid gland is shown in Fig. 40.3. The isthmus is about of variables suggesting thyroid autoimmunity. These tests,
1 cm in greatest transverse and vertical dimensions. such as T3, T4, TSH, TRH, FT4, FT3, TG-Ab, TPO-Ab, and
Knowledge of the embryologic development of the thyroid is TR-Ab, are widely used in the thyroid evaluation [26–33].
essential for understanding a number of abnormalities of the Moreover, as molecular genetics has penetrated all fields
thyroid gland [23, 24]. of medicine and impacts more and more significantly on the
Thyroid hormone has many important biological roles. diagnosis, we summed the full molecular makers of thyroid
Their main functions are mitochondrial metabolism and diseases in this section, including nontoxic goiter, thyroid
membrane transport, which enhance the basal metabolic nodule, thyroiditis, hypothyroidism, hyperthyroidism and
rate. The basal metabolic rate is controlled by increasing the thyrotoxicosis, toxic adenoma and multinodular toxic goiter,
amount of oxygen in the tissue by thyroid hormone and and thyroid carcinoma. The detail information can be seen in
fever. Thyroid hormone for neural development in mammals Fig. 40.4.
is essential for normal growth and sexual maturation. Other
effects include increased heart rate and myocardial contrac-
tility stimulation of adrenergic activity, stimulate carbohy- 40.2.2 Thyroid Carcinoma
drate metabolism and protein synthesis, increased synthesis
40.2.2.1 Classification of Thyroid Carcinoma
Thyroid carcinoma (TC) is classified according to the cell
type of origin (C cells or follicular cells) and expression of
differentiation characteristics [34]. TCs produced by
neuroendocrine-producing C cells are called medullary thy-
roid carcinoma (MTC), and TCs produced by endoderm-
Superior derived follicular cells are called nonmyeloid carcinoma
thyroid artery
(NMTC). NMTC further broke down into four main types
Larynx (PTC, FTC, PDTC, ATC). Papillary (PTC) and follicular
(FTC) TC, as well as other rare types, such as Hürthle cell
(phagocyte) cancer of HCC, belong to the differentiated thy-
roid cancer (DTC) category. Overall, PTC is the most com-
Thyroid gland
mon subtype of TC. Poorly differentiated (PDTC) and
anaplastic (ATC) TC cancer are rare subtypes, which are
Common characterized in a partial or total loss of differentiation.
carotid artery Relative incidence of major types of thyroid cancer in the
USA is shown in Fig. 40.5a. Histological classification of TC
Trachea is closely related to disease outcome. DTC is usually associ-
ated with good prognosis and long-term survival >90%,
although up to 30% of patients may relapse after initial treat-
Inferior ment. ATC has rapid onset and fulminant disease progres-
thyroid artery sion, with an average survival of only 6 months [35]. PDTC
behaves between DTC and ATC, with an average survival
Fig. 40.3 Anatomy of the thyroid gland time of about 3 years.
Fig. 40.4 Molecular markers of thyroid diseases
40.2.2.2 Differentiated Thyroid Carcinoma pected when the nodule is a single nodule in other normal
Characteristic of DTC thyroid glands, in children or adolescents, in men, or in asso-
Differentiated thyroid carcinoma (DTC) includes papillary ciation with ipsilateral lymphadenopathy, especially in pre-
and follicular tissue and variants, more than 90% of all thy- vious cases of external radiation. Childhood exists.
roid cancers. Over the past 30 years, the incidence of thyroid Regardless of the manifestation, the final diagnosis of malig-
cancer has increased in many countries, and it is the fastest nant tumors must rely on the results of FNAC. Although thy-
growing human solid cancer worldwide. Papillary and fol- roid ultrasonography cannot distinguish between benign and
licular thyroid cancer usually have a good prognosis, with a malignant lesions, it can be used to stratify malignant tumors
total mortality rate of less than 10%. This excellent progno- in thyroid nodules and help determine the need for FNAC.
sis is the result of combining the biological characteristics of Most papillary thyroid carcinomas are clinically inert,
most thyroid cancers with effective primary therapies. consistent with their simple genomes, and their copy number
Compared to other malignancies, thyroid cancer is probably changes rarely. Papillary thyroid carcinoma has one of the
the most curable cancer, with high long-term survival rates, lowest cancer mutation densities studied by whole exome
at least in highly differentiated tissue types. However, some sequencing. Although previously thought to be a single
patients are at high risk of recurring disease or even death. entity, papillary thyroid carcinoma encompasses several
Most of these patients can be identified at the time of diagno- tumor types that have mutations in mutually encoded effec-
sis using clear prognostic indicators. tor genes that signal through a mitogen-activated protein
kinase (MAPK) pathway. BRAF V600E accounts for 60% of
Molecular Marker of DTC these mutations, followed by RAS (15%) and chromosomal
Classical Molecular Marker of DTC rearrangement, resulting in illegal expression of the BRAF
The most common manifestation of DTC is the discovery kinase domain or receptor tyrosine kinases (such as RET,
of a cervical thyroid nodule in the USA that occurs in non- NTRK, and ALK) (12%). The remaining 13% mostly have
thyroid or benign thyroid disease. Positive cytology of the no known driver mutations; a subgroup has copy number
nodule confirmed malignancy. Sometimes, especially in abnormalities but no discrete recurrent genetic lesion.
children, one or more metastatic cervical lymph nodes may Different driver gene mutations are associated with different
be the first sign of the disease. Carcinoma should be sus- histological variations of papillary thyroid carcinoma
676 H. Yuan et al.
Fig. 40.5 Pathological Anaplastic thyroid

spectrum of thyroid cancer. carcinoma Thyroid Carcinomas
Poorly differentiated thyroid 1% Hürthle-cell carcinoma
Panel (a) shows the relative carcinoma 2%
incidence of thyroid cancer in 6%
Other
the US major types. Panel (b) Medullary thyroid carcinoma 1%
shows the relative frequency 4%
variation of the pathology of Follicular thyroid carcinoma
papillary thyroid cancer 2%
Papillary thyroid carcinoma

84%
Papillary Thyroid Carcinoma
Follicular variant,
encapsulated without
invasion(RAS)
Follicular variant,
18%
infiltrative(BRAF>RAS)
Microcarcinoma
6%
(all variants
35%)
Tall cell(BRAF)
7%
Classical cariant
(BRAF>RTK fusions)
34%
(Fig. 40.5b) and confer different patterns of gene expression, of genes required for iodide incorporation. RAS-mutated
signaling, and clinical characteristics. Classical or high-cell papillary thyroid carcinoma is associated with papillary thy-
variant papillary thyroid carcinoma with BRAF mutations roid follicular variation.
has a higher rate of lymph node metastasis and recurrence Tumor specific protein (SP70): A Novel Molecular
after thyroidectomy. These cancers also respond poorly to Marker of PTC
radioiodine therapy. Their tolerance to radioactive iodine SP70, a newly developed tumor marker, have been already
appears to be due to the high MAPK pathway output driven identified positively associated with nonsmall cell lung can-
by BRAF V600E oncoprotein, which inhibits the expression cer [NSCLC] [36]. Expression of SP70 was higher in patients
Table 40.5 Clinicopathological characteristics of PTC patients P<0.0001

grouped by SP70 levela 100
SP70 (ng/mL) P-value
Gender 80 P<0.0001
Male 9.4 (8.7, 13.2) 0.656
Female 9.6 (8.0, 11.4)
SP70(ng/ml)
Age 60
≤45 9.6 (8.2, 11.3) 0.936
>45 9.5 (7.7, 11.7) 40
Tumor size
≤10 mm 9.2 (7.5, 10.8) 0.005 20
>10 mm 10.1 (8.6, 12.5)
Metastasis
Yes 10.1 (8.8, 11.9) 0.044 0
No 9.4 (7.6, 11.2)
T
PC
H
BT
Distribution
Unilateral thyroid 9.4 (7.8, 11.2) 0.041
Fig. 40.6 Serum SP70 levels are increased in PTC patients
Bilateral thyroid 10.4 (8.9, 13.4)
Type-B ultrasonic
TI-RADS 0-4a 7.4 (4.9, 9.9)
of MTC may be associated with other endocrine tumors,
TI-RADS 4b-6 9.3 (6.5, 12.1) 0.000 such as pheochromocytoma (PHEO) and/or parathyroid
Source: Research from Lujiang Yi, Erfu Xie, Shiyang Pan, etc. (The
a multiple adenoma disease (PTHAd). According to the phe-
First Affiliated Hospital of Nanjing Medical University, Nanjing, notype, three different syndromes can be distinguished: type
China) 2A multiple endocrine tumor (MEN), which is characterized
Detection method: ELISA (polyclonal antibody coating); Cutoff value: by the association of MTC, PHEO, and PTHAd; MEN2B,
≤7.5 ng/mL
where MTC and PHEO are related to other nonendocrine
diseases, such as multiple mucosa Neuromas, Muffin-like
with NSCLC and that the percentage of SP70 increased in
inertia, and megacolon; the family form of MTC (FMTC)
higher tumor stage patients. Furthermore, serum SP70 levels
and hereditary MTC are not associated with other tumor
are well differentiated between benign thyroid nodules and
formation.
thyroid cancer. There were 410 participants in the study,
Like all thyroid tumors, the clinical manifestations of
including 100 healthy controls (SP70 7.0 ± 1.4 ng/mL), 130
MTC are represented by thyroid nodules, whether isolated or
benign thyroid nodules (SP70 8.2 ± 1.7 ng/mL), and 180
multinodular goiter. Cytological diagnosis has not always
papillary thyroid carcinomas [SP70 9.1 (7.3, 10.8)]; the dif-
been straightforward and can be facilitated by measuring
ference between the three groups is obvious. When the cutoff
serum calcitonin (Ct), which is the most specific and sensi-
value was 9.3 ng/mL, the sensitivity (55%) and specificity
tive serum marker of MTC when> 100 pg/mL. When the
(81%) of thyroid papillary carcinoma were excellent. Serum
tumor is still in the thyroid gland, early diagnosis of MTC is
SP70 levels in PCT patients are significantly increased in
needed to completely cure the patient with the first surgical
tumor size (P = 0.005), lymph node metastasis (P = 0.044),
treatment. The presence of distant metastases at diagnosis
and tumor distribution (P = 0.041) (see in Table 40.5). Serum
and the presence of somatic RET mutations in tumor tissues
SP70 level was significantly higher in PCT patients with
is the most important prognostic factors for poor prognosis.
advanced stage than in those with low stage (P = 0.0001)
MTC is a very rare thyroid tumors, its origin is located
(see in Fig. 40.6). SP70 immunohistochemical is positive in
next to the thyroid follicular or C cells calcitonin, but differ-
thyroid tissue comparing the normal thyroid tissue (see in
ent from follicular cells. Unlike follicular cells, C cells are of
Fig. 40.7). SP70 combined with B-ultrasound grading
neuroendocrine origin; they do not respond to thyroid-
increases the detection rate of PTMC to 20%. SP70 can be
stimulating hormone (TSH), do not produce thyroglobulin
used as a novel biomarker for thyroid carcinoma, especially
(Tg), and cannot take up iodine. However, a new theory on
in papillary thyroid carcinoma.
the origin of the endoderm of mammalian C cell progenitor
cells has recently been proposed, and the expression of
40.2.2.3 Medullary Thyroid Carcinoma
E-cadherin is consistent with the origin of mesenchyme dif-
Clinical Presentation of Medullary Thyroid Carcinoma
ferent from neural c sources, which seems to support this
Medullary thyroid carcinoma (MTC) is a rare neuroendo-
hypothesis.
crine tumor that may be sporadic or familial. In both cases,
In contrast to papillary thyroid cancer (PTC) and follicu-
the pathogenesis is due to somatic or germ line constitutively
lar thyroid cancer (FTC), no differences in gender distribu-
activating mutations in the RET oncogene. The familial form
tion were observed. The clinical manifestations mainly
678 H. Yuan et al.
a b
c d
Fig. 40.7 Expression of SP70 in thyroid tissue. (a) Papillary thyroid carcinoma (HE, ×200), (b) Papillary thyroid carcinoma (Immunohistochemistry,
×200), (c) Benign thyroid disease (HE, ×200), (d) Benign thyroid disease (Immunohistochemistry, ×200)
appeared in the fourth and fifth decades. In the last 30 years, been recognized. Depending on the sporadic or familial
the average age at diagnosis increased from 50 to 54 years, nature of MTC, RET mutations can be somatic or germline.
but it was statistically significant. Children are rarely Spread forms are the most common (75%), while genetic or
affected, and when this happens, the likelihood of facing a familial forms account for the remaining 25%. The genetic
family/hereditary form is very high. Ethnic or environmental form is an autosomal dominant hereditary syndrome with
risk factors for MTC development are unknown. For a long varying degrees of expressiveness and age-related pene-
time, in the genetic alteration of the RET proto-oncogene, trance. Compared with other well-differentiated thyroid can-
mainly the activation of point mutations, the pathogenic cers (i.e., PTC and FTC), the biological behavior of MTC is
mechanism responsible for the development of MTC has more aggressive, although it is not as aggressive as anaplas-
tic cancer (ATC). The 10-year survival rate of MTC patients eficial for successful surgical treatment. Comparison of the
is reported to be about 50%. The cure and survival of these prognosis of the two groups of patients—one group was
patients are positively affected by early diagnosis. A recent diagnosed by serum Ct screening, and the other group was
study showed that the 5-year survival rate for specific dis- diagnosed by cytology or histology—indicates that the
eases increased from 86% to 89%, especially for regional prognosis of the first group of patients is significantly better.
(from 82% to 91%) and distant (from 40% to 51%) MTC However, despite this evidence, routine serum Ct measure-
patients Transfer. ments in nodular thyroid disease are highly adaptable, and it
is clear that there is no consensus when comparing current
Molecular Markers of Medullary Thyroid Carcinoma guidelines. There are major concerns in conducting such
Early diagnosis is highly needed, possibly while the tumor is screening. As for cost-effectiveness, it has recently proven
still in the thyroid gland, as this represents the only possibil- to be acceptable, and the risk of false positives can be over-
ity to cure MTC patients. In most cases, especially in spo- come by calcium stimulation tests, especially at low to
radic cases, the clinical manifestations of MTC are thyroid medium levels (<100 pg/mL). In addition, if serum Ct is
nodules, which may be isolated or appear in the case of mul- elevated but less than 100 pg/mL, it should be interpreted as
tiple nodular goiters. Therefore, the diagnosis is made by a a potential MTC and further diagnostic procedures such as
typical diagnostic examination of a thyroid nodule. immunocytochemistry of Ct on cytological smears and/or
Fine-Needle Aspiration Cytology Ct measurements in needle flushing should be used. A punc-
US-guided fine-needle aspiration cytology (FNAC) is ture of a suspected thyroid nodule should be performed. The
considered the gold standard for preoperative diagnosis of latter method has special diagnostic utility in determining
thyroid nodules. However, a recent multicenter international the nature of cervical lymph nodes (especially before thy-
study involving 12 different referral centers in seven coun- roidectomy) to plan the most appropriate treatment
tries showed that FNAC was able to make a correct preopera- strategy.
tive MTC diagnosis in <50% of 313 analyzed cases. This Other Secretory Products
limitation of the FNAC has had a negative impact when plan- When the disease spreads and there is distant metastasis,
ning for expansion surgery, which is incorrect or inadequate serum carcinoembryonic antigen (CEA) is usually elevated.
in more than 60% of cases. Cases of advanced local disease manifested by cervical USA
Over the years, several series have been reported that with elevated serum CEA levels should be studied by com-
show a high failure rate of FNAC in preoperative diagnosis puted tomography (CT) to better assess the relationship
of MTC. The most reasonable reason for this deficiency is between the disease and the large veins, trachea, and esopha-
related to the fact that the cytological definition of MTC cells gus and develop the most appropriate Treatment plan.
is always ambiguous and may be misunderstood in some However, CEA is most useful in monitoring the progress of
cases. The final cytology may even indicate benign, uncer- the disease because its levels increase when the burden of
tain or even PTC or FTC lesions. Immunocytochemical anal- disease increases rapidly. Serum chromogranin-A may also
ysis of Ct can greatly improve the results of FNAC. However, be elevated in MTC patients, but is highly specific. Recently,
this is not a standard procedure and should only be performed the diagnostic accuracy of serum calcitonin has been proven
under selected conditions, mainly with known elevated to be comparable to that of serum Ct, but further research is
serum Ct levels. Sometimes, negative cytology results may needed before this measurement can be incorporated into
be due to the fact that MTC may be present in one nodule in clinical practice.
the case of multinodular goiter and not submitted to FNAC. In Like many other neuroendocrine tumors, somatostatin
this case, the measurement of serum Ct is more reliable, (SMS), calcitonin gene-related peptide (CGRP), vasoactive
because the measurement of serum Ct increases even in the intestinal peptide (VIP), neuron-specific enolase (NSE), and
presence of the microfocus of MTC. other neuroendocrine substances may abnormally be pro-
Serum Calcitonin and Others duced, but none of these peptides is useful for the diagnosis
Calcitonin (Ct) peptide is the most specific and sensitive of MTC. The difference is that some of them, such as CGRP,
MTC marker before and after thyroidectomy. It is a small VIP, serotonin and prostaglandins, and serum Ct, may cause
peptide hormone of 32 amino acids that is usually produced flushing and diarrhea syndrome.
almost exclusively by C cells. Ct release and secretion are RET genetic analysis found that at least 5–7% of sporadic
mainly regulated by extracellular calcium concentrations. MTCs are hereditary, so not only should family history be
Routine measurement of serum Ct in nodular thyroid dis- considered carefully, especially the occurrence of PHEO and
ease can diagnose suspected sporadic MTC before surgery. PTHAd in other family members, but genetic screening of
Calcitonin screening can determine the early diagnosis of the germline is always indicating RET mutation. This dis-
MTC, usually when the tumor is still in stage I, so it is ben- covery is of great significance to the early discovery of other
680 H. Yuan et al.
gene carriers who are unaware of its status. Currently, RET Treatment outcome: cure
screening is required in recorded genetic cases to screen all
first-degree relatives. Laboratory Test Performed SP70 was used for differen-
Taking EDTA or even a blood sample from a saliva smear tial diagnosis of benign and malignant diseases. Other tests
is sufficient for DNA extraction and genetic analysis. If a were all for auxiliary diagnosis. Several tests such as FT3,
RET mutation is identified, the case can be reclassified as FT4, TSH, and Tg are mainly used to understand whether the
hereditary despite no family history. All first-degree relatives occupancy affects the function of the thyroid gland. The
(i.e., parents, siblings, sons and daughters) should be asked pathological examination was the diagnostical golden stan-
for screening tests. Although there is no standard nursing dard. B-ultrasound is a commonly used screening method.
procedure, RET gene analysis should also be performed in
tumor tissues. Whether it is prognostic value or more accu- Results with Interpretation Guideline B-ultrasound-
rate tissue characterization, this may be very useful if the guided thyroid nodule puncture pathology is the gold stan-
drug is specifically used to suppress the occurrence of dard for the diagnosis of thyroid cancer; however, this test
tumors. It is necessary to Change the RET gene. is invasive, and there is a risk of failure. SP70 is a good
serological diagnostic marker for identifying benign and
40.2.2.4 Typical Case malignant thyroid nodules. Combined with thyroid
Clinical Background A 41-year-old female patient was B-ultrasound grading, it is a relatively noninvasive solution
admitted to hospital for treatment for thyroid nodules 3 for the diagnosis of thyroid cancer. From this case, we can
weeks ago. the scheme of diagnosis and treatment of thyroid cancer in
Fig. 40.10.
B-ultrasound left thyroid nodules, TI-RADS 4C; lymph
node images in the left neck VI area, category 4a; see in This case was from the First Affiliated Hospital of
Fig. 40.8. Nanjing Medical University (also named Jiangsu Province
Hospital).
Initial laboratory values FT3 4.38 pmol/L; FT4
14.10 pmol/L; TSH 2.460 mIU/L; Tg 1.56 ng/mL; PTH
49.3 pg/mL 40.2.3 Nontoxic Goiter
Tumor marker SP70 13.4(↑) ng/mL; AFP 3.85(–) ng/mL; 40.2.3.1 Etiology and Epidemiology
CEA 1.13(–) ng/mL Nontoxic goiter is a common condition associated with
insufficient iodine intake. Other causes include genetic sus-
Histopathological examination (left) papillary thyroid ceptibility, female, age, and smoking [37]. Because thyroid
carcinoma, see in Fig. 40.9. tissue has the advantage of nodular degeneration, most goi-
Treatment radical thyroidectomy (bilateral thyroidec- ters have nodules of different sizes and textures. In some
tomy + left central lymph node dissection) patients with long-term goiter, hyperthyroidism will gradu-
Fig. 40.8 Thyroid tissue ultrasonography

a b
Fig. 40.9 Immunohistochemical analysis of SP70 antigen expressions in thyroid tissue. (a) Papillary thyroid carcinoma (HE, ×200), (b) Papillary
thyroid carcinoma (Immunohistochemistry, ×200)
ally appear due to the autonomic function of one or more without treatment, serum TSH should be monitored at least
nodules [38]. Although the risk of malignancy of nodular once a year. Despite the lack of randomized trials, large-
goiter in unselected patients is low, assessment of suspicious scale epidemiological studies have shown that morbidity and
nodules through thyroid ultrasonography and fine-needle mortality are significantly associated with lower-than-normal
aspiration biopsy is essential to ascertain the nature of the serum TSH levels.
goiter.
Markers of Thyroid Autoimmunity
40.2.3.2 Classification of Goiter All patients with goiter should be tested for serum thyroid
Goiter can be clinically classified as diffuse, solitary nodu- peroxidase (TPO), thyroglobulin, and TSH receptor antibod-
lar, or multinodular thyroid disease; the latter is the most ies. It is common for Hashimoto’s autoimmune thyroiditis
common phenotype. Initially, nodular goiter is nontoxic. and Graves’ disease to stack on classic multinodular goiters
However, some nodules can depart from normal TSH regu- [40]. Therefore, if patients with newly discovered nodular
lation and gain functional autonomy. Such lesions account goiter have elevated serum TSH above the normal range,
for about 10% of all thyroid nodules and are scintigraphic they should increase their doubts about the coexistence of
(or hotter), while cold nodules more or less lose their ability Hashimoto’s autoimmune thyroiditis and anti-TPO. If anti-
to synthesize and secrete thyroid hormones. Therefore, TPO is negative, antithyroglobulin should be measured. In
many nontoxic multinodular goiters are actually a mixture addition, if goiter is subsequently treated with 131I, these anti-
of nodules with different functions. The diagnosis and treat- bodies are an indication of an increased risk of hypothyroid-
ment of goiter patients are recommended as follows ism. TSH receptor antibodies are a marker of Graves’ disease
(Fig. 40.11). and can occur in nodular goiters, which may be persistent for
many years.
40.2.3.3 L aboratory Diagnosis of Nontoxic
Goiter Serum Thyroglobulin
Thyroid Function Tests This is a hallmark of thyroid tissue mass and is mainly used
Obviously, the determination of serum TSH should be used for postoperative monitoring and epidemiological research
as an initial biochemical test, and if the hormone level is not of thyroid cancer. Although there is a positive correlation
in the reference range, the level of serum-free T4 and free T3 between serum thyroglobulin and thyroid size, it has little
should be evaluated. With the development of autonomy, place in diagnostic tests for benign nodular goiter due to the
there will be obvious hyperthyroidism later [39]. Therefore, lack of accuracy at the individual level.
682
Fig. 40.10 The scheme of diagnosis and treatment of thyroid cancer

H. Yuan et al.
Fig. 40.11 Workflow recommendations for goiter patients, including diagnostic strategies and treatment options
Calcitonin Imaging examination At the time of referral and the TSH

Calcitonin is a marker of MTC and can also be used to moni- was not suppressed, total thyroxine (A thyroid Tc-99m
tor the disease. However, it is controversial whether calcito- pertechnetate scintigraphy showed almost homogenous and
nin should be routinely measured in patients with nodular equal uptake in both lobes). The right lobe was considerably
goiter, and there is no consensus on this issue [41]. In detect- enlarged, measuring 6 × 10 cm and protruded across the mid-
ing MTC, measuring basal calcitonin or stimulating calcito- line but not retrosternally. The much smaller left lobe seemed
nin levels is usually more sensitive than fine-needle aspiration of normal size. No hypofunctioning nodules (“cold”) were
biopsy. Due to the low prevalence of MTC in patients with identified, and thus, no fine-needle aspiration was performed.
nodular goiter, in the range of 0.4–1.4%, the determination The diagnosis was diffuse enlargement of the right thyroid
of serum calcitonin in all patients with goiter will lead to lobe.
many false positive values and subsequently many unneces-
sary thyroid cut. Treatment The patient was examined regularly by the same
endocrinologist. For the purpose of volume reduction of the
40.2.3.4 Typical Case thyroid gland, the patient was interested in an attempt of
Clinical Background A 42-year-old woman was referred medical treatment by levothyroxine (Eltroxin, Glaxo
by her general practitioner to the Department of Medical Welcome, Great Britain). The treatment was given in TSH
Endocrinology because of a large goiter, but no clinical or nonsuppressive doses (0.1 mg daily) for 18 months, and TSH
biochemical signs of thyroid dysfunction. The patient had no was between 0.85 and 1.21 (normal range 0.5–4.2 mIU/L).
major focal and major cosmetic problems. The goiter The treatment was without success on volume reduction
appeared at her second pregnancy. evaluated by palpation and subjective symptoms but not veri-
fied by ultrasonography.
Initial laboratory values TSH 1.23 mIU/L (normal range She was then referred to radioiodine treatment, and levo-
0.5–4.2), T4 82 nmol/L (normal range 60–140), T3 thyroxine treatment was discontinued. A second thyroid
2.15 nmol/L (0.9–2.6). Tc-99m pertechnetate scintigraphy demonstrated no change in
684 H. Yuan et al.
size and uptake. Twenty-four-hour I-131 uptake was found to thyroid nodules are cystic lesions (true adenomas) or ade-
be 25% of a test dose (32 mCi). The total calculated thera- nomatic nodules, lacking cysts. Functionally, nodules are
peutic dose was 32 mCi iodine-131, which was given frac- classified as “cold,” “normal,” or “hot,” depending on
tionated in three separate doses to avoid doses above the whether the uptake they show in the flicker scan is reduced,
allowed limit of outpatient radioactive therapy. The patient normal, or increased. Of all the nodules, about 50–85% are
felt well, remained clinically euthyroid, but the goiter was “cold,” up to 40% of them are indifferent to scintillation and
unchanged, and after 10 months, radioiodine treatment was about 10% are “hot” [42] despite the prevalence and clinical
repeated. supply due to environmental iodine varies.
A total of 38 mCi of I-131 was administered and again Thyroid autonomy (i.e., AFTNs) is an almost completely
divided into three doses. Afterward, a third thyroid scintigra- benign disease, and there is little evidence in the literature to
phy was performed, showing an unchanged enlargement of the contrary. In contrast, the differential diagnosis of scintig-
the right thyroid lobe, while the left lobe had disappeared. raphy and CTN includes benign follicular adenoma and ade-
For 3 months, TSH was slightly elevated and normalized noma nodules, as well as papillary thyroid carcinoma and its
spontaneously. Tri-iodothyronine (T3) and thyroxine (T4) variants and follicular thyroid cancer. Although fine-needle
remained within the normal range, and no substitution ther- aspiration cytology is currently the most sensitive and spe-
apy was initiated as the patient had no hypothyroid symp- cific tool for prioritizing the selection of thyroid nodules for
toms although an elevated TSH might stimulate the growth surgery by assessing the malignant criteria of ultrasound, it is
potential of the thyroid cells. Anti TSH receptor antibodies characterized by inherent limitations that lead to “uncertain”
were not measured. cytology. Molecular tests in the form of “exclude” and
“exclude” malignant tests have been proposed to fill this
The Follow-up after Treatment In the subsequent 2½ diagnostic gap. For thermal nodules, the most important
years, there was a small palpable increase in size of the treatment options are radioiodine therapy or surgery. If there
gland, and she then developed local compression symptoms are symptoms or a risk of malignancy, scintigraphy and CTN
with global sensation, coughing, nausea, a feeling of stran- surgery can be carried out.
gulation, and dizziness. She was emotionally unstable and
gained weight, but was still biochemically euthyroid. 40.2.4.2 Laboratory Diagnosis
Sonographic examination of the thyroid gland at this time Fine-Needle Aspiration Cytology
revealed a 6.5 × 7.5 × 10 cm large rounded nodule of the Current guidelines on the differential diagnosis and treat-
right lobe, mostly solid but with sparse cystic areas. The left ment of thyroid nodules suggest clinical evaluation, thyroid-
lobe was reduced in size without focal changes. stimulating hormone (TSH), calcitonin (in Europe), and
Due to local symptoms, a right lobectomy of the thyroid ultrasound for stratifying cancer risk to select thyroid nod-
gland was undertaken. A large right lobe (306 g), protruding ules with a higher risk Fine-Needle Aspiration (FNA) Biopsy
into the mediastinum, was removed. The lobe appeared [43]. By standardized assessment of ultrasound characteris-
homogenous and smooth without signs of malignancy. The tics and thorough stratification of ultrasound malignancies
left lobe could not be identified at all within the operation for thyroid nodules, approximately 50% of thyroid nodules
field. At gross examination, the right lobe was 5 × 6 × 10 cm can be classified as positive or very benign [thyroid imaging
large with an encapsulated, mostly necrotic lesion with reporting and data system (TI-RADS) 2 and 3)], and the risk
haemorrhagic goiter tissue centrally. Microscopically, the of false negatives is 0.3% [44]. The remaining thyroid nod-
lesion was a microfollicular adenoma without malignant fea- ules with high malignant risk (TI-RADS 4A, 4B, 5) should
tures. Surgery was uneventful. undergo subsequent FNA cytology and be evaluated accord-
ing to the Bethesda system score to report thyroid cell pathol-
Treatment Outcome Postoperative thyroid pertechnetate ogy (Bethesda classification) [45].
scintigraphy showed no uptake, not even on the left side, and The Bethesda classification is based on five cytological
thus, thyroxin treatment was initiated. diagnostic categories (non-diagnostic, benign, uncertain,
suspected malignant tumor (SFM) and malignant), and the
This case was from the First Affiliated Hospital of Nanjing uncertainty category is divided into uncertain atypical/
Medical University (also named Jiangsu Province Hospital). important Confirmed follicular lesions (AUS/FLUS) and fol-
licular tumors/suspected follicular tumors (FN/SFN).
According to the state of the National Cancer Institute’s
40.2.4 Thyroid Nodule Thyroid FNA Scientific Conference, the cytology category
differs due to its hidden malignant risk (ROM) and must be
40.2.4.1 Definition and Clinical Manifestations validated for each local environment. These two uncertain
Benign thyroid nodules are quite common in iodine-deficient categories are characterized by an intermediate ROM of
areas. Histologically, the morphological criteria for benign 5–15% (AUS/FLUS) and 15–30% (FN/SFN). Uncertain
(AUS/FLUS, FN/SFN) FNA cytology results are attributed ity and specificity for the seven genomes ranging from
to the inherent limitations of thyroid FNA cytology. Vascular 18–100% to 82–100%, respectively [47].
or cystic infiltration cannot be detected in cytological sam-
ples, which distinguishes follicular adenomas (FAs) or ade- 40.2.4.3 Typical Case
noma nodules from follicular cancer (FTC) and follicular Clinical Background A 37-year-old female patient was
papillary thyroid cancer (fvPTC) standard. In addition, cur- admitted to hospital for “recovering thyroid nodules for more
rent guidelines recommend repeating FNA for AUS/FLUS than 2 years.”
FNA results to increase the chance of a definitive FNA cyto-
logical diagnosis. In addition, the “2015 American Thyroid Physical examination Right thyroid gland can reach nod-
Association Management Guidelines for Adults with Thyroid ules, about 5.0 × 4.0 cm in size, with good toughness, good
Nodules and Differentiated Thyroid Cancer” was published; mobility, and no tenderness.
the diagnostic process is shown in Fig. 40.12.
Imaging examination A plain CT scan of the thyroid gland
“Rule Out” Malignancy and “Rule In” Malignancy showed that the right thyroid gland occupied a diameter of
The “exclusion” test aimed at identifying benign nodules about 3.7 cm.
(that is, malignant tumors that exclude FNA cytological nod-
ules, thereby reducing the number of diagnostic thyroid sur- B-ultrasound right thyroid cystadenoma, size about
geries) is the Afirma Gene Expression Classifier (GEC). 4.9 × 4.2 × 2.9 cm, TI-RADS 3.
In contrast to GEC, which is used to exclude malignan-
cies, detection of cancer-specific mutations can be used to Initial laboratory values TG > 500 ng/mL, SP70 8.1 ng/
detect malignancies in a “regular” method. Early studies mL.
analyzed seven genomes consisting of BRAF, NRAS, HRAS,
KRAS, RET/PTC1, RET/PTC3, and PAX8/PPARG muta- Histopathological examination thyroid puncture pathol-
tions, suggesting that the method can identify approximately ogy: (right thyroid puncture) benign lesions.
60% of cytologically uncertain cancers [46]. At the same
time, it is reported that there are significant differences in the Treatment Ultrasound-guided thyroid ablation was per-
diagnostic parameters of the “rule in” method, with sensitiv- formed in the hospital.
Suspected Thyroid Nodule

TSH Normal or Elevated (R2C)
No nodule or nodule not

meeting FNA size cutoff
Throid /Neck
Sonography (R6.21)
High Intermediate Low Very Low Benign

Suspicion Suspicion Suspicion Suspicion
Pattern Pattern Pattern Pattern Pattern
FNA≥2cm FNA not

FNA≥1.5cm FNA≥1.5cm
(R8D) required
(R8A,B) (R8C)
(R8E,8F,23)
Cytology
Bethesda system
(R9)
Nondiagnostic Benign AUS/FLUS FN/FSN Suspicious Malignant
Repeat FNA No Surgery See Surgery

(R10) (R11,23) Recommendations (R12)
13-17
Fig. 40.12 Evaluation and management algorithm for patients with thyroid nodules based on US model and FNA cytology
686 H. Yuan et al.
Treatment Outcome After treatment, the patient is cured. lomatous or lymphocytic). The rare fibroinflammatory pro-
cess known as Riedel thyroiditis is also included in this
This case was from the First Affiliated Hospital of Nanjing chapter. Diffuse toxic hyperplasia, or Grave’s disease, could
Medical University (also named Jiangsu Province Hospital). qualify for consideration as an inflammatory disease of auto-
immune etiology. The characteristics of different types of
thyroiditis are listed in Table 40.7.
40.2.5 Thyroiditis
40.2.5.2 Classification and Laboratory
40.2.5.1 Introduction Diagnosis of Thyroiditis
Thyroiditis encompasses a heterogeneous group of inflam- Hashimoto’s Thyroiditis
matory diseases of the thyroid [48]. The study of these Epidemiology and Pathogenesis
inflammatory conditions can be challenging because of the Hashimoto’s thyroiditis is defined as an organ-specific
different approaches to classification and variety of syn- autoimmune disease that is characterized by autoimmune-
onyms for a particular type of thyroiditis. Classification can mediated thyroid destruction. Its prevalence in general popu-
be based on whether the clinical course is acute, subacute, lation is 10–12%, making it the most common autoimmune
chronic, or subclinical (Table 40.6). Other approaches to disease. The prevalence of women is higher than that of men,
classification include the type of inflammatory response, the increasing with age, the prevalence of whites is the highest,
etiologic or pathogenic mechanism (e.g., autoimmune, infec- and the prevalence of blacks is the lowest. Hashimoto’s
tious), or the effect on thyroid function. thyroiditis usually occurs in families, which can be seen in a
The most common form of thyroiditis is chronic lympho- high sibling risk rate of 28. Twin studies show genes respon-
cytic thyroiditis, which describes histopathologically the sible for about 73% of TPO-Ab and TG-Ab development
condition called Hashimoto thyroiditis. The chronic lympho- [49]. Therefore, environmental factors will contribute
cytic thyroiditis and Hashimoto thyroiditis are considered 20–30%. Smoking and moderate drinking can prevent
synonymous. Subacute forms of thyroiditis are divided Hashimoto’s thyroiditis to a certain extent. Low selenium or
according to the predominant inflammatory response (granu- vitamin D intake may be related to the higher prevalence of
TPO-Ab, but there is no convincing evidence that supplemen-
tation with selenium or vitamin D may reduce TPO-Ab con-
Table 40.6 Thyroiditis classification and clinical course
centrations. Infection may cause Hashimoto’s thyroiditis, but
Clinical
existing epidemiological studies do not support this effect.
Name Synonyms course
Acute Acute suppurative thyroiditis; Acute
Hashimoto’s thyroiditis is usually a diffuse inflammation
thyroiditis Infectious thyroiditis of the thyroid gland, including destruction of epithelial cells,
Subacute de Quervain thyroiditis; Subacute Subacute infiltration of lymphoid cells, and fibrosis. Thyroid cells are
granulomatous thyroiditis; Painful subacute full of mitochondria and have eosinophilic properties. They
thyroiditis thyroiditis; Postviral thyroiditis; are called Hürthle or Askanazy cells. Clusters of macrophage-
Giant cell thyroiditis; Subacute
nonsuppurative thyroiditis; like cells can be seen in the follicles. Lymphocytes and
Pseudotuberculous thyroiditis; plasma cells are infiltrated in the interstitium, and there are
Struma granulomatosa lymphoid follicles protruding from the germinal center and
Infectious Infectious thyroiditis Subacute different degrees of fibrosis.
granulomatous to
thyroiditis chronic
Laboratory Diagnosis
Hashimoto Hashimoto thyroiditis; Autoimmune Chronic The presence of TPO-Ab and/or TG-Ab in serum was
thyroiditis thyroiditis; Struma lymphomatosum considered positive evidence of the presence of Hashimoto’s
Silent Sporadic thyroiditis; Painless Subacute thyroiditis. The decision value for TPO-Ab (defined as the
thyroiditis thyroiditis; Painless sporadic concentration corresponding to a 0.1% false positive) is
thyroiditis; Painless thyroiditis with
hyperthyroidism; Silent thyrotoxic
15 kU/L and 31 kU/L for TG-Ab. Patients with hypothyroid-
thyroiditis; Subacute lymphocytic ism caused by atrophic or goiter autoimmune thyroiditis
thyroiditis; Atypical subacute have higher concentrations of TPO-Ab and TG-Ab in the
thyroiditis; Spontaneously resolving serum, but these antibodies are usually lower in patients with
hyperthyroidism; Lymphocytic
thyroiditis with spontaneously
Graves’ hyperthyroidism, although usually lower. Serum
resolving hyperthyroidism TPO-Ab and TG-Ab are still sensitive and specific markers
Postpartum Painless postpartum thyroiditis of thyroid autoimmunity. Interestingly, antibodies against
thyroiditis TSHR are also described in Hashimoto’s thyroiditis [50].
40 Endocrine and Metabolic Diseases
Table 40.7 Characteristics of different types of thyroiditis

Hashimoto thyroiditis Postpartum thyroiditis Silent sporadic thyroiditis Infection thyroiditis Traumatic thyroiditis Subacute thyroiditis
Etiology/ Autoimmune Autoimmune Autoimmune Infection Traumatic Uncertain postinfection?
pathogenic
mechanism
Antibodies Present Present Present Absent Usually present Usually absent
(anti-TPO and/ Usually transient if present
or anti-TG)
Thyroid Hypothyroidism Initial hyperthyroidism Initial hyperthyroidism Usually euthyroid Usually euthyroid Initial hyperthyroidism
function Occasional followed by hypothyroidism followed by hypothyroidism About 1/3 followed by hypothyroidism
hyperthyroidism Usual return to euthyroidism Usual return to euthyroidism hypothyroidism Usual return to euthyroidism
Thyroid TPO-Ab↑, TG-Ab↑ TPO-Ab↑, TR-Ab↑ TR-Ab→ TPO-Ab↑, TG-Ab↑ Uncertain T4↓, T3↓, TSH↓
function tests
Clinical course Euthyroid or Usually resolves within Usually resolves within 2–5 Usually dependent on Hypothyroidism in up to Usually resolves within
hypothyroidism at postpartum year 12–30% months ~20% experience underlying disorder 40% several months
time of diagnosis experience persistent or persistent or recurrent and effectiveness of Usually self-limited Minority experience
Steady progression to recurrent hypothyroidism hypothyroidism treatment May require surgical persistent hypothyroidism,
hypothyroidism therapy for tracheal or recurrence, or development of
obstruction esophageal chronic thyroiditis
687
688 H. Yuan et al.
With the widespread use of TPO-Ab and TG-Ab assays, it Sporadic Painless Thyroiditis
became clear that these antibodies also appeared in subjects Epidemiology and Pathogenesis
without goiter. In fact, the vast majority of patients with Sporadic painless thyroiditis is a type of destructive thy-
Hashimoto’s thyroiditis do not have goiters. At the time of roiditis that occurs in nonpregnant women. The disease is
diagnosis, only a few patients with goiter with autoimmune basically the same as postpartum thyroiditis and is character-
hypothyroidism had goiters. ized by a period of hyperthyroidism, hypothyroidism, and
finally normal thyroid function and undergoes the same clin-
Postpartum Thyroiditis ical process. Several synonyms have been used to describe
Epidemiology and Pathogenesis the syndrome, including painless thyroiditis, silent thyroid-
The definition of postpartum thyroiditis (PPT), in women itis, transient thyrotoxicosis, atypical subacute thyroiditis,
not suffering from thyroid disease, occurs only thyrotoxico- and lymphocytic thyroiditis with spontaneous hyperthyroid-
sis or hypothyroidism or thyrotoxicosis secondary to hypo- ism. Silent thyroiditis should be distinguished from subacute
thyroidism in the first year postpartum. PPT is a form of or acute thyroiditis, which is usually caused by a thyroid
“destructive” thyroiditis in which the follicular cells of the virus or bacterial infection. Unlike silent thyroiditis, the clin-
thyroid gland are destroyed, causing preformed thyroid hor- ical course of acute or subacute thyroiditis is characterized
mones to be released into the circulatory system. These dis- by thyroid inflammation, such as neck pain, difficulty swal-
eases usually appear from scratch, but may also be set in the lowing, fever, and elevated signs of inflammation in the
context of preexisting thyroid diseases such as Graves’ dis- serum [52].
ease and Hashimoto’s thyroiditis. The typical clinical course Laboratory Diagnosis
is the initial stage of thyroid toxicity, then the stage of hypo- There are many different diagnoses at each stage of the
thyroidism, and finally the return to normal thyroid status. disease. This is essential to ensure proper treatment of
Patients with postpartum thyroiditis can be distinguished patients with self-limiting diseases and to avoid overtreat-
from patients without subacute or acute infectious thyroiditis ment with antithyroid drugs. In silent thyroiditis, TSH recep-
because it has no laboratory indicators of pain, fever, or acute tor antibodies are negative or elevated to a lesser extent than
inflammation [51]. Graves’ disease, despite the lack of a reliable discriminator.
Thyroid Toxicity PPT is an autoimmune “destructive” In patients with acute and subacute thyroiditis, markers of
thyroiditis. The clinical feature of thyroid toxicity is caused inflammation such as C-reactive protein and erythrocyte sed-
by the release of preformed thyroid hormones stored in the imentation rate are elevated, but it is usually normal in
thyroid gland, because T4 is preferentially stored in the thy- asymptomatic thyroiditis. Ultrasound of the thyroid confirms
roid. However, in postpartum GD, T3 is dominant due to thy- the absence of thyroid nodules, but the thyroid texture of the
roid secretion of T3 and peripheral transformation of T4. thyroiditis is hypoechoic and heterogeneous, which may be
Although the T4/T3 ratio was used to distinguish the two difficult to distinguish from the characteristics of the ultra-
cases in the past, it is no longer commonly used. sound of Graves’ disease. Color Doppler ultrasound will
Laboratory Diagnosis show typical thyroid blood flow patterns in patients with
The TR-Ab test is very sensitive and specific for the Graves’ hyperthyroidism. Serum thyroglobulin can be used
diagnosis of GD—both are estimated to be between 97% to distinguish patients with artificial thyroiditis because thy-
and 99%. The TR-Ab test is very helpful to distinguish roglobulin is elevated in thyroiditis but decreases after thy-
thyroid toxicity PPT from postpartum GD. It is difficult to roid hormone.
distinguish between hypothyroidism and Hashimoto’s thy-
roiditis. Both cases occur late, and most affected women Infectious Thyroiditis
are TPO-Ab positive. However, most people with low thy- Epidemiology and Pathogenesis
roid function do not need thyroxine in the first year after Acute infectious thyroiditis is a rare disease, and its inci-
delivery, and withdrawal medications are safe—their TSH dence is uncertain. Of the 1309 consecutive thyroid surgeries
levels remain asymptomatic within the reference range. performed by a surgeon between 1933 and 1955, six (0.5%)
However, at the end of the first year postpartum, 4–54% of acute supportive infections were reported. The rarity of thy-
these women may require long-term thyroxine treatment roid infections is due to protective nutritional factors and the
because they may have permanent hypothyroidism. On the anatomical characteristics inherent to the thyroid. This
other hand, after withdrawal of thyroxine, symptoms and includes its high iodine content, the production of hydrogen
biochemical thyroid hypofunction in patients with peroxide, the encapsulation of the capsule, the abundant
Hashimoto’s thyroiditis require long-term thyroxine blood supply with an anastomotic arterial network, and the
replacement therapy. rich lymphatic drainage. Infectious thyroiditis usually occurs
acutely, with neck pain, fever, and physical symptoms. Sore pathological results described are considered to be clinically
throat, difficulty in swallowing, and sound disturbances can unrelated and do not pose a risk of thyroidism. However,
also occur. Most patients have a unilateral neck mass that subsequent literature suggests that it is difficult to estimate
moves when swallowed and may fluctuate (indicating the incidence of traumatic thyroiditis due to insufficient
abscess formation). The upper skin may be erythema and awareness, reporting, and clear diagnostic criteria. In the
edema. The patient can avoid neck extension and keep the parathyroidectomy surgery series, the incidence of postop-
neck bent to avoid putting pressure on the thyroid. Infectious erative hyperthyroidism was reported in 31%, of which 4%
thyroiditis can be life-threatening due to complications from had significant thyroid toxicity and symptoms required
airway damage or sepsis. There is a risk of infection spread- medication.
ing to the chest, leading to necrotizing mediastinitis and peri- Diagnosis
carditis. Patients with acute infectious thyroiditis usually Although pain and tenderness have been observed,
have normal thyroid function, although hyperthyroidism trauma-related thyroiditis is usually not tender. Clinical man-
may be caused by the release of thyroxine and triiodothyro- ifestations usually manifest as thyroidism. The development
nine due to destruction of thyroid follicles. More chronic of atrial fibrillation associated with traumatic thyroiditis is
destructive infections can lead to hypothyroidism [53]. described. No specific imaging function has been reported.
Laboratory Diagnosis As with other types of destructive thyroiditis, radionuclide
A preliminary laboratory evaluation of acute infectious scans often show reduced tracers.
thyroiditis usually includes a complete blood count, Traumatic thyroiditis is usually self-limiting, with a com-
C-reactive protein (CRP), and thyroid function tests. plete biochemical remission from 12 days to 3 months in the
Leukocytosis and elevated CRP are usually present. A thy- surgical series. Symptomatic treatment has been used, in
roid function test helps determine the presence of thyroid some cases with glucocorticoids, such as other forms of
dysfunction, and if a thyroid dysfunction is present, it should inflammatory thyroiditis [54].
be monitored by serial testing. Screening for human immu-
nodeficiency virus (HIV) should be considered. Laboratory Subacute Thyroiditis
markers may not be particularly useful in distinguishing Epidemiology and Pathogenesis
acute thyroiditis from invasive thyroid cancer, subacute thy- Subacute thyroiditis which was first described by
roiditis, or the formation of abscesses in nearby anatomy, as Mygind in 1895 affect previously normal thyroid thyroid-
these conditions may each lead to an increase in white blood itis and without abscess formation. In 1905, Swiss surgeon
cell count and CRP. Fritz de Quervain specialized in thyroid diseases, and he
Therefore, imaging and FNA for any mass and/or fluid discovered the pathological characteristics that distinguish
collection are needed. The imaging characteristics of acute subacute thyroiditis from other forms of thyroiditis. The
infectious thyroiditis depend on the stage of inflammation. In giant cell granulomatous change in thyroid tissue is a
the early stages of inflammation, the radiological features of unique pathological finding of this disease. The description
which an abscess has not yet formed are subtle. In the early of this pathology, subacute thyroiditis de Quervain’s thy-
stages of inflammation, ultrasound examination by a skilled roiditis, is commonly referred.
operator may be helpful. CT scan imaging obtained shortly Laboratory Diagnosis
after the onset of inflammation has low specificity, including Subacute thyroiditis is usually associated with elevated
low-density areas of the thyroid lobe and small leaflet swell- CRP. White blood cell counts are usually normal, but may be
ing. Abscesses were not apparent until the acute phase of increased slightly. White blood cell count was significantly
CT. CT imaging of cervical and thoracic venography can increased, should arouse suspicion of suppurative thyroid-
improve anatomical evaluation and is recommended for the itis. Normal discoloration Anemia may occur. Liver function
evaluation of acute infectious thyroiditis. CT of venography test abnormalities may be associated with infection or induce
can define soft tissue enhancement and whether an abscess hyperthyroidism.
extends to the neck or mediastinum. CT scans can also iden- In the acute phase of subacute thyroiditis, most patients
tify piriform sinus fistula as the cause of thyroiditis, espe- have biochemical evidence of hyperthyroidism, elevated
cially if a small operation is performed during CT. serum T3 and T4, and TSH suppression. T3 and T4 ratio is
typically relatively low, reflecting the T4 thyroid follicular
Traumatic Thyroiditis than T3. Acute disease from the outer periphery of T4 to T3
Epidemiology and Pathogenesis deiodination also causes damage to a relatively low-level T3.
It was first described in 1975 as violent palpation of the Almost all patients with serum thyroglobulin subacute thy-
thyroid gland, causing inflammatory reactions and histologi- roiditis were increased, consistent with follicular destruc-
cal changes in multifocal granulomatous folliculitis. The tion, but this is not a regular part of the assessment.
690 H. Yuan et al.
Subacute thyroiditis usually lacks thyroid antibodies, Table 40.8 Classification of hypothyroidism
such as antithyroglobulin, antithyroid peroxidase, and TSH Classification based on the location of the lesion
receptor antibodies, although transient low titer antibodies Primary hypothyroidism
may be found. Positive antithyroid antibodies (antithyro- Acquired
globulin and antithyroid peroxidase) are associated with the Thyroiditis
development of hypothyroidism. These antithyroid antibod- Autoimmune
ies will mostly disappear during recovery stages [55]. Hashimoto’s thyroiditis (goitrous and atrophic forms)
Painless and postpartum thyroiditis (transient, may result in
permanent thyroid failure)
40.2.5.3 Typical Case Other thyroiditis
Clinical Background A 54-year-old nondiabetic woman Subacute (transient, rarely result in permanent thyroid failure)
was referred to our hospital with a 1-year history of severe Riedel’s thyroiditis
diplopia. Thyroid infiltration
Amyloidosis
Initial laboratory values Free triiodothyronine (FT3), Hemochromatosis
2.67 pg/mL (normal range, 1.8–4.6 pg/mL); free thyroxine Sarcoidosis
(FT4), 11.84 pmol/L (normal range, 12–22 pmol/L); thyroid- Cystinosis
Scleroderma
stimulating hormone (TSH), 5.020 mIU/mL (normal range,
Iodine deficiency, goitrogens in foodstuffs, pollutants
0.27–4.2 mIU/mL); antithyroglobulin (A-TG), 1785.00 IU/ Iodine excess
mL (normal range, 0–115 IU/mL); and antithyroid peroxi- Consumptive hypothyroidism
dase (A-TPO), more than 600.00 IU/mL (normal range, Type 3 deiodinase (D3) expression in large tumors
0–34 IU/mL). Additionally, the patient’s thyrotropin receptor Congenital
antibody (TR-Ab) levels were 12 IU/mL (normal range, Agenesia, dyskinesia, ectopia
0.01–30 IU/mL). Dyshormonogenetic goiter
Iodide transport or utilization defect (NIS or pendrin mutations)
Ultrasonography Ultrasonography showed a thyroid gland Iodotyrosine dehalogenase deficiency
Organification disorders (TPO deficiency or dysfunction)
with heterogeneous echogenicity and a nodule in the left
Defects in thyroglobulin synthesis or processing
lobe.
TSH receptor defects and other forms of idiopathic TSH
unresponsiveness
Treatment The patient was referred for an endocrinology Thyroidal Gs protein abnormalities (pseudohypoparathyroidism
consultation, following which she was diagnosed with type 1a)
Hashimoto’s thyroiditis. Subsequently, we rectified the pre- Central hypothyroidism
liminary ophthalmic diagnosis of DEP to TAO based on her Congenital
history and clinical features and findings. She was treated TSH deficiency or structural abnormality
TRH and TRH receptor defects
with 80 mg of prednisolone daily for a week.
Acquired
Pituitary (secondary) or hypothalamic (tertiary) disorders
Cure outcome Her diplopia failed to improve, and a left IR Bexarotene (retinoid X receptor agonist)
recession (9 mm) was performed. Dopamine or severe illness
Classification based on the cause of the lesion
This case was from the First Affiliated Hospital of Nanjing Drug-induced hypothyroidism
Medical University (also named Jiangsu Province Hospital). Iatrogenic
123I
Surgery
40.2.6 Hypothyroidism External irradiation for nonthyroidal malignancy
Drugs blocking synthesis or release of thyroxine
Antithyroid drugs (methimazole, carbimazole, propylthiouracil)
40.2.6.1 Definition and Classification Other drugs: lithium, ethionamide, sulfonamides, iodide
of Hypothyroidism Tyrosine kinase inhibitors (TKIs): sunitinib, regorafenib, and
Hypothyroidism is a clinical symptom characterized by others
reduced synthesis and secretion of the thyroid hormones thy- Cytokines (interferon-α,interleukin-2, others)
roxine (T4) and triiodothyronine (T3). See details in Idiopathic
Table 40.8. The most common cause of hypothyroidism is Classified according to the degree of hypothyroidism
thyroid function loss (primary hypothyroidism), which may Clinical
be autoimmune attack (such as Hashimoto’s thyroiditis), sur- Subclinical
gery, radiation, drugs, and other rare thyroid injuries [56]. A 40.2.6.3 Typical Case
prominent feature of primary hypothyroidism is an increase Clinical Background A 38-year-old female patient with a
in serum thyroid-stimulating hormone (TSH) concentration, history of “hypothyroidism” for 10 years, taking “thyroid
which may stimulate thyroid growth and goiter. Primary tablets” 40 mg once a day, still has symptoms of lethargy.
hypothyroidism is a chronic disease that almost inevitably
leads to permanent thyroid failure, but transient hypothy- B-ultrasound Thyroid ultrasound: bilateral diffuse thyroid
roidism can often be considered as a stage of subacute or lesions. Right thyroid nodules, TI-RADS 3. See the
painless thyroiditis. The lower frequency of hypothyroidism Fig. 40.13.
is due to a decrease in TSH and/or thyroid-stimulating
hormone-releasing hormone (TRH) observed in certain pitu- Initial laboratory values FT3 2.69↓ pmol/L, FT4 5.22↓
itary and hypothalamic disorders, leading to a reduction in pmol/L, TSH > 100.000, mIU/L, hs-CRP 3.55↑ mg/L, FFA
originally normal thyroid stimulation (secondary or central 1.03↑ mmol/L, SHBG 6.0↓ nmol/L, Glu(120')
Hypothyroidism) [57]. 15.96↑ mmol/L, TC 7.32↑ mmol/L, TG 3.07↑ mmol/L,
LDL-C 5.09↑ mmol/L, Glu 9.31↑ mmol/L, UA 472↑ μmol/L,
40.2.6.2 Diagnosis of Hypothyroidism HbA1c 7.7↑ %.
In most cases of central hypothyroidism, serum TSH con-
centrations are low or even undetectable, but may be nor- Tumor marker SP70 6.9 ng/mL.
mal or even slightly higher, although absolute levels seem
to be insufficient compared to low levels of thyroid Treatment Adjusted treatment of “hypothyroidism” drugs,
hormone. lowering blood sugar, lowering blood fat, good disease con-
Diagnosis can be assisted by relevant clinical symptoms trol, and continued oral medication for discharge.
and signs, but clinical examination alone is not reliable. In
addition, clinical assessments are more unreliable in mild Laboratory Test Performed FT3, FT4, and TSH are most
disease—this is the performance of the vast majority of commonly used serological indicators for monitoring thy-
patients. Mild (or subclinical) hypothyroidism is character- roid function. SP70 was used for differential diagnosis of
ized by elevated serum TSH levels and TH concentrations benign and malignant diseases. B-ultrasound can be used to
within the reference range. Prior to biochemical thyroid determine the cause of thyroid dysfunction.
function tests, multiple methods have been applied and only
more severe hypothyroidism has been detected. This chapter Results with Interpretation Guideline The examination
introduces all aspects of the diagnosis and treatment of of serological markers of thyroid function is a common indi-
hypothyroidism. cator for diagnosing thyroid dysfunction, and regular review
In the past, before the biochemical examination of thyroid of relevant indicators can monitor changes in the condition
function, the diagnosis of hypothyroidism mainly depended and adjust treatment options. B-ultrasound can be used to
on clinical symptoms and signs. The advent of TH detection determine the cause of thyroid dysfunction. SP70 is a good
methods has changed the diagnostic rules of hypothyroid- serological diagnostic marker for identifying benign and
ism. In the early 1950s, only one thyroid test was available: malignant thyroid nodules.
indirect estimation of serum total (free and protein-bound)
thyroxine (T4) concentrations using protein-bound iodine This case was from the First Affiliated Hospital of Nanjing
(PBI) technology. Serum TH can be measured as a total Medical University (also named Jiangsu Province Hospital).
(protein-bound) or free (unbound) fraction. Although the
content of free hormones is small compared to total T4 and
T3, it is believed that tiny free fractions of hormones (FT4 and 40.2.7 Hyperthyroidism and Thyrotoxicosis
FT3 are 0.02% and 0.2%, respectively) are responsible for
cellular levels and levels of biological activity. Therefore, the 40.2.7.1 Graves’ Disease
physiological effect of TH is better than the total hormone Epidemiology and Clinical Manifestation
concentration, especially when the binding protein is abnor- Graves’ disease (GD) is a common autoimmune thyroid dis-
mal. The motivation for conducting free hormone tests is the ease that infects 20–30 cases per 100,000 people each year.
abnormally high frequency of binding proteins often encoun- GD is a complex genetic disease, and environmental factors
tered in clinical practice, especially the high TBG status dur- make it susceptible to genetically susceptible people with
ing pregnancy. Currently, most clinical laboratories use multiple susceptible alleles. Thyroid-stimulating hormone
automated immunoassays to estimate serum FT4 and FT3 receptor (TSHR) antibody is an immunological marker of
concentrations. the disease and a fundamental driver of thyroid cell hyper-
692 H. Yuan et al.
Fig. 40.13 Thyroid
ultrasound of the patient
plasia and the resulting hyperthyroidism. In recent years, our tory and thyroid-specific genes play a role in disease. In par-
understanding of the pathogenesis of this disease has evolved ticular, three loci have been identified to have a moderate or
significantly, reflecting the development of human genomics, large impact on Graves’ disease. These are the main histo-
molecular immunology, and murine disease models. The compatibility complexes (MHC), the cytotoxic T lympho-
clinical features of GD are common and present with many cyte antigen 4 (CTLA4) locus, and the tyrosine phosphatase
typical symptoms and signs. Physical examination can reveal 22 protein (PTPN22) gene. Alleles at these loci also make
inflammation of the extremities, atrial fibrillation, signs of significant contributions to other complex autoimmune dis-
orbital thyroid disease, and goiter. GD is accompanied by a eases, including type 1 diabetes, rheumatoid arthritis, celiac
series of extrathyroidal manifestations or appears during the disease, and autoimmune Addison disease [60]. There is
course of the disease. These are related to increased titers of ample evidence that these genes determine the “general” sus-
circulating autoantibodies [58]. The most common extrathy- ceptibility of autoimmunity (and help coexist autoimmune
roidal manifestation is orbital thyroid disease, which may diseases), while more specific “target organ” genes contrib-
threaten vision and requires detailed and careful treatment. ute to organ-specific manifestations of autoimmune diseases.
Recent advances in our understanding of the pathogenesis of Previous studies in the NHANES database have shown that
these diseases may lead to the development of new therapies the prevalence of autoimmune thyroid disease varies by race,
in the coming years. Elevation of one or two serum-free thy- and other studies in different populations (Chinese,
roid hormones and undetectable TSH (in a third-generation Caucasian, etc.) around the world have also supported differ-
assay) confirms the diagnosis of Graves’ disease (thyrotoxi- ences in susceptibility genes between populations.
cosis) (Fig. 40.14). Human Leukocyte Antigen
The HLA complex on chromosome 6 contains sequences
Laboratory Diagnose that encode highly polymorphic genes that are responsible
Graves’ disease is a complex genetic disease with multiple for immune regulation. HLA genes are divided into three
chromosomal sites that can cause the disease to develop. major classes (Class I, Class II, and Class III). Class II
These genes encode proteins in biological pathways that includes histocompatibility genes, which are expressed only
regulate immune system activity or thyroid biology [59]. The on white blood cells and immune-competent cells (HLA-DR).
discrete contributions of individual genes are affected by a The association of GD with the MHC allele on the 6p21
variety of environmental factors and vary among affected chromosome has long been established. In the white popula-
individuals. Advances in our understanding of the genetics of tion of European ancestry, the main association is the haplo-
AITD have led to the recognition that both immunoregula- type HLA-DR3 (DRB1 * 0301-DQB1 * 0201-DQA1 *
Symptoms or signs suggestive of

hyperthyroidism*
check serum thyroid function
Serum TSH < 0.05mU/l

and elevated FT3 or FT4
Yes No
Thyroid eye disease Consider other diagnosis

Thyroid dermopathy • not hyperthyroidism
• if TSH < 0.4mU/l: subclinical hyperthyroidism
No
Serum TSHR Ab
Yes
Positive Negative
Imaging
Diffuse uptake
Radionuclide or US+ scan
Patchy uptake Low or zero uptake
Graves' Toxic nodular

Thyroiditis
hyperthyroidism goiter
Fig. 40.14 Algorithm to confirm Graves’ hyperthyroidism
0501). This haplotype is carried by 25–30% of unaffected dependent activation of competitive CD28 receptors
individuals, but the number of representatives in the GD pop- competing for resting human T cells, which may be related
ulation is always high, of which about 50% carry the haplo- to the environment and to the number of antigen-presenting
type. In many GD subjects, resequencing of the DRB1 gene cells. Not surprisingly, CTLA4 polymorphisms are also sig-
revealed that the key amino acid for disease susceptibility is nificantly associated with disease susceptibility to type 1 dia-
arginine at position 74 [61]. Even so, the odds ratio of DR3 betes and autoimmune Addison disease. CTLA4 is also
haplotype in GD is still about 2, while MHC does not have a involved in the production of TPO and TG antibodies [62].
dominant genetic effect found in GD in other autoimmune Protein Tyrosine Phosphatase Nonreceptor-22
diseases such as type 1 diabetes or rheumatoid arthritis. This PTPN22 encodes the lymphatic tyrosine phosphatase
lacks strong evidence for genetic linkage to 6p21 in many (LYP) molecule and CTLA4, the former involved in the reg-
studies, suggesting that loci other than MHC also have ulation of T cell activation. After contact with the MHC anti-
important roles. Despite the limited data available, other gen, the activated LYP molecule of the coding polymorphism
races carry different haplotypes. Although the risk inferred of arginine 620 codon and tryptophan will more effectively
from HLA is well established, it explains the relatively small inhibit the abnormal signal of T cell antigen receptor (CD3)
proportion of overall genetic susceptibility to GD. kinase. Effect remains to be seen how the variant receptor
Cytotoxic T Lymphocyte Antigen-4 signal transduction in T cells leading to autoimmunity. The
CTLA4 is a T lymphocyte surface protein that plays a role codon 620 of the SNP is also associated with type 1 diabetes
in the regulation of co-stimulatory (“second”) signals in T and other autoimmune diseases. As with other genetic fac-
lymphocyte activation. This is a good candidate, and although tors, the association of PTPN22 with autoimmune thyroid
the exact way in which CTLA4 controls the immune response disease is affected by race [63]. Several additional genes
remains uncertain, CTLA4 is an important regulator of T cell encoding molecules involved in immune regulation have
function and T cell activation (in conjunction with the CD28 been shown to have GD-related allelic variants. Programmed
pathway). Recent work suggests that the quantitative differ- cell death ligand 1 (PD-L1) is another locus that encodes a
ence in CTLA4 expression may be related to CD80/CD86- co-stimulatory molecule. In some studies, the allele is asso-
694 H. Yuan et al.
ciated with GD. This has particular clinical significance, as ing nodules) to a goiter. TA and TMNG are mainly present in
monoclonal antibodies with anticancer effects that stimulate iodine-deficient areas. TMNG may represent the natural evo-
the immune system through co-stimulation blockade lution of diffuse or nodular goiter. Nodular goiter (NGs) are
(“immune checkpoint blockade”) are becoming mainstream clinically identifiable masses. In the absence of thyroid dys-
therapies for melanoma and lung cancer. These treatments function, autoimmune thyroid disease, and thyroid malig-
block the CTLA4 or PD-L1 pathway and cause common side nancies, they constitute entities described as nontoxic
effects due to autoimmune thyroid disease. In addition to NG. NG occurs both locally and is mainly associated with
these genetic variations in the immunomodulatory pathway, iodine deficiency and occasionally. In the early stages of the
GD-specific loci have been identified. These thyroid-specific goiter’s development, the goiter can spread and, over time,
genes include TSHR and TG. not only grow, but also become nodular. Generally, NG can
Thyroid-Stimulating Hormone Receptor be divided into solitary nodular thyroid disease and multi-
After a period of negative studies of the TSH receptor nodular thyroid disease. Thyroid function usually becomes
(TSHR) gene, SNP-tagged alleles were subsequently shown autonomous. The secretion of thyroid hormone becomes
to have a positive association with GD in two different independent of the secretion of thyroid-stimulating hormone
patient cohorts. Single nucleotide polymorphisms (SNPs) in (thyroid autonomy), so the patient gradually develops into
TSHR have been specifically associated with GD in the subclinical hyperthyroidism. Therefore, natural medical his-
Caucasian population. The functional role of these intron tory is clinically characterized by thyroid growth, nodule for-
SNPs remains to be elucidated. One theory is that they pro- mation, and autonomous development of function. Clinical
duce RNA splice variants that increase the level of the forms include TA and TMNG and are responsible for the
TSHR-A subunit. Alternatively, they may result in fewer thy- development of nonautoimmune hyperthyroidism [65].
mus TSHR mRNA transcripts, which may reduce central
tolerance to TSHR [64]. Laboratory Diagnose
Thyroglobulin The diagnosis of TA and TMNG is based on clinical tests,
The thyroglobulin (Tg) gene (8q24) encodes the Tg pro- thyroid function tests, thyroid ultrasound, and scintigraphy.
tein, which is a precursor to the thyroid hormones triiodothy-The evaluation of patients suspected of having TA and
ronine (T3) and thyroxine (T4). In 2003, research showed TMNG includes a careful medical history, whether there is a
insufficient evidence about the association of GD with mic- possible iodine overload, physical examination focuses on
rosatellite markers in the SNP and thyroglobulin (Tg) genes. the neck and upper chest, palpation of the goiter to determine
Tg is a huge exon 48 gene, so further work was undertaken its size and nodules, and assessment of local lymph nodes
to explore and define the huge diversity of haplotypes. In and signs of hyperthyroidism. Standard thyroid function
some Caucasian cohorts, the SNP in the Tg gene is associ- tests (FT3, FT4, and TSH) will confirm significant or sub-
ated with GD, and it has been postulated that there is a high-
clinical hyperthyroidism, but depending on the number of
level interaction between the SNP in Tg exon 33 and autonomous cells, hyperthyroidism may still prevail. In a ret-
HLA-DR. This proposal raises the possibility of making Tg rospective study, the files of patients with nontoxic goiter
polymorphisms favor GD by modulating antigen-presenting were reviewed. Serum TSH was less than 1 mIU/L in 46% of
cells on HLA class II molecules to present Tg peptides to T patients in the single nodular goiter group and 68% in the
cells. The mechanism of action of many of these susceptible multinodular goiter group, indicating partial autonomy. TSH
loci is not fully understood. Further research into the role of
is by far the most commonly used test for preliminary assess-
gene–gene or gene–environment interactions and epigenetic ment of thyroid function. If the TSH concentration is low,
factors may be important to improve our understanding of FT4 must be measured to determine whether the patient has
the genetic basis of this disease. subclinical or significant hyperthyroidism. For patients with
low serum TSH and normal FT4 concentration, serum FT3
40.2.7.2 Toxic Adenoma and Multinodular Toxic should be measured. For management algorithms in TA and
Goiter TMNG, see in Fig. 40.15.
Clinical Aspects and Epidemiology There is no routine indication of thyroid autonomy to
Toxic thyroid adenoma (TA) is a well-encapsulated homoge- measure thyroid antibodies.
neous tumor that secretes thyroid hormones in other normal However, in iodine-deficient areas, it is difficult to distin-
glands without TSH stimulation. As shown by scintigraphic guish between Graves’ disease and TMNG if there is a lack
imaging, the diagnosis includes the ability to take up iodide of extrathyroidal manifestations of autoimmune thyroid dis-
autonomously and the rest of the thyroid to take up reduced ease, and ultrasound examination shows the presence of thy-
or suppressed iodine. Toxic multinodular goiter (TMNG) roid nodules.
covers a range of pathologies, ranging from a single high- In this case, the identification of TSH receptor antibodies
functioning nodule (with additional normal or nonfunction- can help establish the correct diagnosis. Antithyroid peroxi-
Initial laboratory values FT3 6.25 pmol/L, FT4

Thyroid nodule Hyperthyroidism 24.05 pmol/L, TSH 0.012 mIU/L, TPO-Ab 81.5 IU/mL,
TR-Ab 8.77 IU/L.
Treatment Admission to the hospital continued to control

Ultrasound + TSH, fT3 and fT4 thyroid dysfunction and hormone shock therapy for Graves’
ophthalmopathy. After treatment, the swelling of both eyes
was better than before, there was no obvious pain in eye
movement, the hand movement was relieved, and oral drug
Scintiscan
control was continued after discharge.
Test Performed FT3, FT4, TSH, and TPO-Ab are the most
• Check complications of
• ATD + β-blockers Hyperthyroidism and goiter commonly used serological indicators for monitoring thy-
for symptom relief e.g atrial fibrillation, osteoporosis, tracheal roid function. SP70 was used for differential diagnosis of
compression
benign and malignant diseases. CT can be used for screening
Perform ablative therapy
for eye diseases caused by thyroid dysfunction.
Radioiodine Thyroid Results with Interpretation Guideline Serological mark-

therapy surgery
ers of thyroid function are a common indicator for diagnos-
ing thyroid dysfunction, and regular review of relevant
Fig. 40.15 The management algorithm in TA and TMNG indicators can monitor changes in the condition and adjust
treatment options. B-ultrasound can be used to determine the
dase antibodies have been reported to predict the develop- cause of thyroid dysfunction. SP70 is a good serological
ment of hypothyroidism after 131I treatment of diagnostic marker for identifying benign and malignant thy-
hyperthyroidism. In fact, the presence of anti-TPO antibod- roid nodules. Other parts of the disease caused by thyroid
ies is associated with a higher risk of permanent hypothy- dysfunction can be examined by other imaging techniques
roidism, reflecting the coexistence of autoimmune thyroiditis. such as CT.
If iodine contamination is suspected, the amount of iodine
excretion in the urine can be measured. The location, size, This case was from the First Affiliated Hospital of Nanjing
and number of thyroid nodules and goiter can be determined Medical University (also named Jiangsu Province Hospital).
by ultrasound scan. The a priori risk of malignancy in non-
functional nodules may be higher than in normal functioning
tissues. Therefore, based on the ultrasound appearance of 40.3 Dwarfism
thyroid nodules, TMNG fine-needle puncture should be con-
sidered in the area of hypothyroid goiter with normal, nor- Zhaojing Zheng and Juan Geng
mal, and nonfunctional thyroid nodules. If a patient with
normal thyroid function is suspected to have thyroid auton- Overview
omy, a “suppressive” scan can be performed after thyroid Growth is an essential and continuous process in human.
hormone administration to induce exogenous hyperthyroid- Any disturbance or faltering in height is thus, a frequent
ism. As a result, nonautonomous organizations will be sup- cause of concern for the patients and their family. Dwarfism,
pressed, and thyroid autonomy will be covered up. also known as short stature, is a condition in which the height
development is pathogenic short in comparison with the nor-
40.2.7.3 Typical Case mal. Dwarfism could be caused by many different factors. A
Clinical Background The 52-year-old female patient was detailed clinical evaluation including accurate anthropome-
admitted to hospital for treatment because of “hand shake for try is essential to suspect and diagnose the underlying cause.
7 months and eyes for 5 months”. The role of more sophisticated investigations including
molecular diagnosis should not be underestimated for sub-
Imaging examination Multirow CT orbital plain scan: jects with dwarfism. Treatment is directed as per the primary
Bilateral eyeballs protruded, and bilateral extraocular etiology of dwarfism. Growth hormone therapy is a highly
muscles were significantly thickened, Graves eye disease specific and targeted therapy which should be instituted only
may be. under expert consultation.
696 H. Yuan et al.
40.3.1 Clinical Appearance Growth Hormone Deficiency

Growth hormone deficiency (GHD) is a relatively common
40.3.1.1 Disproportionate Dwarfism cause of proportional dwarfism. It occurs when the pitu-
If the body size is disproportionate, some parts of the itary gland does not produce enough growth hormone,
body are small, while the average size of other parts is which is essential for normal child growth. Signs include:
above average. Diseases that will cause disproportionate (1) Height below the third percentile on standard pediatric
dwarfism inhibit bone development. Disproportionate growth charts; (2) Growth rate slower than expected for
dwarfism is characterized by shortened limbs or shortened age; (3) Delayed or no sexual development during the teen-
trunks. age years.
Usually, this means that a person has an average-sized There are four types of isolated GHD according to the
trunk with short limbs and a larger forehead. Facial features severity of the condition, the pathogenic genes involved, and
are often affected, and problems may arise in various parts of the inheritance pattern (Table 40.9). Type IA GHD is charac-
the body. Spinal stenosis, ear infections, and hydrocephalus terized by an absence of growth hormone and is the most
are common. Almost all dwarfism patients have normal severe of all the types. In people with type IA, growth failure
intelligence. is evident in infancy as affected babies are shorter than nor-
mal at birth. Patients with type IB GHD produce very low
Achondroplasia levels of GH. As a result, type IB is characterized by short
The most common cause of dwarfism is a disease called stature, but this growth failure is typically not as severe as in
achondroplasia that causes disproportionate size. The type IA. Growth failure in people with type IB is usually
affected individuals had rhizome shortening of limbs, large apparent in early to mid-childhood. Patients with type II
head deformity and characteristic facial features of forehead GHD have very low levels of GH and short stature that varies
eminence and midfacial retraction. In infancy, hypotonia is in severity. Growth failure in these individuals is usually evi-
typical, and the achievement of developmental sports mile- dent in early to mid-childhood. It is estimated that nearly half
stones is usually abnormal in pattern and delayed. Intelligence of the individuals with type II have underdevelopment of the
and life expectancy are usually close to normal, although pituitary gland (pituitary hypoplasia). Type III is similar to
pressure at the craniocervical junction increases the risk of type II in that affected individuals have very low levels of
infant mortality. Other complications include obstructive GH and short stature that varies in severity. Growth failure in
sleep apnea, middle ear dysfunction, kyphosis, and spinal type III is usually apparent in early to mid-childhood. People
stenosis.
Spondyloepiphyseal Dysplasia Congenita Table 40.9 Genetic forms of isolated GHD

Another cause of disproportionate dwarfism is a rare disor- Type Gene Inheritance Phenotype
der called Congenital spondylitis dysplasia (SEDC). SEDC IA GH1, POC1A, AR Severe postnatal growth
is an autosomal dominant hereditary cartilage dysplasia CRIPT, XRCC4, failure
DNA2, BRCA2, Undetectable serum GH
characterized by a disproportionately short stature (short RNPC3 Anti-GH antibodies with
torso), abnormal callus, and flattened vertebral bodies. GH Rx
Skeletal features are manifested at birth and evolve with IB GH1, GHRHR AR Less severe growth failure
time. Other features include myopia and/or retinal degenera- than type IA
Low, but detectable,
tion with retinal detachment and cleft palate. serum GH
No anti-GH antibodies
40.3.1.2 Proportionate Dwarfism with GH Rx
If all parts of a body are small to the same extent and look II GH1, AD Variability in height
proportionate like a medium-sized body, then the body is POU1F1,GHRHR deficit
Normal or hypoplastic
proportionally small. Medical conditions at birth or early in pituitary
childhood limit overall growth and development. Other pituitary deficits
Proportional dwarfism is characterized by short trunk and can develop
limbs, resulting in a significantly lower height than the aver- III BTK, SOX3, PHEX, X-linked GHD with
GHRHR agammaglobulinemia
age. There may be no significant growth for a long time. GHD with mental
Sexual development is usually delayed or impaired to adult- retardation
hood. This type of dwarfism is caused by endocrine disorders Abbreviations: AR autosomal recessive, AD autosomal dominant, Rx
rather than skeletal dysplasia. treatment
with type III may also have a compromised immune system occur as a result of disorganized molecular and genomic
and are susceptible to frequent infections. changes in the embryonic stage, and thus, it is a unique area
to study growth and developmental abnormalities [67].
Combined Pituitary Hormone Deficiency (CPHD) Other causes of dwarfism are atrophic dysplasia, pseudo-
CPHD refers to the presence of GH deficiency along with chondral hypoplasia, achondroplasia, Noonan syndrome,
additional deficiencies of other pituitary hormones such as Turner syndrome, 3M syndrome, osteogenesis imperfecta
TSH, prolactin, LH, FSH, and/or ACTH. Thus, in addition to (OI), and hypothyroidism. Severe shortness and skeletal dis-
growth retardation of postnatal onset, patients with CPHD tortion also occur in some mucopolysaccharides and other
may manifest with symptoms of hypothyroidism, hypogo- storage disorders. Low gonadotropins may result in a com-
nadotropic hypogonadism, and/or adrenal insufficiency. mensurate but temporary dwarfism. Severe chronic diseases
CPHD typically results from defective anterior pituitary can lead to dwarfism as a side effect. Poor environmental
gland development, with hormone deficits manifesting in conditions, such as malnutrition, can also cause dwarfism.
early childhood. These types of dwarfism are an indirect consequence of a
person’s general unhealthy or malnourished condition, rather
Primary IGF Deficiency (IGFD) and IGF Resistance than an indirect consequence of any particular disease.
The earliest identified form of GH insensitivity (GHI) was
reported by Laron and colleagues almost 50 years ago and
was eventually found to be the consequence of mutations in 40.3.2 Diagnosis
the gene for the GH receptor (GHR) [66]. Multiple muta-
tions and deletions of GHR have now been identified and A basic part of diagnostic procedure for dwarfism is the mea-
shown to result in varying degrees of GHI. surement of height, weight, and head circumference. This is
The growth hormone (GH) promotes body growth by important for identifying abnormal growth, such as delayed
binding to two GH receptors (GHR) on the cell surface. growth or a disproportionately large head. Many distinct
GHR interacts with Janus kinases, signal transducers, and facial and skeletal features are associated with each of sev-
transcriptional activators to stimulate metabolism and eral dwarfism disorders. Thus, it is also very important for
insulin-like growth factor (IGF) synthesis. However, process dwarfism diagnosis to perform appearance observation and
dysfunctions in the GH GHR IGF-1 axis cause dwarfism. If, evaluation. In addition, family history is very helpful for
during the GH process, GHR is not successfully recognized diagnosis of dwarfism. A history of stature in siblings, par-
and/or bound, or GHR fails to transmit the GH signal to IGF- ents, grandparents, or other relatives helps determine whether
1, the GH dysfunction occurs. the average range of height in the patient’s family includes
short stature.
40.3.1.3 Others Imaging technology is always used in the diagnostic pro-
SHOX Deficiency Disorders cedure of dwarfism. Various imaging devices may also reveal
SHOX deficiency phenotype spectrum is caused by the lack delayed maturation of bones, as occurs in growth hormone
of haplotype of SHOX gene, ranging from severe terminal deficiency. A magnetic resonance imaging (MRI) scan may
LWD to mild terminal nonspecific short somatotype. For reveal abnormalities of the pituitary gland or hypothalamus,
adults with a lack of SHOX, the proportion of LWD to short both of which play a role in hormone function. Basically,
stature without LWD characteristics is not known. In LWD, hormone tests are performed to assess levels of growth hor-
the typical clinical trial is short stature, medium length defor- mone or other hormones that are critical for childhood
mity, and Madelung deformity. The short-formed phenotype growth and development for diagnosis of dwarfism.
caused by SHOX deficiency varies greatly in the absence of With increasingly application of next-generation
mesoderm and Madelung malformations, even in the same sequencing (NGS)-based molecular diagnostic methodolo-
family. gies in clinical practice, it is now possible to rapidly detect
genome-wide genetic variations at significantly lower costs.
Primordial Dwarfism A diagnostic algorithm (Fig. 40.16) proposes a comprehen-
Primary dwarfism is a group of hereditary diseases, including sive genetic diagnostic approach to a wide array of clinical
Seckel syndrome, Silver–Russell syndrome, primary dwarf- presentations. The algorithm differentiates patients who
ism I/III, type II, and Meier–Gorlin syndrome (Table 40.10). were born SGA from those with isolated postnatal short
This genetic disorder group is characterized by intrauterine stature. Targeted evaluation of a single gene or panels of
growth retardation and postnatal growth abnormalities which genes is recommended for subgroups of patients who fall
698 H. Yuan et al.
Table 40.10 Subtypes existing in primordial dwarfism

Category (with
Subtypes respect to head size) Chromosomal abnormalities Genes Inheritance Phenotype
Seckel syndrome Microcephalic Increased chromosomal instability ATR, ATRIP, AR Extremely small head
primordial dwarfism at fragile sites CENPJ, TRAIP, with narrow face, dental
(brain size reduced ATRIP, PLK4, alterations, beak-like
to a third of normal CENPE, PCNT protrusion of nose,
volume) CEP152, RBBP8 receding mandible,
intellectual disability
Microcephalic / U4atac AR Dry skin, sparsity of
osteodysplastic hairs and eyebrows
primordial
dwarfism (MOPD)
types I/III
MOPD type II / PCNT, IGF1R AR Prominent nose and
eyes, abnormally small
or missing teeth, and a
high squeaky voice
Meier–Gorlin / ORC1, ORC4, AR/AD Severe intrauterine and
syndrome ORC6, CDT1, postnatal growth
CDC6, CDC45L, retardation,
MCM5, GMNN microcephaly, bilateral
microtia, and aplasia or
hypoplasia of the
patellae
Silver–Russell Normal head size mUPD, mosaic trisomy, IGF2, RSS, AD/AR/ Small triangular face,
syndrome duplications, deletions, CDKN1C, genomic prominent forehead,
microdeletions translocations, HMGA2, imprinting micrognathia, dental
hypomethylation, DNA PLAG1, H19, anomalies
methylation changes, genomic CUL7, OBSL1,
imprinting, epigenetic alterations SGCE, ZFP57,
and unambiguous copy number NF1, CSH1
changes
Abbreviations: AR autosomal recessive, AD autosomal dominant
into distinct diagnostic categories discussed above. For Single-gene testing of COL2A1 can be considered if clini-
those patients who do not fit into a distinct subgroup or for cal findings and/or family history indicate that pathogenic
whom initial genetic testing is inconclusive, genome-wide variants in this particular gene are more likely.
evaluation through exome sequencing and chromosomal A multigene panel that includes COL2A1 and other genes
microarray to detect both sequence variants and CNV is rec- of interest should be considered, particularly in instances
ommended [68]. with diagnostic uncertainty (e.g., prenatal assessment) which
determines the genetic cause of the condition at the most rea-
40.3.2.1 Disproportionate Dwarfism sonable time and cost, while limiting the variation of the
Achondroplasia uncertain meaning and the identification of the pathogen of
Achondroplasia can be diagnosed by characteristic clinical the gene that does not interpret the potential phenotype.
and imaging studies of most affected individuals. For indi-
viduals with diagnostic uncertainties or atypical findings, 40.3.2.2 Proportionate Dwarfism
identification of heterozygote pathogenic variants in FGFR3 Growth Hormone Deficiency
can confirm the diagnosis. The most common cause of IGHD IA is a homozygous dele-
tion of the GH1 gene. GH1 deletions occur by nonallelic
Spondyloepiphyseal Dysplasia Congenita homologous recombination mediated by repeats within the
Spondyloepiphyseal dysplasia congenita is one of the spec- GH gene cluster. Other loss-of-function (LoF) mutations
trums of skeletal disorders caused by mutations in theCO- (frameshift and nonsense) in GH1 also lead to absence of
L2A1gene. This gene provides instructions for making a GH and severe dwarfism [69].
protein that forms type II collagen. Type IB IGHD is caused by mutations in GH1,
Molecular genetic testing approaches can include single- GH-releasing hormone receptor gene (GHRHR), or the
gene testing and use of a multigene panel: ghrelin receptor gene (GHSR). Growth retardation in
Patient with a high likelihood for a

monogenic etiology of short stature
Targeted genetic testing

Does the patient have a
Yes (sequencing, microarray, or
distinct recognizable genetic
karyotype as appropriate) for
syndrome?
known candidate genes
Was the patient small for

gestational age?
Yes No
Does the patient have Does the patient have No

microcephaly? disproportionate short stature
or other skeletal anomaly?
Does the patient have
Yes Yes growth hormone
No deficiency or
Perform a skeletal survey. insensitivity?
Targeted next Testing for Does it suggest a distinct
generation Russell -Silver syndrome?
sequencing panel Syndrome No Yes
and copy number and/ or 3M Yes No
assessment of all syndrome
Male or No Targeted next
genes known to
cause microcephalic Targeted If female Male or Female? generation sequencing
primordial dwarfism genetic panel and copy number
testing Female? assessment of all genes
plus IGF1 and IGF1R
Yes known to cause isolated
If male genes known to cause
isolated GHD, multiple
Karyotype if Karyotype if pituitary hormone
(45,X), (45,X), deficiency or growth
diagnosed as diagnosed as hormone insensitivity
If negative Turner’s Turner's
If negative If negative
syndrome? syndrome?
If negative
If
SHOX testing, negative
consider FGFR3
If negative
If negative
Consider sequencing
NPR2 if one or both
parents has short
stature
Apparent idiopathic short stature
If negative
Consider whole exome sequencing and chromosomal microarray for copy
number assessment
Fig. 40.16 Diagnostic algorithm for the genetic diagnosis of short stature
patients with type IB IGHD is less severe than in those with resulting in production of the 17.5-kDa isoform of GH that
type IA IGHD and patients usually respond well to exoge- has a dominant negative effect on normal GH interaction.
nous GH. IGHD III is an X-linked disorder associated with panhy-
Type IIIGHD is usually caused by mutations affecting the pogammaglobulinemia, and specific mutations in BTK gene
correct splicing of GH1 leading to skipping of exon 3 and have been implicated in this disorder.
700 H. Yuan et al.
Molecular diagnosis is necessary for predicting disease 40.3.3.2 Spondyloepiphyseal Dysplasia

progression, avoiding unnecessary interventions, and for Congenita
genetic counseling and family planning. Spondyloepiphyseal dysplasia congenita is one of the spec-
trum of skeletal disorders caused by mutations in the
Combined Pituitary Hormone Deficiency (CPHD) COL2A1 gene. This gene provides instructions for making a
Testing for deficient secretion of GH, TSH, LH, FSH, PrL, protein that forms type II collagen. This type of collagen is
and ACTH establishes the diagnosis of CPHD. PROP1 is the mainly found in cartilage and transparent gels that fill the
only gene in which pathogenic variants are known to cause eyeballs (vitreous bodies). COL2A1 gene is essential for the
PROP1-related CPHD. normal development of bones and other tissues, which form
the supporting framework (connective tissue) of the human
40.3.2.3 Others body. Mutations in the COL2A1 gene interfere with the
SHOX Deficiency Disorders assembly of type II collagen molecules, which prevent the
Molecular genetic testing approaches include gene-targeted normal development of bone and other connective tissues.
testing and comprehensive genomic testing depending on the
phenotype. 40.3.3.3 Growth Hormone Deficiency
Gene-targeted testing requires the clinician determine IGHD can have different inheritance patterns depending on
which gene(s) are likely involved, whereas genomic testing the type of the condition. IGHD IA and IB are inherited in
does not. Because the phenotype of SHOX deficiency is broad, AR pattern. Parents of individuals with AR disorders each
individuals with the distinctive findings of Leri-Weill dys- carry a copy of the mutant gene, but they usually do not show
chondrosteosis (LWD) described in Suggestive Findings are the symptoms and signs of the disease.
likely to be diagnosed using a combination of chromosomal IGHD II can be inherited in AD pattern, which means a
microarray analysis (CMA) and gene-targeted testing, whereas mutation in one copy of the GH1 gene in each cell is suffi-
individuals with SHOX-deficient short stature in which the cient to cause the disorder. This condition can also result
phenotype may be indistinguishable from many other inher- from new mutations in the GH1 gene and occur in people
ited disorders with short stature particularly in early childhood with no history of the disorder in their family.
are more likely to be diagnosed using genomic testing. IGHD III, caused by mutations in BTK gene, is a condi-
tion of X-linked recessive inheritance. Because it is unlikely
Primordial Dwarfism that females will have two altered copies of this gene, males
Russell–Silver Syndrome are affected by X-linked recessive disorders much more fre-
RSS is a genetically heterogeneous disease that repre- quently than females. A characteristic of X-linked inheri-
sents a phenotype rather than a specific disease for most tance is that fathers cannot pass X-linked traits to their sons.
affected individuals. Therefore, the diagnosis is mainly based
on the consistent clinical features, especially the growth 40.3.3.4 Others
retardation and normal head circumference before and after SHOX Deficiency Disorders
delivery. 30%~60% of patients have DNA methylation dele- No correlation has been established between the severity of
tions in the allelic imprinting control region 1 (ICR1) of the the phenotype and the underlying SHOX pathogenic
11p15 region of chromosome. Five cases were found in variant.
35–50% of RSS patients. About 10% of RSS patients have a Based on a limited number of studies, the frequency of
mother’s single parental dimer (UPD7) on chromosome 7. LWS is greater than SHOX-deficient short stature caused by
Meier–Gorlin Syndrome a SHOX enhancer deletion, suggesting that enhancer dele-
Meier–Gorlin syndrome can be caused by mutations in one tions cause a more severe phenotype. In the French popula-
of several genes. Each of these genes, ORC1, ORC4, ORC6, tion, however, deletions of the downstream enhancer region
CDT1, and CDC6, provides instructions for making one of a of SHOX seem to be associated with a milder phenotype.
group of proteins known as the prereplication complex. Thus, this issue remains unresolved.
40.3.3 Correlation Between Phenotype 40.3.4 Genetic Counseling

and Genotype
40.3.4.1 Achondroplasia
40.3.3.1 Achondroplasia Cartilage hypoplasia is inherited by autosomal dominant
Because nearly all instances of achondroplasia arise second- inheritance. About 80% of patients with chondrodysplasia
ary to identical amino acid substitutions, genotype–pheno- have medium-sized parents and a new pathogenic variant
type correlation related to the primary pathogenic variant is leading to cartilage dysplasia. The risk of such parents with
not possible. achondroplasia is low. Individuals with achondroplasia have
a 50% risk of developing achondroplasia during each preg- for it. But most cases are not identified until after the child
nancy if they have an average size reproductive partner. is born.
When both parents have achondroplasia, the average height
of their offspring is 25%; there is achondroplasia, 50%; and 40.3.5.1 Achondroplasia
homozygous achondroplasia (fatal disease), 25%. If the Preproduction diagnosis can be accidentally performed dur-
reproductive partners of probands and probands are affected ing routine prenatal ultrasound examinations in the third tri-
by different dominant genetic skeletal dysplasia, genetic mester of pregnancy. In high-risk pregnancies, or in
counseling becomes more complex, because it is possible to pregnancies suspected of achondroplasia after ultrasound
inherit two dominant skeletal dysplasia. Prenatal testing for examination, FGFR3 mutations in fetal DNA can be detected
increased risk of gestational dysplasia in pregnancy can be to confirm the diagnosis. Preimplantation genetic diagnosis
performed if an FGFR3 pathogenic variant has been identi- can be performed in a specialized laboratory.
fied in the affected parent or parent.
40.3.5.2 Others
40.3.4.2 Spondyloepiphyseal Dysplasia SHOX Deficiency Disorders
Congenita If the SHOX pathogenic variant has been identified in one or
Heredity is autosomal dominant inheritance; genetic coun- both parents, prenatal diagnostic testing for pregnancies at
seling is possible. increased risk is possible; however, the phenotype of the
SHOX deficiency disorder cannot be accurately predicted
40.3.4.3 C ombined Pituitary Hormone entirely on the basis of prenatal molecular genetic testing
Deficiency results.
Type of transmission varies with the factor and the mutation
involved (recessive transmission for PROP1 and LHX3, Primordial Dwarfism
dominant for LHX4, autosomal or recessive for POU1F1 and MOPD I: Prenatal diagnosis, by ultrasonography at around
HESX1). 20 weeks of gestation, has been reported in affected
families.
40.3.4.4 Others MOPD II: By observing IUGR, the pregnancy of a sick
SHOX Deficiency Disorders child often becomes complicated. Due to IUGR, cesarean
SHOX deficiency is inherited by pseudoautosomal dominant section can be performed at an earlier age. Prenatal diagnosis
inheritance. In pseudo-autosomal dominant inheritance, the is possible if PCNT mutation is detected in the carrier’s
homologous genes located on the short arm (xp) of X chro- parents.
mosome and the short arm (yp) of Y chromosome follow the Silver–Russell Syndrome: Prenatal diagnosis is not usu-
autosomal inheritance rule. Therefore, SHOX pathogenic ally possible (as most cases reported so far are sporadic, the
variants causing SHOX deletion can be located on Y chromo- potential risk of having an affected child is not anticipated
some of the affected male or the X chromosome of the during pregnancy).
affected female.
Each child with an individual with SHOX deficiency has
a 50% chance of inherited SHOX-causing lesions. If both 40.3.6 Typical Clinical Case
parents lack shoes, the offspring have a 50% chance of shoe
deficiency, 25% chance of Langer mid-dwarf, and 25% A 4-year-old boy was referred with a clinical diagnosis of
chance of unconditional. idiopathic short statute. His height was 3SD below normal
range with his mother height of 140 cm and his father of
Primordial Dwarfism 159 cm. Facial dysmorphism and other congenital anomalies
MOPD types 1 and 3: transmitted as autosomal recessive were absent in this boy with normal routine laboratory test-
traits, and genetic counseling is possible. ing results. His parents were not consanguineous.
Silver–Russell syndrome: The recurrence risk is extremely He had a normal result in karyotyping analysis of periph-
low in cases of uniparental disomy of chromosome 7 or epi- eral lymphocytes. Whole exome sequencing was performed
genetic anomalies of the 11p15 region. with Agilent SureSelect Human All Exon V6 kit and Illumina
sequencing platform. After standard bioinformatic process-
ing and proband-only variant filtering strategy, the patient
40.3.5 Prenatal Diagnosis was found to harbor a heterozygous c.5026_5027del variant
and Preimplantation Genetic Diagnosis in ACAN gene, which was categorized as “likely pathogenic”
according to ACMG standard and validated by Sanger
Some types of dwarfism can be identified through prenatal sequencing (Fig. 40.17).
testing if a doctor suspects a particular condition and tests This case was from Shanghai Children’s Medical Center.
702 H. Yuan et al.
Fig. 40.17 Classic case of idiopathic short statute due to ACAN gene sequencing with integrated genome visualization (IGV) browser; the
mutation. The upper row shows the reads harboring the likely patho- lower row shows the chromatography of ACAN c.5026_5027del in the
genic variant c.5026_5027del in ACAN gene through whole exome proband and his parents by Sanger sequencing
40.4 Adrenopathy which are two independent glands in structure, function and
embryonic development. It is also called suprarenal gland
Hong Yuan and Jingyuan Zhao (Fig. 40.18).
Adrenopathy includes various adrenal diseases. Common
The adrenal gland is a major endocrine organ in the human diseases are Cushing syndrome, primary aldosteronism (PA),
body. It is grayish yellow and locates above the kidney. primary chronic adrenocortical hypofunction (Addison’s
There is a pair of adrenal glands in the human body. Each disease), pheochromocytoma, and congenital adrenocortical
gland is divided into two parts: adrenal cortex and medulla, hyperplasia.
40.4.2 Cushing’s Syndrome
40.4.2.1 Pathogeny and Clinical Manifestation

The Etiology of Cushing’s Syndrome
ACTH-dependent Cushing’s Syndrome
1. Cushing’s disease: Accounting for 70% of Cushing’s syn-
drome, most of the patients’ pituitary glands have micro-
adenomas, a few are macroadenomas, and some have not
been found tumors [70].
2. Ectopic ACTH syndrome: Tumors outside the pituitary
gland secrete large amounts of ACTH, accompanied by
adrenal cortical hyperplasia [71].
3. Ectopic CRH syndrome: Some tumors produce CRH and
Fig. 40.18 Anatomy of the adrenal glands; the adrenal glands are stimulate pituitary secretion ACTH, resulting in excessive
divided into two parts: adrenal cortex and medulla
glucocorticoid secretion [72].
40.4.1 Overview Cushing’s Syndrome Independent of ACTH

1. Adrenal cortical adenoma
40.4.1.1 Structure of Adrenal Gland 2. Adrenal cortical carcinoma
The adrenal glands are located above the kidneys on both 3. Bilateral nodular adrenal hyperplasia independent of

sides and are wrapped in the renal fascia together with the ACTH
kidneys, but the adrenal glands have independent fibrous
sacs and fat sacs; the left adrenal gland is half-moon- The Pathogeny of Cushing’s Syndrome at the Molecular
shaped, and the right adrenal gland is triangular or elliptic. Level
The adrenal parenchyma consists of the surrounding cor- Yanan Cao reported in Science that the L205R hot spot
tex and the central medulla. The adrenal cortex accounts mutation in PRKACA gene is closely related to the occur-
for about 90% of the gland. Its structure consists of three rence of adrenocortical adenoma [73], which provides a
layers: the globular zone, the fascicular zone, and the retic- basis for the future development of diagnosis and treatment
ular zone. The cells in the three zones can secrete steroids of Cushing’s syndrome. Gerald et al. also revealed the asso-
(the globular zone mainly secretes aldosterone, the fascic- ciation between mutations in the PRKACA gene and adre-
ular zone mainly secretes glucocorticoid, and the reticular nal tumor [74].
zone mainly secretes androgen). The adrenal medulla is
about 10% of the volume of adrenal gland, located in the The Typical Clinical Manifestations of Cushing’s
center of the adrenal gland, mainly composed of medullary Syndrome
cells, which can be divided into adrenaline cells and nor- 1. Central obesity and moon face: This clinical manifesta-
adrenaline cells. As an endocrine organ, the adrenal gland tion is related to the redistribution of fat caused by
regulates the body’s function mainly by secreting glucocorticoid.
hormones. 2. Polyplasmic appearance: Skin of the patients is very thin,
As an important endocrine organ, a variety of clinical microvessels can be seen easily, and red blood cells and
diseases can cause hyperactivity or insufficiency of adrenal hemoglobin increase.
function, and the examination of adrenaline and its metabo- 3. General muscle weakness and difficulty in standing after
lites can be combined with other type of examinations to squatting is very common.
complete the diagnosis of the diseases. Therefore, we 4. When the skin is slightly damaged, it can cause ecchy-
should have a comprehensive understanding of these com- mosis, purple lines appear on both sides of the lower
monly used examinations, so as to better complete the diag- abdomen, and lateral thighs, and skin pigmentation
nosis of the diseases. Laboratory examination of adrenal deepens.
gland includes two categories: tests that assess the level of 5. Hypertension is the common symptom. The incidence of
adrenal medullary hormone and the level of adrenocortical arteriosclerosis and renal arteriosclerosis increase, and
hormone. steroid diabetes and hypokalemic alkalosis are common.
704 H. Yuan et al.
40.4.2.2 L aboratory Diagnosis of Cushing’s syndrome. It is a qualitative diagnostic test of Cushing’s

Syndrome syndrome.
There is no independent biomarker for the diagnosis of Positive results of above tests indicate that the patients
Cushing’s syndrome; the diagnosis still needs a variety of suffer from Cushing’s syndrome. At this time, clinicians
examinations. The laboratory examination of Cushing’s should arrange other tests to confirm the cause of Cushing’s
syndrome can be divided into two stages. First, it is neces- syndrome.
sary to prove the existence of excessive cortisol. Second, it is
necessary to determine the cause of cortisol elevation through Follow-up Tests
other experiments (Fig. 40.19) [75]. 1. CRH stimulation test: Patients with Cushing’s disease
have higher ACTH level and can be stimulated by
Screening Tests and Confirmatory Test to Diagnosis CRH. After injecting CRH, ACTH increases by more
Cushing’s Syndrome than 50%. Patients with adrenal cortical adenoma can
There are two common tests to evaluate the level of cortisol: secrete a large amount of cortisol autonomously and then
One is midnight plasma cortisol or late-night plasma corti- the feedback inhibits pituitary gland, so ACTH base value
sol, which can help clinicians to measure the plasma cortisol is lower than normal; ACTH increases by less than 50%
and find out its circadian rhythm changes, and the other is after injecting CRH, while ACTH level of patients with
24-h urinary free cortisol test. ectopic ACTH is not affected by CRH.
Low-dose dexamethasone inhibition test (LDDST) can 2. High-dose dexamethasone suppression test (HDDST) can
distinguish pseudo-Cushing’s syndrome from true Cushing’s be used when Cushing’s syndrome has been diagnosed;
Fig. 40.19 Diagnosis
procedure of Cushing’s
syndrome Clinical manifestations of hyperortisolism
Examination of 24-hour urinary free protein

cortisol
Examination of plasma cortisol and its diurnal
changes
Normal
Abnormal LDDST
Other diseases
Can’t be inhibited
Inhibited
Pseudo-chushing
Cushing sydrome
syndrome
Plasma ACTH
Low concentration High concentration

Non-ACTH-dependent cushing sydrome ACTH-dependent cushing sydrome
HDDST1
Inhibited Can’t be inhibited

Cushing disease Ectopic ACTH syndrome
this test can help clinicians to distinguish if the symptoms given a combination of CCB, ARB, and b receptor blockers
are caused by pituitary Cushing’s syndrome (Cushing’s to reduce blood pressure. The patient had a transsphenoidal
disease) or nonpituitary Cushing’s syndrome (ectopic pituitary microadenomas surgery, pathology showed it was
ACTH tumor, adrenocortical adenoma, adrenocortical pituitary adenoma, and immunohistochemistry showed
carcinoma). ACTH (+++).
3. Dexamethasone–corticotropin-releasing hormone test

can help to differentiate Cushing’s syndrome from Treatment Outcome A week later, the level of ACTH and
pseudo-Cushing’s syndrome. F were reviewed, the result was at normal level, and there
4. Plasma ACTH test can distinguish ACTH-dependent and were no abnormalities in thyroid function and gonadal hor-
non-ACTH-dependent Cushing’s syndrome. mone level.
Other Routine Laboratory Tests This case was from the First Affiliated Hospital of Dalian
1. Routine blood test may exist a high WBC count and Medical University.
increased number of neutrophils
2. Glucose tolerance test shows the patients’ glucose toler-
ance is impaired.
40.4.3 Primary Aldosteronism
3. Blood potassium decreased.
40.4.3.1 Pathogeny and Clinical Appearance
40.4.2.3 Typical Medical Case
Pathogeny
Clinical Background A 22-year-old male had an intermit-
Aldosteronoma
tent headache without precipitating cause recent 2 months;
Aldosteronoma is the most common cause of primary
the highest blood pressure was 220/130 mmHg. Oral admin-
aldosteronism. The plasma aldosterone concentration is par-
istration of various antihypertensive drugs had poor effects.
allel to the circadian rhythm of plasma ACTH, but it has no
Since morbidity, his face became round and red consciously,
response to the change of plasma renin.
the gingiva and the skin of palm print became black, intake
Idiopathic Aldosteronism
of water and nocturnal urine increased, emotion fluctuated
Hyperplasia of bilateral adrenal globular zone is com-
easily, no fatigue and limb paralysis, and he gained 10 kg in
mon, sometimes accompanied with nodules.
the past 20 days.
Glucocorticoid-Remediable Aldosteronism (GRA)
It is an autosomal dominant hereditary disease, which
Physical Examination The blood pressure was
usually occurs in teenagers.
180/100 mmHg; BMI was 29.8 kg/m2; central obesity, moon
Aldosterone Tumors
face, plethoric appearance, purple lines in abdomen, and skin
Aldosterone tumors is a rare pathogeny.
darkened in gingiva, palms, nipples, and elbow joints.
Genetics Pathogeny
Supplementary Examination Blood potassium: According to the research, about 40% patients with an
3.35 mmol/L; 24 h urine potassium: 26.2 mmol/24 h; blood adrenal aldosterone-producing adenoma have mutations on a
gas analysis: PH 7.40, BE 3.5 mmol/L. HbA1C: 5.7%; fast- gene: KCNJ5 [76]. Studies on familial hyperaldosteronism
ing blood sugar: 4.2 mmol/L; 2 h postprandial blood glu- also show that the occurrence of type I (FH-I) is related to the
cose: 5.76 mmol/L. formation of chimeric genes by nonreciprocal exchange of
CYP11B2 and CYP11B1 [77] and type III (FH-III) are
Dexamethasone Inhibition Test ACTH-F rhythm disor- closely related to KCNJ5 gene [78, 79]. Other genes com-
der; ACTH, F, UFC increased; F could not be inhibited by monly mutate in Primary aldosteronism are ATP1A1,
small doses of dexamethasone, but was inhibited by large ATP2B3, CACNA1D, and CTNNB1 [80–82].
doses of dexamethasone (inhibition rate is more than 50%).
Clinical Appearance
Adrenal Plain Scan CT and Enhancement CT bilateral Aldosterone can prevent the loss of Na+ and water and dis-
adrenal glands were plump. charge K+, so the main clinical manifestations of the disease
Pituitary MRI pituitary adenoma and pituitary stalk are as follows [83]:
shifted to the left, and optic chiasm did not shift.
1. Hypertension: It is the most common symptom; the

Treatment The patient took potassium chloride sustained- mechanism is water and sodium retention caused by
release tablets to supplement potassium. The patients were aldosterone.
706 H. Yuan et al.
2. Hypokalemia can lead to neuromuscular dysfunction,

urine Na+ 214. 759 mmol/day, 24 h urine CI−
arrhythmia, renal tubular epithelial degeneration, poly- 169.78 mmol/L. ESR 24 mm/h.
uria, and so on.
3. Children may also have growth and development
Supplementary Examination ECG: U wave in Vl-V4;
disorders. 24-h ambulatory blood pressure curve was nondipper; blood
pressure increased slightly, moderately, and severely
40.4.3.2 L aboratory Diagnosis of Primary throughout the day; bilateral renal artery ultrasound showed
Aldosteronism no abnormity; bilateral femoral artery ultrasound showed
Screening Examination hard plaque of left femoral artery; bilateral carotid artery
Aldosterone/renin in blood (ARR) is the recommended ultrasound showed increased resistance index of right carotid
screening test for the diagnosis of primary aldosteronism; if artery; chest X-ray showed no abnormality; echocardiogra-
the ratio increase significantly, primary aldosteronism should phy showed pericardial effusion; sleep breathing monitoring
be considered. At present, this test is considered to be the showed sleep disorder index 25/h; slight obstructive sleep
best screening test for the diagnosis of primary apnea hypopnea syndrome; adrenal CT showed the existence
aldosteronism. of left adrenal adenoma and fatty liver; lesion located in the
right lobe of the liver, considering the possibility of heman-
Confirmation Examination gioma; multiple cysts of the right kidney.
On the basis of the above experiments, confirmation test can
be carried out to confirm the diagnosis of primary aldoste- Treatment The patient underwent surgery. The weight of
ronism (Oral salt loading, saline infusion test, fludrocorti- the adenoma was 22 g. The pathological results confirmed
sone suppression test). that the adenoma was adrenal cortex adenoma.
40.4.3.3 Genetic Testing Treatment Outcome The patient was followed 2 weeks
Genetic testing recommended in several studies of primary after operation, and the patient’s blood pressure was
aldosteronism currently is as follows: For patients with PA 130/80 mmHg without antihypertensive drugs.
under 20 years old, especially those who also have family
history of stroke, gene screening test (FH-I) is recommended, This case was from the First Affiliated Hospital of Dalian
and the most common mutation is CYP11B1/CYP11B2 Medical University.
mosaic. For juvenile PA patients, gene screening for FH-III
is recommended and the most common test is KCNJ5 gene
mutation. 40.4.4 Addison’s Disease
40.4.3.4 Typical Medical Case 40.4.4.1 Epidemiology and Clinical Appearance

Clinical Background A 48-year-old male, whose blood Addison’s disease also known as primary adrenal insuffi-
pressure had been in a high level for 3 years, began to ciency is caused by the insufficiency of steroid hormones
feel fatigue in the past half year. A half year ago, the produced by adrenal glands. It is always associated with the
patient went to the hospital to get a physical examination, development of other autoimmune diseases, such as type I
the blood pressure was 140/90 mmHg, and blood potas- diabetes and thyroid disease.
sium was 2.5 mmol/L. After oral potassium chloride
treatment, the symptoms gradually reduced, but the Pathogeny
patient did not adhere to the treatment. 10 days ago, the 1. Infection: Adrenal tuberculosis is a common cause; in
blood potassium was 2.8 mmol/L, and the blood pressure addition, fungi, cytomegalovirus, and HIV are also pos-
was 140/90 mmHg. sible reasons.
2. Autoimmune adrenalitis.
Physical Examination No special abnormalities. 3. Other rare causes: Leukemia infiltration, metastasis of
malignant tumors, etc.
Laboratory Examination Blood count: WBC 9.38 × 109/L,
L 0.33, NEU 0.60, PLT 259 × 1012/L, RBC 4.7 × 109/L, HGB Clinical Appearance
137 g/L; Urine microalbumin: 37.4 mg/L. 1. Elevated ACTH promotes melanogenesis, which causes
The ratio of urine albumin and SCr: 58.9 mg/g, Urine pigmentation in the whole body.
immunoglobulin G: 2.1 mg/L. 2. Clinical manifestations of hypotension, orthostatic syn-
Urine K+ 23.03 mmol/L, urine Na+ 82.6 mmol/L, urine cope, and hyperkalemia caused by decreased aldosterone
−
CI 65.3 mmol/L, 24 h urine K+ 59.8779 mmol/day, 24 h levels (decreased systemic responsiveness).
3. Decreased anti-infective ability and hypoglycemia caused Physical Examination Temperature: 35.7 °C; heart rate: 62
by decreased cortisol. times/min: respiratory rate: 16 times/min; blood pressure:
4. Decreased sex hormones cause menstrual disorders in 96/63 mmHg; height: 158 cm; weight: 45 kg; skin and
women and sexual dysfunction in men. mucosa color deepened (especially in the frictional part);
axillary hair and pubic hair were sparse; mouth, lip, tongue,
40.4.4.2 L aboratory Diagnosis of Addison’s and cheek mucosa were darkened.
Disease
Cortisol Laboratory Examination Cortisol rhythm: (8 AM)
Cortisol in the blood changes significantly during the day. It 2.76 μg/dL, (4 PM) 2.50 μg/dL; ACTH rhythm: (8 AM)
usually arrives at peak value in the early morning. So blood >1250 pg/mL, (4 AM) 893 pg/mL; PTH: 10.10 pg/mL; After
samples are usually collected in the early morning to detect ACTH excitation, cortisol rhythm: 8 AM 2.5 μg/dL, 4 PM
cortisol, combined with ACTH stimulation test and ACTH 2.26 μg/dL, 0 AM 2.58 μg/dL.
test in the early morning, which can provide strong evidence
for adrenal insufficiency. Supplementary Examination There was no abnormality
in pituitary MRI; no abnormality in bilateral adrenal CT
ACTH plain scan; calcification of abdominal aorta, high-density
ACTH is an indicator that evaluates whether the pituitary shadow in left renal sinus parenchyma, and calcified renal
secretes ACTH normally. If the level of ACTH is below nor- artery can be seen.
mal, it indicates secondary adrenal dysfunction, and when
the ACTH level is abnormally elevated, it indicates the pres- Treatment ACTH intravenous injection and oral
ence of Addison’s disease. prednisone.
Aldosterone Treatment Outcome Symptoms of pigmentation basically

Examination of aldosterone level is also helpful in the diag- disappeared, and cortisol rhythm and ACTH rhythm basi-
nosis of Addison’s disease. If the level of aldosterone pro- cally returned to normal.
duced by the adrenal gland decreases, it indicates that
Addison’s disease may exist. This case was from the First Affiliated Hospital of Dalian
Medical University.
CRH Stimulation Test
This test can be used when the ACTH test is abnormal, which
also help to determine the cause of adrenal insufficiency. 40.4.5 Pheochromocytoma
People with Addison’s disease have a high level of
ACTH. Absent response can be observed in those who have 40.4.5.1 Overview
secondary adrenal insufficiency. Pheochromocytoma originates from the adrenal medulla,
sympathetic ganglion, or other chromaffin tissues. It can
Antiadrenal Antibody continuously or intermittently release large amounts of cat-
Patients with Addison’s disease usually have antiadrenal echolamines, causing persistent paroxysmal hypertension
antibody. and multiple organ dysfunction. Recent years, it has proved
that the occurrence of pheochromocytoma is related to the
Follow-up Tests mutations of pathogenic genes. These genes can be divided
Follow-up tests also should be performed for the further into two groups according to the use in different signal path-
diagnosis, such as renin, electrolytes, BUN, creatinine, and ways: The first group is used in hypoxia pathway, which
glucose. stimulates the expression of hypoxia-related growth factors
by activating hypoxia-inducible factors, thus stimulating the
40.4.4.3 Typical Medical Case growth of tumors, including VHL, SDHx (SDHA, SDHB,
Clinical Background A 61-year-old female has hypergly- SDHC, SDHD, SDHAF2), HIF2A, IDH1, PHD2, KIF1B,
cemia for 7 years and skin darkening for 3 years, and weight and MDH2. The second group promotes tumor growth by
has dropped significantly over the past 6 months. Three years activating MAPK and/or mTOR signal pathways, including
ago, there was no obvious inducement for discomfort such as NF1, RET, MAX, and TMEM127 [84–87].
dark skin, blurred vision, and numbness of limbs. Six months The specific clinical manifestations are as follows:
ago, there was no obvious inducement of fatigue and weight Unstable blood pressure, paroxysmal hypertension, increased
loss.
708 H. Yuan et al.
blood sugar, decreased glucose tolerance, accelerated fat 3 . Adrenal incidentaloma with or without hypertension.
decomposition, constipation, intestinal dilatation, etc. 4. Patients with a family history of pheochromocytoma or
related genetic syndrome.
40.4.5.2 Laboratory Diagnosis 5. Patients with a history of pheochromocytoma.
of Pheochromocytoma
Blood and Urine Catecholamines Patients with family history or syndromic lesions should
Norepinephrine and epinephrine in pheochromocytoma get specific genetic testing. For patients who have nonsyn-
patients are increased, often more than twice the normal dromic lesions or family history, SDHB can be tested first,
value. the positive result may lead to the diagnosis of malignant
tumor, and it also can help in the localization and biochemi-
Urine VMA, MN and NMN cal phenotype of the tumor.
These three are metabolites of catecholamine, so the level in
pheochromocytoma patients also significantly increased, 40.4.5.4 Typical Medical Case
among which MN and NMN have better sensitivity and Clinical Background A 24-year-old female had paroxys-
specificity. mal palpitation, fatigue for more than 1 month, and space-
occupying was found in right adrenal gland 20 days ago. No
Glucagon Provocation Test paroxysmal headache, pale face, no full moon face, buffalo
This test can be carried out during the intermittent period of back, centripetal obesity, acne and mental retardation, and
paroxysmal hypertension. weight loss was 5 kg in 4 months.
40.4.5.3 Genetic Testing Physical Examination T37 °C, R20 times/min, P138
Five kinds of people are recommended to get genetic testing times/min, BP 125/86 mmHg, height 156 cm, weight 43.2 kg,
[88], and the tests used commonly can be seen in Fig. 40.20. BMI 17.8/m2, being confined to a chair.
1. Genetic testing in patients with known or suspected

Laboratory Examination Thyroid function: TT4
familial syndrome. 106.4 nmol/L, TT3 1.86 nmol/L, FT3 4.73 pmol/L, FT4
2. Patients whose symptoms of pheochromocytoma are
17.05 pmol/L, TSH 1.13 mU/L, TG-Ab, TPO-Ab(–). Urine
caused by the use of DAD2 receptor antagonists, opioid, NE: 719.95 μg/24 h; Urine E: 84.88 μg/24 h; Urine DA:
or 5-serotonin reuptake inhibitors. 467.36 μg/24 h; AFP: 96.75 μg/LCEA: 5.10 μg/L. The
Fig. 40.20 Genetic testing

for pheochromocytoma
rhythm of ACTH was normal with no abnormality in capto- Nonclassical CAH

pril test. Genetic testing—RET, VHL, SDHB, SDHC, and This type is milder than classic CAH, and the symptoms can
SDHD—were normal. be irregular or absent menstrual periods, severe acne, and
early appearance of pubic hair.
Supplementary Examination Abdominal ultrasound:
Solid heterogeneous mass (6.4 × 3.9 cm) was found in the 40.4.6.2 Laboratory Examination
right adrenal gland near the retroperitoneum, with clear CAH can cause the decrease in serum cortisol concentration;
boundary and uneven internal echoes. No obvious abnormal- serum 17-OHP and ACTH increased. And α1-24 ACTH
ity was found in the left adrenal gland. stimulation test is the gold standard for identifying
21-hydroxylase deficiency and other steroid synthase
Abdominal CT plain scan A low circular density shadow deficiency.
was seen in the head of pancreas, with uneven density and
multiple spots and strips of calcification. 40.4.6.3 Genetic Testing
There was no obvious abnormality in thyroid ultrasound. Genetic testing is helpful in the diagnosis of CAH, but if
there are typical clinical symptoms and laboratory results,
Treatment The patient was given antihypertensive drugs gene testing is not necessary. In recent years, some genes
and had an operation on pheochromocytoma, and the patho- have been shown to be closely related to the occurrence of
logical result was consistent with the diagnosis of CAH and are currently being used: CYP21A2, CYP11B1,
pheochromocytoma. CYP17A1, HSD3B2, POR [89], STAR [90], PRKAR1A
[91], ARMC5 [92], and CYP11A1 [93]. Genes and its asso-
Treatment Outcome The patient was cured. ciated phenotypes of congenital adrenocortical hyperplasia
are summarized in Table 40.11.
This case was from the First Affiliated Hospital of Dalian
Medical University. 40.4.6.4 Typical Medical Case
Clinical Background An elevated 17-hydroxyprogen was
found in a 1-year-old male during neonatal screening. The
40.4.6 Congenital Adrenocortical Hyperplasia 17-OHP level was detected at birth, 9 months, 10 months,
and 1 year of age, respectively. The results increased gradu-
40.4.6.1 Overview ally from 79 nmol/L to 225 nmol/L.
Congenital adrenal hyperplasia(CAH) is a disease caused by
the mutations of genes and causes the deficiency of the
enzymes which mediate the production of glucocorticoids Table 40.11 Genes and its associated phenotypes of congenital adre-
and mineralocorticoids. So the common clinical manifesta- nocortical hyperplasia
tion of CAH is the symptoms that lack these hormones. Gene Associated phenotypes Inheritance
ARMC5 ACTH-independent macronodular AD
Classical CAH: T21-hydroxylase deficiency (21-OHD) adrenal hyperplasia 2
CPY21A2 gene mutation can lead to 21-hydroxylase dele- CYP11A1 Adrenal insufficiency, congenital, with AD/AR
tion and masculinization. It is the most common type in clas- 46, XY sex reversal
CYP11B1 Adrenal hyperplasia, congenital, due to AD/AR
sical CAH. According to the related clinical manifestations,
11-beta-hydroxylase deficiency
21-OHD can be divided into three types: salt-wasting pheno- CYP17A1 Adrenal hyperplasia, congenital, due to AR
type, simple virilizing type, and nonclassical type. The most 17α-hydroxylase deficiency
serious type is salt-wasting phenotype which lost CYP21A2 Adrenal hyperplasia, congenital, due to AR
21-hydroxylase completely. Neonates are susceptible groups. 21-hydroxylase deficiency
Hyperandrogenism, nonclassic, due to
Besides the masculine manifestations, there are also serious
21-hydroxylase deficiency
digestive tract symptoms and dehydration. HSD3B2 3-Beta-hydroxysteroid dehydrogenase, AR
Simple virilizing type is caused by partial 21-hydroxylase II deficiency
deficiency. Female infants often have masculinization such POR Disordered steroidogenesis due to AR
as clitoral hypertrophy, while male infants have sexual pre- cytochrome p450 oxidoreductase
deficiency, Antley–Bixler syndrome
cocity such as penile enlargement and muscular develop-
PRKAR1A Pigmented nodular adrenocortical AD
ment after several months of birth. disease
Nonclassical type is a syndrome caused by mild STAR Lipoid adrenal hyperplasia AR
21-hydroxylase deficiency, and its clinical manifestations are AD autosomal dominant inheritance, AR autosomal recessive
not obvious. inheritance
710 H. Yuan et al.
Physical Examination Height 78 cm, weight 10.5 kg,

blood pressure 76/40 mmHg, penis 3 cm × 1 cm, testis
1.5 mL, scrotum pigmentless.
Supplement Examination No bilateral adrenal enlarge-

ment was found by B-mode ultrasonography, and the bone
age was in accordance with the actual age.
Family Gene Mutation Detection CYP21 gene detection

indicated that I172N and E3△8 mutations—I172N muta- Uric acid crystals
tions originated from mother and E3△8 deletion originated
from father—conformed to the clinical diagnosis of CAH.
Fig. 40.21 Gout: Uric acid crystals deposited in joints

Diagnosis Congenital adrenocortical hyperplasia (simple
masculinization).
hypertension, and cardiocerebrovascular diseases, which
Treatment Hydrocortisone acetate was given, and the dos- pose a serious threat to health.
age was increased at any time according to the clinical The annual incidence of gout is 2.68 per 1000 people.
manifestations. There is a steady increase in the prevalence of gout with age,
and it occurs 2–6 times more for men than for women due to
Treatment outcome The patients’ height, weight, 17-OHP, estrogen resulting in urate loss in the urine. Ethnicity also
and other hormone levels were normal, and the bone age was strongly affects the gout prevalence and the prevalence rates
consistent with the actual age. of gout in Pacific Islander, New Zealand Maori, and
Taiwanese groups is much higher [94]. The incidence of
This case was from the First Affiliated Hospital of Dalian global gout increases persistently due to impropriate diet
Medical University. habits, lack of exercise, and a higher incidence of obesity and
metabolic syndrome.
40.5 Gout
Hong Yuan and Jingyuan Zhao
Gout is characterized by deposition of monosodium urate
Gout is an inflammatory arthritis caused by an innate immune crystals in joints and tissues, which usually presents with
response to monosodium urate (MSU) crystals deposited in intermittent painful attacks followed by remission in a long
synovial fluid. This chapter stocks up information about period. Acute gout attacks are usually monoarthritis, which
gout, methods of detection, and use of laboratory test results can cause severe inflammation within a few hours, with
in recognizing and characterizing gout. major features of inflammation, including redness, fever,
tenderness, swelling, and dysfunction, that is quickly relieved
by NSAIDs or colchicine. Hyperuricemia may be associated,
40.5.1 Overview but blood uric acid levels are normal in some patients. Tophi
and renal stones are late presentations. Tophi is a mass
Gout is a chronic urate crystal deposition disease, which is formed by a large amount of accumulated MSU crystals,
caused by acute or chronic inflammation and tissue damage which a characteristic clinical manifestation of gout. It is
caused by deposition of monosodium urate (MSU) found in the auricle, subcutaneous tissue, and skin joints.
(Fig. 40.21). Due to elevated blood uric acid levels, urate About 10–25% of gout patients have uric acid stones in the
crystals are deposited in tissues, joints, and kidneys. In kidney. The smaller ones are discharged into the gravel with
severe cases, joint damage and impaired renal function can urine without obvious symptoms. The larger one may cause
occur. Without effective management, gout may turn into a renal colic, hematuria, dysuria, hydronephrosis, pyelone-
chronic stage in some people, accompanied by the develop- phritis, or periarteritis. Gout is also comorbid with other
ment of tophi (organized by urate and immune cells), perma- metabolic-based conditions, such as cardiovascular and kid-
nent bone erosion, and process of disability. Patients with ney disease and type 2 diabetes, although the mechanism is
gout often accompanied by comorbidities like diabetes, not fully understood [95].
40.5.3 Laboratory Diagnosis rate. Blood cell count may be used to distinguish between
septic arthritis and gout by determining whether there is an
40.5.3.1 Blood Uric Acid abnormal increase in the number of leukocytoses.
Adult males and postmenopausal females with uric acid
>420 μmol/L (7.0 mg/dL) and premenopausal women 40.5.3.5 Gene Testing
>350 μmol/L (5.8 mg/dL) can be diagnosed with hyperurice- In fact, the diagnosis of gout after the onset of disease leaves
mia. Note that since large fluctuations of blood uric acid are much to be satisfied in clinical practice. For preonset diagno-
common, blood testing should be repeated. There is a signifi- sis, the genetic diagnosis has been the research focus these
cant dose–effect relationship between the incidence and years. Recently, with in-depth knowledge of genome-wide
blood uric acid levels. When the blood uric acid level association studies (GWAS) associated with blood uric acid
achieved 7.0–7.9 mg/dL, 8.0–8.9 mg/dL, ≥9.0 mg/dL, the levels, researchers demonstrated that most gout-related
incidence of gout was 10.8%, 27.7%, and 61.1%, respec- genetic variation mainly affects renal urate transport and
tively. Although hyperuricemia is a major characteristic fea- reported multiple genetic sites to contribute to hyperuricemia
ture of gout, not all patients with hyperuricemia showed gout and gout genetic susceptibility. Evidence has shown that the
symptom or UA crystals formation; it also should be noted urate-related gene is situated on chromosome 15. GWAS
that SUA may return to normal level during gout flare. If a found 28 sites related to uric acid, including SLC2A9/
diagnosis of gout is made, uric acid testing may be performed GLUT9, ABCG2, LC22A11/OAT4, SLC22A12/URAT1,
regularly to monitor levels. SLC17A1/NPT1, and cofactor PDZK1. They are all located
on genes encoding urate transporters or other functional pro-
40.5.3.2 Urine Uric Acid teins. SLC22A12, SLC2A9, and ABCG2 are recognized
The 24-h urinary uric acid monitoring can help detect every genes that significantly affect uric acid levels.
cause of hyperuricemia. In the absence of a low-lying diet or At present, genetic testing can help clinical diagnosis and
drugs which may affect uric acid excretion, the normal level decisions making on some certain circumstances, like
is 1.2–2.4 mmol (200–400 mg), and uric acid over 3.6 mmol whether a patient with hyperuricemia will develop into gout,
(600 mg) indicates inner uric acid production increase. Renal or whether a patient with gout has potential flare risk and
function tests should be performed on such patients regularly other adverse progression like tophi formation and joints
due to their high risk of renal calculus formation. It may also damage. As we know, serum urate levels are the most impor-
be done to monitor people with gout and help them to choose tant variables in predicting the risk of gout. However, only
the appropriate medicine to lower the uric acid level in the serum urate levels do not reliably enough to predict disease
blood. progression. SLC22A12 gene exon sequencing of gout
patients found that 23% of patients had SLC22A12 muta-
40.5.3.3 Synovial Fluid Analysis tions, suggesting that SLC22A12 is one of the main respon-
The gold standard for gout diagnosis is the identification of sible mutant sites of gout [98]. SLC2A9, which is the most
MSU crystals in the synovial fluid under polarized light significant serum urate genetic factor, accounting for
microscopy. Using a compensator may be helpful to improve approximately 3% of urate level variations and complete
diagnostic efficiency. All the synovial fluid samples obtained loss of GLUT9 results in a more severe urate excretion dis-
from arthrocentesis and undiagnosed inflammatory joints order than URAT1 [99]. GWAS also discovered that the
require crystallization examination. MSU crystals can be missense ABCG2 rs2231142 (Q141K) variant was con-
found in the synovial fluid at every stage of the disease cerned with serum urate concentration of European and East
course almost, no matter during the flare, the critical stage or Asian ancestry. Evidence suggests that the 141K allele also
chronic stage. The leukocytic count, chemistry, culture, and has a positive correlation with increased uric acid and gout
sensitivity of synovial fluid are the further analysis [96]. In morbidity [100].
acute gout, the white blood cell count of synovial fluid may It was useful to create grades of ABCG2 dysfunction
exceed 50,000 cells/mL (most of which are polymorphic). based on Q141 K and Q126X genotype combinations, and
Glucose levels in the synovial fluid are often normal that is individuals with the dysfunctional variants 126X and 141K
different from septic arthritis which glucose levels may be positive having the highest serum urate concentrations and
lower. Yet these two may be present in the one joint simulta- highest risk for gout [101, 102]. Major genetic variations in
neously; hence, bacteriology and Gram staining are also ABCG2 and impact on phenotype are shown in Table 40.12.
indispensable [97]. On the other hand, risk stratification involving genetic
testing can provide more personalized intervention deci-
40.5.3.4 Other Blood Tests sions. Genetic testing to identify genetic variants that predict
Other blood tests usually include complete blood cell count, no response to allopurinol and uric acid excretion drugs can
electrolytes, renal function, and erythrocyte sedimentation provide the possibility of personalized selection of ULT.
712 H. Yuan et al.
Table 40.12 Major genetic variations in ABCG2 and impact on However, treatment failure is also common, usually due
phenotype to poor compliance of regular medications, which empha-
Variant Gout risk Urate Allopurinol response sizes the importance of patient education. When diagnosed
ABCG2 Q126X Increases — — as gout, patients need to be informed by the physician about
(rs72552713) the relevant knowledge of gout, including introduction of the
ABCG2 Q141K Increases Increases Resistance
(rs2231142)
uric acid reduction treatment plan, the long term and impor-
ABCG2 V12M Decreases Decreases — tance of treatment and the possible risks. For the physician,
(rs2231137) the start time of urate-lowering therapy needs to be negoti-
rs10011796 Increases Increases No association ated with patient together, and individualization should be
emphasized due to consideration of the patient’s comorbidi-
Allopurinol is the most widely used ULT agent. GWAS has ties and medicine selection.
determined that ABCG2 141 K allele is related to the adverse
allopurinol response [103]. In addition, human leukocyte
antigen (HLA) variant HLA-B* 5801 is another important 40.5.5 Conclusion
risk factor for severe allopurinol allergy syndrome (AHS), so
it is recommended to perform this variant test before allopu- Gout is a chronic urate crystal deposition disease, which is
rinol usage for high-risk populations (Han, other Asian peo- caused by acute and chronic inflammation and tissue damage
ple) [104]. As profound development has been made in caused by deposition of monosodium urate (MSU), which is
understanding the genetic basis of hyperuricemia and gout, directly related to hyperuricemia caused by sputum metabo-
as well as pharmacogenetics of lowering urate, it is of great lism disorder or uric acid excretion. Although hyperuricemia
significance to improve the clinical diagnosis of gout, to is a major cause of gout, many patients with hyperuricemia
individualize the prognosis, to predict ULT response, and to do not develop into gout or form UA crystals. Risk factors
provide potential new therapeutic targets. for gout include gender, age, ethnicity, and genetic factors,
and their interactions increase the incidence of gout. The
40.5.3.6 Others diagnosis and evaluation of gout are multifaceted, joint
Some other tests, such as basic metabolic panel, may be used puncture, and MSU crystal detection are still the basis for
to evaluate and monitor kidney function; antibodies like RF gout diagnosis. Gout diagnosis is usually based on clinical
(rheumatoid factor) or ANA (antinuclear antibody) may be manifestations, age, comorbidities, symptoms, clinical
ordered to exclude other causes of arthritis symptoms. Blood symptoms, and laboratory results. In 2015, the American
culture and/or synovial fluid culture also should be ordered College of Rheumatology (ACR) and the European Union of
to differentiate septic arthritis. Magnetic resonance imaging Rheumatology (EULAR) jointly launched a new version of
(MRI), ultrasound, CT and dual-energy CT can well indicate the gout diagnostic criteria, which considers the “swelling
urate crystals, lesions and surrounding soft tissue damage, pain or tenderness of a joint or mucous sac” as a condition
and provide favorable evidence for the diagnosis of gout, for entering the diagnostic process. The appearance of uric
thereby improving the sensitivity and specificity of the diag- acid crystals or tophi in the symptomatic joint or mucous sac
nosis of gout. can be used as a sufficient condition for the diagnosis of
gout. The diagnosis process is shown in Fig. 40.22. If this
condition is not met, the results will be scored by clinical
40.5.4 Management symptoms, laboratory tests, imaging examinations, etc., and
≥8 points will be diagnosed as gout. Gout classification cri-
Keeping SUA levels below sedimentation threshold by teria are shown in Table 40.13. The diagnosis of gout requires
dietary adjustment and uric acid-lowering drug use is the a close combination of epidemiology, etiology, and diagnos-
main goal of gout management. Patients education is funda- tics. It is necessary to familiarize with gout itself and poten-
mental to help patients develop a reasonable diet habit and tial susceptibility factor and improve the diagnostic efficiency
lifestyle. Drink more water (no less than 1500–2000 mL/ of gout [105].
day) to alkalinize urine to maintain urine pH at 6.2–6.9,
which is conducive to the dissolution and discharge of urate
crystals. At the same time, maintain a low-purine diet, exer- 40.5.6 Typical Medical Case
cise regularly to keep fitness, and quit smoking. A whole-
some lifestyle not only helps reduce blood uric acid levels Clinical Background A 69-year-old male patient with
but also reduces gout flare. Patients with gout need to take multiple joint swelling and pain, limited mobility for 10
medication for a lifetime to control symptoms. years.
Fig. 40.22 Diagnosis
procedure of gout
Table 40.13 The ACR/EULAR gout classification criteria

Criteria Categories Score
Pattern of joint/bursa involvement during symptomatic episodes(s) ever Ankle or midfoot (as part of monoarticular or 1
oligoarticular episode without involvement of the
first metatarsophalangeal joint
Involvement of the first metatarsophalangeal joint (as 2
part of monoarticular or oligoarticular episode)
Characteristics of symptomatic episode(s) ever: No characteristic 0
• Erythema overlying affected joint (patient-reported or physician-observed) One characteristic 1
• Cannot bear touch or pressure to affected joint Two characteristics 2
• Great difficulty with walking or inability to use affected joint Three characteristics 3
Time course of episode(s) ever: No typical episode 0
Presence (ever) of ≥2, irrespective of anti-inflammatory treatment: One typical episode 1
• Time to maximal pain <24 h Recurrent typical episodes 2
• Resolution of symptoms in ≤14 days
• Complete resolution (to baseline level) between symptomatic episodes
Clinical evidence of tophus: Absent 0
Draining or chalk-like subcutaneous nodule under transparent skin, often with Present 4
overlying vascularity, located in typical locations: joints, ears, olecranon bursae,
finger pads, tendons (e.g., Achilles)
Serum urate: <4 mg/dL (<0.24 mmol/L) −4
Measured by uricase method. Ideally should be scored at a time when the 6–8 mg/dL (0.36 to <0.48 mmol/L) 2
patient was not receiving urate-lowering treatment, and it was >4 weeks from 8 to <10 mg/dL (0.48 to <0.60 mmol/L) 3
the start of an episode (i.e., during intercritical period); if practicable, retest
≥10 mg/dL (≥0.60 mmol/L) 4
under those conditions. The highest value irrespective of timing should be
scored
Synovial fluid analysis of a symptomatic (ever) joint or bursa: (should be MSU negative −2
assessed by a trained observer)
Imaging evidence of urate deposition in symptomatic (ever) joint or bursa: Present (either modality) 4
ultrasound evidence of double-contour sign or DECT demonstrating urate
deposition
Imaging evidence of gout-related joint damage: conventional radiography of Present 4
the hands and/or feet demonstrates at least one erosion
714 H. Yuan et al.
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Neurological Disease
41
Jie Wu, Yutong Zou, Yingchun Xu, Mengxiao Xie,
Zhaojing Zheng, and Juan Geng
The nervous system, containing billions of nerves and spe- the ability of language, abstract representation of concepts,
cialized cells known as neurons, is essentially the electrical transmission of culture, and so on.
wiring of the body. Structurally, the nervous system is com- Neurological disorders refer to a group of heterogeneous
posed of the central nervous system (CNS) and the periph- diseases that impair the function of nerves, muscles, or neu-
eral nervous system (PNS). The CNS contains the brain and romuscular junctions. Common clinical symptoms include
spinal cord. Consisting of nerves that are bundles wrapped cognitive impairment, sensory abnormalities, neuralgia,
by long fibers or axons, the PNS can connect the CNS to myalgia, muscle atrophy, muscle spasm, muscle weakness,
each part of our body. Nerves transmitting signals from the abnormal muscle tone, and abnormal reflexes.
brain are called motor (efferent nerves), while those trans-
mitting information from the body to the CNS are called sen-
sory (afferent nerves). Containing both the efferent and 41.1 Alzheimer’s Disease
afferent components, the spinal nerves are named mixed
nerves. The PNS is divided into the somatic nervous system, Jie Wu, Yutong Zou, and Yingchun Xu
autonomic nervous system, and enteric nervous system. The
autonomic nervous system is further subdivided into sympa-
thetic and parasympathetic nervous systems (Fig. 41.1). 41.1.1 Overview
The nervous system plays leading roles in human activi-
ties and regulates the physiological function. The primary Alzheimer’s disease (AD), a neurodegenerative disease, is
function is to control the body by retrieving complicated the most common form of dementia and comprises around
information from the environment via sensory receptors, 50–60% of all dementia cases. It imposes a huge burden to
sending corresponding signals into the CNS, processing the many families and society. The data from the Alzheimer’s
information to produce appropriate responses, and sending Association indicated that an estimated 5.3 million patients
output signals to muscles or glands to activate corresponding were living with AD in U.S [1]. Worldwide, the numbers are
responses. The signal delivering pathways are diverse. expected to be 65.7 million in 2030 and 115.4 million in
Furthermore, the sophisticated nervous system, mainly con- 2050 [2], increasing at a rate of one new case every 33 s,
trolled by the brain, makes it possible for humans to develop which will result in nearly 1 million new cases per year [3].
The prevalence of dementia takes on significant regional
variations and is lower in developing countries. Characterized
by the neuritic plaques formed by deposition of extracellular
J. Wu (*) · Y. Zou · Y. Xu
Department of Laboratory Medicine, Peking Union Medical aggregates of amyloid-β (Aβ) peptides and the neurofibril-
College Hospital, Beijing, People’s Republic of China lary tangles (NFTs) composed of intracellular hyperphos-
e-mail: wujie@pumch.cn phorylated microtubule-associated tau proteins, AD can
M. Xie (*) cause a lot of clinical symptoms including problems with
Department of Laboratory Medicine, The First Affiliated memory, language, attention, behavior, and so on [4].
Hospital of Nanjing Medical University, Nanjing, Jiangsu,
The pathological changes of AD are frequently accompa-
nied by the loss of neurons and synapses. However, the exact
causes of this disease are still unclear. Nowadays, AD is
University School of Medicine, Shanghai, People’s Republic mainly diagnosed by the history and clinical examination of
of China

718 J. Wu et al.
Fig. 41.1 Diagram of the

Key: Central Nervous System (CNS)
vertebrate nervous system = Structure
Brain and spinal cord
= Function
Integrative and control centers
Peripheral Nervous System (PNS)

Cranial nerves and spinal nerves
Communication lines between the CNS
and the rest of the body
Sensory (afferent) division Motor (efferent) division

Somatic and visceral sensory nerve fibers Motor nerve fibers
Conducts impulses from receptors Conducts impulses from the CNS
to the CNS to effectors (muscles and glands)
Sympathetic division Autonomic nervous Somatic nervous

Mobilizes body systems during system (ANS) System
activity (”fight or flight”) Visceral motor (involuntary) Somatic motor (voluntary)
Conducts impulses from the Conducts impulses from the
CNS to cardiac muscles, CNS to skeletal muscles
Parasympathetic division smooth muscles, and glands
Conserves energy
Promotes “housekeeping”
functions during rest
patients. When needed, physicians also use dedicated neuro- Although the specific molecular mechanisms accounting
psychological testing and medical imaging such as MRI and for the cause of AD are unclear now, most researchers sup-
PET scans of the brain to help clarify the diagnosis and rule port the Amyloid Cascade Hypothesis [8]. This hypothesis
out other similar diseases. It is estimated that even in high- believes that AD is caused in part by the overproduction and
income countries, only 20–50% of dementia cases are recog- lack of clearance of Aβ. As shown in Fig. 41.2, the amyloid
nized and documented in primary care. How to distinguish the precursor proteins (APP) in our brain can be proteolytically
early stage of AD from mild cognitive impairment (MCI), the processed by ɑ-, β-, and γ-secretases, resulting in two differ-
normal aging process, and other formations of dementia is still ent pathways. The nonamyloidogenic pathway is led by
challenging. Moreover, the available treatments have minimal ɑ-secretase and ultimately produces P3, which is proved to
or even no effect on the course of this disease. Clinical labora- be neuroprotective against excitotoxic stimuli. The other
tories are making great efforts to find more sensitive and spe- pathway is led sequentially by β-secretase, which produces
cific tests to improve the diagnosis and treatment of AD. small peptides called Aβ, ranging from 38 to 43 amino acids.
The most dominant forms in senile plaques are Aβ40 and
Aβ42, of which Aβ42 is less soluble and more prone to
41.1.2 Etiology aggregation, thus associated with AD. The accumulation of
Aβ may be a consequence of hyperproduction in genetic
The etiology of AD is uncertain. However, genetics, environ- cases or due to defective clearance in sporadic cases. Proteins
mental factors, lifestyle, and developmental components are playing a crucial role in Aβ clearance include the Aβ prote-
likely to play a role. AD is a progressive neurodegenerative ases, low-density lipoprotein receptor-related protein (LRP),
brain disorder. At the microscopic level, AD is characterized ApoE, and α2-macroglobulin. This hypothesis is supported
by the appearance of senile or neuritic plaques and neurofi- by the observation that patients with Down Syndrome/
brillary tangles in the medial temporal lobes and other cere- Trisomy 21, containing one more copy of chromosome 21,
bral cortical areas and the degeneration of neurons and on which holds the gene for APP, show the symptoms of AD
synapses [5]. Epidemiological studies have suggested sev- as early as 40 years old. Moreover, a detrimental or benefi-
eral risk factors, including age, diabetes, obesity, low educa- cial APP mutation at the β-secretase cleavage site can change
tional and occupational attainment, reduced brain size, low the onset of AD (Fig. 41.2).
mental ability in early life, and reduced mental and physical However, this hypothesis is updated in 2009, suggesting
activity during late life [6, 7]. that β-secretase may be a major culprit but not the only one
41 Neurological Disease 719
Fig. 41.2 The amyloid

cascade hypothesis β
APP α
γ
α−secretase β−secretase
sAPPα C83 sAPPβ C99
γ-secretase γ-secretase
P3 Ab (eg:Ab40 Ab42)
senile plaques bloodstream

LRP apoE
α2-macroglobulin
synaptic and
fragments
neuronal loss
LRP:Low-density lipoprotein receptor-related protein
for the development of AD. Other studies show that the Table 41.1 The clinical appearances of three stages of AD
degree of dementia in AD is more correlated with the Preclinical AD
presence of NFTs than amyloid [9]. Some studies show that 1. The preclinical deficits have happened across multiple cognitive
the intracellular hyperphosphorylated tau protein can tangle domains
2. No obvious clinical presentation of cognitive impairment
with other threads of tau protein and finally form the NFTs
MCI
seen in AD [10]. In recent years, several clinical trials of
1. Memory loss is the predominant symptom
therapeutic drugs targeting Aβ have failed, and researchers 2. The patients perform less than 1SD below normal on cognitive
have begun to question the proposed beta-amyloid theory. In measures, which is clearly defined but only mild memory
addition, studies have reported that the herpes virus may be impairment
contributing to Alzheimer’s disease. There are also other Dementia (AD)
1. Obvious memory impairment is the most pervasive feature and
competing hypotheses trying to explain the mechanism of
the leading symptom of AD. The loss of short memory is usually
AD. The exact mechanism still needs to be further explored. the first presentation. And as the disease develops, they can lost
their semantic memory, implicit memory, and long-term memory
2. The patients take on approximately 4 SD below normal on
cognitive measures
3. Language problems are common, especially a shrinking
vocabulary and decreased word fluency, which can lead to lose
In 2011, the course of AD was divided into three phases, the ability of speech ultimately
preclinical AD, MCI, and dementia, according to the National 4. The problems with visual difficulties especially abnormal near
vision, color vision, and impaired reading and writing skills can
Institute of Aging, Alzheimer’s Association (NIA-AA) diag-
be developed
nostic criteria [11]. The specific clinical appearances are 5. A variety of neuropsychiatric symptoms such as apathy, depressive
shown in Table 41.1. symptoms, anxiety, and irritability are frequently present at early
A person with preclinical AD will go on to be diagnosed stage, and some can throughout the course of disease
6. The patients can have subtle problems with the executive functions
with AD and show poorer cognitive performance after a
of attentiveness, planning, thinking, etc. at the early stage. As
follow-up period [12]. The early identification of preclini- progressive deterioration develops, they can lose the ability of living
cal AD is important for the diagnosis and management of alone, for example, urinary incontinence may happen
AD. MCI is a transitional stage between healthy people and
very mild AD, which does not meet the commonly accepted neuronal loss occurs primarily in the hippocampus and cor-
criteria for dementia or AD. This is also an important clini- tices tightly associated with cognition and memory, AD
cal stage and needs to be recognized to predict the occur- patients suffer from significant memory impairment, which
rence and development of AD. Because the synaptic and is the most pervasive feature and the leading symptom of
720 J. Wu et al.
AD [13]. Furthermore, AD patients can have problems with Workgroup set a new diagnostic guideline of AD, which
language, praxis, vision, neuropsychiatric disturbances, emphasized both cognitive and neuropsychiatric symptoms.
executive dysfunctions, and so on. With progressive dete- The detailed graphic from Mckhann and colleagues is
rioration, they can even be bedridden and unable to perform shown in Table 41.2. In 2012, the “ABC” score based on the
the activities of daily living, thus requiring tremendous assessment of AD-related neuropathological changes was
assistance from their caregivers and close care in special- proposed. As shown in Table 41.3, the score consists of
ized facilities, which would be a huge financial burden for three scales—A for Aβ plaque score, B for Braak NFT
themselves, their families, and society. Other complica- stages, and C for CERAD neuritic plaque score. It is
tions seen in advanced AD include malnutrition, dysphagia, expected that low-level changes will be observed in the pre-
aspiration pneumonitis/pneumonia, deep venous thrombo- clinical stage, medium-level changes will be observed in
sis, and various infections, contributing to the mortality of MCI, and high-level changes will be observed in patients
patients with AD. with AD. Since then, the IWG-2 updated the clinical entity
of prodromal AD in 2014 and introduced improved bio-
41.1.4 Clinical Diagnosis

Table 41.2 The diagnosis of AD by the NIA-AA Workgroup
At present, AD is usually diagnosed by neuropsychological Probable AD
testing and neuroradiological imaging. When a patient shows Inclusion criteria
memory impairment, initial screening tests should be done in Onset of symptoms occurs over months to years with clear-cut
progression of cognitive deficits, which are established by either
clinic. Examiners usually apply screening tests such as the reports or observation. The cognitive profiles of symptoms are as
Mini-Mental State Examination, the Functional Activities follows: An amnestic presentation with prominent deficits in the
Questionnaire, and sometimes other more specific tests, such learning and recall of information, which is the most common, and
as the verbal fluency tests and the Digit Symbol Substitution a nonamnestic presentation in which language (word finding),
visuospatial (object agnosia, face recognition, simultanagnosia, and/
Test [14]. However, these tests are not sensitive enough for or alexia), or executive dysfunction (impaired reasoning, judgment,
the diagnosis of dementia, especially for mild dementia. In and problem solving) are most prominent
recent years, neuroimaging techniques such as the MRI, Exclusion criteria
Positron Emission Tomography (PET), and Single Photon 1. Evidence of substantial cerebrovascular disease as suggested by a
Emission Computed Tomography (SPECT) have been history of stroke or the presence of multiple or extensive infarcts
or severe white matter hyperintensity. Evidence should predate
widely used in addition to the neuropsychological tests, onset or worsening of dementia symptoms
sometimes leading to overdiagnosis and overtreatment of 2. The presence of core features of other dementias not typically
AD. Although there is no effective treatment to cure AD, associated with AD: For example, dementia with Lewy bodies,
early diagnosis and management of dementia may improve early onset of balance problems and/or visual hallucinations,
behavioral variant frontotemporal dementia, early onset of
health care, help patients and family plan for the future, and dramatic changes in behavior predating obvious memory
generally improve quality of life. As a detectable and mea- impairment, the semantic and nonfluent/agrammatic variants of
surable component in body fluids, biomarkers may help cli- primary progressive aphasia (PPA), and prominent language
nicians and researchers make early diagnosis of AD and impairments predating prominent memory impairment
3. E vidence of other neurologic or non-neurologic disorders that
further understand the pathogenesis. could account for the symptoms (such as substantial exposure to
heavy metals, Wernicke-Korsakoff syndrome, and Wilson disease)
41.1.4.1 Diagnostic Criteria 4. Concurrent use of medication that could account for a substantial
The National Institute of Neurological and Communicative component of the symptoms listed under inclusion criteria
Possible AD dementia
Disorders and Stroke and the Alzheimer’s Disease and
Inclusion criteria
Related Disorders Association (NINCDS-ADRDA) criteria,
Not only meets criteria for AD dementia but also has either:
proposed in 1984, were the earliest diagnostic criteria for 1. Atypical course with sudden onset of symptoms or insufficient
AD and have been confirmed with high accuracy postmor- historical evidence to determine temporal pattern of onset or
tem test [15]. However, these criteria believed that AD was lacking objective evidence of progressive decline
2. Etiology is obfuscated by the presence of exclusion criteria under
a neuropathologic disease, and the diagnosis could only be
probable AD. Possible AD dementia with evidence of the AD
made postmortem. In 2007, the International Working pathophysiological process is reserved for individuals who meet
Group (IWG) for New Research Criteria for the Diagnosis the criteria for a nonAD dementia, but who either show
of Alzheimer’s Disease established a method incorporating biomarker evidence of AD pathophysiology or meet the
neuropathologic criteria for AD. Dementia unlikely to be due to
biomarkers in the diagnosis of AD [16]. It changed the con-
AD is reserved for individuals who, although meeting criteria for
cept of AD from a clinical pathological disease to a clinical probable or possible AD, have strong evidence for an alternative
biological entity that can be identified in vivo. In 2011, the diagnosis that rarely or never is associated with the presence of
National Institute on Aging and the Alzheimer’s Association AD dementia
Table 41.3 The “ABC” score by National Institute on Aging-Alzheimer’s Association

Amyloid accumulation Thal phase Braak NF stage CERAD plaque score
No 0 = A0 None = C0 0, I, or II = B0 or B1
III or IV = B2
V or VI = B3
Low 1 or 2 = A1 C0, sparse = C1, B0 or B1 or B2 or B3
Moderate = C2 and frequent = C3 B0 or B1
3 = A2 Any C
4 or 5 = A3 C0 or C1 or C2 or C3
Intermediate A1 C2 or C3 B2 or B3
A2 Any C
A3 C0 or C1
A3 C2 or C3 B2
High A3 C2 or C3 B3
markers for AD. However, the diagnosis of “definite AD” Table 41.4 Classification of multiple laboratory diagnosis biomarkers
still requires more valuable biomarkers. for AD
Test classification Test item
41.1.4.2 Laboratory Diagnosis Cerebrospinal fluid biomarkers Amyloid-β
Cerebrospinal fluid (CSF) biomarkers of Aβ and tau proteins Total tau protein(T-tau)
Phosphorylated tau protein
are accurate in detecting the neuropathological changes of
(P-tau)
AD. However, the use of CSF biomarkers is limited by inva- Blood-based biomarker Amyloid-β
siveness. Therefore, blood-based biomarkers are eagerly candidates Tau protein
needed and explored. Besides Aβ, tau, and neurofilament Neurofilament light (NFL)
light (NFL), blood based biomarkers also include classical Classical genetic testing APP
genetic testing, potential genetic risk genes, and candidate PSENA1 and PSEN2
APOE
epigenetics biomarkers (see Table 41.4). Other potential genetic risk SORL1, CLU, BIN1, CD33,
genes of AD TOMM40, etc.
Cerebrospinal Fluid (CSF) Biomarkers Candidate epigenetics DNA methylation
Amyloid-β biomarkers Histone modifications
As the main protein component of plaques, amyloid-β (Aβ) microRNAs
is generated by proteolytic cleavage of the amyloid precursor
protein (APP). There are several N- and C-terminally trun- Blood-Based Biomarker Candidates
cated forms of Aβ, of which the two major C-terminal vari- Aβ1–42:Aβ1–40 Ratio
ants are a shorter form ending at Val-40 (Aβ40) and a longer In the past, the ultrasensitive digital enzyme-linked sand-
one ending at Ala-42(Aβ42). Aβ42 is recognized as the ini- wich immunoassay (ELISA) was used to measure Aβ42 and
tial and predominating form of Aβ. It aggregates more rap- Aβ40 in plasma. However, the immunoprecipitation mass
idly than Aβ40. Patients with AD consistently exhibit a spectrometry (IP-MS) assay was developed to quantify Aβ42
moderate to marked decrease in CSF Aβ42 to about 50% of and Aβ40 in plasma in recent years. The ratio of Aβ42 to
control levels, as the aggregation of Aβ into plaques (hence, Aβ40 in plasma was reduced in amyloid PET-positive com-
retention of the peptide in the brain parenchyma) results in a pared with PET-negative cases [18]. Additionally, the ratio of
reduced availability of Aβ to diffuse into the CSF [17]. a specific APP fragment (APP669–711) to Aβ42 in plasma
can be used to identify Aβ-positive individuals with high
Tau Protein sensitivity and specificity using MS assay.
Primarily located in the neuronal axons, tau protein is a
microtubule-associated protein. Its levels in CSF could Phosphorylated Tau Protein (P-Tau)
reflect the intensity of the neuronal damage and degenera- High levels of plasma total tau protein were found to be asso-
tion. There is a moderate to significant increase in tau protein ciated with not only the cognitive decline but also the risk of
in CSF of AD patients. However, high tau levels in CSF are mild cognitive impairment (MCI). The correlation, however,
not specific for AD and can also be found in other CNS dis- is independent of the Aβ levels in the brain. Furthermore,
orders with neuronal degeneration or damage, such as acute phosphorylated tau protein (P-tau) is recognized as a more
stroke and Creutzfeldt-Jakob disease. specific biomarker for AD pathogenesis than total tau. A
722 J. Wu et al.
semisensitive electrochemiluminescence assay has been located near the beta proteolytic cleavage site could lead to
developed for the determination of tau protein phosphory- impaired cleavage of APP and reduction of Aβ40 and Aβ42
lated at threonine 181 [19]. Using this assay, researchers levels in vivo, thereby reducing the risk of AD. In contrast to
found higher plasma P-tau levels in AD patients than those in this, another variant at the same position showed protective
controls. Plasma P-tau levels correlated with Aβ and tau-PET function only in the heterozygous state, suggesting that the
and had a higher correlation with AD compared to the plasma mixing of wild type and mutant APP would affect the aggre-
T-tau test, which is a promising result that needs to be gation of Aβ peptides.
replicated. PSENA1 and PSEN2: The presenilin genes PSENA1 (on
chromosome 14q24.3) and PSEN2 (on chromosome 1q31–
Neurofilament Light (NFL) q42) encode the proteins that carry the active site of the
Serum or plasma NFL concentrations were found to be cor- γ-secretase. Mutations in both genes impair the cleavage of
related with that in CSF, suggesting that most NFL signals APP mediated by the γ-secretase, leading to an increased
in the blood are CNS-derived, at least in the absence of sig- ratio of Aβ42 to Aβ40 through increasing the production of
nificant peripheral nerve diseases. Serum NFL concentra- Aβ42, decreasing the production of Aβ40, or both. Mutations
tion can effectively identify the occurrence of in PSEN1 are the most frequent cause of autosomal domi-
neurodegeneration diseases in familial AD and correlates nant AD known to date. Like APP, PSEN1 is also associated
with longitudinal measurements of disease intensity/neuro- with complete penetrance; thus, people with a PSEN1 muta-
sis in AD. However, the high NFL levels in either plasma or tion will develop AD under normal lifespan conditions. With
CSF are not specific for AD. Instead, elevated NFL levels complete penetrance, the PSEN1 mutations induce the most
have been found in many neurodegenerative diseases, such severe form of AD, and the onset of AD may happen as early
as frontotemporal dementia and progressive supranuclear as 25 years of age. However, the PSEN2 mutations show a
palsy. Therefore, as a possible future application, plasma penetrance of 95%, which means that not every carrier of a
NFL could be used as a screening biomarker at the first PSEN2 mutation will develop AD. Compared with the
clinical evaluation of patients with cognitive impairment, PSEN1 mutation, carriers of the PSEN2 mutation show an
such as in the primary care setting. Plasma NFL can be older age of onset of AD.
used as a simple, noninvasive, and inexpensive screening
tool, mainly to exclude neurodegeneration. Genes Implicated in LOAD
APOE: As a lipid-binding protein, APOE (on chromosome
Classical Genetic Testing 19q13) is expressed in humans as three common isoforms
AD is traditionally subdivided into early onset (EOAD) and encoded by three alleles, APOE ε2, ε3, and ε4. It partici-
late onset (LOAD). EOAD, which is also called autosomal pates in nerve growth and regeneration, immune regulation,
dominant AD, has an onset before 65 years old and accounts repair response to tissue injury, and activation of lipolytic
for 1–5% of all cases. Having an onset after age 65, LOAD is enzymes. In a large cohort of autopsy data and a lot of other
the predominant form of AD. Likewise, AD can be divided studies, APOE gene status exhibited a definite association
into familial AD and sporadic AD. Different AD types have with AD, especially LOADS [21]. It is estimated that a
different signs and symptoms. single APOEε4 allele can cause a two- to threefold increase
in AD risk and two copies a fivefold or more increase, with
Genes Implicated in EOAD a dose-dependent effect on the age of onset. Each inherited
To our knowledge, three genes involved in the amyloid cas- APOEε4 lowers the onset age by 6–7 years. It is also asso-
cade hypothesis have shown a close association with AD, ciated with lower cognitive performance and progression
especially autosomal dominant EOAD. Mostly autosomal from MCI to dementia. Additionally, studies have shown
dominantly inherited, AD-linked mutations in these three that the APOEε4 allele accounts for 50–70% of the popula-
genes show high penetrance (>85%), affect the γ-secretase tion attributable risk for AD, and the presence of an ε2
cleavage of APP, and shift Aβ from Aβ40 to the more toxic allele may play a protective role against the development of
and aggregation-prone Aβ42. AD. However, it is neither necessary nor sufficient to cause
APP: The APP gene (on chromosome 21q21.3) encodes the disease.
the precursor of Aβ, which is the substrate of γ-secretase.
Mutations of APP gene or genes involved in its metabolism Other Potential Genetic Risk Genes of AD
can influence the progress of AD [20]. For example, the APP Except for these four classical genes mentioned above, about
E693G increases the aggregation propensity of Aβ and fibril 20 other genetic risk loci, such as SORL1, CLU, BIN1,
formation, which can induce the occurrence and develop- CD33, and TOMM40, have been found by using genome-
ment of AD. In addition, a study in the Icelandic population wide association studies (GWAS), massive parallel rese-
showed that a rare protective variant (p.A673T) in APP quencing (MPS), and meta-analysis.
SORL1: Located on chromosome 11q23.2–q24.2, TOMM40: As one of the genes around the APOE locus,
sortilin-related receptor 1 (SORL1) can determine whether Translocase of Outer Mitochondrial Membrane 40
APP is sorted in the retromer recycling-endosome pathway (TOMM40) encodes a mitochondrial protein (Tom40) that
or transferred to the endosome-lysosomal pathway, where it forms a complex with Tom22, Tom7, Tom5, and Tom6 and is
is cleaved to produce Aβ [22]. This mechanism may be the involved in the integration of protein precursors of the outer
reason why the SORL1 gene has close associations with both mitochondrial membrane. A SNP in TOMM40, rs2075650,
EOAD and LOAD. Some common and rare variants in has been shown to be associated with AD, especially with an
SORL1, such as T2134 and T588I, can cause reduced traf- increased risk of developing LOAD. The patients with
ficking of the mutant SORL1 protein from the endoplasmic rs2075650-G make an average of AD onset of 6 years earlier
reticulum/Golgi network to the cell surface. Therefore, more than those with rs2075650-A allele.
APP is misdirected into the late endosome pathway, where Other susceptibility genes include CASS4, CD2AP,
the APP is exposed to β-secretase and γ-secretase. This pro- CELF1, CR1, EPHA1, EXOC3L2, FERMT2, HLA cluster,
cess can increase the production of Aβ, especially Aβ42 [22]. INPP5D, MEF2C, MS4A cluster, NME8, PICALM, PTK2B,
CLU: Like ApoE, Clusterin, encoded by CLU (also SLC24A4, and ZCWPW1. All these genes are associated
named APOJ), is a lipoprotein highly expressed in both the with AD mainly due to effects on three pathways, including
periphery and the brain. It participates in the process of lipid immune response, APP processing, and lipid metabolism
transportation. Acting as an extracellular chaperone, CLU and endocytosis. Although these genes have been screened
directly interacts with soluble Aβ and forms a complex to by GWAS and proven in many studies, future research is still
cross the blood-brain barrier. It also influences the aggrega- needed to explore the specific mechanism and significant
tion and receptor-mediated clearance of Aβ. The soluble Aβ variants.
is transferred into insoluble forms such as oligomers, result-
ing in the suppression of toxicity and deposition of Aβ. Some Candidate Epigenetics Biomarkers
rare coding and common regulatory variants of CLU within DNA Methylation
a single locus may also exist and have independent effects on As one of the best-characterized chromatin modifications,
AD. However, the particular coding variants accounting for DNA methylation is mediated by DNA methyltransferases
the observed genetic relativity with CLU and the specific (DNMTs). The most predominant form is methylated on
association are unclear and need further study. the cytosine-phosphate-guanine (CpG) islands, which leads
CD33: As a member of a family of cell surface immune to the silence of genes [24]. Many studies have shown that
receptors, the protein encoded by the CD33 gene can pro- the overall methylation level of DNA in zones II and III of
mote cell-cell interactions and regulate functions of cells. the cerebral cortices, especially the hippocampi, is
It is expressed on monocytes and microglial cells, espe- decreased in AD patients [25]. Additionally, some research-
cially in the brain. Microglial cells expressing CD33 have ers have found that the methylation level of glycogen syn-
impaired Aβ phagocytosis, thus causing a reduction of the thase kinase 3β (GSK3β), which phosphorylates tau in the
uptake and clearance of Aβ. CD33 in an all-female cohort brain, raises too and may play a potential role in the patho-
has been proved to be the only gene region that has a sig- genesis of AD.
nificant AD-associated single-nucleotide polymorphism
(SNP) and shows a significant aggregate association with Histone Modifications
cognitive decline. On the other hand, a protective allele in The main histone modifications—acetylation and methyla-
CD33 has been found, which can increase the uptake of tion—mainly happen in arginine and lysine residues and
Aβ and reduce amyloid plaque burden and insoluble Aβ are catalyzed or removed by a specific set of enzymes.
levels. Also, large eight-SNP linkage disequilibrium (LD) Recently, the role of histone acetyltransferases in neural
blocks in CD33, such as rs2455069, have been found to development and disease has been revealed. The most
exhibit significant univariate associations with cognitive important evidence comes from the results of treating AD
decline [23]. patients with inhibitors of histone deacetylases (HDAC).
BIN1: Bridging integrator 1 (BIN1) is involved in diverse Some studies demonstrated that HDACI (coded as both
cellular processes, including membrane trafficking, actin W2 and I2) decreased the expression of γ-secretase com-
dynamics, and clathrin-mediated endocytosis, and thus can ponents while increased the expression of Aβ degradation
affect the production and clearance of Aβ. BIN1 is highly enzyme [25].
expressed in the human brain and is associated with later
onset and shorter duration of disease in AD patients. Studies MicroRNAs
have shown that P-tau is elevated in the CSF of AD patients. In recent years, increasing evidence suggests that there is a
It is assumed that BIN1 mediates AD risk through Tau dysregulation of microRNAs (miRNAs) in neurodegenera-
pathology. tive disorders (NDDs). For example, miR-125b, miR-146,
724 J. Wu et al.
miR-29, miR-106, miR-9, miR-107, miR-181, etc. have been patients to another ChEI, or adding memantine. Decisions
reported to be dysregulated in AD and may serve as potential regarding the duration and termination of treatment should
diagnostic biomarkers for AD. Targeting the mRNA of the be carefully considered and should take into account patient
key innate immune and inflammation-related regulatory pro- needs, the presence of adverse events, comorbidities, com-
teins, microRNA-125b and microRNA-146a are signifi- pliance, and effectiveness. If a lack of efficacy or presence of
cantly increased in AD patients. Also, microRNA cluster adverse events happens in the mild to moderate stage, a
miR-29a/b-1, which contributes to the derepression of switch in therapy needs to be considered. The appearance of
β-secretase 1 (BACE1) expression, increases in CSF of AD hallucinations and delusions before termination of ChEI may
patients and may be selected as a candidate biomarker for be predictive of clinical deterioration. ChEI disruption may
AD. also be related to cognitive impairment. However, when
patients with AD reach the end of their disease, antidementia
Imaging Diagnosis drugs should be discontinued, at which point they are usually
Imaging technology plays an important role in the clinical eligible for hospice care. At this time, most or all nonpallia-
evaluation of patients with suspected AD. Recent observa- tive care should be discontinued.
tions suggest that at least one structural scan (CT or MRI) For AD management, treatments for neuropsychiatric fea-
should be applied for the evaluation of the regional atrophy tures are very important. It is estimated that over time, more
in the medial temporal region, providing positive diagnostic than 90% of AD patients will eventually develop psychiatric
information [26]. With the development of the automated or behavioral changes. Although no drug is currently
hippocampal segmentation algorithms, the hippocampal vol- approved for the treatment of these symptoms, antipsychot-
ume could be more accurately measured and easily applied ics and antidepressants are often used off-label in AD
in routine clinical settings. F-fluorodeoxyglucose (FDG) patients.
PET is a good method to determine the glucose uptake by In recent years, more attention has been paid to the devel-
neurons and glial cells and is sensitive to abnormal synaptic opment of putative disease-modifying therapies that target
function. If abnormal hypometabolism is noted involving the the pathobiological processes involved in AD. These thera-
temporoparietal and posterior cingulate regions, then AD is peutic drugs are designed to slow the progression of AD, not
suggested. If significant hypometabolism is identified in just to address the symptoms. Unfortunately, to date, all
more anterior regions or more asymmetric, or both, then the completed phased clinical trials have failed to meet the
study is more suggestive of frontotemporal dementia. desired primary endpoints. Overall, the development and
Currently, the most innovative imaging technology for the optimization of future therapies remain a challenge until a
clinical evaluation of AD is PET with ligands for Aβ. As a better understanding of the underlying disease mechanisms
necessary but not sufficient biomarker for the diagnosis of becomes available [27].
AD, the amyloidosis in the brain diagnosed by the amyloid
PET has a high negative predictive value but a moderate pos-
itive predictive value. 41.1.6 Conclusion
As a chronic neurodegenerative disease, AD is prevalent and

41.1.5 Management poses significant health and financial burden to the patients,
their families, and society. It has a broad clinical presentation
Although no treatments can reverse the course of AD cur- and cannot be treated effectively. Accurate early diagnosis is
rently, the five FDA-approved AD drugs, including four cho- crucial for the evaluation and management of AD patients,
linesterase inhibitors (ChEIs) and one N-methyl-d-aspartate but the present diagnostic methods are not enough. As a
(NMDA) receptor partial antagonist, show advantages in detectable component in biological fluids, suitable, sensitive,
terms of cognition, behavior, and daily function. Treatment and specific biological markers may help improve the timely
should be considered when AD is first diagnosed. Based on diagnosis of AD and promote the understanding of AD
the current treatment guidelines, ChEIs are recommended pathophysiology. In this review, we provide an overview of
for mild to moderate symptoms, and memantine or the com- the classical and candidate biomarkers that are closely rele-
bination of memantine and ChEI for moderate to severe vant to the incidence and development of AD. However,
symptoms. For patients with symptomatic progression dur- there is still no available marker in the clinical laboratory for
ing ChEI monotherapy, it is necessary to consider increasing the preclinical stage of AD, calling for more efforts to be
their current treatment dose (if applicable), switching made.
41.1.7 Typical Medical Case able therapies, the expected survival of GBM patients
remains frustrating. The high degree of infiltration makes
Clinical Background A 69-year-old female patient was complete surgical resection impossible. This, together
treated because of “memory impairment for more than three with the notoriously known radio- and chemoresistance,
years with an accelerated decline over the past year.” accounts for GBM’s high recurrence rates and mortality
of nearly 95%.
Cognitive Evaluation The scores in almost all domains of

41.2.2 Classification
the CERAD-Plus-Neuropsychological Assessment Battery
were below the normal value adjusted by age and education
Gliomas are classified by cell type, grade, and location.
by at least −1.37 standard deviations. She scored 24 out of
30 points in the Mini-Mental State Examination.
41.2.2.1 By Type of Cell
Gliomas are named after specific cell types that share histo-
Laboratory Tests Liver and renal function, thyroid, thia-
logical characteristics but do not necessarily originate from
mine level, vitamin B12, and folate were all within the nor-
them. The main types of gliomas are:
mal range. APOE gene was ε4/ε4.
1 . Ependymomas: ependymal cells.
Imaging Examination Atlas-based volumetric MRI analy-
2. Astrocytomas: astrocytes (Glioblastoma multiforme is a
sis: significant reduction, especially temporal and parietal
malignant astrocytoma and the most common primary
lobe volumes.
brain tumor in adults.)
3. Oligodendrogliomas: oligodendrocytes.
Fluorodeoxyglucose-PET (FDG-PET): The glucose
4. Brainstem glioma: develop in the brain stem.
metabolism in cerebral parietal and temporal cortical areas
5. Optic nerve glioma: develop in or around the optic nerve.
showed an asymmetric reduction; glucose metabolism in the
6. Mixed gliomas: such as oligoastrocytomas, containing
right hemisphere was more significantly reduced. In addi-
different types of gliomas.
tion, glucose metabolism in the posterior cingulate cortex
was significantly reduced.
41.2.2.2 By Grade
Gliomas are further classified according to their grade, which
Diagnosis The patient presented with a 3-year history of
is determined by pathological evaluation of the tumor. The
cognitive decline with impairment in daily activities. She
neuropathological evaluation and diagnosis of brain tumor
scored low in almost all domains of the CERAD and exhib-
specimens are based on WHO Classification of Tumors of
ited temporoparietal atrophy and hypometabolism on neuro-
the Central Nervous System (Table 41.5).
imaging. A clinical diagnosis of AD was thus made.
This case was from Peking Union Medical College Hospital. 1. Low-grade [WHO grade II] gliomas are well-differentiated
(not anaplastic). These tend to show a benign trend and
predict a better prognosis for the patient. However, they
41.2 Glioma have a uniform recurrence rate and increase in grade over
time.
Mengxiao Xie 2. High-grade [WHO grades III–IV] gliomas are undifferen-
tiated or anaplastic. These are malignant and have a poor
prognosis.
41.2.1 Overview
Of numerous grading systems used, the most common is
Glioma is a tumor of glial cells originating in the brain or the WHO grading system for astrocytoma, in which tumors
spine. Gliomas comprise about 30% of central nervous are graded from I (least advanced disease—best prognosis)
system tumors and account for 80% of malignant brain to IV (most advanced disease—worst prognosis).
tumors. The World Health Organization (WHO) classifies
gliomas according to histological criteria into four grades 41.2.2.3 By Location
of ascending malignancy. Glioblastoma multiforme Gliomas can be classified based on whether they are located
(GBM, WHO grade IV) is one of the deadliest human can- above or below a membrane in the brain called the tentorium
cers, with conventional therapy only offering palliation. cerebelli. The tentorium cerebelli separates the cerebrum
Despite concerted efforts and advances in currently avail- (above) from the cerebellum (below).
726 J. Wu et al.
Table 41.5 WHO classification of central nervous system tumors

Type Grade Description
Astrocytoma II Found diffusely infiltrating into surround neural tissue, increased hypercellularity, and no mitosis
Oligodendroglioma II Occur in the white matter and cortex of the cerebral hemispheres, low mitotic activity, and no
necrosis
Oligoastrocytoma II Diffuse mixed tumor with mixed glial background
Anaplastic-astrocytoma/ III Highly infiltrating tumors with increased mitotic activity and no necrosis or vascular proliferation
Oligodendroglioma
Glioblastoma IV Infiltrating glial neoplasm with necrosis and microvascular proliferation and high rate of mitosis
1. The supratentorial gliomas are above the tentorium, in the ment typically involves the frontal lobe. Patients with bilat-
cerebri, and more common in adults (70%). eral frontal lobe involvement will demonstrate impairment
2. The infratentorial gliomas are below the tentorium, in the earlier in the disease course. Additionally, bilateral frontal
cerebelli, and more common in children (70%). lobe dysfunction can manifest as a loss of executive function.
3. The brainstem gliomas are in the pons of the brainstem. Other subtle mental status changes include forgetfulness,
The brainstem is divided into three parts (pons, midbrain, loss of insight, indifference to social interactions, decreased
and medulla). The pons controls critical functions such as initiative, or blunted affect. Occasionally, these symptoms
breathing, making these surgeries extremely dangerous. are erroneously attributed to dementia or old age. A further
hindrance to proper diagnosis comes from the patient as he
or she will conceal or deny these changes due to embarrass-
41.2.3 Signs and Symptoms ment or health concerns. Patients often admit to decreased
energy, altered sleep patterns, and a loss of interest in activi-
Symptoms of gliomas are decided by which part of the cen- ties they used to enjoy.
tral nervous system is affected. A brain glioma can cause Personality changes, such as apathy, anhedonia, and inat-
headaches, vomiting, seizures, and cranial nerve disorders tentiveness, are common. More extreme changes, ranging
due to elevated intracranial pressure. Optic nerve glioma can from depression and emotional lability to impulsivity and
cause visual loss. Spinal cord gliomas can cause pain, weak- disinhibition, can also be observed. Confusion and dementia
ness, or numbness in the limbs. Gliomas do not metastasize typically occur later in the disease course and are associated
through the bloodstream, but they can spread through the with other neurologic deficits.
cerebrospinal fluid and cause “drop metastases” to the spinal Seizures may be either the initial presenting symptom of
cord. glioma or may develop subsequently as the disease pro-
The most common manifestation of brain tumors is a gresses. New-onset seizures in adults should raise suspicion
headache, which is present in over half of the patients with for underlying focal structural abnormality, especially neo-
the illness. Headache was the initial symptom in 37% of plasm. The incidence of seizures has been reported in
patients with supratentorial tumors and 56% of infratentorial 25–50% of brain tumors.
tumors. Patients with large tumors causing elevated intracra- Numerous studies have shown that most patients with
nial pressure or mass effect are more likely to suffer from low-grade gliomas experience epileptic seizures as the pre-
tumor-related headaches. senting symptom. The prevalence of seizures among high-
Nausea and vomiting associated with gliomas are usually grade glioma (III or IV) patients is higher than that among
secondary to increased intracranial pressure from tumor low-grade gliomas as high as 30% of patients with GBM
mass, tumor-associated edema, or obstructive hydrocepha- present with epileptic seizures at the time of diagnosis [28].
lus. An elevated intracranial pressure causes irritation of the Just like the overall symptomatology of gliomas, the seizure
area postrema, a medullary center on the floor of the 4th ven- semiology varies by the focus of origin.
tricle that controls vomiting. Indicators of increased ICP as The most popular signs are strabismus and decreased
an underlying cause of vomiting include association with visual acuity. There is a significant variation in symptoms
changes in body position or maneuvers that increase ICP depending on whether the glioma is associated with neurofi-
(e.g., sneezing or coughing). bromatosis [29]. In NF patients, proptosis is more common,
Mental status changes are common features of intracra- whereas in sporadic cases, nystagmus and hydrocephalus are
nial tumors. Psychomotor retardation, or the slowing of more common [30]. Anterior optic pathway gliomas occur
thought processes and physical motor activity, is among the before retinal fibers reach the chiasm, whereas posterior
most common presenting symptom of gliomas. These symp- lesions occur either in or posterior to the chiasm. Most ante-
toms are initially subtle and usually only identifiable to the rior gliomas are classified as low-grade astrocytomas and are
patient, family members, or close friends. Cognitive impair- most commonly found in children.
41.2.4 Causes and Pathogenesis 41.2.4.2 Adult Stature and Body Weight

Two large cohort studies observed a significant linear trend
The causes of gliomas are unknown. Gliomas cover all between self-reported adult height and increasing glioma
tumors that originate from glial cells in the brain and account risk [35]. A prospective study that measured height directly,
for most of the primary malignant brain tumors. Astrocytomas however, did not find an association. The relationship
and oligodendrogliomas are the two primary types of glio- between adult obesity and glioma risk is not clear. Multiple
mas. GBM is the most common type of astrocytoma and cohort studies have found no association between adult BMI
accounts for about 50% of gliomas diagnosed in adults. The or other body fat measurements, including waist circumfer-
majority of GBMs occur de novo (~95%), and a small pro- ence and waist-to-hip ratio, with glioma risk. However, in
portion of GBMs progress from low-grade or anaplastic one study, obesity at the age of 18 was significantly associ-
astrocytoma (~5%). ated with an increased risk of glioma in later life.
41.2.4.1 Genetic Factors 41.2.4.3 A llergies and Other Medical

Familial clustering and strong associations with rare genetic Conditions
syndromes suggest that heredity plays a role in the onset of Immunologic factors may be involved in the pathogenesis of
glioma. Rare autosomal dominant genetic conditions associ- gliomas. Studies have consistently observed a significant
ated with familial glioma include tuberous sclerosis (involv- negative correlation between the history of allergies and the
ing TSC1 and TSC2), neurofibromatosis types 1 and 2 risk of gliomas. Several studies have noted a significant
(involving NF1 and NF2), Turcot syndrome (involving DNA dose-response relationship with a greater risk reduction
repair genes APC, hMLH1, hMSH2, PMS2, and PTEN), and observed with the increasing number of allergic conditions.
Li-Fraumeni syndrome (involving TP53) [31]. A meta-analysis of eight observational studies totaling 3450
Multiple studies have observed familial aggregation of glioma cases found that the risk of gliomas associated with
glioma. In a population-based study in Utah that examined any history of allergies, asthma, or eczema was significantly
familial clustering for different glioma tumor types, among reduced by 30–40% [36].
first-degree relatives of glioma probands of the same histol- History of seizures and epilepsy has consistently been
ogy type, the prevalence of GBM and astrocytoma is signifi- associated with glioma [37]. It is unclear whether epilepsy
cantly higher than expected [32]. Familial clustering is and seizures are etiologically relevant to the onset of glioma
observed in about 5% of glioma cases, and rare genetic syn- or instead represent early symptoms of the evolving tumor.
dromes are present in about 1% of cases [33]. Given that Many studies have considered craniocerebral injury as a
there is a low prevalence of rare inherited mutations among potential risk factor for glioma [38].
cases, the genetic risk of gliomas is likely to be the result of
the common inheritance of multiple low-risk variants. 41.2.4.4 Dietary Factors
Common polymorphisms in genes involving DNA stabil- Large consumption of cured meat during pregnancy is related
ity and repair, cell cycle, and carcinogen metabolism related to the development of childhood brain tumors [39]. However,
to gliomas have been studied [34]. Variants significantly no consistent association has been demonstrated between
associated with glioma risk have been identified in one or dietary NOC (N-nitroso compounds) exposure and glioma in
more studies among the DNA repair genes XRCC7, XRCC1, adults. Several case-control studies have shown a significant
PARP1, MGMT, ERCC1, and ERCC2 and the cell cycle positive association with consumption of cured or processed
gene EGF. In genome-wide association studies (GWAS), meat, whereas others have no correlation. A meta-analysis
common genetic variants in CCDC26, PHLB1, telomere incorporating nine observational studies suggested that indi-
maintenance genes TERT and RTEL1, and cell cycle genes viduals with a high intake of cured meat have an increased
CDKN2A/B and EGFR were associated with glioma risk. risk for glioma.
The associations with genetic variants differ by glioma Vitamins C and E can inhibit the formation of endoge-
histology, suggesting that there are different etiologic path- nous NOC. In a large case-control study involving 802 gli-
ways for the recognized glioma subtypes. TERT, RTEL1, oma cases, a significant negative correlation was observed
and CDKN2A/B are most strongly associated with astrocy- between dietary vitamin C intake and glioma [40].
tomas and GBM, whereas variants in CCDC26 and PHLB1 Prospective studies, however, did not observe an association
are primarily associated with astrocytomas and oligodendro- between dietary or supplemental vitamin C and E intake and
gliomas [31]. An uncommon variant in TP53 found in glioma risk [33]. The findings for fruit and vegetable con-
approximately 1% of Caucasians is associated with a more sumption have also been inconsistent. According to an analy-
than twofold excess risk in all subtypes of glioma. Additional sis of the INTERPHONE study, which included 1185 glioma
rare variants conferring susceptibility will likely be cases, it was found that the intake of green leafy vegetables
discovered with the completion of next-generation
and orange-yellow vegetables was significantly negatively
sequencing-based studies. related to the risk of glioma and was closely related to astro-
728 J. Wu et al.
cytoma and GBM. Prospective studies, however, have not affect arginine at position 132 of the amino acid sequence,
supported these observations. Further research is needed to which belongs to an evolutionarily highly conserved region
determine whether dietary modifications can reduce the risk at the isocitrate binding site. The most frequent mutations in
of glioma. this region were R132H mutations (>90%), but other vari-
ants were also found (R132S, R132C, R132G, and R132L).
41.2.4.5 Ionizing Radiation Interestingly, nonR132H mutations were isolated in different
Exposure to ionizing radiation is the only recognized envi- histological and molecular subtypes of gliomas.
ronmental risk factor for gliomas. Several studies have Histologically, nonR132H mutations occasionally occur in
shown that adults have a significantly increased risk of brain classic oligodendroglioma and significantly more frequently
tumors receiving radiation therapy for childhood cancer in other grade II and III gliomas. Genetically, nonR132H
[38]. In a cohort of 14,361 children who were 5-year cancer mutations occur in TP53-mutated tumors and accumulate
survivors, the children who received therapeutic radiation with different (based on gene expression profiling) intrinsic
had almost a sevenfold increased odds of primary glioma molecular subtypes, while LOH does not actually exist in 1p
compared to children who did not receive radiation [41]. and 19q tumors, respectively.
Studies of children receiving low-dose ionizing radiation for Importantly, IDH1 mutations are associated with
tinea capitis and hemangiomas have also found that the risk improved prognosis. Therefore, the IDH1 mutation state is
of brain tumors is too high [42]. In several studies, the excess likely to be used as a molecular marker for prognosis in the
risk for brain tumors was greatest among children exposed to near future. In addition, two studies examined whether IDH1
radiation at younger ages. mutation status can predict response to glioma treatment. In
Few studies have explored whether ionizing radiation a group of patients with undifferentiated low-grade astrocy-
from medical diagnostic procedures increases glioma risk. toma, there was no difference in response to TMZ between
No link to glioma risk has been demonstrated with occupa- IDH1 mutant and wild-type tumors after radiotherapy. In
tional exposure to ionizing radiation [43]. patients with anaplastic oligodendroglioma treated with
radiotherapy alone or adjuvant PCV radiotherapy, IDH1
mutations have reportedly not been predictive of the
41.2.5 Molecular Markers in Gliomas response.
The complex biological and molecular heterogeneity of 41.2.5.3 MGMT Promotor Methylation
these tumors makes it difficult to develop effective treat- The O6-methylguanine-DNA methyltransferase (MGMT)
ments. Recent research has focused on deciphering the gene encodes a nuclear repair enzyme alkyltransferase that
molecular biology of gliomas. removes alkylated adducts from the O6 position of thymine.
By doing so, the enzyme is involved in maintaining the
41.2.5.1 Loss of Heterozygosity (LOH) of 1p19q integrity of the DNA. More specifically, the product of the
Loss of 1p19q heterozygosity (LOH) is a chromosomal aber- MGMT gene protects cells from alkylating and methylating
ration and is closely related to classical OD (oligodendro- agents (such as BCNU [N,N [prime]-bis (2-chloroethyl)-
cytic cells). Due to two important characteristics, determining N-nitrosourea], carba Hydrazine, and TMZ). In gliomas,
the status of 1p19 in gliomas is clinically relevant. First, glio- CpG islands located on the MGMT gene promoter are often
mas with LOH of 1p19q usually grow more slowly than most methylated, resulting in the “epigenetic” silencing of the
other gliomas and therefore have a better prognosis. Second, gene. Theoretically, this methylation will give greater sensi-
the presence of this mutation predicts a therapeutic response tivity to alkylating and methylating agents. In everyday prac-
to alkylating agents such as PCV (procarbazine, lomustine, tice, the meaning of MGMT identity is more difficult to
and vincristine). However, many gliomas benefit from alkyl- explain. Several studies have shown that epigenetic silencing
ating agents to some extent. Nevertheless, the status of 1p19q of the MGMT gene is clinically important because it is asso-
is a strong prognostic factor, and the presence of 1p and 19q ciated with increased survival and a better response to com-
is currently being tested in tumor patients with oligodendro- bined chemoradiation in GBM. The results show that the
cyte characteristics. effect of MGMT silencing is particularly pronounced when
TMZ is used as chemotherapy. Other studies have shown that
41.2.5.2 IDH1 and IDH2 Mutations the time to manage TMZ seems to be crucial. One study
Recently, somatic mutations in the gene encoding isocitrate showed that the positive effect of methylated MGMT genes
dehydrogenase 1 (IDH1) have been found in gliomas. IDH1 on survival was only seen when TMZ was administered dur-
mutations occur mainly in low-grade gliomas and secondary ing radiotherapy, while other studies showed a positive effect
GBM and are therefore considered to be early events in glio- when TMZ was administered before or after radiotherapy.
mas. Interestingly, glioma-specific mutations in IDH1 always Interestingly, a recent study showed that methylation of the
MGMT promoter in GBMs could better predict the response 41.2.5.5 Important Factors in Glioma Biology
to radiotherapy and could even serve as a prognostic marker
for patients who did not receive adjuvant alkylation chemo- RTK/RAS/PI(3K), P53
therapy. This study suggested that MGMT might be a gener- GBMs have changes in three gene alternations, including
ally favorable prognostic factor in GBM rather than a mutations, amplifications, or deletion. Phosphatidylinosital-
predictor of alkylation chemotherapy response. 3-kinase (PI3K) regulates apoptosis and cell cycle via p53,
Another difficulty in explaining the role of MGMT is that cyclin-dependent kinases, and retinoblastoma 1 signaling.
there are several different methods for detecting the methyla-Changes in PI3K/growth factor receptor/MAPK signaling
tion status of the MGMT promoter. These different methods include amplification of the platelet-derived growth factor
lead to variable results and can be difficult to compare. MGMTreceptor (PDGFR), the epidermal growth factor receptor
activity can be measured using immunohistochemical meth- (EGFR/erbB1), or the HER2/erbB2 receptor gene or muta-
ods, real-time polymerase chain reaction, or promoter meth- tions of the MET or EGFR genes. EGFR mutations, espe-
ylation analysis methods. Similarly, changes related to cially the EGFRvIII mutation in which most of the
measurements on frozen tissue versus paraffin samples as well extracellular domain is deleted, occur in 25–30% of
as possible differences between observers can also lead to dif-
GBM. Downstream signaling from growth factor receptors
ferences in results. Despite these, the status of MGMT is con-can be activated by deletion or mutation in the neurofibroma-
sidered an important biomarker for gliomas. tosis 1 gene (NF1), mutations in PIK3CA (the gene for
PI3K), mutations in KRAS, and deletion or loss of heterozy-
41.2.5.4 Germline Mutation of TP53 gosity of PTEN, a PI3K inhibitor. Although about one third
Brain tumors are usually accompanied by hereditary cancer of GBM may have deletion or mutation of TP53, loss of p53
syndromes such as Li-Fraumeni-like syndrome (LFLS), function can also be acquired by amplification of MDM4 or
Li-Fraumeni syndrome (LFS1), CHEK2-related syndrome MDM2 [44]. Finally, deletion or mutation of the cell cycle
(LFS2), Ollier syndrome, Maffucci syndrome, von Hippel- inhibitors CDKN2A, CDKN2B, and CDKN2C, loss of the
Lindau syndrome, or tuberous sclerosis complex. The latter RB1 gene or cyclin-dependent kinases, and less frequent
two syndromes are usually associated with tumors with spe- amplifications in cyclins can disrupt cell cycle regulation.
cific histopathology, hemangioblastoma, and subependymal Translocations and fusion proteins may be important in acti-
giant astrocytoma, respectively. In contrast, other genetic vating growth factor signaling. About 2% of GBMs have
syndromes are related to a variety of glial tumors and cho- fusions of EGFR with PSPH, which regulates the prolifera-
roid plexus tumors. TP53 is the most important genetic fac- tion of neural stem cells, while another 4% have fusions of
tor affecting the occurrence of human brain tumors without a EGFR with SEPT14, which activates STAT3 signaling [45].
doubt. Furthermore, about 3% of GBM have translocations that
Germline mutations of TP53 are among the most famous result in a fusion of the fibroblast growth factor receptor
genetic etiologies of human cancers, including brain tumors. genes, FGFR1 or FGFR3, with a transformed acidic coiled-
Germline mutations of TP53 in human cancer cases consis- coil TACC1 or TACC3 containing a protein gene. There is
tent or not consistent with Li-Fraumeni syndrome have been in vitro and in vivo evidence that tumors with these fusions
studied. The latest database of TP53 germline mutations can be suppressed with appropriate tyrosine kinase inhibi-
showed that out of 1053 recorded TP53 germline mutations, tors. However, it is unclear whether they have a prognostic or
138 occurred in patients with brain tumors. Even if there are predictive effect in humans.
no characteristic tumors of Li-Fraumeni syndrome, such as
soft tissue sarcoma, breast cancer, and adrenocortical can- IDH Mutation
cers, there are cases of familial aggregation. Especially in In 2008, a common point mutation in the metabolic gene
brain tumors alone, among the immortal lymphocytes of 51 IDH1 was identified in 12% of glioblastoma samples using
patients with brain glioma, TP53 mutations were found in 6 multiple combinations of whole-exome sequencing. Further
cases. They raised that diversity (in the brain and other research found that this mutation is present in secondary
organs) and a familial history suggest the presence of a TP53 GBM and 80% of grade 2–3 gliomas. Although mutations in
germline mutation, and they noted that 6 subjects in 9 pedi- IDH2 are also rare in gliomas, they are mutually exclusive
grees were in their 30 s when their glial tumors were diag- with mutations in IDH1. To date, all mutations identified
nosed. These reports are instructive when we encounter cases have been a single amino acid missense mutation in IDH1 at
of brain tumors in adolescents and young adults in practice. arginine 132 (R132) or similar residue in IDH2 (R172). Even
Ranging from oligodendroglioma to astrocytoma and GBM, though initial research showed that the mutant IDH functions
it is reported that there are over 20 different germline mis- as a dominant inactivator by heterodimerizing to wild-type
sense mutations in the germline of patients with various bio- IDH1 and weakening its activity, more recent in vitro studies
logical grades of gliomas. have shown that the protein of mutated IDH1has the ability
730 J. Wu et al.
to convert α-ketoglutarate (α-KG) to R(−)-2-hydroxyglutarate (MMPs), membrane- type matrix metalloproteases

(2-HG). These findings show that mutant IDH is an onco- (MT-MMPs), and adamalysins (ADAMS). The expression
gene and 2-HG is an “oncometabolite.” New evidence indi- and secretion of these proteases can be regulated by TGF-
cates that 2-HG competitively inhibits the activity of many beta and NF-κB.
α-KG-dependent dioxygenases by antagonizing α-KG,
including without limitation histone demethylases (e.g., pro-
lyl hydroxylases, collagen prolyl-4-hydroxylase, and the ten- 41.2.6 Treatment
eleven translocation (TET) family of DNA hydroxylases).
TCGA analysis of GBM indicates an association between Treatment type for brain gliomas depends on the cell type,
increased promoter methylation (G-CIMP) and IDH muta- the location, and the degree of malignancy. Usually, treat-
tion, which usually leads to transcriptional silencing of the ment is combined with surgery, radiation therapy, and
related genes. Two recent independent studies have shown chemotherapy.
that the G-CIMP phenotype is not associated with IDH; IDH
mutation alone is actually responsible for the G-CIMP 41.2.6.1 Surgical Resection
hypermethylation phenotype in diffuse gliomas. Surgical resection is indicated in almost all glioma patients
during their diseases. Surgical resection has three main pur-
Hypoxia, Pseudohypoxia, and Angiogenesis poses, including obtaining a histological diagnosis, relieving
Gliomas usually present in hypoxic or pseudohypoxic condi- symptoms, and improving patient survival. In some cases,
tions. An extreme example of hypoxia is pseudopalisading surgery can even lead to a cure. Recently, multiple data have
necrosis in GBM when the blood supply to the tumor is demonstrated the importance of aggressive surgical resection
insufficient. Hypoxia leads to the upregulation of hypoxia- for the improved prognosis of glioma. Particularly, increas-
inducible factor (HIF1a), and HIF1a can induce stem cell ing the extent of resection has been shown to improve sur-
survival processes and new angiogenesis. HIF1a is overex- vival in both low-grade and high-grade gliomas directly.
pressed in gliomas, especially high-grade gliomas. The deg- Low-grade gliomas (WHO I and II) are a group of tumors
radation of HIF1a by the ubiquitin-proteasome system is that are diverse clinically, histologically, and molecularly.
also inhibited by changes in prolyl hydroxylases driven by WHO grade I tumors include pilocytic astrocytoma and sub-
2-hydroxyglutarate produced by mutant IDH. Therefore, ependymal giant cell astrocytoma, and the pilocytic astrocy-
IDH mutation can lead to a situation in which HIF1a protein toma is more common. Surgical resection is the treatment of
levels are high (consistent with hypoxia) with a normal oxy- choice for WHO grade I gliomas, which carry a good
gen tension. This situation is called “pseudohypoxia.” It may prognosis.
be a major biological driver in these tumors, along with other WHO grade II gliomas include oligodendrogliomas, dif-
consequences of altered proline hydroxylation. Blood vessel fuse fibrillary astrocytoma, and oligoastrocytomas, all of
formation in gliomas usually takes the form of angiogenesis which have similar invasive and malignant potential. Grade
and is driven by vascular endothelial growth factor (VEGF). II gliomas that are symptomatic and surgically accessible
VEGF is overexpressed in brain tumors, with increased should undergo maximal cytoreductive surgical resection as
expression corresponding to increased grade [46]. the treatment of choice. In low-grade gliomas, predictors of
CXCR4 signals can also support angiogenesis. incomplete tumor resection include tumor involvement of
Alternatively, in a process called angiogenesis, gliomas can the corticospinal tract, large tumor volume, and oligodendro-
occupy normal vessels, which is usually a process mediated glioma histopathologic type. The 5-year survival of gliomas
by angiopoietin signaling, or new blood vessels can be has been reported up to 95–97% following gross total resec-
formed from bone marrow-derived endothelial cells. Glioma tion in these lesions.
cells may even transform into malignant endothelial cells. It For both grade I and grade II tumors, maximal surgical
has been suggested that blocking angiogenesis, and specifi- resection is the only best treatment for improving survival.
cally inhibiting VEGF signaling, may lead to increased gli- Adjuvant chemotherapy or radiation treatment may be used
oma invasiveness, although this hypothesis remains when there is progressive tumor growth following resection
controversial. or progressive neurological symptoms in unresectable
Invasion of the normal brain is one of the hallmarks of tumors.
diffuse gliomas and one of the features that make them High-grade gliomas (WHO III and IV) include anaplastic
incurable by surgery alone. Gliomas migrate along the sec- astrocytoma (Grade III) and glioblastoma (Grade IV). These
ondary structures of Scherer, such as white matter trajecto- tumors are malignant and have a significantly poorer progno-
ries, neuronal trajectories, vasculature, or the subvertebral sis than the low-grade gliomas. The fastest way to relieve the
space. The secretion of multiple proteases promotes the mass effect exerted by the tumor is resecting a high-grade
migration of gliomas, including matrix metalloproteases glioma. In the latest multicenter study of 408 patients with
malignant gliomas, 53% improved neurologically after sur- group study in North America included 203 patients with
gery, while permanent new deficits were seen in only 8%. low-grade gliomas and randomized these patients into groups
While there are no comparable potentials for symptomatic receiving either 50.4 or 64.4 Gy. The results of this study
improvement after tumor biopsy, radiotherapy, or chemo- showed that patients receiving 64.4 Gy of radiation had a
therapy in high-grade gliomas, the benefits of surgical resec- lower but not statistically significant overall survival rate (65
tion are evident. Many studies have demonstrated that vs. 72%). Due to the lack of improvement in overall survival
patients with malignant gliomas survive longer after a com- among patients receiving adjuvant RT for low-grade glio-
plete versus a partial resection or a biopsy. Meanwhile, some mas, RT is not currently recommended in most standard pro-
retrospective data, however, suggested that surgical resection tocols at the initial presentation. Radiation may be useful in
played no major role in the survival of at least some patients recurrent disease or various subsets of patients with low-
with glioblastoma. In conclusion, patients with a resectable grade gliomas and is still being studied. The treatment of
malignant glioma should probably undergo tumor removal high-grade gliomas, WHO grades III and IV tumors, has
as complete as possible. Surgical resection will often provide always been particularly challenging. In the late 1970s,
fast symptom relief, and the literature supports the concept Walker et al. performed a landmark randomized controlled
of an oncological benefit derived from extensive tumor study and provided conclusive evidence that adjuvant RT is
resections. A partial resection may still provide some of the superior to surgery alone. Whole-brain radiation therapy
benefits of complete tumor removal, although to a lesser (WBRT) was administered to patients with malignant glio-
degree. mas after maximum surgical resection in doses varying from
5000 to 6000 rads. Those who received adequate treatment
41.2.6.2 Radiation Therapy saw a rise in median survival from 17 weeks with surgery
Radiation therapy (RT) is considered the next step in the alone (the previous standard of care) to 37.5 weeks with the
treatment of glioma following surgical resection. The princi- addition of WBRT, a 120% rise in median life expectancy.
pal goal of RT is to destroy residual tumor cells that are not Further studies verified the findings of Walker and colleagues
removed with surgery, therefore preventing or postponing and continued to demonstrate the benefits of RT in the treat-
tumor recurrence. RT, as an adjuvant therapy in addition to ment of high-grade gliomas. The dose of 45 Gray (Gy)
surgical resection, is most effective on smaller lesions. administered to the whole brain showed improvement in sur-
Resection of maximal tumor bulk results in smaller residual vival, although further dose escalation was limited by poor
volumes, which are more responsive to RT, and thereby brain tissue tolerance. Doses of <45, 50, 55, and 60 Gy were
increases the efficacy of RT. Typically, RT is considered for compared. Maximum survival was observed at doses of
patients after initial biopsy or resection and for patients at a 60 Gy [47]. The addition of a 10 Gy boost failed to demon-
higher risk of early malignant transformation. strate any change in outcome [48]. Further dose escalation to
RT has been used in the treatment of low-grade gliomas 90 Gy using 3D conformal technique was feasible, although
for several decades. Despite its long history in low-grade failure was still predominantly secondary to local recurrence
gliomas, the optimal timing and dose of RT remain unclear. [49]. It is of note that the treatment for unresectable high-
Maximum surgical resection followed by close observation grade gliomas is similar to that of resectable tumors with the
has remained the standard of care, as the data for radiation in obvious exception of surgery.
low-grade gliomas have not been significantly positive. The
European Organization for Research and Treatment of 41.2.6.3 Chemotherapy
Cancer (EORTC) 22,845 trial randomized 314 patients to In addition to surgical resection and RT, the other main treat-
undergo immediate postoperative radiotherapy with 54 Gy ment for malignant glioma is chemotherapy. Chemotherapy
or delay radiotherapy until the time of progression; the study is another adjuvant therapy for glioma and can be given dur-
showed a statistically significant increase in progression-free ing radiation, following radiation treatment, or at progres-
survival in patients who received adjuvant radiation therapy sion. As with RT, chemotherapy is more effective in treating
compared to the control group that did not receive smaller tumors. Therefore, maximizing the extent of surgical
RT. However, the overall survival in the group of patients resection is important to provide a more desirable response
receiving radiation was the same as in the control group. Two to chemotherapy. There is a substantial overlap in the chemo-
large randomized trials investigated the potential benefit of therapeutic regimens utilized in high-grade and low-grade
higher radiation doses for low-grade gliomas. EORTC 22844 gliomas. Common chemotherapies include temozolomide
included 343 patients randomized to 45 or 59.4 Gy adjuvant (an alkylating agent), bevacizumab (an antiangiogenic
RT and failed to demonstrate a clear dose-response relation- agent), and PCV (procarbazine/lomustine [CCNU]/vincris-
ship for RT as adjuvant treatment for low-grade gliomas. The tine). Currently, there is still controversy over the ideal che-
overall survival and progression-free survival were nearly motherapy agent, the ideal time of administration, and the
identical, approximately 60 and 50%, respectively. An inter- optimal duration of chemotherapy treatment.
732 J. Wu et al.
41.2.7 Typical Medical Case dystrophy, myotonic dystrophy, facioscapulohumeral mus-

cular dystrophy, oculopharyngeal muscular dystrophy, distal
Clinical Background A 73-year-old male with a 2-year myopathy, Emery-Dreifuss muscular dystrophy, and limb-
history of right limb weakness presents with a progressive girdle muscular dystrophy—which are the most heteroge-
worsening of the weakness over the past 2 months. neous group. Results gained from genetic testing are essential
for establishing an accurate diagnosis and providing reliable
Head MRI Scan Left frontal parietal space occupying genetic counseling and prenatal diagnosis. Muscle atrophy is
lesion. a symptom characterized by the loss of normal muscle mass.
It is caused by a decline in the total number of muscle cells
Initial Laboratory Values WBC 4.7 × 10^9/L, Neutrophils or by a substantial decline in the substance of individual
2.5 × 10^9/L, RBC 4.14 × 10^12/L, Hb 123 g/L, PT 11.6 s, muscle cells. Spinal muscular atrophy (SMA) is a genetic
APTT 30.1 s. disorder of motor neurons in the anterior horns of the spinal
cord and brainstem that results in muscle atrophy and weak-
Muscle Strength Test Right upper limb muscle strength ness. SMA is an autosomal recessive disease linked to dele-
level 0; right lower limb muscle strength level I. Left upper tions of the SMN1 gene on chromosome 5q. SMN2 gene,
and lower limbs muscle strength level V. Left muscular ten- whose product can mitigate disease severity, leads to the
sion had been reduced. variability in severity and age of onset of disease [51].
Histopathological Examination The exon 6 sequence

132R (CGT) of IDH1 is wild type. IDH2 exon 5 sequence 41.3.2 Clinical Appearance
140R (CGG) and 172R (AGG) are wild type. Exons 9, 11,
13, and 17 of C-KIT are wild type. Exons 18, 19, 20, and 21 Muscular dystrophy is a clinically, genetically, and biochem-
of EGFR are wild type. ically heterogeneous group of disorders that share clinical
and dystrophic pathological features on muscle biopsy. They
Results with Interpretation Guideline A glioma is a type are characterized by progressive muscle weakness that
of tumor that starts in the glial cells of the brain or the spine. affects limb, axial, and facial muscles to a variable degree. In
The diagnosis of glioma mainly depends on clinical symp- specific forms, other muscles, including respiratory muscles,
toms, CT, MRI, and postoperative pathology. Histological cardiac smooth muscles, and swallowing muscles, can also
and molecular pathological examinations can help to deter- be affected. In rare variants, the disorder is associated with
mine the pathological grading and molecular subtypes of involvement of other organs or tissues, such as the brain,
gliomas. Gliomas are prone to recurrence, and treatment inner ears, eyes, or skin. The severity, age of onset, rate of
requires multidisciplinary collaboration. progression, and consequent complications and prognosis
vary greatly in different forms of the disorder [52].
Final Diagnosis Several tests were performed for screening There are nine major groups of muscular dystrophies,
this disease, such as CT and MRI. According to the patho- which vary in symptoms and severity. These disorders are
logical examination results, this disease was diagnosed as a characterized by the extent and distribution of muscle weak-
left parietal lobe high-grade glioma. ness, age of onset, rate of progression, severity of symptoms,
and family history. Although some forms of muscular dys-
This case is from the First Affiliated Hospital of Nanjing trophies appear in infancy or childhood, others may not
Medical University (also named Jiangsu Province Hospital). appear until middle age or later.
Muscle atrophy is also known as muscle wasting and rep-
resents a debilitating condition when muscle mass decreases
41.3 Muscular Dystrophy/Muscular due to several factors. Atrophy of a muscle can occur mainly
Atrophy in two ways—disuse or denervation—and it occurs in a vari-
ety of pathologies (Fig. 41.3). Malnutrition, alcohol-
Zhaojing Zheng and Juan Geng associated myopathy, aging, obesity, and diabetes can lead to
different degrees of muscle atrophy.
41.3.1 Overview 41.3.2.1 Muscular Dystrophy
Muscular dystrophies are a heterogeneous group of inherited Duchenne Muscular Dystrophy (DMD) and Becker
disorders that share similar clinical features and dystrophic Muscular Dystrophy (BMD)
changes on muscle biopsy [50]. They can be subdivided into DMD is more frequent, occurs earlier, and is more severe
several groups: Duchenne and Becker, congenital muscular than BMD. DMD and BMD primarily affect males. In DMD,
Fig. 41.3 Clinical conditions associated with muscular atrophy. AIDS: acquired immunodeficiency syndrome
walking is often delayed. Cognitive functions can be altered. The lifespan of someone with this type also varies, depending
Diagnosis is generally made at the age of 5 when children on the symptoms. Some patients with congenital muscular dys-
present with a waddling gait, talipes equinus, calf pseudohy- trophy die in infancy, while others live until adulthood.
pertrophy, and a positive Gowers’ sign. The inability to walk
appears by 10–12 years of age. Scoliosis, cardiomyopathy, Myotonic Dystrophy
and restrictive respiratory failure progressively appear. BMD Myotonic dystrophy is also called Steinert’s disease or dys-
appears later, between the ages of 5 and 15 years with a proxi- trophia myotonica. This form of muscular dystrophy causes
mal motor deficiency of variable progression. Heart involve- myotonia, an inability to relax the muscles after they con-
ment can be the initial sign [53]. Other clinical forms also tract. Myotonia is exclusive to this type of muscular
exist (isolated cardiomyopathy, exercise intolerance, and dystrophy. It is the most common form of muscular dystro-
symptomatic forms of muscular dystrophy of Duchenne and phy that begins in adulthood. Myotonic dystrophy is charac-
Becker in female carriers). terized by progressive muscle wasting and weakness. People
with this disorder often have prolonged muscle contractions
Congenital Muscular Dystrophy and are unable to relax certain muscles after use. For exam-
Congenital muscular dystrophy (CMD) is a clinically and ple, patients may have difficulty in releasing their grip on a
genetically heterogeneous group of inherited muscle disorders. doorknob or handle. They may also have slurred speech or
Muscle weakness typically presents from birth to early infancy. temporary locking of their jaw. Other signs and symptoms of
The salient features include hypotonia, muscle weakness, and myotonic dystrophy include clouding of the lens of the eye
reduced deep tendon reflexes, with or without joint contrac- (cataracts) and abnormalities of the electrical signals that
tures. Feeding and respiratory insufficiency are common due to control the heartbeat (cardiac conduction defects). In affected
associated bulbar and respiratory muscle involvement. men, hormonal changes may lead to early balding and an
Additional features may include microcephaly, eye anomalies, inability to father a child (infertility). The features of this
cerebral malformation, joint laxity, muscle atrophy or hyper- disorder often develop in the second to third decades,
trophy, adducted thumbs, and skin changes. While symptoms although they can occur at any age. The severity of the condi-
vary from mild to severe, the majority of people with congeni- tion varies widely among affected people, even among mem-
tal muscular dystrophy are unable to sit or stand without help. bers of the same family.
734 J. Wu et al.
Facioscapulohumeral Muscular Dystrophy features of this disorder are joint deformities called contrac-
Facioscapulohumeral muscular dystrophy (FSHD)—origi- tures. Contractures restrict the movement of certain joints,
nally named Landouzy-Dejerine—is a usually autosomal most often the elbows, ankles, and neck, and usually become
dominant inherited form of muscular dystrophy that initially noticeable in early childhood. Most affected individuals also
affects the skeletal muscles of the face (facio), scapula (sca- experience muscle weakness and wasting that worsen slowly
pulo), and upper arms (humeral). This type of muscular dys- over time, beginning in muscles of the upper arms and lower
trophy affects the muscles in the face, shoulders, and upper legs and later also affecting muscles in the shoulders and
arms. FSHD may lead to difficulty in chewing or swallow- hips. Most individuals with EDMD die in mid-adulthood
ing, slanted shoulders, a crooked appearance of the mouth, from heart or lung failure.
and a wing-like appearance of the shoulder blades. A smaller
number of people with FSHD may develop hearing and 41.3.2.2 Muscular Atrophy
respiratory problems. FSHD tends to progress slowly.
Symptoms usually appear during the teenage years, but Spinal Muscular Atrophy
sometimes may not become apparent until the 40s. Most Spinal muscular atrophy (SMA) is a genetic disorder charac-
people with this condition live a full life span. terized by weakness and wasting (atrophy) in muscles used
for movement (skeletal muscles). It is caused by a loss of
Limb-Girdle Muscular Dystrophy specialized nerve cells, called motor neurons, which control
Limb-girdle muscular dystrophy (LGMD) refers to a heteroge- muscle movement. The weakness tends to be more severe in
neous group of autosomal muscular dystrophies with progres- the muscles that are close to the center of the body (proxi-
sive weakness affecting predominantly the hip and shoulder mal) compared to muscles away from the body’s center (dis-
girdles. The facial and distal muscles are generally spared early tal). The muscle weakness usually worsens with age. Poor
in the disease. The disorders are labeled consecutively by the weight gain with growth failure, restrictive lung disease, sco-
alphabet according to when the individual genes were identi- liosis, joint contractures, and sleep difficulties are common
fied. LGMD affects both males and females. Most people with complications.
this form of muscular dystrophy are disabled by age 20.
However, many have a normal life expectancy. Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a progressive nervous
Oculopharyngeal Muscular Dystrophy system (neurological) disease that destroys nerve cells and
Oculopharyngeal muscular dystrophy (OPMD) is a genetic causes disability. In ALS, motor neurons die (atrophy) over
condition characterized by muscle weakness that begins in time, leading to muscle weakness, a loss of muscle mass, and
adulthood, typically after age 40. OPMD causes weakness in an inability to control movement. ALS often begins with
the facial, neck, and shoulder muscles. Other symptoms muscle twitching, weakness in a limb, or slurred speech.
include drooping eyelids, trouble swallowing, voice changes, Eventually, ALS affects control of the muscles needed to
vision problems, heart problems, and difficulty walking. move, speak, eat, and breathe. There is no cure for ALS, and
OPMD occurs in both men and women. Individuals usually the disease is ultimately fatal. ALS often starts in the hands,
receive a diagnosis in their 40s or 50s. feet, or limbs and then spreads to other parts of the body. As
the disease advances, more nerve cells are destroyed, and the
Distal Myopathy muscles progressively weaken. This eventually affects chew-
Distal myopathy (or distal muscular dystrophy) is a general ing, swallowing, speaking, and breathing. ALS does not usu-
term for a group of rare progressive genetic disorders charac- ally affect the bowel or bladder control, sensory functions, or
terized by wasting (atrophy) and weakness of the voluntary higher cognitions. Patients can remain actively engaged with
distal muscles. The distal muscles are away from the center their families and friends.
of the body and include muscles of the forearms, hands,
lower legs, and feet. Conversely, the proximal muscles are
close to the center of the body, such as muscles of the torso, 41.3.3 Diagnosis
shoulder, upper arms, hips, and thighs. Although the onset
can occur at any age from infancy to adulthood, most forms Patients with muscular dystrophy/muscular atrophy often
develop later in life and are slowly progressive. Inheritance present with a diverse spectrum of symptoms and features,
is autosomal dominant or recessive. part of which is suggestive of the underlying etiology. In
general, clinical biochemistry shows markedly elevated
Emery-Dreifuss Muscular Dystrophy serum levels of creatine kinase (CK). In addition, electromy-
Emery-Dreifuss muscular dystrophy (EDMD) is a condition ography, genetic testing, and muscle biopsy are used to
that primarily affects muscles used for movement (skeletal establish the diagnosis of muscular dystrophy with the
muscles) and the heart (cardiac muscle). Among the earliest abnormal pattern of electrophysiological signals and patho-
genic morphology of the muscle tissue. However, genetic WES is demonstrated to have a higher diagnostic yield for
testing is increasingly used to confirm the diagnosis. the molecular diagnosis of CMD. Also, the WES data allow
Muscular dystrophy and muscular atrophy are character- for the routine performance of CNV detection, further
ized by genetic heterogeneity and allele heterogeneity; thus, improving the performance of WES in the molecular diagno-
the diagnosis is usually established with the presence of sis of CMD.
pathogenic variants found in causative genes. With the wide
application of next-generation sequencing (NGS), targeted DM
gene panel and whole-exome sequencing (WES) are increas- Myotonic dystrophy (DM) is an autosomal dominant, multi-
ingly used in molecular diagnosis of Muscular dystrophy and system disorder characterized by myotonic myopathy with
muscular atrophy in clinical practice with different testing associated abnormalities in other organs. DM is clinically
algorithms. heterogeneous, with at least two types recognized in practice
so far: DM type 1 (DM1; Steinert disease), which can be
41.3.3.1 Muscular Dystrophy subdivided into four clinical subgroups and DM type 2
(DM2; proximal myotonic myopathy [PROMM] or Ricker
DMD and BMD syndrome). Both DM1 and DM2 are caused by unstable
DMD and BMD are caused by mutations in the dystrophin- DNA repeats in untranslated regions of different genes: a
encoding DMD gene. Usually, deletion/duplication analysis (CTG)n repeat in the 3′-UTR of DMPK gene and a (CCTG)
of DMD is performed first followed by sequence analysis if n repeat in intron 1 of CNBP (formerly ZNF9) gene, respec-
no pathogenic variant is found. Large deletions and duplica- tively [55].
tions are most common, but exonic deletion/duplication and The first step in the molecular analysis of DM1 or DM2 is
single nucleotide variation (SNV) have been found as well. to analyze whether an individual has two alleles with an
However, genome-wide chromosomal microarray analysis abnormal number of repeats that can easily be detected by
(CMA) is also used to detect DMD deletion/duplication in conventional PCR and gel electrophoresis analysis. If only
some laboratories. one allele size is determined using this method, subsequent
With significantly lowered cost of high throughput techniques such as repeat-primed PCR and Southern blotting
sequencing, targeted multigene panel sequencing and WES of genomic DNA or long-range PCR products are usually
are increasingly applied in clinical molecular diagnosis of used to detect possible repeat expansions.
DMD and BMD. In addition, WES-based copy number vari-
ation (CNV) prediction is increasingly used in clinical FSHD
molecular diagnosis of inherited diseases, including DMD FSHD is likely caused by inappropriate expression of the
and BMD. With the added values of CNV prediction and double homeobox-containing gene DUX4 in muscle cells.
detection of the absence of heterozygosity (AOH), which is DUX4 lies in the macrosatellite repeat D4Z4 on chromosome
implicated in the pathogenesis of some common inherited 4q35, which has a length between 11 and 100 repeats in nor-
diseases, WES is expected to become a first-tier diagnostic mal alleles. Approximately 95% of individuals with FSHD
test for patients with genetic disorders, such as DMD and have a D4Z4 allele of 1 ~ 10 repeats. The shortening of the
BMD, in the near future. D4Z4 allele causes chromatin relaxation at the D4Z4 locus
and the DUX4 promoter, thereby derepressing DUX4. This
CMD common form of FSHD is designated FSHD1. Molecular
Genetic testing is often required to establish or confirm the genetic testing measures the length of the D4Z4 allele. About
diagnosis of CMD and subtype classification. Traditionally, 5% of individuals with FSHD show chromatin relaxation at
the sequential molecular genetic testing strategy is frequently D4Z4 without having a D4Z4 contraction. In these individu-
applied in clinical practice. In some instances, the clinical als with the so-called FSHD2, pathogenic variants in the
examination may assist in prioritizing the order in which chromatin modifier SMCHD1 cause the chromatin relaxation
genes are tested [54]. at D4Z4. To confirm/establish the diagnosis of either FSHD1
Multigene panel testing by NGS is increasingly used to or FSHD2, physicians can follow the testing algorithm dem-
find the underlying genetic culprit for the diseases. However, onstrated in Fig. 41.4 [56].
there are considerable variations in genes included in differ-
ent panels and test performances between different laborato- LGMD
ries. In addition, with the accelerated discovery of novel LGMD is a clinically and genetically heterogeneous disease
CMD genes geared by population-scale genetic variants entity with different subtypes. LGMD1 includes several
database and other infrastructure, multigene panels must be forms of the disorder with an AD inheritance pattern.
periodically updated to cover all the potential disease genes, Mutations in LMNA gene cause LGMD1B. LGMD1C is a
thus negatively affecting its clinical application. group of muscle disorders called caveolinopathies caused by
736 J. Wu et al.
LGE or PFGE
EcoRI,(or EcoRI/HindIII)/ BlnI 1–10U 4q No XapI 1–10U 4q No D4Z4 probe
detected detected
p13E-11 probe
D4Z4 1–10U 4q
Low Methylation methylation No detected
Yes
Yes
Confirmation
Yes
FSHD
Yes
Linear Gel Electrophoresis (LGE), Pulsed Field Gel Electroporesis (PFGE)
Fig. 41.4 Southern Blotting for molecular diagnostic analysis of FSHD
mutations in CAV3 gene. LGMD2 includes different forms classification and differential diagnostic algorithms to
of the disorder with an AR inheritance. Calpainopathy, or increase the possibility of reaching a final genetic diagnosis
LGMD2A, is caused by mutations in CAPN3 gene. LGMD2A for the patient [58].
is the most common form of LGMD, accounting for about
30% of all cases. Dysferlinopathies, also called LGMD2B, EDMD
are caused by mutations in DYSF gene. Sarcoglycanopathies There are three major forms of EDMD with distinct clinical
are caused by mutations in SGCA, SGCB, SGCG, and SGCD presentations and pathophysiological features. X-linked
genes. These sarcoglycanopathies are known as LGMD2D, EDMD (XL-EDMD) is caused by disease-causing mutations
2E, 2C, and 2F, respectively. A TTN gene mutation causes in EMD and FHL1. AD-EDMD and AR-EDMD are associ-
LGMD2J, which has been identified only in the Finnish pop- ated with pathogenic variation in LMNA. For all forms of
ulation. Mutations in ANO5 gene cause LGMD2L. Mutations EDMD, the diagnosis is based on clinical findings and fam-
in several other genes cause distinct forms of LGMD called ily history. The diagnosis of X-linked EDMD also relies on
dystroglycanopathies, including LGMD2I, 2K, 2M, and 2N, failure to detect emerin or FHL1 protein in various tissues
respectively. There are also other rare forms of LGMD, and and molecular genetic testing of EMD or FHL1. The diagno-
the underlying molecular mechanism for some of them sis of AD-EDMD and AR-EDMD relies on molecular genetic
remains to be determined. testing of LMNA.
The single-gene testing approach could be used if the
OPMD condition to measure the expression level of emerin is avail-
OPMD is suspected based on clinical criteria, and the diag- able, in conjunction with an assessment of the inheritance
nosis is confirmed by molecular genetic testing of PABPN1. pattern. For example, if emerin is absent or reduced (muscle,
Confirmatory diagnosis depends on the detection of an skin, and lymphoblastoid cell lines), EMD should be tested
expansion of a GCN trinucleotide repeat in the first exon of first, whereas in the typical clinical presentation and X-linked
PABPN1. Normal alleles contain ten GCN trinucleotide inheritance, EMD should be analyzed first, followed by
repeats. Autosomal dominant alleles range in size from 12 to FHL1.
17 GCN repeats; autosomal recessive alleles comprise 11 A multigene panel including EMD, FHL1, LMNA, and
GCN repeats [57]. other genes of interest and WES may be considered when-
ever necessary.
Distal Myopathy
Advanced molecular diagnosis technologies have made it 41.3.3.2 Muscular Atrophy
possible to clarify and delineate an ever-growing number of
distinct new disease entities in the group of distal m
yopathies. SMA
Recent developments have increased the number of separate Patients with SMA have a loss of function involving both
diseases identified by molecular genetics to more than 20 SMN1 copies. Genetic testing for homozygous deletion will
different entities [58] (Table 41.6). For clinicians, this means confirm the disease in 95% of patients irrespective of disease
more demanding diagnostic challenges requiring an updated severity. Essentially, all other patients with SMN-related
Table 41.6 Distal myopathies with identified genetic cause Interpretation of the significance of an SOD1 variant regard-
Disease Gene ing the disease severity and progression depends on the spe-
1. Early-onset autosomal dominant cific variant identified because of the wide variability in
a. Laing distal myopathy (MPD1) MYH7 genotype-phenotype correlations. Failure to detect an SOD1
b. KLHL9-mutated distal myopathy KLHL9 variant does not rule out FALS.
c. POLG1-mutated distal myopathy POLG1 SETX testing is appropriate in kindreds with adolescent-
d. BICD2-mutated distal myopathy BICD2 onset SMA with pyramidal features. VAPB testing should be
2. Early onset autosomal recessive forms
performed in the context of clinical symptoms of primarily
a. Distal nebulin myopathy NEB
adult-onset SMA. FUS/TLS, TARDBP, and ANG testing
b. Distal nemaline myopathy NEM2
c. ADSSL1 distal myopathy ADSSL1 should be considered for SOD1-negative individuals with
3. Early adult-onset autosomal recessive FALS. ALS2 testing is appropriate for those with childhood-
a. Miyoshi myopathy DYSF onset upper motor neuron (UMN)-predominant ALS. VCP
b. Distal anoctaminopathy ANO5 testing should be considered for individuals with a family
c. Distal myopathy with rimmed vacuoles (GNE GNE history of ALS with or without symptoms of inclusion body
myopathy) myopathy, Paget’s disease, and frontotemporal dementia.
4. Adult-onset autosomal dominant OPTN testing may be considered for individuals with a fam-
a. Desminopathy DESM
ily history consistent with AD or AR inheritance, including
b. Distal ABD filaminopathy FLNC
c. Myopathy, distal, and Tateyama type CAV3
simplex cases that do not have a variant in other common
d. DNAJB6 distal myopathy DNAJB6 ALS-related genes.
e. HSPB8 distal myopathy HSPB8 Nowadays, more and more laboratories shift to another
5. Late adult-onset autosomal dominant approach in molecular diagnosis for patients with
a. Sequestosome1 (SQSTM1) distal myopathy SQSTM1 ALS. Multigene panel and WES are increasingly used to
b. Tibial muscular dystrophy (Udd myopathy) TTN delineate the underlying genetic defects for ALS. Of course,
c. VCP-mutated distal myopathy VCP it is necessary to update the multigene panel design along
d. ZASPopathy (Markesbery–Griggs) LDB3 with the discovery of more novel ALS-associated genes with
e. Matrin-3 distal myopathy (VCPDM, MPD2) MATR3
stringent validation and optimization. In addition, the impor-
f. Distal myotilinopathy TTID
tance of CNV prediction from WES data and validation by
g. Alpha-B crystallin-mutated distal myopathy CRYAB
h. Myopathy, myofibrillar, 3 MYOT
alternative techniques should not be underestimated in the
i. Welander distal myopathy TIA1 comprehensive molecular diagnosis of ALS.
SMA will be compound heterozygotes with a single SMN1 41.3.4 Correlation between Phenotype
deletion and a frameshift, nonsense, or missense mutation in and Genotype
the other SMN1 copy. Therefore, if homozygous SMN1 dele-
tion is not evident in a patient with suspected SMA, SMN1 41.3.4.1 Muscular Dystrophy
dosage analysis and sequencing of the remaining SMN1 gene
should be performed [59] (Fig. 41.5). DMD/BMD
When a pathogenic variant is identified, the diagnosis of a
ALS dystrophinopathy is established, but the distinction between
About 90–95% of ALS cases are sporadic. An estimated DMD and BMD can be difficult in some cases. For example,
5–10% of ALS is familial (FALS) and caused by mutations deletion of exons 3–7, the most extensively investigated
in one of the several genes. The pattern of inheritance varies deletion associated with both phenotypes, has been found in
between affected individuals depending on the genes males with DMD and BMD. The reading frame rule states
involved. ALS can be inherited in an AD, AR, or X-linked that pathogenic variants that do not alter the reading frame
pattern. Genetic counseling and risk assessment depend on (in-frame deletions/duplications) usually correlate with the
the accurate determination of the specific genetic diagnosis. milder BMD phenotype, whereas those that alter the reading
It is necessary to note that a perfect genetic testing strategy frame (out-of-frame) tend to correlate with the more severe
for ALS does not currently exist in clinical practice. DMD phenotype. Therefore, the type of deletion/duplication
SOD1 testing is appropriate in any individual with ALS can distinguish between the DMD and BMD phenotypes
who has another affected family member or an incomplete with 91–92% accuracy in young children who represent sim-
family history, including the early death of a close relative plex cases and in cases where a muscle biopsy is not needed
from any cause. Approximately 20% of individuals with to address the issue of BMD versus DMD. Although excep-
FALS have an identified pathogenic variant in SOD1. tions to the “reading frame rule” have been reported to occur
738 J. Wu et al.
Fig. 41.5 Approach to

molecular diagnosis of SMA
Clinical symptoms of suspected SMA:
weakness occurring predominantly at the
proximal muscles; decrease or loss of
reflexes; tongue fasciculations; limb
fremitus; other aspects
SMA diagnosis by molecular methods
SMN1 gene deletion test

Homozygous gene deletion mutation? YES
NO
5q SMA
NO SMN1 gene dosage test
Non-5q SMA
Heterozygous gene deletion mutation?
YES YES
NO
SMN1 gene sequencing
Sequence mutation?
at a rate below 10%, more recent studies suggest that this DM

may only hold true for the DMD phenotype, and the rate of Different subtypes of DM have different genotype-phenotype
exception may be higher with the BMD phenotype for both correlations. For patients with DM1, longer CTG repeat
deletions and duplications. Correlation of clinical features expansions correlate with an earlier age of onset and more
with genetic mutations is thus very important. In males with severe disease. Small but abnormal repeats (50–99) are often
DMD and BMD, phenotypes are best correlated with the associated with a mild or asymptomatic phenotype.
expression level of dystrophin, which is largely determined Nevertheless, for patients with DM2, no significant correla-
by the reading frames generated from the deleted allele. tion exists between CCTG repeat size and age of onset of
weakness or other measures of disease severity (e.g., age of
CMD cataract extraction). A correlation does exist between the
Most mutations resulting in typical complete laminin a2 repeat size and the age of the individual with DM2 at the
deficiency are functionally null mutations leading to the time that the repeat size is measured, indicating that the
absence of the laminin a2 protein on immunostaining and a repeat length increases with age.
more severe nonambulatory phenotype. Compound hetero-
zygosity for a null mutation and an in-frame deletion or exon FSHD
skipping mutations may lead to a milder phenotype with par- A correlation has been reported between the degree of the
tial deficiency of laminin a2. pathogenic contraction of the D4Z4 locus and the age of
In contrast, in-frame deletions affecting the N-terminal onset of symptoms, age at loss of ambulation, and muscle
G-domain, critical for binding of laminin isoforms to strength, particularly in affected females. Individuals with a
a-dystroglycan and various integrins, affect the function of large contraction of the D4Z4 locus tend to have an earlier
this molecule profoundly, leading to a severe phenotype, onset of the disease and more rapid progression than those
even though laminin a2 may be partially present in the base- with smaller contractions of the D4Z4 locus.
ment membrane by immunohistological staining.
Rare homozygous missense mutations have been associ- OPMD
ated with laminin a2 deficiency. Affected siblings may dem- The mean age at diagnosis and the severity of the clinical
onstrate intrafamilial variability of the onset and severity of symptoms correlate with the number of PABPN1 (GCN)
clinical manifestations and the degree of laminin a2 defi- repeats. Homozygous patients showed the worse phenotype,
ciency observed on muscle biopsy immunostaining. suggesting a gene–dose effect in addition to the repeat num-
ber expansion. Altogether, this suggests that the determina- severity of the disease, that is, the homozygous exon 7 dele-
tion of the size of the triplet expansion is an important test to tion is observed with the same frequency in all phenotypes.
perform in patients with OPMD, notably in aged patients It seems that a large deletion involving neighboring genes
with bulbar signs, after exclusion of autoimmune myasthenia such as NAIP and SERF1A causes the severe phenotypes of
gravis and amyotrophic lateral sclerosis. SMA. Also, PLS3 gene, located on chromosome Xq23, can
play a role as a positive modifier of the SMA phenotype.
Distal Myopathy
There is some genotype-phenotype correlation. The first ALS
families identified with distal myopathy had missense muta- Age of onset of ALS1 is poorly correlated with genotype;
tions on both chromosomes, while the severe nemaline intrafamilial variability can be extensive in patients with
myopathy is caused by more disruptive nebulin mutations. SOD1 pathogenic variants. Clinical presentation can occa-
Recently, the distal myopathy phenotype has also been asso- sionally be correlated with SOD1 pathogenic variants.
ciated with nonsense mutations on one allele that cause the Symptomatic patients with p.Ala4Val and p.Val148 variants
nemaline myopathology. often have few UMN findings. Reduced penetrance has been
documented, most notably with the p.Ile113Thr and p.
EDMD Asp90Ala pathogenic variants.
The majority of EMD pathogenic variants are null variants Pathogenic variants in FUS have recently been identified
that result in a complete absence of emerin expression in in approximately 4% of SOD1-negative FALS cases. The
nuclei; however, intra- and interfamilial variability in the most common variants are p.Arg521Cys and p.Arg521Gly,
severity of the phenotype associated with null variants has which are characterized by diffuse ubiquitin positivity, indi-
been observed. The few missense variants that have been cating cytoplasmic retention and aggregation of the mutated
identified are associated with decreased or normal amounts protein. Individuals with FUS pathogenic variants show pre-
of emerin and result in a milder presentation. dominantly lower motor neuron disease.
LMNA pathogenic variants do not show a clear genotype- In ALS10, a spinal onset is reported in 77% of individuals
phenotype correlation in EDMD. Individuals with early harboring TARDBP pathogenic variants, and lower motor
skeletal muscle involvement frequently have pathogenic neuron-predominant disease is seen in 39%.

missense variants, whereas those with later-onset muscle Classic ALS patients with noncoding GGGGCC hexanu-
symptoms often have frameshifts, presumably leading to cleotide repeat expansion in C9orf72 commonly present
truncated protein or nonsense-mediated decay. Marked intra- with progressive muscular atrophy without upper motor neu-
and interfamilial variability is observed for the same LMNA ron signs.
pathogenic variant.
Homozygous and compound heterozygous LMNA patho-
genic variants appear to lead to a more severe muscular phe- 41.3.5 Genetic Counseling
notype. Three of the five reported individuals with
homozygous LMNA pathogenic variants lost ambulation Individuals with a family history of a neuromuscular condi-
within the third to fifth decades of life. tion often have many concerns when contemplating parent-
Severe EDMD has been reported in individuals with hood. Genetic counseling can help patients and their families
pathogenic variants in both genes. A range of clinical presen- understand their conditions (about the inheritance, chance of
tations (i.e., CMT2, CMT2-EDMD, and isolated cardiomy- recurrence, and family planning options, etc.) and adapt to
opathy) was found in a large family in which pathogenic the medical and psychosocial implications.
variants in EMD and LMNA co-segregate. In the case of pregnancy, some prenatal tests are carried
out during pregnancy to screen the fetus for common abnor-
41.3.4.2 Muscular Atrophy malities and may be ordered to investigate for specific condi-
tions. The tests, however, are only available for some
SMA neuromuscular conditions.
All types of SMA result from mutations in the SMN1 gene.
The significant variations in the age of onset and the severity
of clinical symptoms are caused by modifier genes. Since a 41.3.6 Prenatal Diagnosis
full-length product of SMN1 is necessary for lower motor and Preimplantation Genetic Diagnosis
neuron function, the loss of SMN1 is essential for the patho-
genesis of SMA, and the SMN2 copy number variation modi- Due to the high genetic and allelic heterogeneity in MD, pre-
fies the severity of phenotype. The presence of three or more natal diagnosis of MD should be made with caution.
copies of SMN2 is correlated with a milder phenotype. No Technically, multiple analytic methods could be used, such
correlation exists between the loss of SMN1 exon 7 and the as Sanger sequencing, MLPA, CMA, etc. Recently, WES has
740 J. Wu et al.
been increasingly applied for this purpose. However, prena- and disease expression, and high genetic heterogeneity.
tal diagnosis can only be carried out if there is a precise According to the underlying mechanism of diseases, epi-
genetic diagnosis of the family’s condition. A normal test lepsy could be classified into different types, including auto-
result does not guarantee that the baby will be healthy. The somal dominant nocturnal frontal lobe epilepsy, autosomal
test only looks for the particular type of MD in the family but dominant epilepsy with auditory features, SCN1A seizure
not all other possible problems. disorders, etc. Genetic information is essential for the diag-
Newer tests are being developed and can test for the free nosis, prognosis, and management of epilepsy. In this chap-
fetal DNA (ffDNA) in a blood sample from the mother. This ter, clinical characteristics, diagnosis (esp., molecular
is known as noninvasive prenatal diagnosis (NIPD). NIPD is diagnosis), the correlation between phenotype and genotype,
currently used to determine the sex of a fetus when it is med- genetic counseling, prenatal diagnosis and preimplantation
ically important to know this as well as its Rhesus blood genetic diagnosis, and a classic clinical case are discussed.
group. It is hoped that NIPD will soon be able to diagnose
conditions such as DMD.
For couples at risk of having a child affected by MD, 41.4.1 Overview
another possible option is to perform preimplantation genetic
screening (PGS) or preimplantation genetic diagnosis Seizures are typically paroxysmal and episodic. Caused by
(PGD). While PGD has the advantage of avoiding the termi- abnormally excessive cortical neuronal activities, seizures
nation of fetuses affected by the condition, it also has a num-
result in sudden onset and transient behavioral, somatosen-
ber of drawbacks. These include the modest success rate of sory, motor, or visual symptoms and signs. Seizures can
becoming pregnant after IVF and the substantial social, occur spontaneously or may be provoked by certain factors
financial, and emotional burdens of the combined IVF and (e.g., trauma, brain hemorrhage, toxic-metabolic distur-
PGD process. Apart from couples who need IVF so they can bances, or drug exposures). Some individuals may have
conceive a child, the number of people using PGD is small. recurrent provoked seizures without having epilepsy, sug-
gesting the presence of an underlying heightened tendency
toward spontaneous recurrent seizures. Provoked seizures do
41.3.7 Typical Clinical Case not recur when provoking factors are altered or corrected.
In 2014, the International League Against Epilepsy
A 4-year-old boy was referred for his walking difficulty. On (ILAE) adopted a new practical definition for epilepsy as a
physical examination, he was found to have several bean- disease with either recurrent unprovoked seizures (i.e., two
sized lymph nodes in his neck and a positive Gowers’ sign. or more unprovoked seizures occurring at least 24 h apart) or
Biochemistry showed that his serum concentration of CK a heightened tendency toward recurrent unprovoked seizures
was significantly elevated (18,433 μ/L), the liver amino- (i.e., a single seizure, accompanied by evidence from clini-
transferases were moderately increased (AST 555 μ/L, ALT cal, electroencephalographic, or neuroimaging tests that a
504 μ/L), while serum levels of myoglobin and lactate dehy- heightened risk [at least 60%] exists for future seizures over
drogenase (LDH) were within normal ranges. the next 10 years), or when an epilepsy syndrome is diag-
A sequential strategy was adopted for the molecular diagnosed. This new definition recognizes the practical impor-
nosis of this case. At first, DMD gross deletion/duplication tance of treating some patients after a single seizure, since
was excluded with the MLPA kit. Whole-exome sequencing they may also experience the consequences of epilepsy, such
(WES) was then performed using an Agilent SureSelect as lower quality of life, loss of psychosocial functioning, and
Human All Exon V6 kit and Illumina sequencing platform. injury.
After standard bioinformatic processing and proband-only The principal clinical symptoms and signs of epilepsy
variant filtering strategy, the patient was found to harbor a include ictal (during a seizure), postictal (immediately fol-
hemizygous c.6986dupA variant in the DMD gene, which lowing seizure termination), and interictal (between seizure
was categorized as “pathogenic” according to ACMG stan- episodes) manifestations. Behavioral alterations accompany-
dard and validated by Sanger sequencing (Fig. 41.6). ing epileptic seizures are diverse, ranging from subjective
This case was from Shanghai Children’s Medical Center. feelings reported by the patient to objectively witnessed
behavioral arrests, unresponsiveness, or involuntary move-
ments. The nature of the ictal behavioral disturbance depends
41.4 Epilepsy upon the location of seizure onset in the brain and its rate and
pattern of propagation involving neuronal networks in neigh-
Zhaojing Zheng and Juan Geng boring or distant brain regions [60].
Depending on seizure origin in the brain, epilepsies are
Epilepsy is a complex group of diseases characterized by classified into generalized and focal epilepsies (Fig. 41.7).
diverse clinical features and symptoms, variable penetrance Generalized epilepsies consist of abnormal brain activity
Fig. 41.6 Classic case of DMD due to DMD pathogenic variant grated genome visualization (IGV) browser; the lower row shows the
c.6986dupA. The upper row indicates the reads occurring c.6986dupA chromatograph of PKD1 c.6986dupA by Sanger sequencing
variation in the DMD gene through whole-exome sequencing with inte-
involving both hemispheres at the onset, whereas focal epi- ized epilepsy may have tonic-clonic seizures, absence sei-
lepsies involve seizures with a localized origin (that may zures, myoclonic seizures, or atonic seizures. Persons with a
then spread). Determining a patient’s particular type of epi- focal epilepsy may have focal aware seizures, focal seizures
lepsy is important, because antiepileptic drug (AED) choice with impaired awareness, or focal seizures that spread and
is directed in part by epilepsy type. Persons with a general- become tonic-clonic seizures [61].
742 J. Wu et al.
Fig. 41.7 Operational
classification of seizures Seizure
Focal onset Generalized Onset Unknown Onset
Aware Motor
Motor
Tonic-clonic
Tonic-clonic
Epileptic spasms
Moto Onset Clonic
Nonmotor
Automatisms Tonic
Behavior arrest
Atonic Myoclonic
Clonic Myoclnoc-tonic-clonic
Myoclonic-atonic Unclassified
Epileptic spasms
Hyperkinetic Atonic
Myoclonic Epileptic spasms
Tonic Nonmotor (absence)
Nonmotor Onset Typical
Autonomic Atypical
Behavior arrest Myoclonic
Cognitive Eyelid myoclonia
Emotional
Sensory
Focsal to bilateral tonic-clonic
41.4.2 Diagnosis cause of inherited syndromes associated with epilepsy [62].

In some circumstances, brain magnetic resonance spectros-
The most common etiologies of epilepsy vary across the copy may identify metabolic abnormalities even when the
lifespan. In children, genetic predisposition, congenital mal- MRI is normal.
formations, and stroke are the most common causes. Genetic testing has the highest likelihood of providing
Traumatic brain injury, infection, scarring, and tumors unambiguous information for subjects with epilepsy who
become significant causes in young adults. In older adults, have: (1) family history of epilepsy; (2) dysmorphic features,
stroke, neurodegenerative disease, and cerebrovascular dis- cognitive impairment, or nonacquired neuroimaging abnor-
ease are the most common causes [62, 63]. mality; or (3) one of the following syndromes: epileptic
Electroencephalography (EEG) is a basic diagnostic tool encephalopathy, myoclonic seizures, and febrile convul-
for assessment of interictal cortical function and classifica- sions. Such individuals are most likely to harbor a disease-
tion of epilepsy, which in turn provides valuable information causing mutation in an epilepsy-associated gene.
for the further investigation of potential genetic (and nonge- Nonetheless, it is important to note that, with the increasing
netic) causes. Epileptic discharges are often affected by the application of more comprehensive testing (e.g., moving
sleep cycle and circadian rhythms. Therefore, a 24-h or lon- from karyotype to chromosome microarray analysis [CMA]
ger recording of EEG capturing the natural sleep state may and from single gene testing to whole exome sequencing
provide greater detection of epileptic discharges than a rou- [WES]/whole genome sequencing [WGS]), pathogenic
tine short-term EEG. In general, EEG findings correlate genetic variations are more efficiently identified in individu-
more closely with the types of seizures than with the under- als who do not conform to any of these syndromes. These
lying causes. Nonetheless, some EEG patterns suggest spe- include subjects with apparently sporadic, nonsyndromic,
cific genetic epilepsy. generalized, and focal epilepsy. Using a combination of
Beyond the value in excluding nongenetic disorders, these methods, typically sequentially, overcomes the limita-
brain MRI may provide insights into the classification and tions of individual approaches (Table 41.7) [63, 64].
Table 41.7 Clinically available genetic testing modalities in epilepsy

Test Indication Scope Advantage Disadvantage
Single-gene Syndromes usually associated with Targeted Faster and less expensive than Not all commercially
testing specific gene(s) gene-panel testing available tests include
deletion and duplication
testing as well as sequencing
Gene-panel Syndrome associated with several Semitargeted If several genes may be candidates Not all commercially
testing epilepsy-related genes or syndrome not for a syndrome, this option is available tests include
clearly related to specific gene more time-efficient than serial deletion and duplication
single-gene testing and faster than testing as well as
WES and WGS sequencing; Expensive
WES Suspected genetic disease without a Untargeted Currently, difficult to derive copy Incidental findings;
specific test available; number information from WES Expensive;
Disorders with genetic heterogeneity data. Coverage of specific genes
that would require sequencing of Not guaranteed because of
multiple genes simultaneously; Limitations in capture
Genetic disorder is suspected, but technology
specific genetic tests are negative
Special prenatal circumstances
WGS Epilepsy syndrome without specific Untargeted Also enables evaluation for copy Incidental findings;
gene associations number abnormalities Expensive;
Chromosome
rearrangements and
tissue-restricted somatic
mosaicism are reluctant to
WGS
CMA Drug-resistant epilepsy, especially with Targeted and May provide information Rarely, can miss a
intellectual disability and/or autism. untargeted homozygosity in consanguineous chromosomal rearrangement
Suspected chromosomal microdeletion individuals, thereby possibly such as a ring chromosome
or duplication syndrome suggesting specific genes. abnormality
Relatively fast (few weeks)
Karyotyping Suspected monosomy, trisomy, or Untargeted Fast (few days); can detect Lower resolution than CMA
chromosomal rearrangement; maternal aneuploidy and balanced
history of recurrent pregnancy losses; translocations not detectable by
generally recommended if strong CGH
suspicion and CMA result is negative
41.4.3 Genetic Testing Algorithm that might be associated with the onset of seizures, e.g., trau-
matic brain injury, encephalitis, vasculitis, hypoxia, abscess,
Genetic testing and clinical biochemistry will provide neoplasm, metabolic disturbances [including those condi-
insights into the cause, treatment, and prognosis for every tions resulting in hypoglycemia, hyponatremia, and amino-
patient with epilepsy. Indeed, clinical research underway acidopathies], and drugs [including alcohol] withdrawal or
includes WGS for unselected patients with epilepsy, includ- toxicity), through disease and family history, imaging, and
ing those with and without a family history of similar disor- laboratory analysis.
ders. At present, however, the expense and frequent lack of
insurance reimbursement, often further complicated by 41.4.3.2 Family History
ambiguous results (e.g., variations of unknown significance), It is essential to review the family history in detail, specifi-
makes it important to select subjects for whom genetic anal- cally inquiring about other family members with the same
ysis is most likely to yield clinically useful information syndrome, other seizure disorders, or other neurologic dis-
(Fig. 41.8) [63]. eases. A first-degree relative with a similar disorder suggests
a single-gene disorder for which genetic testing has the high-
41.4.3.1 Exclude Nongenetic Disorders est likelihood of informative results. The mode of inheritance
Acquired brain injury as the primary (or contributing) cause (AD, AR, X-linked, or maternal transmission) refines the
of seizures should be excluded from the cascading investiga- candidate list of epilepsy syndromes and causative genes. A
tion procedures for potential genetic factors that cause or family history of disparate seizure syndromes suggests that
contribute to epilepsy. This is true even for individuals who either the gene mutation results in variable clinical presenta-
have a family history of epilepsy. During the diagnostic tions (variable expressivity) or that family members do not
workup for epilepsy, it is also necessary to exclude factors share the same cause and type of epilepsy.
744 J. Wu et al.
Medical history
Physical and neurologic examination
Routine laboratory testing
Electroencephalo-graph
Neuroimaging examination
Malformed features, Classification of syndromes

inherent abnormalities, or Myoclonic seizures, Febrile
Abnormal neuroimaging
history of infertility and seizures, Epileptic
miscarriage encephalopathy, etc
Abnormal structure (except for Whether syndromes are in

abscess or neoplasm) Karyotype
Leukodystrophy accord with congenital
Tuberous sclerosis, Chromosome microarray
malformations and so on disorder
Yes No
Specific biochemical, 1.Chromosome microarray analysis

Specific gene or syndrome-
Laboratory examination enzyme and gene 2.Analysis of epilepsy-related genes
specific gene testing
examination 3.Whole exome or genome sequencing
Detection of specific
enzymes of genes
Fig. 41.8 Approach to genetic evaluation in subjects with epilepsy
41.4.3.3 G eneral Physical and Neurologic (15p-), 1p36 monosomy, Wolf–Hirschhorn syndrome, ring
Evaluations 20 chromosome syndrome, Miller–Dieker syndrome, or
It is important to assess early developmental growth, head 18p-syndrome. CNV-associated disorders should also be
circumference and shape, vision and hearing, general health, taken into consideration when the patient is observed to have
and the occurrence of dysmorphic features. Epilepsy syn- multiple dysmorphisms. Certainly, features or symptoms of
dromes associated with intellectual disabilities and dementia tuberous sclerosis (facial or subungual angiolipomas and
are too numerous to review in this context. Intellect should hypopigmented macules) would prompt imaging and
be evaluated carefully in all subjects to determine if the cog- disease-specific evaluation. Cleft lip or cleft palate in an indi-
nition is affected by: (1) the underlying cause of epilepsy, (2) vidual with epilepsy whose mother also has epilepsy could
the direct consequences of frequent seizures. or (3) medica- be part of a genetic syndrome or potentially attributable to
tion. It is important to consider the possibility that cognitive anticonvulsant toxicity [67].
impairment and dysmorphic features in children of mothers
with epilepsy could be due to fetal anticonvulsant toxicity 41.4.3.4 Inherited Metabolic Disorders
rather than a manifestation of the spectrum of the parent’s Inherited metabolic disorders are those genetic disorders for
seizure disorder. The possibility of identifying a causative which biochemical abnormalities are detectable by analytic
gene mutation is higher in epilepsy subjects who have devel- methods performed in clinical laboratories. The distinction
opmental delay, cognitive impairment, and particularly between inherited metabolic disorders causing epilepsy and
dementia compared to subjects who do not have cognitive other genetic causes of epilepsy is useful in the sense that
impairment [65, 66]. some inherited disorders have clinically measurable biomark-
The possibility that epilepsy is a symptom of abnormal ers that can be used for diagnosis and sometimes as therapeu-
brain development involving the cortex and subcortical tic monitoring parameters. Inherited metabolic disorders are
structures should be considered if dysmorphic features are particularly important in the differential diagnosis of newborn
present in the patient. This could be due to specific gene and infantile-onset epilepsy because many have specific treat-
mutation or more commonly to chromosome abnormality ments or interventions. Cerebrospinal fluid (CSF) examina-
such as trisomy 21 (Down syndrome), cri-du-chat syndrome tion may be recommended in the course of excluding
nongenetic causes of refractory epilepsy and may be useful Familial epilepsies include those in whom transmission
for subjects who remain undiagnosed. CSF examination may occurs in AD or AR mode. However, many familial types of
identify cerebral folate deficiency [68], neurotransmitter epilepsy are characterized by multiple affected family mem-
abnormalities suggestive of pyridoxine-dependent epilepsy, bers but without a clear inheritance pattern and may not eas-
[69], and lactate and pyruvate elevation consistent with mito- ily be grouped into one of these categories.
chondrial disturbance. For AD transmission, a mutation in one of the two alleles
is sufficient to cause disease. This is the case in many of the
known familial epilepsy syndromes such as genetic/general-
41.4.4 Correlation between Phenotype ized epilepsy with febrile seizures plus (GEFS+) due to
and Genotype mutations in SCN1A or SCN1B; autosomal dominant noctur-
nal frontal lobe epilepsy (ADNFLE) due to mutations in
Genetic risk factors predisposing to epilepsies may be dif- CHRNA4, CHRNB2, or CHRNA2; or the benign familial
ferent in how they associate with a given phenotype. For neonatal and infantile epilepsies due to mutations in KCNQ2,
some genes, the connection is very strong. For example, a KCNQ3, SCN2A, or PRRT2.
disruptive mutation in the SCN1A gene has a very high like- Recessive epilepsies are caused by mutations in both
lihood of Dravet syndrome, a condition in which the pheno- alleles of a gene. These alleles can be affected through homo-
type is well defined, and the clinical course is relatively zygous mutations or through compound heterozygous muta-
consistent among patients. For other genes, the correlation tions. Recessive epilepsies are usually severe disorders, and
between phenotype and genotype may not be consistent and prominent examples are the progressive myoclonus epilep-
straightforward. For example, mutations in STXBP1 were sies such as Unverricht-Lundborg disease or Lafora disease
first identified in patients with Ohtahara syndrome, while and many neurometabolic disorders. In many cases, the
some mutations in STXBP1 can also cause a broader range recessive epilepsy is secondary to a primary storage or meta-
of phenotypes, including intellectual disability without bolic defect.
seizures. However, disease-causing mutations can also be transmit-
Epilepsy-associated CNVs also have a wide phenotypic ted through the X chromosome. For X-linked epilepsies,
spectrum that includes autism spectrum disorder (ASD), they can either be dominant (XLD), affecting mainly girls
intellectual disability, and schizophrenia. In many cases, (e.g., CDKL5) or recessive (XLR), affecting primarily boys
however, the phenotypic spectrum of particular genes is (e.g., ARX gene).
not fully established. Notably, the phenotypes associated Disease-causing mitochondrial mutations are associated
with specific genes may be specific for mutations in par- with a subset of patients with epilepsy. Mitochondrial muta-
ticular domains or even single base pairs. For example, a tions are transmitted maternally [71].
triplet repeat expansion in ARX gene is associated with If the type of epilepsy is inherited in an AD manner,
infantile spasms without brain malformations, while other such as autosomal dominant nocturnal frontal lobe epi-
mutations may result in brain malformations or X-linked lepsy (ADNFLE), most individuals have an affected par-
intellectual disability without seizures. In addition, f ent. The proportion of cases caused by de novo pathogenic
SCN2A truncation mutations are shown to result in intel- variants is unknown, as the frequency of subtle signs of the
lectual disability, whereas epileptic encephalopathies disorder in parents has not been thoroughly evaluated, and
appear to be exclusively correlated with missense muta- molecular genetic data are insufficient. Penetrance is esti-
tions [70]. mated at 70%, and the risk to each offspring of inheriting
the pathogenic variant is 50%; thus, the chance that the
offspring will manifest ADNFLE is (50% × 70%) 35%.
41.4.5 Genetic Counseling Prenatal testing for pregnancies at increased risk of the
disorder is possible.
Genetic counseling is essential to ensure that the affected Lafora disease is a subtype of epilepsy of AR inheritance.
individuals with epilepsy and their families can assess the Heterozygotes (carriers) are asymptomatic and not at risk of
recurrence risk, prognosis, and family planning under developing the disorder. At conception, each sib of an
informed consent. The focus of genetic counseling might dif- affected individual has a 25% chance of being affected, a
fer depending on whether it involves a teenager or adult with 50% chance of being an asymptomatic carrier, and a 25%
epilepsy, the parents of a young child with epilepsy, or an chance of being unaffected and not a carrier. Carrier testing
at-risk family member. The approach to counseling should, for at-risk relatives, prenatal diagnosis for at-risk pregnan-
therefore, be tailored to the individual, but several crucial cies, and preimplantation genetic diagnosis are possible if
elements of genetic counseling should always be included. the pathogenic variants in the family are known.
746 J. Wu et al.
For example, the advice given to the family of a young prenatal testing to be the choice of the parents, discussion of
girl with epilepsy and intellectual disability with a PCDH19 these issues is appropriate.
mutation and that given to the family of a child with Dravet For example, the STXBP1 pathogenic variant has been
syndrome resulting from an SCN1A mutation can be mark- identified in an affected family member, and prenatal testing
edly different. PCDH19 mutations can be inherited in XLD, for a pregnancy at increased risk and preimplantation genetic
such that male carriers transmit the mutant allele only to diagnosis are possible.
their daughters. Therefore, PCDH19-associated epilepsy is
limited to female patients, and the degree of intellectual dis-
ability is variable due to X chromosome inactivation. Dravet 41.4.7 Typical Clinical Case
syndrome is usually caused by a de novo mutation, but it is
critically important to note the hidden risk of recurrence A 22-month-old boy was referred for persistent seizures. The
because Dravet syndrome can be secondary to a low-level boy was reported to have his first febrile seizure at 9 months
parental mosaicism in a minority of patients [72–75]. after birth. The seizures recurred thereafter more than 10
times, and the last three occurred when the boy was afebrile.
The spasm duration was typically within 3–5 min.
41.4.6 Prenatal Diagnosis Whole exome sequencing was performed using an Agilent
and Preimplantation Genetic Diagnosis SureSelect Human All Exon V6 kit and Illumina sequencing
platform. After standard bioinformatic processing and proband-
Differences in perspective may exist among medical profes- only variant filtering strategy, the patient was found to harbor a
sionals and within families regarding the use of prenatal test- de novo heterozygous c.301C > T variant in SCN1A gene,
ing, particularly if the testing is being considered for the which was categorized as “pathogenic” according to ACMG
purpose of pregnancy termination rather than early diagno- standard and validated by Sanger sequencing (Fig. 41.9).
sis. While most centers would consider decisions regarding This case was from Shanghai Children’s Medical Center.
Fig. 41.9 Classic case of epilepsy due to SCN1A pathogenic variant grated genome visualization (IGV) browser; the lower row shows the
c.301C > T. The upper row indicates the reads occurring c.301C > T chromatograph of SCN1A c.301C > T by Sanger sequencing
variation in SCN1A gene through whole exome sequencing with inte-
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Reproductive Organ Cancer
42
Jinhai Tang, Xiangjun Cheng, Jieshi Xie, Zheng Cao,
Yanhong Zhai, and Boyan Song
42.1 Breast Cancer dence of cervical cancer [2]). The incidence of breast cancer
is the highest in countries such as Australia/New Zealand,
Jinhai Tang and Xiangjun Cheng Northern Europe (e.g., UK, Sweden, Finland, and Denmark),
Western Europe (Belgium, Netherlands, and France),
Southern Europe (Italy), and North America [2].
42.1.1 Overview About 5–10% of breast cancer cases are related to heredi-
tary and genetic factors, including a personal or family his-
Breast cancer is the most commonly diagnosed cancer in tory of breast or ovarian cancer, and genetic mutations
women and the leading cause of cancer death. About one (BRCA1, BRCA2, and other genes susceptible to breast can-
million new cases are diagnosed each year in China [1]. cer). Studies on immigration have shown that nonhereditary
According to the latest data released by the World Health factors are the main driver of observed differences in interna-
Organization in 2017, the number of breast cancer deaths in tional and interethnic incidence. A comparison of migrating
China reached 49,011, accounting for 0.52% of the total low-risk populations to high-risk populations shows that the
deaths. The age-adjusted mortality rate is 5.76 deaths per incidence of breast cancer has increased for successive
100,000 people, ranking China 179 globally. Worldwide, generations.
there will be an estimated 2.1 million newly diagnosed breast However, it is limited to understanding geographic or
cancer cases for women in 2018, accounting for a quarter of temporal changes in the incidence associated with a specific
all cancers in women. And there will be approximately 0.63 cause. Over the past few decades, the incidence of breast
million breast cancer death in 2018 [2]. Recently, the inci- cancer has been rising in most countries in transition, such as
dence of this cancer is increasing in developing countries. South America, Africa, and Asia. These trends may reflect a
Breast cancer is the most frequently diagnosed cancer in combination of demographic factors related to social and
most countries (154 of 185 countries) and is also the leading economic development, including delayed births and fewer
cause of cancer deaths in more than 100 countries; the main children, higher levels of obesity and lack of physical activ-
exceptions are Australia/New Zealand, Northern Europe, ity, and increased breast cancer screening and awareness. In
North America (where lung cancer is the top one), and many some developed countries, including the United States,
countries in sub-Saharan Africa (due to the increased inci- Canada, the United Kingdom, France, and Australia, the
decline of incidence in the early 2000s was partly due to the
reduced use of postmenopausal hormone therapy after the
J. Tang (*) · publication of the Women’s Health Initiative trial link
Department of General Surgery, The First Affiliated Hospital
Posthormonal use to increase the risk of breast cancer [3].
People’s Republic of China The main risk factors for breast cancer are not easily changed
e-mail: jhtang@njmu.edu.cn because they result from long-term endogenous hormone
X. Cheng exposure; although prevention by promoting breastfeeding,
Department of Laboratory Medicine, The First Affiliated especially for longer durations, may be beneficial.
Hospital of Nanjing Medical University, Nanjing, Jiangsu,
J. Xie · Z. Cao (*) · Y. Zhai (*) · B. Song
Department of Laboratory Medicine, Beijing Obstetrics and
Gynecology Hospital, Capital Medical University, Beijing,

752 J. Tang et al.
Fig. 42.1 Anatomy of the

breast Sebaceous
gland
Lactiferous Montgomery
Second rib tubercle
duct
Pectoralis major
muscle
Pectoralis minor
muscle
Intercostal muscles
Retromammary fat
Areola
Nipple
Lobes of Lactiferous ducts

mammary gland
Intraglandular fat
Subcutaneous fat
Suspensory (Cooper’s)
ligaments
42.1.2 Anatomy of the Breast 42.1.3 Histological Classification
The breasts are modified skin gland that are located at the The classification of breast tumor was confirmed by the World
front of the chest and also partially outside the chest. The Health Organization (Table 42.1). All types of breast cancer are
upper part of each breast extends to the second rib, the lower classified based on their histological and/or cytological appear-
part extends to the sixth rib cartilage, the middle extends to ance. Regardless of the type of cancer, some general findings
the sternum, and the lateral extends to the midaxillary line. It should be recorded, including the location, size, shape, consis-
illustrates the lobular system of the breast and the anatomic tency, color, overall appearance of the margins, and the adja-
location of some common pathological changes (Fig. 42.1). cent breasts (skin, nipples) and extramammary structures
The catheter system contains numerous leaflets with acinar (fascia, muscle), and the number of malignant lesions.
cells. Each leaflet is fed into a terminal catheter, which in
turn is fed into a segmented catheter. The segmented catheter
eventually enters the collection catheter, where about 15–20 42.1.4 Surveillance and Diagnosis
converge under the areola through a separate orifices and
reach the nipple surface. 42.1.4.1 Imaging Technologies
The three most common causes of female breast mass are and Applications in Early Diagnosis
cysts, fibroadenomas, and cancer. and Prognosis for Breast Cancer
Cysts and fibroadenomas come from lobule lesions, There is growing interest in developing imaging tests to
and carcinomas developed in the terminal ducts. Nipple screen for breast cancer, especially in high-risk groups where
adenomas also come from segmental ducts, near the open- traditional techniques are inadequate. Mammography is con-
ings in the nipple. Paget’s disease of the breast is the shed- sidered to be the most effective method for early detection of
ding of skin in the nipple-areola complex. This usually breast cancer. The limitations of this approach are the impe-
indicates the presence of potential breast cancer. Ductal tus for efforts to improve existing mammography techniques
carcinoma in situ and invasive carcinoma each account for and develop new technologies that improve breast cancer
about half. detection capabilities. Ultrasound is expected to be a method
42 Reproductive Organ Cancer 753
Table 42.1 Histological classification of carcinoma of breast [adapted Table 42.2 BI-RADS mammography categories according to the
from WHO] American College of Radiology
Invasive ductal carcinoma, not otherwise specified (NOS) 8500/3 BI-RADS Assessment categories
Mixed type carcinoma Category 0 Need additional imaging evaluation
Pleomorphic carcinoma 8022/3 Category 1 Negative. Keep screening
Carcinoma with osteoclastic giant cells 8035/3 Category 2 Benign finding. Keep screening
Invasive lobular carcinoma 8520/3 Category 3 Probably benign finding. Short interval of follow-up
Tubular carcinoma 8211/3 is suggested
Invasive cribriform carcinoma 8201/3 Category 4 Suspicious abnormality. Biopsy should be
Medullary carcinoma 8510/3 considered
Mucinous carcinoma and other tumours with abundant Category 5 Highly suggestive of malignancy.
mucin Appropriate action should be taken
Mucinous carcinoma 8480/3
Cystadenocarcinoma and columnar cell mucinous 8480/3
carcinoma formed by women when they are asymptomatic, which
Signet ring cell carcinoma 8490/3
means they are in the preclinical stage. This is the purpose of
Invasive papillary carcinoma 8503/3
screening mammograms, and the goal is to be able to detect
Invasive micropapillary carcinoma 8507/3
Apocrine carcinoma 8401/3
any abnormalities with higher sensitivity (Table 42.2).
Metaplastic carcinomas 8575/3 Diagnostic mammography is an X-ray examination of
Pure epithelial metaplastic carcinomas 8575/3 women who have breast discomfort (e.g., a lump or nipple dis-
Mixed epithelial/mesenchymal metaplastic carcinomas 8575/3 charge) or found abnormalities during mammography screen-
Lipid-rich carcinoma 8314/3 ing. Diagnostic mammography is more complicated and
Adenoid cystic carcinoma 8200/3 time-consuming than screening mammography, and can be
Acinic cell carcinoma 8550/3 used to determine the exact size and location of breast abnor-
Glycogen-rich clear cell carcinoma 8315/3 malities and to image surrounding tissue and lymph nodes. In
Inflammatory carcinoma 8530/3
general, several other views of the breast are imaged and inter-
Lobular carcinoma in situ 8520/2
preted during a mammogram diagnosis, especially in women
Ductal carcinoma in situ 8500/2
Microinvasive carcinoma
with breast implants or a personal history of breast cancer.
Although mammography is still the gold standard, it does
have limitations, especially in women with dense breasts.
for detecting cancer in women with dense breast tissue, New imaging technologies are emerging to overcome these
which is often problematic for conventional film mammog- limitations and enhance cancer detection capabilities and
raphy. Ultrasound also plays an important role in breast improve patient prognosis.
imaging. As an aid to diagnostic mammography, it can be
used for biopsy guidance, assessment of accessible masses, 42.1.4.3 Ultrasound
and continuous assessment of benign masses. Magnetic reso- Ultrasound has become the main diagnostic method after
nance imaging (MRI) is an accepted diagnostic method for mammography. Its diagnostic and guidance role has
many breast-related indications and is sensitive to tumors. expanded and developed, and recently, we have begun to
On the contrary, its specificity is poor. If the area does show reevaluate the process of ultrasound as an auxiliary examina-
enhancement, it may or may not be a tumor. To address this, tion tool for women at high risk, dense breasts, or both after
further imaging or biopsy may be required. Digital mam- mammography. Ultrasound: As a detection program for
mography systems use digital detectors to convert X-ray breast cancer, ultrasound cannot replace mammograms for
photons into digital signals for display on a high-resolution breast cancer screening. One of the advances in medical and
monitor. These systems provide features that conventional imaging research over the past two decades has been the sig-
film X-ray mammography cannot provide. PET-CT plays an nificant expansion of breast ultrasound’s ability to assess
important role in detecting local disease recurrence and dis- breast disease. Ultrasound has become an essential compo-
tant metastases in patients with breast cancer. nent of breast cancer diagnosis and prognosis.
Without a perfect screening program, women’s risk of
breast cancer varies widely, and screening can lead to unnec- 42.1.4.4 Magnetic Resonance Imaging
essary procedures and alerts, so ideally screening should be MRI is one of the most relevant breast cancer diagnostic tools
tailored to the individual’s cancer risk. today (Table 42.3). It is widely used to screen patients with an
increased risk of breast cancer, such as BRCA- positive
42.1.4.2 Mammography patients; it is also widely used to select the best treatment.
There are two types of mammograms: screening and diagno- MRI has the highest sensitivity in breast cancer imaging, but
sis. Screening mammograms are X-rays of the breasts per- its low specificity remains its biggest drawback.
754 J. Tang et al.
Table 42.3 Uses of MRI in breast cancer 42.1.4.6 Genomic Biomarkers

MRI in breast cancer BRCA1 was the first gene that has been shown to be suscep-
MRI screening tible to hereditary breast cancer. Subsequently, BRCA1
BRCA carriers
(located at 17q21) has also been confirmed to also indicate
Untested first-degree relatives of BRCA carriers
Individuals with more than 20% lifetime risk of breast cancer ovarian cancer [4, 5]. Many cohorts with exposure data and
Extent of disease evaluation other participant details have been used to identify genetic
Risk of change to a more extensive treatment due to false additional biomarkers associated with breast cancer [6]. The Cooperative
disease Oncology Genetic Environment Study (COGS) is a large-
MRI-guided biopsy is recommended prior to changing the
treatment
scale genotyping study funded by the European Commission.
Use in selected patients More than 150,000 samples have been genotyped in this
Study of the contralateral breast study. A family-based high-permeability susceptible gene
Risk of change to a more extensive treatment due to false additional was identified through association studies, and then a low-
disease
permeability gene was identified. Carriers of such genes and
MRI-guided biopsy is recommended prior to changing the
treatment single nucleotide polymorphisms (SNPs) are susceptible to
Low positive predictive value breast cancer. Pharoah and Caldas found that a set of 70
Evaluation of axillary metastasis genes can predict the prognosis of breast cancer. Genomic
High sensitivity and specificity in detecting axillary node markers include SNPs, mutations, additions and deletions,
metastases
UPSIO-enhanced MRI has the highest sensitivity and specificity
recombination, and copy number changes. Different research
Not a replacement for SLNB groups of M. Verma and D. Barh 395 have performed
Evaluation after neoadjuvant chemotherapy genome-wide association studies (GWAS) to identify breast
Best imaging technique in correlation between the preoperative cancer susceptibility genes that may be useful for breast can-
measurements and the pathological findings cer screening in high-risk groups [7–9]. In another study,
High specificity and a low sensitivity in predicting pathological
complete remission 2702 women with invasive breast cancer of European descent
Suitable for selecting patients for neoadjuvant chemotherapy and 5726 controls were conducted [10]. The SNPs identified
in this study were mainly located in 1p11.2, 2q35, 3p, 5p12,
8q24, 10q23, 13, 14q24.1, and 16q regions. The affected by
Detection of breast cancer by MRI is based on tumor these SNPs are involved in actin cytoskeleton regulation,
angiogenesis. In tumors, the capillaries of the tumor and sur- glycan degradation, alpha-linolenic acid metabolism, circa-
rounding stroma uncontrollably proliferate, forming abnor- dian rhythm regulation, and drug metabolism.
mal blood vessels with increased permeability. The increase
in permeability leads to the rapid penetration of contrast agent 42.1.4.7 Epigenomic Biomarkers
into the interstitial space, which leads to an increase in the and Methylation Biomarkers
signal in MRI, which can describe the shape and nature of the The term epigenome is used to define the overall epigenetic
tumor. Due to this tumor angiogenesis, contrast-enhanced state. The basic biological characteristics of a DNA frag-
breast MRI is the most sensitive imaging technique. It is cur- ment (such as gene density, replication time, and recombi-
rently used to detect invasive breast malignancies. nation) are related to its GC content. The promoter region
is rich in CpG content. A genomic region of about 0.4 kb
42.1.4.5 B reast Cancer Biomarkers for Risk with a GC content of about 50% is called a CpG island. In
Assessment, Screening, Detection, mammals, CpG islands are 200–300 bp. Promoters of tis-
Diagnosis, and Prognosis sue-specific genes located in CpG islands are usually
Early detection of disease can prevent breast cancer mortality. unmethylated. However, during breast cancer development,
Over the past few decades, progress has been made in identi- these CpG sites begin to methylate. Cytosine methylation
fying invasive and noninvasive biomarkers. Genetic biomark- can regulate gene expression by preventing the association
ers, based on mutations and single nucleotide polymorphisms of certain transcription factors with their associated DNA
(SNPs) associated with breast cancer, have potential uses in recognition sequences. Methyl CpG binding protein (MBP)
screening high-risk populations to identify individuals who can bind to methylated cytosines and mediate inhibitory
may have the disease. Among epigenetic markers, hyper- signals, or MBP can interact with chromatin-forming pro-
methylation and specific microRNA (miR) analysis of teins to modify the surrounding chromatin, thus linking
selected genes can be used for cancer detection, diagnosis, DNA methylation to chromatin modification. DNA meth-
and prognosis. Some new methods will be introduced that ylation at cytosine 5 is performed by DNA methyltransfer-
make currently applicable techniques and analytical methods ase (DNMT). These enzymes are necessary to initiate and
suitable for clinical use. The ultimate goal of the test is to maintain methylation.
identify (a) biomarkers that can be analyzed in noninvasively Cancer cells accumulate abnormal patterns of DNA meth-
collected samples, (b) cheap analytical methods, and (c) bio- ylation, leading to a malignant breast cancer phenotype. The
markers that show high sensitivity and specificity. distribution of methylated genomes is not very clear, and
many GWAS identifications have been performed to identify 42.1.4.9 B reast Circulating Tumor Cells
biomarkers associated with breast cancer risk [8, 11, 12]. Potential Biomarkers for Breast Cancer
Using methylated DNA immunoprecipitation combined with Diagnosis and Prognosis Evaluation
high-throughput sequencing (MeDIP-seq), methylation lev- Circulating tumor cells (CTC) are considered as an indicator
els were compared in normal and breast cancer cell samples, of tumor invasion. CTCs have recently been detected in
and in breast cancer samples, especially in the CpGrich breast cancer patients and they have become targets for
region, overall are insufficiently methylated. The location of assessing breast cancer progression, prognosis, and diagno-
these CpG-rich regions is independent of the transcription sis. CTCs are heterogeneous population with a phenotype
start sites of various genes. Using this method, the pattern of from epithelium to mesenchyme. CTCs express various
methylation during epithelial-to-mesenchymal transition markers according to the stage of epithelial-mesenchymal
was also evaluated and used for disease stratification. Methyl transition, including epithelial cell adhesion molecules, cyto-
receptivity in malignant breast tissue is approximately 2–3 keratin, and MUC-1. CTCs are usually detected and con-
times higher than in matched controls. firmed in two steps, including enrichment and identification.
However, methyl acceptability varies widely among These methods have become powerful tools for diagnosing
patients. Quantitative analysis of 5meC levels showed a sig- and predicting response to systemic therapies.
nificant decrease compared to normal tissues. BRCA1 and CTCs have been shown to have prognostic and diagnostic
BRCA2 cancers have slightly lower levels of hypomethyl- effects in breast cancer patients and are associated with PFS,
ation, but are significant. Genome-wide hypomethylation is DFS, and OS. Early breast cancer patients with CTCs have a
associated with satellite sequence hypomethylation. Defined high risk of metastasis. Recent results have also demon-
area (Sa2 encoding) chromosome 1 and satalpha are specifi- strated a correlation between the presence of CTCs and the
cally hypomethylated. On chromosome 5, the region con- histological grade difference of the primary tumor. Evaluation
taining the SATr-1 coding sequence also showed insufficient of CTC during treatment can provide information on the effi-
methylation. cacy of the treatment and the risk of relapse. In addition,
analysis of the molecular characteristics of CTCs can pro-
42.1.4.8 miR Biomarkers vide information on therapeutic and chemoresistance target
miR is a key regulator of many gene expression regulators. proteins. However, before CTCs can be used as a powerful
Tissue-specific miRs have been reported in different groups tool for breast cancer diagnosis and prognosis, further devel-
[13]. These RNAs are small and have a unique stem-loop opment is needed, including identifying specific markers for
structure. Many miRs can be cyclically separated. Due to breast cancer CTCs, developing high sensitivity and specific-
their smaller size and stability (due to the secondary struc- ity methods for detecting CTCs, and exploring the molecular
ture), these circulating miRs provide a rich source of diagnos- characterization of CTCs. In terms of CTCs markers related
tic biomarkers for breast cancer. The relationship between to cancer progression, recurrence, and metastasis. However,
more than 300 miRs and breast cancer has been evaluated in the rapid increase in breast cancer CTCs research will make
inflammatory breast cancer cells [14]. The most promising CTCs a powerful tool for breast cancer diagnosis and prog-
miRs are miR-29a, miR-30b, miR-342-5p, and miR-520a-5p. nosis in the near future.
Analysis of these functions miRs reveals their role in cell pro-
liferation and signal transduction pathways. Whenever a sub- 42.1.4.10 Tumor-Specific Protein 70 (SP70)
type analysis of breast cancer cells is required, these markers Owing to the lack of accuracy, most current tumor markers
can be used to identify inflammatory breast cancer cells. The are limited in clinical. Some candidate markers have been
promoter region of the miR coding region was evaluated by reported in recent years. SP70 was reported to be a new pro-
combining 5-methylcytosine immunoprecipitation with miR tein mainly expressed in nonsmall cell lung cancer (NSCLC)
slice microarray analysis. Several miR promoters were found with the relative molecular mass (Mr) of 70 kDa. It could be
to be hypermethylated, particularly miR-31, miR-130a, miR- a sensitive biomarker for monitoring timely response to che-
let7a-3/let 7-b, miR-155, and miR-137 [13]. motherapy in patients with advanced NSCLC. Not only that,
Mitchell and colleagues have demonstrated the advan- it was found to be highly expressed in breast cancer tissues,
tages of using miR to detect cancer due to the stability of and significantly associated with the tumor stage, metastasis,
miR even in fixed tissues. miR-155 predicts the prognosis of early recurrence, and viral load.
triple-negative breast cancer (higher miR-155 expression is We continuously compared the interplay between the
associated with higher angiogenesis and aggressiveness) common clinical serum tumor markers and tumor size, clas-
[15]. In summary, miR can be used for breast cancer screen- sification, and lymphatic metastasis. CEA is only associated
ing and risk assessment before the disease develops. with tumor stages (P = 0.01), while CA15-3 is associated
Furthermore, a group of miRs can be used for the detection with tumor sizes (P = 0.025). CA125 showed no obviously
and diagnosis of breast cancer. To follow up on breast cancer associations with these clinical characteristics in breast can-
treatment, miR analysis can also be applied on breast cancer cer patients. However, levels of SP70 were positively related
prognosis and survival assessment. to advanced tumor stages (III–IV), lager tumor sizes, and
756 J. Tang et al.
*
a b
100 120
80 100
60 80
40 60
20 40
10 40
CA125 (ng/ml)
8
CEA (ng/ml)
30
6
20
4
10
2
0 0
N0 N1 T1 T2 I-II III-IV N0 N1 T1 T2 I-II III-IV
* * *
c * d
70
60 40
50 30
40
30 20
30
CA153 (ng/ml)
15
SP70 (ng/ml)
20
10
10
5
0
0
N0 N1 T1 T2 I-II III-IV N0 N1 T1 T2 I-II III-IV
Fig. 42.2 Comparison of serum biomarkers levels among breast cancer Pathological parameters; N0: No Lymphnode metastasis; N1: Lymphnode
metastasis; T1: Maximum diameter of tumor ≤ 20 mm; T2: Maximum diameter of tumor >20 mm; Tumor staging: I–II, III–IV; *P < 0.05
lymphatic metastasis, which are shown in Fig. 42.2 (P = 0.012, cancer antigen 15.3 (CA15.3) or cancer embryonic antigen
P = 0.01, and P = 0.002). The data from this study came from (CEA). When breast cancer is detected at a local stage and
the First Affiliated Hospital of Nanjing Medical University the tumor size is less than 10 mm, its 5-year survival rate is
from March to October 2017, and the reference interval of 98%. If the lesion is large, it usually spreads to nearby lymph
SP70 (ELISA) in the study was ≤7.5 ng/mL. nodes (regional disease), and the 5-year survival rate drops to
50–80%. If the cancer has spread (metastasis) to distant
42.1.4.11 Other Protein Markers organs, such as the lung, bone marrow, or liver, the 5-year
To date, some proteins have been proposed as possible mark- survival rate is less than 25%.
ers for early detection of breast cancer. These include the Therefore, it is essential to develop more sensitive diag-
carbohydrate antigen CA15.3 [16], carcinoembryonic anti- nostic tools that can not only complement mammography, but
gen (CEA), clusterin, and alpha-1-antichymotrypsin. also detect and diagnose breast cancer earlier than current
However, due to the lack of specificity and/or sensitivity to methods. The ideal screening gap protocol would involve the
early disease, none of these markers is useful for detecting development of a set of highly specific and sensitive biomark-
early breast cancer. Despite satisfactory progress in screen- ers that can be used to screen high-risk populations, detect
ing and treatment of breast cancer, about 40% of patients still relapses, and monitor treatment using simple blood-based
succumb to the disease. The development of distant metasta- tests that can be performed by general physicians. At present,
ses is the leading cause of these deaths. Once metastatic the exploration of CTCs and the development of biomarkers
lesions are detected by “classical” methods, breast cancer is based on genomics, transcriptomics, metabolomics, and pro-
usually incurable: clinical manifestations of spread, imaging teomics are currently expected to become better markers for
methods, and serum marker assays, such as those based on screening various stages of breast cancer.
Fig. 42.4 MRI
monitoring of breast cancer because they are convenient,

cost-effective, available, and repeatable, such as CEA,
CA15-3, and new marker SP70.
This case was from the First Affiliated Hospital of Nanjing
Fig. 42.3 Mammography
Medical University (also named Jiangsu Province Hospital).

42.2 Ovarian Cancer
Clinical Background A 44-year-old female patient came to
hospital for a small mass in the right breast. A day ago, the Jieshi Xie and Zheng Cao
patient found “right breast mass”, no pain, no history of nip-
ple discharge, and then went to the hospital for related exam-
ination. Mammography showed that the right breast occupied 42.2.1 Overview
space and the right axillary lymph node was enlarged.
Mammography target: right extramammary superior quad- The ovaries are important female reproductive organs,
rant asymmetric dense shadow, BI-RADS 4A (Fig. 42.3). responsible for the production of oocytes and female hor-
MIR showed that the background enhancement of the paren- mones. They are a pair of substantive organs, located in the
chymal early background of the double-milk gland: a little; ovary fossa on the lateral wall of the pelvic cavity
Right breast 12-point nodule, BI-RADS 6; the small nodules (Fig. 42.6). The state of the ovaries changes with age: in
on the left and the outside, taking into account the intramam- childhood, ovaries are small and smooth; during sexual
mary lymph node, BI-RADS 2 (Fig. 42.4). maturity, they become larger and scarred because of ovula-
tion; the ovaries begin to shrink at the ages of 30–40. Until
Laboratory Data CEA 2.24 ng/mL, CA125 17.91 U/mL, about 50 years old, ovaries gradually undergo atrophy
CA15-3 12.15 U/mL, SP70 12.9 ng/mL(↑). The pathological along with menopause.
report was invasive ductal carcinoma, and immunohisto- Ovarian cancer is the third most common gynecologic
chemical staining of SP70 was performed, see in Fig. 42.5. malignant tumor worldwide and the deadliest gynecologi-
cal malignancy in an accident. The statistics demonstrate
Treatment Resection Results with interpretation guideline, that more than 200,000 women are diagnosed with ovarian
Guidelines from the National Comprehensive Cancer cancer, and 125,000 die from this malignancy worldwide
Network (NCCN) recommend surveillance being currently each year [17]. Although the prognosis is mostly excellent
primarily imaging-based (B ultrasound and mammography). at an early-stage of ovarian cancer, the early-stage symp-
Breast cancer can be diagnosed by biopsy. However, tumor toms are subtle and generalized. Most patients are diag-
markers still play an important role in the diagnosis and nosed with the advanced-stage disease along with widely
758 J. Tang et al.
a b
Fig. 42.5 Pathological results. (a) hematoxylin-eosin staining (×100); (b) immunohistochemical staining (×100)
be diagnosed at an earlier stage and younger age. The former

is inclined to the nonwhite, obese women with a family his-
tory of breast or ovarian cancer, and relative to the mutation
of DICER1 and FOXL2. The latter’s incidence increases
around puberty and white women have a lower incidence
relatively. Epithelial ovarian cancer accounts for more than
90% of malignant epithelial neoplasms, predisposes to post-
menopausal women. White women have a higher incidence
(12.8 per 100,000) compared with black women (9.8 per
100,000). The incidence of epithelial ovarian cancer has
been declining from 2003 to 2012 in America. Reproduction
and hormone are the traditional risk factors of ovarian can-
cer, in addition to endometriosis, taller height and high BMI
in adolescence may also increase the risk [17].
Fig. 42.6 The anatomical structure of the ovary

Most cases of ovarian cancer are epithelial ovarian cancer,
which presents an atypical symptom at early stage of disease
disseminated intraperitoneal metastasis. The clinical char- generally. Therefore, it is difficult to diagnose with the epi-
acteristics of ovarian cancer result in a poor 5-year survival thelial ovarian cancer at the early stage. The significant
rate. Some studies show that only 25% of ovarian cancers symptoms usually emerge in the last 6–12 months of life and
are detected at clinical stage I, of which more than 90% of the severity of symptoms is increasing. In this period, a
patients can be cured. While most the ovarian cancers are majority of patients may present with vague bloating,
diagnosed in late stages, and the 5-year survival rate abdominal or pelvic pains, seroperitoneum, early satiety,
dropped to less 30% [18]. gastrointestinal distress, change in bowel habits, and urinary
Ovarian cancer can be classified to nonepithelial ovarian symptoms. Moreover, some patients experience emaciation
cancer and epithelial ovarian cancer. Sex-cord stromal ovar- and anemia, and 67–92% of patients suffer significant fatigue
ian neoplasms and ovarian germ cell tumors belong to the throughout disease after diagnosis [17, 19]. Ovarian tumor
nonepithelial ovarian cancer. They represent only 1.2% and torsion, rupture, and infection are the serious complications
5% of ovarian cancer cases, respectively. Both of them can of ovarian cancer.
The ovarian neoplasms often involve bilateral ovaries and most in serous carcinomas (85%) and the least in mucinous
can be examined by gynecological palpation. The neoplasms cancers (12%). In papillary, endometrioid, clear cell, and
are usually solid or cystic solid with an uneven surface and undifferentiated adenocarcinomas, the abnormal expression
poor activity. Gynecological triad examination may reveal of CA125 is 68%, 65%, 40%, and 38%, respectively.
palpable hard nodules in the rectouterine fossa. Sometimes, Although the abnormal expression of CA125 can be detected
the apparent lymph nodes in groin, armpit, or supraclavicular in most ovarian tissues, the sensitivity of CA125 is limited.
bone can be palpated. On the other hand, the specificity of CA125 is unsatisfactory.
At present, transvaginal ultrasonography is the most com- In menopausal women, the specificity of CA125 assay is
mon method for ovarian cancer initial screening because of about 99%, but it does not attain 99.6% for specificity
its availability, high resolution, and harmlessness. However, required to achieve a positive predictive value of 10%. In
it is difficult for transvaginal ultrasonography to distinguish premenopausal women, many benign diseases (peritonitis,
the benign and malignant cysts, which limits the accuracy of liver cirrhosis, menstruation, pregnancy, endometriosis, ade-
prediction severely. Moreover, the ultrasound operator’s nomyosis, and salpingitis) can induce CA125 expression
expertize has an important effect on the reports which may increased. Benign ovarian cysts and tumors, uterine fibroids,
lead to unstable sensitivity (85–100%) and specificity (50– inflammation of the pleura, peritoneum, or pericardium can
100%). Nowadays, there is no test that can diagnose early also promote the increase of CA125 regardless of age. The
ovarian cancer accurately alone. The cases which cannot be base value of CA125 usually exceeds 35 U/mL in some
confirmed by ultrasonography must be performed with other women without any recognizable disease. Besides, other
examination, such as Doppler ultrasound (sensitivity 84%, cancers, such as breast cancer and lung cancer, can also lead
specificity 82%), Magnetic Resonance Imaging (MRI, sensi- to a high expression of CA125, which complicates the diag-
tivity 76%, specificity 97%), contrast-enhanced MRI (sensi- nosis for ovarian cancer [21]. From the above, CA125 is not
tivity 81%, specificity 98%), and CT (sensitivity 81%, sufficiently specific for early diagnosis of ovarian carcinoma.
specificity 87%) [20]. Nowadays, to elevate the diagnostic accuracy of ovarian can-
cer, CA125 assay is usually combined with ultrasonography
or other biomarkers.
Human Epididymis 4 (HE4)
42.2.3.1 Tumor Markers HE4 is a whey-acidic-protein (WAP) expressed in different
organ tissues, found by Kirchhoff in 1991. It has been con-
CA125 firmed that HE4 exhibits high expression in endometrial can-
CA125 is a heavily glycosylated high-molecular-weight cer, pulmonary cancer, breast adenocarcinomas, and
membrane-bound cell surface mucin, composed by a repeat- mesotheliomas. As HE4 expression in ovarian cancer is sig-
ing peptide epitope of the mucin 16 (MUC16). It is highly nificantly higher than normal ovarian tissues, it is considered
expressed in many epithelial origin tumors, which imply it as a potential biomarker of ovarian cancer. Normally, the
may play a significant role in tumorigenesis. CA125 expres- serum concentration of HE4 is increasing steadily with age,
sion is minimal in normal ovarian tissues but increases from 41.1 to 82.1, pmol/mL HE4 is reduced during preg-
greatly in ovarian carcinomas. Therefore, CA125 is usually nancy which is different from CA125 [22]. Lu and colleagues
used as a serum biomarker to monitor ovarian cancer pro- demonstrated that HE4 was associated with ovarian carci-
gression and recurrence. It has been confirmed that CA125 noma cell maturation and played an important role in tumor
can promote the proliferation of cancer cell, impede the cell adhesion and motility [23]. Moore and colleagues
function of human natural killer cells, and then assist cancer implied over-expressed HE4 can promote ovarian cancer
cells to evade immunological surveillance and promote growth and chemoresistance [24]. Lin and colleagues con-
ovarian tumorigenesis [18]. A serum concentration of sidered HE4 could distinguish malignant ovarian cancer
CA125 > 35 U/mL is indicative of potential malignancy. from benign ovarian diseases effectively [25].
The postoperative serum concentration of CA125 > 65 U/ FDA has authorized HE4 as ovarian cancer biomarker to
mL is associated with worse 5-year survival. monitor ovarian cancer disease progression and recurrence.
As a serum biomarker of ovarian cancer, CA125 can be All of the endometrioid ovarian cancer and 93% serous ovar-
detected increased in half of the patients at early stage of ian cancer overexpress HE4. Diagnostic sensitivity of HE4 is
disease, but almost 90% patients in late-stage ovarian cancer. 82.5% in serum from ovarian cancer, higher than CA125,
Overall, almost 80% of ovarian diseased tissues abnormal with 95% specificity. In urine sample, HE4 sensitivity is up
express CA125 observably, and the amount of CA125 to 86.6% and 89.0% with 94.4% specificity for stage I/II and
expression varies with histotype. CA125 expression is the stage III/IV, respectively. Generally, both HE4 and CA125
760 J. Tang et al.
are complementary. Moore and colleagues suggested that filtration rate was increased by 42% in early-stage and 75%
simultaneous detection of HE4 and CA125 is better than in late-stage in urine samples from the same donors. It was
either alone in ovarian cancer diagnosis [24]. When com- implied that urine may be the better sample for MSLN detec-
bined with CA125, HE4 sensitivity can elevate from 72.9% tion [21].
to 76.5% at 95% specificity in urine and serum samples [26].
Therefore, Combined HE4 and CA125 assays can improve Osteopontin
the accuracy of ovarian cancer diagnosis, exclude other Osteopontin is an acidic calcium binding glycoprotein syn-
benign ovarian diseases. Generally, both HE4 and CA125 thesized by osteoblast and vascular endothelial cells. It is an
are abnormally increased in serum of ovarian cancer; in important component of the extracellular matrix. Osteopontin
benign ovarian tumors or other benign diseases, the serum has been identified as a potential biomarker of ovarian cancer
concentration of CA125 elevates alone usually; high concen- by the high-throughput cDNA microarray system. Highly
tration of HE4 and normal CA125 suggest the presence of expression of osteopontin was observed in epithelial ovarian
epithelial ovarian cancer or other types of cancer (e.g., endo-
cancer and the amount of osteopontin in borderline and inva-
metrial cancer). sive ovarian cancer was more than benign tumors.
Osteopontin takes effect on tumor cells metastasis and tumor
Mesothelin progression in a stressful environment. Besides, it enhances
Mesothelin (MSLN) is a glycosylphosphatidylinositol- tumor cells survival by increasing Akt activation and HIF-1
anchored membrane glycoprotein which is located on the alpha expression. Osteopontin is also complementary to
cell surface of mesothelial cells lining the pericardium, other ovarian cancer biomarkers. The sensitivity of osteo-
pleura, and peritoneum in normal physiological conditions. pontin alone yields to 81.3%, but the value enhances to
Human MSLN is composed of 16 exons and produces three 93.8% in combination with CA125, at 33.7% specificity.
predominant variants (variant 1, variant 2, and variant 3) by Detecting osteopontin, insulin-like growth factor, prolactin,
alternative splicing. It has been provided by Hellstrom that and leptin simultaneously raises diagnostic sensitivity to
MSLN1 primarily expresses on the cell surface and soluble 96% at a specificity of 94% [21]. Further, superpose macro-
MSLN derived from cleavaged variant 1 can be released to phage inhibitory factor and CA125 test can achieve a sensi-
body fluid in some patients suffering from several tumor tivity of 95.3% at 99.4% specificity. The histological types,
types [18, 27]. Normal mesothelin cells express MSLN in grades or stages of ovarian cancer barely affect the amount of
trace amount. While the expression of MSLN increases osteopontin. At cut-off value of 252 ng/mL, the osteopontin
markedly in human cancers (ovarian cancer, pancreatic ade- specificity is 80.4%, and the sensitivities of early and late
nocarcinoma, and mesotheliomas). Especially, almost all of stages ovarian cancer are 80.4% and 85.4%, respectively
serous borderline and cyst adenocarcinoma display the high [26]. So far, more data of early-stage cases are needed to
concentration of MSLN in urine samples. Therefore, MSLN evaluate the application of multiple marker combinations.
has been identified as a tumor-associated marker. Notably, a fragment derived from osteopontin has been
Furthermore, MSLN can combine with CA125 and plays an found in ovarian cancer patients’ urine.
important role in tumor cells adhesion, chemoresistance, and
tumor progression. As tumor marker, MSLN shows the Kallikreins
oncogenic properties, promotes ovarian cancer cells invasion Human kallikrein family consists of 15 members all of which
and adhesion through MAPK/ERK and JNK pathways, and have serine proteases activity and are involved in tumor cells
induces drug resistance through MAPK/ERK and PI3K/ growth, apoptosis, metastasis, and angiogenesis in many
AKT pathways [18]. cancers. In ovarian cancer, there are 12 kallikreins overex-
Scholler and colleagues found 77% of serum samples pression on mRNA and/or protein level. Kallikrein 4 is
from advantage stage ovarian cancer exhibited elevated closely related to ovarian cancer progression, usually
expression of MSLN at a specificity of 100% [28]. Synthetic detected in the late stage of serous ovarian cancer. Kallikrein
data including early and late stages revealed that MSLN 7 mRNA elevates in 66.7–78.1% of malignant tumor cells
expression increased in 60% of ovarian cancer sera at 98% generally. Kallikrein 11 overexpresses in 70% serum sample
specificity [26]. Importantly, MSLN and serum CA125 are from ovarian cancer patients at 95% specificity. Kallikrein
complementary; the combination of the two tumor biomark- 10 expression is also up-regulated obviously in some sub-
ers can improve the detected fraction of ovarian cancer com- types of ovarian carcinoma [26]. In Rosen’s study, the tissue
pared with either one alone. In a recent study focusing on arrays were used to identify potential biomarkers which
ovarian cancer by Donna, serum MSLN was elevated by could complement CA125. Kallikrein 10 and kallikrein 6
12% in early-stage (stage I/II) and 48% in late-stage (stage expressions were observed in the ovarian cancers lacked
III/IV) ovarian cancer respectively, at 95% specificity. CA125 expression. Although several normal tissues can
However, MSLN expression normalized by the glomerular express kallikreins, kallikrein 10 in 56% ovarian cancer
serum is higher than healthy women and serum kallikrein 11 MicroRNAs

overexpresses in 70% ovarian cancer, at a specificity of 95%. MicroRNA (miRNA) demonstrates different expression
Therefore, kallikrein 6, kallikrein 10, and kallikrein 11 have levels in different ovarian cancer histological types. The
been identified as the potential biomarkers of ovarian cancer difference also exists between normal and tumor tissues.
[21, 26]. Up-regulated miR-21, miR-141, miR-200, miR-203, and
miR-205 were detected in serous and endometrioid sub-
B7-H4 types. Both miR-145 and miR-222 expressions are down-
B7-H4 expression is located in the membranous and cyto- regulated in clear cell carcinomas. In ovarian cancer serum,
plasmic in serous ovarian cancer, endometriod carcinomas, up-regulated miRNA panel includes miR-21, miR-29, miR-
and clear cell cancer. Positive signals of stained B7-H4 are 92a, miR-93, and miR-126, whereas miR-99, miR-127, and
observed in 60% of stage I and 90% of stage II ovarian tis- miR- 155 expressions are reduced. Some miRNAs have
sues. It was confirmed that 45% early-stage patients and been identified as markers for ovarian cancer diagnosis,
67% late-stage patients demonstrated high expression of such as miR-21, miR-141, miR-203, miR-205, miR-214,
B7-H4 at 97% specificity. B7-H4 and CA125 are comple- and let-7f. The functions of miRNAs in cancers are compli-
mentary. B7-H4 can be detected in the patients whose cated. For example, miR-148b promotes tumor progres-
CA125 expression is low. When B7-H4 is combined with sion; miR-200a, miR-200b, and miR-429 are associated
CA125, the sensitivity of diagnosis rises to 65% which is with relapse; miR-200 and miR-182 are connected with
higher than either B7-H4 (45%) or CA125 (52%) alone. prognosis. Given that a large number of miRNAs are
B7-H4 is considered as a common biomarker for ovarian involved in complex processes of ovarian cancer, further
cancer of early stage [21, 26]. studies are needed urgently [26].
Interleukins Exosomes
Interleukins play a role in a variety of cancers. IL-6 exhibits Exosomes are small extracellular membrane vesicles
high expression in half of the primary ovarian cancer patients. deriving from endosomal cellular. They are considered as
The concentrations of IL-6 and IL-8 antibodies increase in messengers between cellular and transfer functional con-
ovarian cancer serum. Detecting levels of the antibodies of tents including proteins, DNAs, mRNAs, metabolites, and
IL-8 (IgG) in stage I/II ovarian cancer patients and logistic so on. Exosomes derived from ovarian cancer cells support
regression analysis of the data can diagnose the ovarian can- tumor growth and progression through binding to stroma
cer at early stage with 65.5% sensitivity at a specificity of cells. In this process, they primarily promote angiogenesis,
98% [26]. IL-6 and IL-8 are cytokines secreted abundantly immunosuppression and marrow progenitors’ recruitment.
by omental adipocytes which mediate the metastasis of ovar- It has been identified that exosomes-derived ovarian can-
ian tumor cells to omentum. Th17 cell, the primary source of cer contain a large number of functional proteins, such as
IL-17, has been observed in cancer tissue. IL-6 can promote major histocompatibility complex I, tetraspanins CD63
the differentiation of Th17 cells. To improve the diagnostic and CD81, lysosomal- associated membrane proteins 1
performance of early biomarkers of ovarian cancer, IL-6 and and 2, and tumor susceptibility gene 101 protein. Besides,
IL-8 are combined with CA125 and three other markers. The the specific proteins and nucleic acids of exosomes can
diagnostic sensitivity for early stage is 84%, at 95% specific- help determine the donor cell. Therefore, miRNA, pro-
ity. A combination of serum IL-8, IL-8 antibodies, and teins, and metabolites contained in exosomes may be
CA125 makes early stage ovarian cancer diagnostic sensitiv- potential biomarkers of tumors. The detection of serum
ity rise to 88% achieved at 98% specificity [21]. exosome inclusions may contribute to improving the diag-
nostic accuracy of ovarian cancer and other tumor types
Vascular Endothelial Growth Factor (VEGF) [26, 28].
VEGF has been identified as an important factor for tumor
angiogenesis in cancers. VEGF has been detected in ovarian Circulating Cell-Free DNA
cancer patients’ ascites, serum, and tumor tissues. Compared Noninvasive cell-free DNA quantitative analysis has been
with normal controls or benign lesion, serum VEGF of ovar- used as a clinical tool for ovarian cancer. Serum cell-free
ian cancer patients was elevated significantly. To predict DNA content elevates markedly in ovarian cancer patients
ovarian cancer better, VEGF was included in a panel of five compared with healthy women and patients with benign
markers by Gorelik, and the sensitivity turned out to be 84%, ovarian tumors. However, we still cannot confirm the corre-
at 95% specificity. In addition, VEGF exists in 81% ovarian lation between plasma cell-free DNA concentration and
cancer tissues lacking CA125, implying that VEGF and ovarian tumor size, stage, and sites at present. Surely, the
CA125 can be used as a combination to improve the diagno- serum cell-free DNA concentration in stage III and IV ovar-
sis of ovarian cancer [21]. ian cancer is higher than stage I and II ovarian cancer. Data
762 J. Tang et al.
from nine studies were used for meta-analysis to evaluate the MCJ. Although the specificity of single methylated gene
diagnostic accuracy of cell-free DNA in ovarian cancer. The detection is insufficient, a panel of methylation genes detec-
results revealed that the sensitivity was 70% and the specific- tion may meet ovarian cancer diagnostic requirement.
ity was 90%. The cell-free DNA diagnostic sensitivity was Excitingly, Seeber and Van Diest have illustrated some single
unsatisfactory, though the specificity was acceptable. It is methylated genes (FBXO32, IGFBP-3, and HOXA11) which
suggested that serum cell-free DNA detection can be used as play an important role in ovarian cancer prognosis are asso-
an adjuvant diagnosis to improve the diagnostic specificity ciated with disease progression, poor prognosis, and high
of ovarian cancer [29]. mortality rate [28].
In addition to gene methylation, the alterations of histone
42.2.3.2 Genetic Test modifications and its related enzymes are also observed in
ovarian cancer, which suggested the anomalous modifica-
Genetic Mutation tions of histone may play a crucial role in antioncogenes
The genetic factor is still one of the strongest risk factors for silence.
ovarian cancer. Studies implemented in twins suggested the
effect of inherited genetics on ovarian cancer was more cru-
cial than lifestyle and environmental factors. The famous 42.2.4 Management
susceptibility genes of ovarian cancer are BRCA1 and
BRCA2 gene mutations. About 15% advantage serous epi- In summary, many new markers are under investigation for
thelial ovarian cancer can be detected gremlin mutation in the diagnosis of ovarian cancer. Some newly developed
BRCA genes, and almost all of the women with BRCA1 markers are promising, but further validations are still
mutation will develop the serous histologic disease. By the required. At present, serum CA125 detection combined with
age of 70, the risk rates for ovarian cancer are 39–46% for pelvic ultrasound is mainly used to screen ovarian cancer.
the women with BRCA1 mutation and 10–27% for BRCA2 However, there is no evidence-based ovarian cancer screen-
mutation carriers. In addition, some intermediate-risk sus- ing program for the general population. Genetic counseling
ceptibility genes have been identified including RAD51C, and genetic detection are significative for ovarian cancer in
RAD51D, FANCM, BRIP1, and DNA mismatch repair high-risk populations. The women, with a family history of
genes. The risk evaluation of DNA mismatch repair genes ovarian cancer, require genetic counseling and BRCA
for ovarian cancer exhibited the risk is 4–20% in MLH1 car- genetic detection. The National Comprehensive Cancer
riers, 7.5–20% and 13.5% in MSH2 and MSH6 carriers, Network (NCCN) recommends prophylactic double-
respectively [17]. Currently, identified susceptibility alleles adnexectomy to reduce the risk of ovarian cancer after com-
are limited. There are still more alleles and mutations to be pletion of pregnancy for those with identified genetic
discovered. mutations. Continued efforts are needed to discover and vali-
date effective test used to early diagnosis and general popu-
Epigenetic Regulation lation screening of ovarian cancer.
Besides genetic mutation, gene methylation is another inher-
ited risk factor for ovarian cancer. A prospective study
implied abnormal methylation in BRCA promoter could be 42.2.5 Conclusion
the strongest risk factor for ovarian cancer except for BRCA
gene mutation. Several technologies have been developed to Transvaginal ultrasonography and CA125 are the traditional
evaluate DNA methylation in genome-wide or at a specific screening procedures for ovarian cancer. Currently, ovarian
site. It was implied that DNA methylation changes could be cancer early diagnosis is still the most effective measure to
served as a marker for ovarian cancer. Studies have reported reduce the mortality rate. In the last two decades, many
that hypermethylation of CpG island gene promoter is serum biomarkers have been identified and evaluated for
detected in ovarian cancer frequently. OPCML, tumor sup- early stage ovarian cancer diagnosis. Current researches
pressor gene, also displays hypermethylation in its promoter. mainly focus on new biomarkers exploitation, which can be
Importantly, some specific gene promoter methylation has detected and diagnose disease before clinical symptoms
been matched to ovarian cancer clinical symptoms. The appear without compromise on specificity and sensitivity.
methylation of SOX1, SFRP, and PAX1 gene promoters in There is a large heterogeneity of ovarian cancer between dif-
malignant ovarian cancer is higher compared with benign ferent patients, so no single biomarker can be used for initial
and borderline ovarian cancer. Other investigators reported screen alone. So far, at least 29 serum tumor biomarkers have
some gene promoter hypomethylation was associated with been used to combine with CA125, and the combination has
ovarian cancer, such as Claudin-4, trag3, IGF-2, and increased the sensitivity and specificity. Among these bio-
arterial phase venous phase plain scan
Fig. 42.7 Abdominal CT scan of the patients
markers, HE4, KLK6, KLK7, and miRNA are prominent,

worthy of further study. Most biomarkers of ovarian cancer
are derived from serum.
Clinical Background A 83-year-old woman, 40 years

menopause. 11 days ago, there was no obvious cause of
lower abdomen swelling pain, no nausea, vomiting and other
discomfort, and no significant relief after exhaust or
defecation.
Abdominal Ultrasonography A 11.4 cm pelvic mass was

found.
Abdominal CT Scan Right pelvic capsule solid space occu- Fig. 42.8 Histopathological examination of SP70
pation, mixed with intratumoral hemorrhage, see in Fig. 42.7
mined. Staining of undefined cancer tissues can be performed
Laboratory Data CA125 27.4 U/mL, CA199 1781.4 U/ with markers, such as SP70, CA125, CEA, etc., to improve
mL, CEA 5.4 ng/mL, CA50 451.9 IU/mL, CA724 14.9 IU/ diagnostic accuracy.
mL, CA242 93.20 IU/mL.
This case was from Nanjing First Hospital, Nanjing
Histopathological Examination Mucinous cyctic carci- Medical University.
noma of the ovary. Immunohistochemical ER (25%1+), PR
(−), CEA (1+), P53 (−), EMA (1+), WT (−), CA125 (−),
SP70 (3+), see in Fig. 42.8. 42.3 Cervical Cancer
Treatment Resection. Yanhong Zhai and Boyan Song
Results with Interpretation The diagnosis of ovarian can-

cer is currently mainly based on CT or MRI monitoring and 42.3.1 Overview
confirmed by pathological examination. The purpose of
pathological examination is to confirm the diagnosis and The cervix is located at the lower part of the uterus and
verify the preoperative diagnosis. Second, after the diagnosis approximately conical, about 2.5–3 cm in length. The upper
is clear, the next treatment plan and prognosis can be deter- end is connected to the uterus and the lower end is connected
764 J. Tang et al.
Mucinous adenocarcinoma is the most common type of cer-

vical cancer which is mainly derived from the columnar
mucous cells of the cervical canal. Adenocarcinoma can be
divided into three differentiated classes-well, moderately,
and poorly differentiated classes. Malignant adenoma is a
highly differentiated mucosal adenocarcinoma of the cervi-
cal canal. For this disease, many cancerous glands are vary-
ing in size and morphology which protrude into the deep
interstitial of the human cervix. The glandular epithelial cells
have no atypicality and are often accompanied by lymph
node metastasis. Adenosquamous carcinoma accounts for
3–5% of cervical cancer. The cancer tissues contain adeno-
carcinoma and squamous cell carcinoma, which are formed
by the simultaneous differentiation of cells into glandular
cells and squamous cells.
42.3.1.2 HPV Introduction

Papillomavirus DNA is about 8 kb in length, double-spiral,
and surrounded by histones. It mainly encodes six early
Fig. 42.9 Anatomical structure of the cervix
genes and two late genes [33]. Early genes include E1, E2,
E4, E5, E6, and E7. Late genes include L1 and L2.
to the vagina (Fig. 42.9). The cervix contains glands that Currently, more than 130 HPV genotypes have been identi-
secrete mucus to regulate sperm entry into the uterus. fied and are divided into mucosal and skin types. Among
Cervical cancer is a severe disease that causes death in the mucosal types, 40 subtypes are transmitted through
women. There are approximately 530,000 new cases world- sexual contact and can be further divided into high-risk and
wide each year, resulting in the death of 270,000 patients low-risk types. It is reported that almost all types of cervi-
[30]. The incidence of cervical cancer ranks the sixth and the cal cancer are associated with HPV infection, of which
second among women in developed countries and develop- 70% of cervical cancers are associated with HPV-16 and
ing countries, respectively. In spite of the high incidence HPV-18 infections [34].
rate, cervical cancer can be diagnosed in the early stage by The HPV virus is mainly transmitted through sexual con-
pathological and genomic techniques, to be treated effec- tact while skin damage can also result in HPV infection. For
tively [31]. Cervical cancer has no obvious clinical symp- most women under the age of 30 years old, HPV is cleared
toms in the early stage, while can occur vaginal bleeding, within 8 weeks and viral load is reduced to undetectable lev-
apocenosis, and other symptoms as the disease progresses. els within 2 years [35]. In postmenopausal women, HPV
Risk factors of cervical cancer include human papillomavi- infection rates range from 14 to 38%. Since the persistent
rus (HPV) infection, multiple sexual partners, younger age infection of HPV, a high risk of cervical cancer, the fre-
of first delivery, multiple pregnancies and deliveries, among quency of screening should be related to the age of the
which the sustained HPV infection is the leading cause of patients.
malignant transformation and increased risk of cancer [32]. Despite of high-risk HPV infection, other factors are
In worldwide, 17.5% of women without cervical lesions are important for the malignant transformation of cells. For
infected with HPV. example, immunosuppression promoting persistent infection
of HPV, and coinfection of HIV with HPV leading to malig-
42.3.1.1 Staging and Classification nant transformation of cells. Studies have found that women
Cervical cancer is mainly divided into squamous cell carci- infected with HIV were 2–5 times more likely to be infected
noma, adenocarcinoma, and adenosquamous carcinoma. with HPV, and are 3–5 times more likely to develop CIN or
According to the degree of histological differentiation, squa- cervical cancer [36].
mous cell carcinoma can be divided into three grades, where Genetic susceptibility also plays an important role in the
grade I refers to a well-differentiated squamous cell carci- infection of HPV. Many studies have focused on the role of
noma; grade II is a moderately-differentiated squamous cell HLA in the persistent infection of HPV. In a HLA-wide
carcinoma; grade III is a poorly differentiated squamous cell analysis, the DQ/DR gene was involved in the development
carcinoma. Adenocarcinoma makes up 15–20% of the total of cervical cancer, while HLA-DRB1*13 played a role in
number of cervical cancer and is mainly divided into muci- the prevention from the development of cervical cancer
nous adenocarcinoma and malignant adenoma [31]. [37, 38].
HPV Infection Procedure in the Cervix Aberrantly expression of E6 and E7 genes in high-risk

HPV usually infects squamous epithelial basal cells. The E6 HPV can immortalize keratinocytes and make genomes
and E7 gene encoding products of the virus bind to and unstable. Oncogenesis is mainly due to unstable genomes
inhibit the function of intracellular tumor suppressor genes and disorder of cell proliferation regulation caused by virion
(such as P53, RB, etc.). These tumor suppressor genes are proto-oncogene expression.
important for cell cycle regulation and apoptosis, inhibition
of which will lead to malignant transformation of cells [39]. Self-limiting and Persistent Infection of the Virus
Infections of HPV often occur at damaged sites of endothe- Most HPV infections are cleared in a short period time with-
lial cells, especially in the squamocolumnar junction. Infections out causing cancer. It is still controversial whether the virus
are usually located in cells surrounding the wound, which last is eliminated or is kept at a low level. One hypothesis is that
for a long time and are less noticeable [40]. HPV is unlikely to HPV infects cells in the vicinity of the basement membrane
cause cell death or systemic viremia, and its proliferation is to continuously release HPV. Another hypothesis is that
influenced by the differentiation of epithelial cells [41]. HPV primarily infects keratinocytes with a higher degree of
The HPV is mediated through the intracellular vesicles differentiation. Similarly, the location of HPV infection is
and L1 into the host cells. HPV-infected epithelial cells bind also very important. Recent studies have shown that
to heparan sulfate proteoglycan (HSPG) in the basement CK7 + p63-cells are located in the squamocolumnar part and
membrane through L1, which changes the shape of the viral provide a proper environment for cervical cancer develop-
capsid and exposes the amino terminus of L2, and thus inter- ment [47].
nalizes the virus into the cells.
Ingestion of the Virus and Delivery of the Genome 42.3.2 Clinical Appearance

to Nucleus
After the virus enters into the cell, L2 leaded intracellular Patients suffering from cervical cancer usually feature abnor-
virus to enter into the nucleus. After L1 is degraded, L2 mal leucorrhea, vaginal discharge, vaginal bleeding, foreign
mediates viral genome to enter into the trans-Golgi appara- body in cervix uteri, and others. In the advanced stage of
tus [42, 43]. The entry of the L2 genomic complex into the cancer, there are different secondary symptoms due to the
nucleus depends on cell cycle progression and the cell mem- extent of lesions, such as frequent urination, urgent urina-
brane rupture phase of cell mitosis [44]. Then, L2 genomic tion, constipation, swelling of the lower limbs, etc. When the
complex binds to the medullary nucleus of the promyelo- mass is compressed or involves the ureter, ureteral obstruc-
cytes, and once it enters into the nucleus, the early replication, hydronephrosis, and uremia can be induced, even
tion of the virus starts [45, 46]. resulting in symptoms of systemic failure such as anemia
and cachexia in the advanced stage.
Virus Transcription and Life Cycle Colposcopy showed no obvious lesions in microinvasive
In the early phase, E1 and E2 genes are involved in DNA carcinoma. Polypoid and cauliflower-like neoplasms were
replication in HPV, forming 50–100 copies per cell [46]. In spotted in exogenous cervical cancer. The endogenous type
the productive phase, the HPV has a unique spatiotemporal was characterized by hypertrophy of the cervix, hard mass,
regulation mechanism with the differentiation of squamous and swelling of the cervix. Tissue necrosis and detachment
epithelial keratinocytes. HPV switches to a high copy were found in the advanced stage of cancer, forming ulcers
(>103per cell) mode as basal cells differentiate into the epi- or cavities. When the vaginal wall was implicated, growth of
dermis and detach from the basement membrane. Replication neoplasms or hardening of the vaginal wall can be seen.
and expression of HPV virus assembly-associated genes In liquid-based cytology, squamous epithelial lesions or
(such as capsid-associated proteins) are only expressed in glandular epithelial abnormalities were detected.
terminally differentiated epidermal cells, while the virus-
loaded epidermal cells are detached.
In order to utilize the DNA replication system of the host 42.3.3 Laboratory Diagnosis
cells, HPV brings the host keratinocytes into the S phase,
during which the protein E5, E5 stimulates epidermal growth 42.3.3.1 Papanicolaou Test (Pap Test)
factor receptor (EGFR) and causes cells to exit G0–G1 The Pap test has been used to screen cervical cancer for more
phase, E6 binds to and activates the proteasome, degrades than 60 years. In the Pap test, sampled cells are smeared on a
P53 to promote cell survival and cell dedifferentiation, and glass slide, followed by fixation with 95% alcohol, pasteuri-
up-regulates the expression of MYC and thus promotes cell zation, and observation morphology of cells under a micro-
survival, and E7 binds to a variety of proteins, including Rb, scope. Based on the Pap smear method, a Thin-Cytologic
etc. to cross the restriction point [33]. Test (TCT) has been developed. Although this method is
766 J. Tang et al.
simple and cheap, the interpretation of the results is subjec- Chemotherapy is mainly applied in advanced-stage patients
tive and relies on experienced physicians, which limits its or those with recurrent metastasis. Chemotherapy drugs
sensitivity and accuracy [41]. mainly include cisplatin, paclitaxel, etc.
42.3.3.2 Molecular Marker

The sustained HPV infection is important for the occurrence 42.3.5 Conclusion
and development of cervical cancer. Detection of HPV DNA
is more sensitive than cytology and can be used to classify Cervical cancer is a major disease that threatens women’s
infected HPV, which includes fluorescent PCR, gene chip health. Tumor markers for cervical cancer play a significant
technology, and so on. Also besides, there are detection sys- role in the diagnosis, prognosis, and detection of this disease.
tems for HPV mRNA. Detecting E6 and E7 RNA can iden- As the advancement of genomic and proteomic technology,
tify 14 high-risk HPVs [48]. more and more tumor markers for cervical cancer will be
identified, which will significantly improve the diagnosis
Squamous Cell Carcinoma Antigen (SCCA) and therapeutic effects for cervical cancer.
SCCA has been widely used in the screening and early diag-
nosis of cervical cancer. The serum SCC is significantly ele-
vated in patients with cervical squamous cell carcinoma [49]. 42.3.6 Typical Medical Case
The level of SCCA in the serum of patients with cervical can-
cer is closely related to tumor stage, tumor size, depth of Clinical Background The patient was 38 years old, G2P1.
interstitial infiltration, and lymph node metastasis. SCCA She went to outpatient for over 2 months due to pruritus vul-
plays an important role in the monitoring of disease during vae. The cervical diameter was 3 cm, smooth, showing con-
and after treatment of cervical cancer. If the SCCA value does tact bleeding. The corpus uteri were in a horizontal position,
not decrease or increase instead after the treatment, it means normal size, with no obvious tenderness. Colposcopy results
a poor curative effect. In addition, the pretreatment SCCA showed that the cervical canal may be related to the low-level
value can also serve as an important reference for the need for glandular intraepithelial lesion (LG-CGIN) combined with a
adjuvant therapy after surgery. The decrease of SCCA value low-level squamous intraepithelial lesion (LSIL- CIN1).
after treatment is closely related to chemotherapy response, Higher risk of lesions may be caused, and it is recommended
where a significant decrease of SCCA means a good progno- to perform diagnostic cervical conization. The patient refused
sis. In most patients with recurrent cervical cancer, the first surgical treatment because she had fertility willingness. The
symptom is an increase in SCCA. SCCA is an important patient was informed for reexamination in 3–6 months. One
marker for cervical cancer diagnosis and disease monitoring. year later, the patient returned to the hospital of reexamina-
tion due to repeated abnormal vaginal discharge. After exami-
Serum Fragments of Cytokeratin (CYFRA) nation, the cervix had less erosion, and a neoplasm with a size
Cytokeratin is a major component of the epidermal cell cyto- of 0.5 cm × 0.5 cm was spotted in the cervical canal.
skeleton. CYFRA is the content of soluble keratin 19 frag-
ments in serum, which previously was used as a tumor Colposcopy When the patient was treated for the first time,
marker for lung cancer [50, 51]. It was reported that 42–52% colposcopy showed low-level squamous intraepithelial
of patients with squamous cervical cancer showed a CYFRA lesions of the cervix, developing chronic cervicitis; low-level
increase in blood [52]. Compared with SCCA, CYFRA is glandular intraepithelial lesion (LG-CGIN) combined with a
more popular in assessing the prognosis and therapeutic low-level squamous intraepithelial lesion (LSIL-CIN1) was
effect for cervical cancer. considered for the cervical canal, with P16 (+), and Ki67 8%.
When the patient was treated for the second time, cervical
biopsy under colposcopy + cervical canal neoplasms exci-
42.3.4 Management sion + cervical canal curettage was performed, and tissues
were sent for pathology. Pathological report of cervical ade-
According to the patient’s clinical-stage, age, medical level, nocarcinoma suggested the high possibility of common type
and equipment conditions, comprehensive consideration endometrial adenocarcinoma of the cervix; a small number
should be given to formulating individualized treatment of adenocarcinoma cell nests were seen in the neoplasms of
schemes, including surgery, radiation therapy, chemother- the cervical canal; a small amount of dissociative adenocar-
apy, and others. Surgical treatment can preserve the ovarian cinoma cell tissues were occasionally spotted in multiple
and vaginal function of patients, mainly for early cervical blood clots within the cervical canal.
cancer patients. Radiation therapy is mostly adapted for Laboratory values:
early to middle and advanced-stage patients, mainly includ- The results of the TCT examination at the first treatment
ing intracavity irradiation and external beam radiation. showed low-level squamous intraepithelial lesions. HPV
classification: HPV16 positive, other 12 high-risk HPV 13. Vrba L, et al. miRNA gene promoters are frequent targets of

positive. aberrant DNA methylation in human breast cancer. PLoS One.
2013;8(1):e54398.
The results of TCT examination at the second treatment 14. Van der Auwera I, et al. Integrated miRNA and mRNA expression
showed atypical glandular cells (AGC), with HPV16 and the profiling of the inflammatory breast cancer subtype. Br J Cancer.
other 12 high-risk HPV positive. HPV-DNA: 161.38 pg/mL. 2010;103(4):532–41.
15.
Kong W, et al. Up-regulation of miRNA-155 promotes
tumour angiogenesis by targeting VHL and is associated with
Results with Interpretation Guideline Existing studies have poor prognosis and triple-negative breast cancer. Oncogene.
shown that persistent infection of HPV-16 and HPV-18 can 2014;33(6):679–89.
lead to cervical adenocarcinoma. In this case, persistent infec- 16. Dede DS, Arslan C, Altundag K. Serum levels of CEA and CA
tion of HPV-16 caused continuous deterioration of the patient’s 15-3 in triple-negative breast cancer at the time of diagnosis. Med
Oncol. 2010;27(4):1429.
lesions, eventually resulting in cervical adenocarcinoma com- 17. Mallen AR, Townsend MK, Tworoger SS. Risk factors for ovarian
bined with high-level squamous epithelial lesions. This case carcinoma. Hematol Oncol Clin North Am. 2018;32:891–902.
suggested that detection of HPV infection is conducive to alert- 18. Hilliard TS. The impact of mesothelin in the ovarian cancer tumor
ing cervical cancer and further risk of malignancy. microenvironment. Cancers (Basel). 2018;10:277.
19. Miller D, Nevadunsky N. Palliative care and symptom management
for women with advanced ovarian cancer. Hematol Oncol Clin
Final Diagnosis The patient received total laparoscopic North Am. 2018;32:1087–102.
hysterectomy with bilateral adnexectomy, plus pelvic cavity, 20. Sharma S, Raghav R, O’Kennedy R, et al. Advances in ovarian can-
and aorta abdominal lymphadenectomy. The pathological cer diagnosis: a journey from immunoassays to immunosensors.
Enzym Microb Technol. 2016;89:15–30.
report suggested cervical adenocarcinoma combined with 21. Badgwell D, Bast RJ. Early detection of ovarian cancer. Dis

high-level squamous intraepithelial lesions (HSIL/CIN2). Markers. 2007;23:397–410.
22. Jia LT, Zhang YC, Li J, et al. The role of human epididymis protein
This case was from Jiangsu Women and Children Health 4 in the diagnosis of epithelial ovarian cancer. Clin Transl Oncol.
2016;18:233–9.
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Prenatal Diagnosis and Preimplantation
Genetic Diagnosis 43
Chengcheng Liu, Xiaoting Lou, Jianxin Lyu,
Jian Wang, and Yufei Xu
Nowadays, millions of individuals suffer from dominant or tilized in vitro, thereby preventing the transmission of these
recessive genetic mutations that cause highly severe or life- disorders to offspring. The Timeline map below provides an
threatening phenotypes in the world. In total, the Online overview of the evolution of both prenatal and preimplanta-
Mendelian Inheritance in Man (OMIM) database currently tion genetic testing (Fig. 43.1).
reports more than 4600 phenotypes with a genetic cause. In In 1966, karyotyping of cultured cells obtained from
fact, every individual carries alleles that in a homozygous amniotic fluid sampling, the first human prenatal genetic
state could cause recessive disorders. Besides, approximately test, was performed providing a chromosomal view of the
0.2% of the human population are carriers of a balanced fetus. In the following year, the first prenatal diagnosis of a
translocation that often causes infertility or recurrent miscar- chromosomal abnormality was achieved. Later, the introduc-
riages (owing to embryonically lethal segmental aneuploi- tion of chromosome-banding techniques in the 1970s led to
dies in the conceptuses), or severe birth defects in offspring. an increase in resolution and enabled the detection of seg-
To eschew the transmission of pathogenic genetic variants mental chromosomal imbalances. Over the next 40 years,
and to enable early discovery of genetic disorders, prenatal karyotyping became gold standard for prenatal diagnosis for
genetic testing (PGT) is introduced. Genetic testing can the genome-wide detection of genomic rearrangements,
potentially provide an accurate diagnosis to a fetus with despite its intrinsic limitations (including invasive procedure
developmental anomalies detected by ultrasonography and to obtain a tissue sample, culturing of cells, visual screening
enable its parents to make an informed decision about the for numerical or structural chromosome anomalies, and lim-
pregnancy. For couples who are known carriers of mutant ited resolution). Hence, there has been an unceasing pursuit
alleles, preimplantation genetic diagnosis (PGD) enables the for improving DNA-based molecular genetics techniques.
detection of genetic disorders in embryos that have been fer- From 1990s, as the development of molecular biology, a
series of technologies, such as fluorescence in situ hybridiza-
tion (FISH), quantitative fluorescence PCR (QF-PCR), mul-
C. Liu (*)
Department of Laboratory Medicine, The First Affiliated Hospital tiplex ligation-dependent probe amplification (MLPA) as
of Nanjing Medical University, Nanjing, Jiangsu, People’s well as array comparative genomic hybridization (aCGH)
Republic of China were introduced subsequently (Table 43.1). Figure 43.2
X. Lou shows the application of cell-free fetal DNA in aneuploidy
Key Laboratory of Laboratory Medicine, Ministry of Education screening in prenatal diagnosis, which is extensively used in
of China, School of Laboratory Medicine and Life Science, clinical practice at present.
Wenzhou Medical University, Wenzhou, Zhejiang,
People’s Republic of China Preimplantation genetic diagnosis (PGD) was intro-
duced at the beginning of the 1990s as an alternative to pre-
J. Lyu (*)
Zhejiang Provincial People’s Hospital, Affiliated People’s natal diagnosis, to avoid termination of pregnancy in
Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, couples with a high risk for offspring affected by a sex-
People’s Republic of China linked genetic disease. At that time, embryos obtained
e-mail: ljx@hmc.edu.cn in vitro were tested to ascertain their sex, and only female
J. Wang (*) · Y. Xu embryos were transferred to uteri. Since then, techniques
Department of Molecular Genetic Diagnostics, Shanghai for genetic analysis at the single-cell level, involving
Children’s Medical Center, Shanghai Jiao Tong University
School of Medicine, Shanghai, People’s Republic of China assessment of first and second polar bodies from oocytes or
e-mail: labwangjian@shsmu.edu.cn blastomeres from cleavage-stage embryos, have evolved.

770 C. Liu et al.
Fig. 43.1 Timeline of prenatal and preimplantation genetic diagnostics. cell-free fetal DNA; FISH fluorescence in situ hybridization; IVF in vitro
Key milestones in the implementation of genetic tests for invasive (gray) fertilization; MLPA multiplex ligation-dependent probe amplification; NGS
and noninvasive (blue) prenatal and preimplantation diagnosis. aCHG next-generation sequencing; PGD pre-implantation genetic diagnosis;
array comparative genomic hybridization; ADO allele drop out; cfDNA QF-PCR quantitative fluorescence PCR; WES whole-exome sequencing
Table 43.1 Prenatal diagnostic tests

Tests Invasive Specimen Item Utilities
Biochemical markers N Maternal serum AFP, uE3, β-HCG, Inhibin A ONTDs, trisomy 18, trisomy 21
NIPT N cffDNA, fNRBCs MPSS, TMPS, SNP-based MPS Aneuploidies, deletions, duplications
Karyotype Y AF, CV NA Aneuploidies, chromosome abnormalities
Molecular DNA Y AF, CV RFLP, QF-PCR, MLPA Mutations and aneuploidy
testing
FISH Y AF, CV NA Aneuploidies, deletions, or duplications
CMA Y AF, CV aCGH, SNP microarray Uniparental disomy, aneuploidies, DNA copy number
NGS Y AF, CV WGS, WES Discovery of novel mutations
Note: + possible; − not possible; +/− possible but requires specific work-up; NA not available; AF amniotic fluid; CV chorionic villus; CVS chorionic
villus sampling; AFP alpha-fetoprotein; uE3 unconjugated estriol; β-HCG β-human chorionic gonadotropin; DIA dimeric inhibin A; ONTDs open
neural tube defects; NIPT non-invasive prenatal testing; cffDNA cell-free fetal DNA; FISH fluorescence in situ hybridization; fNRBCs fetal nucleated
red blood cells; MPS massively parallel sequencing; MPSS massively parallel shotgun sequencing; TMPS targeted massively parallel sequencing;
RFLP restriction fragment length polymorphisms; QF-PCR quantitative fluorescent PCR; MLPA multiplex ligation-dependent probe amplification;
FISH fluorescence in situ hybridization; CMA chromosomal microarray analysis; aCGH array comparative genomic hybridization; SNP Microarray
single nucleotide polymorphism microarray; NGS next-generation sequencing; WGS whole-genome sequencing; WES whole-exome sequencing
43 Prenatal Diagnosis and Preimplantation Genetic Diagnosis 771
Fig. 43.2 Cell-free fetal

DNA aneuploidy screening
methods. Two major strategies
are widely implemented in
routine cell-free fetal DNA
(cfDNA) aneuploidy testing;
random and targeted
sequencing. Chr
chromosome; NGS next-
generation sequencing
FISH has been introduced for the analysis of chromosomes revolutionized prenatal genetic testing; that is, genetic test-
and PCR for the analysis of genes in cases of monogenic ing from conception until birth. Newly developed technolo-
diseases (Fig. 43.3). gies, such as Genome-wide single-cell arrays and high
throughput sequencing analyses, are significantly improving
our ability to detect embryonic and fetal genetic abnormity,
43.1 Noninvasive Prenatal Testing and have substantially improved embryo selection for in vitro
fertilization (IVF). Moreover, both invasive and noninvasive
Chengcheng Liu mutation scanning of the genome are helping to identify the
genetic causes of prenatal developmental disorders. These
advances are changing clinical practice and pose novel chal-
43.1.1 Overview lenges for genetic counseling and prenatal care [2].
Traditional invasive procedures to detect such genomic
Genomic abnormalities are a leading cause of birth defects abnormalities could pose a relative risk to both mother and
and pregnancy complications, including in utero growth unborn fetus potentially. The discovery of free fetal DNA
retardation and risk of miscarriage [1]. In the past decade, it (ffDNA) in maternal circulation marked the beginning of the
has been observed that the development of technologies have Noninvasive prenatal testing (NIPT) era, and allowed the
772 C. Liu et al.
a
PB
Female pronucleus
Male pronucleus
Zygote 6- or 8-cell embryo Blastocyst
PB1 and PB2 Blastomeres

Trophectoderm cells
PGD PGS
(monogenic) (chromosomal)
b
FISH Multiplex qPCR aCGH NGS
Fluorescence
Read counts
Probe
Target
Cycles Chr1 Chr2 Chr3
c
Aneuploidy
or mutation
Embryo selection
No aneuploidy Select for transfer
or mutation
Fig. 43.3 Preimplantation genetic diagnosis and screening. (a) The screening. (b) The methods routinely applied in diagnostic services. (c)
developmental stages of an in vitro fertilization (IVF)-derived embryo Embryos are prioritized for transfer to the uterus. PB polar body
and the type of cells used for pre-implantation genetic diagnosis and
development of the leading noninvasive prenatal tests [3]. cuss here the principle, methods of detection as well as appli-
NIPT is a method that determines the genomic status of a cations of NIPT.
fetus in utero by analyzing circulating fetal DNA in maternal
plasma or serum. Recently, a series of clinical studies have
shown that human cell-free detection of trisomies 21, 18, and 43.1.2 Laboratory Examination
13 in singleton pregnant subjects has resulted in fewer false-
positive and higher positive prediction rates than using the 43.1.2.1 C ell-Free Fetus DNA(cffDNA) and Its
previous standard screening procedures. Furthermore, cou- Detection in Maternal Plasma
pled with the use of more advanced and refined molecular Cell-free DNA (cfDNA) is composed of small fragments of
technologies, NIPT is becoming a promising clinical tool for extracellular DNA that circulate freely in the maternal
the screening and diagnosis of subchromosomal abnormali- plasma. The cfDNA in gravida is consist of two parts: the
ties and monogenic diseases (e.g., cystic fibrosis, sickle cell majority is maternal in origin and the rest, only accounting
disease, and β-thalassemia) [1]. In this section, we will dis- for around 10% on average, is from the fetus (cffDNA) that
is released from the placenta at around 4 weeks’ gestation. Different diagnostic purposes require different sequencing
The cffDNA fragments which represent the entire fetal depths. Deeper sequencing allows one to discover less com-
genome are pregnancy specific as they are rapidly eliminated mon nucleic acid sequence variants, for instance, to distin-
from the maternal circulation after delivery. Therefore, anal- guish a paternally inherited pathogenic cytosine (C) variant
ysis of cfDNA offers significant potential for use in prenatal in fetal DNA from the majority of sequence reads from
diagnosis. However, analysis is less reliable when the pro- maternal DNA that carries the normal adenine (A) allele,
portion of cffDNA in the maternal circulation is below 4%. which could be missed at low sequencing depth because of
The fetal fraction increases with advancing gestation and, random error. In contrast, the sequencing depth required for
while this often reaches levels sufficient for testing for some chromosomal aneuploidy detection is lower. Sequencing
applications by around 7 weeks’ gestation, in the majority of coverage, another important parameter, is sometimes used
women. In order to ensure high accuracy, many tests are not interchangeably with depth, but it has also been specifically
performed until later gestations [4]. defined as the percentage of target bases that are sequenced a
NIPT is generally defined as the screening, testing, and given number of times (the “breadth” of sequencing). In
diagnosis of fetal chromosomal or genetic conditions by recent years, the ability to deep sequencing cfDNA in the
analysis of cell-free fetal nucleic acids in maternal plasma maternal plasma has enabled various newer developments in
or serum. In 1997, the presence of cffDNA was reported by the field of NIPT [5]. At present, two primary approaches
Lo and colleagues firstly. Conventionally, the detection of used for MPS-based NIPT are massively parallel shotgun
fetal chromosomal or genetic conditions requires sampling sequencing (MPSS) and targeted massively parallel sequenc-
of fetal or placental tissues by amniocentesis or chorionic ing (TMPS). In both methods, sequence reads can be used to
villus sampling, which are invasive procedures with a small detect a slight excess of fetal genomic material coming from
but definite risk of fetal loss and maternal morbidity. As a the chromosome of interest. Nevertheless, MPSS produces a
result, noninvasive means of prenatal testing are demand- large number of sequence reads from all chromosomes while
ing in the past few decades. Before the discovery of cffDNA TMPS generates a larger proportion of reads from the chro-
in maternal plasma, circulating fetal cells had been the pri- mosomes of interest.
mary focus of NIPT research, but the rarity of these cells Quantifying the fetal DNA fraction is important so as to
and the difficulty in isolating them limited the development ensure that the threshold of detection is reached for these
of this approach. Thus, the research focus switched to the testing methods, achieving high sensitivity and specificity,
analysis of circulating cell-free fetal nucleic acids. cffDNA which are required for a diagnostically applicable clinical
is a minor cell-free DNA species admixing with cell-free test. There are various methods for quantifying cffDNA, the
maternal DNA that is dominant in circulating cfDNA. On first of which involves the analysis of size distribution of cir-
average, 10–20% of cfDNA in maternal plasma is origi- culating DNA. Compared with cfDNA original from gravida
nally from fetus. This percentage varies among individuals in maternal plasma, these cffDNA fragments are relatively
and generally increases with gestational age until delivery, short, 99% being shorter than 300 bp. A ratio of short to long
after which fetal DNA is rapidly cleared from the maternal read-lengths generated by whole-genome sequencing can
plasma within hours. cffDNA, likely from apoptotic tro- accurately predict the fetal DNA fraction relative to maternal
phoblasts in placenta, can be detected as early as 5 weeks of cfDNA [1].
gestation, whereas cell-free maternal DNA has a predomi-
nant hematopoietic origin. Unlike intact genomic DNA, 43.1.2.2 Fetal Nucleated Red Blood Cells
cfDNA in maternal plasma exists in fragments, and cffDNA cffDNA-based NIPT is widely used in the clinical practice
is generally shorter than cell-free maternal DNA. The peak for screening for fetuses with trisomy 21, 18, and 13 in high-
size of cell-free maternal DNA is 166 base pairs (bp), risk gravidas presents no risk of miscarriage and provides an
whereas for fetal DNA it is 143 bp. The distribution of fetal economical, convenient, and effective method compared to
and maternal DNA across the entire genome is relatively other invasive technologies. However, the limitations of
even [5]. cffDNA-based NIPT remain: (1) it cannot eliminate chromo-
Among all molecular techniques used for characterization somal anomalies like mosaicism, duplication, and deletion;
of cell-free nucleic acids, massively parallel sequencing (2) limited data are currently available on the use of NIPT in
(MPS) has significantly accelerated the evolution of this twins and multiple pregnancies; (3) cell-free DNA cannot be
field since it emerged, as it enables millions to billions of used to distinguish specific abnormalities such as
nucleic acid molecules in plasm to be sequenced in a high- Robertsonian translocation and high-level mosaicism; (4)
throughput and comprehensive manner at single nucleotide samples from gravidas with low-level mosaicism or solid
resolution. An important parameter of data generated by tumor as well as a high body mass index (BMI) or early ges-
MPS is the sequencing depth, which is the number of reads tational age will result in variations of circulating cffDNA
that a certain base in the reference genome is sequenced. impacting prenatal testing results [6].
774 C. Liu et al.
genomics-based noninvasive prenatal testing (gNIPT) to

analyze the fetal genome [17]. Table 43.2 shows the charac-
teristics of commonly reported chromosomal aneuploidies in
literature. Two updated meta-analysis showed that MPSS
and TMPS perform similarly in terms of clinical sensitivity
and specificity for the detection of fetal T21, T18, and T13.
However, as to the replacement of invasive tests, the perfor-
mance of gNIPT observed is not sufficient to replace current
invasive diagnostic tests [17, 18]. In addition, the fNRBCs
presented exciting performances in the diagnosis of chromo-
somal aneuploidy.
Although NIPT presents high sensitivity and accuracy
Fig. 43.4 Schematic showing NIPT using fetal nucleated red blood in aneuploidy diagnosis, the existence of infrequent con-
cells (fNRBCs)
fined placental mosaicism (CPM) may cause a discrep-
ancy between NIPT and Amniotic Chromosomal Test,
Due to inherent drawbacks of cffDNA in NIPTs, atten- probably false-positive results [19]. Hayata K et al. expe-
tion is attracted to circulating fetus-derived cells in the rienced a case of advanced maternal age in which a fetus
maternal bloodstream. Till now, four types of fetal nucle- was found to be positive for trisomy 18 at re-examination
ated cells have been reported: trophoblasts, fetal nucleated following indeterminate NIPT. Later, amniotic fluid chro-
red blood cells (fNRBCs), hematopoietic progenitor cells, mosomal test revealed a normal karyotype, and CPM was
and lymphocytes [7–9]. Among these, fNRBCs are the pre- observed in a SNP microarray analysis of the placenta.
ferred choice for NIPTs owing to their unique characteris- Finally, the child was born with no defects or complica-
tics. First, fNRBCs have intact nuclei containing the total tions [20].
fetal genome for prenatal analysis. And second, fNRBCs
have distinct cell markers, such as epsilon hemoglobin 43.1.3.2 Subchromosomal Abnormalities
transferrin receptor (CD71), thrombospondin receptor As the methods for detecting aneuploidy through NIPT
(CD36), GPA, and antibody 4B8/4B9, enabling isolation of becomes more refined and more reliable, the possibility to
these rare cells from large volumes of maternal blood [10– detect abnormalities beyond the chromosome copy number
15]. Recently, a highly sensitive and rapid isolation method becomes true. Subchromosomal abnormalities (mainly
has been developed for NIPT using fNRBC-specific anti- including deletions or duplications) in the genome have far-
body (anti-CD147) and a capture-releasing material that is reaching influences on the developing fetus. Several studies
composed of biotin-doped polypyrrole nanoparticles [6, have shown the potential of extending NIPT to detect fetal
16]. Figure 43.4 describes the principle of NIPT using deletion/duplication syndromes from maternal plasma.
fNRBCs. However, the detection of subchromosomal abnormalities
requires deep sequencing, which is costly and time-
consuming, and thus, is limited to only a handful of patients
43.1.3 Clinical Application [21, 22]. Recently, a work by Yin and colleagues showed that
NIPT could reliably detect subchromosomal deletions/dupli-
43.1.3.1 Chromosomal Aneuploidy cations in women carrying high-risk fetuses by using less
Aneuploidies are chromosomal abnormalities characterized sequencing depth at a lower cost. The study, which used a
by a different (additional or missing) number of chromo- semiconductor sequencing platform (SSP), demonstrated
somes than that normally present in humans (23 pairs). These that increased concentration of cfDNA and increased
chromosomal anomalies are among the most common types sequencing depth could improve the detection of these
of genetic disorders and they cause significant morbidity or abnormalities [23]. Besides, cell-based NIPT method also
death in both childhood and adulthood. Due to the serious has the capacity to reliably diagnose fetal subchromosomal
health consequences of various aneuploidies and given their abnormalities [24].
incurable nature, prenatal screening is an option essential for Although the accuracy of this method was less than that
pregnant women. Traditionally, pregnant women, who were of chromosomal microarray analysis, especially for dele-
found to be at high risk of fetal aneuploidy after the prenatal tions/duplications smaller than 5 Mb, it may offer the
screening, were suggested to undergo an invasive diagnostic advantage of unbiased testing, and is substantially more
test (e.g., amniocentesis) which could cause a procedure- accurate than current blood-based biomarker screening
related risk of miscarriage. The discovery of circulating tests (e.g., AFP, HCG, and estriol) for aneuploidy. At a
cfDNA in maternal blood has enabled the development of sequencing depth of 3.5 M reads, this report detected 75%
Table 43.2 Characteristics of reported chromosomal aneuploidies

Other
Aneuploidies name(s) Prevalence Clinical features Prognosis
Trisomy 21 Down 1 in 800 Intellectual disability (mild to moderate), heart defect, Mean and median life expectancies
syndrome newborns gastroesophageal reflux, celiac disease, hypothyroidism, are estimated to be 51 and 58 years
a characteristic facial appearance, hypotonia, cognitive old
delays, delayed development (speech, language, motor),
mood disorders, autism spectrum disorder
Trisomy 18 Edwards 1 in 5000 Intellectual disability (severe), intrauterine growth Many individuals with trisomy 18 die
syndrome newborns retardation, low birth weight, heart defects and before birth or within their first
abnormalities of other organs, abnormal head, small jaw month. Only 5 to 10 percent of
and mouth, clenched fists with overlapping fingers children with this condition live past
their first year
Trisomy 13 Patau 1 in 16,000 Intellectual disability (severe), heart defects, brain or Many infants with trisomy 13 die
syndrome newborns spinal cord abnormalities, microphthalmia, extra fingers within their first days or weeks of life.
or toes, cleft lip with or without cleft palate, hypotonia Only 5 to 10 percent of children with
this condition live past their first year
45,X Turner 1 in 2500 Female, infertile, short stature, ovarian hypofunction, Mortality in 45,X women is threefold
syndrome newborn webbed neck, low hairline, lymphedema of hands and higher than in the general population
girls feet, skeletal abnormalities, kidney problems, heart with an average life span of 69 years
defect
47,XXX Trisomy X 1 in 1000 Female, normal sexual development,able to conceive Mortality significantly increased with
newborn children, learning disabilities, delayed development a median survival age of 70.9 years
girls (speech, language, motor), hypotonia, behavioral and compared to 81.7 years for euploid
emotional difficulties, seizures, kidney abnormalities females
47,XXY Klinefelter 1 in 650 Male, infertile, small testes, delayed or incomplete Life expectancy is slightly shorter
syndrome newborn puberty, gynecomastia, decreased muscle mass and bone (approximately 2 years) than euploid
boys density, cryptorchidism, hypospadias, micropenis, tall men
stature, delayed development (speech, language, motor),
mood disorders, autism spectrum disorder, metabolic
syndrome
47,XYY Jacob’s 1 in 1000 Male, normal sexual development, able to father Mortality increased with a reduction
syndrome newborn children, learning disabilities, delayed development of life span of 10.3 years compared to
boys (speech, language, motor), hypotonia, mood disorders, euploid men
increased belly fat, macrocephaly, macrodontia, pes
planus, clinodactyly, hypertelorism, scoliosis
48,XXXX Tetrasomy NA Female, mental retardation, small head, wide-set eyes, NA
X expressionless face, epicanthic folds, absent
menstruation, weak eye muscles, webbed neck, mild to
severe mental retardation, growth restriction, hypotonia,
ovarian failure
48,XXXY XXXY 1 in 17,000 Male, infertility, intellectual disability (mild), tall NA
syndrome to 50,000 stature, learning difficulties, delayed development
newborn (speech, language, motor), mood disorders, hypotonia,
boys hyperextensibility, elbow abnormalities, fifth finger
clinodactyly, pes planus, ocular hypertelorism,
upslanting palpebral fissures, epicanthal folds, short
penis, cryptorchidism, small testes, gynecomastia
48,XXYY XXYY 1 in 18,000 Male, infertility, tremor, dental problems (delayed teeth The standardized incidence ratio and
syndrome to 40,000 appearance, thin tooth enamel, crowded and/or standardized mortality ratio for
males misaligned teeth, multiple cavities), peripheral vascular non-Hodgkin lymphoma in patients
disease, deep vein thrombosis, allergies, asthma, type 2 with a 48,XXYY constitution was
diabetes, seizures, and congenital heart defects. Other 36.7 (95% CI = 4.4 to 132.5) and 32.6
clinical features are likely similar to that in 48, XXXY (95% CI = 6.7 to 95.4).
NA not available
more abnormal karyotypes compared to aneuploidy alone. 43.1.3.3 Single Gene Mutations
In the long term, sensitivity could be further improved NIPT is not confined to detect common chromosome disor-
with greater sequencing depth. Thus, as no specific prena- ders as well as clinically significant copy number variations.
tal screening currently exists for subchromosomal abnor- Monogenic disorders, generally caused by deletion/inser-
malities routinely, it could represent just a powerful tions and even a single base mutation, require base-level
extension of NIPT. resolution in determination. Nevertheless, the level of circu-
776 C. Liu et al.
lating cffDNA is very low in maternal plasma at 11–17 weeks Therefore, other approaches are required. One strategy
of gestation. For example, the average size of the circulating was based on epigenetic markers. Some genes have been
cffDNA could be as short as 166 bp, and its concentration identified to display a differential methylation pattern in
could be as low as 25 genome copies/mL. Thus, conventional maternal blood cells and in fetal placenta. For example, the
diagnostic approaches faced great challenges [25, 26]. maspin gene (SERPINB5) promoter is unmethylated in the
Via approaches such as relative mutation dosage (RMD) placenta but hypermethylated in maternal blood cells, while
for β-thalassemia, hemophilia, and sickle cell disease, NIPT the RASSF1A gene (a tumor suppressor gene) is unmethyl-
has already been proved to be have the possibility of per- ated in maternal cells and hypermethylated in the placenta,
forming in monogenic diseases [27–30]. Other approaches allowing us to distinguish maternal from fetal DNA [40–42].
include SNPs for whole-genome sequencing, direct linear Another strategy of identifying female fetuses from maternal
amplification and quantification for Wilson’s disease, rela- plasma was to use paternally inherited short tandem repeats
tive haplotype dosage for congenital adrenal hyperplasia (STRs) located on the X chromosome [43].
(CAH), and β-thalassemia. Furthermore, with the develop- As NIPT can be performed without posing a risk to the
ment of digital PCR and NGS technology, novel detections, pregnancy, it could lead to an increase in such requests.
such as paternally inherited mutant alleles, mutations arising However, ethical concerns about the use of NIPT for non-
de novo, genotyping, and quantitation of circulating fetal medical traits and objectification of the child have been
DNA in maternal plasma become reality. When the informa- raised and thus should not be neglected [44, 45].
tion on parental and proband haplotypes was provided, doc-
tors can construct fetal haplotypes and then generally 43.1.3.5 Hemolytic Disease of the Newborn
diagnose the genetic disease. RhD antigen is an important blood type antigen expressed on
the surface of red blood cells of human beings. Among the
43.1.3.4 Fetal Sex Determination population, the majority are RhD positive in blood type,
The first application of cffDNA in maternal plasma was while very few people present RhD negative. When a woman
aimed to determine the fetal gender. It is extremely important whose blood type is RhD negative is pregnant, it is likely that
for a mother who is a carrier of an X-linked disorder (such as the fetus might have a RhD-positive blood type. When the
Duchenne muscular dystrophy or hemophilia), because preg- fetal RhD-positive cells enter maternal circulation, an
nancies with male fetuses are primarily at high risk. Besides, immune response, which is termed as “sensitisation,”
it is also beneficial for pregnant women who are at risk of launches leading to the production of anti-D antibodies
conditions associated with ambiguous development of exter- against the RhD antigen. The response can happen at any
nal genitalia (e.g., congenital adrenal hyperplasia), because time during the pregnancy, but it is most common in the third
early maternal treatment with dexamethasone can protect a trimester and during childbirth [46]. The process of sensiti-
female fetus from serious virilization [31, 32]. zation itself has few adverse effects on the mother and does
As for detection of male fetus-specific DNA in maternal not usually affect the first fetus. However, in the next preg-
plasma, the most commonly used technology is qPCR, which nancy with an RhD-positive fetus in women already sensi-
can amplify the single copy SRY (sex-determining region Y) tized, the anti-D antibodies in maternal plasma may cross the
gene, the single copy sequence DYS14, and the multicopy placenta into the fetal blood resulting in hemolytic disease of
DAZ gene [33–35]. But when the analytical tests were per- the fetus and newborn. This can cause severe fetal anemia
formed ahead of 7 weeks of gestation, it was found that the that leads to a series of fetal complications, e.g., heart failure,
results were unreliable because of the low cffDNA level [36]. fluid retention and swelling (hydrops), hyperbilirubinemia,
However, identification of a female fetus using NIPT is kernicterus, and even perinatal death [47].
based on the null results of Y chromosome-specific sequences In NIPT of fetal RhD status, a real-time qPCR method is
and therefore may lead to the potential of false-negative used to detect cffDNA, which are small fragments of extra-
results if the amount of male fetal DNA is too low to be cellular DNA derived from the placenta in the maternal
detected [37]. Besides, organ transplantation status could plasma. Furthermore, via application of automated plat-
also affect the determination. For example, Neofytou M and forms, high-throughput NIPT is capable of performing large
colleagues presented an exceptional case where NIPT con- numbers of tests at the same time, and is therefore suitable
tradicts the ultrasound-based sex determination. The preg- for large-scale population screening of pregnant women. As
nant woman was a recipient of a liver transplant from a male a result of the unclear fetal RhD status, once RhD-negative
donor. Thus, graft-derived cell-free male DNA released into women conceive a fetus, anti-D immunoglobulin should be
the maternal circulation interfered with the NIPT-based sex used to prevent the pregnant mothers from sensitization via
determination. Hence, NIPT is not suitable for gravidas blocking the production of anti-D antibodies. If the fetus is
underwent an organ transplant [38]. In addition, maternal RhD-negative, the risky therapy seems to be unnecessary.
mosaicism should also be considered in practice [39]. With the help of NIPT, fetal RhD status can be determined
and may enable anti-D immunoglobulin to be retreated from quantification has been documented as a promising bio-
RhD-negative women who are carrying an RhD-negative marker for the prediction of pre-eclampsia [55]. Rolnik’s
fetus. In addition, these women may not need extra treatment research shows that the lower the cffDNA, the higher were
of anti-D immunoglobulin following potentially sensitizing the risks for preeclampsia, but its capacity to act an as inde-
events, and there may no longer be a requirement for sero- pendent first-trimester marker in an algorithm for screening
logic cord testing at birth. High-throughput NIPT has already for pre-eclampsia requires further research [56]. On the con-
been used in this way in some European countries [48, 49]. trary, other literatures also reported pre-eclampsia may be
A lately published systematic review has shown that high- associated with elevated cffDNA in maternal serum [57–59].
throughput NIPT testing presents high diagnostic perfor- Thus, well-designed further research remains needed.
mance for the detection of fetal RhD status in RhD-negative
women, with very low false negative and false positive rates
in women tested at or after 11 weeks’ gestation. After all, 43.1.4 Summary
high-throughput NIPT which could be used as a routine
screening test for fetal RhD status in RhD-negative women The discovery of circulating cffDNA was inspired by reports
can largely remove unnecessary exposure to prophylactic of circulating tumor DNA in cancer patients [3]. This opened
anti-D treatment [50]. a tremendous era of research into the use of cfDNA for
developing noninvasive methods for extracting fetus-specific
43.1.3.6 Other Applications information during early pregnancy. Chromosomal aneu-
Indeed, NIPT can rapidly screen fetal abnormality in genes, ploidy is a leading cause of fetal miscarries and birth defects,
meanwhile, it could also be extensionally used for maternal so attentions are primarily focused on how to use fetal
health status monitoring, such as tumor and pre-eclampsia. cfDNA as a potential noninvasive source of genetic informa-
Similar to placental DNA, tumor DNA can also be detected tion. And this could facilitate the detection of aneuploidies.
in plasma, and therefore cell-free tumor DNA can be ana- With the progress of biotechnology, especially the now
lyzed for characterization and monitor cancers. Plasma DNA available deep sequencing and single molecule PCR meth-
analysis allows for pre-symptomatic detection of tumors in ods, scientists are exploring more specific fetal genomic
pregnant women undergoing routine NIPT. Amant F and col- abnormalities, including monogenic diseases and large chro-
leagues reported that during NIPT in over 4000 prospective mosomal insertion/deletion mutations. Although promising,
pregnancies by parallel sequencing of maternal plasma cell- NIPT still draws skepticism because some recent reports
free DNA, 3 aberrant genome representation (GR) profiles claim that compared to ultrasonography techniques, up to
were observed that could not be interpreted by the maternal 8% of NIPT studies have underdiagnosis. Nevertheless, the
or fetal genome. Those 3 patients were performed whole- variety of potentially life-altering genetic diseases has cer-
body diffusion-weighted magnetic resonance imaging tainly not been exhausted, and consequently, the possibility
(MRI), which revealed an ovarian carcinoma, a follicular of delivering an accurate prenatal diagnosis of such condi-
lymphoma, and a Hodgkin lymphoma, each confirmed by tions remains a promising research avenue [1].
subsequent pathologic and genetic investigations. In addi-
tion, the copy number variations in the subsequent tumor
biopsies were accordant with the previous NIPT plasma GR 43.1.5 Typical Medical Case
profiles [51]. Also, Cohen and colleagues reported a proof-
of-concept method describing the early diagnosis of ovarian Clinical Background The patient was a healthy pregnant
cancer using sub-chromosomal changes in plasma DNA in woman, whose age, height, and weight were 34 years,
the nonpregnant women, identified with a routine NIPT plat- 164 cm, and 71 kg, respectively. The history of gestation was
form, but the debate still exists [52, 53]. And the mutual gravida 2, para 1.
influence between circulating tumor DNA and circulating
fetal DNA needs further investigated [54]. Imaging Examination The ultrasound examination at a
Hypertensive disorder during pregnancy, especially pregestational age of 26 weeks indicates that besides low-lying
eclampsia, is one of the major risk factors of increased mor- placenta, the fetus has a left ventricular bright spot and a
bidity and mortality of pregnancy and perinatal period all small amount of pericardial effusion.
over the world. Early prediction of pre-eclampsia, the need
for modern obstetrics, can timely prevent the progress of the Test Performed NIPT results indicated that chromosome
disease and reduce related fetal and maternal morbidity and 21 was abnormal. Then, prenatal diagnosis was performed,
mortality. In addition to the screening of fetal aneuploidies, including karyotyping and chromosome microarray
Rhesus-D status, fetal sex, single gene disorders, the cffDNA analysis.
778 C. Liu et al.
NIPT Z-score of chromosome 21 was −6.876 suggesting respiratory chain structural proteins. Unlike the nuclear
occurence of deletions, and the deletion was approximately genome (nDNA), mtDNA lacks an intron–exon structure.
18 Mb (Fig. 43.5a). Furthermore, mtDNA follows a maternal inheritance while
the nDNA is inherited in a Mendelian manner. Lastly, each
Chromosomal Microarray-Based Analysis Chromosomal cell contains different copies of mtDNA, according to differ-
microarray-based analysis results showed about 19.2 Mb ent cell types the number can vary from a few hundred to
deletions in 21q11.2-q22.11 (15,016,486-34,251,578)x1 tens of thousands [62, 63]. The mitochondrial genomes have
(Fig. 43.5b, c). their own functions of replication, transcription, and transla-
tion, controlled by the Displacement Loop (D-Loop).
Chromosome Karyotype Analysis Karyotype analysis of D-Loop is a ~ 1.1 kb single non-coding region. There are
amniotic fluid showed chromosome structural abnormalities about 1500 mitochondrial proteins in total, which are
46, XN, del(21)(q11.2q22.1). The analysis of chromosome encoded by both mitochondrial genome and nuclear genome
karyotype of the parents showed no obvious abnormalities (13 of them are encoded by mtDNA). Among the 1500 pro-
(Fig. 43.5d). teins, only about 150 proteins are directly involved in
OXPHOS and ATP production. Assembly of the complexes,
Final Diagnosis The deletion region (21q11.2-q22.11) con- maintenance and expression of mitochondrial DNA, protein
tains some disease-causing genes, including LIPI, LPDL, synthesis, and mitochondrial dynamics counts for the rest.
PRED5, PRSS7, ENTK, APP, AAA, CVAP, AD1, SOD1,
ALS1, MRAP, FALP, C21orf61, GCCD2, FGD2, C21orf59, 43.2.1.2 Mitochondrial Diseases
CILD26, SYNJ1, PARK20, EIEE53. The deletion of two or Mitochondrial diseases are a group of clinical heterogeneous
more genes in this region is critical. To a great extent, it will genetic disorders, can be caused by deleterious variants both
have birth defects if the fetus is born. Finally, the parents in mitochondrial genome and nuclear genome. Mitochondrial
opted for termination of pregnancy. disease is one of the most common subtype of hereditary
disease, the prevalence of mitochondrial diseases in children
This case was from The First Affiliated Hospital of Air is estimated to be 5 ~ 15 in 100,000 [64, 65]; while which is
Force Medical University. ~12.5 in 100,000 among adults [66]. According to a cohort
study in England, nuclear genome mutant rate is estimated to
be 2.9 per 100,000 and mitochondrial genome mutant rate is
43.2 Mitochondrial Deafness around 9.6 per 100,000 [66]. Mitochondrial diseases can be
observed at any age, such as neonates, kids, teenagers, and
Xiaoting Lou and Jianxin Lyu adults; as mitochondria are ubiquitous in the human body,
mitochondrial diseases can be associated with any tissues or
organ with high energy demands (e.g., heart, brain, skeletal
43.2.1 Introduction muscle, cochlea). As describe, there are hundreds of and
thousands of mitochondrial DNA moleculars, normally, all
43.2.1.1 Mitochondria and Mitochondrial moleculars are the same in the tissues which is called homo-
Genome plasmy. However, when a tissue contains both mutant
Mitochondrion is a two phospholipid bilayers-bounded mtDNA and wildtype mtDNA, they are given the name of
organelle, exists in almost all eukaryotic cells except the heteroplasmy. The distribution of mutant and wild-type
erythrocytes. Mitochondrial complex V (ATP synthetase) moleculars determines the clinical manifestation of the
provides almost 90% of the adenosine triphosphate (ATP) patient, usually there exist a critical “threshold” (minimum
required for the body. Hence, mitochondria are usually called percentage of mutant mtDNA). Over the threshold, the mito-
as the “powerhouse” of the cells [60]. Mitochondrion is an chondria cannot function properly, causing mitochondrial
essential cellular organelle, which participates in a lot of piv- diseases. The severity of mitochondrial disease may vary
otal metabolic pathways not only the well-known oxidative throughout the lifetime, which partly induced by “Mitotic
phosphorylation (OXPHOS) but also fatty acid oxidation, segregation” of cells. During the division, the mutant mtDNA
amino acid metabolism, lipid metabolism, urea cycle, Krebs moleculars in daughter cells can vary a lot, which finally will
cycle, gluconeogenesis, and ketogenesis [61]. make accordingly changes in the clinical phenotype.
Mitochondria are the only organelles having their own With the development of next-generation sequence
genome in animal. Human mitochondrial genome (mtDNA) (NGS), the original method of sanger sequence is gradually
is a ~ 16.6 kb circular, double-stranded DNA (Fig. 43.6). get substituted with long-range polymerase chain reaction
Mitochondrial genome includes 37 genes, 2 rRNA genes, 22 (LR-PCR) and then NGS technology. The LR-PCR plus
tRNA genes, and 13 mRNA genes encode for mitochondrial NGS helps a lot in the diagnosis of mitochondrial genome-
Fig. 43.5 Analysis result of

fetal chromosome 21. (a)
Result of fetal chromosome
21 analyzed by NIPT. The red
dot area refers to the deletion
part chr21; (b) Result of fetal
chromosome 21 analyzed by
chromosome microarray
showing approximate
19.2 Mb deletions in
21q11.2-q22.11 of
chromosome 21; (c)
Microarray profile of
chromosome 21 showing the
region of deletions and the
corresponding genes in
OMIM; (d) The fetal
karyotype was 46,XN,del(21)
(q11.2q22.1)
780 C. Liu et al.
ing the balance, which can be divided into three anatomical

portions external (outer), middle, and inner region (Fig. 43.7).
There are several methods to describe and categorize the
hearing loss. Based on the anatomic defects, hearing loss can
be classified into conductive hearing loss (CHL), sensorineu-
ral hearing loss (SNHL), mixed hearing loss (both CHL and
SNHL exist); based on the etiology, hearing loss can be
mainly classified into genetically inherited hearing loss and
environment-induced hearing loss. As reported, among the
congenital hearing loss, genetically inherited hearing loss
count 50%, while among the prelingual hearing loss, genetic
count for 80%. Therefore, we need to focus more on geneti-
cally inherited hearing loss [73].
The inheritance pattern of hereditary hearing loss includ-
ing autosomal recessive, autosomal dominant, X-linked, and
mitochondrial inheritance. In this chapter, we will focus on
the mitochondrial inherited hearing loss and deafness. The
same as other mitochondrial DNA induced disease, mito-
chondrial deafness is maternally inherited, mainly affect the
tissues with high energy demand (e.g., cochlea), the onset
age is variable which will range from 5 to 50 years old, and
the hearing impairment degree also variable from mild to
profound but mostly progressive. Mitochondrial hearing loss
is belonging to the sensorineural hearing loss, not conductive
(Fig. 43.8). According to if the hearing loss is occurred with
other symptoms affecting other parts of the body, mitochon-
Fig. 43.6 Mammalian mtDNA
drial impairment can be classified into syndromic and non-
syndromic [73].
related diseases, with the accurate detection of the hetero-
plasmy of the sample. However, the effective treatment
methods still lacked here for mitochondrial diseases.
Although there are some investigations on mice model have 43.2.2 Clinical Appearance
received good news [67–69]. For example, Michele and his
colleagues applied the hypoxia treatment in a mouse model Hearing impairment is a frequent sign of mitochondrial dis-
with Leigh syndrome received evidence of reversion of disease with extremely variable onset and severity. The clinical
ease to a certain degree [70]. In the future, further investiga- phenotypic expression of mitochondrial hearing loss
tions and correlated therapeutic clinical trials are required to (mtDNA related) can be highly variable with several causes,
follow up. the mutation load of mtDNA (heteroplasmy); the nuclear
background, as He reported in 2013, the predisposition to
43.2.1.3 Deafness and Mitochondrial Deafness aminoglycoside antibiotics of mt.1477C>G mutation can be
Deafness is one of the major human health concerns. modulated by the nuclear gen modifier MTO2 [74]; the mito-
According to the World Health Organization, hearing impair- chondrial DNA haplogroup plays a role in the mitochondrial
ment affects about 5% population worldwide [71]. It is well disease clinical manifestation, Hudson and his colleagues
recognized that a normal hearing ability is critical for speech studied more than 3600 patients found the clinical expres-
and language skills development, however, the impairment sion of Leber Hereditary Optic Neuropathy can be affected
of hearing will impair the voice, speech, and language, which by the mitochondrial genome haplogroup group [75]; the
will furthermore do harm to the communication ability. environmental factors.
Conventionally, higher numbers of decibels (dB) indicating Cochlea, which is high energy required, is highly sensi-
worse hearing: normal hearing was defined when hearing tive to mitochondrial dysfunction. Accordingly, mitochon-
thresholds ≤25 dB in both ears, disabling hearing in adult drial disease is presumably occurred with sensorineural
defined when hearing thresholds >40 dB, and disabling hear- hearing loss. Mitochondrial sensorineural hearing loss could
ing in child defined when hearing thresholds >35 dB [72]. present in both syndromic hearing loss and non-syndromic
Ear is a critical organ, for detecting the sound and maintain- hearing loss forms.
structure of the human ears
Medical and birth history

Audiometric assessment of hearing loss
Three-generation pedigree and family
medical history
Physical examination
Suspect acquired hearing loss?
Yes No
Provide CMV testing, imaging, or Provide pre-test genetic counseling and genetic testing as clinically indicated:
other testing based on suspected • If syndromic hearing loss is suspected, consider targeted gene testing based
etiology (e.g., rubella, meningitis) on suspected diagnosis;
• If nonsyndromic hearing loss is suspected, consider single-gene tests such as
GJB2 and GJB6 gene panel tests, or NGS testing based on history and findings
Acquired etiology confirmed? Provide imaging or other testing as appropriate for suspected diagnosis
No or inconclusive Genetic etiology confirmed?

Yes
Reconsider potential acquired and
genetic etiologies
Yes No or inconclusive
Provide additional testing and
imaging based on findings
Provide treatment as clinically indicated
Provide follow-up counseling, including genetic counseling, as needed,

based on genetic and other test results and findings
Provide rolorrals to specialists, as needed, based on genetic and other

test results and findings
Provide follow-up care at periodic intervals based on genetic and other

test results and findings, and patient needs
Fig. 43.8 Schematic view of the evaluation and diagnosis of hearing loss from ACMG guideline
782 C. Liu et al.
43.2.2.1 M itochondrial Syndromic Hearing mutation, m.8344A>G mutation in MTTK gene is most com-
Loss mon. Patients with MERRF usually first experience the
As mentioned, mitochondrial sensorineural hearing loss myoclonus (muscle twitches) then followed the weakness,
could occur both in syndromic and non-syndromic forms. ataxia, and epilepsy, which is a canonic feature of
The auditory deprivation is categorized as mitochondrial MERRF. Ragged red fibers can be seen in most muscle biop-
syndromic hearing loss if mitochondrial sensorineural hear- sies of the patients. This syndrome was first described by
ing impairment present with other symptoms (e.g., neuro- Fukuhara in 1980 [81]. Most MERRF share the common
muscular signs, retinopathy, and kidney diseases). Here, we mitochondrial disease symptoms including learning disabil-
will mention some typical mitochondrial syndromic hearing ity, short stature, optic atrophy, and hearing loss. Hearing
impairment. loss in MERRF manifested with learning disability, short
stature, optic atrophy, and hearing loss. And hearing loss in
Kearn-Sayre Syndrome MERRF is the type of bilateral sensorineural hearing impair-
Kearn-Sayre syndrome (KSS; OMIM #530000) is a condi- ment which can appeared suddenly or gradually [82, 83].
tion caused by mitochondrial dysfunction. KSS appears
when mitochondrial oxidative phosphorylation could not 43.2.2.2 Mitochondrial Non-Syndromic
provide enough energy because the certain deletion of cer- Hearing Loss
tain genes in mitochondrial genome is inherited in a mito- The same as mitochondrial syndromic hearing loss men-
chondrial pattern (maternal inheritance). Depending on the tioned before, mitochondrial non-syndromic hearing loss
epidemiological study, KSS appears around 1–3 in 100,000 generally is sensorineural hearing loss with the impairment
[76]. The patient was first reported by Kearn in 1965 [77], to cochlea (a critical inner ear structure). Most mitochondrial
characterized by ophthalmoparesis and pigmentary retinopa- non-syndromic hearing loss are caused by mutations in
thy. KSS is an early onset disease, symptoms presumably MT-RNR1 and MT-TS1 (Table 43.3). MT-RNR1 encodes
appear before 20 years old. KSS is a kind of rare mitochon- mitochondrial 12S ribosomal RNA, and MT-TS1 encodes the
drial dysfunction induced progressive neuromuscular dis- serine transfer RNA. Both 12S ribosomal RNA and serine
ease with multi-system and multi-organ affected (e.g., central transfer RNA play a key role in mitochondrial protein
nervous system, musculoskeletal system, retina, kidney, synthesis.
liver, heart, and inner ear). As reported, hearing loss in
patients with Kearn-Sayre syndrome almost present in a MT-RNR1 Related Mitochondrial Hearing Loss
high-frequency hearing deteriorating form [78]. Due to the Sensorineural Hearing Impairment Induced by
energy deficits among KSS patients, the cochlea cannot work Aminoglycosides
properly, especially the external hair cells in basal coil who As well recognized, aminoglycoside antibiotics are widely
are responsible for the high-frequency sound [78]. used for Gram-negative bacilli infections. However, amino-
glycosides like gentamicin, kanamycin, tobramycin, and
Mitochondrial Encephalopathy with Lactic Acidosis, paromomycin may put patients at the risk of renal toxicity
and Stroke-like episodes and ototoxicity. Individuals with pathogenic variants in
Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke- MT-RNR1 can be associated with the predisposition to
like episodes (MELAS; OMIM#540000) is a syndrome due aminoglycosides-induced ototoxicity. Concisely, aminogly-
to the abnormality of mitochondrial oxidative phosphoryla- cosides destroy the bacteria by binding to the small subunit
tion function, usually caused by m.3243A>G mutation [79]. (30S) of the bacterial 70S ribosome, which will destroy the
MELAS is a multi-systemic syndrome with variable signs protein synthesis (translation). The difference between the
and symptoms includes encephalopathy, seizure, dementia, structures of 70S ribosomes (prokaryotes, each consisting of
lactic acidosis, muscle weakness, exercise intolerance, 30S and 50S subunits) and 80S ribosomes (eukaryotes, each
stroke-like episodes, learning disability, auditory depriva- consisting of 40S and 60S subunits) helps aminoglycoside
tion, and recurrent vomiting [79]. Most clinical appearance antibiotics combat the bacteria without doing harm to the
will first present between 2 and 40 years old. Hearing impair- human mammalian cells. However, the mutation in MT-RNR1
ment is not rare in patients with MELAS, according to a (e.g., mt.A1555G) will make the human mitochondrial 80S
cohort study of 45 patients, the frequency of hearing loss is ribosomes more similar to the 70S ribosomes, which will
almost 77% [80]. Hearing deficits in MELAS are mostly per- facilitate the aminoglycosides to bind to human ribosome
formed in a mild and progressive form. site [109]. Once the aminoglycosides are bound, they will
hold a long half-life in the hair cells (HCs) in the inner ear
Myoclonic Epilepsy with Ragged Red Fibers (even several months) finally lead to an ototoxicity.
Myoclonic epilepsy associated with ragged red fibers Hearing loss induced by aminoglycosides generally
(MERRF; OMIM#545000) is a multi-system involved mito- appears bilateral, severe to profound, most irreversible but
chondrial disorder, can be induced by mitochondrial DNA not progressive, and with a variable onset time (a few days to
Table 43.3 Mitochondrial mutations associated with hearing loss

Variations (nucleotide Clinical Aminoglycoside
Genes change) Product significance/status Additional features ototoxicity References
rRNA genes
MT-RNR1 m.827A>G 12S rRNA Drug response Non-syndromic SNHL + [84]
m.961delT/insC 12S rRNA Pathogenic Non-syndromic SNHL + [85]
m.961T>G 12S rRNA Pathogenic Non-syndromic SNHL + [86]
m.1095T>C 12S rRNA Drug response Non-syndromic SNHLor with + [87]
Parkinson, and neuropathy
m.1494C>T 12S rRNA Drug response Non-syndromic SNHL + [88]
m.1555A>G 12S rRNA Drug response Non-syndromic SNHL + [89]
tRNA genes
MT-TS1 m. 7444G>A tRNASer(UCN) Pathogenic Non-syndromic SNHL + [90]
m. 7445A>C tRNASer(UCN) Pathogenic Non-syndromic SNHLor with PPK / [91]
m. 7445A>G tRNASer(UCN) Pathogenic Non-syndromic SNHLor with PPK / [92–95]
m. 7471_7472insC tRNASer(UCN) Pathogenic Ataxia, dysarthria, myoclonus / [96]
m. 7505T>C tRNASer(UCN) Pathogenic [97]
m. 7510T>C tRNASer(UCN) Pathogenic Non-syndromic SNHL / [98, 99]
m. 7511T>C tRNASer(UCN) Pathogenic Non-syndromic SNHL / [100,
101]
m. 7512T>C tRNASer(UCN) Pathogenic Progressive myoclonic epilepsy, / [102]
ataxia, and hearing impairment
MT-TL1 m.3243A>G tRNALeu(UUR) Pathogenic MIDD, MELAS, PEO / [103]
m.3256T>C tRNALeu(UUR) Pathogenic MERRF / [104]
MT-TI m.4269A>G tRNAIle Pathogenic Cardiomyopathy / [105]
MT-TQ m.4336A>G tRNAGln Uncertain Migraine / [106]
significance
m.4336T>C tRNAGln Pathogenic Alzheimer, Parkinson, migraine / [107]
MT-TH m.12201T>C tRNAHis Pathogenic / [108]
SNHL sensorineural hearing loss; PPK palmoplantar keratoderma; MIDD maternally inherited diabetes and deafness; MELAS myoclonic epilepsy,
lactic acidosis, and stroke-like episodes; PEO progressive external ophthalmoplegia; MERRF mitochondrial encephalomyopathy with ragged red
fibers; rRNA ribosomal RNA; tRNA transfer RNA
weeks after the usage of aminoglycosides) [92]. However, ated with many health conditions, include MERRF,
the vestibular signs are not common under this situation Palmoplantar keratoderma with deafness [93], non-
according to a big cohort in China [110]. syndromic hearing loss, and other symptoms and signs. Non-
syndromic hearing loss induced by MT-TS1 has a wide array
Sensorineural hearing impairment without of clinical appearances [92]. The severity of hearing impair-
aminoglycosides exposure ment varies from mild, moderate, to profound. Most patients
MT-RNR1 pathogenic variants would render individuals pre- share the onset time of childhood, and with a progression of
disposition to aminoglycoside antibiotics; however, muta- the deterioration.
tions in MT-RNR1could also lead to hearing impairment
even deafness independent with an aminoglycosides expo-
sure. As reported, m.1555A>G is a hotspot in mitochondrial 43.2.3 Diagnosis
non-syndromic hearing loss [111]. M.1555A>G induced
sensorineural hearing loss has variable clinical manifesta- Diagnosis is the very first step for later treatment, manage-
tion, onset time, ranging from congenital to late-onset; sever- ment, and genetic counseling. Teamwork is welcomed for
ity, ranging from mild, moderate to profound. The Vestibular the identification and evaluation of mitochondrial hearing
signs are also rare here. According to Zhu [112], the severity impairment, otolaryngologists, audiologists, clinical geneti-
of the sensorineural hearing loss induced by m.1555A>G cists, and other specialists are needed.
mutation is consistent with the mutant load, a higher mutant
load is correlated to a worse hearing ability.
43.2.3.1 M
itochondrial Sensorineural Hearing
MT-TS1 Related Mitochondrial Hearing Loss Loss
MT-TS1 is located on mitochondrial genome 7446-7514,
encoded for transfer RNA serine 1 (tRNASer(UCN)), tRNASer(UCN) Initial Diagnosis
is critical for mitochondrial oxidative phosphorylation pro- The diagnosis of mitochondrial sensorineural hearing loss is
tein synthesis. The pathogenic variants in MT-TS1 are associ- suggested in a proband with the followings:
784 C. Liu et al.
A. Family history Table 43.4 Hearing loss graded by level of severity

Clinical geneticists are required to collect the three- Degree of hearing loss Hearing loss range (dB)
generation family history information of the proband. Normal −10 to 15
More attention should be paid to the relatives with hear- Minimal 16 to 25
ing loss, physicians are supposed to obtain the informa- Mild 26 to 40
tion by performing the direct test or reviewing the Moderate 41 to 55
medical records. As the maternal inheritance of mito- Moderately severe 56 to 70
Severe 71 to 90
chondrial genome-related diseases, we should be more
Profound 91+
careful about the maternal relatives.
B. Clinical examination
Further clinical examinations are required here if rel- of mitochondrial genome mutation, so the panel is sup-
evant symptoms and signs exist, or evidence indicates posed to have the ability to identify the heteroplasmic
other organ involvement. For example, renal tests, car- level of the pathogenic variant.
diac tests, and ophthalmologic evaluation are always C. Mitochondrial genome sequencing
required here. If both target testing and multigene panel do not give
C. Audiometric testing any satisfactory result, and clinical evaluation indicates
Auditory Brainstem Response (ABR) Test and maternal inheritance pattern, complete mitochondrial
Brainstem Auditory Evoked Response (BAER) Test. genome sequencing should be considered. Mitochondrial
These two tests are designed to check if the brain will whole- g enome long-range PCR plus next-generation
respond to sound properly. Otoacoustic Emissions (OAE) sequencing now is widely applied, with this method, patho-
is a test that helps check how the inner ear responds to genic variants on mitochondrial genome can be easily iden-
sound. Behavioral Audiometry Evaluation is a test that tified including the heteroplasmic level of the sample.
could check all regions of ear, examine how an individual
responds to a sound overall. 43.2.3.2 Differential Diagnosis
The results of mitochondrial deafness supposed to behave Aminoglycosides-Induced Ototoxicity

as follows, severity: moderate to profound hearing loss (clas- Aminoglycosides is a class of commonly used antibiotics
sification based on severity using decibel (dB) system, with the side effects of nephrotoxic, ototoxicity, and vestibu-
Table 43.4); mild-to-moderate high-frequency hearing loss; lar toxicity. As reported, individuals with the MT-RNR1
the family history indicates a maternal inheritance pattern of mutation will predispose them to aminoglycoside-induced
hearing loss or other relative symptoms (hearing loss some- ototoxicity, however, hearing impairment will also happen
times is sub-clinical cannot be noticed). when there are no MT-RNR1 pathogenic variants as amino-
glycosides could generate free radicals within the inner ear.
Establishing the Diagnosis
In order to establish the diagnosis of mitochondrial hearing
loss either syndromic or non-syndromic, clinicians are 43.2.4 Management
required to finish the initial evaluation first. If the initial
results indicate a high possibility of mitochondrial deafness, a 43.2.4.1 Treatment of Manifestations
molecular genetic test is required to establish the diagnosis. Appropriate and timely evaluation and treatment are required
for the auditory deprivation patients, a team of specialists
A. Targeted testing including an audiologist, an otolaryngologist, a clinical
If an individual develops a hearing disability after the geneticist, and others are needed here. As for the later reha-
exposure to aminoglycoside antibiotics, the target testing bilitation, hearing aids, cochlear implantation, speech ther-
could be performed first, including MT-RNR1 m.1555A>G, apy, and language training are required. For the patients
MT-RNR1 m.1494C>T, MT-TS1 m.7445A>C, MT-TS1 suffered from severe to profound hearing loss, an earlier
m.7445A>T, and MT-TS1 m.7445A>G. cochlear implantation (>12 months) is benefit for the attain-
B. Multigene panel ment of language skills and social functions. Electric acoustic
There are several mitochondrial deafness-related mul- stimulation (EAS) is good for patients with residual hearing
tigene panels, however, clinical geneticists are trained to left in the lower frequencies. Lastly, the enrollment of appro-
choose or design an appropriate panel according to the priate educational programs may be important for the chil-
evaluation results. The heteroplasmy is a critical feature dren’s knowledge acquisition.
43.2.4.2 Prevention of Primary Manifestations Amniocentesis and chorionic villus sampling (CVS) are the
Firstly, before applying the treatment of aminoglycoside two main methods in PND. PND can be processed, if the
antibiotics, clinicians are required to inquire or review medi- disease-causing mutation is identified in the family. If the
cal records and family history of relevant hearing loss. If a mutation load in mother is at very high percentage, there is
patient has relatives with a history of aminoglycosides- even no need to perform genetic testing based on the mater-
induced hearing deficits, especially when he/she is a maternally inherited pattern. However, because of the mitotic seg-
nal relative, aminoglycosides treatment should be substituted. regation, we should be careful that the mutant load of the
When sensorineural hearing impairment happens when an mtDNA in amniocentesis and CVS is not reliable to corre-
individual is accepting the aminoglycosides treatment, the spond to that of other fetal or adult tissue and the disease
treatment ought to stop as soon as possible. severity [113].
43.2.4.3 Prevention of Secondary 43.2.5.2 P reimplantation Genetic Diagnosis

Complications in Mitochondrial Deafness
Auditory deprivation may cause severe sequelae with proper As mentioned, prenatal diagnosis needs to be concerned
early auditory intervention. Sequelae like poor language because of the mitotic segregation, there may be a poor cor-
development (including speech production and communica- relation between the mutation load of mtDNA in fetal sam-
tion skills), reading development may do harm to individu- ples and disease severity. Preimplantation genetic diagnosis
als’ social adaptation. Thus, timely auditory intervention (PGD) is the genetic profiling of embryos before implanta-
(including auditory amplification, otologic surgery, and tion, a way to help the doctor to identify genetic defects
cochlear implantation) would be good for the hearing loss, within embryos. In vitro fertilization (IVF) is the common
especially prelingual hearing loss. process to create the embryos used in PGD. The obtained
embryos are analyzed and only the ones with the mutation
43.2.4.4 Surveillance load below the certain threshold has the chance to be trans-
Firstly, audiometric testing needs to be done annually (in ferred. Some certain genetic disorders can be prevented to
order to evaluate the stability and progression of hearing pass to the child with PGD. PGD was first reported in 1990
impairment). A complete document is required to help the and was first performed by Handyside [114, 115]. To our
physician track the progression. Moreover, as mentioned knowledge, PGD may provide the mtDNA mutation carriers
before, a teamwork may help the identification, the related a chance to conceive a healthy baby. However, we are sup-
physical examinations are also required to be done by a pedi- posed to know that the application of PGD may only reduce
atrician or a physician. not eliminate reproductive risk.
43.2.4.5 Agents and Circumstances to Avoid

If an individual carries the pathogenic variant of MT-RNR1 43.2.6 Conclusion
m.1555A>G, MT-RNR1 m.1494C>T, MT-TS1 m.7445A>C,
MT-TS1 m.7445A>T, and MT-TS1 m.7445A>G, he/she This chapter focuses on mitochondrial deafness, including a
should not be exposed to aminoglycoside antibiotics and simple introduction, clinical appearance, diagnosis, and
unnecessary environmental noise. If one of maternal rela- management. In the introduction part, we give a brief back-
tives, have the mutations mentioned here, exposure should ground of mitochondria, mitochondrial genome, deafness,
also be avoided. and mitochondrial deafness. Mitochondrial deafness,
belongs to sensorineural hearing loss due to the inner ear
43.2.4.6 Evaluation of Relatives at Risk structure, cochlea deficit. Hearing deficits is one of the most
Individuals at risk for hereditary deafness and hearing loss common features in mitochondrial diseases both syndromic
should receive early detection and could benefit from avoid- and non-syndromic. Benefit by the advances of the next-
ing aminoglycosides and early management. generation sequencing (NGS), the diagnosis of genetic dis-
eases becomes more precise. Genetic diagnosis is critical for
the following, prognosis, genetic counseling, and manage-
43.2.5 Prenatal Diagnosis ment. Auditory deprivation may cause severe sequelae with
and Preimplantation Genetic Diagnosis proper early auditory intervention. Sequelae like poor lan-
in Mitochondrial Deafness guage development (including speech production and com-
munication skills), reading development may do harm to
43.2.5.1 P renatal Diagnosis in Mitochondrial individuals’ social adaptation. Thus, timely auditory inter-
Deafness vention (including auditory amplification, otologic surgery,
Prenatal diagnosis (PND) is a procedure used prior to birth to and cochlear implantation) would be good for hearing loss,
help diagnose if there is any defect in your developing baby. especially prelingual hearing loss. For the patients suffered
786 C. Liu et al.
from severe to profound hearing loss, an earlier cochlear

implantation (>12 months) is benefit for the attainment of
language skills and social functions.
43.2.7 Internet Resources
The following organizations are good resources for informa-

tion on mitochondrial deafness:
1. Online Mendelian Inheritance in Man (OMIM)

https://www.omim.org/
2. MITOMAP
http://www.mitomap.org/
3. Hereditary Hearing Loss Homepage
https://hereditaryhearingloss.org/
4. Mitochondrial disorders
https://neuromuscular.wustl.edu/mitosyn.html
5. ACMG Guideline
https://www.acmg.net/docs/ACMG_Guideline_for_
Clinical_Eval_and_Etiologic_Dx%20of_Hearing_Loss_
Fig. 43.9 The anatomy of the retina. The optic disc, the macula, and
GIM_Apr20 artery can be observed from the fundus examination
layer of neurons and connected with bipolar cells. The trans-

43.3 Hereditary Vascular Retinopathy mitted messages are carried out of the eye along their axons,
which constitute the optic nerve fibers. The RPE and retinal
Jian Wang and Yufei Xu
neurosensory layer, which is formed by the differentiation of
Diseases involving the retinal vascular system constitute the outer and inner layers of the optic cup, respectively [116].
the most frequent causes of visual impairment and blindness. There is a potential gap between the two layers. Normally,
There are many causes of retinal vascular disorders, includ- the retinal neurosensory layer is attached to RPE. The adhe-
ing the effects of ocular and systemic diseases on retinal ves- sion between the two layers is weak, which is the anatomical
sels, which can be divided into hereditary and non-hereditary basis for their easy detachment.
factors. Regional variations in retinal vascular function are From the center of the optic nerve radiate the major blood
important for the diagnosis and management of a number of vessels of the retina (Fig. 43.9). There are two sources of
retinal diseases. This chapter summarizes the content of blood supply to the retina: the central retinal artery and the
hereditary vascular retinopathy, including the clinical fea- choroidal blood vessels. The choroid receives 65–85%
tures, examination, management, and genetic counseling of blood flow and is essential for the outer retina (particularly
several major disorders. the photoreceptors) and the remaining 20–30% flow through
the central retinal artery from the optic nerve head to nour-
ish the inner retinal layers. The central retinal artery has
43.3.1 Overview four main branches in the human retina. The arterial intra-
retinal branches then supply three layers of capillary net-
43.3.1.1 The Anatomy of the Retina works (i.e., the radial peripapillary capillaries, and the inner
The retina is a thin, light-sensitive layer of tissue, which is and outer layer of capillaries). Retinal blood vessels consist
the basis of forming various visual functions. It begins at the of non-fenestrated endothelial cells surrounded by contrac-
ora serrata and ends at the optic papilla. Ten layers of cells in tile pericytes, which are vital for hemodynamic autoregula-
the retina can be observed microscopically. In general, the tion in response to changing metabolic requirements of the
retina is composed of four main layers: (1) The retinal pig- retina [117].
mented epithelium (RPE) is next to the choroid, (2) The The tight junctions and pericytes between the endothelial
light-sensitive cells (the layer of rods and cones) are above cells of the retinal capillary wall constitute the inner retinal
the epithelium, (3) A layer of nerve cells (neurons) are called barrier. The tight junctions between the RPE cells constitute
the bipolar cells, (4) The ganglion cells are the innermost the outer retinal barrier [116]. Due to the existence of inter-
nal and external retinal barriers, the retinal neurosensory anemia, leukemia, and hemoglobin abnormality can cause
layer, normally, keeps dry and transparent. If any of these different retinopathies. (5) Metabolic diseases, such as dia-
barriers are destroyed, blood plasma and other components betic retinopathy. (6) Retinal vascular abnormalities and
in the blood vessels will leak into the retinal neurosensory developmental abnormalities: such as Coats disease, retinop-
layer, causing retinal edema or detachment. RPE, Bruch’s athy of premature infants, and retinal cavernous
membrane and choroidal capillary, called the pigment hemangioma.
epithelium-Bruch membrane-chorio-capillaris complex,
constitute a unified functional network. It plays an important 43.3.1.3 The Examination of the Retina
role in maintaining photoreceptor microenvironment. Many The regional lesions of retinal vascular diseases reflect the
disorders of the ocular fundus are associated with the pathophysiology of these conditions, and in cases where a
impaired complex. causal relation can be established between pathophysiology
and the distribution of lesions, this information may help to
43.3.1.2 The Physiology of the Retinal Vessels predict the course of and in devising new therapeutic inter-
Both the inner and the outer retinal vascular supply display ventions for the disease. A prerequisite for studying regional
regional differences in structural and functional ways, which lesions in vascular retinopathy is to have access to methods
are reflected in the disease patterns occurring in these regions that allow the resolution of these regional variations in reti-
[116]. The inner retinal vascular supply has been more exten- nal vascular structure and function. The fundus is the only
sively studied than the outer, due to its availability for exami- part of the body where blood vessels can be observed directly
nation through the optics of the eye in vivo. The purpose of [118]. Therefore, fundus examination is helpful to the diag-
the inner retinal vascular supply is to meet the metabolic nosis and to understand the severity of the lesions (Fig. 43.10).
demands of the inner down to the outer plexiform layer, and Clinical diagnoses are made by noting how morphological
thus its distribution will reflect the organization of these lay- lesions in the retina vary in shape, size, location, and dynam-
ers. The retinal vascular system has no autonomic innerva- ics, and subsequently concluding the presence of a specific
tion, indicating that the retinal blood flow is regulated by disease entity.
pressure autoregulation, metabolic autoregulation, or vaso- The following approach can be used to record the mor-
motion [118]. The retinal vascular system has a limited phological changes, to identify the site of a retinal vascular
capacity to increase the blood flow when the retinal metabo- occlusion, and to assess whether retinal diseases are pri-
lism is enhanced, and the high oxygen extraction imposes a marily due to changes in the larger retinal vessels or the
vulnerability of the retina to conditions where its metabolic microcirculation [117]. (1) Ophthalmoscope is a traditional
demand exceeds the supply from the blood. This may be the and routine method for observing fundus morphology.
reason why capillary ischemia is one of the main signs of Indirect ophthalmoscope has a large field of vision and
retinal vascular disease. The choroidal blood flow not only strong stereoscopic sense. It is widely used in the clinic to
meets the metabolic demand of the outer retina, but also acts observe distant periphery combined with scleral compres-
to divert the heat generated when light passes the photore- sion. (2) The slit lamp combined with various preplaced-
ceptors and is absorbed in the pigmented layers of the retina mirror or three-mirror lens examinations can also delicately
and the choroid [117]. This results in a low extraction of observe various parts of the fundus. (3) Ultrasound can be
metabolites and has the phenomena that the metabolic envi- used to detect the posterior segment of the eye when the
ronment of the choroidal veins and the outer retina is almost refractive medium is opaque, and to diagnose and differen-
similar to that of the arterial blood. tiate the posterior segment mass. (4) Fundus fluorescein
There are many reasons for retinopathy caused by retinal angiography can be used to understand the condition of the
vascular changes [117], including the effects of ocular and retina and choroidal circulation system (Fig. 43.10).
systemic conditions on retinal vessels, which can be sum- Indocyanine green angiography (ICGA) can provide a
marized as follows. (1) Mechanical obstruction of retinal clearer and more intuitive understanding of choroidal cir-
vessels: retinal vascular obstruction due to internal embolism culation dynamics. (5) Optical coherence tomography
or thrombosis, or external compressions, such as retinal (OCT) is a non-contact and noninvasive imaging technique
artery occlusion or retinal vein occlusion. (2) Retinal vascu- with high resolution, which is of great value in the diagno-
lar inflammatory disease or immune complex invasion of sis, differential diagnosis, disease tracking, and therapeutic
vascular wall, such as Eales disease, giant cell arteritis, and evaluation of posterior segment diseases ( especially macu-
acute retinal necrosis. (3) Systemic vascular disease: hyper- lar diseases). (6) Electroretinogram (ERG) can diagnose
tension or arteriosclerosis can cause various retinopathy, retinopathy in different layers according to the abnormality
such as malignant hypertension and pregnancy-induced of various waves. Electrooculogram (EOG) mainly reflects
hypertension syndrome. (4) Systemic hematopathy, such as the functional status of RPE [118].
788 C. Liu et al.
OS, FA, 1:42.39 55º

OS, FA, 0:29.20 55º
Fig. 43.10 Fundus fluorescein angiography of the left eye
43.3.2 von Hippel-Lindau Syndrome creas. Of these, retinal hemangioblastomas sometimes are
present as the primary manifestation, further lead to vision
43.3.2.1 Overview loss, visual impairment, and even blindness. The incidence
von Hippel-Lindau (vHL) syndrome (OMIM #193300) is of vHL syndrome is estimated at approximately one in
an autosomal dominant inherited tumor predisposition syn- 36,000 births [119]. Although the majority of tumors occur
drome, which is characterized by a variety of benign and in adulthood, children patients account for a significant
malignant neoplasms, involved predominantly the retina, proportion and are vulnerable to the delayed diagnosis and
cerebellum, spinal cordf kidney, adrenal gland, and pan- its sequelae [118].
43.3.2.2 Clinical Appearance fundoscopy and routine ophthalmoscopy. The abnormal reti-
Tumors occurring in different regions result in specific phe- nal function could be caused by quiescent retinal angiomas
notypes. Even among family members, the manifestations [123]. Associated audiological evaluation could evaluate for
and severity are quite different. hearing loss. Serum and urinary catecholamine metabolites
The most characteristic tumor type in vHL is hemangio- test can assist in the diagnosis of pheochromocytomas.
blastoma. Hemangioblastomas of the cerebellum, retina and Imaging examinations such as ultrasound, CT, or MRI are
spinal cord, pancreatic and renal cysts, clear cell renal cell useful in identifying vHL lesions of the brain, spinal cord,
carcinoma, neuroendocrine tumors, and pheochromocytoma abdomen, and middle ear.
are the most frequent. Although it is a benign tumor made of Identification of a heterozygous germline pathogenic
newly formed blood vessels, it may cause complications variant in vHL gene through genetic testing approaches
such as ataxia and loss of vision when the tumor grows in the include single-gene testing, multigene panel, whole-exome/
central nervous system (CNS) and retina [120]. genome sequencing [122].
Retinal hemangioblastomas are identified in around 70%
of affected individuals. The tumors are most often located in 43.3.2.4 Diagnosis
the vessels of the temporal periphery, optic disc, and poste- The diagnosis of vHL syndrome is established in a patient
rior pole. Patients could be asymptomatic or show a visual with the clinical manifestations and/or by identification of a
field defect/vision loss caused by retinal exudation, hemor- germline pathogenic variant in vHL gene through molecular
rhage, or detachment [121]. The probability of vision loss genetic testing.
may increase with age, although the number of tumors is A clinical diagnosis of vHL can be established in one of
steady. two scenarios: (1) in a proband with a family history of vHL
CNS hemangioblastomas remain the main cause of death. syndrome and with one or more of the manifestations of spi-
The most common sites are infratentorial space (mainly the nal, cerebellar or retinal hemangioblastoma, pheochromocy-
cerebellar hemispheres) and the pituitary stalk. For instance, toma, RCC, or multiple renal and pancreatic cysts; (2) in a
patients present with headache, vomiting, or ataxia, indicat- sporadic case with two or more characteristic lesions of
ing the lesions occurred in infratentorial regions [118]. hemangioblastomas, RCC, pheochromocytomas, or other
Spinal hemangioblastomas, most commonly occur in the tumors mentioned in Clinical Characteristics Section [120].
cervical or thoracic region, usually present with pain, sen- When a proband who has suspected characteristics
sory and motor loss resulting from cord compression. regardless of a vHL family history should be performed
Cysts are also common manifestations and mostly genetic testing. vHL protein is primarily responsible for the
occurred in the kidneys, pancreas, and genital tract. Multiple degradation of hypoxia-inducible factor, which plays an
and bilateral renal cysts are frequent. Most pancreatic lesions important role in oxygen regulation in the cells. Mutations in
are simple cysts and have no malignant potential and rarely the vHL tumor suppressor gene prevent the production or
cause endocrine or exocrine insufficiency. Biliary obstruc- cause abnormal production of the vHL protein, resulting in
tion may be caused by the cysts that happened in the head of the uninhibited upregulation of hypoxia-inducible factor and
the pancreas. associated downstream growth factors and consequently the
Tumors in other regions can be observed in patients with formation of cysts and vascular tumors [118].
vHL [122]. Around 69% of patients with vHL will develop
renal cell carcinoma (RCC), which is also the dominant fac- 43.3.2.5 Genotype–Phenotype Correlations
tor of death. Pheochromocytoma, located in one or both Several researchers suggested the molecular etiology of
adrenal glands, can be asymptomatic or cause headaches, pheochromocytomas is distinguished among vHL lesions.
hypertension. Neuroendocrine tumors, which have been in Thus, four vHL subtypes (type 1, type 2A, type 2B, type 2C)
about 5%–17% of vHL syndrome patients, are often non- are classified according to the risk of pheochromocytoma
functional. However, malignant behavior has been found. and other tumors. Table. 43.5 summarizes the genotype–phe-
Endolymphatic sac tumors lead to vertigo, tinnitus, sudden notype studies to date [124].
hearing loss in 10%–16% of patients. Bilateral epididymal
and broad ligament cystadenomas could cause infertility. 43.3.2.6 Differential Diagnosis
43.3.2.3 Examination Isolated Hemangioblastoma or RCC

Physical examination is usually limited since the diagnosis is The application of molecular genetic testing could effec-
generally made based on accessory examination findings. tively rule out vHL syndrome with a high degree of certainty.
Retinal hemangioblastomas and others such as retinal However, it is worth noting that somatic mosaicism of a vHL
detachment, macular edema, or cataracts can be detected on gene variant still could be thought about.
790 C. Liu et al.
Table 43.5 Genotype–phenotype correlations of vHL syndrome ment papillary cyst adenomas are symptomatic or threaten-
vHL ing fertility, they require surgical treatment.
subtype Phenotypes Genotype Regular surveillance and early detection could prevent
Type 1 Low risk for pheochromocytoma Truncating or secondary complications, like neurologic symptoms, the
missense variants demand for kidney transplantation besides hearing or sight
that disrupt the
folding of the loss [126]. To those vHL probands and at-risk relatives, regu-
protein lar surveillance includes regular assessment for neurologic
Type High risk for pheochromocytoma, Missense variant symptoms, visual and auditory functions, blood pressure
2A retinal and CNS hemangioblastoma detection, plasma urine for fractionated metanephrines, thin-
Type High risk for pheochromocytoma, Missense variant slice MRI in comparison with the internal auditory canal,
2B retinal and CNS hemangioblastoma
pancreatic cysts, neuroendocrine abdominal ultrasound/MRI scan, and MRI of the brain and
tumors, RCC spine.
Type High risk for pheochromocytoma Missense variant Tobacco, chemicals and industrial toxins, and contact
2C sports should have refrained as they have been regarded to
damage vHL-involved organs [126].
Pheochromocytoma Several innovative treatments target the upregulated sig-
Several other genes like RET, SDHA, SDHB, or TMEM127 are nal pathways like VEGF receptor inhibitors (ranibizumab
associated with pheochromocytoma. Patients with pheochro- and bevacizumab) have been applied in patients to treat reti-
mocytoma should be distinguished from multiple endocrine nal and CNS hemangioblastomas [127]. Sunitinib, a tyrosine
neoplasia type 2, hereditary paraganglioma-pheochromocy- kinase inhibitor, has also been demonstrated to effectively
toma syndrome, and neurofibromatosis [122]. treat RCC. Preclinical investigation of premature termina-
tion codon 124 (PTC124) effects is ongoing.
RCC
Patients with RCC and/or family history may be considered 43.3.2.8 Genetic Counseling
as hereditary leiomyomatosis and renal cell cancer and Birt- VHL syndrome is inherited by autosomal dominance. About
Hogg-Dubé (BHD) syndrome. 79% of probands have an affected parent and others resulted
from a de novo vHL gene variant [128]. There is also a report
43.3.2.7 Management about mosaicism. The offspring of vHL syndrome patients
The major cause of mortality was attributable to CNS heman- are at a 50% risk. Prenatal testing like a preimplantation
gioblastoma and RCC. In recent years, owing to the early genetic diagnosis for a pregnancy in danger is necessary for
diagnosis, recognition of tumors, and multidisciplinary man- the presence of pathogenic variant. If the pathogenic variant
agement, the risks have been significantly decreased. is identified with certainty, molecular genetic testing can be
Most CNS hemangioblastomas can be surgically removed used to screen the genetic status within high-risk families to
and some asymptomatic lesions can follow with yearly imag- determine if the follow-up monitoring is needed.
ing studies. Surgical treatment was approved to be relatively
safe and effective. Retinal hemangioblastomas except for
optic nerve are suggested to be applied with prospective 43.3.3 Retinal Vasculopathy with Cerebral
treatment including diathermy, xenon, laser, and cryocoagu- Leukodystrophy
lation, in order to avoid progressively blindness [125].
Compared with the ophthalmoscopy and conventional angi- 43.3.3.1 Overview
ography, ultra-widefield fluorescein angiography is more Retinal vasculopathy with cerebral leukodystrophy (RVCL)
useful in the evaluation of retinal hemangioblastoma. (OMIM #192315) was primarily referred to in various names
Early surgery is the better option for large lesions of RCC, (known as hereditary vascular retinopathy, hereditary endo-
while closely monitoring and cryoablation is suitable for theliopathy, and cerebroretinal vasculopathy, retinopathy,
small lesions. Individuals who undergo the necessary bilat- nephropathy, and stroke), and was considered to represent
eral nephrectomy should receive renal transplantation. different neurovascular syndromes. Nowadays, owing to the
Pancreatic cysts and neuroendocrine tumors are usually not identification of pathogenic heterozygous C-terminal frame-
hormonally active and have no malignant behavior. Close shift mutations in TREX1 gene, it unified these three syn-
monitoring is needed and surgical removal is necessary for a dromes into one single autosomal dominant disorder [129].
high risk of metastasis. Then vestibular function of individu- Due to systemic vascular involvement, RVCL is regarded as
als with small endolymphatic sac tumors can be protected by a neurovascular syndrome. It affects the microvessels of the
early intervention [122]. When epididymal or broad liga- eyes, brain, kidneys, and liver.
43.3.3.2 Clinical Appearance bility [133]. Studies on histopathology showed ischemic

RVCL means a small vessel vasculopathy, which especially necrosis with minimal inflammation, luminal narrowing, and
involved the retina and brain, resulting in CNS degeneration multi-laminated basement membranes. The lesions have
including progressive vision loss, motor damage, cognitive prominent small infarcts in the white matter of the brain,
decline, and stroke. Vascular retinopathy and brain lesions which can be combined to form pseudotumors.
usually related to progressive visual impairment and pro- Fluorescein angiography can be used to detect retinal
gressive neurological function impairment, respectively, abnormalities even in asymptomatic family members. When
were the most diagnostic disease-specific discovery [130]. patients presented with symptoms mainly during later stages
The retinal vasculopathy is featured by retinal capillary of the disease, positive manifestations included block of
occlusion beginning in the macula, retinal microangiopathy, branches of large retinal arteries, avascular areas in the reti-
retinal hemorrhages, areas of capillary non-perfusion, and nal periphery, and sometimes proliferative retinopathy with
even occlusion of large retinal vessels leading to a wide extensive avascular areas, even near the optic disc [132].
range of avascular areas, which could induce a neurovascular Other laboratory examinations like liver function (serum
response [130]. In the early stages, retinopathy was pre- levels of gamma-glutamyltranspeptidase, alkaline phospha-
sented with telangiectasias, micro-aneurysms, and cotton tase, transaminases, etc.) and renal function (urine protein,
wool spots (edema, swelling, and degeneration of the nerve serum creatinine, etc.) is helpful for early detection of sys-
fibers). In the later stages, it is manifested by perifoveal cap- temic lesions [134]. Studies of histopathology in biopsy and
illary obliteration and neovascularization. The findings from autopsy have reported that nodular regenerative hyperplasia
histopathological examination of the retina were in accor- was the predominant feature when the lesions affected the
dance with scattered microinfarcts, including the thickened liver. Other findings included micro- and macro-vesicular
hyalinized walls of the retinal arteries and focal areas of dis- steatosis, periportal inflammation, and portal and bridging
ruption of the ganglion cells and inner nuclear layer of the fibrosis were also reported. Pathologic examination of the
retina besides vascular changes [131]. Also, some lesions kidney revealed that renal arteriolosclerosis, arterioloneph-
may progress to retinal hemorrhage and neovascularization. rosclerosis, and focal or diffuse glomerulosclerosis.
The retinopathy cause symptoms of a gradual decrease of Identification of a heterozygous germline pathogenic
visual acuity, transient visual disturbances, and visual field variant in TREX1 gene through genetic testing approaches
defects in adulthood. Later, especially around 60 years old, include single-gene testing, multigene panel, whole-exome/
most patients suffer from severe visual impairment and even genome sequencing [129].
blindness.
The other predominant features are cerebral white mat- 43.3.3.4 Diagnosis
ter and contrast-enhancing mass lesions, which cause focal The relatively non-specific clinical and imaging findings
and global neuropsychiatric symptoms, progressive neuro- lead to the challenging of diagnosis. Stam AH, and col-
logical decline, and even premature death. The cause of leagues show that the combination of vascular retinopathy,
death primarily results from the complications of neuro- neurological decline, positive family history related to the
logical decline. Symptoms of brain injury included focal described white matter lesions on brain MRI should prompt
neurological deficits, migraine, seizures, psychiatric distur- clinical monitoring of the syndrome and genetic detection
bances, and cognitive impairment [132]. The findings of [135]. To aid the clinical diagnose of RVCL, they proposed
neuroimaging revealed punctate, hyperintense, rim-enhanc- operational diagnostic criteria (Table 43.6). Demonstration
ing mass white matter lesions with calcifications or nodular of C-terminal frameshift variants in TREX1 gene would con-
enhancement [133]. firm the diagnosis. TREX1 (three prime repair exonuclease
Substantial numbers of affected individuals have 1) encodes a 3′-5’ DNA exonuclease involved in clearing
Raynaud’s phenomenon, micronodular cirrhosis, and glo- cytosolic nucleic acids, which plays an important role in
merular dysfunction, indicating the involvement of systemic multistep processes of DNA replication, repair, and recombi-
vessels. These phenotypes are more common in RVCL nation that required the excision of nucleotides from DNA
patients than the major population, showing that are also 3-prime termini [136]. Variants may impair DNA degrada-
belong to the clinical spectrum [118]. Systemic features tion and homeostasis causing the disruption of cell death
involved liver disease, nephropathy, anemia, hypertension, mechanisms.
mild Raynaud’s phenomenon and gastrointestinal bleeding.
43.3.3.5 Genotype–Phenotype Correlations
43.3.3.3 Examination Mutations in TREX1 gene are associated with four different,
Neuroimaging studies including brain MRI or CT show although overlaying, clinical phenotypes. The molecular
contrast-enhancing lesions in the white matter of the cere- pathology of RVCL seems different from that of Aicardi-
brum and cerebellum suggested abnormal vascular permea- Goutieres syndrome (AGS), familial chilblain lupus (FCL),
792 C. Liu et al.
Table 43.6 Proposed diagnostic criteria of RVCL 43.3.3.6 Differential Diagnosis

Strength of RVCL is a rare disorder, which is difficult to diagnose due to
evidence Description the systemic involvement of complex manifestations and
Major diagnostic Vascular retinopathy radiological findings. Young adults with systemic microangi-
criteria Features of focal and/or global brain opathy involving the brain, retina, kidney, and other organs
dysfunction associated on MRI with typical
lesions should be suspected. The cerebral lesions observed in
Positive family history patients with RVCL may be mistaken for a tumor or multiple
Identification of a heterozygous C-terminal sclerosis. Thus, RVCL sometimes could be misdiagnosed,
frameshift variant in TREX1 gene which leads to unnecessary diagnostic procedures such as
Supportive CT/MRI indicated white matter lesions biopsies [125].
features Microvascular liver disease Regarding certain brain MRI researches, these white mat-
Microvascular kidney disease
ter lesions may cause neurologic phenotypes. This situation
Possibly Anemia consistent with blood loss and/or
associated chronic disease
should remind physicians of RVCL in the differential diag-
features Microscopic gastrointestinal bleeding nosis of demyelinating lesions associated with multiple scle-
Hypertension rosis or certain gliomas. The results of genetic tests can be
Migraine helpful in differential diagnosis.
Raynaud’s phenomenon
43.3.3.7 Management
and systemic lupus erythematosus (SLE) [131]. Although Due to the rarity of disease reports, there are no unified
white matter brain disease and intracranial calcification can be guidelines for management. The main management is
presented in both AGS and RVCL, these diseases are clinically focused on the treatment of manifestations. So far, no effec-
different. The heterozygous p.D200N and p.D18N variants are tive treatments for the vascular retinopathy associated with
respectively associated with AGS1 and CHBL with the evi- the TREX1 gene variants have been reported. Raynaud’s phe-
dence of exonuclease enzyme analysis. Studies showed that nomenon is usually asymptomatic without treatment. Several
either p.D200N or p.D18N mutant proteins entirely lacked for studies have presented the affected individuals who received
degrading dsDNA and degraded ssDNA that were about 2 different medical treatments. At present, it is not clear which
times lower than wild-type protein. Besides, the mutant pro- way can treat these kinds of tumor-like lesions optimally.
teins suppressed the dsDNA degradation activity of wild-type DiFrancesco and colleagues reported one case treated with
TREX1, supporting an interpretation for the dominant pheno- immunosuppression did not receive substantial improvement
type [130]. Contrarily, the homozygous p.R114H mutation was [129]. Although they also applied cyclophosphamide in the
reported to cause AGS while heterozygous p.R114H mutation course, further information was absent. The combination of
was reported to cause SLE, leading to the dysfunctional dsDNA rituximab and administration of intravenous cyclophospha-
and ssDNA degradation activities. The p.R114H homodimer mide monthly has also successfully applied in a patient with
lacked inhibitory activity against wild-type protein, indicating RVCL and the clinical course was reported to be apparently
the recessive inheritance mode of the mutation in stable during the 24-month follow-up period. One report
AGS. Frameshift mutations in the C-terminus of the gene are suggested that natalizumab may be beneficial for RVCL
considered to be related to SLE, and a C terminus frameshift patients while no documented data was recorded.
mutation are discovered in patients with typical AGS. Loss of The management of leukodystrophies caused by other eti-
function recessive mutations in TREX1 gene are related to AGS ologies is based on different approaches, including specific
and SLE [129]. Mutant proteins of p.Asp18 and p.Asp200 resi- therapies, symptomatic therapies like hematopoietic stem
dues, were reported to result in skin and neurological pheno- cell transplantation, and supportive therapies like occupa-
types compatible with AGS or FCL. Mutations in TREX1 gene, tional physical and speech training. Specific therapies have
which are associated with AGS, FCL, and SLE, appear to share been increasingly developed for the metabolic leukodystro-
a common pathological mechanism of the induction of a type I phies and the life expectancy and quality of patients have
interferon response [137]. been shown to be significantly improved.
Contrary to the mutations presented in AGS, FCL and Also, a study proposed that plasma exchange, intravenous
SLE, RVCL-associated TREX1 gene variants have been immunoglobulin, or chronic immunosuppression has no
reported exclusively to develop as frameshift mutations in benefit, and a healthy lifestyle and prevention of related risk
the C-terminus. Mutations in TREX1 gene, which are associ- factors for cerebrovascular disease may delay the onset of
ated with RVCL, may result in altered cellular distribution age and severity of the disease. The evaluation of these thera-
with pathological mechanism of gain-of-function or toxic peutic options of these RVCL patients is needed in a longitu-
effect [134]. dinal follow-up.
43.3.3.8 Genetic Counseling Studies have found that the failure of peripheral retinal
RVCL is inherited in an autosomal dominant manner with vascularization is unifying common feature presented in
plausible 100% of penetrance [122]. There has been no case all patients. However, it usually does not cause any clini-
report of RVCL cases caused by compound heterozygous or cal symptoms by itself. The resulting visual problems and
homozygous variants in TREX1 gene. Each offspring from other phenotypes caused by retinal ischemia such as the
RVCL patients is at a 50% risk of inheriting the pathogenic development of hyperpermeable blood vessels, neovascu-
variant of TREX1 gene and being affected. Prenatal diagno- larization, vitreoretinal traction, and falciform retinal
sis and preimplantation genetic diagnosis of high-risk preg- folds. These complications lead to visual impairment and,
nancies are possible when pathogenic variants of TREX1 in some cases, cause partial or total retinal detachment
gene are found in affected family members. [118]. Rare retinal features included peripheral brush
margin anastomosis, peripheral fibroangioma, retinal hia-
tus, exudation, retinoschisis, and giant retinal tears have
43.3.4 Familial Exudative Vitreoretinopathy also been described. Hence, the severity of the disorder is
associated with the complications. Expressivity, accord-
43.3.4.1 Overview ingly, may be asymmetric and vary greatly from mild
Familial exudative vitreoretinopathy (FEVR) is a hereditary (absence of symptoms) to severe (early onset of blind-
retinal disorder, which is characterized by the defective ness). Severely affected individuals manifest clinical
development of the retinal vasculature. To date, seven genes symptoms in the first decade of life, while mild patients
have been reported to be responsible for this disease, includ- may receive the diagnosis of FEVR through the profes-
ing FZD4, NDP, EVR3, LRP5, TSPAN12, CTNNB1, and sional ophthalmological examination without any dis-
ZNF408 genes [117]. Its expressivity varies greatly even comfort [138].
within families, from asymptomatic to severe. Severely Other features included low bone mineral density and
affected patients usually manifested as blind in infancy, susceptibility to fracture. These osteological features are
while mildly affected patients may present with few visual more frequent in the affected individuals with pathogenic
problems, which can only be seen through fluorescein angi- variants in LRP5 gene, and bone mass is reported to have
ography that there may be non-vascular areas around the decreased in these patients [138]. While preliminary studies
retina. This peripheral avascularity is the main abnormality of FEVR patients indicated that these findings are not
in FEVR patients and is the result of incomplete retinal observed in patients with FZD4 gene pathogenic variants or
angiogenesis. In the past, it was generally considered a rare other forms of FEVR.
disease, because the clinical status was determined by visual
impairment or other clinical symptoms. Now, it has been 43.3.4.3 Examination
found that penetrance is 100% in view of the fact that all The manifestations of FEVR patients are somehow similar to
affected individuals have the same characteristics from fluo- those of other vitreoretinal diseases in children and adults.
rescein angiography [122]. Thus, the frequency of FEVR Thus, careful examination is the key to distinguish FEVR
was likely to be underestimated. from other diseases.
Full ophthalmic examinations are essential in suspected
43.3.4.2 Clinical Appearance individuals, including the measurement of visual acuity,
FEVR is characterized by the absence of peri-retinal blood examination of slit lamp, and fundus fluorescein angiogra-
vessels. The main pathology is considered as a premature phy. The findings of retinal avascularity may be missed but
arrest of retinal angiogenesis or abnormality of retinal vascu- they are easier to be detected with the help of the indirect
lar differentiation, leading to incomplete peripheral retinal ophthalmoscope and scleral indentation. Based on ophthal-
vessels. Shukla and colleagues reported that affected indi- moscopic findings, the severity of FEVR could be catego-
viduals who have normal vision were at the most mildest rized into the following five stages [122], which has been
spectrum with only an asymptomatic area of peripheral reti- summarized in Table 43.7.
nal avascularity. Retinal exudates, retinal traction, and even The pathogenic changes of retinal vessels can be observed
developed with rhegmatogenous retinal detachment were more vividly with fundus fluorescein angiography. And, a
found in some FEVR patients. Severe phenotypes, including study suggested that the application of fundus fluorescein
reduced vision, strabismus, and leukocoria, usually occur in angiography can detect 100% of FEVR patients. The classic
the first decade of life. In the most serious situations, blind- features from fundus fluorescein angiogram would show the
ness occurred in affected infants shortly after birth [122]. lesions with no blood vessel area around retina, dilated and
Thus, this area of retinal avascularity is believed to exist at truncated capillaries with leakage of fluorescein dye, and the
birth and to have further sequelae that may stabilize in early preserved straightened retinal vessels near the avascular
adulthood and/or later life. zone. In severe cases, it may be observed the extensive vit-
794 C. Liu et al.
Table 43.7 Clinical classification of FEVR pathogenic variants in these genes are responsible for only
Stage Ophthalmoscopic findings part of the FEVR patients, there remain unknown genes
1 Avascular areas in the retinal periphery without extra- associated with FEVR to be identified. Thus, failure to iden-
retinal vascularization tify pathogenic variants in these seven genes does not rule
2 Avascular areas in the retinal periphery with extra-retinal out the diagnosis.
vascularization, and with/without exudate
3 Subtotal retinal detachment (primarily exudative,
primarily tractional), not involving fovea 43.3.4.5 Genotype–Phenotype Correlations
4 Subtotal retinal detachment (primarily exudative, Several studies indicated that affected individuals with
primarily tractional), involving fovea FEVR who carried pathogenic variants within LRP5 gene
5 Total retinal detachment might show signs of osteoporosis on a dual X-ray absorpti-
ometer more frequently than those who carried variants in
reoretinal traction, retinal fold, neovascularization, and other other genes [122]. One study suggested that there existed
retinal features mentioned before. synergistic action between the FZD4 and the LRP5 patho-
Germline pathogenic variants in one of the seven disease genic variant in the FEVR phenotypes, due to the findings
causing genes (FZD4, NDP, EVR3, LRP5, TSPAN12, that patients with pathogenic variants in both genes in cis had
CTNNB1, and ZNF408) can be identified by genetic testing more severe phenotypes than patients with the same FZD4
methods including single-gene testing, multi-gene panel, pathogenic variant only. The affected individuals who car-
whole-exome/genome sequencing. In a cohort of Chinese ried the LRP5 gene variants showed broader phenotypic
FEVR pedigrees, the study analyzed six FEVR-associated spectra that varied from stage 2 to stage 5. While patients
genes and identified variants including 16.1% in LRP5 gene, carrying NDP gene variants were correlated with severe phe-
9.7% in NDP gene, 6.5% in FZD4 gene, and 3.2% in notypes, which may be stage 4 or above. It is controversial
TSPAN12 gene [135]. that the pathogenic missense variants in the C-terminus
region of NDP gene may be related to the milder phenotypes
43.3.4.4 Diagnosis of FEVR [138].
The diagnosis of FEVR is established in patients with the
typical clinical manifestations and/or by identification of 43.3.4.6 Differential Diagnosis
germline pathogenic variants in FEVR-related genes through
molecular genetic testing, which is compatible with the pro- Retinopathy of Prematurity
band’s family history. Retinopathy of Prematurity (ROP) is also characterized by
The main clinical evidence is the examinational findings the failure of the peripheral retinal system development. The
(i.e., bilateral peripheral retinal avascularity) from fundus manifestations of retinal neovascularization and cicatricial
fluorescein angiography, or the indirect ophthalmoscope. sequelae, which are similar to those of affected individuals
When a proband with or without a family history of FEVR with FEVR, mainly occur in premature infants due to prema-
who have suspected manifestations should be performed ture delivery before retinal vascularization is completed. As
genetic testing. Pathogenic variants in one of seven genes a sporadic disorder, a history of premature birth and a lack of
(FZD4, NDP, EVR3, LRP5, TSPAN12, CTNNB1, and family history are helpful in the differential diagnosis.
ZNF408) are known to be associated with FEVR [122]. Due Retinal neovascularization and scar sequelae are similar to
to the various inheritance mode of these genes, FEVR can be those of affected individuals with FEVR.
inherited by autosomal dominant (the most common), auto-
somal recessive, or X-linked. Thus, gene panel or whole- Coats Disease
exome sequencing would be the better option to order. Coats disease is characterized by severe retinal telangiecta-
Among the seven genes, most of them encode proteins as sia, retinal exudates, and retinal detachment that resembles
part of the norrin/beta-catenin signaling pathway, in which FEVR phenotypes. Most affected the unilateral eye and were
the ligand (norrin) binds to receptor complexes composed of predominantly common in male children. Fundus fluores-
frizzled-4, co-receptor low-density lipoprotein receptor- cein angiography could detect abnormal vessels presented
related protein-5 and auxiliary protein-12. Variants may throughout the fundus.
impact the expression and/or function of the associated
encoded protein, leading to reduction of the binding affinity Persistent Hyperplastic Primary Vitreous
of frizzled-4. In the absence of norrin binding, the activation Persistent Hyperplastic Primary Vitreous (PHPV), known as
of this signaling pathway is arrested or the transduction of “persistent fetal vasculature, is a developmental eye malfor-
β-catenin signaling is impaired, which resulting in phosphor- mation, which is characterized by the retrolental fibrovascu-
ylation of cytoplasmic β-catenin and directional degradation lar membranes. It is attributed to the failure of primary
through ubiquitin-proteasome pathway [139]. However, vitreous degeneration in utero [122]. This abnormality is
usually unilateral and observed in full-term infants, compa- regression and the accelerated fibrosis, indicating it can be
nied by microphthalmia, cataract, glaucoma, and congenital used as an alternative treatment for FEVR [141]. The long-
retinal nonattachment. Microphthalmia are more common term evaluation of new therapies remains to be confirmed
compared with FEVR. with more data.
Individuals who are at high risk such as have a positive
Norrie Disease family history should be carried out a routine ophthalmo-
Norrie Disease (ND) is a known X-linked recessive condi- logical examinations to assess the development of retinal
tion characterized by early blindness in childhood due to exudates, neovascularization, and traction. Regular surveil-
degenerative and proliferative changes of the neuroretina. lance should be performed as follows [122]: (i) ophthalmo-
Retinal findings of this disorder can mimic the manifesta- logic examination, such as indirect ophthalmoscopy; (ii)
tions of FEVR. Patients with Norrie disease may also present fundus fluorescein angiography, especially in those asymp-
with progressive mental features, sensorineural deafness, tomatic affected individuals, which may improve the detec-
growth failure, seizures, etc. Funduscopic examination is tion rate of peripheral avascularity. The frequency of the
helpful in distinguishing between the two disorders. follow-up depends on the assessment of the professional
ophthalmologists.
Toxocariasis
As an acquired disorder, patients with toxocariasis usually 43.3.4.8 Genetic Counseling
have an infectious history of the roundworm Toxocara canis. Because of the various inheritance mode of FEVR (i.e., auto-
In certain forms, it may present with retinal traction and reti-
somal dominant, autosomal recessive, and X-linked man-
nal fold due to the development of a peripheral granuloma- ners) and the difficulty of distinguishing by clinical
tous mass. ophthalmologic examination, the genetic counseling is more
complicated.
43.3.4.7 Management If it is inherited in an autosomal dominant mode, the off-
In spite of the presence of retinal avascularity, patients may spring of affected individuals have a 50% risk of inheriting
not manifest any symptoms, and in general, did not receive the pathogenic variant. The situation of asymptomatic indi-
treatment. However, it has the risk of causing retinal periph- viduals with FEVR needs to be cautious. In an autosomal
ery lesions including ischemia and neovascularization. These recessive manner, the parents of a proband are at a 25% risk
lesions could be cured by preventive cryotherapy or argon of having another affected child, an even chance to have a
laser photocoagulation, which have the potential to induce carrier child [122]. When it is inherited in an X-linked man-
regression of the new vessels. The application of the above- ner, the pathogenic variant would be passed from the male
mentioned techniques is also beneficial to prevent retinal patients to all their potential carrier daughters. Carrier
detachment, which may result from retinal exudate or hiatus. females have an even chance to transmit the pathogenic vari-
However, the prognosis of exudative retinal detachments is ant to boys who would be affected, or daughters who would
reported to be poor [140]. Rhegmatogenous retinal detach- be carriers. If the pathogenic variants have been identified in
ments, which are derived from retinal traction, can be the pedigrees, it is possible to carry out carrier testing, prena-
repaired by conventional surgery with satisfactory prognosis. tal pregnancy detection, and preimplantation genetic diagno-
Current methods do not prevent peripheral retinal avascular sis for their high-risk relatives.
arcs, which are believed to be abnormal in retinal
development.
Although the long-term follow-up information is not 43.3.5 Typical Medical Case
available yet, patients with FEVR (especially those who
have pathogenic variants in LRP5 gene) presented with bone Clinical Background The proband is a 15-month-old
mineral density reduction may be expected to benefit from Chinese boy, born at 40-week gestation with a birth weight
medicine like bisphosphonates and calcium agent used to of 3600 g via cesarean section. The proband was the first
treat osteoporosis in order to prevent secondary complica- child of the couple. Neonatal clinical examination at the
tions [122]. local hospital did not show any abnormalities. At 3 months
Vascular endothelial growth factor (VEGF) inhibitors of age, his parents noticed that his eyes did not react to light.
have been widely applied in acquired retinal diseases with At 9 months of age, he had developmental delay and mild
neovascularization or exudation components. Some reviews thumb adduction and hypertonia of the extremities.
suggested this kind of treatment may be beneficial for those
patients with FEVR. Also, several studies reported the appli- Physical Examination (at the Age of 15 Months) His head
cation of intravitreal injection of bevacizumab acquired circumference was 44.0 cm (< −2SD). He had low-set ears,
change of neovascularized tissue, which benefits from rapid a high palate arch, and hypertonia, He still had no reaction to
796 C. Liu et al.
Fig. 43.11 Sequencing traces of the pedigree. Left side showed the results of Sanger sequencing. Right side showed the family tree
the light and appeared to have a complete lack of vision. He Follow-Up The proband was followed up to 3.5 years old.
could raise his head but still could not sit or walk without He still could not walk alone or speak. Although the option
support. No language development was found. of surgery was proposed by the clinician, his parents refused
the suggestion due to private reasons. The proband was
Ophthalmologic Examination Results of an ultrasound of blindness and even showed leukocoria. Although the variant
the eyes showed a retinal detachment and lens and vitreous was supposed to be de novo and the risk to the sibs of the
opacities in both eyes. Intraocular pressures were as follows: proband appears to be low, we should still be cautious
oculus dexter: 15.3 mm Hg; oculus sinister: 14.7 mm Hg. because of the possibility of germline mosaicism. When his
Falciform retinal folds and fundus hemorrhages were also mother was pregnant again, we recommended prenatal test-
reported. Fundus fluorescein angiography in both eyes was ing of amniotic fluid DNA, and the result showed that the
normal. Ultrasound examination of the eyes at 4 and 7 months fetus does not carry the mutation. As far as we know, the
revealed persistent retinal detachments in both eyes. second child is 3-month old and no abnormalities have been
Magnetic resonance imaging of the proband’s brain, electro- found.
encephalogram, abdominal ultrasound, and hearing exami-
nation revealed no abnormalities. His genetic/metabolic The case was from Shanghai Children’s Medical Center.
workup, including karyotype, liver function tests, alkaline
phosphatase, glucose, lactate, electrolytes, and urinalysis
were all normal. References
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Transplant Matching
44
Binwu Ying and Lijuan Wu
Organ transplantation is one of the most effective methods to (now the West China Hospital of Sichuan University). After
solve end-stage organ failure. Transplant matching is an the 1980s, the efficacy of organ transplantation was greatly
important factor affecting the success and long-term survival improved due to advances in surgical techniques, improve-
of organ transplantation. Since the waiting list for organ ments in preservation methods, development of high-speed
transplants is growing, improving the survival rate of organs transportation, establishment of transplant centers, and espe-
in vivo and prolonging its survival time could markedly cially the use of new immunosuppressive agents with few
relieve the demand for organs. Good transplant matching is side effects and strong potency. Currently used transplant
vital for the later survival of the transplanted organs with organs include kidney, heart, liver, pancreas and islets, para-
important clinical significance. thyroid glands, cardiopulmonary, bone marrow, and cornea;
in the initial clinical or experimental stage, there are heart,
lung, small intestine, adrenal gland, thymus, testis as well as
44.1 Overview liver cells, fetal liver cells, spleen cells Infusion, etc.
The transplant immune response is an immune response
Transplantation is a technology to transfer cells, tissues, or against the graft antigen, and involves both humoral and cel-
organs in the same individual or to another individual by sur- lular immunity, and has a strong correlation with natural
gery or other methods which would replace dysfunctional immunity. In hyperacute rejection, the main cause of graft loss
cells, tissues, or organs. An individual who donates a trans- is humoral immunity, while in acute rejection, cellular immu-
plant, is called a donor and the person who receives a trans- nity is predominant. Due to the characteristics of organ trans-
plant, is called a recipient. According to the relationship plantation itself, dangerous signal molecules such as HMGB1,
between the donor and the recipient, it can be divided into which are caused by surgical wounds, can activate APC and
autologous transplantation, that is, the donor and recipient of other cells that participate in both natural and acquired immu-
the organ are the same person; homologous transplantation, nity, and accelerate the process of immune rejection. In addi-
that is, the donor and the recipient are not the same person, tion to the recipient’s immune cells, the donor’s immune cells
but the donor (i.e. the identical twin) has the same genetic are also involved in the transplant immune response, and the
material; allogeneic transplantation, that is, human-to-human donor’s APC can activate the same reactive T cells in the recip-
transplantation; xenotransplantation, that is, transplantation ient through a direct recognition mechanism, causing a strong
between different species of animals. According to the dif- rejection. According to different subjects of immune rejection,
ferent grafts, they are divided into cells, tissues, and organs. allograft rejection includes host versus graft reaction (HVGR)
Transplantation does not include the use of synthetic poly- and graft versus host reaction (GVHR). Depending on the
meric materials in the body. Transplant medicine is a cutting- degree of tissue compatibility between the graft and the host,
edge discipline. The earliest established Institute in China of as well as the immune status of the recipient, transplant rejec-
Organ Transplantation of Wuhan Medical College was offi- tion is mainly manifested in three different types.
cially established in 1980, then, the establishment of the
transplant center was Zhejiang Medical University, Zhong
Shan Medical University, and Huaxi Medical University 44.1.1 Hyperacute Rejection
B. Ying (*) · L. Wu The hyperacute rejection reaction typically occurs 24 hours

Department of Laboratory Medicine, West China Hospital, Sichuan after transplantation. It is currently believed that this r ejection
University, Chengdu, Sichuan, People’s Republic of China is mainly caused by antibodies to the ABO blood group anti-

802 B. Ying and L. Wu
body or to the class I major histocompatibility antigen. This also familiar in the traditional sense, namely the first group
antibody binds to the vascular endothelial cells of the trans- of classical HLA genes.
planted kidney, directly destroys the target cells by activating Classical HLA genes often show a high degree of poly-
complement, or a variety of complement cleavage fragments morphism, that is, most of the loci have a large number of
generated during complement activation, resulting in platelet alleles, and are directly involved in the regulation of the
aggregation, neutrophil infiltration, and activation of the human immune response, and are divided into HLA-I genes
coagulation system. It eventually leads to severe ischemia and HLA-II genes [4, 5]. HLA-I genes include A, B, C, and
and graft necrosis. Once hyperacute rejection occurs, it will other loci, located at the far end of the HLA gene complex,
lead to graft failure. Therefore, appropriate donors are and the product is called HLA-I molecule or class I antigen,
selected by ABO and HLA matching before transplantation which the heavy chain encoded by each gene locus and the
to prevent the occurrence of hyperacute rejection. light β2-M encoded by the non-HLA gene of chromosome
15 are combined. The HLA-II gene is located at the near-tip
point of the complex and is at least divided into three sub-
44.1.2 Acute Rejection regions of DR, DQ, and DP. The product is called HLA-II
molecule or class II antigen that is a heterodimer composed
Acute rejection is one of the most common types of rejection of an α chain (35 kD) and a β chain (28 kD), both of which
[1], usually occurring within days to months after transplan- are encoded by a class II gene. HLA-II genes are more com-
tation, and progresses rapidly. The cellular immune response plex in their number and composition than class I genes.
is the main cause of acute rejection of transplants, and CD4+
T cells and CD8+ T cells are the main effector cells. Even
with the application of human leukocyte antigen (HLA) 44.2.2 Genetic Characteristics of HLA
matching and immunosuppressive drugs before transplanta-
tion, 30% to 50% of transplant recipients will develop acute 44.2.2.1 Phenotype, Monotype, and Genotype
rejection. Most acute rejection can be alleviated by the use of The HLA antigen is encoded by the alleles on the chromo-
effective immunosuppressive agents. some, and an individual’s HLA antigen specificity can usu-
ally be detected by a known typing reagent or a committed
cell, and the antigen-specific type detected by this method
44.1.3 Chronic Rejection is called a phenotype. However, the antigenic phenotype
does not reflect the pattern of allele combinations on the
Chronic rejection usually occurs several months to several chromosome of the individual. The combination of HLA
years after organ transplantation [2]. The main pathological alleles on a single chromosome is called monotype or hap-
feature is the proliferation of vascular endothelial cells in lotype. If this combination is extended from class I and
transplanted organs, narrowing the arterial lumen, and grad- class II genes to class III genes, it is often called extended
ual fibrosis. Chronic immune inflammatory response is the type (extended haplotype). The HLA genotype of an indi-
main cause of the above-mentioned histopathological vidual consisting of two monotypes, i.e., the pattern of
changes. There are currently no ideal treatments for chronic HLA allele combinations on the two chromosomes within
rejection [1–3]. the individual. Monotypes and genotypes can only be deter-
mined by phenotypic analysis of individual members of the
family. The phenotype of each individual can be deter-
44.2 HLA and Transplantation Matching mined in a variety of combinations, i.e., different geno-
types. Understanding the individual’s monotype and
44.2.1 Overview genotype is important in allogeneic organ transplantation
and forensic paternity testing.
The human major histocompatibility complex (MHC)
(Fig. 44.1), also known as HLA, is located on the short arm 44.2.2.2 HLA Genetic Model
of chromosome 6, and is the main gene system that regu-
lates the human-specific immune response and determines Monotype Inheritance
the individual susceptibility to disease [4]. The correspond- According to the HLA phenotypic analysis of each member
ing DNA is about 3500 kb long. According to the function of the family, the HLA inheritance pattern is transmitted
and product structure, they are divided into three groups: from the parent to the offspring in a single unit, which is
classical HLA gene, immune function-related genes, and characterized by linkage inheritance. The offspring can ran-
immune-independent genes. It is closely related to blood domly obtain an HLA monotype from each of the parental
transfusion and acute rejection of transplantation, and is counterparts to form a new genotype of the progeny. Among
44 Transplant Matching 803
2. DNA Denaturation,
neutralization(10 min). 3. Adding
magnetic beads.
4. Hybridization(60°C,
8. Data collection. 15 min).
1. Amplification (90 min).
5. Wash buffer →
Oscillation →
Centrifugation →
Removing supernatant.
7. Wash buffer. 6. Adding SAPE
(60°C, 5 min).
Fig. 44.1 Human major histocompatibility complex
the siblings in the same family, the probability of two mono- Linkage Disequilibrium
types being identical is 25%, the probability of a single type Linkage disequilibrium refers to the phenomenon of non-
being the same is 50%, and the probability of two monotypes random distribution of single-type genes, so some genes
being completely different is 25%. Therefore, when select- (A1-B8 in whites, A2-B46 in southern China) always appear
ing the appropriate donors and recipients for clinical alloge- together, and their single-type frequency (actual value) is
neic organ transplantation, it is much easier to find the donor significantly higher than the theoretical value (for the prod-
and recipient HLA antigens in the family than the unrelated uct of various allele frequencies, such as the gene frequency
donors. However, it is worth noting that when the parental of the A1 gene frequency × B8), while others are less fre-
single type is passed to the offspring, an exchange may occur quently present. This non-free combination phenomenon is
between the two types. This has been encountered in many called linkage disequilibrium (Fig. 44.2).
cases in our previous practice, which should also be noted in
HLA typing [4].
44.3 Tissue Matching Technology
Codominant Inheritance and Related Experiments
The codominant inheritance refers to the antigen encoded by
each pair of alleles expressed on the cell membrane, no 44.3.1 HLA Typing
recessive genes, and no allelic rejection. If the two HLA
monotypes of a single body are different, there are two Organ transplantation is an important means of clinical treat-
alleles at each HLA locus, and all are reflected in the ment of end-stage organ failure and saving patients’ lives.
phenotype. With the improvement of organ transplantation technology,
Fig. 44.2 Experimental
analysis path of organ
transplantation matching
the progress of basic research on transplantation immunity, tive means for HLA genotyping research, and the HLA
and the rational application of immunosuppressive drugs, the typing technology has been rapidly developed. On the one
success rate of organ transplantation and graft survival rate is hand, with the development and application of immuno-
significantly improved. A large number of studies have magnetic beads and monoclonal antibody typing reagents
shown that human leukocyte antigen (HLA) matching is one for isolating and purifying T lymphocytes [7] and B lym-
of the important factors affecting rejection and graft phocytes [8], the traditional serological typing technology
survival. has been improved and developed. On the other hand,
In the early 1960s, Snell and Dausset and colleagues dis- molecular biology techniques such as sequence-specific
covered the immunogenetic histocompatibility antigen sys- primer polymerase chain reaction (PCR-SSP), sequence-
tem and found an antiserum source that recognizes HLA specific oligonucleotide probe hybridization (PCR-SSO),
antigens. Later, Rood and Leeuwen applied computer meth- and gene chip technologies have been widely used in HLA
ods to analyze the complex components of HLA antiserum typing research. Because DNA typing technology directly
and proposed the concept of alleles. On this basis, Terasaki classifies HLA gene polymorphisms from the genetic
invented micro-complement-dependent cytotoxicity (CDC) level, the method is accurate and sensitive, and it can
experiment and applied it to HLA serological typing, thereby detect some genotypes that cannot be detected by serologi-
achieving standardization of HLA typing and promoting the cal methods. Therefore, the serological typing techniques
rapid development of HLA serological research. CDC have been replaced by genotyping techniques in many
method becomes an important method and means of immu- laboratories. However, serological typing technology is
nogenetics and histocompatibility research. the basis and main means of HLA research, and many lab-
In recent years, with the rapid development of molecu- oratories still use it as the main method of HLA-I class
lar biology technology [6], it has provided new and effec- antigen typing.
44.3.1.1 HLA Serological Typing Technology differences in many HLA alleles are confirmed at the nucleo-
HLA-I antigens are expressed on both T lymphocytes and B tide level. DNA-based HLA genotyping has higher resolu-
lymphocytes, while HLA-II antigens are mainly expressed tion and accuracy than traditional serological typing methods.
on B lymphocytes and activated T lymphocytes. Therefore, It is known that the genetic difference of HLA individuals is
HLA class I antigen typing can be used directly. For lympho- determined by the DNA molecules encoding the gene prod-
cytes, purified T lymphocytes can also be used, while HLA-II ucts. Therefore, in theory, using molecular biology tech-
antigens can only be purified using purified B lymphocytes niques to perform donor–recipient matching at the DNA
[9]. HLA serological typing mainly uses the principle of level can reduce rejection and improve graft survival. The
CDC technology. That is, the anti-HLA antibody can bind to rate will make more sense.
the surface of the living lymphocyte membrane with the cor-
responding HLA antigen; in the presence of complement, the 44.3.1.3 T he Basis of HLA Genotyping–
lymphocyte membrane is holed. If the lymphocyte mem- Polymerase Chain Reaction
brane does not carry the corresponding HLA antigen, this The Polymerase Chain Reaction (PCR) technique is mainly
effect is not obtained. Then there is no such effect. The dead based on the semi-reserved replication mechanism of DNA
lymphocytes that destroy the cell membrane can be observed in cell division in vivo, and the nature of in vitro DNA
and judged by various methods. The most commonly used double-stranded and single-strand transition at different tem-
method is the staining method, which is stained with eosin or peratures, controlling the temperature of the in vitro synthe-
cone blue in the early stage. Under the phase contrast micro- sis system, and base-pairing the DNA template with the
scope, the dead cells become larger due to the dye entering corresponding primers. In principle, specific binding occurs,
the human body. Living cells are not colored, and the refrac- and under the action of DNA polymerase, the primer strand
tive index is strong. Now ethidium bromide (EB) and acri- can extend along the DNA template, thereby reproducing
dine orange (AO) double staining is mainly used. Under the exactly the same product as the template DNA. The entire
fluorescence phase contrast microscopy, dead cells become amplification process is divided into three phases: ① DNA
red fluorescence due to EB entering the nucleus and DNA template denaturation. DNA double-stranded at 95 °C high
binding. AO can quickly bind to lipids and pass the cell temperature to form single-stranded DNA; ② annealing.
membrane lipid bilayer to enter the living cells and is mainly During the temperature decrease, the primers added to the
enriched in lysosomes in the cytoplasm, so that the viable reaction system are complementary to the corresponding
cells can be labeled and green fluorescence is excited after single strand of the template DNA; ③ extend. At the appro-
excitation. The micro-lymphocyte toxicity test technique is priate temperature and DNA polymerase, the primer extends
an internationally accepted HLA typing standard technology from the 5′ end to the 3′ end along the template, and the
designated by the National Institutes of Health (NIH). newly synthesized primer strand can serve as a template for
The traditional HLA typing serum is mainly from human the next round of cycling and a new amplified fragment. The
serum and placental serum, and often has defects such as low template DNA is denatured, annealed, and extended in mul-
specificity, strong cross-reaction, low titer, difficulty in tiple cycles of three stages to form a specific, uniform DNA
obtaining some rare antibodies, complicated antibody fragment. These amplified DNA fragments of interest can be
screening, and difficult transportation and storage. In order used for nucleotide sequence analysis, single-strand confor-
to solve many problems in serological typing technology, in mation polymorphism analysis, and restriction fragment
the late 1980s, Terasaki and colleagues began to develop length polymorphism analysis.
HLA typing monoclonal antibodies to replace standard anti-
serum, and officially launched HLA monoclonal antibody 44.3.1.4 R estriction Fragment Length
typing plate reagents in 1992. Because monoclonal antibod- Polymorphism Typing
ies have the advantages of high specificity, high titer, and low There are multiple restriction endonuclease sites in different
cross-reactivity, the accuracy of typing is significantly parts of the HLA nucleotide base sequence, and the target
improved. Therefore, the current clinical HLA serological gene is amplified by PCR, and then the amplification product
typing is mainly based on monoclonal antibody technology. is digested and cleaved with a set of restriction endonucle-
ases. The cleavage site in the sequence produces DNA frag-
44.3.1.2 HLA Genotyping Technology ments of various lengths, which can be directly analyzed by
Since the discovery of the in vitro gene amplification gel electrophoresis, or transferred to a nitrocellulose filter to
technology-polymerase chain reaction (PCR) by Cetus in hybridize with the corresponding DNA probe, and subjected
1985, molecular biology technology has developed rapidly. to autoradiography to analyze. Due to the different base
At the same time, more and more molecular biology tech- sequences of different alleles, the restriction endonuclease
niques have been applied to HLA typing research, making sites have different distributions. Therefore, after digestion,
HLA research into a new stage of DNA genotyping, and the the fragments can be produced in different numbers and
lengths, and different DNA bands appear during electropho- olution of the product is high; Specificity: specific primers
resis. According to this, the specificity of the HLA gene can designed for known allele sequences, which determine the
be identified. specific amplification from the first cycle of PCR, unique
Restriction Fragment Length Polymorphism (RFLP) is amplification conditions (such as the two annealing tempera-
the earliest method for HLA genotyping. It is cumbersome tures, endogenous control primers, the use of primer pairs
and time-consuming, and it is prone to incomplete restriction between the groups to determine the results, etc.) further
enzyme digestion of the target gene or the length of the frag- improved the specificity; wide range of applications: accord-
ment is similar. The difficulty of the analysis limits the wideing to the different uses and requirements of HLA genotyp-
application of this technology in the field of HLA typing. ing, both low-resolution PCR-SSP typing primers can be
designed, and medium-resolution and high-resolution PCR-
44.3.1.5 Single Strand Conformation SSP typing primers can be designed to meet different require-
Polymorphism Typing ments. Different requirements for HLA genotyping in
The PCR-SSCP typing technique was pioneered by Orita laboratory and transplant clinical; simple and rapid of the
and colleagues. It is a method of analyzing a single-stranded results analysis: the amplification products of PCR-SSP
DNA polymorphism based on single-stranded DNA obtained technology only need to be subjected to conventional aga-
by denaturation of a PCR amplification product by non- rose gel electrophoresis, and the results can be determined
denaturing gel electrophoresis. The principle is that single- according to the specific amplification bands that appear.
stranded DNA forms a certain spatial conformation when
subjected to neutral polyacrylamide gel electrophoresis 44.3.1.7 PCR-Sequence Specific
without denaturing agents. Single-stranded DNA of the same Oligonucleotide Probe Hybridization
length can cause conformational differences due to differ-
ences in base sequence and even single bases, which in turn The Basic Principle of PCR-SSO
causes different migration speeds and mobility. Through Using PCR technology, the target gene was amplified by
PCR amplification, including the occurrence of a single base HLA allele or inter-group specific primers, and then hybrid-
substitution site and DNA fragments on both sides, Single ized with the synthetic oligonucleotide probe. The probe was
Strand Conformation Polymorphism (SSCP) analysis after hybridized with the PCR product under certain conditions,
denaturation can distinguish the single base changes in the strictly following the principle of base complementation,
target DNA, and effectively detect point mutations and DNA which has a high degree of specificity. The probe may be
polymorphisms and detect new alleles. labeled with radionuclide (such as 32P) and detected by
autoradiography, or may be labeled with nonradioactive
44.3.1.6 Sequence-Specific Primers Typing label (such as digoxin, biotin, and fluorescein) and the cor-
The PCR-SSP typing technique was first created by the responding markers can be detected.
Swedish scientist Olerup and colleagues in the early 1990s. The molecular hybridization pattern can be divided into
It was mainly used for HLA-II genotyping in the early stage forward hybridization and reverse hybridization. Forward
and now has been widely used in HLA-I and HIA-II hybridization is the transfer of PCR amplification products to
genotyping. solid phase carriers, such as cellulose nitrate membrane or
According to the known nucleotide sequence polymor- nylon membrane, and the identification of amplification
phism of HLA coding gene, a series of corresponding fragments is used by southern hybridization using H-specific
sequence-specific primers (SSP) were designed, and the sequence-specific oligonucleotide probes (SSO). Reverse
3′-end base of the primer was strictly complementary to the hybridization, on the contrary, is to put the specific oligonu-
polymorphic sequence of the target gene. Therefore, each cleotide probes of each allele sequence of HLA on the same
type of allele has a specific primer corresponding to it. A membrane, and then hybridize the product of site-specific or
type-specific DNA fragment of each allele was amplified by intergroup-specific PCR amplification with biotin. After
PCR, and a corresponding specific amplification product washing the membrane and detection, the hybridization sig-
band appeared on agarose gel electrophoresis. Homozygous nal is shown, so as to determine the allele type. Due to the
produces a specific amplified band, and heterozygotes have high polymorphism of HLA allele, the number of SSO
two specific amplified bands. Even a single base difference probes required is huge, and there is sequence sharing
can be accurately distinguished and, therefore, can be used between alleles, so it will be very difficult to use forward
for high-resolution HLA genotyping. SSP features: High hybridization technology for typing detection of a large sam-
resolution: Each pair of primers is designed according to the ple size. Therefore, the reverse hybridization technology has
principle of complementary bases of alleles, and only a spe- become the main clinical application for HLA typing
cific nucleotide sequence fragment is amplified, and the res- detection.
PCR-SSO Reverse Hybridization Typing tetramethylbenzidine were developed, the probe hybridiza-
Using reverse PCR-SSO gene hybridization technology, the tion position was recorded, the genotyping results were
unlabeled sequence-specific oligonucleotide (SSO) probes obtained by the corresponding analysis software.
were spotted on the same membrane, and then the corre-
sponding sites PCR products were mixed with bio-sand Flow Cytometry-SSO Typing Method
Hybridization. After membrane washing appropriately, col- Flow cytometry-SSO is an HLA typing technique that com-
oration with avidin-labeled alkaline phosphatase and its sub- bines PCR reverse sequence-specific oligonucleotide probe
strate, coloration, and then judge the HLA allelic type of the technique with flow cytometry. The principle of Flow
sample to be tested according to the hybridization signal. cytometry-SSO is to use PCR technology to amplify the tar-
This method is characterized by high efficiency, specificity, get gene with specific primers at HLA alleles or between
accuracy, and rapidity, and is suitable for genotyping with groups, and then hybridize with oligonucleotide probes of
large sample size. Currently, a reverse PCR-SSO genotyping each HLA gene attached to fluorescent labeled microparticle
kit is available. Taking HLA-DRB genotyping reagent as an magnetic beads. Each microparticle magnetic bead is not
example, it mainly includes the steps of target gene amplifi- only attached with specific HLA probes, but also color dif-
cation, gene hybridization, substrate color development, and ferences. Finally, the corresponding HLA alleles can be
result analysis. detected by laser in the liquid chip system of dedicated flow
Firstly, the HLA-DRB1 gene with high polymorphism cytometry LABScanTM100, LuminexTM200, and so on
was amplified by primers, and the amplified fragment was (Fig. 44.3).
272 bp in length. After the amplification, the amplified prod-
uct is denatured into a single strand, and added to a nylon 44.3.1.8 Gene Chip Typing
membrane containing a sequence-specific oligonucleotide
probe. The biotin-labeled amplification product is bound to Basic Principle
the probe on the membrane to pass through the linkage. The Gene chip is a technology, which can not only detect normal
avidin-horseradish peroxidase and its substrates H2O2 and DNA loci, but also detect certain genetic diseases and find
Patient preparing for

organ transplanation
ABO, Rh HLA
HLA antibody MIC antibody
blood group genotypimg
Waiting for organ transplant

registration form
Establishing patient library
Conforming to the principle of

Organ donor blood transfusion;
HLA antigen matching; Reselecting donor
No DSA
Blood group,
HLA genotyping
Cross-matching of
donor and recipient
Transplantation Rejecting
Regular monitoring after Transplantation
HLA antibody MIC antibody Pathogen Drug concentration Biochemistry,

monitoring blood routine
Fig. 44.3 Experiment operation process of flow-type fluorescent microspheres reverse SSO typing
new mutation loci from the gene level. Domestic scholars 44.3.2 HLA Antibody Detection
such as Li et al. have developed gene chip technology for
HLA genotyping. Kidney transplant recipients may develop antibodies in the
Gene chips are miniaturized by traditional reverse dot blot body due to transplant failure, pregnancy, blood transfusion,
hybridization with solids as supports such as glass, plastic, or infection, and other reasons. In addition to anti-A and B blood-
silicon. Specific oligonucleotide probes are put and fixed on type antibodies, HLA antibodies are the main antibodies that
the phase support, and thousands to hundreds of thousands affect the survival of the graft [10, 11]. It has been suggested
of probes can be fixed per square centimeter, so that a large that sensitization to HLA antigens is the only cause of clini-
number of base sequences can be detected in a short time. cally relevant positive cross-matching results. According to
The probe is designed according to the unique nucleotide the theory of transplanted humor proposed by Professor Pual
sequence of different subtypes of HLA, and that can be made I. Terasaki, HLA antibodies have the following effects: ① lead
into an HLA gene chip. After the PCR sample is labeled with to hyperacute rejection of grafts; ② lead to graft C4d deposi-
fluorescein by PCR amplification, it is hybridized with the tion combined with early graft failure; ③ is an indicator to pre-
immobilized probe on the chip. Laser scanning can automat- dict the sensitization state leading to acute rejection; ④ lead to
ically determine the HLA allele type of sample DNA by chronic graft rejection; and ⑤ new HLA antibodies after trans-
automatically analyzing the fluorescence signal value gener- plantation predict subsequent graft loss. Before transplanta-
ated by hybridization. tion, the recipient should regularly detect the presence of HLA
antibodies, antibody levels, and antibody specificity in the
Advantages of Gene Chip serum to determine whether there is an HLA antibody corre-
High Sensitivity Gene chip technology has high sensitivity sponding to the donor HLA antigen, which can provide a basis
through secondary amplification effect (i.e., PCR amplifica- for the selection of appropriate donors.
tion of the target gene DNA and fluorescence The laboratory usually uses three levels of detection: HLA
amplification). mixed antigen plate (LATM) as the qualitative screening to
determine the presence of HLA antibodies in the recipient
High Efficiency Thousands of different oligonucleotide serum. When LATM was positive, HLA antigen plate (LAT)
probes can be immobilized on one chip, so that all HLA was used to determine the level of HLA antibody (the degree
alleles may be detected at the same time. of sensitization). Finally, HLA monoclonal antigen plate
(IHD) detection was performed to determine the specificity of
High Specificity Due to the automation and programming positive antibodies. The measurement method is as follows.
of experimental methods, the manual operation error is
reduced and the accuracy and specificity of typing are 44.3.2.1 Complement Dependent Cytotoxicity
improved. CDC method is a method for the determination of antibody
specificity and reaction degree in unknown serum by using
Low Testing Cost Large sample size and automated testing standard complement-dependent cytotoxicity method after
can reduce the cost. lymphocytes from 30 to 90 different individuals were cryo-
preserved on Terasaki plate. The standard CDC method was
used to detect the antibody specificity and response degree in
44.3.1.9 H LA Typing Based on Sequence-Based the unknown serum.
Typing The main disadvantages of the method are:
Sequence-Based Typing (SBT) is HLA typing method based
on direct determination of DNA base sequence. The above 1. Non-HLA autoantibodies were detected. The improve-
typing methods can only detect the phenotype of HLA, but ment measures including incubation at 37 °C to avoid
cannot determine the nucleotide sequence of the phenotype. cold antibody reactions and add serum dithiothreitol
The essence of biological polymorphism is the nucleotide (DTT) to the serum to inactivate IgM antibody. However,
sequence encoding gene products rather than the phenotype. the strong autoantibodies of SLE patients are IgG anti-
Individuals with the same phenotype do not necessarily have bodies, and DTT cannot solve the problem.
the same DNA sequence. Due to the high polymorphism of 2. The CDC requires the combination of complement and
HLA, it is difficult to determine all alleles by the above lysis of target cells to detect antigen–antibody reactions.
methods. Direct analysis of HLA base sequence is the most However, some antibodies against common epitopes can-
accurate and reliable method. In recent years, SBT has not effectively activate complement, namely cytotoxicity
evolved from manual sequencing to automated sequencing test negative-absorption test positive phenomenon. The
and is equipped with a range of database analysis software. improvement measure is to add anti-human globulin
SBT can not only recognize and classify sequences, but also (AHG), which increases the sensitivity of the test by
find new sequences. enhancing the binding efficiency of complement C1q.
44.3.2.2 ELISA cells have been found in patients who have rejected the graft.
The purified HLA antigen was coated on the microplate and Similarly, antibodies in endothelial cell lines are not neces-
the antibody reaction was determined by the sandwich sarily polymorphic, but antibodies are produced after endo-
method. The main advantage of ELISA is that the board thelial damage. Superovulation caused by endothelial cell
reader is easy to obtain and relatively inexpensive. The spec- antibodies has been reported. Concealed superovulation
ificity of the antibody can be detected by many reactions in a (ultra-urgent rejection occurs after the incision is closed) is
96-well (60-well) assay, and ELISA does not require fixation likely to be associated with non-HLA antibodies, which are
of the complement. Main advantages of ELISA: less damaging than classical HLA antibodies and have a
slower rate of microcirculatory thrombosis. Clinically,
1 . More sensitive than CDC and the specificity is high. patients with negative HLA antibody were found to have pri-
2.
Can simultaneously detect HLA-I and HAL-II mary non-function of transplanted kidney (PNF) after sur-
antibodies. gery, which was confirmed by pathological examination of
3. Not affected by anti-lymphocyte therapy. the graft as hyperacute rejection. Ozawa et al. reported a kid-
4. Easy to use. ney transplant between 15 HLA identical twins. All the
recipients had rejected the transplanted kidney. The results of
44.3.2.3 Flow Fluorescent Microsphere Method antibody detection were anti-Lewis antibody positive in 2
The purified HLA-I and HLA class II antigens are coated on cases, anti-MICA antibody positive in 8 cases, anti-
the microparticles, and the anti-human globulin labeled with endothelial antibody positive in 3 cases, and the other 6 cases
the fluorescent marker shows the HLA antibody. The laser did not find non-HLA antibodies. Some antibodies may be
beam is used to excite the fluorescent label to detect the reac- beneficial to the graft, such as autoantibodies.
tion intensity. The specificity of HLA antibodies can be Preoperative anti-Fab antibodies have proven to be benefi-
determined using microparticles coated with different HLA cial, and recently, anti-Fab autoantibodies against IgA have
antigens, and HLA-I and HLA-II antibodies can also be dis- been shown to improve graft survival. They may be able to
tinguished in one trial. The method can detect low titer HLA antagonize the activity of common cytotoxic antibodies. Anti-
antibodies. The latest single-specific immune microbeads idiotypic antibodies to HLA may also have antagonistic effects.
technology and liquid chip technology (Luminex) are
employed to read the results, which can identify antibody
specificity accurately. 44.3.5 Cross-Matching Experiment of Donor
and Recipient
44.3.3 MICA Antibody Analysis Rejection is an important cause of organ transplantation fail-
ure. Hyperacute rejection and accelerated rejection caused
MICA antigen is an antigen expressed by major histocom- by the existence of preexisting antibodies (including HLA
patibility complex class I chain-related genes (MIC gene), antibodies) against donor-specific antigens in recipients are
which has 30% homology with the molecular heavy chain of the key factors that must be avoided during transplantation
HLA-I antigen and is expressed on the surface of endothelial [14]. Therefore, the cross-matching between donors and
cells and fibroblasts [12]. Like HLA antibodies, MICA anti- recipients before transplantation is very important for the
bodies can also be induced during pregnancy, blood transfu- success of transplantation. At present, the more commonly
sion, and transplantation. The presence of MICA antibodies used cross-matching method is the complement-dependent
in recipients can lead to acute or chronic rejection of organ micro-lymphocyte toxicity test (CDC), which involves the
transplantation. At present, MICA antibody is detected by co-incubation of the recipient’s serum with the donor’s lym-
ELISA, flow cytometry, liquid chip technology (Luminex) in phocytes and fresh complement for a period of time before
the laboratory. Luminex is currently the most widely used staining. Finally, according to the percentage of dying cells
method due to its superior sensitivity and reproducibility for staining, the compatibility between the recipient and the
purification and recombinant alloantigen. donor can be evaluated.
44.3.4 Non-HLA Antibodies 44.3.6 Monitoring of Drug Concentration

After Transplantation
Non-HLA antibodies associated with kidney transplantation
mainly include antibodies to vascular endothelial cells and It is of great clinical significance to regularly detect the blood
Lewis blood group antigens [13, 14]. Antibodies that affect concentration of immunosuppressants (cyclosporine A,
the graft may be directed against non-HLA antigens. tacrolimus, etc.) after transplantation, so as to achieve the
Antibodies against epithelial, mononuclear, and endothelial purpose of safe, effective, and rational drug use.
44.3.7 Detection of Pathogens Associated of HLA on kidney transplantation is the group with the deep-
with Infection After Transplantation est study and the largest number of accumulated cases, and it
is also representative of the HLA compatible effect in the
Due to the use of immunosuppressants after transplantation, study of substantial organ transplantation [16].
the recipient’s immunity is inhibited, which leads to an
increased risk of infection. According to the specific situa-
tion of the recipient, the detection of relevant pathogenic 44.4.2 HLA and Liver Transplantation
microorganisms can be carried out, including various viruses
(cytomegalovirus, Epstein-Barr virus, BK-JC virus, etc.), HLA-I antigens have high density in hepatic biliary epithe-
fungi, tuberculosis, Pneumocystis, and other bacteria. lial cells, venous epithelial cells, and mesenchymal–epithe-
lial cells, and have low density on hepatocytes. HLA class
II antigens are not expressed in normal hepatocytes, but in
44.3.8 Routine Blood Cell Analysis hepatic portal vein epithelial cells, mesenchymal cells, and
and Biochemical Index Detection sinusoidal cells. During acute rejection, the expression of
HLA-I antigen on hepatocytes and expression of HLA-II
After transplantation, regular blood cell analysis and bio- antigens in biliary epithelial cells and hepatic portal vein
chemical indexes examination are helpful for doctors to epithelial cells increased significantly. Liver transplanta-
understand the functional recovery of the transplanted organs tion is mostly an emergency operation. The recipients wait
of the recipients and whether the drugs used after transplan- for the liver transplantation for a short period of time, the
tation are toxic and have side effects. liver has limited storage time, and the cold ischemia time is
significantly shorter than the kidney transplantation. There
is also a significant difference between liver immunology
44.4 C
linical Significance of HLA Matching and kidney transplantation. If liver transplantation is not
in Organ Transplantation easy to hyperacute rejection, it is generally believed that
hyperacute rejection does not occur; liver transplantation is
The debate about the clinical significance of HLA matching not sensitive to immune rejection, and chronic rejection is
in organ transplantation has gradually become unified. mostly random.
1. HLA matching is necessary for organ transplantation, and

the degree of HLA compatibility is still one of the main 44.4.3 HLA and Heart Transplantation
factors affecting the long-term survival of grafts. At the
same time, the influence of other factors cannot be Heart transplantation is mostly an emergency transplant with
ignored. limited waiting time for recipient [16]. Nonimmune factors
2. Kidney transplantation class I antigens mainly affect
are the main risk factors for early death. Based on the clinical
long-term survival, especially HLA-B antigen. Class Q research results of foreign scholars in recent years, the
antigens have an impact on long-term survival and short- impact of HLA compatibility on heart transplantation mainly
term survival. In general, HLA-DR antigen is the most includes the following aspects.
important in cadaveric kidney transplantation. HLA compatibility can reduce the incidence of cardiac
3. The level of fineness of HLA typing in bone marrow transplant rejection. The effects of HLA compatibility on
transplantation is higher. cardiac allograft rejection include a reduction in early acute
4. For other substantial organ transplants, including heart rejection and a decrease in the incidence of overall
transplantation, liver transplantation, and pancreas trans- rejection.
plantation, the clinical values of HLA matching have HLA compatibility can improve the survival rate of heart
been gradually considered and valued. But the first con- transplantation. From large sample retrospective studies,
sideration is the compatibility of ABO blood group [15]. most scholars believe that HLA compatibility can improve
short-term survival and improve long-term survival.
However, some results only show an improvement trend
44.4.1 HLA and Kidney Transplantation without statistical difference. Overall analysis of HLA com-
patibility on the survival of transplanted heart, HLA-DR
HLA-I antigens are expressed in all tissues of the kidney, antigen has the most obvious effect, HLA-B antigen also has
while HLA-II antigens are expressed only in some tissues a certain relationship, and the effect of HLA-A antigen seems
such as glomeruli, renal tubules, and endothelium. The effect to be small.
44.4.4 HLA and Lung Transplantation 4. Edgerly CH, Weimer ET. The past, present, and future of HLA typ-
ing in transplantation. Methods Mol Biol. 2018;1802:1–10.
5. Montgomery RA, Tatapudi VS, Leffell MS, et al. HLA in transplan-
The effect of HLA compatibility on lung transplantation is tation. Nat Rev Nephrol. 2018;14:558–70.
basically the same as heart transplantation. Due to the small 6. Madden K, Chabot-Richards D. HLA testing in the molecular diag-
number of transplants, there is a lack of large sample retro- nostic laboratory. Virchows Arch. 2019;474:139–47.
7. Macdonald WA, Chen Z, Gras S, et al. T cell allorecognition via
spective clinical study. Scholars believe that DR compatibil-
molecular mimicry. Immunity. 2009;31:897–908.
ity can reduce acute rejection of lung transplantation, reduce 8. Vallin P, Desy O, Beland S, et al. Clinical relevance of circulating
antirejection therapy, and reduce the incidence of pathogenic antibodies and B lymphocyte markers in allograft rejection. Clin
bacteria infection, as well as shorten hospital stay and reduce Biochem. 2016;49:385–93.
9. Dragun D, Muller DN, Brasen JH, et al. Angiotensin II type
costs. But in fact, HLA compatibility in lung transplantation 1-receptor activating antibodies in renal-allograft rejection. N Engl
is difficult to achieve. The main reason is the organ preserva- J Med. 2005;352:558–69.
tion time and the limitations of the recipient sample pool, 10. Picascia A, Grimaldi V, Napoli C. From HLA typing to anti-HLA
plus the influence of other nonimmune factors on preopera- antibody detection and beyond: the road ahead. Transplant Rev
(Orlando). 2016;30:187–94.
tive selection, the probability of achieving HLA-A.B.DR 11. Loupy A, Lefaucheur C, Vernerey D, et al. Complement-binding
compatibility is very low. A.B.DR single or combination anti-HLA antibodies and kidney-allograft survival. N Engl J Med.
analysis, the more mismatches, the greater the relative risk, 2013;369:1215–26.
any one of the matches is an independent positive effect, 12. Luo L, Li Z, Wu W, et al. Role of MICA antibodies in solid organ
transplantation. Clin Transpl. 2014;28:152–60.
which significantly affects the survival rate of lung trans- 13. Zhang Q, Reed EF. The importance of non-HLA antibodies in
plantation [17]. transplantation. Nat Rev Nephrol. 2016;12:484–95.
14. Jackson AM, Lucas DP, Melancon JK, et al. Clinical relevance
and IgG subclass determination of non-HLA antibodies identi-
fied using endothelial cell precursors isolated from donor blood.
References Transplantation. 2011;92:54–60.
15. Campbell P. Clinical relevance of human leukocyte antigen anti-
1. Justiz Vaillant AA, Waheed A, Fan J, et al. Acute transplantation bodies in liver, heart, lung and intestine transplantation. Curr Opin
rejection. Treasure Island, FL: StatPearls; 2019. Organ Transplant. 2013;18:463–9.
2. Justiz Vaillant AA, Waheed A, Mohseni M. Chronic transplantation 16. Robson KJ, Ooi JD, Holdsworth SR, et al. HLA and kidney

rejection. Treasure Island, FL: StatPearls; 2019. disease: from associations to mechanisms. Nat Rev Nephrol.
3. Tong A, Sautenet B, Chapman JR, et al. Research priority set- 2018;14:636–55.
ting in organ transplantation: a systematic review. Transpl Int. 17. Ju L, Suberbielle C, Li X, et al. HLA and lung transplantation.
2017;30:327–43. Front Med. 2019;13:298–313.
Paternity Testing
45
Binwu Ying and Juan Zhou
Paternity testing refers to the analysis of human genetic crying, one woman, afraid of hurting the boy, released her
markers using the tools of medicine, molecular biology, and grip, while the other woman did not care and successfully
genetics as well as identification of the genetic relationship got the boy. Bao Zhen then determined that the woman who
between the alleged biological parents and their child accord- let go was the true mother of the baby because she could not
ing to the principles of genetics. Parentage determination is bear to hurt the boy and let him keep crying in pain.
needed in many situations [1]. For instance: In 1900, Karl Landsteiner discovered the ABO blood group
system. The genetic law of serological blood group typing
1. A mother accusing a man of being her child’s biological provided an essential scientific basis for paternity testing, but
father it alone was far from adequate. Later, with the analysis of vari-
2. A husband suspecting that the child is not his own ous other proteins and enzymes, the accuracy of paternity test-
3. An accusation of babies switched at birth in the hospital ing gradually improved. After the advent of deoxyribonucleic
4. Confirmation of lost children and their relatives acid (DNA) fingerprinting technology, DNA profiles started to
5. Inheritance dispute be used in paternity testing. This revolutionized the identifica-
6. Relationship confirmation in cases dealing with the immi- tion ability and dramatically improved the diagnostic accuracy
gration of children born out of transnational marriages of parentage determination.
7. Determination of sexual assault resulting in pregnancy Nowadays, forensic medicine methods of paternity testing
8. Biological identification of illegal children include the identification of blood types, comparison of physical
9. Evidence in cases of child abduction features, examination of skin texture, examination of genetic
diseases, distinction of earwax, examination of taste blindness,
Determining parentage has been a problem since ancient and the inference of pregnancy duration, production period, and
times. In ancient China, forensic medical technicians reproductive capacity. With the discovery of new genetic mark-
attempted to determine paternity by assessing the compati- ers, additional methods of paternity testing have been devel-
bility of the blood samples collected from the involved indi- oped. At present, we can perform accurate DNA profiling by
viduals. If the two person’s blood can be merged, then they testing blood (fresh or stains), body fluids (fresh or stains), shed
are family; otherwise, they are not. Trial judges also some- epithelial cells, hair, nails, etc. We can also test the unborn fetus
times applied the practice of forensic psychology to help using amniotic fluids or chorionic villi. Using this technology
resolve paternity disputes. A traditional Chinese historical for paternity test, certain probability can reach above 99.99%,
drama described a story of how Judge Bao Zheng in China’s and the accuracy of excluding biological father is higher.
Song Dynasty once solved a case when two women both
claimed that an infant boy was their son. Bao Zheng sepa-
rated the two women, drew a circle on the ground in the 45.1 Overview
middle, put the boy inside, and asked the women to pull the
baby out, claiming that whoever got the baby first would be 45.1.1 Alleged Father (AF) /Alleged
the mother. In the beginning, the two women were deter- Mother (AM)
mined to pull the boy to their side. When the baby started
In paternity testing, a man/woman who is required to clarify
the genetic relationship with a child is called a controversial
B. Ying (*) · J. Zhou
Department of Laboratory Medicine, West China Hospital, Sichuan father/mother, assumed father/mother, or alleged father
University, Chengdu, Sichuan, People’s Republic of China (AF)/alleged mother (AM).

814 B. Ying and J. Zhou
45.1.2 Genetic Marker etes via meiosis followed by cellular differentiation, with
each sperm and ovum normally containing only 23 unpaired
Genetic marker refers to genetic characteristics including chromosomes. With fertilization, the mature gametes then
both physical characteristics and blood genetic characteris- fuse to form a zygote, thus combining the two sets of chro-
tics, inherited from parents and controlled by genes on pairs mosomes, one inherited from each parent. A gene, made up
of chromosomes. of DNA in humans, is the basic physical and functional unit
Physical hereditary features, such as hair, skin, eye color, of heredity. A variant form of a gene at a genetic locus on a
ear hair, face shape, brachydactyly, and polydactyly, can chromosome is called an allele. Some genes can have a vari-
occasionally help resolve parentage disputes. For example, if ety of different alleles, which are said to be polymorphic.
a boy has a black hairy mole in front of his right ear and the Humans inherit two alleles for each gene, one from each par-
AF has a same black mole in the same location, the AF is ent. Each pair of alleles represents the genotype of a specific
very likely the child’s biological father, because the chance gene. Nucleotides, each containing a nitrogenous base, are
of a random coincidence of black moles in front of the right the basic building blocks of DNA. The haploid human
ear is tiny. nuclear genome comprises about 3 billion DNA base pairs.
Blood hereditary features include the many genetic mark- The random interchange and combination of genetic mate-
ers and polymorphisms of the four main components of rial before germ cell formation makes it never possible for
blood, namely, the red blood cells (RBC), white blood cells two people in the world to have the same genome, and this
(WBC), platelets, and plasma. To date, more than 260 eryth- forms human genetic polymorphism.
rocyte homologous antigens, 50 erythrocyte enzymes, nearly The theoretical basis for judging the parent-child relation-
100 plasma proteins, and 124 leukocyte homologous anti- ship is the Mendel’s Law of Segregation and the Law of
gens have been found to be useful for paternity identifica- Independent Assortment [2]. The genes in the somatic cell
tion. There are 539 genetic markers involving 56 blood group nucleus appear in pairs and determine the genetic traits of the
systems, out of which 107 have been tested clinically organism. The Law of Segregation refers to the fact that
(Table 45.1). when germ cells form gametes through meiosis, pairs of
Since the advent of DNA fingerprinting technology and alleles randomly segregate from each other so that each gam-
the establishment of polymerase chain reaction (PCR) ete contains only one allele for each gene. For example, in
method, in laboratories with better conditions, multi- the MN blood group system, during gametes formation,
parameters combining blood groups, enzyme types, and alleles M and N of the heterozygote gene MN are split into
DNA polymorphisms have been used, significantly improv- different gametes; which allele enters which gamete is
ing the probability of paternity exclusion. entirely up to chance. The Law of Independent Assortment
states that genes of different traits are passed onto the off-
spring independently of each other, such that the inheritance
45.1.3 Genetic Law of genes at one location does not influence the inheritance of
genes at another location in a genome, as long as there is no
In humans, DNA and histone proteins are tightly packaged genetic linkage among these loci. This law is the theoretical
into chromosomes in the nucleus of cells. A typical human basis for calculating the cumulative discriminant probability
somatic and germ cell has 23 pairs of chromosomes (46 of genetic markers in personal identification statistics.
chromosomes)—22 paired autosomes plus the 23rd pair of Individual identification or paternity testing should select
sex chromosomes. The germ cell gives rise to haploid gam- genetic markers that conform to the Law of Independent
Assortment. Usually, these markers are located on different
Table 45.1 Blood genetic markers for resolving paternity disputes chromosomes or at distant locations on the same chromo-
some. They need to be confirmed to have no genetic linkage
Genetic markers System
and can be inherited independently through population sur-
Known Applied Known Applied
Blood components number number number number veys. Multiplication principle can be used to calculate the
Erythrocyte 260 26 24 10 cumulative discriminant probability of multiple genetic
homologous markers only when independent phenotype or gene fre-
antigen quency is used independently.
Erythrocyte 55 23 13 7 In addition, maternal inheritance and paternal inheritance
isozyme
are the other two major inheritance laws closely related to
Plasma protein 100 37 18 13
Leukocyte 124 21 1 1 paternity testing [2]. A typical example of maternal inheri-
homologous tance is mitochondrial DNA, which mainly transmits genetic
antigen information to the next generation through the ova, making the
Total 539 107 56 31 mitochondrial DNA sequences of the offspring accordant with
45 Paternity Testing 815
those of his/her mother. Maternal inheritance can be used to AF’s genotype. If there is no obligatory gene, we can exclude
trace the history of human maternal evolution. Also, since the the hypothetical father-child relationship. If the AF has the
mitochondrial genetic markers are only derived from the OG, the result cannot rule out the AF’s biological relation-
mother, they are particularly valuable in the absence of pater- ship. For example, if the mother is FGA-22/23 and the child
nity testing and for the identification of siblings of the off- is 22/25, it can be determined from the comparison that the
spring or siblings of the same mother. Paternal inheritance OG is FGA-25. If the AF1 is FGA-21/24 and AF2 is FGA-
refers to the fact that most of the Y chromosome DNA does 23/25, we can conclude that AF1 does not possess the OG
not recombine with the X chromosome. These non- FGA-25, so his biological relationship with the child can be
recombinant genetic markers are transmitted directly from the excluded; in contrast, AF2 has FGA-25, and it does not
father to the son, which are highly conservative and character- exclude the paternal relationship with the child.
istic. The study of Y chromosome non-recombinant DNA
polymorphism in a population can not only trace the evolu-
tionary history of human paternity but also play an important 45.2 S
everal Common Paternity Testing
role in personal identification and paternity testing. Techniques
The child’s genetic characteristics (markers) are the combi-

45.1.4 Basic Principles of Paternity Testing nation of genes provided by both parents and determined
from the moment of fertilization. Therefore, when perform-
The basic principles of paternity testing are as follows: ing a paternity test, we can test the genetic markers of the
father, the mother, and the child and judge whether they con-
1. If we affirm that a child’s allele is from the biological form to the genetic law. At present, the standard methods of
father and AF does not have this allele, we can exclude paternity testing in forensic medicine include ABO blood
him as the biological father of the child. Obviously, the group test, DNA fingerprint analysis, and STR technology
more genetic markers examined, the more likely non- application.
biological fathers will be excluded.
2. If we affirm that some alleles of the child are from the
biological father, and AF also carries these alleles, we 45.2.1 Paternity Exclusion by Red Blood
cannot rule out that he is the biological father of the child. Cell Type
At this point, we can calculate how big is the theoretical
power if we judge that he is the biological father of the Three alleles control the ABO blood group system: a reces-
child. sive allele i and codominant alleles IA and IB. The alleles
make six genotypes IAIA, IAi, IBIB, IBi, IAIB, and ii and four
In a family, the inheritance law can be summarized as phenotypes A, B, AB, and O.
follows: Blood type inheritance follows Mendelian inheritance. In
a family, the children’s blood type genes must come from the
1. A child cannot carry alleles that neither of the parents has. parents. However, children’s blood types are not entirely the
2. A child must have one of the pairs of alleles in each same as the parents’, because what the children inherit from
parent. parents is the gene that controls blood types instead of the
3. Except when both parents have the same allele, a child blood type (phenotype). Children’s blood types are not
cannot have two identical alleles. unique, but rather a highly variable probability event.
4. When one or both of the parents is/are homozygote(s), the However, except for some cases that blood type A person
allele of the homozygous gene must be inherited in the married with blood type B person, other blood type combi-
child. nations can all result in an impossible event with a probability
of 0. Red blood cells can only be used for paternity exclu-
The basic principles of paternity testing with biallelic sion, but not for paternity affirmation.
genetic markers can be extended to genetic markers of mul-
tiple alleles, such as the short tandem repeats (STR) multi-
plex system. 45.2.2 Paternity and Family Relationship
In most of the paternity testing cases, the mother-child Identification by DNA Fingerprint
relationship is definite, requiring the identification of the Techniques
assumed father-child relationship. Firstly, from the compari-
son of the mother and child genotypes, it can be determined There are three main types of DNA molecules, single-copy
that which allele of the child may come from the father sequences, moderately repetitive sequences, and highly
(obligatory gene, OG). Then, we can judge and observe the repetitive sequences, among which, there is a kind of repeti-
tive sequence with less than 300 nucleotides in length. individual identification, paternity test, archaeology, and
Because of being highly repetitive, the sequence appears gene diagnosis as the ideal DNA genetic markers [3]. STR
near the main DNA band as satellite band after ultracentrifu- gene loci generally follow the Mendelian dominant genetic
gation, so it is also called satellite DNA, and the repeat law in gene transmission due to their polymorphism and dif-
sequence unit is called “small satellite DNA.” Small satel- ferences in core sequence repetition numbers among indi-
lites are highly variable, but in “small satellite DNA,” a short viduals. In addition, short DNA fragments lead to less
sequence called “core sequence” is the same in all individu- possibility of mismatch and preferential amplification, thus
als. If the core sequences are connected in series as a molec- improving the success rate and sensitivity of PCR amplifica-
ular probe to hybridize with DNA of different individuals, tion. Moreover, the required amount of samples is small
the unique hybridization map will appear. They have incred- (0.1 ng template DNA), the amplification results are stable
ibly high selectivity and specificity like human fingerprints, and repeatable, and automatic typing can be carried out. It
so it is called “DNA fingerprints.” Due to the multisite char- can be used for paternity testing, individual identification,
acteristic, high variability, and simple and stable heredity of and gene ID project. Compared with traditional paternity
DNA fingerprinting, it has attracted people’s attention since tests performed using red blood cell types or HLA-A, B, and
its emergence, showing great practical value. The high vari- DR, this method has a higher probability of affirming pater-
ability and somatic stability of DNA fingerprints make DNA nity (99.99%) and more gene loci of excluding paternity
fingerprinting extremely valuable for suspect identification (≥3). Now, it has become the main technical tool for pater-
as well as kinship establishment in forensic medicine. nity testing and individual identification in forensic laborato-
High-resolution DNA fingermarks usually consist of 15 ries, and it has greatly improved the ability of paternity
to 30 bands, like the bar codes on products. Most of the identification.
bands in the DNA fingerprinting region are independently
inherited and follow the Mendelian inheritance law. Each
band in the offspring’s DNA fingerprints can be found in one 45.3 J udgement and Analysis of Paternity
of the parents’ DNA fingerprint. Therefore, DNA fingerprint Testing Results
analysis can be used for paternity identification. If the DNA
pattern of the child and the test man does not match on one 45.3.1 Parent-Child Relationship Exclusion
or more DNA probes, the man is 100% ruled out as the bio-
logical father. If the child’s DNA pattern completely matches Paternity testing is most important in cases judging the
the test man’s, we can calculate that there is a 99.9% or parental relationship between the AF and the child. In pater-
higher chance that he is the biological father. Herein, DNA nity testing, when the mother is the biological mother, if the
fingerprint analysis is more accurate than ABO blood type in child’s genetic marks are not provided by the biological
paternity testing. However, this technology is complicated in mother, it must be provided by the biological father. If the AF
the operational process, time-consuming, and may be inap- cannot provide the genetic markers, he is not the child’s bio-
propriate for various situations. It requires a large number of logical father, and the parental relationship is excluded,
materials. It does not allow for determining the location of which calls paternity exclusion. If the AF can provide the
each band in the chromosomes and the independence child’s OG, on the other hand, the possibility of him being
between individual sites. Furthermore, it is difficult in geno- the child’s biological father cannot be ruled out.
typing standardization. All these disadvantages make DNA In paternity testing, the parent-child relationship can be
fingerprinting difficult in application. In recent years, STR ruled out mainly in two cases [2]:
technology has gradually replaced DNA fingerprint technol-
ogy in forensic paternity testing [3]. 1. The child carries an allele that is absent in both his/her
The human genome is composed of about 3 billion base pairs. biological mother and the AF. For example, if the mother
STR generally refers to repetitions of 2–7 bp core sequences. is FGA-22/23, the AF is FGA-22/25, and the child is
For example, D5S818:5’-GGGTGATTTTCCTTTTGGT FGA-22/24, the parental relationship between the AF and
(AGAT)7-14TGTGGCTATGATTGGAATCA-3′ shows length the child is ruled out. In this case, neither the mother nor
polymorphism because of the difference of the repetition the AF can provide the OG FGA-24 of the child.
number of the core sequence between individuals. STR gene 2. The child does not have the gene that the AF would defi-
loci are widely distributed in the human genome, with short nitely pass along to his offspring. For example, if the bio-
sequence fragments, generally ranging from 100 to 500 bp. logical mother is FGA-22/23, the AF is FGA-25/25, and
Simultaneous multiple gene amplification can be used in the child is FGA-22/24, the parental relationship is also
PCR analysis for STR. It is not only accurate but also highly ruled out because the child does not have the allele FGA-
convenient and efficient. Therefore, STR is widely applied in 25, which is expected in all offspring of the AF.
How many genetic markers that do not meet the heredity genetic markers. At present, the commonly used DNA
laws are needed to make a paternity exclusion conclusion? genetic marker systems are codominant inheritance, and
Most experts and scholars believe that the parent-child rela- the number of alleles is large. For a genetic marker system,
tionship can be ruled out when more than three genetic mark- let pi represent the i-th allele frequency in the population,
ers do not conform to the heredity law. One incongruent pj represents the j-th allele frequency in the population,
genetic marker could be caused by mutation or unequal and the allele i is not equal to the allele j, then the EP of the
amplification. Two incongruent genetic markers are indeed genetic marker is:
suspicious, but it has been reported that in a true triplet fam-
ily, two STR loci variants were found when testing more than

2

EP pi 1 pi 1 / 2 pi 2 pj2 4 3pi 3pj
twenty STR loci. When there are more than three indepen-
dent genetic markers that do not meet the heredity laws, the i 1 i 1 j i 1
excluded conclusion can be reasonably made with confi-
dence. Assuming the mutation rate of a genetic marker is 45.3.1.3 C umulative Excluding Probability
0.002, the probability of simultaneous mutation of three of Paternity (CEP)
genetic markers is only 8 × 10−9; thus the probability of false The above formula for calculating the EP is used for a par-
exclusion is very low. In our practice, there were very few ticular locus. Since more than one locus is used for paternity
cases in which we reported paternity exclusion based on only testing, it is necessary to know the total genetic markers used
three genetic markers. We found more than five incongruent for a man who is not the father of a child in order to deter-
genetic markers in most of the exclusion cases. The more mine the efficacy at excluding the innocent AF, that is, the
incongruent genetic markers, the lower the probability of an total cumulative excluding probability of paternity (CEP).
erroneous exclusion. Therefore, for each genetic marker sys- The CEP is calculated as:
tem, there is an excluding probability of paternity when only
men with certain genetic markers can be excluded and others CEP 1 1 EP1 1 EP 2 1 EP3
cannot be excluded among all possible combinations of
1 EPk 1 1 EPk
mother and child.
where EPk is the EP value of the k-th genetic marker.
45.3.1.1 Excluding Probability of Paternity (EP) Check a variety of genetic markers, determine the EP value
The systemic efficacy of genetic markers for paternity test- according to the genetic method of various genetic markers,
ing is often quantitatively assessed using the excluding prob- and then calculate the total CEP value according to the for-
ability of paternity (EP). The EP refers to the probability that mula. Table 45.2 shows an example of the excluding proba-
a man who is not the father of a child can be excluded from bility of paternity of 15 commonly used STR loci in the
the genetic markers. When a man who is not a child’s father Chengdu Han population. As shown, the more genetic mark-
is mistakenly accused of a biological father, it can theoreti- ers used, the higher the CEP, and the stronger the discrimi-
cally be denied based on genetic marker detection. However, nating ability is.
when the genetic marker has a poor discriminating ability,
the genetic markers of a random man and a child without
blood relationship may coincide with the genetic law, so it is
not certain that he has no parent-child relationship with the Table 45.2 EP and CEP of 15 STR loci in Chengdu Han population
child. Different genetic markers have different polymor- Locus EP CEP
phisms; the probability that an assumed man cannot be D5S818 0. 482 0.482
excluded due to chance is high or low. Therefore, it is neces- FGA 0. 690 0. 839,420
sary to know the EP when it is applied to a man who is mis- D8S1179 0. 718 0. 954,716
takenly accused of a child’s father by applying a genetic D21S11 0. 626 0. 983,064
marker [2]. For example, if the EP of the HLA-B system is D7S820 0. 498 0. 991,498
0.8071, it means that 80.71 non-parents can theoretically be CSF1PO 0. 466 0. 995,460
D3S1358 0. 428 0. 997,403
excluded out of 100 non-parents by detecting HLA-B
TH01 0. 466 0. 998,613
antigen.
D13S317 0. 626 0. 999,481
D16S539 0. 539 0. 999,761
45.3.1.2 C alculation of Excluding Probability D2S1338 0. 672 0. 999,922
of Paternity (EP) D19S433 0. 581 0. 999,967
The EP is related to the genetic pattern and degree of poly- vWA 0. 608 0. 999,987
morphism of the genetic markers. It mainly depends on the TPOX 0. 214 0. 999,990
number of alleles and allelic frequency distribution of the D18S51 0. 757 0. 999,998
45.3.1.4 E rrors in Excluding Parent-Child 45.3.2 Affirmation of Parent-Child

Relationship and Its Solutions Relationship
As the number of genetic markers detected increases, genetic
mutations with low frequency are gradually disclosed. Lack At present, more and more genetic markers are used for
of this knowledge makes it prone to false paternity exclu- paternity testing, especially the widespread use of STR
sion. Genetic mutations include gene mutations, alternative genetic markers. It has changed the condition that the blood
alleles, weak antigens, silent genes, gene deletions, blood type test can only be used for parental exclusion and cannot
type variants, gene exchanges, CIS-AB effects, chimeras, affirm parental rights. When the genetic marker test results
mosaic antigens, and physiological and pathological varia- do not violate the genetic law between the parent and the
tions [2, 4, 5]. offspring, there may be a biological relationship. At this
point, the parent-child relationship index and the parent-
Gene Mutation child relationship probability can be calculated to understand
In the process of meiosis, the exchange and recombination of the possibility of the existence of a biological relationship
genes and the mutation of genes due to the effect of uncertain between them and to determine whether there is a biological
factors can occur. This is an important reason for the genetic relationship.
markers of the parents and offspring to be incompatible with
the genetic law. It may affect the accuracy of paternity test- 45.3.2.1 Paternity Index
ing and thus mislead the detection and trial of the case. The paternity index (PI) is the likelihood ratio of the two
Therefore, genetic markers with lower mutation rates should probabilities required to determine the parent-child relation-
be used in the process of paternity testing. ship, that is, the ratio of the probability that the AF is the
child’s biological father (X) to the probability that a random
Alternative Allele man is the child’s biological father (Y). In other words, PI
Among the erythrocyte blood types, there are some alterna- assesses how much more likely the AF who possesses the
tive alleles, such as CW, CX, Rh26-c, EW, ET-E, V, VS-e, Mg- OG than a random man who also has the OG is to be the
M, Fy3, Fy4-Fya, or Fyb. For example, suppose that both the child’s biological father.
mother and the daughter are the N type, and the AF is the M
type. Under normal circumstances, the AF should be 45.3.2.2 Relative Chance of Paternity
excluded. However, if the AF is the MMg type and the child The above-calculated PI value is an absolute value. In order
is the NMg type, no antigen is detected by anti-M or anti-N to express the relative chance of paternity (RCP) in a proba-
serum alone, and the AF could be mistakenly judged as the bilistic form, the PI value must be converted into a relative
M type (MM) while the child as the N type (NN). In reality, value RCP. After calculating the PI value, RCP = [PI / (PI
however, the MMg type father cannot be excluded because he +1)] × 100%. According to the practice of paternity testing at
can provide the Mg allele to the child. home and abroad, it can be assumed that the father and the
child have a biological relationship when the RCP value is
Weak Antigen greater than 99.99%. If the RCP value does not reach 99.99%,
The weak blood type antigen is challenging to detect and is it can be assumed that the parent and the child do not have a
easily misidentified as a negative antigen. The following biological relationship. If the RCP value is less than 99.99%,
antigens of the fetus or babies are not well developed, and the number of detection sites should be increased until the
the antigenicity is very weak, including A1, I, P1, Leb, Xga, RCP is greater than 99.99%.
and Hp. The A1 type is easily mistaken for the A2 type, the P1
type for the P2 type, and the HP type for the HpO type.
45.3.3 Forensic Criteria of Paternity Testing
Gene Exchange
Several gene exchanges of MNSs have been discovered, and 45.3.3.1 The Standard of Paternity Exclusion
one Rh gene exchange has been found, but the probability is The genetic marker is tested by standardized experiments.
very low. The male assumed as the father who could not provide nec-
essary alleles to the child can be excluded paternity; in the
CIS-AB Effect absence of a mutation, we can conclude that he is not the
The A and B genes can be located on one chromosome as a biological father of the child. In order to avoid potential
unit and pass on to the offspring. The AB blood type parents mutational effects, the exclusion of paternity should be based
can produce O type children, and dozens of such cases have on at least three genetic markers. In no case can the paternity
been reported in the literature. be excluded based on only one genetic marker.
45.3.3.2 The Standard of Paternity Affirmation the correct results. In order to ensure that each result is cor-
When genetic markers were tested by standardized experi- rect and reliable, negative and positive controls must be set.
ments and the AF could not be excluded paternity, the pater- The positive control is a verification of the reliability of the
nity can be affirmed; that is, the AF can be judged to be the reagents, equipment, and sample results. Practices have
child’s biological father if the following two indicators are proved that the accurate positive control results are the pre-
met at the same time after calculating the probability [6]: requisite for a correct judgment of the test samples. Qualified
laboratories should also regularly participate in laboratory
1. The experimentally detected CEP of genetic markers is comparisons and laboratory proficiency testing activities.
equal to or greater than 99.99%. This not only allows labs to assess their capabilities but also
2. Assuming that the father’s cumulative PI is equal to or provides opportunities to communicate with and learn from
greater than 10,000; that is, under the condition that the each other, making progress together.
pre-probabilities are the same, the father’s relative chance Appraisal of the paternity testing results is accurate and
of paternity is assumed to be equal to or greater than authoritative only under strict quality control, standardized
99.99%. The PI is the ratio of two probabilities, and it is operation, and technical standards [4–6].
usually converted, when used as evidence provided to the
courts, into a conditional probability, the RCP, according
to Bayes’ theorem. 45.4 Collection and Preservation
of Paternity Test Samples
RCP PI / PI 1 100%
Paternity testing nowadays uses samples containing DNA for
identification. Samples are mainly divided into three catego-
For example, the PI of a paternity test is calculated to be ries, including routine samples (blood, bloodstain, hair with
2497. Under the condition that the pre-probabilities are the follicles, buccal swab), special samples (nails, pure spots,
same, then: cigarette butts, amniotic fluid, chewing gum, toothbrush,
RCP 2497 / 2497 1 100% 99.96% bone, menstrual blood), and difficult samples (embryo, slice
or paraffin-embedded tissue, mixed spots). Different types of
According to the above-mentioned paternity criteria, if materials have different degrees of change in their denatur-
the RCP value is greater than 99.99% (equivalent to PI ation, degradation, and corruption due to their characteristics
≥10,000), it is assumed that the parent-child relationship and environmental factors, bringing certain difficulties to the
should be unambiguous. inspection and identification work. Therefore, before the
samples are delivered to the laboratory, the samples should
be kept appropriately, and the inspection time should be
45.3.4 Laboratory Standards of Paternity shortened as much as possible [4–6].
Testing
The quality control of the paternity test is to standardize the 45.4.1 Collection of Paternity Test Samples
laboratory and ensure that the test results are correct and reli-
able. More and more laboratories now recognize the impor- 45.4.1.1 Blood/Bloodstain
tance of this work. The International Association of Forensic Mark the date of sample collection and sample identity on a
Genetics paternity test committee recommended the interna- clean envelope bag, such as father, mother, and child. First,
tional standards for paternity testing laboratories in 2002. As use an alcohol swab to disinfect, collect 2–5 mL venous
a qualified paternity test laboratory, it should firstly comply blood as blood specimens, and five drops of blood collected
with the ISO 17025 guidelines, so that the legal documents it from fingertips or earlobes (for children can also take blood
produces are authoritative. The guidelines stipulate that the from soles) on a medical gauze or filter paper as bloodstain
laboratory management personnel and authorized signatory specimens. After being naturally dried (not allowed to blow
should have specific qualifications and be recognized by the dry or sundry), each is placed in a labeled envelope.
authority. Secondly, the appraisers should have the qualifica- Note: During the collection process, one should not touch
tion to engage in test appraisal, relevant professional knowl- the bloodstain sample. Ensure that the sample is consistent
edge, strong instrument operation ability, and data analysis with the identity of the person marked on the envelope. If a
and interpretation skills. At the same time, sound equipment person has received a blood transfusion or bone marrow
and equipment files should be established. Due to our depen- transplant in the past year, blood/blood mark samples are not
dence on highly automated instruments and equipment, rou- recommended; hair or buccal swab samples can be used as
tine services and regular verifications are crucial to achieving an alternative.
45.4.1.2 Hair women and fetuses. Collect 3–5 mL of amniotic fluid sam-
Mark the date of sample collection and sample identity on a ples, which are clear and transparent. They should not con-
clean envelope bag, such as father, mother, and child. Pull off tain the blood components of the mother. The samples should
at least five hairs from the head, eyelashes, or body hair, with be sent for inspection as soon as possible, especially in the
clear hair follicles visible at the end of the hair. Immediately summer. It is better to provide a blood sample of the mother
put the newly removed hair into the envelope that has been as a control.
marked.
Note: Make sure that the hair follicles can be seen at the
end of the hair with the naked eye. Avoid touching the hair 45.4.2 Preservation of Paternity Test Samples
follicles of the sample with hands. Do not touch the hair fol-
licles of the hair during the collection process. Specimens After the samples are collected, each sample must be indi-
that have fallen on the ground or have been unplugged for a vidually packaged to avoid sample loss and cross-
long time should be rejected. contamination during the process of inspection. The
packaging of the sample should be firm, clean, and easy to
45.4.1.3 Oral Swab label. Various types of paper bags, plastic centrifuge tubes,
Mark the date of sample collection and sample identity on a jars, etc. can be used. Liquid or wet samples should be stored
clean envelope bag, such as father, mother, and child. Avoid frozen at −20 °C as soon as possible after collection. Freezing
touching the cotton swab with hands. Rinse the mouth with is a simple and effective way to preserve DNA test samples,
water before collecting. Take a special cotton swab in the but freezing cannot be sterilized. After thawing, microbial
right cheek of the mouth and slowly rotate it 20 times. Take growth will still occur. Drying can inhibit the growth and
out another special cotton swab and repeat the above steps to reproduction of microorganisms; most of the samples can be
contact the left cheek in the mouth and slowly rotate 20 made into dry stains for long-term preservation, and it is bet-
times. Each subject needs to repeat the above steps to collect ter to keep the dry stains in the −20 °C refrigerator. Ultraviolet
six swabs. After the collection is completed, the cotton sticks light can degrade DNA more quickly, and the materials
for each specimen are dried in the shade (not in the sun, or should be kept away from direct sunlight. The tissue soaked
hot air), and then placed in the prepared envelope. in the formaldehyde solution is difficult to extract DNA, and
it can generally be stored in 75% ethanol solution.
45.4.1.4 Saliva and Saliva Spots
After the saliva is provided, let the saliva naturally flow out
1–2 mL, collect it in a clean test tube or a small beaker, and References
boil for 5 minutes in a water bath or store it frozen. Saliva
spots are common in the remaining cigarette butts, pipes, 1. Peng R, Bin P, Suyun H. The application of paternity testing in the
field of forensic evidence. Legal System and Society. 2019;5:224–5.
chewing gum, melon shells, straws, beverage containers, bite 2. HouYiping. Forensic biological evidence. 3rd ed. Beijing: People's
marks, toothpicks, and toothbrushes. They are not visible to Medical Publishing House; 2009.
the naked eye and are not easy to find. Suspicious items can 3. Veselinović I. Microsatellite DNA analysis as a tool for forensic
be sent to the laboratory for inspection. paternity testing (DNA paternity testing). Med Pregl. 2006;59:241–3.
4. HouYiping LY. Forensic DNA genotyping. Beijing: Science Press;
2007.
45.4.1.5 Amniotic Fluid Samples 5. Zheng X. Forensic DNA analysis. Beijing: Chinese People's Public
Amniotic fluid samples must be collected by an experienced Security University Press; 2002.
obstetrician in a hospital to ensure the safety of pregnant 6. Chengtao L, Yiping H, Li L, Suhua Z, Yacheng L, Hongyu

S. Specification of parentage testing. GB/T. 2018:37223–2018.
Appendixes
Appendix A: Tests of Infectious Disease 2. Take the serum in the acute phase and the recovery phase
of the patient for detection. If the serum antibody titer in
Infectious diseases refer to local tissues and systemic inflam- the recovery period is 4 times or more than the acute
matory reactions caused by bacteria, viruses, fungi, and very phase, it is meaningful.
common pathogens (mycoplasma, chlamydia, tuberculosis,
etc.) that invade the human body. Among the world’s deaths A.2 Aspergillus Antigen (Galactomannan, GM)
from diseases, infectious diseases account for 45%. In addi- Detection Tests
tion to clinical manifestations, the diagnosis of infectious Sample Required Serum, BALF, CSF (300 μL)
diseases is more important by means of the detection of Detection Method EIA
pathogens or their markers. Clinical microbiological testing Reference Ranges index < 0.50
faces enormous challenges. Traditional artificial culture Interpretation
takes a long time, while morphological examination technol-
ogy requires high experience and is easy to miss. Finding 1. Sera with an index < 0.50 are considered to be negative
fast and accurate markers is the key to infectious disease sur- for galactomannan antigen, index ≥ 0.50 are considered
veillance and effective treatment. With the all-round devel- to be positive for galactomannan antigen.
opment of new technologies such as molecular biology 2. A negative result may indicate that the patient’s result is
detection technology and serology, pathogen variants and below the detectable level of the assay. Negative test from
“new pathogens” are known to be rapidly and accurately dis- samples cannot rule out the diagnosis of invasive aspergil-
covered, which brings great convenience to clinical diagno- losis. Repeat testing is recommended if the result is nega-
sis. Infection diagnostic markers can be broadly classified tive, but the disease is suspected.
into two categories: rapid antigen-antibody detection and 3. Regular screen (twice-weekly) of samples from patients
rapid nucleic acid detection. With the introduction of new at risk for invasive. Aspergillosis is recommended to
foreign technologies and testing platforms, automated and increase the sensitivity and early positivity of the test.
digital related detection technologies have been applied and 4. For all positive results, it is recommended to reprocess the
widely used in the diagnosis of infectious diseases. specimens for testing to increase the accuracy of the
result.
A.1 Adenovirus Antibody Tests
Sample Required Serum (100 μL) A.3 Aspergillus Antibody Detection Test
Detection Method EIA, CLIA, ECL Sample Required Serum, BALF, CSF, Urine (200 μL)
Reference Ranges Negative Detection Method EIA GICA
Interpretation Reference Ranges Negative
Interpretation
1. Adenovirus infection specimens include throat swab

specimens, nasal washes, and stool specimens. However, 1. A negative blood test means that it is likely that no infec-
adenovirus infection is significant through serological tion is present. However, a negative screening test means
diagnosis. At least 12 types of adenovirus have been asso- only that there is no evidence of disease at the time of the
ciated with illnesses such as pneumonia, and acute hem- test.
orrhagic cystitis. Asymptomatic infections can make 2. Antibodies may not be detected for several weeks after
serologic responses difficult to interpret. exposure to aspergillus. After aspergillus infection, as

S. Pan, J. Tang (eds.), Clinical Molecular Diagnostics, https://doi.org/10.1007/978-981-16-1037-0
822 Appendixes
long as the antigen does not disappear, the antibody can A positive broad-range PCR/sequencing result indicates
exist in the serum for a long time. that bacterial nucleic acid of the specified organisms was
3. The combination of the two methods can greatly improve detected, which may be due to bacterial infection or envi-
the sensitivity and specificity of detection, and shorten the ronmental or contaminating nucleic acids in the specimen.
clinical infection of deep aspergillus. Especially the diag- A negative broad-range PCR/sequencing result indicates the
nostic time of subacute and chronic aspergillosis, and GM absence of detectable bacterial (including mycobacterial)
test will be negative soon after antifungal treatment, but nucleic acids in the specimen, but does not rule-out false-
aspergillosis IgG antibody is still positive as long as the negative results that may occur due to sampling error,
fungal infection is not completely cleared. sequence variability underlying the primers, the presence of
4. Continuous monitoring of the level of aspergillosis IgG bacterial nucleic acids in quantities less than the limit of
antibody is helpful for doctors to evaluate the curative detection of the assay, or inhibition of PCR. If PCR testing
effect. The combination of the two methods can greatly appears to be negative but there is evidence of PCR inhibi-
improve the sensitivity and specificity of detection. tion, testing will be repeated. If inhibition is again detected,
the result will be reported as “PCR inhibition present.”
A.4 BK Virus Nucleic Acid Detection Tests
Sample Required Serum/Urine A.6 Candida Antigen (Mannan) Test
Detection Method Real-time fluorescence quantitative Sample Required Serum, plasma, whole blood, CSF, BALF
PCR (100 μL)
Reference Ranges Negative Detection Method ELISA
Interpretation Reference Ranges <0.5 ng/mL
Interpretation
1. The virus can be used as a monitoring of organ and tissue
transplantation, and its infection diagnostic index pro- 1. Mannan antigen is a specific secretion of Candida and can
vides objective indicators for efficacy observation and be used as a diagnostic basis for Candida infection.
success or failure of transplantation. 2. Mannan <0.50 ng/mL are considered to be negative for
2. It has clinical guiding significance for autoimmune dis- Candida antigen and ≥0.50 are considered to be positive
eases and patients who use immunosuppressive agents. for Candida antigen.
3. A negative blood test means that it is likely that no infec-
A.5 Broad-Range Bacterial PCR and Sequencing tion is present. However, a negative result may indicate
Sample Required Varies that the patient’s result is below the detectable level of the
Detection Method PCR and Sequencing assay.
Reference Ranges No bacterial DNA detected 4. Repeat testing is recommended if the result is negative,
Clinical Information but the disease is suspected. Regular screen of samples
Cultures from patients with suspected bacterial infection from patients at risk of Candida infection is recommended
involving normally sterile sites may fail to provide bacte- to increase the sensitivity and early positivity of the test.
rial (including mycobacterial) growth for identification due
to the presence of a fastidious or slow-growing bacterium A.7 Candida Antibody Test
or as a result of antecedent antimicrobial chemotherapy. Sample Required Serum, CSF (100 μL)
Broad- range bacterial PCR amplification followed by Detection Method EIA, CLIA, ECL, GICA
Sanger sequencing of the amplified product may poten- Reference Ranges Negative
tially detect bacterial (including mycobacterial) nucleic Interpretation
acids in such situations, enabling a diagnosis. Ideal speci-
mens are those in which bacteria (includes mycobacteria) 1. A negative blood test means that it is likely that no infection
are visualized by microscopy. Heart valves from patients is present. However, a negative screening test means only
with endocarditis with positive Gram stains are, for exam- that there is no evidence of disease at the time of the test.
ple, especially suitable. Useful For: Detecting and identify- 2. Antibodies may not be detected for several weeks after
ing bacteria (including mycobacteria) from normally sterile exposure to the Candida. If a person knows he or she has
sources, including synovial fluid; body fluids such as pleu- been exposed, or if suspicion of infection remains high,
ral, peritoneal, and pericardial fluids, cerebrospinal fluid then repeat testing at a later date may be required.
(CSF); and both fresh and formalin-fixed paraffin-embed- 3. It is also important for those who are at increased risk of
ded (FFPE) tissues. Candida infection to have screening tests performed regu-
Interpretation larly to check for possible infection.
Appendixes 823
A.8 Chlamydia Trachomatis (CT) Detection Tests completely cleared. Better therapeutic observation indica-
Sample Required Cotton swab for genital and urethral tors were normalization of CSF sugar, chloride and white
secretions blood cell counts and negative culture.
Detection Method Nucleic acid amplification test
Reference Ranges Negative A.10 Cryptococcus Antibody Test
Interpretation Sample Required Serum, CSF (100 μL)
Detection Method EIA, CLIA, ECL, GICA
1. When the nucleic acid test result is positive, there is CT- Reference Ranges Negative
related pathogenic infection, but the following factors Interpretation
need to be excluded:
(a) The dead pathogen could still be detected, because 1. A negative blood test means that it is likely that no infec-
there are still a small number of dead pathogens in the tion is present. However, a negative screening test means
affected area under the effective drug treatment after only that there is no evidence of disease at the time of the
infection. It is suggested that the test should be done test.
2 weeks after drug withdrawal. If the condition is 2. Antibodies may not be detected for several weeks after
monitored during drug use, it should be combined exposure to the Cryptococcal.
with clinical symptoms. If necessary, the diagnosis 3. If a person knows he or she has been exposed, or if suspi-
should be confirmed by culture method. cion of infection remains high, then repeat testing at a
(b) The target of PCR is nucleic acid. Improper operation later date may be required.
can result in contamination among samples, so that 4. It is also important for those who are at increased risk of
false positives may occur. It is necessary to transport Cryptococcal infection to have screening tests performed
and operate the samples strictly in accordance with regularly to check for possible infection.
the principle.
2. When the test result is negative, there is no CT infection, A.11 Clostridioides (Clostridium) Difficile Toxin
but the following factors need to be excluded: Molecular Detection
(a) Eliminating false-negative phenomena caused by Sample Required Feces
PCR inhibitors, attention should be paid to the identi- Detection Method PCR
fication of the results. Reference Ranges Not applicable
(b) Gene mutations caused by drug resistance can also Clinical Information
lead to the failure of amplification and false-negative Clostridioides (Clostridium) difficile is the cause of C
results. When clinical signs and symptoms are obvi- difficile-associated diarrhea (CDAD), an antibiotic-
ous and multiple PCR tests are negative, the occur- associated diarrhea, and pseudomembranous colitis (PMC).
rence of this situation should be considered. In these disorders bacterial overgrowth of C difficile devel-
ops in the colon, typically as a consequence of antibiotic
A.9 Cryptococcus Capsulatum Polysaccharide usage. Clindamycin and broad-spectrum cephalosporins
Antigen Test have been most frequently associated with CDAD and PMC,
Sample Required Serum, plasma, whole blood, CSF, BALF, but almost all antimicrobials may be responsible. Disease is
Urine (40 μL) related to production of toxin A and B. Treatment typically
Detection Method GICA, LA, EIA, LFA involves withdrawal of the associated antimicrobials and, if
Reference Ranges Negative symptoms persist, orally administered and intraluminally
Interpretation active metronidazole, vancomycin, or fidaxomicin.
Intravenous metronidazole may be used if an oral agent can-
1. Capsular polysaccharide antigen is a specific secretion of not be administered. In recent years, a more severe form of
Cryptococcus and can be used as a diagnostic basis for CDAD with increased morbidity and mortality has been rec-
Cryptococcus infection. ognized as being caused by an epidemic toxin-hyperproducing
2. A negative blood test means that it is likely that no infec- strain of C difficile (NAP1 strain). Many toxin-
tion is present. However, a negative result may indicate hyperproducing isolates also contain the binary toxin gene
that the patient’s result is below the detectable level of the and are resistant quinolones. This test does not differentiate
assay. between toxin-hyperproducing and non-toxin-
3. Repeat testing is recommended if the result is negative, hyperproducing strains. Traditionally, diagnosis relied upon
but the disease is suspected. The decrease of antigen titer (1) clinical and epidemiologic features, (2) culture (which is
is an effective index for the treatment of Cryptococcus, labor intensive and time consuming), (3) cytotoxicity assays,
but it keeps a high titer level after Cryptococcus has been which are labor intensive and time consuming, and (4) toxin
824 Appendixes
detection immunoassays (which are insensitive). The infection. Useful For: Aids in diagnosing Coxiella burnetii
described PCR assay detects the regulatory gene (tcdC) infection (e.g., Q fever).
responsible for production of toxins A and B. This test is Interpretation
used for rapid diagnosis of CDAD and PMC enabling prompt A positive test is diagnostic of Coxiella burnetii disease.
treatment that may reduce hospital stays for inpatients with A negative result does not negate the presence of the organ-
CDAD. ism or active disease, as false-negative results may occur due
Interpretation to inhibition of PCR, sequence variability underlying the
A positive PCR result for the presence of the gene regulat- primers and probes, or the presence of C burnetii in quanti-
ing toxin production (tcdC) indicates the presence of ties less than the limit of detection of the assay.
Clostridioides (Clostridium) difficile and toxin A and/or
B. A negative result indicates the absence of detectable C A.14 Coxsackievirus 16 (CoxA16) Detection Tests
difficile tcdC DNA in the specimen, but does not rule-out C Sample Required Throat swab
difficile infection. False-negative results may occur due to Detection Method Real-time fluorescence quantitative
inhibition of PCR, sequence variability underlying the prim- PCR
ers or probes, or the presence of C difficile in quantities less Reference Ranges Negative
than the limit of detection of the assay. Interpretation
A.12 Coxsackievirus (COX) Antibody Tests 1. Coxsackievirus can be divided into two groups, A and
Sample Required Serum (100 μL) B. There is a type 23 virus in group A and a type 6 virus
Detection Method EIA, CLIA, ECL in group B.
Reference Ranges Negative 2. Coxsackievirus is a common type of virus that infects the
Interpretation human body through the respiratory tract and digestive
tract. Infection during pregnancy can cause non-paralytic
1. The COX virus can be divided into Group A and Group B, polio inflammatory lesions and cause intrauterine infec-
which is used for screening COX infections mainly in the tion and teratogenicity.
children and pregnant women. Anti-COX positive can 3. Coxsackievirus group A, type 16 and enterovirus 71 are
also be seen in pediatric pneumonia, muscle weakness, common pathogens of hand, foot, and mouth disease.
mild paralysis.
2. Most children with hand, foot, and mouth disease are A.15 Cytomegalovirus (CMV) Antibody Tests
associated with COX A virus infection. Sample Required Serum (100 μL)
3. COX B viruses cause a wide variety of illnesses, includ- Detection Method EIA, CLIA, ECL
ing pleurodynia, meningitis, rash, pulmonary infection, Reference Ranges Negative
and a generalized systemic infection. Interpretation
A.13 Coxiella Burnetii (Q fever) Molecular Detection 1. Anti-CMV is mainly used to diagnose acute infections of
Sample Required Blood CMV or recent infections. Infected CMV in the popula-
Detection Method PCR tion is especially common, and should pay attention to the
Reference Ranges Not applicable specific analysis of clinical conditions.
Clinical Information 2. Anti-CMV IgM is usually present in the serum at the
Coxiella burnetii, the causative agent of Q fever, is a small early post-infection and can last for half a year.
obligate intracellular bacterium that is distributed ubiqui- 3. Anti-CMV IgG titer in the acute phase is 4 times higher
tously in the environment. The agent is acquired through than that in the recovery phase, suggesting the recent
aerosol exposure and generally causes mild respiratory dis- CMV infection or subsequent CMV infection.
ease. A small number of these acute cases will advance to a
chronic condition, which typically manifests as endocarditis. A.16 Cytomegalovirus (CMV) Nucleic Acid Detection
If left untreated, cases of Q fever endocarditis are fatal. Tests
Current diagnostic methods of Q fever endocarditis include Sample Required Serum
serologic studies and histopathologic examination of excised Detection Method Real-time fluorescence quantitative
cardiac tissue. These current methods are subjective and PCR
non-specific, limiting usefulness in patient diagnostics. Reference Ranges <5.00E+2
Evaluation of infected tissue, blood, or serum using PCR has Interpretation
been shown to be an effective tool for diagnosing C burnetii
Appendixes 825
1. Cytomegalovirus often causes infection in patients with Results > or =0.01 IU/mL suggest a vaccine response. A
pneumonia and organ transplantation, providing a diag- diphtheria toxoid booster should be considered for patients
nostic basis for early diagnosis and differential diagnosis with antidiphtheria toxoid IgG values between 0.01 and less
of HCMV infection. than 0.1 IU/mL.
2. Quantitative determination of CMV-DNA can help detect
the efficacy of antiviral drugs in HCMV-infected patients. A.18 Epstein–Barr Virus (EBV) Antibody Tests
3. It can be used for organ transplantation, immunodefi- Sample Required Serum (100 μL)
ciency patients, monitoring of HCMV infection in anti- Detection Method EIA, CLIA, ECL
tumor therapy, use of immunosuppressants after organ Reference Ranges Negative
transplantation, immune system damage caused by anti- Interpretation
tumor treatment in immunodeficiency and malignant
tumors, and detection of CMV-DNA in these patients. It 1. EBV-related antibody tests include EBV-EA, EBV-VCA,
helps to take appropriate treatment measures in time to and EBNA.
avoid serious consequences. 2. Anti-EBV positive can be used to different acute from
4. Can be used for the etiology of stillbirth, teratogenic, chronic or reactivated infections with Epstein–Barr virus,
abortion, low birth weight, infant hepatitis syndrome. and may be seen commonly in infectious mononucleosis,
5. For the study of the relationship between CMV and
malignant lymphoma in children and some patients with
tumor: CMV is now considered to be associated with the nasopharyngeal carcinoma.
occurrence of tumors such as cervical cancer, testicular 3. Anti-EBV IgM showed a positive reaction at the early
cancer, prostate cancer, Kaposi meat cancer, fibroblast stage, and reached a peak in the blood of the patient.
cancer, Wilms tumor, and colon cancer. 4. Anti-EBV IgG positive was considered as a previous his-
tory of infection, but anti-EBV IgM positive can prove
A.17 Diphtheria Toxoid IgG Antibody recent infection.
Sample Required Serum
Detection Method PCR A.19 Epstein–Barr Virus (EBV) Nucleic Acid
Reference Ranges Vaccinated: Positive (> or = 0.01 IU/ Detection Tests
mL), Unvaccinated: Negative (<0.01 IU/mL), Reference Sample Required Serum
Values apply to all ages. Detection Method Real-time fluorescence quantitative
Clinical Information PCR
Diphtheria is an acute, contagious, febrile illness caused Reference Ranges <5.00E+2
by the bacterium Corynebacterium diphtheriae. The dis- Interpretation
ease is classically characterized by a combination of local-
ized inflammation in the upper respiratory tract with the 1. Epstein–Barr virus is the main cause of infectious mono-
formation of a diphtheric pseudomembrane over the oro- nucleosis, nasopharyngeal carcinoma, Burkitt’s lym-
pharynx, including the tonsils, pharynx, larynx, and poste- phoma, immunodeficiency or defective B lymphocyte
rior nasal passages. Corynebacterium diphtheriae produces malignancy, Hodgkin’s disease, and post-transplant
a potent diphtheria exotoxin that is absorbed systemically malignant lymphoma. Both are positive.
and can lead to cardiac failure and paralysis of the dia- 2. Epstein–Barr virus DNA detection is a secondary diagno-
phragm. The disease is preventable by vaccination with sis of EB virus infection, combined with other tumor
diphtheria toxoid, which stimulates antidiphtheria toxoid markers, can effectively improve the detection rate of
antibodies. In the USA, diphtheria toxoid is administered nasopharyngeal cancer, early detection, early diagnosis,
to children as part of the combined diphtheria, tetanus, and and early treatment of nasopharyngeal carcinoma.
acellular pertussis (TDaP) vaccine. A patient’s immuno-
logical response to diphtheria toxoid vaccination can be A.20 Fungus (1–3)-β-d-Glucan Test (G test)
determined by measuring antidiphtheria toxoid IgG anti- Sample Required Serum, BALF, CSF (200 μL)
body using this enzyme immunoassay technique. An Detection Method Chromogenic Method
absence of antibody formation post-vaccination may relate Reference Ranges <60 pg/mL (except cryptococcus and
to immune deficiency disorders, either congenital or zygomycete)
acquired, or iatrogenic due to immunosuppressive drugs. Interpretation
Useful For: Determining a patient’s immunological
response to diphtheria toxoid vaccination aids in the evalu- 1. No deep fungal infection was found below 60 pg/mL
ation of immunodeficiency. except Cryptococcus and conjugating bacteria. Between
Interpretation 60 and 100 pg/mL, for observation period, continuous
826 Appendixes
detection should be carried out. Above 100 pg/mL, it is 1. Used for rapid detection of group B streptococcal antigen
suspected to be a deep fungal infection, which is recom- in vaginal fornix swab or urethral swab, providing basis
mended to be treated in combination with clinical for clinical diagnosis, and used for screening of group B
symptoms. streptococcal infection before and after delivery
2. G test can only detect (1–3)-beta-d-Glucan content, but 2. Positive test results indicated the presence of group B
cannot distinguish fungal species, and cannot detect streptococcus.
Cryptococcus and Zygomycete.
3. When positive results occur, continuous detection is rec- A.24 Hepatitis A Virus (HAV) Antibody Tests
ommended to increase the accuracy of experimental Sample Required Serum
results. Detection Method EIA, CLIA, ECL
Reference Ranges Negative
A.21 Flu Virus Nucleic Acid Detection Tests Interpretation
Sample Required Throat swab
Detection Method Real-time fluorescence quantitative 1. Anti-HAV positive combined with clinical symptoms can
PCR be used for a diagnostic indicator for hepatitis A.
Reference Ranges Negative 2. Anti-HAV IgM antibodies can be detected on the day of
Interpretation onset, which is an important indicator for early diagnosis
of acute hepatitis A.
1. Human influenza viruses can be classified into three cat- 3. Anti-HAV IgG occurs after IgM shows positivity and per-
egories based on their antigenicity: influenza A virus, sists for a long time to protect the body from HAV re-
influenza B virus, and influenza C virus. infection. Therefore, this diagnostic method is basically
2. Influenza virus variants have variations in antigenic varia- used to detect epidemiological investigations of popula-
tion, temperature-sensitive variation, host range, and sen- tion immunity.
sitivity to non-specific inhibitors, but the most important
are antigenic variations. The antigenic variation is differ- A.25 Hepatitis B Surface Antibody (HBsAb) Tests
ent from other viruses, and the surface antigens HA and Sample Required Serum (100 μL)
NA are susceptible to mutation. There are two forms of Detection Method EIA, CLIA, ECL
variation, namely antigenic transformation and antigenic Reference Ranges Negative
drift. Interpretation
3. Among the three influenza viruses that infect humans,
influenza A virus has a strong variability, followed by 1. HBsAb is a protective antibody against hepatitis B virus
type B, while the antigenicity of influenza C virus is very immunity.
stable. 2. Its positive indicates that it has been previously infected
with hepatitis B virus, but has already ruled out the virus,
A.22 Group A Streptococcus Antigen Tests or has been vaccinated with hepatitis B vaccine to p roduce
Sample Required Pharyngeal swab; Bacteria protective antibodies, or injection of hepatitis B surface
Detection Method DIGFA, ELISA immunoglobulin.
Reference Ranges Negative 3. Weakened titer or no hepatitis B surface antibody is

Interpretation unable to prevent hepatitis B virus infection.
1. The detection was used to detect the etiology of upper A.26 Hepatitis B e-antigen Antibody (HBeAb) Tests
respiratory tract infection caused by group A streptococ- Sample Required Serum (100 μL)
cus. Positive test results indicated the presence of group A Detection Method EIA, CLIA, ECL
streptococcus. Reference Ranges Negative
2. The test does not distinguish between dead or live bacte- Interpretation
ria, and patients who have been infected or are currently
receiving treatment may also be positive. 1. HBeAb is one of the indicators of human hepatitis B virus
serological markers.
A.23 Group B Streptococcus Antigen Tests 2. HBeAb positive is mainly found in the asymptomatic
Sample Required Leucorrhea; Bacteria hepatitis B surface antigen carriers and chronic hepatitis
Detection Method DIGFA, ELISA B patients. This indicates that the virus replication in the
Reference Ranges Negative body is inhibited to varying degrees, indicating that the
Interpretation blood HBV is reduced and the infectivity is reduced.
Appendixes 827
Surface antibodies have the ability to resist hepatitis B 2. Anti-HCV is positive used for the early diagnosis of hepa-
virus in humans, and other antibodies cannot effectively titis C.
kill hepatitis B virus. 3. Anti-HCV negative cannot completely exclude HCV

infection, because antibodies are not a direct indicator of
A.27 Hepatitis B core antigen Antibody (HBcAb) Tests HCV detection.
Sample Required Serum (100 μL)
Detection Method EIA, CLIA, ECL A.30 Hepatitis C Virus Nucleic Acid (HCV-RNA)
Reference Ranges Negative Detection Tests
Interpretation Sample Required Serum
Detection Method Real-time fluorescence quantitative
1. HBcAb is mostly used in the diagnosis of chronic active PCR
HBVinfection and in the determination of the clinical sta- Reference Ranges <1.00E+3
tus of HBV infected individuals in conjunction with other Interpretation
HBV serological markers and in differential diagnosis of
hepatitis, which includes anti-HBc IgM and anti-HBc 1. HCV is the main cause of post-transfusion hepatitis and
IgG. sporadic non-A, non-B hepatitis. HCV infection can lead
2. Anti-HBc IgM is an early antibody to HBV infection, and to various liver diseases such as chronic hepatitis, cirrho-
is also considered an infectivity marker. sis, and hepatocellular carcinoma.
3. High levels of anti-HBc IgG in serum indicate a recent 2. HCV-RNA detection is used for early diagnosis of hepati-
attack of HBV or an acute onset of chronic infection. tis C virus infection, to identify the activity and degree of
replication of hepatitis C virus.
A.28 Hepatitis B Virus Nucleic Acid Quantitative 3. HCV-RNA quantification can timely estimate the

Detection Tests response, timely adjust the course of treatment and guide
Sample Required Serum medication, and provide objective indicators for efficacy
Detection Method Real-time fluorescence quantitative observation and prognosis.
PCR 4. To make up for the high miss detection rate of ELISA
Reference Ranges <5.00E+2 method, HCV-RNA can be used as an indicator for the
Interpretation diagnosis of HCV infection.
5. Anti-HCV-positive and no HCV-RNA in serum suggests
1. HBV-DNA is the direct basis for the existence of hepatitis a previous infection, and detection of HCV-RNA in serum
B virus. HBV-DNA is a marker of hepatitis B virus replica- does not mean that the liver has no virus replication.
tion, and it is an indicator for evaluating hepatitis B virus 6. The immune function is low, and HCV-RNA can still be
replication level, infectious power, and drug efficacy. detected by anti-HCV negative. Such patients are suitable
2. Diagnosis of hepatitis B virus infection, HBV-DNA posi- for detection by HCV core antigen or antigen-antibody
tive is a sign that the patient is infectious, and HBV-DNA combined detection reagent.
supplements the two pairs of hepatitis B.
3. HBV-DNA is a sensitive indicator for judging the efficacy A.31 Hepatitis D Virus (HDV) Antibody Tests
of hepatitis B antiviral drugs, and it is an indicator for Sample Required Serum (100 μL)
evaluating hepatitis B virus replication level and Detection Method EIA, CLIA, ECL
infectiousness. Reference Ranges Negative
4. HBV-DNA can detect occult chronic hepatitis B. Interpretation
A.29 Hepatitis C Virus (HCV) Antibody Tests 1. Determination of anti-HDV can be used as one of the
Sample Required Serum (100 μL) methods for identifying delta infections and conducting
Detection Method EIA, CLIA, ECL epidemiological investigations. Hepatitis D virus antibod-
Reference Ranges Negative ies are classified into anti-HDV IgG and anti-HDV IgM.
Interpretation 2. Anti-HDV IgG positive is only measured in HBsAg posi-
tive serum, and acted as a reliable indicator for the diag-
1. Anti-HCV can be currently the main indicator for the nosis of hepatitis D.
diagnosis of in patients with acute and chronic hepatitis C 3. Anti-HDV IgM appears earlier and can be used for early
infections. Most of the HCV-Ab of the acute patients are diagnosis of hepatitis D.
IgM, and the chronic patients are mostly IgG. The detec- 4. Serum anti-HDV was negative in all HBsAg-negative

tion of anti-HCV IgG is of great significance for the patients.
screening of blood donors.
828 Appendixes
A.32 Hepatitis E Virus (HEV) Antibody Tests A.35 HIV Nucleic Acid Detection Tests
Sample Required Serum (100 μL) Sample Required Serum
Detection Method EIA, CLIA, ECL Detection Method Real-time fluorescence quantitative
Reference Ranges Negative PCR
Interpretation
1. At present, two kinds of HEV antibodies are Anti-HEV
IgG and Anti-HEV IgM. 1. Auxiliary diagnosis: When the results of HIV antibody
2. Anti-HEV IgM appears earlier and disappears quickly test cannot be clearly diagnosed, the results of RNA
which lasts for a shorter period of time, so it is easy to determination can help provide evidence of early or end
miss the diagnosis in the window period. stage of HIV infection. For infants born to HIV-infected
3. Anti-HEV IgG titer in the acute phase is 4 times higher mothers younger than 18 months of age, two HIV nucleic
than that in the recovery phase, suggesting that HEV is acid tests at different times are positive to make a
newly infected and has diagnostic significance. diagnosis.
4. Anti-HEV IgG positive and Anti-HEV IgM negative indi- 2. Guide antiviral therapy and efficacy judgment: If condi-
cate that the hepatitis E virus has been infected in the past. tions are available, it is necessary to make a baseline viral
load test before treatment, which is convenient for observ-
A.33 Hemorrhagic Fever Antibody Tests ing the virus suppression effect after antiviral therapy. At
Sample Required Serum (100 μL) the beginning of treatment for 16 weeks, the viral load of
Detection Method EIA, CLIA, ECL all patients with primary antiviral therapy should be lower
Reference Ranges Negative than the lower limit of detection. If the viral load is still
Interpretation below the lower limit of detection after 6 months of treat-
ment, the cause should be combined with the clinical need
1. Epidemic hemorrhagic fever is a group of natural epi- (dependency, compliance, drug interactions, etc.) to con-
demic diseases caused by arboviruses. Immunological sider whether treatment failures and adjustments to treat-
detection methods mainly detect two related antibodies: ment options. After the start of treatment, the viral load
IgM and IgG. should be tested every 6 months to evaluate the treatment
2. IgM antibodies can be used as an indicator for early effect. If the load can change, the treatment plan should be
diagnosis. adjusted in time according to the clinical search reasons.
3. IgG antibodies can be detected after 2 days from the
course of the disease. The positive duration is long A.36 Haemophilus Influenzae Type B Antibody
although the IgG antibody appears slightly later. So when Sample Required Serum
the test results are positive, clinical symptoms and past Detection Method PCR
history should not be ignored, and comprehensive analy- Reference Ranges > or = 0.15 mg/L, Reference values
sis should be carried out. apply to all ages.
Clinical Information
A.34 Human Immunodeficiency Virus (HIV) Antibody Haemophilus influenzae type B (HIB) is an encapsulated
Tests gram-negative cocco-bacillary bacterium that can cause dev-
Sample Required Serum (100 μL) astating disease in young children including meningitis, bac-
Detection Method EIA, CLIA, ECL teremia, cellulitis, epiglottitis, pneumonia, and septic
Reference Ranges Negative arthritis. One of the great advances in modern medicine has
Interpretation been the development of an effective vaccine against HIB. A
patient’s immunological response to HIB vaccine can be
1. Detection of HIV antibodies in the blood is currently the determined by measuring anti-HIB IgG antibody using this
most commonly used laboratory method and indirect EIA technique. Useful For: Assessing a patient’s immuno-
indicator for detecting HIV infection. logical (IgG) response to Haemophilus influenzae type B
2. If the primary screening test is positive, confirm that the (HIB) vaccine assessing immunity against HIB aiding in the
test is positive before it can be diagnosed as HIV evaluation of immunodeficiency.
infection. Interpretation
3. HIV antibody negative does not mean no infection, HIV An anti-Haemophilus influenzae type B (HIB) IgG anti-
antibodies persist for a long time and can be detected body concentration of 0.15 mg/L is generally accepted as the
throughout the life of the virus except for the early tran- minimum level for protection at a given time; however, it
sient “window period” after viral infection. does not confer long-term protection. A study from Finland
Appendixes 829
suggested that the optimum protective level is 1.0 mg/L post- A.39 Helicobacter Pylori Antigen
immunization. Furthermore, studies have shown that the Sample Required Feces
response to HIB vaccine is age-related. By testing pre- and Detection Method PCR
postvaccination patient serum specimens, this test may be Reference Ranges Negative
used to aid diagnosis of immunodeficiency. Clinical Information
Helicobacter pylori is well recognized as the cause of
A.37 Human Papillomavirus (HPV) Antibody Tests chronic active gastritis, duodenal ulcer, and non-ulcer dys-
Sample Required Serum (100 μL) pepsia. Currently accepted methods for the diagnosis of
Detection Method EIA, CLIA, ECL Helicobacter pylori infection include the urea breath test
Reference Ranges Negative (UBT/Helicobacter pylori Breath Test) and culture or histo-
Interpretation logic examination or direct urease testing of biopsy speci-
mens obtained at the time of gastroduodenoscopy. Each of
1. HPV is a general term for many types of viruses, which these tests has its drawbacks, including lack of specificity
can be sexually transmitted. The level of HPV antibodies (serology) or high cost, complexity, and inconvenience for
can be paralleled with the clinical development of the the patient. The utility of this test in asymptomatic individu-
disease. als is not known, but testing for Helicobacter pylori in such
2. In the early stage of HPV infection, anti-HPV may not be individuals is not generally recommended. See Helicobacter
detected in serum due to the short infection time and HPV pylori Diagnostic Algorithm in Special Instructions. Useful
has not induced the production of anti-HPV. For: Aiding in the diagnosis of Helicobacter pylori infection
3. Anti-HPV occurs later in the condyloma acuminata, and Monitoring the eradication of Helicobacter pylori after ther-
antibody positive may only reflect infected with this virus. apy (in most situations, confirmation of eradication is not
mandatory).
A.38 Human Papillomavirus Nucleic Acid Detection Interpretation
Tests Positive results indicate the presence of Helicobacter
Sample Required Cervical exfoliated cells pylori antigen in the stool. Negative results indicate the
Detection Method Real-time fluorescence quantitative absence of detectable antigen, but do not eliminate the pos-
PCR sibility of infection due to Helicobacter pylori.
Interpretation A.40 Helicobacter Pylori Antibody Tests
Sample Required Serum (20 μL)
1. Human Papillomavirus is a genus of papillomavirus A Detection Method DIGFA, ELISA
belonging to the genus Papillomavirus, which is a spheri- Reference Ranges Negative
cal DNA virus that can cause squamous epithelial prolif- Interpretation
eration of human skin mucosa.
2. At present, more than 130 kinds have been isolated, and 1. Positive serological detection of Hp antibody does not
different types have caused different clinical manifesta- represent present infection, nor can it be used for re-
tions. According to the different parts of the invading tis- examination after eradication treatment.
sues, they can be divided into: 2. Serological detection of Hp antibody is used for screening
(a) Low-risk skin type: including HPV1, 2, 3, 4, 7, 10, specific population and evaluation of eradication indica-
12, 15, etc. related to common warts, flat warts, warts, tions, early gastric cancer screening, and gastric extinc-
etc.; tion. Hp infection has unique application value in the
(b) High-risk skin types: HPV5, 8, 14, 17, 20, 36, 38 are diagnosis and epidemiological investigation of diseases.
associated with sickle epidermal dysplasia, and other 3. Serological detection of Hp antibody can be used as a
malignant tumors associated with possible HPV supplement to other detection methods, which is of great
infection include: vulvar cancer, penile cancer, anal significance for rapid and accurate detection of Hp
cancer, prostate cancer, bladder cancer; infection.
(c) Low-risk mucosa such as HPV-6, 11, 13, 32, 34, 40,
42, 43, 44, 53, 54, and so on with genital, anal, oro- A.41 Helicobacter Pylori Detection Tests
pharynx, esophageal mucosa; Sample Required Biopsy specimens of gastric mucosa
(d) High-risk type HPV-16, 18, 30, 31, 33, 35, 39 and Detection Method Nucleic acid amplification test
cervical cancer, rectal cancer, oral cancer, tonsillar Reference Ranges Negative
cancer. Interpretation
830 Appendixes
1. The detection was used to detect Helicobacter pylori

myelitis and septic arthritis in children aged 6–36 months. K
nucleic acid in gastric mucosa biopsy specimens qualita- kingae may also cause endocarditis, involving both native
tively. Helicobacter pylori (Hp) is a worldwide pathogen and prosthetic valves, in patients of any age and is consid-
of human infection, which has a high infection rate in the ered part of the HACEK (Haemophilus species,
population. Aggregatibacter species, Cardiobacterium hominis,
2. Some patients with Hp infection develop chronic gastritis, Eikenella corrodens, and Kingella species) group of organ-
duodenitis, peptic ulcer, gastric cancer, mucosa-associated isms, known for causing culture-negative endocarditis. K
lymphoma, and other diseases. kingae produces a repeat-in-toxin (RTX) toxin. Diagnosis of
K kingae infection may be challenging due to the fastidious
A.42 Herpes Simplex Virus (HSV) Antibody Tests nature of the organism in culture. Evaluation of blood by
Sample Required Serum (100 μL) PCR is a useful tool for the diagnosis of some cases of K
Detection Method EIA, CLIA, ECL kingae infection. Useful For: Aiding in the diagnosis of
Reference Ranges Negative Kingella kingae infection.
Interpretation Interpretation
A positive test is strongly suggestive of Kingella kingae
1. HSV is divided into HSV-I and HSV-II, mainly causing disease. A negative result does not negate the presence of the
herpetic meningitis, neonatal herpes, and so on. Infections organism or active disease, as false-negative results may
outside the reproductive organs were mostly caused by occur due to inhibition of PCR, sequence variability underly-
HSV-I, while HSV infection in the reproductive organs ing the primers and probes, or the presence of K kingae in
was mainly caused by HSV-II. quantities less than the limit of detection of the assay.
2. Anti-HSV IgM positive or Anti-HSV IgG antibody titers
increased fourfold suggests a recent HSV infection. In A.45 KPC (blaKPC) and NDM (blaNDM) Surveillance
patients without a history of disease, a positive result of Sample Required Blood
this test may indicate that the primary infection is Detection Method PCR
asymptomatic. Reference Ranges Not applicable
A.43 Influenza Virus Antibody Tests The Centers for Disease Control and Prevention recom-
Sample Required Serum (100 μL) mends active surveillance to detect unrecognized colonized
Detection Method EIA, CLIA, ECL patients who may be a potential source for carbapenem-
Reference Ranges Negative resistant (drug-resistant) Enterobacteriaceae (CRE) trans-
Interpretation mission. Such surveillance testing may be focused in certain
high-risk settings or patient groups (e.g., ICUs, long-term
1. The population is generally susceptible to influenza virus, acute care, patients transferred from areas or facilities with
and there are the extremely frequent antigenic variations high CRE prevalence) or by infection control to investigate
in the influenza viruses. Influenza virus antibody could an outbreak. Nonsusceptibility to carbapenems in gram-
only be used as the evidence for a screening indicator, not negative bacilli by means of the enzyme KPC (Klebsiella
a definitive diagnosis. pneumoniae carbapenemase) or NDM (New Delhi
2. If the antibody titer in the recovery period is more than 4 metallo-beta-lactamase) is becoming more common. The
times higher than the acute phase, flu can be diagnosed. genes blaKPC and blaNDM encode KPC and NDM enzyme
During the outbreak of influenza, those who are positive production, respectively. PCR is a sensitive, specific, and
for antibody screening should pay special attention. rapid means identifying patients colonized by CRE harbor-
ing blaKPC or blaNDM. Useful For: Identifying carriers of
A.44 Kingella Kingae Molecular Detection carbapenem-resistant Enterobactericeae harboring KPC
Sample Required Blood (Klebsiella pneumoniae carbapenemase) or NDM (New
Detection Method PCR Delhi metallo-beta-lactamase) genes.
Reference Ranges Not applicable Interpretation
Clinical Information This PCR detects and differentiates blaKPC and blaNDM
Kingella kingae is a fastidious short Gram-negative bacil- in surveillance specimens (perirectal/rectal swabs or stool).
lus that may colonize the oropharynx of young children. A positive KPC (Klebsiella pneumoniae carbapenemase)
Colonization may occasionally lead to invasive disease via and/or NDM (New Delhi metallo-beta-lactamase) PCR indi-
hematogenous dissemination, primarily in children younger cates that the patient is colonized by a Gram-negative bacil-
than 4 years of age. This most commonly results in bone and lus (or Gram-negative bacilli) harboring a carbapenemase
joint infection; K kingae is the most frequent cause of osteo- gene, blaKPC and/or blaNDM, respectively. A negative
Appendixes 831
result indicates the absence of detectable DNA; however, A.49 Neisseria Gonorrhoeae (NG) Detection Tests
false-negative results may occur due to inhibition of PCR, Sample Required Cotton swab for genital and urethral
sequence variability underlying primers and probes, or the secretions
presence of the blaKPC or blaNDM genes in quantities less Detection Method Nucleic acid amplification test
than the limit of detection of the assay. Reference Ranges Negative
Interpretation
A.46 Legionella Antibody Tests
Sample Required Serum (100 μL) 1. Gonorrhea is a common bacterial sexually transmitted
Detection Method IFA, MAT, ELISA disease (STD) that can cause serious complications if not
Reference Ranges Negative detected and treated. Gonorrhea testing identifies if the
Interpretation bacteria Neisseria gonorrhoeae is the cause of a person’s
infection.
1. After Legionella spp. infection, Legionella spp. specific 2. NG DNA detection has important value in early diagno-
IgM antibody in serum can be detected in about 1 week sis, treatment, and prevention of chronic infection of gon-
while specific IgG antibody can be detected in about 2 orrhea. Although bacterial culture is the golden standard,
weeks. it is tedious and time consuming. Real-time fluorescent
2. In some patients, antibody titers did not increase after 2 PCR can solve the problem of rapid diagnosis of Neisseria
months of infection. Detection of antibody titers in a sin- gonorrhoeae infection, especially for early diagnosis of
gle serum is often difficult to determine whether the dis- genitourinary tract infection and detection of asymptom-
ease is present or a previous infection. atic carriers.
A.47 Mycoplasma Pneumoniae (MP) Detection Tests A.50 Neisseria Gonorrhoeae (NG) by Nucleic Acid
Sample Required Sputum, Pharyngeal swab Amplification
Detection Method Nucleic acid amplification test Sample Required Blood
Reference Ranges Negative Detection Method PCR
1. Mycoplasma pneumoniae (MP) infection tends to be
Gonorrhea is caused by the bacterium Neisseria gonor-
younger in recent years. Infants aged 0–4 account for rhoeae, which is a common sexually transmitted infection
50–65%. (STI). Many infections in women are asymptomatic and the
2. This detection is suitable for quantitative detection of true prevalence of gonorrhea is likely much higher than
Mycoplasma pneumoniae (MP) DNA in sputum, swab reported. The organism causes genitourinary infections in
and other specimens. It can be used for assistant diag- women and men and may be associated with dysuria and
nosis of Mycoplasma pneumoniae (MP) infection and vaginal, urethral, and/or rectal discharge. Potential compli-
monitoring the efficacy of drug treatment for infected cations include pelvic inflammatory disease in women and
patients. gonococcal epididymitis and prostatitis in men. Gonococcal
bacteremia, pharyngitis, and arthritis may also occur.
A.48 Mycobacterium Tuberculosis Antibody Tests Infection in men is typically associated with symptoms that
Sample Required Serum (40 μL) would prompt clinical evaluation. Given the risk for asymp-
Detection Method DIGFA, ELISA tomatic infection in women, screening is recommended for
Reference Ranges Negative women at increased risk of infection (e.g., women with pre-
Interpretation vious gonorrhea or other STI, inconsistent condom use, new
or multiple sex partners, and women in certain demographic
1. This method is mainly used for effective detection of spe- groups, such as those in communities with high STI preva-
cific antigen proteins, carbohydrates, and lipids on the lence). Routine bacterial culture was previously considered
surface of Mycobacterium tuberculosis. the gold standard test for diagnosis of Neisseria gonorrhoeae
2. A large number of clinical practices have shown that infection. However, organisms are labile in vitro, therefore,
the detection of tuberculosis antibody has the charac- precise specimen collection, transportation, and processing
teristics of increasing active tuberculosis and decreas- conditions are required to maintain organism viability, which
ing during recovery period, and is not affected by BCG is necessary for successful culturing. In comparison, nucleic
vaccination. acid amplification testing (NAAT) provides superior sensi-
tivity and specificity and is now the recommended method
for diagnosis in most cases. Immunoassays and nonamplifi-
832 Appendixes
cation DNA tests are also available for Neisseria gonor- metallo-beta-lactamase) PCR indicates the isolate carries
rhoeae detection, but these methods are significantly less blaVIM. A negative result indicates the absence of detect-
sensitive and specific than NAAT. Improved screening and able DNA.
performance of NAAT testing have resulted in an increased
number of accurately diagnosed cases. Improved detection A.52 Respiratory Pathogen Panel PCR
rates result from both the increased performance of the assay Sample Required Blood
and the patients’ easy acceptance of urine testing. Early Detection Method PCR
identification of infection enables sexual partners to seek Reference Ranges Negative (for all targets)
testing and treatment as soon as possible and reduces the risk Clinical Information
of disease spread. Prompt treatment reduces the risk of com- Respiratory infections are common and generally cause
plications in women. Useful For: Detection of Neisseria self-limited illnesses in healthy, immunocompetent hosts.
gonorrhoeae. Viruses account for a significant percentage of respiratory
Interpretation diseases, but bacteria may be associated with respiratory
A positive result indicates the presence of rRNA of infections. Although respiratory illnesses are frequently
Neisseria gonorrhoeae. A negative result indicates that mild, viruses may cause significant morbidity and mortality
rRNA for Neisseria gonorrhoeae was not detected in the in immunocompromised hosts (e.g., transplant recipients,
specimen. The predictive value of an assay depends on the patients with underlying malignancies). Influenza viruses
prevalence of the disease in any particular population. In (type A and type B) and respiratory syncytial virus (RSV)
settings with a high prevalence of sexually transmitted dis- are 2 common causes of viral respiratory illness, with peak
ease, positive assay results have a high likelihood of being incidence in the winter and spring months in the Northern
true-positives. In settings with a low prevalence of sexually hemisphere. Both viruses can cause a clinically indistin-
transmitted disease, or in any settings in which a patient’s guishable syndrome, characterized by fever, cough, head-
clinical signs and symptoms or risk factors are inconsistent ache, and general malaise. RSV is a leading cause of
with gonococcal or chlamydial urogenital infection, posi- respiratory illness in young children. Early diagnosis of
tive results should be carefully assessed and the patient influenza and RSV is important so that (1) necessary infec-
retested by other methods (e.g., culture for Neisseria gon- tion control precautions can be taken if the patient is hospi-
orrhoeae), if appropriate. talized, and (2) antiviral therapy can be considered if the
patient is hospitalized or considered at high-risk for severe
A.51 OXA-48-like (blaOXA-48-like) and VIM disease. Human metapneumovirus is also a cause of respira-
(blaVIM) in Gram-Negative Bacilli Molecular Detection tory illness in both children and adults. Human rhinovirus
Sample Required Blood and coronavirus (serotypes HKU1, NL63, 229E, OC43) are
Detection Method PCR the causative agents of the common cold, with symptoms
Reference Ranges Not applicable including runny nose, sore throat, and malaise. Infections
Clinical Information with rhinovirus and coronaviruses are extremely common,
In the USA, Klebsiella pneumoniae carbapenemase due to the large number of serotypes of these viruses. Most
(KPC) is the most common carbapenemase, followed by infections are mild and self-limiting; however, immunocom-
New Delhi metallo-beta-lactamase (NDM). OXA-48-like promised hosts may suffer more severe illnesses, including
and VIM carbapenemases predominate in other parts of lower respiratory tract disease. Parainfluenza viruses and
the globe, but do occur in the USA. The genes blaOXA- adenovirus are also common causes of viral infection, espe-
48-like and blaVIM encode OXA-48-like and VIM cially in young children. Parainfluenza viruses are most
enzyme production, respectively. PCR is a sensitive, spe- common during the spring, summer, and fall months, with
cific, and rapid means of identifying these genes. This test symptoms including fever, runny nose, and cough. However,
detects the genes encoding OXA-48-like (oxacillin- parainfluenza viruses may also cause more severe lower
hydrolyzing beta-lactamase) and VIM (Verona integron- respiratory disease, such as croup or pneumonia.
encoded metallo-beta-lactamase) types of beta-lactamases Adenoviruses may infect a range of organ systems, with
in bacterial isolates. Useful For: Assessing pure isolates sequelae ranging from cold-like symptoms (sore throat), to
of Gram-negative bacilli for mechanism of carbapenem pneumonia, conjunctivitis (pink eye), or diarrhea. Similarly
resistance. to the viruses described above, parainfluenza viruses and
Interpretation adenoviruses generally cause mild, self-limited infections
This PCR detects and differentiates blaOXA-48-like and but may cause severe disease in immunosuppressed patients.
blaVIM. A positive blaOXA-48-like (oxacillin-hydrolyzing Respiratory infections may also be caused by bacterial
beta-lactamase) PCR indicates that the isolate carries pathogens, including Bordetella pertussis, Chlamydophila
blaOXA-48-like. A positive VIM (Verona integron-encoded pneumoniae, and Mycoplasma pneumoniae. Bordetella per-
Appendixes 833
tussis is the causative agent of pertussis, or whooping cough, is an extremely common cause of pediatric gastroenteritis
a disease characterized by prolonged cough that may be in winter. The clinical manifestation is acute gastroenteri-
associated with an inspiratory whoop and post-tussive vom- tis, which is osmotic diarrhea. The course of the disease is
iting. Mycoplasma pneumoniae is a cause of upper respira- usually about 1 week.
tory infection, pharyngitis, tracheobronchitis, and 2. The test result is often positive for IgM antibody, suggest-
pneumonia. Chlamydophila pneumoniae is a rare cause of ing the current infection. More than 90% of adult anti-
pneumonia. Useful For: Rapid detection of respiratory infec- rotavirus lgG antibodies are positive, suggesting previous
tions caused by the following: Adenovirus—Coronavirus infections.
(serotypes HKU1, NL63, 229E, OC43)—Human
metapneumovirus-Human rhinovirus/enterovirus-Influenza A.54 Rubella Virus (RV) Antibody Tests
A (H1, H1-2009, H3)—Influenza B—Parainfluenza virus Sample Required Serum (100 μL)
(serotypes 1–4)—Respiratory syncytial virus (RSV)— Detection Method EIA, CLIA, ECL
Bordetella pertussis—Chlamydophila pneumoniae— Reference Ranges Negative
Mycoplasma pneumoniae. Interpretation
Interpretation
Results are intended to aid in the diagnosis of illness and 1. The peak incidence of RV infection is from spring to
are meant to be used in conjunction with other clinical and early summer, used as one of the common indicators for
epidemiological findings. A negative result should not rule- prenatal screening widely. Antibodies tested by RV
out infection in patients with a high pretest probability for a include two types: IgG and IgM.
respiratory infection. The assay does not test for all potential 2. Anti-RV lgM is produced about 4 days after the rash, and
infectious agents of respiratory disease. Specimens collected Anti-RV IgG appears later than the lgM.
too early or too late in the clinical course may not yield the 3. When both antibodies are positive, it shows a recent acute
organism causing disease. Negative results should be consid- infection with the RV. When both antibodies are negative,
ered in the context of a patient’s clinical course and treatment it is necessary to consider the window period and review
history, if applicable. For immunocompromised patients them regularly.
who have a negative FilmArray respiratory panel test from a
nasopharyngeal sample, but a high suspicion for infection, A.55 Shiga Toxin Molecular Detection
there may be additional value in testing a bronchoalveolar Sample Required Feces
lavage specimen (RESLR/Respiratory Pathogen Panel, PCR, Detection Method PCR
Miscellaneous Sources). Positive results do not distinguish Reference Ranges Not applicable
between a viable or replicating organism and the presence of Clinical Information
a nonviable organism or nucleic acid, nor do they exclude the Shiga toxins (also known as Shiga-like toxins, Vero tox-
potential for coinfection by organisms not included in the ins, or Vero-like toxins) are encoded by some strains of
panel. Nucleic acid may persist in some patients for days to Escherichia coli, most notably O157:H7. Shiga toxin can
weeks, even following appropriate therapy. Detection of 1 or also be produced by other serogroups of enterohemorrhagic
more organisms included in this test suggests that the virus E coli (EHEC), as well as Shigella dysenteriae type 1.
or bacteria is present in the clinical sample; however, the test Generally, Shiga toxin-producing organisms cause bloody
does not distinguish between organisms that are causing dis- diarrhea although this is not universal. Unlike some bacterial
ease and those that are present but not associated with a clini- gastrointestinal infections, antimicrobial therapy is contrain-
cal illness. Coinfections (e.g., detection of multiple viruses dicated, as antimicrobials may exacerbate disease. Treatment
or bacteria or viruses and bacteria) may be observed with this is primarily supportive (e.g., hydration). A complication of
test. In these situations, the clinical history and presentation infection by an organism producing Shiga toxin is hemolytic
should be reviewed thoroughly to determine the clinical sig- uremic syndrome (HUS). The percentage of people that
nificance of multiple pathogens in the same specimen. develop HUS varies among outbreaks of E coli O157:H7, but
generally ranges from 3 to 20%. HUS is characterized by a
A.53 Rotavirus (RV) Antibody Tests triad of findings: hemolytic anemia, thrombocytopenia, and
Sample Required Stool kidney failure. Most people recover completely, however,
Detection Method EIA, CLIA, ECL some require permanent dialysis, and some die as a result of
Reference Ranges Negative complications. Several diagnostic methods available for the
Interpretation detection of EHEC lack sensitivity, are labor intensive, or
have a long turnaround time. There are more than 160 sero-
1. Rapid detection of rotavirus from stool specimens of
groups of EHEC; the first serogroup to be associated with
patient suspected of having viral gastroenteritis. Rotavirus HUS was O157:H7. This is also the serogroup that is most
834 Appendixes
commonly implicated in outbreaks. EHEC O157:H7 is ated with infection by certain rheumatogenic serotypes (M1,
detectable as nonfermenting colonies when cultured on sor- M3, M5, M6, M18, and M19), while glomerulonephritis fol-
bitol MacConkey (SMAC) agar, but the majority of non- lows infection by nephritogenic serotypes (M2, M12, M49,
O157:H7 Shiga toxin-producing E coli strains ferment M57, M59, and M60). Glomerulonephritis and rheumatic
sorbitol and, therefore, are undetectable by this method. The fever occur following the infection, after a period of latency
Vero cell line is susceptible to the Shiga toxin, but the assay following the infection, during which the patient is asymp-
can take up to 48 h and is non-specific. Commercial enzyme- tomatic. The latency period for glomerulonephritis is
linked immunosorbent assay (ELISA) antigen detection kits approximately 10 days, and for rheumatic fever the latency
have a sensitivity of 90% when compared to culture, but an period is 20 days. Useful For: Demonstration of acute or
overnight enrichment step is necessary for adequate sensitiv- recent streptococcal infection using both antistreptolysin O
ity. PCR detection of stx, the gene encoding Shiga toxin, and anti-DNase B titers.
directly from stool specimens is a sensitive and specific tech- Interpretation
nique, providing same-day results. PCR assay identifies non- Elevated values are consistent with an antecedent infec-
O157:H7 Shiga toxin-producing bacteria, extending the tion by group A streptococci.
utility beyond strains identifiable on SMAC agar. Useful For:
Sensitive, specific, and rapid detection of the presence of A.57 Streptococcus Pneumoniae Antigen
Shiga toxin-producing organisms such as Escherichia coli Sample Required Spinal Fluid
O157:H7 and Shigella dysenteriae type 1 in stool. Detection Method PCR
Interpretation Reference Ranges Negative, Reference values apply to
A positive PCR result indicates the likely presence of all ages
Shiga toxin-producing Escherichia coli in the specimen. Clinical Information
Although Shigella dysenteriae serotype 1 may produce a Streptococcus pneumoniae is the most frequently encoun-
positive result, it is extremely rare in the USA. A negative tered bacterial agent of community-acquired pneumonia,
result indicates the absence of detectable Shiga toxin DNA in and can also be an agent of bacterial meningitis. Because of
the specimen, but does not rule out the presence of Shiga the significant morbidity and mortality associated with pneu-
toxin-producing E coli, as false-negative results may occur mococcal pneumonia, septicemia, and meningitis, it is
due to inhibition of PCR, sequence variability underlying the important to have diagnostic test methods available that can
primers and probes, or the presence of the Shiga toxin gene provide a rapid diagnosis. In instances where empirical anti-
in quantities less than the limit of detection of the assay. biotics are being considered prior to culture confirmation,
Shiga toxins are encoded on mobile genetic elements and antigen testing may be useful. Useful For: Rapid diagnosis of
can theoretically be lost by their bacterial host. pneumococcal meningitis Note: According to the College of
American Pathologists (CAP, IMM.41830), cerebrospinal
A.56 Streptococcal Antibodies Profile fluid (CSF) samples collected to make an initial diagnosis
Sample Required Feces and submitted for detection of Streptococcus pneumoniae
Detection Method PCR antigen testing should also be submitted for routine bacterial
Reference Ranges ANTISTREP-O TITER culture. Mayo Clinic Laboratories recommend that CSF bac-
terial cultures can be performed at the originating site.
• <5 years: < or = 70 IU/mL Interpretation
• 5–17 years: < or = 640 IU/mL A positive result supports a diagnosis of pneumococcal
or = 18 years: < or = 530 IU/mL ANTI-DNase B TITER meningitis. A negative result suggests that pneumococcal
• <5 years: < or = 250 U/mL antigen is absent in the cerebrospinal fluid (CSF). However,
• 5–17 years: < or = 375 U/mL infection due to Streptococcus pneumoniae cannot be ruled
or = 18 years: < or = 300 U/mL out since the antigen present in the specimen may be below
the lower limit of detection of the test. If pneumococcal men-
Clinical Information ingitis is suspected, bacterial culture and Gram-stain analysis
A number of bacterial antigens have been identified in on CSF should be performed.
cultures of group A streptococci. These extracellular prod-
ucts are primarily enzymatic proteins and include streptoly- A.58 Streptococcus Pneumoniae Antigen
sin O, streptokinase, hyaluronidase, deoxyribonucleases Sample Required Spinal Fluid
(DNases A, B, C, and D), and nicotinamide adenine nucleo- Detection Method PCR
tidase. Infections by the group A streptococci are unique Reference Ranges Negative, Reference values apply to
because they can be followed by the serious non-purulent all ages
complications of rheumatic fever and glomerulonephritis. Clinical Information
Recent information suggests that rheumatic fever is associ-
Appendixes 835
Streptococcus pneumoniae is the most frequently encoun- A.61 Toxoplasma (TOX) Antibody Tests
tered bacterial agent of community-acquired pneumonia, Sample Required Serum (100 μL)
and can also be an agent of bacterial meningitis. Because of Detection Method EIA, CLIA, ECL
the significant morbidity and mortality associated with pneu- Reference Ranges Negative
mococcal pneumonia, septicemia, and meningitis, it is Interpretation
important to have diagnostic test methods available that can
provide a rapid diagnosis. In instances where empirical anti- 1. TOX is transmitted to humans through the feces of cats,
biotics are being considered prior to culture confirmation, and most people develop antibodies without any clinical
antigen testing may be useful. Useful For: Rapid diagnosis of disease. Self-limiting lymphadenitis is the most common
pneumococcal meningitis. Note: According to the College of clinical manifestation of symptomatic infections, and
American Pathologists, cerebrospinal fluid (CSF) samples infections in AIDS patients are more pronounced.
collected to make an initial diagnosis and submitted for 2. Anti-TOX IgG and lgM were positive, or the Anti-TOX
detection of Streptococcus pneumoniae antigen testing IgG titer was more than 1:512, indicating a recent infec-
should also be submitted for routine bacterial culture. tion of the TOX.
Interpretation 3. Both Anti-TOX IgG and IgM were negative, indicating
A positive result supports a diagnosis of pneumococcal perhaps no TOX infection.
meningitis. A negative result suggests that pneumococcal
antigen is absent in the cerebrospinal fluid (CSF). However, A.62 Tuberculosis Detection Tests
infection due to Streptococcus pneumoniae cannot be ruled Sample Required Sputum, cerebrospinal fluid, pleural effu-
out since the antigen present in the specimen may be below sion, ascites, Bronchoalveolar lavage fluid
the lower limit of detection of the test. If pneumococcal men- Detection Method Nucleic acid amplification test
ingitis is suspected, bacterial culture and Gram-stain analysis Reference Ranges Negative
on CSF should be performed. Interpretation2
A.59 Streptococcus Pneumonia Antigen Tests 1. Rapid diagnosis of Mycobacterium tuberculosis infec-
Sample Required Urine; Cerebrospinal fluid tion. Because of long incubation period, it is difficult to
Detection Method DIGFA, ELISA diagnose Mycobacterium tuberculosis infection by cul-
Reference Ranges Negative ture method in clinic. However, it can be done by PCR
Interpretation method. Hemorrhage, lymphadenopathy, tuberculous
meningitis, tuberculous pleuroperitonitis, etc.
1. It is used to detect streptococcus pneumoniae antigen in 2. The monitoring of anti-tuberculosis treatment. In anti-
the urine of pneumonia patients and cerebrospinal fluid tuberculosis treatment, regular detection by PCR can
(CSF) of meningitis patients. evaluate the efficacy of anti-tuberculosis drugs.
2. Positive test results indicated the presence of Streptococcus 3. Attention should be paid to the clinical “false positive”
pneumoniae. problem. The pathogen nucleic acid is detected by
PCR. No matter whether or not Mycobacterium tubercu-
A.60 Syphilis Specific Antibody Tests losis is a living bacterium, it can be detected by PCR. After
Sample Required Serum (100 μL) one course of antibiotic treatment, it is suggested to do the
Detection Method EIA, CLIA, ECL PCR test again after 2 weeks in order to avoid clinical
Reference Ranges Negative false positive.
Interpretation
A.63 Ureaplasma Urealyticum (UU) Detection Tests
1. It is important for those who are at increased risk of syph- Sample Required Cotton swab for genital and urethral
ilis infection to have screening tests performed regularly secretions
to check for possible infection. Detection Method Nucleic acid amplification test
2. Negative test results represent that it is possible that infec- Reference Ranges Negative
tion does not exist at this time of the test. Antibodies may Interpretation
not be detected for several weeks after exposure to the
bacteria. 1.
Ureaplasma urealyticum belongs to the order
3. Positive test can be diagnosed as syphilis, which can also Mycoplasma, Mycoplasma family and one species of
be used to follow treatment response in patients being Ureaplasma genus in Membranaceae. It is divided into
treated for syphilis infection, and not be used to screen for two biota (Biovar) with 14 standard serotypes.
syphilis infection. 2. It is one of the common pathogens of genitourinary tract
infection. It not only causes non-gonococcal urethritis,
836 Appendixes
but also is considered to cause a variety of urogenital dis- several years, which means that the IgG serves as an indi-
eases, such as prostatitis, epididymitis, infertility, sponta- cator of previous infection.
neous abortion, and birth tract infection.
3. It can be used for assistant diagnosis of Ureaplasma urea- A.67 Nucleic Acid Detection Tests of 2019-nCoV
lyticum (UU) infection and monitoring the curative effect Sample Required Nasal and pharyngeal swabs, bronchoal-
of drug treatment for infected patients. The test results are veolar lavage fluid (BAL), sputum, bronchial aspirates.
only for clinical reference, and cannot be used as a basis Detection Method Real-time fluorescence quantitative
for the diagnosis or exclusion of cases alone. PCR
A.64 Vibrio CholeraeO1/O139 Antigen Tests Interpretation
Sample Required Stool; Bacterial suspension
Detection Method DIGFA, ELISA 1. Two target genes, including open reading frame 1ab

Reference Ranges Negative (ORF1ab) and nucleocapsid protein(N), were simultane-
Interpretation ously amplified and tested during the real-time RT-PCR
assay.
1. Vibrio cholerae causes virulent infectious cholera, so it is 2. Quality control is very important. It is recommended to
very important to identify it quickly and accurately. The set up one weak positive quality control and three nega-
test is used to detect the presence of vibrio cholerae in the tive quality controls for each batch of testing. The internal
stool or cultured bacterial fluid of the patient. standard gene can accurately monitor the entire process of
2. Positive test results indicated the presence of Vibrio chol- extraction, reverse transcription, and quantitative fluores-
eraO1/O139. cence detection.
3. False-negative results will appear in 2019-nCOV nucleic
A.65 Widal’s Tests acid detection tests, and the suspected patient should be
Sample Required Serum (200 μL) combined with symptoms, medical history, and other lab-
Detection Method Agglutination oratory tests for comprehensive evaluation.
Reference Ranges H < 1:160; O < 1:80; Type A < 1:80; 4. Unreasonable sample collection, transport and process-
Type B < 1:80; Type C < 1:80. ing, and low RNA content in the sample can cause detec-
Interpretation tion failure or inaccurate results; variation in the target
Widal’s reaction is an agglutination test with the standard sequence region of the test or sequence changes caused by
solution of Salmonella paratyphoid A and B and the serum of other reasons may lead to inaccurate results; unverified
patients with known typhoid and paratyphoid bacteria H (fla- other interference factors or nucleic acid response inhibi-
gellum) and O (bacterium) which is used for assistant diag- tors may also lead to inaccurate results.
nosis of typhoid and paratyphoid fever or for
immunoagglutination test for epidemiological investigation.
Appendix B: Cancer Tests
A.66 Antibody Tests of 2019-nCoV
Sample Required Serum, Plasma, Whole Blood Cancer, as the first of the three most difficult and serious
Detection Method EIA, CLIA, ECL, IGLT problems, has been perplexing the medical profession with
Reference Ranges Negative many problems, such as high incidence, high mortality, high
Interpretation recurrence rate, rejuvenation, and so on. Through surgery,
radiotherapy and chemotherapy and other traditional treat-
1. Both IgM and IgG are immunoglobulin which are pro- ment methods cannot solve the clinical situation, overcom-
duced by the immune system to provide protection against ing cancer urgently needs new treatment direction and train
the 2019-nCoV. Anti-2019-nCoV positive combined with of thought.
clinical symptoms can identify the 2019-nCoV. The World Health Organization (WHO) clarifies that can-
2. The level of IgM antibody begins to rise 1 week after the cer is the result of abnormal differentiation of human cells
initial infection, some patients with negative results in and accumulation of gene mutation. Therefore, it is very
nucleic acid test show positive in IgM test, indicating that important to realize early screening, intervention and clinical
the IgM detection is one of the effective methods for the diagnosis of tumor, guidance of drug use, monitoring of
diagnosis of 2019-nCoV. postoperative course of disease, risk assessment of recur-
3. Anti-2019-nCoV IgG appears later than IgM (usually in rence, and so on through the detection and analysis of gene
14 days after infection) and can last for 6 months or even molecules in human cells.
Appendixes 837
This chapter mainly introduces the clinical molecular Detection Method PCR, RT-PCR, Southern Blot, FISH
diagnosis and strategy of tumor chemotherapy and risk Reference Ranges Wild type
assessment. Main package includes molecular screening of Interpretation
tumor targeted medical treatment, conventional chemother- The iconic, characteristic genetic alterations of ALCL
apy drugs, tumor risk assessment, and molecular diagnosis (anaplastic large cell lymphoma) is t(2;5)(p23;q35),on chro-
of blood tumors. mosome 5, ALK is fused to the NPM, about 75% of the
ALCL of ALK (+), and the translocation causes the NPM to
B.1 ABCC2 (ATP-Binding Cassette, Subfamily C, translocate to the ALK gene to produce the NPM-ALK
Member 2) fusion gene. So as to produce an NPM-ALK fusion protein
Sample Required Tissues that can have a sustained tyrosine kinase activity, leading to
Detection Method Gene Chip, FISH, q-PCR the occurrence of a tumor. The findings of the NPM-ALK
Reference Ranges Wild type fusion gene are of great significance to the prognosis of
Interpretation ALCL.
Decreased ABCC2 function can affect normal liver func-
tion, including its ability to secrete endogenous complexes B.5 APC (Adenomatous Polyposis Coli)
(such as bilirubin, steroids). Secondly, the change of ABCC2 Sample Required Serum, Plasma, Cell supernatant, Tissues
function can affect the clearance of important drugs in clinic, Detection Method ELISA, RT-PCR,
including chemotherapeutic drugs, antibiotics, and so on. At Immunohistochemistry, PCR-RFLP, PCR-SSCP, HPLC,
the same time, it also affects the clearance of many toxins secondary-generation sequencing
and their complexes. ABCC2 is associated with multiple Reference Ranges Negative/wild type
chemotherapeutic drug resistance. Interpretation
B.2 AFP (α-Fetoprotein) 1. APC gene inactivation happens in the early stage and sta-
Sample Required Serum bilizes in the whole process of occurrence and develop-
Detection Method CLIA, ELISA, RIA ment of colon cancer. The deletion of APC gene is closely
Reference Ranges 10–20 ng/mL (CLIA); <25 ng/mL related to the genetic susceptibility of colon cancer. Loss
(ELISA); <20 ng/mL (RIA) of heterozygosity (LOH) of APC gene has been reported
Interpretation in sporadic colorectal cancer in China.
2. Germline mutation of APC gene exists in typical familial
1. AFP concentration elevated greater than 400 ng/mL in adenomatous polyposis syndrome. Moreover, APC muta-
patients with cirrhosis highly supports diagnosis of HCC. tion has been reported deeply involved in the transforma-
2. Elevated AFP can be detected in other diseases, such as tion of gastric adenoma to adenocarcinoma.
chronic liver diseases, acute and chronic hepatitis, preg-
nancy, in non-hepatic malignancies such as nonsemino- B.6 AS (Angiostatin and Endostatin)
matous germ cell tumors of the ovary and testicles, as Sample Required Serum
well as in other malignancies such as gastric, lung, biliary, Detection Method ELISA
and pancreatic cancers. Reference Ranges Negative
Interpretation
B.3 AMF (Autocrine Motility Factor)
Sample Required Serum 1. Angiostatin (AS) and endostatin are anti-angiogenic com-
Detection Method ELISA pounds which are proteolytic fragments of plasminogen
Reference Ranges Negative and collagen XVIII, respectively. They are reported to
Interpretation serve as anti-angiogenic agents both in vitro and in vivo.
Autocrine motility factor (AMF) is a family of thermo- 2. Prior results indicated that they are able to obviously
soluble proteins with molecular weight of 55–66 kDa. It is inhibit the formation of micrangium and hamper tumor
secreted by cancer cells and binds to AMF receptor (gp78) growth and metastasis formation in several animal tumor
on the same cell and stimulates cell growth and motility by models. Therefore, application of these naturally occur-
autocrine. MAPK/ERK or PI3K/AKT pathways are thought ring anti-angiogenic molecules in a clinical setting may
to be involved in cancer metastasis mediated by AMF. be especially attractive and some of them have been in
phase IV clinical trial.
B.4 ALK (Anaplastic Lymphoma Kinase)
Sample Required Bone marrow (EDTA anticoagulated, B.7 ASXL1 (Additional Sex Combs Like 1)
2 mL), Peripheral blood (EDTA anticoagulated, 2 mL), Sample Required Peripheral blood (EDTA anticoagulated,
Tissues 2 mL)
838 Appendixes
Detection Method Mutation-specific PCR, Gene Therefore, Bcl-6 gene mutation can be used as an indepen-
sequencing dent monitoring index to indicate a good prognosis in clini-
Reference Ranges Wild type cal follow-up.
Interpretation
ASXL1 mutations were associated with poor prognosis in B.11 Bcl-1/CCND1 Gene Rearrangement
MDS. Sample Required Bone marrow (EDTA anticoagulated),
Peripheral blood (EDTA anticoagulated), Tissues
B.8 BAG-1(Bcl-2-Binding Anti-apoptosis Gene) Detection Method PCR, FISH, Southern Blot
Sample Required Serum, Tissues Reference Ranges Negative
Detection Method FISH, Gene Chip Interpretation
Reference Ranges Wild type/negative The abnormal t(11;14) of MM (multiple myeloma) is
Interpretation closely related to the expression of CCND1. A large number
of case studies have found that the prognosis of MM patients
1. BAG-1 displays low or absent expression in normal tis- with t(11;14) translocation is poor and insensitive to chemo-
sue, but significantly higher expression in tumors, such as therapy. Whether it is new or recurrent, it has advanced to the
endometrial carcinoma, breast cancer, and esophageal III stage, and there are a large number of plasma cells in the
carcinoma. circulation.
2. BAG-1expression is correlated with pathological grade,
clinical stage, distant metastasis, and prognosis of the B.12 BCR-ABL Fusion Gene
tumor. Sample Required Bone marrow (EDTA anticoagulated,
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL)
B.9 B2cl-2 Detection Method FISH, RT-PCR
Sample Required Bone marrow (EDTA anticoagulated, Reference Ranges Wild type
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3mL), Interpretation
Tissues The detection of BCR-ABL fusion gene can provide
Detection Method Southern, PCR-SSP, FISH molecular level basis for the diagnosis of CML leukemia.
Reference Ranges Wild type In addition, BCR-ABL positive patients were much more
Interpretation likely to recur after bone marrow transplantation than BCR-
The high expression of Bcl-2 in FL patients is mainly ABL negative CML patients. Moreover, the tyrosine kinase
manifested in molecular genetics. In patients with FL with produced by the coding of BCR-ABL fusion gene is the
t(14;18) chromosome translocation, the clinical develop- target of molecular targeted therapeutic drug, so patients
ment process is relatively slow. If accompanied by other with BCR-ABL fusion gene can be treated with targeted
gene abnormalities, the clinical development process is drugs under the guidance of doctors to improve the survival
greatly accelerated. Only t(14;18) chromatin translocation rate.
did not affect the survival rate of FL patients, but it was
reported that if chromosome 17 was involved at the same B.13 BRC2A1/BRCA2 (Breast Cancer Susceptibility
time, the prognosis of the patients was poor. This may be Gene)
related to the influence of chromosome 17 abnormality on Sample Required Serum, Tissues
p53 gene at 17p13. Detection Method FISH, Gene Chip
Reference Ranges Wild type/negative
B.10 Bcl-6 Interpretation
Sample Required Bone marrow (EDTA anticoagulated,
2–3 mL), Peripheral blood EDTA anticoagulated, 2–3 mL), 1. Mutations in BRCA1 or BRCA2 lead to improperly

Tissues repair of damaged DNA, which increases the risk for
Detection Method Western blot, Mutation Specific-PCR, breast cancer and ovarian cancer.
Immunohistochemistry 2. Poly (ADP-ribose) polymerase (PARP) is a family of pro-
Reference Ranges Wild type teins involved in DNA repair and programmed cell death.
Interpretation Its inhibitor can hence promote apoptosis of tumor cells
3q27 translocation involving Bcl-6 is the most representa- and enhance the therapeutic effect of radiotherapy or che-
tive and common DLBCL translocation, the detection rate is motherapy such as alkylating agent and platinum drug.
30–40%. Bcl-6 gene rearrangement has a characteristic clini- Olaparib, a PARP inhibitor, has been found to not only
copathological correlation in adult DLCBL and is associated prolong the PFS but also improve the objective remission
with primary lymph node and non-bone marrow invasion. rate (ORR) of metastatic breast cancer patients with
Appendixes 839
HER2-negative but germline BRCA mutation compared 1. CA15-3 is a tumor marker for many types of cancer, most
with standard chemotherapy. Olaparib was thus approved notably breast cancer. Generally, the higher the level ele-
by FDA in January 2018 as the first PARP inhibitor for vated, the more malignant the tumor presented.
breast cancer. 2. Elevated CA15-3 in conjunction with alkaline phospha-
tase (ALP) was found to correlate with an increased risk
B.14 BRAF Gene Mutation of early recurrence in breast cancer. CA 15-3 together
Sample Required Tissues with another epitope on the same protein antigen product
Detection Method Hybridization, AS-PCR of the breast cancer-associated MUC1 gene can be
Reference Ranges Wild type detected to increase in patients with benign conditions,
Interpretation such as ovarian cysts, breast disease, and liver disease.
Elevations may also be seen in cirrhosis, sarcoidosis, and
1. BRAF gene is an oncogenic gene, the BRAF gene
lupus.
encodes a RAF kinase protein that transmits cell signals,
the protein is part of the RAS-RAF-MEK-ERK signaling B.18 CA19-9 (Carcinoma Antigen 19-9)
pathway. MAPK/ERK signaling pathway can regulate Sample Required Serum
cell growth, proliferation, differentiation, migration, and Detection Method CLIA, RIA, ELISA
apoptosis. Therefore, the signal transduction of this signal Reference Ranges <37 U/mL
pathway is very important for the normal development of Interpretation
cells.
2. BRAF gene can be used as an independent index to evalu- 1. A significant or sustained increase in postoperative serum
ate the prognosis of patients. The prognosis of patients level of CA19-9 suggests recurrent or progressive
with V600E BRAF gene mutation is more worse. disease.
2. In people with pancreatic masses, CA19-9 can be useful
B.15 Bmi-1 (B-Cell-Specific Moloney Murine to distinguish benign from malignant pancreatic patholo-
Leukemia Virus Insertion Site 1) gies. In addition, CA19-9 can be used as a prognostic
Sample Required Serum, Tissues indicator and a marker to assess response to systemic
Detection Method FISH, Gene Chip therapy as well.
Reference Ranges Wild type/negative
Interpretation B.19 CA125 (Carcinoma Antigen 125)
Aberrant expression of Bmi1 appears to be involved in Sample Required Serum
several types of cancer, such as breast, ovarian, bladder, Detection Method CLIA, RIA, ELISA
prostate cancer as well as hematological malignancies. Its Reference Ranges <35 U/mL
amplification and overexpression are especially pronounced Interpretation
in myelodysplastic syndrome (MDS) and are positively pro-
portional to the score of international prognostic scoring sys- 1. CA125 can be elevated in patients with gynecologic can-
tem (IPSS). cers or non-gynecologic malignancies such as pancreatic
and lung cancer, or other benign conditions such as pelvic
B.16 CA (A Catecholamine) inflammatory disease, endometriosis, pregnancy, cirrho-
Sample Required Urine sis, and hepatitis.
Detection Method HPLC, Fluorimetric determination of 2. CA125 is the most frequently used biomarker for ovarian
trihydroxy indole cancer detection. Around 90% of women with stage III or
Reference Ranges <591 nmol/24 h IV ovarian cancer have elevated serum levels of CA125,
Interpretation which can be used to detect ovarian cancer patients with
Detection of epinephrine in urine can indirectly under- symptoms.
stand the secretion of catecholamine in vivo. Extremely high 3. Monitoring CA-125 blood serum levels has also been pro-
levels of catecholamine can be seen in pheochromocytoma. posed to reflect how ovarian cancer is responding to treat-
ment and to predict prognosis after treatment. Also, an
B.17 CA153 (Carcinoma Antigen 15-3) increase in CA-125 levels after debulking surgery and
Sample Required Serum chemotherapy is a strong predictor of the recurrence with
Detection Method CLIA, RIA, ELISA 95% accuracy.
Reference Ranges <25 U/L
Interpretation B.20 CALR Mutation
Sample Required Bone marrow (EDTA anticoagulated),
Peripheral blood (EDTA anticoagulated)
840 Appendixes
Detection Method Mutation-Specific PCR 1. The importance of chromosome 11q13 translocation

Reference Ranges Wild type and increased expression of CCND1 protein in mantle
Interpretation cell lymphomas has been recognized internationally
CALR mutations are the second most common genetic and can be regarded as a molecular characteristic differ-
abnormality (after JAK2 mutations) associated with essential ent from other B-cell lymphomas, which is highly sen-
thrombocythemia or primary myelofibrosis. They are present sitive and relative. CCND1 egg self-expression and
in about 20–25% of adults with ET and 25–30% of adults with Bcl-1 gene rearrangement are also helpful in the diag-
PMF. Although rare and not well understood in children, 50% nosis of mother cell type and multiple lymphocytic
of pediatric PMF patients had CALR mutations. The CALR polyposis.
mutation is acquired after birth as opposed to inherited. It is 2. Overexpression of cyclin D1 has been found in many
caused by the addition or removal of small amounts of genetic malignant tumors such as NPC, laryngeal cancer, breast
material to a region of the gene called exon 9. This leads to an cancer, esophageal cancer, lung cancer, and so on.
abnormal calreticulin protein. It is not yet understood how the Significantly high expression of cyclin D1 protein is an
mutant protein leads to signs and symptoms of MPN. In addi- independent prognostic factor in head and neck squamous
tion to helping diagnose MPNs, CALR mutation testing can cell carcinoma after surgical resection.
provide information about a person’s prognosis. Studies have 3. Tumors in NPC patients with negative expression of

shown that compared to individuals with the JAK2 mutation, cyclin D1 were prone to recur locally than those with high
those with the CALR mutation had a milder disease course, expression.
fewer signs and symptoms of blood clotting (thrombotic epi- 4. In addition, the 3-year survival rate of cyclin D1-negative
sodes), and better survival. expression was 30% while that of cyclin D1-positive
expression was 70%, which suggests that the level of
B.21 Cathepsin cyclin D1 expression is a promising prognostic molecule
Sample Required Serum, Plasma in patients with NPC.
Detection Method ELISA, CLIA
Reference Ranges B.23 CD44
Interpretation Sample Required Serum, Plasma, Peripheral blood (EDTA
anticoagulated, 2–3 mL), Tissues
1. Cathepsin B (CB) can directly or indirectly activate and Detection Method ELISA, Flow cytometry
dissolve enzymes of ECM such as collagen, laminin, and Reference Ranges Negative
basement membrane in the process of metastasis of can- Interpretation
cer cells to promote the infiltration of cancer cells into
deep tissues, thereby opening channels for expanding of 1. CD44 plays a vital role in lymphocyte homing, migration
cancer cells. High expression of CB has been found in and binding to hypervascular endothelial cells.
various human tumors, such as gastric, lung, intestinal, 2. CD44 splice variants containing variable exons are desig-
breast, ovarian, prostate, kidney cancer, and other cancer nated CD44v which is expressed by metastatic tumor
cells. cells.
2. Cathepsin D (CD) is a recently discovered mitogen in all 3. CD44 is associated with the pathologic activities of can-
kinds of cells and is able to degrade various substrates cer cells, such as proliferation, invasion, metastasis, and
such as fibronectin and laminin. This ability indirectly prognosis. It has been widely studied as a potential thera-
destroys the basement membrane of blood vessels, and peutic target.
activates CB, triggers chain reaction, induces activation
of uPA. Different from other cathepsins, CD has some B.24 CEA (Carcinoembryonic Antigen)
protease activity at neutral pH. High levels of CD in tumor Sample Required Serum
cells appear to be correlated with greater invasiveness and Detection Method CLIA, ELISA, RIA
metastasis. Reference Ranges <2.5 ng/mL in nonsmokers and <5 ng/
mL in smokers
B.22 CCND1/Cyclin D1 Interpretation
Sample Required Serum, Plasma, Bone marrow (EDTA
anticoagulated, 2–3 mL), Peripheral blood (EDTA anticoag- 1. Serum CEA level > 20 ng/mL frequently indicates exis-
ulated, 2–3 mL), Tissues tence of a digestive system tumor.
Detection Method ELISA, CLIA, Western blot, RT-PCR, 2. CEA is useful for monitoring treatment efficacy and eval-
Immunohistochemistry uation prognosis, especially when combined with other
Reference Ranges Negative tumor markers, such as CA242 and CYFRA 21-1.
Interpretation
Appendixes 841
3. The continuous determination of serum CEA level is the B.28 CIN (Chromosomal Instability)
best non-invasive method for monitoring the recurrence Sample Required Serum, Tissues
of invasive colorectal cancer and lung cancer. Detection Method Karyotype analysis
B.25 CEBPA Mutation Interpretation
Peripheral blood (EDTA anticoagulated), Tissues 1. CIN is mostly resulted from chromosome segregation
Detection Method Mutation-Specific PCR, Gene disorders, telomere dysfunction, and defective DNA dam-
sequencing age repair mechanism, resulting in aneuploidy, chromo-
Reference Ranges Wild type some genome amplification, and high frequency of loss of
Interpretation heterozygosity (LOH).
2. CIN is a common occurrence in solid and hematological
1. CEBPA mutations were found in between 7 and 15% of cancers, especially CRC. The most common single gene
patients with acute myeloid leukemia. The three different mutations are APC and K-Ras genes, involving Wnt sig-
types of mutations seen in these AML patients include naling pathway, K-Ras signaling pathway, p53 and its
germline N-terminal mutation, N-terminal frameshift downstream signaling pathway.
mutation, and C-terminal mutation. These mutations are
most frequently found in acute myeloid leukemia M1 or B.29 CIMP (CpG Island Methylator Phenotype)
acute myeloid leukemia M2. Many reports link CEBPA Sample Required Tissues
mutations with a favorable outcome in acute myeloid leu- Detection Method Gene sequencing, FISH
kemia. Patients with CEBPA mutations have longer Reference Ranges Negative
remission duration and survival time than those without Interpretation
the mutations.
2. The higher levels of CEBPA methylation are directly pro- 1. CIN is mostly resulted from chromosome segregation
portionate with treatment response. The complete disorders, telomere dysfunction, and defective DNA dam-
response rate increased proportionately with the level of age repair mechanism, resulting in aneuploidy, chromo-
CEBPA methylation. some genome amplification and high-frequency of loss of
heterozygosity (LOH).
B.26 Chromosome 13 Deletion 2. CIN is a common occurrence in solid and hematological
Sample Required Bone marrow (EDTA anticoagulated), cancers, especially CRC. The most common single gene
Peripheral blood (EDTA anticoagulated), Tissues mutations are APC and K-Ras genes, involving Wnt sig-
Detection Method FISH naling pathway, K-Ras signaling pathway, p53 and its
Reference Ranges Wild type downstream signaling pathway.
Interpretation
Chromosome 13 deletion exists at various stages of MM B.30 Circulating miRNAs
(multiple myeloma), but not in normal people. Through Sample Required Serum
FISH, about 50% of patients with initial MM had del (13). In Detection Method PCR, Gene sequencing
most cases, del (13) showed chromosome 13 monomers, a Reference Ranges Wild type/negative
few showed cumulative 13q14 stroma deletions, and the Interpretation
common deletions of double alleles were very rare.
1. Most of the miRNAs that promote tumorigenesis are

B.27 Chromosomal 1 Aberration located in the amplification region of tumor genes, while
Sample Required Bone marrow (EDTA anticoagulated), most of the miRNAs that inhibit tumor are located in the
Peripheral blood (EDTA anticoagulated), Tissues region of gene deletion, indicating that miRNAs play
Detection Method FISH multiple roles in tumor formation and development.
Reference Ranges Negative Studies have found that different types of miRNA are up-
Interpretation regulated or down-regulated in the serum of patients with
In general, there is a certain relationship between various certain diseases or are specifically expressed in tissues,
cytogenetic abnormalities of MM (multiple myeloma). 1p21 such as mir-211 is highly specifically expressed in the
deletion was significantly correlated with 1q21 amplification lung, mir-122 is highly specifically expressed in the liver,
and chromosome 13 deletion. and mir-124 is highly expressed in the brain.
842 Appendixes
2. Compared with traditional protein biomarkers, miRNA B.34 CTC (Circulating Tumor Cell)
has the following advantages: low complexity, no post- Detection Method FACS
processing modification, high affinity “capture” reagents Sample Required Peripheral blood (EDTA anticoagu-
can be synthesized, tissue restricted expression profile lated, 2–3 mL)
and signal amplification can be performed. Changes in Reference Ranges Negative
circulating miRNA expression profiles have been reported Interpretation
in a variety of cancers, and their expression patterns
appear to be tissue-specific. Due to their size, abundance, 1. CTM and mesenchymal phenotype CTC have stronger
tissue specificity, and relative stability in circulation, metastatic potential. EMT is a key step in the process of
plasma circulating miRNAs show great potential as new helping CTC entering the peripheral blood circulation.
biomarkers for non-invasive tumors. When the tumor cells enter the blood, they will migrate in
the form of single cells or CTM. When the flow reaches
B.31 CircRNA (Circular RNAs) the appropriate location, the tumor cells will reverse
Sample Required Serum, Plasma, Tissues metastasis through the blood vessel wall, metastasis from
Detection Method Gene sequencing, Gene Chip the blood to other tissues, proliferation and formation of
Reference Ranges Wild type new lesions.
Interpretation 2. Tumor metastasis is the main cause of death of tumor
patients. Tumor cells invade into the surrounding tissues
1. circRNAs can be divided into the following three catego- of primary tumor cells, enter the blood and lymphatic sys-
ries: exon circRNAs, intron circRNAs (ciRNAs), and tem, and form CTC of circulating tumor cells, which are
exon-intron intron circRNAs (EIciRNAs). transferred to the remote tissues, exudate, adapt to the
2. CircRNAs play different roles in different cancers. Some new microenvironment, and finally settle down to form
circRNAs may be carcinogenic while others may play a metastasis.
role in tumor inhibition.
B.35 ctDNA (Circulating Tumor DNA)
B.32 C-MAF Detection Method PCR, Gene sequencing
Sample Required Bone marrow (EDTA anticoagulated), Sample Required Peripheral blood (EDTA anticoagu-
Peripheral blood (EDTA anticoagulated), Tissues lated, 2–3 mL), Tissues
Detection Method PCR, FISH, Southern Blot Reference Ranges Wild type/negative
Reference Ranges Wild type Interpretation
Interpretation
The expression level of C-MAF gene was very low in nor- 1. The ctDNA concentration is proportional to the tumor
mal MM (multiple myeloma) cell lines and even in non- load size, but the tumor stage, location, and size cannot be
t(14;16) MM cell lines, but only in MM cells with t(14;16). determined.
2. ctDNA testing, especially the detection of ctDNA muta-
B.33 C-MYC tions, can monitor tumor progression and prognosis. Tests
Sample Required Bone marrow (EDTA anticoagulated, have shown that the level of ctDNA can be used as an
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL), independent indicator to evaluate the prognosis of some
Tissues tumors.
Detection Method Western blot, RT-PCR, 3. It can detect specific mutations in tumor patients’ tissues
Immunohistochemistry and plasma, accurately classify tumors, and guide clini-
Reference Ranges Wild type cians to target treatment.
Interpretation 4. ctDNA test can reflect whether the tumor has recurrence
Burkitt lymphoma is a highly malignant B-cell lymphoma. and metastasis and whether there is minimal residual
Its molecular genetic characteristics are chromatin transloca- disease.
tion involving MYC gene, the most common of which is 5. ctDNA can reflect the information of whether anticancer
t(8;14)(q24;q32), which involves the MYC gene of 8q24 and treatment is effective and whether drug resistance occurs,
the IgH gene of 14q32. It can also be found occasionally in so that clinicians can timely adjust the treatment plan,
t(2;8)(p11;q24) and t(8;22)(q24;q11), which involve the k reduce expensive ineffective treatment, and achieve indi-
light chain of MYC gene and Ig, respectively. These transloca- vidualized medication and treatment.
tions lead to the high expression of MYC gene and the signifi-
cant increase of cell proliferation activity.
Appendixes 843
B.36 CYP2D6 (Cytochrome P450, Family 2, Subfamily Reference Ranges Negative/Wild type
D, Polypeptide 6) Interpretation
Sample Required Peripheral blood (EDTA anticoagulated,
2–3 mL) 1. Its normal expression can lead to strong adhesion between
Detection Method AS-PCR cells, limiting the transfer of cells.
Reference Ranges Wild type 2. LOH of DCC is one of the most frequent genetic abnor-
Interpretation malities that occur in advanced CRC. The lower the
The (CytochromeP450, family2, subfamily D, polypep- degree of differentiation and the later the Dukes stage, the
tide 6) of mammary cancer patients treated with was closely more obvious the loss of DCC protein expression is.
related to the concentration of active metabolites in the Therefore, the detection of DCC expression in colon can-
blood, which significantly affected the recurrence rate and cer has certain reference value for early diagnosis, evalu-
survival rate of the patients. Therefore, FDA recommended ation of treatment and prognosis of colon cancer.
that patients first detect CYP2D6 genotype before receiving
tamoxifen drug treatment to predict the efficacy of the B.40 DCP (Dextral-γ-Hydroxy-Prothrombin)
tamoxifen drug in 2006. Sample Required Serum
Detection Method ELISA, RIA, CLIA
B.37 CYFRA21-1 (Cytokeratin 19 Fragment, Also Reference Ranges <40 ng/mL
Known as KRT19) Interpretation
Detection Method CLIA, ELISA, RIA 1. DCP is useful to monitor therapeutic effect as well as recur-
Reference Ranges <4 ng/mL rence of tumors. An important characteristic of DCP is that its
Interpretation half-life is 2.5 days, so it falls back quickly after resection.
2. DCP displays high specificity and stability that it is was
1. CYFRA21-1 is useful in determination of tumor stage, only detectable in 91% of HCC patients but not found in
prediction of patient prognosis and the efficacy of s urgical other liver diseases and the DCP level did not change with
resection of tumors, and reflection of tumor burden. The the administration of vitamin K.
diagnostic cutoff level, sensitivity, and specificity of
Cyfra21-1 were 2.17 ng/mL, 60.36%, 81.03%, B.41 DNA Methylation in Peripheral Blood
respectively. Sample Required Peripheral blood (EDTA anticoagulated,
2. Cyfra21-1 is an independent prognostic factor for non- 2–3 mL)
small cell lung cancer, especially squamous cell carci- Detection Method PCR, Gene sequencing
noma. Serum level of Cyfra21-1 is not affected by Reference Ranges Negative
smoking and blood collection skills, so the false positive Interpretation
rate is low.
1. Gene methylation occurred in p16, RASSF1A, e-cadherin,
B.38 DAPK (Death-Associated Protein Kinase) h-cadherin, MGMT, DAPK, DCC, COL1A2, TAC1, SST,
Sample Required Peripheral blood (EDTA anticoagulated, and GALR1.In 65% of head and neck cancers, more than
2–3 mL), Tissues two tumor suppressor genes were methylated, and patients
Detection Method ELISA, Western blot, PCR, HPLC with multiple gene methylation had a worse prognosis than
Reference Ranges Negative those without or with less than two genes methylation. This
Interpretation indicates that DNA hypermethylation is closely related to
1. Methylation of CpG islands in the promoter region results the occurrence, development, and prognosis of tumor tis-
in silence of DAPK gene and thus inhibits apoptosis, sues. Specific gene hypomethylation can cause oncogene
which may lead to the formation of tumors. This methyla- activation and promote tumor growth and metastasis.
tion is an early event of tumorigenesis, which can possi- 2. Common DNA methylation markers are: (1) Cyclin-
bly be detected before cell canceration. DAPK promoter dependent kinase inhibitors (p16INK4a); (2) O6-methyl
methylation was found in many cancers, such as NSCLC, guanine-DNA methyltransferase (MGMT); (3) glutathione
HCC and cervical cancer, as well as some benign condi- s-transferase1(GSTP1); (4) MLH1 genes; (5) breast cancer
tions, such as in bronchial epithelial cells of smokers. I susceptibility gene (BRCA1); (6) SEPT9 genes; and so on.
B.39 DCC (Deleted in Colorectal Carcinoma) B.42 DNMT3A (DNA

Sample Required Serum, Plasma, Tissues (Cytosine-5)-Methyltransferase 3A)
Detection Method ELISA, Immunohistochemistry, Sample Required Peripheral blood (EDTA anticoagulated,
PCR-RFLP, PCR-SSCP, HPLC 2–3 mL), tissues
844 Appendixes
Detection Method Mutation-Specific PCR, existed in normal nasopharyngeal epithelium, representing

Immunohistochemistry, Gene Sequencing, FISH an early event of NPC.
Reference Ranges Wild type
Interpretation B.46 E-cadherins
Sample Required Serum, plasma, tissues
1. DNMT3A is responsible for de novo DNA methylation. Detection Method ELISA, Western blot,
Such function is to be distinguished from maintenance Immunohistochemistry
DNA methylation which ensures the fidelity of replica- Reference Ranges Negative
tion of inherited epigenetic patterns. Interpretation
2. DNMT3A mutations were most commonly seen in
It was found that the expression of E-CD was down-
AML. These mutations most often occur at position regulated in many tumors and negatively correlated with the
R882 in the protein and this mutation may cause loss of grade and metastasis of tumors.
function.
3. DNMT3A mutations are associated with poor overall
B.47 Endosome
survival. Sample Required Serum, fluid, tissues
Detection Method Total internal reflection fluorescence
B.43 DPYD (Dihydropyrimidine Dehydrogenase) microscopy
Sample Required Tissues Reference Ranges Negative
Detection Method Immunohistochemistry, FISH, q-PCR Interpretation
Reference Ranges Wild type 1. Endosome refers to a membrane-bound organelle in

Interpretation eukaryotic cells, belonging to a vesicular structure.
The detection of DPYD gene mutation is often used to Endosomes can be divided into early endosome, late
predict the efficacy of drugs. When the DPYD activity is sig- endosome, and recycling endosome according to the dif-
nificantly increased, the catabolism of 5-FU increases and ferent time stages of endocytosis, and they can be distin-
the synthesis metabolism decreases, which can easily lead to guished by protein markers such as GTP binding protein
drug resistance to 5-FU treatment. rabs, and the three endosomes are also different in mor-
phology. Once vesicles in endocytosis are released, they
B.44 E2A-PBX1 Fusion Gene first fuse with the primary endosome, then grow into the
Sample Required Bone marrow (EDTA anticoagulated, secondary endosome and fuse with the lysosome. Many
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL) viruses enter the cell through this way. Defects in the
Detection Method FISH, RT-PCR endosomal system cause abnormal expression or localiza-
Reference Ranges Wild type tion of tumor-related proteins, which is closely related to
Interpretation the occurrence and development of tumors. EGFR is a
The detection of E2A-PBX 1 fusion gene can provide well studied receptor tyrosine kinases, and its overexpres-
molecular basis for the diagnosis of ALL leukemia. The clin- sion is associated with the development of various
ical symptoms of patients with this translocation are danger- cancers.
ous. In addition, pre B-ALL patients with EZA-PBXI fusion
gene had a better prognosis, so E2A-PBX 1 was a marker for B.48 ERCC1 (Excision Repair Cross Complementing
predicting the good prognosis of Pre B-ALL. Patients with group 1)
positive gene can be monitored and detected for minimal Sample Required Tissues
residue. Detection Method Gene sequencing
B.45 EBERs (EB Virus Encoded RNAs) Interpretation
Sample Required Tissues
Detection Method Situ hybridization 1. ERCC1 is an important member of nucleic acid repair
Reference Ranges Negative, only occasionally positive family, a key member of NER system, involved in DNA
in normal nasopharyngeal and atypical hyperplasia epithelial strand cutting and damage identification. ERCC1 gene is
tissue also the first cloned human DNA damage repair gene. The
Interpretation expression of ERCC1 was found in all tumor cells, and
EBER1 was always positive in the majority of keratinized the expression level of ERCC1 was very different. The
squamous cell carcinoma and non-keratinous carcinoma expression of ERCC1 directly affected the physiological
cells. This result indicates that latent infection of EB virus process of DNA repair.
Appendixes 845
2. Clinical studies have confirmed that ERCC1 is involved 1. The positive expression rate of ER in patients with stage I
in the occurrence of platinum and other chemotherapeutic and II was significantly higher than that in patients with
drugs, and its expression level is negatively correlated stage III, IV.
with the efficacy and survival time of platinum chemo- 2. The decreased expression of ER in advanced breast can-
therapy in various tumors, that is to say, patients with low cer is mainly due to the loss of normal structure caused by
expression level are sensitive to platinum drugs and cell depolarization and proliferation, which may also be
patients with high expression level show drug resistance. related to the occurrence of estrogen-independent breast
Therefore, the clinical treatment guidelines for NCCN cancer.
non-small cell lung cancer in the USA clearly point out
that when the expression of ERCC1 gene mRNA is high, B.51 EGFR (The Epidermal Growth Factor Receptor)
it suggests that patients are resistant to platinum chemo- Sample Required Serum, Tissues
therapeutic drugs. Detection Method AS-PCR, ARMS, RIA, HRMA
B.49 Exosome Interpretation
Detection Method Ultracentrifugation, centrifugal filtra- 1. EGFR is a member of the ErbB family of receptors, a
tion, Immunomagnetic separation subfamily of four closely related receptor tyrosine
Reference Ranges Negative kinases: EGFR (ErbB-1), HER2/neu (ErbB-2), Her 3
Interpretation (ErbB-3), and Her 4 (ErbB-4).
2. Mutation or amplification of EGFR could result in cancer.
1. Exosome is utricle bubble secreted by cells, a diameter of PI3K-AKT and RAS-MEK-ERK signaling activated by
50~200 nm double lipid package structure, with maternal EGFR mutation promote the growth, proliferation, and
cells biological information characteristic molecules. migration of cancer cells.
Exosomes promote virus infection and transmission. For
example, HIV virus can find the optimal cells through B.52 EML4-ALK (Echinoderm Microtubule-
exosomes for infection and transmission. Exosomes also Associated Protein-Like 4-Anaplastic Lymphoma
play an important role in cancer. Cancer cells tend to Kinase)
release exosomes higher than normal cells. Exosomes Sample Required Serum, Tissues
secreted by cancer cells play an important role in the Detection Method ELISA, CLIA, RIA
occurrence and progression of cancer. They can promote Reference Ranges negative
the proliferation, spread, metastasis, and deterioration of Interpretation
cancer cells and help cancer cells escape from the clear-
ance mechanisms in the body or in vitro. 1. ALK (Anaplastic lymphoma kinase) is a member of the
2. Exosome has the following three main advantages: (1) Its insulin receptor subfamily of the tyrosine kinase receptor
levels are higher than the CTC in body fluids. It is more (RTK) family.
than 10/mL blood and more sensitive than the CTC; (2) It 2. Mutation, acquisition of additional gene copies and for-
has a lipid bilayer structure, which can protect the internal mation of a fusion gene with any of several other genes of
nucleic acid molecules from degradation; (3) Due to its ALK gene can all lead to tumors. The EML4-ALK fusion
small size and strong permeability, it is easy to enter body gene is a key driven gene which is responsible for around
fluids and thus be detected. Fully recognizing and utiliz- 3–5% of NSCLC. Therefore, it has been considered as a
ing the characteristics and advantages of exosomes, novel target for the treatment of NSCLC. Clinical trials
screening and finding new markers are expected to over- have confirmed that ALK heterotopic lung cancer patients
come the problems of tumor heterogeneity, which will are highly sensitive to the ALK inhibitors, such as crizo-
have important clinical significance in early diagnosis, tinib and brigatinib.
treatment monitoring, and prognosis evaluation of tumors.
In addition, exosomes are expected to be used as drug car- B.53 FGF (Fibroblast Growth Factor)
riers and may have great potential in tumor therapy. Sample Required Serum, Plasma
Detection Method ELISA
B.50 ERs (Estrogen Receptors) Reference Ranges
Sample Required Tissues, Serum Interpretation
Detection Method Immunohistochemical, Western blot The overexpression of FGF in the peripheral and invasive
Reference Ranges Negative marginal cells of the tumor tissue, while the low level of FGF
Interpretation in tumor tissue with early stage, indicates the heterogeneity
846 Appendixes
of the tumor cell genes, that is, there are differences in inva- and the 5-year survival rate of gp78 positive patients is sig-
sion and metastasis related expression in the same tumors, nificantly lower than that of negative ones.
and cells with high expression of fibroblast growth factor
gene will invade and metastasize. Inhibitors of the FGF/ B.57 HCG (Human Chorionic Gonadotropin)
FGFR system have been shown to act as “two compartment” Sample Required Serum, Urine
targeting drugs implicated for the therapy of FGF/FGFR- Detection Method CLIA, Colorimetry
driven tumors. Reference Ranges 0–3 mIU/m (serum), 0–312 U/L
(urine)
B.54 FGFR3/MMSET Fusion Gene Interpretation
Peripheral blood (EDTA anticoagulated), Tissues 1. Usually, β-HCG concentration < 500 mIU/mL presented in
Detection Method PCR, FISH, Southern Blot patients with seminoma while > 1000 mIU/mL is more
Reference Ranges Negative often seen in nonseminomatous germ cell tumors (NSGCT).
Interpretation 2. Elevation of β-HCG can be seen in 60–80% of patients
Many studies have found that the presence of t(4;14) of with NSGCTs 10–20% with stage I seminoma and up to
MM (multiple myeloma) cell lines and the overexpression of 30–50% of disseminated seminoma.
FGFR3 gene in the patient, whereas in the absence of t(4;14) 3. β-HCG may also be elevated in select non-trophoblastic
of the MM cell line it is difficult to detect the expression of solid tumors such as tumors of the small and large bowel,
FGFR3, and the use of anti-FGF antibodies can significantly hepatomas, lung, and stomach cancers, HCC, renal cell
inhibit the growth of the MM cell line in which t(4;14) is carcinoma (RCC), and bladder carcinoma. In addition, as
present. Some scholars have suggested that after the occur- β-HCG is not able to cross the blood–brain barrier, pres-
rence of, t(4;14), FGFR3 mutates under the action of IGH ence of β-HCG in the cerebrospinal fluid and the ratio is
promoter, which leads to the progress of MM. In addition, it more than 1:60 strongly indicates the brain metastasis of
was found that 20% of MM patients with t(4;14) lacked the the malignant tumor.
expression of FGFR3, but they still expressed MMSET, so it
was believed that the activation of MMSET gene also played B.58 HER2 (Human Epidermal Growth Factor
an important role in the occurrence of MM. Receptor-2)
Sample Required Tissues, Serum
B.55 FLT3 (Fms-Like Tyrosine Kinase 3) Detection Method FISH, IHC, CISH
Sample Required Bone marrow (EDTA anticoagulated, Reference Ranges Negative
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL) Interpretation
Detection Method Array-CGH, SNP-array, Mutation
Specific-PCR 1. Overexpression of the HER2 has been reported to occur
Reference Ranges Wild type in approximately 15–30% of breast cancers which is
Interpretation closely related to increased recurrence and a poor progno-
FLT3-ITD has its unique clinical manifestations. The sis of breast cancer.
AML patients with FLT3-ITD positive are insensitive to 2. Trastuzumab (marketed as Herceptin) is a monoclonal
chemotherapy and resistant to chemotherapy, and the antibody drug targeted HER2. It has been found that
prognosis of these patients is poor. The length of long tumors with HER2+ acquired accelerated apoptosis and
FLT3-ITD repeat sequence is related to the poor progno- decreased tumor volume after administration of trastu-
sis, which indicates that FLT3-ITD gene mutation can be zumab in breast cancer patients, which provides direct
used as an index to predict the prognosis of AML. The evidence that HER2 is the driving gene of breast cancer.
frequency of FLT3 gene mutation in AML patients is very
high, so the detection of FLT3 gene mutation can provide B.59 hENT1
molecular level basis for the diagnosis of AML Sample Required Tissues
leukemia. Detection Method Immunohistochemistry, FISH, q-PCR
B.56 Gp78 Interpretation
Sample Required Tissues There was significant difference in the expression of
Detection Method Immunohistochemistry hENTI1between normal tissues and corresponding tumor
Reference Ranges Negative tissues. Because of the polymorphism of hENTl gene, the
Interpretation low expression of hENTl in normal tissues can reduce the
The expression of gp78 in esophageal cancer and colon plasma clearance rate of GEM, while the low expression of
cancer with lymph node metastasis is significantly increased, GEM gene in tumor tissues will lead to drug resistance,
Appendixes 847
while the low expression of GEM gene in normal tissues and of IgH gene rearrangement technique in the early diagnosis
normal tissues will greatly reduce the efficacy of GEM and and differential diagnosis of malignant lymphoma is of great
affect the prognosis. hENT1 deficient tumor cells have a significance, especially for those cases which cannot be
high resistance to GEM. diagnosed by routine pathomorphological examination.
B.60 IDH1/IDH2 (Isocitric Dehydrogenase 1/2) B.63 JAK2 (Janus Kinase 2)

Detection Method SSCP, Gene sequencing, Sample Required Bone marrow (EDTA anticoagulated),
Immunohistochemistry Peripheral blood (EDTA anticoagulated)
Sample Required Tissues Detection Method PCR, RT-PCR, Gene sequencing
Reference Ranges Wild type Reference Ranges Wild type
Interpretation Interpretation
The protein encoded by IDH gene is isocitrate dehydro-
genase. There are three types of human IDH: IDH1, IDH2, 1. JAK2 gene 1849G>T (V617F) mutation is common in
and IDH3, IDH1 localization in cytoplasm and peroxi- patients with myeloproliferative tumors in hemolym-
some, IDH2 and IDH3 localization in mitochondria. This phoid tumors, and there is a certain difference in clinical
kind of protease can oxidize isocitrate to oxalyl succinic characteristics between JAK2 V617F positive patients
acid and then convert it to α-ketoglutaric acid. It was first and JAK2 V617E negative patients. The mutations of
found that the mutation of IDH1 was closely related to JAK2 gene 1849G>T (V617F), JAK2 gene 1849G>T
glioma, and then it was found that the mutation was asso- (V617F), and JAK2 gene 1849G>T (V617F) in patients
ciated with prostate, paraganglioma, and IDH1/2 mutation with true polycythemia were 95–100%, 1849G>T
in acute myeloid leukemia. The tumorigenic mechanism is (V617F), and 1849G>T (V617F), respectively.
that the mutant IDH can convert α-ketoglutaric acid into 2. According to the close relationship between JAK2 muta-
2-hydroxyglutaric acid, and the latter can inhibit the target tion and myelodysplastic diseases, in the revised World
of the former, resulting in abnormal expression of these Health Organization (WHO) classification system in
targets leading to cancer. 2008, JAK2 gene mutation became the main diagnostic
index in chronic myeloproliferative neoplasms (MPN).
B.61 Integrins Detection of JAK2 gene mutation is of great significance
Sample Required Serum, Plasma, Peripheral blood (EDTA in the diagnosis of chronic myeloproliferating diseases
anticoagulated, 2–3 mL), Tissues and leukemia. If there is no mutation in V617F of JAK2
Detection Method ELISA, Western blot, gene, but the mutation in exons 12 or 13 of JAK2 gene is
Immunohistochemistry, Flow cytometry detected in the gene, it can also be judged that the person
Reference Ranges Negative is a PV (polycythemia vera) patient combined with clini-
Interpretation cal symptoms.
Integrin mainly mediates the adhesion between cells and
ECM, which enables cells attached to form integration, B.64 KIT Gene Mutation
hence its name. And this adhesion and attachment are closely Sample Required Tissues
related to tumor invasion and metastasis. Detection Method Gene sequencing, AS-PCR, ARMS
B.62 IgH Interpretation
Sample Required Bone marrow (EDTA anticoagulated, It has been confirmed that the location of KIT gene muta-
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL), tion can affect the response of tumor patients to tyrosine
Tissues kinase inhibitors like Imatinib. So detect the mutation state
Detection Method Southern, PCR-SSP, FISH of KIT gene can assist GISTS diagnosis, further diagnosis of
Reference Ranges Wild type CD177 negative patients, diagnosis of familial gastrointesti-
Interpretation nal stromal tumor, guidance of chemotherapy, prediction of
The proliferation of lymphoid cells is monoclonal, and chemotherapy effect.
the possibility of disease is considered if only one gene rear-
rangement fragment is detected. IgH gene rearrangement is B.65 K-RAS Gene Mutation
often used as marker in FL detection. The detection of gene Sample Required Tissues
rearrangement can not only distinguish whether lymphoid Detection Method Gene sequencing, Hybridization,
tissue is neoplastic hyperplasia or reactive hyperplasia, but AS-PCR
also make it possible to accurately judge the origin of cells Reference Ranges Wild type
and perfect the classification of lymphoma. The application Interpretation
848 Appendixes
1. K-RAS is a proto-oncogene, about 35 kb long and located B.68 MDR1 (Multidrug Resistance Protein 1)
on chromosome 12. It is a member of the RAS gene fam- Sample Required Tissues
ily and encodes K-ras protein (21 kDa). It is the down- Detection Method FISH, q-PCR
stream molecule of the functional signal of epidermis Reference Ranges Wild type
growth factor receptor. It is related to tumor formation, Interpretation
proliferation, migration, diffusion, and angiogenesis. The high expression and polymorphism of MDR1 gene
Detection of K-ras gene mutation is an important index to make tumor cells resistant to purple Taxanes drugs. Taxanes
deeply understand the situation of cancer genes, to under- drugs are not recommended in patients with high expression
stand the development and prognosis of various cancers, of MDR1mRNA.
and to evaluate the curative effect of radiotherapy and
chemotherapy. B.69 MET
2. The mutation of K-ras gene occurs in the early stage of Sample Required Bone marrow (EDTA anticoagulated,
malignant transformation of tumor, and the K-ras gene of 2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL)
primary and metastatic foci is highly consistent. It is gen- Detection Method FISH, SISH, q-PCR
erally believed that the status of K-ras gene will not Reference Ranges Wild type
change as a result of treatment. The K-ras gene mutation Interpretation
was found in 20% of non-small cell lung cancer (NSCLC). MET amplification (defined as ≥6 copies by FISH) has
The mutation rate of K-ras in patients with colorectal can- been reported in 2–4% of denovo untreated NSCLC and in
cer was 30~35%. up to 20% of EGFR/TKI-resistant NSCLC patients, whereas
MET exon 14 skipping mutations are present in 3–4% of
B.66 LDH (Lactate Dehydrogenase) lung adenocarcinoma cases. MET amplification might
Sample Required Serum, Urine account for progression after treatment with EGFR and other
Detection Method Colorimetry, Continuous monitoring targeted tyrosine kinase inhibitors. Combination of MET
method inhibitor (Tivantinib or Onartuzumab) with EGFR inhibitor
Reference Ranges 100–300 U/L (serum), 560–2050 U/L (Erlotinib) has been demonstrated as a potential treatment
(urine) regimen to overcome acquired resistance caused by MET
Interpretation amplification. Meaningful clinical response to MET inhibi-
tors, as Crizotinib, has been reported and longer survival in
1. LDH expression may act as a general indicator in the NSCLC patients with MET exon 14 skipping mutations is
prognosis of cancers because its level corresponding with associated with MET inhibitors.
tumor burden with levels >200 U/L usually correlating
with bulky disease. B.70 MSI5 (Microsatellite Instability)
2. In addition, it can be used to monitor response to treat- Sample Required Serum, Tissues
ment and to find recurrence and metastasis of tumors, Detection Method Karyotype analysis
even to indicate immune suppression in cancer. Reference Ranges Negative
3. LDH has been identified as a promising target in cancer Interpretation
treatments through inhibiting its activity and thus prevent-
ing carcinogenic cells from proliferating. 1. The presence of MSI strongly indicates that DNA mis-
match repair (MMR) is not functioning normally. MSI
B.67 LncRNA (Large Intergenic Non-coding RNA) may exhibit one of the following three states: MSI-High
Sample Required Serum, Plasma, Tissues (MSI-H), MSI-Low (MSI-L), or Microsatellite Stable
Detection Method Gene sequencing, Gene Chip (MSS) in colon cancers. CRC with MSI-H was reported
Reference Ranges wild type to have a better prognosis, but a less sensitivity to the
Interpretation 5-FU-based chemotherapy compared to patients with
MSI-L or MSS.
1. LncRNA expression is time-specific and spatially spe- 2. In May 2017 the FDA approved an immunotherapeutic
cific, which further confirmed that lncRNA expression called Keytruda® (pembrolizumab) (PD-1 inhibitor) for
has a strict regulatory mechanism. Studies have shown the treatment of solid tumor patients with MSI-H or
that the abnormal expression of lncRNA is closely related dMMR. This is the first anti-tumor therapy that is inde-
to the proliferation, apoptosis, invasion, migration, and pendent of tissue type and tumor location or differentia-
drug sensitivity of tumor cells. tion stage but merely according to whether it is MSI-H,
2. LncRNA is a kind of new potential biomarkers and targets which is a real milestone.
for cancer treatment.
Appendixes 849
B.71 MLL (Mixed Lineage Leukemia Genes) 1. They are correlated with the cleavage of cell surface
Sample Required Bone marrow (EDTA anticoagulated, receptors, the release of apoptotic ligands (such as the
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL) FAS ligand), and chemokine/cytokine inactivation.
Detection Method array-CGH, SNP-array, RT-PCR 2. MMPs are also capable of destroying local tissue structure,
Reference Ranges Wild type promoting tumor proliferation; ruining basement membrane
Interpretation barrier, facilitating tumor metastasis; and remodeling ECM,
Usually, with the exception of MLL-AF 10, AML patients accelerating tumor angiogenesis. Among MMPs, MMP-2
with MLL gene rearrangement have poor prognosis. MLL and MMP-9 are considered to be important in metastasis.
gene rearrangement can be used as an index to predict the
prognosis of AMI. In addition, the detection of MLL gene B.75 MPL (Myeloproliferative Leukemia Virus
rearrangement can also provide molecular level basis for the Oncogene)
diagnosis of AML leukemia. Sample Required Bone marrow (EDTA anticoagulated),
Peripheral blood (EDTA anticoagulated)
B.72 MLL-AF4 Fusion Gene Detection Method PCR, RT-PCR, Gene sequencing
Detection Method FISH, RT-PCR
Reference Ranges Wild type 1. Mutations in the W515L (1544G>T) and W515K (1543–
Interpretation 1544TG>AA) sites of the tenth exon of the MPL gene
The detection of MLL-AF4 fusion gene can provide occurred in approximately 5–15% of primary myelofibro-
molecular level basis for the diagnosis of B-ALL leukemia. sis (PMF) and 2–5% of primary thrombocytosis (ET).
In addition, MLL-AF4 fusion gene in B-ALL is considered The MPL gene encodes a transmembrane receptor tyro-
to be a marker of poor prognosis in infants and adults, but for sine kinase, which is a thrombopoietin (TPO) receptor,
adult B-ALL, the existence of MLL-AF4 fusion gene seems which is responsible for modulating a glycoprotein cyto-
to enhance the efficacy of high-dose Ara-C, so M LL-A F4 is kine produced by the platelets.
a marker for predicting the prognosis of B-ALL. Patients 2. The most common mutations in MPL gene are W515K
with positive gene can be monitored and detected for mini- and W515L, which have been proved to be able to con-
mal residue. tinuously activate JAK/STAT signaling pathway and con-
tribute to carcinogenicity in the absence of platelet auxin.
B.73 Mucoprotein MPL gene mutation is negative in most patients with
Sample Required Serum, Fluid, Tissues V617F mutation of JAK2 gene, but patients have the same
Detection Method ELISA, Western blot, symptoms as bone marrow proliferating tumors.
Immunohistochemistry 3. Detection of MPL gene mutation or quantitative detection
Reference Ranges 20~40 mg/L of MPL gene mutation can be used to diagnose and moni-
Interpretation tor myeloproliferative tumors, as well as to predict the
non-mutation of JAK2 gene in a group of patients with
1. Mucoproteins can be found throughout the body, includ- primary myelofibrosis or primary platelet hyperplasia.
ing the gastrointestinal tract, airways, reproductive
organs, and mammary gland, lubricating and protecting B.76 MVD (Microvessel Density)
normal epithelium and mediating signal transduction and Sample Required Tissues
cell adhesion as well. Detection Method Immunohistochemistry
2. In tumors, MUC is usually aberrantly expressed and is in Reference Ranges
correlation with invasion, metastasis, and prognosis of Interpretation
tumors.
1. MVD refers to the counts of microvessels per unit volume
B.74 MMP in tumors. Elevated counts of MVD are closely related to
Sample Required Serum, fluid histological grade, clinical stage, lymph node metastasis,
Detection Method Zymography, Western blot, recurrence, and prognosis of tumors. Highly increased
Immunohistochemistry, PCR MVD found in bladder, colorectal, gastric, and NSCLC is
Reference Ranges Negative prone to metastasis.
Interpretation 2. MVD is considered to be a reliable predictor of metastatic
potential in many tumors. It is suggested that application
of MVD during pathological examination would provide
an important evidence for clinical evaluation of prognosis
850 Appendixes
and treatment. In addition, tumor MV can be considered Reference Ranges Negative/wild type
as a potential predictive marker for targeted angiogenesis Interpretation
therapy.
1. The expression of NES1 gene is absent in breast cancer,
B.77 NPM1 Mutation which can be used as an early indicator of breast cancer.
Sample Required Bone marrow (EDTA anticoagulated), 2. The expression of NES1 is significantly decreased in

Peripheral blood (EDTA anticoagulated), Tissues patients with acute lymphoblastic leukemia (ALL) and
Detection Method Mutation-Specific PCR, Capillary prostate cancer, and is in correlation with remission,
electrophoresis, Gene sequencing, FISH, disease-free survival (DFS), and overall survival (OS) of
Immunohistochemistry tumors. Inactivation of NES1 gene is an early event in the
Reference Ranges Wild type occurrence of ALL, and methylation of exon 3 is an indi-
Interpretation cator of poor prognosis.
NPM1 gene is up-regulated, mutated, and chromosomally
translocated in many tumor types. Chromosomal aberrations B.80 P53
involving NPM1 were found in patients with non-Hodgkin Sample Required Bone marrow (EDTA anticoagulated,
lymphoma, acute promyelocytic leukemia, myelodysplastic 2 mL), Peripheral blood (EDTA anticoagulated, 2 mL),
syndrome, and acute myelogenous leukemia. In solid tumors Tissues, Serum, tissue
NPM1 is frequently found overexpressed. Of high impor- Detection Method FISH, Gene Chip, PCR, PCR-RFLP,
tance is NPM involvement in acute myelogenous leukemia, PCR-SSCP
where a mutated protein lacking a folded C-terminal domain Reference Ranges Wild type/negative
(NPM1c+) has been found in the cytoplasm in patients. This Interpretation
aberrant localization has been linked to the development of
the disease and is associated with improved clinical out- 1. Wild type p53 gene is a kind of anti-oncogene whose
comes. In the context of clonal hematopoiesis of undeter- inactivation plays a pivotal role in tumor formation. P53
mined significance harboring a DNMT3A mutation, protein that leads to tumor formation or cell transforma-
subsequent NPM1 mutations drive progression into overt tion is the product of mutated P53 gene.
myeloproliferative neoplasm. 2. P53 expression levels are related to the high proliferation
of cells and the degree of differentiation of tumor cells.
B.78 NSE (Neuron-Specific Enolase) Tumors with lower differentiation and higher degree have
Sample Required Serum higher levels of p53 genes. Moreover, breast cancer
Detection Method RIA, ELISA, CLIA patients with positive p53 protein and negative ER have
Reference Ranges <1.5 μg/L worse prognosis and short survival time.
Interpretation 3. 30–45% of patients with large cell lymphoma were

accompanied by abnormal chromosome 17, including the
1. It is a specific tumor marker of neuroendocrine tumors loss of short arm or whole chromosome 17 of chromo-
including neuroblastoma, medullary carcinoma of the some 17. This abnormality was related to poor prognosis
thyroid, and small cell lung cancer (SCLC). and shortened survival time. P53 gene mutation was
2. Level of NSE in patients with SCLC is significantly
detected in 30 and 25% of DLBCL, and FL patients, and
higher than that in NSCLC which, hence, can be used for the frequency of p53 mutation in BL was as high as
differential diagnosis and is useful for monitoring the 35–45%. Almost all BL are involved in chromosome
therapeutic effect of SCLC after radiotherapy and translocation related to C-MYC gene, so the activation of
chemotherapy. p53 and C-MYC may be very important for the formation
3. NSE can also be chosen to distinguish between neuro- of BL.
blastoma and nephroblastoma. NSE significantly elevated
in the former rather than the latter, the change in which is B.81 PD-1/PD-L1
not obvious. It also has high clinical value in the early Sample Required Tissues
diagnosis, monitoring the state of illness, and prediction Detection Method Immunohistochemistry
recurrence of neuroblastoma. Reference Ranges Negative
Interpretation
B.79 NES1 (Nesfatin 1) PD-L1 is expressed in many types of tumors. At present,
Sample Required Serum, plasma (ELISA); Serum, Tissues, the expression of PD-L1 is the best biomarker of immuno-
Urine (CLIA) suppressive response. The benefits of checkpoint inhibitors
Appendixes 851
are higher in patients with PD-L1 positive tumors than in Interpretation

patients with PD-L1 negative tumors. The expression of PR is based on the expression of ER
and is capable of promoting and synergizing the effect of
B.82 PDGFRA (Platelet-Derived Growth Factor estrogen on ER. It has been proved that breast cancer patients
Receptor Alpha) with simultaneously positive of ER and PR have a better
Sample Required Tissues prognosis. Endocrine therapy for breast cancer such as
Detection Method Gene sequencing, AS-PCR, ARMS tamoxifen and anastrozole is recommended when these two
Reference Ranges Wild type receptors are detected in breast cancer tissues.
Interpretation
The protein encoded by PDGFRA(platelet-derived B.86 PSA (Prostate-Specific Antigen)
growth factor receptor alpha) gene is called platelet-derived Sample Required Serum
growth factor receptor α. PDGFRA can be activated after Detection Method RIA, ELISA, CLIA
binding to its corresponding ligand PDGF, and then the Reference Ranges ≤4 ng/mL
phosphorylation pathway of phosphatidylinositol, cAMP, Interpretation
and many kinds of proteins can be activated to regulate cell
division and proliferation. When the gene activation is abnor- 1. PSA is a valuable tool to evaluate the prognosis, to moni-
mal, it will lead to tumorigenesis and promote tumor angio- tor treatment of patients with prostate cancer. The serum
genesis. The mutation of PDGFRA is closely related to GIST level of PSA is proportional to the clinical stage and the
of gastrointestinal stroma. volume of cancerous tumor of prostatic cancer in
untreated patient. A PSA level > 20 ng/mL highly indi-
B.83 PI3KCA (Phosphatidylinositol-4, 5-Bisphosphate cates of the existence of bone metastases.
3-Kinase Catalytic Subunit Alpha) 2. In addition, several calculated parameters such as PSA-
Sample Required Tissues density (PSAD), PSA-velocity (PSAV), and PSA-
Detection Method Gene sequencing, AS-PCR, ARMS doubling time (PSADT) have been developed for prostate
Reference Ranges Wild type cancer.
Interpretation
The mutation of PI3KCA gene is closely related to the age B.87 RASSF1A (Ras Association Domain Family 1A)
of mammary cancer, the mutation of PI3KCA gene is an inde- Sample Required Serum, Plasma, Bronchoalveolar lavage
pendent risk factor for poor prognosis of mammary cancer. fluid
Detection Method PCR-RFLP, PCR-SSCP, HPLC, Gene
B.84 PML-RARα Fusion Gene sequencing
Detection Method FISH, Mutation Specific-PCR
Reference Ranges Wild type 1. Inactivation of this gene is very common in a variety of
Interpretation cancers, which suggests the tumor suppressor function of
The existence of PML-RARα fusion gene fusion gene is this gene.
very important to the diagnosis, curative effect, and progno- 2. The hypermethylation of CpG island promoter region is
sis of APL. The positive expression of the PML-RARα closely related to the silence of RASSF1A and is consid-
fusion gene indicates a great potential for recurrence, while ered the early and more significant, compared with gene
the persistence of the PML-RARα fusion gene indicates a mutation or deletion, event involved in the occurrence of
longer survival of the patient. The presence of PML-RARα NPC. Other than early diagnosis, this gene is useful for
fusion gene can be used as an index to predict the prognosis detecting invasion, metastasis, and treatment of NPC as
of AML. The gene-positive patients can be used for monitor- well.
ing the curative effect and the detection of the micro-residue.
In addition, the detection of the PML-RARa fusion gene can B.88 RBM15-MKL1 Fusion Gene (RNA-Binding
also provide a molecular level basis for the diagnosis of Motif Protein 15-Megakaryoblastic Leukemia-1)
AML leukemia and a follow-up treatment target point. Sample Required Bone marrow (EDTA anticoagulated,
B.85 PR (Progesterone Receptor) Detection Method Array-CGH, SNP-array, Mutation
Sample Required Tissues, Serum Specific-PCR
Detection Method Immunohistochemical, Western blot Reference Ranges Wild type
Reference Ranges Negative Interpretation
852 Appendixes
AML patients with RBM15-MKL1 fusion gene have low B.92 SPLUNC1 (Short Palate Lung and Nasal
remission rate, high recurrence rate, and poor prognosis after Epithelium Clone 1)
treatment, which can be used as an index to judge the prog- Sample Required Serum, Plasma
nosis of AML. In addition, the detection of DEK-CAN fusion Detection Method ELISA, Western blot,
gene can also provide molecular level basis for the diagnosis Immunohistochemistry
of AML autoimmune disease. Reference Ranges Negative
Interpretation
B.89 ROS-1 (c-Ros Proto-Oncogene 1)
Sample Required Serum, Tissues 1. SPLUNC1 get connection with many human respiratory
Detection Method FISH, Gene Chip diseases during participation in these biological processes.
Reference Ranges Wild type/Negative 2. It plays an intrinsic immune protective role in the very
Interpretation early stage of nasopharyngeal carcinoma (NPC) and can
be considered as a very promising marker for early diag-
1. ROS1 rearrangements are mutually exclusive of ALK nosis, efficacy of treatment, and prognosis of NPC and
rearrangement and EGFR mutation. ROS1 gene rear- NSCLC.
rangement has been shown an incidence of approximately
1% in patients with NSCLCC, making it a distinct molec- B93. SP70 (Tumor Specific Protein 70)
ular subset of NSCLC with a therapeutic target. Sample Required Serum, Tissues, Pleural effusion
2. Crizotinib, used in ALK rearrangement, was also
Detection Method ELISA, Immunohistochemistry,
approved for the treatment of metastatic NSCLC patients Specific circulating lung cancer cell (SCLCC) assay,
with ROS1-positive. Attentions must be paid that there is Immunomagnetic Beads Capture Based Liquid Biopsy
drug resistance in ROS1 + lung cancer due to possible Reference Ranges 0–7.5 ng/mL (ELISA, coat with anti-
mechanisms including kinase domain mutations in ROS1 SP70 polyclonal antibody), 0–4.5 ng/mL (ELISA, coat with
and bypass signaling via RAS and EGFR. anti-SP70 monoclonal antibody)
Interpretation
B.90 RUNX-ETO Fusion Gene
Sample Required Bone marrow (EDTA anticoagulated, 1. SP70 plays an extremely important role in the malignant
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL) proliferation, invasion, and metastasis of tumor cells. It is
Detection Method FISH, SNP-array, RT-PCR a tumor marker for many types of cancer, most notably
Reference Ranges Wild type non-small cell lung cancer (NSCLC).
Interpretation 2. It can be used for dynamic monitoring of NSCLC to aid
The existence of RUNX1-ETO indicates a good progno- in the determination of disease progression or therapeutic
sis. The existence of RUNX 1-ETO fusion gene can be used effect. Recent study suggests that SP70 can also be an
as an index to predict the prognosis of AML, and it also has auxiliary diagnostic reagent for other tumors such as liver
certain guiding significance for the choice of treatment plan. cancer, colorectal cancer, pancreatic cancer, ovarian can-
Patients with positive gene can be monitored and detected cer, breast cancer and thyroid carcinoma, etc.
for minimal residue. In addition, the detection of RUNX1-
ETO fusion gene can also provide molecular level basis for B.94 STMN1 (Stathmin 1)
the diagnosis of AML leukemia. Sample Required Tissues
Detection Method FISH, q-PCR
B.91 SF3B1 (Splicing Factor 3B Subunit 1) Reference Ranges Wild type
Sample Required Bone marrow (EDTA anticoagulated, Interpretation
2mL), Peripheral blood (EDTA anticoagulated, 2 mL), The level of STMN1 gene expression in tumor cells was
Tissues negatively correlated with the efficacy of drugs. In patients
Detection Method Gene sequencing, Mutation-Specific with non-small cell lung cancer who received Cisplatin, in
PCR, FISH Treatment of Untreated, patients with low STMN1 mRNA
Reference Ranges Wild type expression had a better drug response rate. Patients with low
Interpretation gene expression level are more effective in the treatment of
SF3B1 mutations have been shown to be associated with anti-microtubule drugs.
a favorable prognosis and are correlated with the presence of
ring sideroblasts (RS). B.95 Survivin
Sample Required Serum, Plasma
Appendixes 853
Reference Ranges Negative Detection Method Mutation Specific-PCR, Southern

Interpretation Blot, FISH, Karyotypic Analysis
1. The survivin protein is highly expressed in fetal tissue Interpretation
and most human tumors, but is absolutely absent in termi- In the normal and reactive lymphoid tissues, the Bcl-10
nally differentiated human cells. protein is mainly expressed in the cytoplasm of the germi-
2. Survivin functions to prevent cells from suicide (that is, nal center B-cells, whereas in t(1;14) positive MALT
apoptosis or programmed cell death) through inhibition (extranodal marginal zone B-cell lymphoma of mucosa-
of caspase activation. Survivin is enriched in a variety of associated lymphoid tissue), the Bcl-10 is expressed in the
cancer cells, such as lung cancer, colon cancer, pancreatic tumor cell nucleus. About 50% of t(1;14) negative MALT
cancer, prostate cancer, and breast cancer. Survivin level lymphomas also showed the nuclear expression of Bcl-10,
is proportional to progress of disease and histological but the intensity of Bcl-10 expression was lower than that
stage. These data suggest survivin might act as a bio- of t(1;14) positive cases, and the nuclear expression of Bcl-
marker for bad prognosis of cancer. 10 was significantly correlated with the clinical stage and
t(11;18) of t(11;18). In addition, Bcl-10 was highly and
B.96 t(11;18)(q21;q21)/API2-MALT1 selectively expressed in the B nucleus of spleen marginal
Sample Required Bone marrow (EDTA anticoagulated, zone of Bcl-10 transgenic mice. Under normal circum-
2 mL), Peripheral blood (EDTA anticoagulated, 2 mL), stances, Bcl-10 is mainly expressed in cytoplasm and is a
Tissues necessary factor for the maturation and function of T and
Detection Method Mutation Specific-PCR, Southern B-cells. In t(1;14) positive cells, Bcl-10 was strongly
Blot, FISH, Karyotypic Analysis expressed in the nucleus. Most of t(1;14) translocation pos-
Reference Ranges Wild type itive MALT lymphomas were advanced and insensitive to
Interpretation Hp therapy.
1. t(11;18) has a specific correlation with the MALT (extra- B.98 t(14;18)(q32;q21)/IgH-MALT1

nodal marginal zone B-cell lymphoma of mucosa- Sample Required Bone marrow (EDTA anticoagulated,
associated lymphoid tissue) lymphoma and is closely 2 mL), Peripheral blood (EDTA anticoagulated, 2 mL),
related to the MALT lymphoma, and no detection of Tissues
B-cell lymphoma and other non-Hodgkin lymphoma in Detection Method Mutation Specific-PCR, Southern
the splenic margin. In MALT lymphoma, the incidence of Blot, FISH, Karyotypic Analysis
chromosome translocation varies with the location of Reference Ranges Wild type
tumorigenesis. The results of foreign studies showed that Interpretation
t(11;18) had the highest incidence in lung and stomach 66 cases of MALT (extranodal marginal zone B-cell lym-
(38% and 24%, respectively), followed by conjunctiva phoma of mucosa-associated lymphoid tissue) lymphoma
and orbit (19% and 14%, respectively), but lacked this were analyzed and the liver (4/4), the skin (3/11), the eye-
chromosome translocation in thyroid, skin, liver, and attached (3/8), and the salivary glands (2/11) were found.
other rare parts of MALT lymphoma. There are also dif- There is a translocation of this chromosome in the MALT
ferences in the occurrence of this chromosome transloca- lymphoma. In the case of the MALT lymphoma of the gas-
tion in MALT lymphoma in China. trointestinal tract (19), the lung (7), the thyroid (4), and the
2. These results suggest that the occurrence of t(11;18) may breast (2), the presence of the chromosomal translocation is
be affected by pretumor diseases related to MALT lym- not seen.
phoma. It was found that, t(11;18)(q21;q21) positive
cases did not respond to HP eradication therapy in 111 B.99 t(3;14)(p14;q32)/IgH-FOXP1
cases of gastric MALT lymphoma treated with HP radical Sample Required Bone marrow (EDTA anticoagulated,
therapy, which may be due to the fact that this chromo- 2mL), Peripheral blood (EDTA anticoagulated, 2 mL),
some translocation has the survival advantage of tumor tissue
cells and makes its growth no longer depend on HP medi- Detection Method Mutation Specific-PCR, Southern
ated immune stimulation. Blot, FISH, Karyotypic Analysis
B.97 t(1;14)(p22;q32)/Bcl-10-IgH Interpretation
Sample Required Bone marrow (EDTA anticoagulated, The results of 91 cases of MALT (extranodal marginal
2 mL), Peripheral blood (EDTA anticoagulated, 2 mL), zone B-cell lymphoma of mucosa-associated lymphoid tis-
Tissues sue) lymphoma showed that the incidence was about 10%.
854 Appendixes
Among them, the incidence of thyroid MALT lymphoma a constant region (C region). The major difference of the
was the highest, followed by ocular adnexal MALT lym- amino acids in the V region of the TCR is shown in the
phoma and skin MALT lymphoma, but no chromosome three complementarity determining regions (CDR) and
translocation was found in stomach, saliva gland, lung the fourth high-transformation region (HV4). The three
MALT lymphoma, spleen and lymph node marginal zone CDR and HV4 regions collectively determine the specific
cell lymphoma. antigen recognition of the TCR. According to the compo-
sition of heterodimer, TCR can be divided into αβ type
B.100 t(15;17)(q22;q21)/PML-RARA and γδ type. The antigen receptor on most T cells is αβ
Sample Required Bone marrow (EDTA anticoagulated, type TCR (95%), and only a few of them are divided into
2 mL), Peripheral blood (EDTA anticoagulated, 2 mL), γδ type (5%).
Tissues 2. Because of the diversity of gene rearrangement, the rear-
Detection Method PCR, RT-PCR, Southern Blot, FISH, ranged TCR gene junction sequence is different in each
Karyotypic Analysis lymphocytes, so each patient has its own specific TCR
Reference Ranges Wild type gene rearrangement. This specific TCR gene rearrange-
Interpretation ment sequence can be used as a molecular marker of
At the molecular level, as a result of the t(15;17) translo- malignant cloning and can assist in the diagnosis and
cation, the gene for retinoic acid receptor alpha (RARA) on tracking of residual tumor cells by PCR detection of TCR
17q21 fuses with a transcription factor gene (promyelocytic gene rearrangement.
leukemia or PML) on 15q22, giving rise to a PML/RARA 3. The detection of TCR gene rearrangement in patients
gene fusion product [2]. This PML/RARA fusion gene tran- with T cell lymphoma can provide objective basis for the
script is known to play a pivotal role in the pathogenesis of intensity and duration of clinical chemotherapy, early pre-
APL and the sensitivity to all-trans retinoic acid (ATRA) [3]. diction of recurrence and guidance of bone marrow trans-
Approximately 70–80% of patients with newly diagnosed plantation. TCR gene rearrangement can also judge the
APL carrying PML/RARA achieve long-term remission; source of recurrent cloning, and its clinical application
however, some patients still have a poor outcome. value is very high.
B.101 t(16;16)(p13.1;q22)/CBFB-MYH11 B.103 TET2 (Tet Methylcytosine Dioxygenase 2)

Sample Required Bone marrow (EDTA anticoagulated, Sample Required Bone marrow (EDTA anticoagulated),
2 mL), Peripheral blood (EDTA anticoagulated, 2 ml), Peripheral blood (EDTA anticoagulated)
tissue Detection Method PCR, RT-PCR, Gene sequencing
Detection Method PCR, RT-PCR, Southern Blot, FISH, Reference Ranges Wild type
Karyotypic Analysis Interpretation
Reference Ranges Wild type There are several types of TET2 gene mutations, such as
Interpretation deletion, insertion, transcoding mutation, nonsense mutation,
t(16:16) is a rare chromosomal abnormality in AML and missense mutation, and passivation. This change often leads
constitutes 10% of patients with core binding factor leuke- to truncated translation so that the potential tumor inhibitor
mias and less than 1% of all cases of AML evaluated at our TET2 cannot be fully expressed, thus promoting the occur-
center. Presence of t is associated with an excellent prognosis rence of tumor. This mutation is more common in patients
despite presence of additional cytogenetic or molecular with myeloproliferative tumors. The mutation rate of TET2
alterations if treated with a high-dose Ara-C containing gene is 10–15% in primary myelofibrosis (PMF), 5% in
program. essential thrombocythemia (ET), and 14% in polycythemia
vera (PV). The detection of TET2 gene mutation is helpful to
B.102 TCR Rearrangement assist in the diagnosis of myeloproliferative neoplasms.
Sample Required Bone marrow (EDTA anticoagulated,
2 mL), Peripheral blood (EDTA anticoagulated, 2 mL), B.104 TEL-PDGFR-β Fusion Gene
tissue Sample Required Bone marrow (EDTA anticoagulated,
Detection Method PCR, RT-PCR, Southern Blot, FISH 2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL)
Reference Ranges Negative Detection Method FISH, RT-PCR
Interpretation Reference Ranges Wild type
Interpretation
1. The TCR is a specific surface marker for T cells. The The detection of TEL-PDGFR-β fusion gene can provide
TCR has 4 subunits, α, β, γ, δ of 4 peptide chains. Each molecular basis for the diagnosis of AML leukemia. In addi-
peptide chain consists of a variable region (V region) and tion, the prognosis of TEL-PDGFR-β positive patients is
Appendixes 855
poor, so the existence of this fusion gene can be used as a B.108 TPS (Tissue Polypeptide Specific Antigen)
judgment of CML. So the existence of this fusion gene can Sample Required Serum
be used as an indicator of prognosis of CML. Moreover, Detection Method ELISA, RIA
patients with TEL-PDGFR-β fusion gene can be treated with Reference Ranges <120 U/L
PDGFR inhibitors and chemotherapeutic drugs under the Interpretation
guidance of doctors. Serum concentration of TPS is a more specific indicator
to evaluate the degree of tumor cell division and proliferation
B.105 TEL-RUNX1 Fusion Gene activity, which is conducive to the early diagnosis, treatment,
Sample Required Bone marrow (EDTA anticoagulated, disease monitoring, and prognosis of cancer.
Detection Method FISH, RT-PCR B.109 TPMT (Thiopurine S-Methyltransferase)
Reference Ranges Wild type Sample Required Tissues, Peripheral blood (EDTA antico-
Interpretation agulated, 2–3 mL)
TEL-RUNX1 fusion gene can provide molecular basis for Detection Method Immunohistochemistry, FISH,
the diagnosis of ALL leukemia, in addition, the prognosis of q-PCR
TEL-RUNX1 positive patients is better, so the existence of Reference Ranges Wild type
this fusion gene can be used as an index to judge the progno- Interpretation
sis of ALL. Finally, because the inhibitory effect mediated TPMT gene is located in the 6p22.3 position of human
by TEL-RUNX1 can be alleviated by HDAC blockers, chromosome, with a total length of 26832bP. There are 11
patients with this fusion gene can be treated with chemo- exons. mRNA is 3258nt long and encodes a protein com-
therapeutic drugs and HDAC blockers under the guidance of posed of 245 amino acid residues. Studies have shown that
doctors. the genotype of TPMT is closely related to the toxic and side
effects of purine drugs. Gene polymorphism leads to a
B.106 The Loss of Heterozygosity of Chromosome decrease in enzyme activity, which increases the risk of toxic
1p, 19q and side effects of purine drugs.
Sample Required Tissues
Detection Method FISH, aCGH, LOH, MPLA B.110 Trisomy 12
Reference Ranges Wild type Sample Required Bone marrow (EDTA anticoagulated,
Interpretation 2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL)
The loss of heterozygosity of chromosome 1p, 19q mainly Detection Method FISH, RT-PCR
occurred in WHO II oligodendroglioma, astrocytoma, Reference Ranges Wild type
VWHO III anaplastic oligogliomas, but almost impossible to Interpretation
detect in other type glioma. The loss of heterozygosity of The detection of 12 chromosome trisomy can provide
chromosome 1p, 19q is an independent and significant prog- molecular level basis for the diagnosis of B-CLL autistic
nostic factor. disease. In addition, the reaction of 12 chromosome trisomy
positive patients to chemotherapy is obviously poor.
B.107 TPA (Tissue Polypeptide Antigen) Therefore, 12 chromosome trisomy can be used as an inde-
Sample Required Serum pendent index of B-CLL further classification, which has
Detection Method ELISA, RIA important clinical significance for the prognosis of B-CLL.
Reference Ranges <80 μg/L
Interpretation B.111 TSLC1 (Tumor Suppressor in Lung Cancer 1)
Sample Required Serum, Tissues
1. Serum TPA can be detected in almost 70% of patients with Detection Method FISH, Gene Chip
malignant tumors, but it will decrease after treatment. An Reference Ranges Wild type/negative
increase of TPA later indicates a recurrence of tumors. Interpretation
2. Clinically, it is used to assist the diagnosis of malignant
tumors with rapid proliferation and to monitor the treat- 1. It has been reported that the prognosis of patients with
ment efficacy of tumors. negative expression of TSLC1 in lung adenomas is poorer,
3. TPA is well known to differentiate cholangiocarcinoma which is worse in males than in females.
(TPA elevated) from hepatocellular carcinoma (TPA not 2. TSLC1 deletion, more common in patients with clinically
elevated). advanced meningiomas, results in a significant decrease
4. TPA may rise in patients with lung cancer, acute hepatitis, in survival rate. 50% of patients with esophageal cancer
pancreatitis, or pneumonia. had decreased expression of TSLC1 which is closely
856 Appendixes
related to grade of lesions, tumor invasion and metastasis, B.115 VEGF (Vascular Endothelial Growth Factor)
and cell mobility. The lack of TSLC1 expression is an Sample Required Serum, Plasma
important factor in tumor growth and invasion. Detection Method ELISA
Reference Ranges 0–160 ng/mL
B.112 TSP-1 (Thrombospodin-1) Interpretation
Detection Method ELISA, CLIA 1. Vascular endothelial growth factor (VEGF) is the most
Reference Ranges ELISA (180 ng/mL) direct angiogenic active protein and is a vascular endothe-
Interpretation lial cell-specific cytokinin and angiogenic factor which
can bind to endothelial cell tyrosine kinase receptor and
1. TSP-1 is capable of resisting angiogenesis, inducing the KDR factor.
apoptosis of vascular endothelial cells. It can reduce the 2. VEGF is involved in several biological processes, such as
vascular density by acting on the CD36 receptor on the survival, metastasis or further differentiation. Hence,
vascular endothelial cells, leading to the necrosis of the VEGF is a promising target for the treatment of cancer. A
tumor cells. monoclonal antibody named bevacizumab is a VEGF tar-
2. TSP-1 can also regulate the growth of the tumor cells and geted inhibitor which can inhibit the biological activity of
induce the apoptosis of the cells through the TGF-1- human VEGF, as a result promotes endothelial cell mito-
dependent mechanism but inhibits angiogenesis through genic activity, increase vascular permeability and angio-
TGF-1- independent way. It has been found that exoge- genesis activity, so as to achieve the anti-tumor effect.
nous gene TSP-1 could prevent the metastasis of dormant Bevacizumab was the first anti-VEGF drug approved in
microtumor after radiotherapy, and it could also inhibit 2004 and was usually combined with standard chemo-
the metastasis of tumor by inducing apoptosis of tumor- therapy regimen in clinic. Around 10–15% of patients
associated microvascular endothelial cells. benefit from bevacizumab therapy; however, biomarkers
for bevacizumab efficacy are still underwent observation.
B.113 TYMS (Thymidylate Synthetase)
Sample Required Tissue B.116 XRCC1 (X-Ray Repair Cross Complementing
Detection Method Immunohistochemistry, FISH, q-PCR Group 1)
Reference Ranges Wild type Sample Required Tissues
Interpretation Detection Method FISH, q-PCR
TYMS gene polymorphisms such as 3R alleles lead to Reference Ranges Wild type
elevated TS expression levels, promote over-proliferation of Interpretation
cells, avoid aging and apoptosis, and reduce chemotherapy
with 5-FU efficacy. 1. XRCC1 is X-ray repair complementing defective repair
in Chinese hamster cells 1 X-ray repair cross comple-
B.114 uPA (Urokinase-Type Plasminogen Activator) menting group 1). The protein encoded by XRCC1 can
Sample Required Serum effectively repair DNA single strand breaks caused by
Detection Method Chromogenic substrate, ELISA ionizing radiation and alkylates. XRCC1 encodes pro-
Reference Ranges teins interacting with DNA ligase III, polymerase β and
Interpretation Poly (ADP-ribose) polymerase, which is involved in the
base excision repair pathway of DNA. It has been con-
1. Urokinase-type plasminogen activator (uPA), also known firmed that lead exposure can induce oxidative stress to
as urokinase, is a serine protease present in humans and cause DNA damage, so XRCC1, as an important DNA
other animals. The synthesis and secretion of uPA are damage repair gene, is related to a variety of tumors and
influenced by many factors, such as proto-oncogene lead poisoning. The polymorphism of some sites on
V-Src, Ras, and cytokines including TNF, INF, IL-1, XRCC 1 gene is closely related to cancer.
TGF-β, IGF-1, FGF and MMP, etc. 2. Current research has shown that the content of malondial-
2. Elevated expression level of uPA is found to be correlated dehyde (MDA) and superoxide dismutase (SOD) activity
with tumor malignancy. uPA stimulates cancer cells to pro- in lead workers increases with the increase of lead content
duce plasminogen which is further catalyzed by uPA to and increases with the increase of MDA level of DNA
activated plasmin. Plasmin is capable of degrading a large damage, which indicates that lead contact can induce oxi-
amount of extracellular matrix proteins and then facilitates dative stress and lead to DNA damage. XRCC 1, as an
tissue invasion and, thus, contributes to metastasis. This important DNA damage repair gene, is associated with
makes uPA a promising drug target, and, hence, inhibitors lead poisoning.
have been sought to be used as anticancer agents.
Appendixes 857
B.117 13q14 Deletion Detection Method Next-generation sequencing (NGS)/

Sample Required Bone marrow (EDTA anticoagulated, Massively parallel sequencing (MPS)
2–3 mL), Peripheral blood (EDTA anticoagulated, 2–3 mL) Sample Required Blood
Detection Method FISH, Mutation Specific-PCR Reference Ranges Negative
Reference Ranges Wild type Interpretation
Interpretation
Detection of 13q14 deletion can provide molecular basis 1. Mutations in MCCC1 and MCCC2 cause
for the diagnosis of CLL leukemia. In addition, the prognosis 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC
of CLL patients with 13q14 deletion is better, so the exis- deficiency).
tence of fusion gene can be used as an index to predict the 2. This condition is inherited in an autosomal recessive

prognosis of CLL. pattern.
3. Affected infants appear normal at birth but usually exhibit
characteristics in infancy. Signs and symptoms can range
Appendix C: Tests of Genetic Disease from mild to life-threatening, including difficulty feeding,
recurrent vomiting and diarrhea, lethargy, and hypotonia.
Human genetic diseases are diseases or defects caused by Without treatment, this disorder can cause developmental
changes in genetic material and are transmitted vertically in delays, seizures, and coma.
a certain way. According to the latest data, the birth popula-
tion is about 20 million annually in China and it is estimated C.3 ACAT1 Sequence Analysis (Prenatal Diagnosis)
that 6% of births have different genetic diseases. About 1.2 Genes ACAT1 (11q22.3)
million children with genetic diseases are added every year. Sample Required Blood
As epidemics and dystrophic diseases are basically con- Detection Method Bi-directional Sanger Sequence
trolled, the spectrum of diseases has changed, and the inci- Analysis
dence of hereditary diseases has gradually increased, which Reference Ranges Negative
has attracted people’s attention. Interpretation
According to the difference of genetic material, genetic
diseases are mainly divided into three categories: genetic 1. Mutations in the ACAT1 gene can cause beta-ketothiolase
hereditary diseases, chromosomal genetic diseases, and deficiency.
somatic hereditary diseases. Among them, genetic hereditary 2. This condition is inherited in an autosomal recessive

diseases are divided into single gene hereditary diseases and pattern.
polygenic hereditary diseases according to hereditary meth- 3. Affected children may experience ketoacidotic attacks,
ods. This chapter focuses on the molecular genetic diagnosis caused by infections, fasting, or increased consumption of
of genetic diseases. protein-rich foods. The signs and symptoms include vom-
iting, dehydration, breathing difficulties, extreme fatigue,
C.1 2-Methylbutyrylglycinuria and occasionally seizures. These episodes sometimes lead
Gene ACADSB (10q26.13) to a coma.
Sample Required Blood
Detection Method CNV by q-PCR, Bi-directional Sanger C.4 Acrocallosal Syndrome
Sequence Analysis Gene KIF7 (15q26.1)
Reference Ranges Negative Sample Required Blood
Interpretation Detection Method PCR with allele specific hybridiza-
tion, Bi-directional Sanger Sequence Analysis
1. Mutations in ACADSB have been found to cause Reference Ranges Negative
2-Methylbutyryl-CoA dehydrogenase (MBD) deficiency. Interpretation
2. MBD deficiency is an autosomal recessive metabolic dis-
order of impaired isoleucine degradation. 1. Mutations in the KIF7 gene have been found to cause
3. This condition is usually clinically asymptomatic which acrocallosal syndrome.
is often identified by neonatal screening. It is reported that 2. This condition is inherited in an autosomal recessive

some patients may have delayed development and neuro- pattern.
logic signs. 3. The signs and symptoms include agenesis of the corpus
callosum, polydactyly, as well as unique facial features,
C.2 3-Methylcrotonyl-CoA Carboxylase (3-MCC) such as hypertelorism and a high, prominent forehead,
Deficiency (3-MCC) and cephalomegaly.
Genes MCCC1 (3q27.1), MCCC2 (5q13.2)
858 Appendixes
C.5 Adenosine Deaminase Deficiency 1. Mutations in the MAN2B1 gene can cause alpha-

Gene ADA (20q13.12) mannosidase. In the European population, 27% of patho-
Sample Required Blood genic alleles variant is c.2248C>T (p.Arg750Trp).
Detection Method NGS/MPS 2. Alpha-mannosidosis is inherited in an autosomal reces-
Reference Ranges Negative sive manner.
Interpretation 3. Signs and symptoms of alpha-mannosidosis disease can
range from mild to severe. Patients may experience intel-
1. Mutations in the ADA gene have been found to cause ade- lectual disabilities, unique facial features, and skeletal
nosine deaminase (ADA) deficiency. Among more than abnormalities. Affected individuals may also have corneal
70 ADA pathogenic variants which have been identified opacities, hearing loss, speech impairments, ataxia,
in people with ADA deficiency, approximately 60% is myopathy, delay in developing motor skills, immunodefi-
missense, 20% is splicing, 9% is small deletion, 7% is ciency, hepatosplenomegaly, and hydrocephalus.
nonsense, and 3% is deletion of one or multiple exons.
2. ADA deficiency is inherited in an autosomal recessive C.8 Alpha Thalassemia (HBA1/HBA2)
manner. Genes HBA1 (16p13.3), HBA2 (16p13.3)
3. This is a systemic purine metabolic disorder, which
mainly affects the development, viability, and function of Detection Method Multiplex Ligation-dependent Probe
lymphocytes. The main symptoms include pneumonia, Amplification (MLPA), Bi-directional Sanger Sequence
chronic diarrhea, and a widespread rash. Analysis, GAP-PCR
C.6 Alkaptonuria (HGD Single Gene Test) Interpretation
Gene HGD (3q13.33)
Sample Required Blood 1. Deletion of HBA1 and/or HBA2 is the most common
Detection Method NGS/MPS cause of α-thalassemia.
Reference Ranges Negative 2. The α-thalassemia is inherited in an autosomal recessive
Interpretation manner which has two clinically significant forms.
3. Hb Bart syndrome, due to the loss of all four alpha-globin
1. Mutations in the HGD gene can cause alkaptonuria. alleles, is the most severe form of alpha-thalassemia. It is
Missense accounts for the majority of pathogenic variants; characterized by fetal systemic edema, pleural and peri-
nonsense, frame shift, and splice site variants also occur. cardial effusions, and severe hypochromic anemia, with-
c.1102A>G (p.Met368Val) is the most prevalent patho- out ABO or Rh incompatibility.
genic variant in Europe. A founder variant, p.Cys120Trp, 4. HbH disease is caused by the loss of three of the four
can be seen in the Dominican Republic. There is evidence alpha-globin alleles, characterized by microcytic hypo-
of mutational hotspots (e.g., c.342+1G>A) and a founder chromic anemia, splenomegaly, and mild jaundice.
effect (e.g., the frequent pathogenic variant p.Gly161Arg) Sometimes affected individuals may have thalassemia-
in the Slovak population. like bone changes.
2. This condition is inherited in an autosomal recessive

pattern. C.9 Alstrom Syndrome
3. It is a genetic disorder that causes urine to turn black Gene ALMS1 (2p13.1)
when exposed to air. The main characteristics of alkap- Sample Required Blood
tonuria are the presence of homogentisic acid in the urine, Detection Method NGS/MPS
ochronosis, and arthritis in early adulthood, especially in Reference Ranges Negative
the spine and large joints. Other features include blue- Interpretation
black pigmentation, heart problems, and so on.
1. So far, more than 200 ALMS1 pathogenic variants in the
C.7 Alpha-Mannosidosis ALMS1 gene have been identified to cause Alstrom syn-
Gene MAN2B1 (19p13.13) drome. Almost all are nonsense or frameshift variants
Sample Required Blood which are associated with complete lack of protein
Detection Method NGS/MPS expression.
Reference Ranges Negative 2. This condition is inherited in an autosomal recessive

Interpretation pattern.
3. Typical signs and symptoms include obesity, progressive
loss of vision and hearing, acute infantile-onset cardio-
myopathy and/or adolescent- or adult-onset restrictive
Appendixes 859
cardiomyopathy, and type 2 diabetes mellitus. This disor- are missense, nonsense, or frameshift variants.
der can also cause non-alcoholic fatty liver disease, β-thalassemia is rarely caused by a genetic deletion.
chronic progressive kidney disease, and acanthosis 2. The β-thalassemia is inherited in an autosomal recessive
nigricans. manner.
3. It is characterized by microcytic hypochromic anemia.
C.10 Apparent Mineralocorticoid Excess Individuals with major condition suffer from severe ane-
Genes HSD11B2 (16q22.1) mia and hepatosplenomegaly and usually come to hospi-
Sample Required Blood tal within the first 2 years of life.
Detection Method PCR with allele specific hybridiza-
tion, Bi-directional Sanger Sequence Analysis C.13 Bloom Syndrome
Reference Ranges Negative Gene BLM (15q26.1)
Interpretation Sample Required Blood
1. Mutations in the HSD11B2 gene can cause apparent min- tion, Bi-directional Sanger Sequence Analysis
eralocorticoid excess (AME). Reference Ranges Negative
Interpretation
pattern.
3. It is a form of low-renin hypertension, characterized by 1. Mutations in the BLM gene cause bloom syndrome

low aldosterone, metabolic alkalosis, hypernatremia, and (BSyn). Identified pathogenic variants mainly include:
hypokalemia. nucleotide insertions and deletions (33%), nonsense vari-
ants (33%), intron variants (16.7%), and missense vari-
C.11 ARVC Panel ants (16.7%).
Genes DSC2 (18q12.1), DSG2 (18q12.1), DSP (6p24.3), 2. This condition is inherited in an autosomal recessive

JUP (17q21.2), PKP2 pattern.
(12p11.21), RYR2 (1q43) 3. The signs and symptoms of BSyn include severe antepar-
Sample Required Blood tum and postnatal growth defects, immune abnormalities,
Detection Method NGS/MPS sensitivity to sunlight, and insulin resistance. Patients
Reference Ranges Negative have a high risk of developing many tumors that occur at
Interpretation an early age.
1. Mutations in several genes can cause arrhythmogenic

C.14 Brugada Syndrome panel
right ventricular cardiomyopathy (ARVC). Similar to that Genes CACNA1C (12p13.33), CACNB2 (10p12.33-12.31),
in foreign countries, the most common disease-causing GPD1L (3p22.3), HCN4 (15q24.1), KCNE3 (11q13.4),
genes in Chinese population is PKP2 gene (42%), fol- SCN1B (19q13.11), SCN3B (11q24.1), SCN5A (3p22.2),
lowed by DSG2 (11%), DSP (6%), and DSC2 (3%). SLMAP (3p14.3)
2. Most familial cases of the disease have an autosomal Sample Required Blood
dominant pattern of inheritance. Rarely, ARVC has an Detection Method PCR with allele specific hybridiza-
autosomal recessive pattern of inheritance. tion, Bi-directional Sanger Sequence Analysis, NGS/MPS
3. This condition causes progressive fibrofatty replacement Reference Ranges Negative
of the myocardium, increasing the risk of arrhythmia and Interpretation
sudden death, especially during strenuous exercise. The
common symptoms include palpitations, dizziness, and 1. Mutations in several genes can cause Brugada syndrome.
syncope. The most commonly mutated gene is SCN5A (30%).
Pathogenic variants in SCN5A include p.Arg282His,
C.12 Beta-Thalassemia p.Thr512Ile, p.Arg1232Trp, and p.Thr1620Met.
Gene HBB (11p15.4) 2. This condition is inherited in an autosomal dominant

Sample Required Blood pattern.
Detection Method MLPA, Bi-directional Sanger 3. Typical characteristics include cardiac conduction abnor-
Sequence Analysis malities which can lead to sudden death. Other manifesta-
Reference Ranges Negative tions may include sudden infant death syndrome, the
Interpretation sudden unexpected nocturnal death syndrome, and other
conduction defects (intraventricular conduction delay,
1. Nearly 300 alleles in the HBB gene which can cause sick sinus syndrome, right bundle branch block, and so
β-thalassemia have been characterized. The vast majority on).
860 Appendixes
C.15 Catecholaminergic Polymorphic Ventricular 1. Mutations in the HMGCL gene cause 3-Hydroxy-3-

Tachycardia: Full Panel (CPVT Full) methylglutaryl-CoA lyase (HMG-CoA lyase) deficiency.
Genes CASQ2 (1p13.1), RYR2 (1q43) 2. This is a rare autosomal recessive disorder.
Sample Required Blood 3. The main characteristics of this condition include meta-
Detection Method NGS/MPS bolic acidosis without ketonuria, hypoglycemia, and ele-
Reference Ranges Negative vated organic acid metabolites in urine. Other
Interpretation manifestations are dicarboxyuria, hepatomegaly, hyper-
ammonemia. Affected individuals may also experience
1. Mutations in RYR2 and CASQ2 genes can cause catechol- lethargy, coma, irritability, and vomiting.
aminergic polymorphic ventricular tachycardia (CPVT).
RYR2 gene mutations cause about 65% cases, while C.18 Cohen Syndrome
mutations in the CASQ2 gene account for 3–5% cases. Gene VPS13B (8q22.2)
2. When CPVT results from mutations in the RYR2 gene, it Sample Required Blood
has an autosomal dominant pattern of inheritance. When Detection Method MLPA, Bi-directional Sanger
CPVT is caused by mutations in the CASQ2 gene, the Sequence Analysis
condition has an autosomal recessive pattern of inheri- Reference Ranges Negative
tance with the exception of p.Lys180Arg, which is associ- Interpretation
ated with a dominant CPVT phenotype.
3. This condition is characterized by arrhythmia (ventricular 1. More than 100 novel pathogenic variants in VPS13B have
tachycardia) which typically begin in childhood. Affected been subsequently identified which can cause Cohen syn-
children can experience light-headedness, dizziness, and drome. In the Finnish population, the most common
syncope. pathogenic variant is c.3348_3349delCT (75%).
2. Cohen syndrome is inherited in an autosomal recessive
C.16 Citrullinemia Types 1 and 2: Gene Sequencing pattern.
Panel (RAPID Testing) 3. The signs and symptoms include stunted growth, trunk
Genes ASS1 (9q34.11), SLC25A13 (7q21.3) obesity, cheerful disposition, acquired microcephaly,
Detection Method NGS/MPS unique facial features, progressive choroid dystrophy and
Sample Required Blood myopia, hypotonia, arthrochalasis, and neutropenia.
Interpretation C.19 Congenital Adrenal Hyperplasia NGS and
Deletion/Duplication Panel
1. Mutations in the ASS1 gene are responsible for type I Genes CYP11B1 (8q24.3), CYP21A2 (6p21.33)
citrullinemia (CTLN1). The most prevalent pathogenic Sample Required Blood
variant is p.Gly390Arg (in exon 15). Mutations in the Detection Method Microarray, NGS/MPS
SLC25A13 gene cause adult-onset type II citrullinemia Reference Ranges Negative
(CTLN2). Interpretation
2. Citrullinemia is inherited in an autosomal recessive

manner. 1. Mutations in the CYP11B1 and CYP21A2 gene can cause
3. CTLN1 includes acute neonatal form, milder late-onset congenital adrenal hyperplasia. Among them, mutations
form, asymptomatic or hyperammonemia form, and a of the CYP11B1 gene results in 11-β-hydroxylase defi-
form in which females have severe symptoms during or ciency, and mutations of the CYP17A1 gene results in
after pregnancy. 17-α-hydroxylase deficiency.
4. CTLN2 mainly causes neurological symptoms, including 2. This condition is inherited in an autosomal recessive

irritability, restlessness, memory loss, aggression, hyper- pattern.
activity, convulsive seizures, and coma. 3. It is characterized by defective synthesis of cortisol and/or
aldosterone, leading to adrenal cortical cell proliferation.
C.17 CoA-3-Hydroxy-3-Methylglutaryl Lyase 11-β-hydroxylase deficiency and 17-α-hydroxylase defi-
Deficiency ciency can lead to a compensatory increase in adrenocorti-
Gene HMGCL (1p36.11) cotropin, which leads to adrenal hyperplasia. 11-β
Detection Method PCR with allele specific hybridiza- hydroxylase deficiency can lead to masculinization in
tion, Bi-directional Sanger Sequence Analysis females due to excess androgen. 17-α hydroxylase defi-
Sample Required Blood ciency can cause sexual hormone synthesis disorder, result-
Reference Ranges Negative ing in delayed puberty, primary amenorrhea, and so on.
Interpretation
Appendixes 861
C.20 Crigler–Najjar Syndrome zures shortly after birth, almost always in the first few
Gene UGT1A1 (2q37.1) months of life. They exhibit visual and hearing impair-
Sample Required Blood ment and have severe intellectual disabilities. Patients
Detection Method Bi-directional Sanger Sequence may have unusual facial features.
Analysis
Reference Ranges Negative C.23 Dilated Cardiomyopathy (DCM) Panel
Interpretation Genes MYH7 (14q11.2), MYBPC3 (11p11.2), TNNT2
(1q32.1), DSP (6p24.3), TTN (2q31.2), LMNA (1q22), MYH6
1. Mutations in the UGT1A1 gene cause Crigler–Najjar
(14q11.2), MYPN (10q21.3), RBM20 (10q25.2), SCN5A
syndrome. (3p22.2), ANKRD1 (10q23.31), RAF1 (3p25.2), DES (2q35),
2. CF is inherited in an autosomal recessive pattern. DMD (Xp21.2-21.1)
3. It is characterized by hyperbilirubinemia, leading to yellow- Sample Required Blood
ing of the skin and whites of the eyes, apparently at birth or in Detection Method NGS/MPS
infancy. Severe unconjugated hyperbilirubinemia can cause Reference Ranges Negative
kernicterus. There are two types of Crigler–Najjar syndrome. Interpretation
Type 1 (CN1) is much more severe than Type 2 (CN2).
1. Mutations in more than 30 genes have been found to
C.21 Cystic Fibrosis (CF) cause familial dilated cardiomyopathy (DCM). Mutations
Gene CFTR (7q31.2) in one gene, TTN, account for approximately 20% cases
Sample Required Blood of DCM.
Detection Method PCR with allele specific 2. In 80–90% cases, DCM is inherited in an autosomal dom-
hybridization inant pattern. In rare instances, this condition is inherited
Reference Ranges Negative in an autosomal recessive pattern or X-linked pattern.
Interpretation 3. Symptoms of DCM typically begin in mid-adulthood, but
can occur at any time from infancy to late adulthood.
1. More than 1000 variants in the CFTR gene have been Characteristics of DCM can include arrhythmia, dyspnea,
reported to cause cystic fibrosis (CF), almost all of which fatigue, syncope, and swelling of the legs and feet. Sudden
are missense variants or small deletions (1–84 bp). cardiac death occurs first in some cases.
p.Phe508del is the most common pathogenic variant,
accounting for 30–80% of pathogenic variants. C.24 Donnai–Barrow Syndrome
2. CF is inherited in an autosomal recessive pattern. Gene LRP2 (2q31.1)
3. This is a multisystem disorder that affects the epithelial Detection Method NGS/MPS
cells of the respiratory tract, exocrine pancreas, intestinal Sample Required Blood
tract, hepatobiliary system, and exocrine sweat glands. Reference Ranges Negative
Signs and symptoms include pulmonary disease (e.g., Interpretation
progressive obstructive lung disease with bronchiectasis),
pancreatic dysfunction and malnutrition, recurrent sinus- 1. Mutations in the LRP2 gene cause Donnai–Barrow syn-
itis and bronchitis, and male infertility. Pulmonary dis- drome (DBS). The homozygous or compound heterozy-
ease is the leading cause of CF morbidity and mortality. gous LRP2 variants include small deletions or insertions
and conserved splice site, nonsense, and missense vari-
C.22 D-Bifunctional Protein Deficiency ants inherited from each heterozygous carrier parent.
Gene HSD17B4 (5q23.1) 2. This condition is inherited in an autosomal recessive

Detection Method NGS/MPS pattern.
Sample Required Blood 3. The main clinical features of this disease are corpus cal-
Reference Ranges Negative losum dysplasia, giant cranium, large anterior fontanium,
Interpretation large forehead, wide distance from the eyes, high myopia,
severe neurological deafness, congenital diaphragmatic
1. Mutations in the HSD17B4 gene cause D-bifunctional hernia, omphalocele/umbilical hernia, low molecular
protein deficiency. weight proteinuria.

manner. C.25 Ehlers-Danlos syndrome Comprehensive Panel:
3. This disorder causes severe biochemical abnormalities Dominant & Recessive
and is usually fatal in the first 2 years of life. Affected Genes ADAMTS2 (5q35.3), ATP7A (Xq21.1), B3GALT6
babies are born with poor muscle tone. Most develop sei- (1p36.33), B4GALT7 (5q35.3), C1R (12p13.31), C1S
862 Appendixes
(12p13.31), CHST14 (15q15.1), COL12A1 (6q13-14.1), Detection Method MLPA, NGS/MPS, Bi-directional
COL1A1 (17q21.33), COL1A2 (7q21.3), COL3A1 (2q32.2), Sanger Sequence Analysis
COL5A1 (9q34.3), COL5A2 (2q32.2), DSE (6q22.1), Reference Ranges Negative
FKBP14 (7p14.3), FLNA (Xq28), PLOD1 (1p36.22), Interpretation
PRDM5 (4q27), SLC39A13 (11p11.2), ZNF469 (16q24.2)
Sample Required Blood 1. Mutations in the APOB, LDLR, LDLRAP1, and PCSK9
Detection Method NGS/MPS, Bi-directional Sanger genes cause familial hypercholesterolemia (FH). The
Sequence Analysis LDLR gene mutation accounts for about 90%. So far,
Reference Ranges Negative more than 1200 mutations have been found in related
Interpretation genes, but only 79% are pathogenic. More than 100 muta-
tions in LDLR have been found in China. In addition, the
1. Mutations in any of several genes are associated with APOB gene mutation accounts for about 5% of the FH
Ehlers-Danlos syndrome (EDS). The main pathogenic gene mutation, and the PCSK9 gene mutation accounts
genes are collagen synthesis related genes, including type for about 1%.
I, III, and V collagen. The most clinically significant is 2. FH caused by mutations in the LDLR, APOB, or PCSK9
the fatal vascular EDS caused by mutations in the gene has an autosomal dominant inheritance pattern.
COL3A1 gene. More than 90% of patients with clinically When FH is caused by mutations in the LDLRAP1 gene,
diagnosed vascular EDS carry COL3A1 mutations, nearly the condition is inherited in an autosomal recessive
half of which are new mutations. pattern.
2. This condition is mostly autosomal dominant, and a few 3. The clinical features of FH are significantly increased lev-
are autosomal recessive. els of cholesterol and low-density lipoprotein cholesterol,
3. Three main signs of EDS include weak skin and blood xanthomas and early onset and progressive atheroscle-
vessels, too strong skin elasticity which can pull out very rotic cardiovascular disease.
long fold lead to thinning skin, and too large joint activity
to do automatic, passive joint hyperextension. EDS is C.28 Familial Hyperinsulinemic Hypoglycemia
often secondary to infection and sometimes can be associ- (Familial Hyperinsulinism) NGS Panel
ated with congenital heart disease. Genes ABCC8 (11p15.1), GCK (7p13), GLUD1 (10q23.2),
HADH (4q25), INSR
C.26 Factor V Leiden Thrombophilia (FVL) (19p13.2), KCNJ11 (11p15.1), SLC16A1 (1p13.2)
Gene F5 (1q24.2) Sample Required Blood
Sample Required Blood Detection Method NGS/MPS
Detection Method Allele specific primer extension Reference Ranges Negative
(ASPE) Interpretation
Interpretation 1. Mutations in at least nine genes have been found to cause
familial hyperinsulinism (FHI). Mutations in the ABCC8
1. A particular mutation in the F5 gene can cause Factor V gene (p.Phe1387del, c.3989-9G>A, p.Val187Asp, and
Leiden thrombophilia. The c.1691G>A (p.Arg534Gln) p.Glu1506Lys) are the most common known cause of the
variant is frequent in the population which is necessary disease.
for the inactivation of activated protein C (APC) factor 2. FHI can be inherited in an autosomal recessive or autoso-
V. Other variants in F5 at APC cleavage sites, p.Arg306Thr mal dominant manner.
and p.Arg306Gly may contribute when combined with 3. Affected individuals have abnormally high insulin levels,
other genetic or acquired risk factor. leading to frequent episodes of hypoglycemia. These epi-
2. Factor V Leiden thrombophilia is inherited in an autoso- sodes are characterized by lethargy, irritability, or diffi-
mal dominant manner. culty feeding in infants and young children. Repeated
3. This disorder is characterized by a poor anticoagulant episodes of hypoglycemia increase the risk of serious
response to APC and an increased risk for venous throm- complications, such as breathing difficulties, seizures,
boembolism (VTE). intellectual disability, vision loss, brain damage, and
coma.
C.27 Familial Hypercholesterolemia (HF)
Genes APOB (2p24.1), LDLR (19p13.2), LDLRAP1 C.29 Familial Mediterranean Fever (FMF)
(1p36.11), PCSK9 (1p32.3) Gene MEFV (16p13.3)
Sample Required Blood Sample Required Blood
Appendixes 863
Detection Method Bi-directional Sanger Sequence and most severe form. In the USA, p.Gln188Arg is the
Analysis most common pathogenic variant. Galactosemia type II
Reference Ranges Negative results from mutations in the GALK1 gene, while muta-
Interpretation tions in the GALE gene account for galactosemia type III.
2. Galactosemia is inherited in an autosomal recessive

1. Mutations in MEFV cause Familial Mediterranean fever manner.
(FMF). It has been shown that the p.Pro369Ser and p. 3. Galactosemia type I can lead to life-threatening complica-
Arg408Gln variants in exon 3 are in linkage disequilib- tions, including feeding problems, arrest of growth, hepa-
rium. Four pathogenic variants (p.Met694Val, p. tocellular damage, bleeding, and E. coli sepsis in infants.
Met694Ile, p.Met680Ile, and p.Val726Ala) are reported Infants with type II may experience cataracts. The signs
to have an associated phenotype and cause disease-related and symptoms of type III range from mild to severe and
symptoms. may include cataracts, growth and mental retardation,
liver disease, and kidney problems.
pattern.
3. FMF type 1 is characterized by recurrent short episodes C.32 Gaucher Disease: GBA Gene Sequencing (GBA
of inflammation and serositis including fever, peritonitis, Gene Sequencing)
synovitis, pleuritis, and, rarely, pericarditis and meningi- Gene GBA (1q22)
tis. Amyloidosis is the first clinical manifestation of FMF Sample Required Blood
type 2 in an otherwise asymptomatic individual. Detection Method Bi-directional Sanger Sequence
Analysis
C.30 Friedreich Ataxia Reference Ranges Negative
Gene FXN (9q21.11) Interpretation
Detection Method NGS/MPS
Sample Required Blood 1. Mutations in the GBA gene cause Gaucher Disease (GD).
Reference Ranges Negative Nearly 12% of pathogenetic alleles are recombined by
Interpretation GBA and GBAP. RecNciI is the most common recombi-
nant allele. In Ashkenazi Jewish individuals with type 1
1. Mutations in the FXN gene can cause Friedreich ataxia. GD, the variants p.Asn409Ser, c.84dupG, c.115+1G>A,
These inactivating pathogenic variants can be divided and p.Leu483Pro account for 90%, while 50–60% in non-
into three types: the GAA repeat amplification, nonsense Jewish individuals. The occurrence of deleterious alleles
or frameshift variants leading to translational abnormality p.Leu483Pro (41%) p.Phe252Ile (14%) among the
or premature termination, and loss-of-function missense Japanese and p.Leu483Pro (54%) and RecNciI (25%) in
and splicing variants. the Chinese may explain the higher incidence of neuro-
2. Friedreich ataxia is inherited in an autosomal recessive pathic disease.
manner. 2. GD is inherited in an autosomal recessive pattern.
3. This is a genetic condition that affects the nervous system 3. Clinical manifestations of GD vary greatly. The signs and
and causes movement problems. People with this condi- symptoms include growth retardation, progressive
tion experience unique clinical features, such as child- enlargement of the liver and spleen, pathological fracture,
hood onset, progressive ataxia, with pyramidal tract sign, fish scale skin, central nervous system disorders (con-
difficulty in pronunciation, deep paresthesia, scoliosis, sciousness change, language barrier, walking difficulties,
arched foot and heart damage. seizures), lung diseases (cough, difficulty breathing, pul-
monary hypertension), and eye involvement (eye move-
C.31 Galactosemia: Gene Sequencing Panel (RAPID ment disorder, difficulty in horizontal fixation, and
Testing) strabismus).
Genes GALE (1p36.11), GALK1 (17q25.1), GALT (9p13.3)
Sample Required Blood C.33 Glucocorticoid Resistance Generalized
Detection Method NGS/MPS Genes NR3C1 (5q31.3)
Reference Ranges Negative Sample Required Blood
Interpretation Detection Method PCR with allele specific hybridiza-
1. Mutations in the GALT, GALK1, and GALE genes can Reference Ranges Negative
cause galactosemia. Mutations in the GALT gene cause Interpretation
classic galactosemia (type I) which is the most common
864 Appendixes
1. Mutations in the NR3C1 gene can cause generalized glu- C.36 Hemophilia A
cocorticoid resistance. Gene F8 (Xq28)
2. This condition is inherited in an autosomal dominant
pattern. Detection Method MLPA, Bi-directional Sanger
3. Systemic glucocorticoid resistance is characterized by
Sequence Analysis
elevated plasma cortisol concentrations, elevated urinary Reference Ranges Negative
free cortisol, and resistance to dexamethasone inhibition Interpretation
of the adrenal gland, without the clinical features of
Cushing syndrome. Common symptoms include hypo- 1. Changes in the F8 gene are responsible for hemophilia A.
glycemia, high blood pressure, and metabolic alkalosis. F8 intron 22 inversions can cause severe hemophilia A
Overproduction of adrenal androgens in women can lead (45%). An inversion between a 1-kb sequence in intron 1,
to infertility, male-pattern alopecia, hirsutism, and irregu- an inverted repeat 5′ to F8, single-nucleotide variants
lar menstruation. leading to new stop codons as well as most frameshift
variants are all associated with severe phenotype.
C.34 Glucose-6-Phosphate-Dehydrogenase Deficiency Depending on the specific changes and location, splice
Gene G6PD (Xq28) site variability can lead to mild or moderate disease, but
Sample Required Blood also serious type. Missense mutations occur in nearly
Detection Method Bi-directional Sanger Sequence 20% of people with severe hemophilia A, but can be
Analysis found in almost all people with mild or moderate disease.
Reference Ranges Negative A single-base change in the 5′ promoter region of F8 is
Interpretation reported to be associated with mild disease.
2. Hemophilia A is inherited in an X-linked manner.
1. More than 200 mutations in the G6PD gene have been 3. It is characterized by a lack of activity of Factor VIII,
identified to cause glucose-6-phosphate dehydrogenase resulting in prolonged exudation after injury, tooth extrac-
deficiency. These changes can reduce the amount of the tion or surgery, and delayed or recurrent bleeding before
enzyme produced in cells or disrupt the normal structure the wound is completely healed.
and function of the enzyme. The two most common types
of genetic mutations in the Chinese population are 1376M C.37 Hemophilia B
and 1388M. Gene F9 (Xq27.1)
2. Glucose-6-phosphate dehydrogenase deficiency is inher- Sample Required Blood
ited in an X-linked recessive pattern that occurs almost Detection Method MLPA, Bi-directional Sanger
exclusively in men. Sequence Analysis
3. This condition is characterized by hemolytic anemia.
Affected individuals can experience pale complexion, Interpretation
jaundice, dark urine, fatigue, shortness of breath, and
rapid heartrate. 1. Mutations in the F9 gene cause hemophilia B. Severe
hemophilia B is caused by loss-of-function variants. Mild
C.35 GRACILE Syndrome or moderate type is primarily related to missense changes.
Genes BCS1L (2q35) 2. Hemophilia B is inherited in an X-linked manner.
Sample Required Blood 3. This condition is mainly characterized by hemorrhage,
Detection Method PCR with allele specific hybridiza- and joint cavity and muscle hemorrhage are characteristic
tion, Bi-directional Sanger Sequence Analysis features of severe cases.
Interpretation C.38 Hereditary Myopathy with Lactic Acidosis due to
ISCU Deficiency, Sequencing ISCU Gene
1. Mutations in the BCS1L gene cause GRACILE
Gene ISCU (12q23.3)
syndrome. Sample Required Blood
2. GRACILE syndrome is inherited in an autosomal reces- Detection Method PCR with allele specific hybridiza-
sive manner. tion, Bi-directional Sanger Sequence Analysis
3. This is a serious illness that starts before birth character- Reference Ranges Negative
ized by growth retardation, amino aciduria, cholestasis, Interpretation
iron overload, lactic acidosis, and premature death.
Appendixes 865
1. Mutations in the ISCU gene cause myopathy with defi- sweating, palpitations, pale face, fear, or anxiety. PGL is
ciency. So far, homozygous for c.418+382G>C, a patho- more prone to develop metastatic disease than PCC.
genic splice variant in intron 4 originating from a founder
haplotype in northern Sweden are the majority of affected C.40 HFE-Associated Hereditary Hemochromatosis:
individuals. However, compound heterozygous splice Targeted Gene Sequencing
variant which are also common for Swedish and a patho- Gene HFE (6p22.2)
genic c.149G>A missense variant in exon 3 can cause this Sample Required Blood
condition, too. Detection Method Bi-directional Sanger Sequence
2. Myopathy with deficiency of ISCU is inherited in an auto- Analysis
somal recessive manner. Reference Ranges Negative
3. This mitochondrial myopathy is typically characterized Interpretation
by lifelong exercise intolerance, which primarily affects
muscles used for exercise (skeletal muscles). Mild exer- 1. Mutations in the HFE gene cause HFE hemochromatosis.
cise leads to rapid heartbeat (tachycardia), shortness of Most persons with HFE hemochromatosis phenotypes
breath, muscle weakness, and pain. However, patients have homozygosity for p.Cys282Tyr, compound hetero-
usually have normal muscle strength at rest. Long-term or zygosity for p.Cys282Tyr and p.His63Asp, or compound
repeated physical activity can lead to more serious symp- heterozygosity for p.Cys282Tyr.
toms and signs, including destruction of muscle tissue 2. HFE hemochromatosis is inherited in an autosomal reces-
(rhabdomyolysis) and myoglobinuria. Most patients do sive manner.
not get worse with time. 3. This condition is characterized by inappropriately high
absorption of iron by the small intestinal mucosa. Persons
C.39 Hereditary Paraganglioma-Pheochromocytoma with clinical HFE hemochromatosis may experience end-
Syndrome Sequencing Panel with CNV Detection organ damage caused by iron overload. Individuals with
Genes FH (1q43), MAX (14q23.3), MEN1 (11q13.1), NF1 biochemical HFE hemochromatosis have increased
(17q11.2), RET (10q11.21), SDHA (5p15.33), SDHAF2 transferrin-iron saturation and the only evidence of iron
(11q12.2), SDHB (1p36.13), SDHC (1q23.3), SDHD overload is elevated serum ferritin concentration. For
(11q23.1), TMEM127 (2q11.2), VHL (3p25.3) patients with non-expressing p.Cys282Tyr homozygotes,
Sample Required Blood neither clinical manifestations of HFE hemochromatosis
Detection Method Bi-directional Sanger Sequence nor iron overload are present.
Analysis, NGS/MPS
Reference Ranges Negative C.41 Hypertrophic Cardiomyopathy Short Panel
Interpretation Genes ACTA1 (1q42.13), DES (2q35), FLNC (7q32.1), GLA
(Xq22.1), LAMP2 (Xq24), MYBPC3 (11p11.2), MYH7
1. Mutations in these genes increase the risk of developing (14q11.2), MYL2 (12q24.11), MYL3 (3p21.31), PRKAG2
different types of hereditary paraganglioma-(7q36.1), PTPN11 (12q24.13), TNNC1 (3p21.1), TNNI3
pheochromocytoma (PCC/PGL). SDHB gene mutations (19q13.42), TNNT2 (1q32.1), TPM1 (15q22.2), TTR
are the most common (10.3%), followed by SDHD (18q12.1)
(8.9%), VHL (7.3%), RET (6.3%), and NF1 (3.3%). Sample Required Blood
SDHB gene mutation is a risk factor for malignant PCC/ Detection Method NGS/MPS
PGL. VHL gene mutation leads to Von Hippel-Lindau Reference Ranges Negative
(VHL) syndrome. Mutation of the RET gene results in a Interpretation
penetrance of MEN2, PCC of about 50%. Mutations in
the NF1 gene result in neurofibromatosis type 1 and 5% 1. Mutations in one of several genes can cause familial
of PCC. In addition, hereditary PGL is divided into 1, 2, hypertrophic cardiomyopathy (HCM). The most com-
3, 4, and 5 types, which correspond to SDHD, SDHAF2, monly involved genes are MYH7, MYBPC3, TNNT2, and
SDHC, SDHB, and SDHA gene mutations, respectively. TNNI3.
2. This condition is inherited in an autosomal dominant 2. This condition is inherited in an autosomal dominant pat-

pattern. tern. Rarely, both copies of the gene are altered, leading to
3. PGL/PCC syndromes are characterized by paraganglio- more severe signs and symptoms.
mas and pheochromocytomas. Symptoms of PGL/PCC 3. It is characterized by cardiac hypertrophy, mainly leads to
result from either mass effects or catecholamine hyperse- left ventricular hypertrophy. This condition usually refers
cretion. The manifestations include sustained or paroxys- to the two-dimensional echocardiographic of the ventric-
mal blood pressure, headache, intermittent excessive ular septum or left ventricular wall thickness 15 mm, or
866 Appendixes
≥13 mm in adults with a clear family history, ≥11 mm in variant in exon 22 of MED12. XLOS has been reported to
children, without left ventricular enlargement. Diagnosis be caused by p.Arg1148His, p.Ser1165Pro, and p.
should exclude increased load, such as hypertension, aor- His1729Asn hemizygous pathogenic variants in MED12.
tic stenosis, and congenital aortic valvular wall thickening It is reported that p.Ser1967GlnfsTer84, p.Gln1974His,
caused by left ventricular wall. In addition, cardiac hyper- p.Ala1383Thr, p.Arg815Gln, and p.Ile1023Val hemizy-
trophy in some rare HCM phenotypic diseases is also gous pathogenic variants in MED12 can cause non-
consistent with the diagnosis of HCM, accounting for specific intellectual disability distinct from FGS1, LS, or
5–10% of clinical diagnosis of HCM. The poor prognosis XLOS.
of HCM includes sudden cardiac death (SCD), heart fail- 2. This condition is inherited in an X-linked recessive

ure, and stroke, which are the leading causes of SCD in pattern.
young people (≤35 years old). 3. Patients with FGS1 and LS experience cognitive impair-
ment, hypotonia, unusual facial features, and abnormali-
C.42 Immunodeficiency, X-linked, with Hyper-IgM ties of the corpus callosum. The signs and symptoms
Gene CD40LG (Xq26.3) include intellectual disability, blepharophimosis, and
Sample Required Blood facial coarsening.
tion, Bi-directional Sanger Sequence Analysis C.44 Krabbe Disease
Reference Ranges Negative Gene GALC (14q31.3)
Interpretation Sample Required Blood
1. About 170 pathogenic variants throughout five exons of tion, NGS/MPS, Bi-directional Sanger Sequence Analysis
the CD40LG gene have been published which can cause Reference Ranges Negative
X-linked hyper-IgM syndrome (HIGM1). The most com- Interpretation
mon are in the TNF-homology domain (exon 5). Missense
variants account for 26%, nonsense variants account for 1. Mutation of GALC gene causes Krabbe disease. The most
20%, the others are small deletions/insertions, splicing common variant is 30-kb deficiency, which accounts for
variants, and partial- or whole-gene deletions or large about 45% of the variants in people of European descent.
insertions. Another 15% of abnormal alleles in patients of European
2. HIGM1 is inherited in an X-linked recessive manner. descent were associated with infantile Krabbe disease,
3. This disorder is characterized by low serum IgG and IgA including p.Thr529Met, p.Tyr567Ser, and c.1472delA,
concentrations and normal or elevated serum IgM con- and 7.4-kb deletion was also observed in some patients.
centrations. Affected babies are prone to recurrent upper- Even if the 30-kb deletion appears as the second gene, the
and lower-respiratory tract bacterial infections, presence of pathogenic variant p.gly286asp will cause the
opportunistic infections, and recurrent or persistent diar- late-onset form of GALC deficiency.
rhea associated with failure to thrive. Anemia, neutrope- 2. Krabbe disease is inherited in an autosomal recessive
nia, and thrombocytopenia are also common. pattern.
3. It is a serious neurological disease caused by demyelin-
C.43 Invitae MED12-Related Disorders Test ation in the nervous system, characterized by abnormal
Gene MED12 (Xq13.1) cells in the brain called spherical cells, which usually
Sample Required Blood have more than one nucleus.
Reference Ranges Negative C.45 L1CAM Sequencing
Interpretation Gene L1CAM (Xq28)
1. Mutations in the MED12 gene cause MED12-related dis- Detection Method NGS/MPS
orders. The phenotypic spectrum of MED12-related dis- Reference Ranges Negative
orders includes FG syndrome type 1 (FGS1), Lujan Interpretation
syndrome (LS), X-linked Ohdo syndrome (XLOS), and
so on. A recurrent pathogenic p.Arg961Trp missense 1. So far, about 350 different pathogenic variants in the
variant in exon 21 is the most common pathogenic variant L1CAM gene have been reported to cause L1 syndrome.
in FGS individuals. A pathogenic p.Gly958Glu missense All types of pathogenic variants are found. The nonsense
variant in exon 21 of MED12 may also cause FGS. LS is and frameshift variants can cause truncation of the L1
caused by a recurrent pathogenic p.Asn1007Ser missense protein.
Appendixes 867
2 . L1 syndrome is inherited in an X-linked pattern. 1. Mutations in the HPRT1 gene cause Lesch–Nyhan syn-
3. L1 syndrome is also known as CRASH syndrome accord- drome. Pathogenic variants are spread nearly randomly
ing to its clinical manifestations, including dysplasia of throughout HPRT1. Each family generally has a unique
the corpus callosum, mental retardation, adduction of the pathogenic variant. Some pathogenic variants that have
thumb, spastic paraplegia, and hydrocephalus. The most occurred independently in more than one family are
consistent manifestation is mental retardation (IQ 20–50). known. In the 271 cases studied, 218 pathogenic DNA
Severe cases presented with hydrocephalus and resulted variants (primarily missense and nonsense variants and
in fetal or neonatal death, while mild cases presented with small deletions/insertions) have been found.
mild mental retardation without other abnormalities. 2. Lesch–Nyhan syndrome is inherited in an X-linked

pattern.
C.46 Leber Congenital Amaurosis 3. Patients often have neurological and behavioral disorders,
Genes AIPL1 (17p13.2), CEP290 (12q21.32), CRB1 including abnormal involuntary muscle movements, such
(1q31.3), CRX (19q13.33), GUCY2D (17p13.1), IMPDH1 as dystonia and chorea. As a result, patients often need to
(7q32.1), KCNJ13 (2q37.1), LCA5 (6q14.1), LRAT (4q32.1), use wheelchairs. Due to abnormal purine metabolism,
NMNAT1 (1p36.22), RD3 (1q32.3), RDH12 (14q24.1), children with Lesch–Nyhan syndrome show self-injury
RPE65 (1p31.3), RPGRIP1 (14q11.2), SPATA7 (14q31.3), and behaviors of hurting others immediately after birth.
TULP1 (6p21.31)
Sample Required Blood C.48 Liddle Syndrome (Pseudoaldosteronism)
Detection Method Bi-directional Sanger Sequence Genes SCNN1B (16p12.2), SCNN1G (16p12.2)
Analysis, NGS/MPS Sample Required Blood
Reference Ranges Negative Detection Method Bi-directional Sanger Sequence
Interpretation Analysis
1. Mutations in the LCA1, LCA2, LCA8, and LCA10 genes Interpretation
are the most common causes of Leber congenital amauro-
sis (LCA), with other mutations accounting for a smaller 1. Mutations in the SCNN1B or SCNN1G gene can cause
proportion. About 30% of all people with LCA have an Liddle syndrome.
unknown cause. 2. This condition is inherited in an autosomal dominant

2. LCA is usually inherited in an autosomal recessive pat- pattern.
tern. When LCA is caused by mutations in the LCA7 or 3. Clinical symptoms of Liddle syndrome are similar to pri-
LCA11 genes, the disease is inherited in an autosomal mary aldosteronism, mainly hypertension, hypokalemia,
dominant pattern. and alkalosis. The patient presented with headache, mus-
3. LCA is clinically divided into two types, infant type and cle weakness and soft palate, polyuria, polydipsia, con-
juvenile type. Patients often have poor visual function, vulsions, paresthesia, and retinopathy.
often with nystagmus, slow or almost no pupil response,
photophobia, high hyperopia, and keratoconus. Visual C.49 Long QT Syndrome Panel
acuity rarely exceeds 20/400. A typical finding is that Genes AKAP9 (7q21.2), ANK2 (4q25-26), CACNA1C
Franceschetti’s oculo-digital sign, including eye poking, (12p13.33), CALM1 (14q32.11), CAV3 (3p25.3), KCNE1
pressing, and rubbing. The appearance of the fundus var- (21q22.12), KCNE2 (21q22.11), KCNH2 (7q36.1), KCNJ2
ies greatly. All patients can be accompanied by psycho- (17q24.3), KCNJ5 (11q24.3), KCNQ1 (11p15.5-15.4),
neurotic symptom, such as mental retardation, hearing SCN4B (11q23.3), SCN5A (3p22.2), SNTA1 (20q11.21)
obstacle, epilepsy, and some older patients can experience Sample Required Blood
keratoconus or spherical cornea. Detection Method Bi-directional Sanger Sequence
Analysis
C.47 Lesch–Nyhan Syndrome Reference Ranges Negative
Gene HPRT1 (Xq26.2-26.3) Interpretation
Detection Method CNV by q-PCR, Bi-directional Sanger 1. Mutations in several gene can cause long QT syndrome
Sequence Analysis (LQTS). Among them, KCNQ1 (LQTS1), KCNH2
Reference Ranges Negative (LQTS2), and SCN5A (LQTS3) genes can explain about
Interpretation 75% of patients, and the remaining pathogenic genes can
explain 5–10% of patients.
868 Appendixes
2. This condition mainly follows an autosomal dominant C.52 McKusick–Kaufman Syndrome

pattern of inheritance. Gene MKKS (20p12.2)
3.
LQTS is characterized by episodes of fainting Detection Method PCR with allele specific hybridiza-
(SYNCOPE) and varying degree of ventricular arrhyth- tion, Bi-directional Sanger Sequence Analysis
mia as indicated by the prolonged QT interval. The inher- Sample Required Blood
ited forms are caused by mutation of genes encoding Reference Ranges Negative
cardiac ion channel proteins. The two major forms are Interpretation
ROMANO- WARD SYNDROME and JERVELL-
LANGE NIELSEN SYNDROME. 1. Pathogenic variants in all of the coding exons of the
MKKS gene can cause McKusick–Kaufman Syndrome
C.50 Maple Syrup Urine Disease Panel (MKS).
Genes BCKDHA (19q13.2), BCKDHB (6q14.1), DBT 2. MKS is inherited in an autosomal recessive pattern.
(1p21.2), DLD (7q31.1) 3. This condition is characterized by the combination of
Sample Required Blood postaxial polydactyly, congenital heart disease, and
Detection Method NGS/MPS hydrometrocolpos in women and genital malformations
Reference Ranges Negative in man (hypospadias, cryptorchidism, and chordee).
Interpretation
C.53 Methylmalonic Acidemia: Gene Sequencing
1. Mutations in the BCKDHA, BCKDHB, and DBT genes Panel (RAPID Testing)
can cause maple syrup urine disease (MSUD). MSUD Genes MCEE (2p13.3), MMAA (4q31.21), MMAB
can be divided into three main types: type 1A (45%), type (12q24.11), MMADHC (2q23.2), MMUT (6p12.3)
1B (35%), and type 2 (20%). Type 1A is caused by muta- Sample Required Blood
tions in the BCKDHA gene (c.1312T>A). Type 1B is Detection Method NGS/MPS
caused by mutations in the BCKDHB gene (c.548G>C, Reference Ranges Negative
c.832G>A, c.1114G>T). Mutations in the DBT gene can Interpretation
cause MSUD type 2.
2. MSUD is inherited in an autosomal recessive manner. 1. Mutations in the MMUT, MMAA, MMAB, MMADHC,
3. The condition gets its name from the distinctive sweet and MCEE genes cause methylmalonic acidemia.
odor of affected infants’ urine. It is also characterized by Mutations in the MMUT gene that prevent the production
poor feeding, vomiting, lethargy, abnormal movements, of any functional enzyme result in a form of the condition
and delayed development. Maple syrup urine disease can designated mut0, can cause about 60% cases. Mut0 is the
lead to seizures, coma, and death without treatment. most severe form of methylmalonic acidemia with the
poorest outcome.
C.51 Marfan Syndrome-FBN1 Gene (Fib) 2. Methylmalonic acidemia is inherited in an autosomal

Gene FBN1 (15q21.1) recessive pattern.
Sample Required Blood 3. Methylmalonic acidemia usually occurs in early infancy
Detection Method Bi-directional Sanger Sequence and its effects can range from mild to life-threatening.
Analysis Affected infants may experience vomiting, dehydration,
Reference Ranges Negative hypotonia, stunted growth, lethargy, enlarged liver, and
Interpretation failure to gain weight and thrive. Long-term complica-
tions include feeding problems, intellectual disability,
1. More than 1,800 FBN1 gene-related mutations have been chronic kidney disease, and pancreatitis.
reported to cause Marfan syndrome. The proportion of
FBN1 gene mutations detected in MFS patients meeting C.54 MORM Syndrome
clinical diagnostic criteria is 70–93%. Gene INPP5E (9q34.3)
pattern. Detection Method Uni-directional Sanger sequencing
3. It is a disorder that affects the connective tissue in many Reference Ranges Negative
parts of the body. Marfan syndrome can affect many sys- Interpretation
tems, often causing abnormalities in the heart, blood ves-
sels, eyes, bones, and joints.
Appendixes 869
1. Mutations in the INPP5E gene cause MORM syndrome. C.57 Niemann–Pick Disease
The causative locus has been mapped to chromosome Genes SMPD1 (11p15.4), NPC1 (18q11.2), NPC2 (14q24.3)
region 9q34. Sample Required Blood
2. It is transmitted in an autosomal recessive manner. Detection Method Bi-directional Sanger Sequence
3. MORM syndrome is associated with intellectual deficit, Analysis
truncal obesity, retinal dystrophy, and micropenis. Reference Ranges Negative
Interpretation
C.55 Myotonia Congenita: Gene Sequencing
Gene CLCN1 (7q34) 1. Mutations in the SMPD1, NPC1, and NPC2 genes cause
Sample Required Blood Niemann–Pick disease (NPD) which can be divided into
Detection Method NGS/MPS four main types: type A, type B, type C1, and type C2.
Reference Ranges Negative Types A and B are caused by mutations in the SMPD1
Interpretation gene. Mutations in the NPC1 or NPC2 gene cause
Niemann–Pick disease type C1 and type C2.
1. More than 275 different pathogenic variants in CLCN1 2. NPD is inherited in an autosomal dominant manner.
gene have been identified to be associated with autosomal 3. Niemann–Pick disease is mainly caused by the deposition
recessive myotonia congenita. Pathogenic variants caus- of neuromyelin, cholesterol, and other phospholipids in
ing dominant myotonia congenita are often located in the liver and spleen endothelial cells and brain caused by
exon 8. The p.Arg894Ter pathogenic variant is probably enzyme deficiency. Clinical manifestations include liver,
the most common semi-dominant pathogenic variant with splenomegaly, neurological symptoms, paralysis, blind-
homozygotes being more severely affected than ness, and mental retardation. Saffron erythema can also
heterozygotes. be seen in the macular deposits of the macula. The disease
2. The two main types of myotonia congenita are known as is progressive and often dies several years after the onset
Thomsen disease and Becker disease. Thomsen disease is of the disease.
inherited in an autosomal dominant pattern, while Becker
disease is inherited in an autosomal recessive pattern. C.58 Nijmegen Breakage Syndrome: Gene Sequencing
3. Myotonia congenita is characterized by muscular stiff- Gene NBN (8q21.3)
ness from childhood. It may involve all striated muscle Sample Required Blood
groups, including external eye muscles, facial muscles, Detection Method NGS/MPS
and tongue muscles, which are usually hypertrophic. Reference Ranges Negative
Muscle stiffness can be alleviated by repeated contrac- Interpretation
tions (“warm-up” phenomenon).
1. Mutations in the NBN gene cause Nijmegen breakage
C.56 Nemaline Myopathy Sequencing Panel syndrome.
Genes ACTA1 (1q42.13), CFL2 (14q13.1), KBTBD13 2. It is transmitted in an autosomal recessive manner.
(15q22.31), KLHL40 (3p22.1), KLHL41 (2q31.1), LMOD3 3. This condition is characterized by short stature, micro-
(3p14.1), NEB (2q23.3), TNNT1 (19q13.42), TPM2 (9p13.3), cephaly, unique facial features, recurrent respiratory
TPM3 (1q21.3) infections, intellectual disability, and an increased risk of
Sample Required Blood cancer.
Reference Ranges Negative C.59 Oculocutaneous Albinism (OCA)
Interpretation Genes OCA2 (15q12-13.1), RPP38-DT (10p13), SLC24A5
(15q21.1), SLC45A2
1. Mutations in many genes (ACTA1, CFL2, KBTBD13,
(5p13.2), TYR (11q14.3), TYRP1 (9p23)
KLHL40, KLHL41, LMOD3, NEB, TNNT1, TPM2, Sample Required Blood
TPM3) can cause nemaline myopathy (NM). Mutations Detection Method MLPA, NGS/MPS
in NEB gene account for about 50% all cases of NM and Reference Ranges Negative
ACTA1 gene mutations account for 15–25% of all cases. Interpretation
2. This condition is inherited in an autosomal dominant or
autosomal recessive manner. 1. Mutations in several genes, including TYR, OCA2,

3. It is characterized by weakness, hypotonia, and depressed TYRP1, and SLC45A2 can cause oculocutaneous albinism
or absent deep tendon reflexes. Muscle weakness in face, (OCA). Mutations in the TYR gene can cause type 1,
neck flexors, and proximal limb muscles is usually most mutations in the OCA2 gene lead to type 2, TYRP1 muta-
severe. tions cause type 3, and mutations in the SLC45A2 gene
870 Appendixes
lead to type 4. Mutations in the MC1R gene can alter the 2 . CPHD is inherited in an autosomal recessive manner.
appearance of patients with type 2. 3. CPHD is associated with deficiencies of growth hormone.
2. OCA is inherited in an autosomal recessive pattern. Lack of these hormones can affect the development of
3. It is a group of conditions that affect coloring (pigmenta- many parts of the body. Growth retardation and short stat-
tion) of the skin, hair, and eyes. Affected individuals typi- ure are evident in early childhood. Patients may have
cally have very fair skin and white or light-colored hair. hypothyroidism, which can include weight gain and
OCA type 1 is characterized by white hair, very pale skin, fatigue. Other features of pituitary hormone deficiency
and light irises. Type 2 is usually not as severe as type 1; include delayed or absent puberty and a lack of the ability
the skin is usually milky white and hair may be light yel- to have children (infertility). The condition may also be
low, golden, or light brown. Type 3 includes an albinism linked to a lack of the hormone cortisol, which impairs
called rufous OCA, which usually affects dark-skinned the body’s immune system and makes individuals more
people. Patients have reddish-brown skin, ginger or red susceptible to infection.
hair, and hazel or brown irises. Type 3 is often associated
with milder vision abnormalities than the other forms of C.62 Protein S Deficiency, AD
OCA. Type 4 has similar signs and symptoms as type 2. Gene PROS1 (3q11.1)
C.60 Phenylketonuria Detection Method MLPA, Bi-directional Sanger
Gene PAH (12q23.2) Sequence Analysis
Sample Required Blood Reference Ranges Negative
Detection Method PCR with allele specific hybridiza- Interpretation
Reference Ranges Negative 1. Mutations in the PROS1 gene are associated with protein
Interpretation S deficiency.
2. Protein S deficiency is inherited in an autosomal domi-
1. So far, more than 900 different pathogenic variants in nant pattern.
PAH gene have been identified to cause phenylketonuria 3. People with mild protein S deficiency are at risk of devel-
(PKU). The majority of pathogenic variants in PAH are oping deep vein thrombosis, a clot that occurs in the deep
missense, nonsense, frameshift, and splice variants. Large veins of the arm or leg. In severe cases of protein S defi-
deletions account for less than 1% of all pathogenic vari- ciency, a baby may develop purpura pimples (life-
ants in most populations, but 3% in the Czech threatening blood clotting) shortly after birth.
population.
2. Phenylketonuria is inherited in an autosomal recessive C.63 Protein C Deficiency (PROC)
manner. Gene PROC (2q14.3)
3. PKU causes intolerance to the dietary intake of the essen- Sample Required Blood
tial amino acid phenylalanine and produces a spectrum of Detection Method MLPA, Bi-directional Sanger
disorders. If there is no effective therapy, most people Sequence Analysis
with severe PAH deficiency (known as classic PKU) Reference Ranges Negative
develop serious and irreversible intellectual disability. Interpretation
C.61 PROP1-Related Combined Pituitary Hormone 1. Mutations in the PROC gene can cause protein C

Deficiency: Gene Sequencing deficiency.
Gene PROP1 (5q35.3) 2. Protein C deficiency is inherited in an autosomal domi-
Sample Required Blood nant pattern.
Detection Method NGS/MPS 3. People with mild protein C deficiency are at risk of devel-
Reference Ranges Negative oping deep vein thrombosis, a clot that occurs in the deep
Interpretation veins of the arm or leg. In severe cases of protein C defi-
ciency, a baby may develop purpura pimples (life-
1. Pathogenic variants in PROP1 gene are known to cause threatening blood clotting) shortly after birth.
PROP1-related combined pituitary hormone deficiency
(CPHD). The common recurrent PROP1 deletion in C.64 Prothrombin-Related Thrombophilia (Factor II)
which three AG repeats are reduced to two AG repeats Gene F2 (11p11.2)
(c.301_302delAG) accounts for 55% of alleles in familial Sample Required Blood
cases and 12% of alleles in simplex cases of CPHD. Detection Method SNP Detection
Appendixes 871
Reference Ranges Negative 1. Mutations in several genes can cause pulmonary arterial
Interpretation hypertension (PAH). Most heritable PAH (75%) is caused
by a pathogenic variant in BMPR2; pathogenic variants in
1. A particular mutation in the F2 gene can cause
other genes (ACVRL1, KCNK3, CAV1, ENG, SMAD9,
prothrombin-related thrombophilia. The F2 gene plays a BMPR1B) are account for 1–3%.
critical role in the formation of blood clots in response to 2. This condition is inherited in an autosomal dominant

injury. All individuals reported to date with manner.
prothrombin-
related thrombophilia have had a parent 3. It is characterized by widespread obstruction and oblitera-
who is heterozygous or homozygous for the F2 20210G>A tion of the smallest pulmonary arteries. Initial symptoms
allele. include dyspnea, fatigue, syncope, chest pain, palpita-
2. Prothrombin-related thrombophilia is inherited in an
tions, and leg edema.
autosomal dominant manner.
3. It is characterized by venous thromboembolism most
C.67 Pyridoxamine 5′-Phosphate Oxidase Deficiency
commonly seen in adults, that is, deep venous thrombosis Gene PNPO (17q21.32)
of the leg or pulmonary embolism. Sample Required Blood
C.65 Pseudohypoaldosteronism Type II: Gene tion, NGS/MPS
Sequencing Panel Reference Ranges Negative
Gene CUL3 (2q36.2), KLHL3 (5q31.2), WNK1 (12p13.33), Interpretation
WNK4 (17q21.2)
Sample Required Blood 1.
Mutations in the PNPO gene cause Pyridoxal
Detection Method NGS/MPS 5′-phosphate-dependent epilepsy (PNPOD).
Reference Ranges Negative 2. PNPOD is inherited in an autosomal recessive pattern.
Interpretation 3. This is a condition where seizures begin shortly after birth
or in some cases before birth. Seizures usually include
1. Mutations in the WNK1, WNK4, CUL3, or KLHL3 gene irregular involuntary myoclonus, abnormal eye move-
can cause pseudohypoaldosteronism type 2 (PHAII). ments, and convulsions. Most babies with this condition
CUL3 alterations causing PHAII occur within intron 8, are born prematurely and may have a temporary, poten-
exon 9, or intron 9 and disrupt splicing of exon 9. tially toxic lactic acidosis. In addition, some babies suffer
Dominant KLHL3 pathogenic variants that cause PHAII from fetal distress.
are missense alternations. Recessive KLHL3 pathogenic
variants occur throughout the gene and include frameshift C.68 Pyruvate Carboxylase Deficiency
and premature termination variants. All reported WNK4 Gene PC (11q13.2)
pathogenic variants are missense alterations. Sample Required Blood
2. This condition is usually inherited in an autosomal domi- Detection Method PCR with allele specific hybridiza-
nant pattern. Some cases caused by mutations in the tion, Bi-directional Sanger Sequence Analysis
KLHL3 gene are inherited in an autosomal recessive Reference Ranges Negative
pattern. Interpretation
3. PHAII is characterized by hyperkalemia although glo-
merular filtration rate (GFR) is normal and hypertension 1. Missense, nonsense, frameshift, and splice site variants in
is common. Other related manifestations in both children PC can cause pyruvate carboxylase deficiency (PCD).
and adults include hyperchloremia, metabolic acidosis, Mosaicism is reported to be observed in five individuals.
and suppressed plasma renin levels. Notably, two substitutions—c.1892G>A (p.Arg631Gln)
and c.2549C>T (p.Ala847Val)—were found in a mosaic
C.66 Pulmonary Hypertension Comprehensive Panel state on the same allele in three patients.
Genes ACVRL1 (12q13.13), BMPR1B (4q22.3), BMPR2 2. This condition is inherited in an autosomal recessive

(2q33.1-33.2), CAV1 (7q31.2), EIF2AK4 (15q15.1), ENG pattern.
(9q34.11), FOXF1 (16q24.1), KCNK3 (2p23.3), SMAD9 3. PCD is characterized by failure to thrive, stunted growth,
(13q13.3) recurrent seizures, and metabolic acidosis in most
Sample Required Blood patients. This disease has three clinical types. Most type
Detection Method NGS/MPS, Bi-directional Sanger A (infantile form) dies in infancy or infancy. Type B
Sequence Analysis (severe neonatal form) presents with hepatomegaly, pyra-
Reference Ranges Negative midal tract syndrome, abnormal movement, and death in
Interpretation the first 3 months of life. Type C (intermittent/benign
872 Appendixes
form) has normal or mildly delayed neurodevelopment 1. Mutations in more than 60 genes are known to cause non-
and episodic metabolic acidosis. syndromic retinitis pigmentosa. Of them, more than 20
are related to the autosomal dominant form of the disor-
C.69 Pyruvate Kinase Deficiency with Hemolytic der. Mutations in the RHO gene, the most common cause
Anemia of autosomal dominant retinitis pigmentosa, account for
Gene PKLR (1q22) 20–30% cases. At least 35 genes are involved in the auto-
Sample Required Blood somal recessive form of the condition. Mutations in the
Detection Method PCR with allele specific hybridiza- most common gene, USH2A, are responsible for 10–15%
tion, Bi-directional Sanger Sequence Analysis cases of autosomal recessive retinitis pigmentosa.
Reference Ranges Negative Mutations in at least 6 genes can cause the X-linked form.
Interpretation Most of these cases are caused by mutations in the RPGR
and RP2 genes.
1. Mutations in the PKLR gene may cause pyruvate kinase 2. This condition can be inherited in an autosomal domi-
deficiency. nant, autosomal recessive, or X-linked pattern.
2. Pyruvate kinase deficiency is inherited in an autosomal 3. Retinitis pigmentosa is a group of related eye diseases
recessive pattern. that mostly cause progressive vision loss.
3. This condition is characterized by chronic hemolytic ane-
mia. Some patients have few or even no symptoms. Severe C.71 Salla Disease
cases can be life-threatening in infancy, and such affected Gene SLC17A5 (6q13)
babies may need regular blood transfusions to survive. Sample Required Blood
This disease may get worse due to infection or Detection Method NGS/MPS
pregnancy. Reference Ranges Negative
Interpretation
C.70 Retinitis Pigmentosa NGS Panel
Genes ABCA4 (1p22.1), AGBL5 (2p23.3), AIPL1 (17p13.2), 1. The SLC17A5 gene mutation causes various forms of
ARHGEF18 (19p13.2), ARL6 (3q11.2), BBS1 (11q13.2), sialic acid storage disease. The most common in Finnish
BBS2 (16q13), BEST1 (11q12.3), C8orf37 (8q22.1), CA4 patients is p.Arg39Cys pathogenic variant.
(17q23.1), CDHR1 (10q23.1), CERKL (2q31.3), CLRN1 2. Sialic acid storage disease is inherited in an autosomal
(3q25.1), CNGA1 (4p12), CNGB1 (16q21), CRB1 (1q31.3), recessive manner.
CRX (19q13.33), CYP4V2 (4q35.1-35.2), DHDDS (1p36.11), 3. The allelic disorders of free sialic acid metabolism,

EYS (6q12), FAM161A (2p15), FLVCR1 (1q32.3), FSCN2 including Salla disease, intermediate severe Salla disease,
(17q25.3), GNPTG (16p13.3), GUCA1B (6p21.1), HGSNAT and infantile free sialic acid storage disease (ISSD), are
(8p11.21-11.1), IDH3B (20p13), IMPDH1 (7q32.1), IMPG2 neurodegenerative disorders caused by increased lyso-
(3q12.3), KLHL7 (7p15.3), LRAT (4q32.1), MAK (6p24.2), somal storage of free sialic acid. Salla disease, the mildest
MERTK (2q13), MFRP (11q23.3), NEK2 (1q32.3), NR2E3 phenotype, is characterized by normal appearance and
(15q23), NRL (14q11.2-12), OFD1 (Xp22.2), PCARE neurological manifestations at birth, followed by slow
(2p23.2), PDE6A (5q32), PDE6G (17q25.3), PRCD progression of neurological deterioration leading to mild
(17q25.1), PROM1 (4p15.32), PRPF3 (1q21.2), PRPF31 to moderate psychomotor retardation, paralysis, sagging,
(19q13.42), PRPF4 (9q32), PRPF6 (20q13.33), PRPF8 and seizures. ISSD, the most severe phenotype, is charac-
(17p13.3), PRPH2 (6p21.1), RBP3 (10q11.22), RDH12 terized by severe developmental delay, enlarged face,
(14q24.1), REEP6 (19p13.3), RGR (10q23.1), RHO (3q22.1), liver, spleen, and heart enlargement.
RLBP1 (15q26.1), ROM1 (11q12.3), RP1 (8q11.23-12.1),
RP2 (Xp11.3), RP9 (7p14.3), RPE65 (1p31.3), RPGR C.72 Sandhoff Disease
(Xp11.4), SAG (2q37.1), SEMA4A (1q22), SNRNP200 Genes HEXB (5q13.3)
(2q11.2), SPATA7 (14q31.3), TOPORS (9p21.1), TTC8 Sample Required Blood
(14q31.3), TULP1 (6p21.31), USH2A (1q41), ZNF408 Detection Method PCR with allele specific hybridiza-
(11p11.2), ZNF513 (2p23.3) tion, NGS/MPS
Sample Required Blood Reference Ranges Negative
Detection Method NGS/MPS Interpretation
Interpretation 1 . Mutations in the HEXB gene cause Sandhoff disease.

manner.
Appendixes 873
3. Sandhoff disease is a progressive neurodegenerative dis- Reference Ranges Negative

ease characterized by an accumulation of GM2 ganglio- Interpretation
sides, especially in neurons.
1. Mutations in any of several genes are associated with
C.73 Spinal Muscular Atrophy familial thoracic aortic aneurysm and dissection (familial
Genes GEMIN2 (14q21.1), SMNDC1 (10q25.2), SMN1 TAAD). Mutations in the ACTA2 gene have been identi-
(5q13.2), SMN2 (5q13.2) fied in 14–20% of affected individuals, and TGFBR2
Sample Required Blood gene mutations have been found in 2.5% of patients.
Detection Method Quantitative PCR Mutations in several other genes account for a small pro-
Reference Ranges Negative portion of cases.
Interpretation 2. This condition is inherited in an autosomal dominant

pattern.
1. Mutations in the SMN1 gene cause all types of spinal 3. The first feature of familial TAAD is usually aortic dila-
muscular atrophy (SMA). The number of copies of the tion. Aneurysms usually have no symptoms, but they can
SMN2 gene is related to the severity of the condition and cause pain in the chin, neck, chest or back, swelling in the
helps determine which type of disease develops. arms, neck or head, difficulty or pain in swallowing,
2. SMA is inherited in an autosomal dominant manner. hoarseness, shortness of breath, wheezing, chronic cough-
3. SMA is characterized by muscle weakness, which is sym- ing, or hemoptysis.
metric, proximal > distal, and progressive, and atrophy.
The onset of weakness ranges from prenatal to puberty or C.76 Waardenburg Syndrome Panel
early adulthood. Common complications include poor Genes EDN3 (20q13.32), EDNRB (13q22.3), KIT (4q12),
weight gain with growth failure, restrictive lung disease, MITF (3p13), PAX3
scoliosis, joint contractures, and sleep difficulties. (2q36.1), SNAI2 (8q11.21), SOX10 (22q13.1)
C.74 Standard Short QT Syndrome Detection Method NGS/MPS
Genes KCNH2 (7q36.1), KCNJ2 (17q24.3), KCNQ1 Reference Ranges Negative
(11p15.5-15.4) Interpretation
Detection Method Bi-directional Sanger Sequence 1. Mutations in the PAX3 gene can cause Waardenburg syn-
Analysis drome (WS) types I and III. WS type II is caused by
Reference Ranges Negative mutations in the MITF or SNAI2 gene. Mutations in the
Interpretation SOX10, EDN3, or EDNRB gene can cause WS type IV. In
some cases, the genetic cause of WS is not clear.
1. Mutations in the KCNH2, KCNJ2, and KCNQ1 genes can 2. WS is inherited in an autosomal dominant manner.
cause short QT syndrome (SQTS). 3. It is characterized by heterochromia iridis, hearing loss,
2. SQTS appears to have an autosomal dominant pattern of dystopia canthorum, and changes in the color of the skin
inheritance. and hair.
3. This is a condition that can cause arrhythmia. The part of
the heartbeat known as the QT interval for these patients C.77 WAS Sequence Analysis
is abnormally short. It can lead to a variety of signs and Gene WAS (Xp11.23)
symptoms, from dizziness and fainting (syncope) to car- Sample Required Blood
diac arrest and sudden death without treatment. From Detection Method Bi-directional Sanger Sequence
early infancy to old age, these signs and symptoms may Analysis
appear at any time. Reference Ranges Negative
Interpretation
C.75 Thoracic Aortic Aneurysm Panel
Genes ACTA2 (10q23.31), CBS (21q22.3), COL3A1 1. More than 350 pathogenic variants spanning all twelve
(2q32.2), FBN1 (15q21.1), FBN2 (5q23.3), FLNA (Xq28), exons of the WAS gene have been identified to cause a
MYH11 (16p13.11), MYLK (3q21.1), SKI (1p36.33-36.32), spectrum of disorders of hematopoietic cells, including
SLC2A10 (20q13.12), SMAD3 (15q22.33), TGFB2 (1q41), Wiskott–Aldrich syndrome, X-linked thrombocytopenia,
TGFBR1 (9q22.33), TGFBR2 (3p24.1) and X-linked congenital neutropenia. Missense variants
Sample Required Blood or nonsense variants account for about 50%.
874 Appendixes
2. The WAS-related disorders are inherited in an X-linked C.80 X-Linked Agammaglobulinemia

recessive manner. Gene BTK (Xq22.1)
3. These disorders usually appear in infancy. Male patients Sample Required Blood
present with thrombocytopenia with intermittent mucosal Detection Method Uni-directional Sanger sequencing
hemorrhage, bloody diarrhea, intermittent or chronic ery- Reference Ranges Negative
thema and purpura, eczema and recurrent infections, Interpretation
especially ear infections.
1. More than 600 different pathogenic variants in the BTK
C.78 Wilson Disease gene can cause X-linked agammaglobulinemia (XLA).
Gene ATP7B (13q14.3) Premature stop codons, splice defects, or frameshift vari-
Sample Required Blood ants account for two-thirds of them, the others are amino
Detection Method PCR with allele specific hybridiza- acid substitutions. For more than 3% of individuals, two
tion, Bi-directional Sanger Sequence Analysis or even more pathogenic variants appeared.
Reference Ranges Negative 2. XLA is inherited in an X-linked recessive manner.
Interpretation 3. XLA is characterized by recurrent bacterial infections in
affected men in the first 2 years of life. Lung infections
1. Mutations in the ATP7B gene can cause Wilson disease. (pneumonia and bronchitis), otitis media, conjunctivitis,
p.His1069Gln is the most common pathogenic variant in sinusitis, and chronic diarrhea are the most common
populations of European. The most common pathogenic infections.
variant in the Asian population is p.Arg778Leu which is
in exon 8. C.81 X-linked Severe Combined Immunodeficiency
2. Wilson disease is inherited in an autosomal recessive Gene IL2RG (Xq13.1)
manner. Sample Required Blood
3. This is a disorder of copper metabolism. Patients can Detection Method NGS/MPS
experience hepatic and/or neurologic, or psychiatric dis- Reference Ranges Negative
turbances ranging from 3 years old to over 50 years old. Interpretation
Different patients have different symptoms. Due to the
deposition of copper on the Descemet’s cornea, patients 1. More than 200 pathogenic variants spanning all eight
often have Kayser–Fleischer rings that reflects the high exons of the IL2RG gene have been identified which can
storage of copper in the body. cause X-linked severe combined immunodeficiency
(SCID). It is reported that mutational hot spots are at
C.79 Wolfram Syndrome Comprehensive NGS Panel codons p.Arg224, p.Arg226, p.Arg285, and p.Arg289.
Gene WFS1 (4p16.1), CISD2 (4q24) 2. X-linked SCID is inherited in an X-linked recessive

Detection Method NGS/MPS manner.
Sample Required Blood 3. This immune system disorder occurs almost exclusively
Reference Ranges Negative in men. Boys with X-linked SCID are prone to relapses
Interpretation and persistent infections due to the lack of necessary
immune cells to fight off certain pathogens. Many affected
1. Mutations in the WFS1 gene cause more than 90% of babies develop chronic diarrhea, a fungal infection, and
Wolfram syndrome type 1 cases. A certain mutation in the skin rashes called thrush. They may grow more slowly
CISD2 gene was identified to cause Wolfram syndrome than others, and usually do not live beyond infancy with-
type 2. out treatment.
2. Wolfram syndrome is inherited in an autosomal dominant
manner. C.82 Zellweger Syndrome Panel
3. The main features of Wolfram syndrome are hyperglyce- Genes PEX1 (7q21.2), PEX10 (1p36.32), PEX12 (17q12),
mia caused by diabetes and progressive loss of vision PEX13 (2p15), PEX14 (1p36.22), PEX16 (11p11.2), PEX19
caused by optic atrophy. Patients also often have pituitary (1q23.2), PEX2 (8q21.13), PEX26 (22q11.21), PEX3
dysfunction, leading to diabetes insipidus, sensorineural (6q24.2), PEX5 (12p13.31), PEX6 (6p21.1)
deafness, urinary tract problems, hypogonadism, neuro- Sample Required Blood
logical or psychiatric disorders. Detection Method PCR with allele specific hybridiza-
tion, Bi-directional Sanger Sequence Analysis/NGS/MPS
Interpretation
Appendixes 875
1. Mutations in at least 12 genes (PEX1, PEX2, PEX3,

Common polymorphic loci
PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, 5792C>T and −444A>C
PEX19, and PEX26 genes) have been identified to cause Detection method
Zellweger syndrome (ZS). These variants span the full The GP1BA gene polymorphism detection method can be
gamut of DNA nucleotide substitutions and indel varia- sequenced using the second generation.
tion (small and large) associated with human disease. The
only common variants (p.Ile700TyrfsTer42 and p. D.1.2 Clopidogrel
Gly843Asp) are found in PEX1. Drug introduction
2. ZS is inherited in an autosomal recessive manner.
3. Patients with ZS develop symptoms during the neonatal 1. It is used to prevent and treat heart, brain, and other arte-
period. These babies experience low tension, eating prob- rial circulatory disorders caused by high platelet aggrega-
lems, hearing and vision loss, and epilepsy. They may tion, such as recent onset stroke, myocardial infarction,
have skeletal abnormalities, unique facial features (flat and confirmed peripheral arterial disease.
face, broad nose, and high forehead), and generally do not 2. It is a platelet aggregation inhibitor that selectively inhib-
live 1 year old. its the binding of ADP to the platelet receptor and inhibits
the activation of the ADP-mediated glycoprotein GPIIb/
IIIa complex, while inhibiting platelet aggregation. Non-
Appendix D: Pharmacogenomics ADP-induced platelet aggregation can also be inhibited.
A large number of research results show that the majority of Gene profile
individual drug response differences are mainly caused by
genetic factors. The gene type of patient drug metabolizing 1. The gene that affects the drug treatment and evaluates the
enzymes, drug transporters, and drug-targeting proteins can dose of the drug is CYP2C19.
affect pharmacokinetics and pharmacodynamics, leading to 2. CYP2C19 is an important member of the second subfam-
individual differences in drug effects. Through genetic test- ily of CYP450 enzyme, and is an important drug metabo-
ing, we can obtain the genotype of patients, achieve “quanti- lizing enzyme in the human body, which is expressed in
tative drug” treatment, reduce adverse drug reactions, release the liver. The CYP2C19 locus is located on chromosome
the pain and economic burden of patients, achieve the perfect 10q24.2 and consists of 9 exons. CYP2C19 has 92%
combination of pharmacogenomics and clinical drug ther- sequence homology with CYP2C9.
apy. The diversity of drug effects depends on the genetic
polymorphism. Common polymorphic loci
681G>A, 636G>A and −806C>T
D.1 Anticoagulation Therapy Detection method
D.1.1 Aspirin CYP2C19 gene polymorphism detection methods usually
Drug introduction use real-time PCR.
1. Aspirin (Aspirin, acetylsalicylic acid) is a white crystalline D.1.3 Warfarin

or crystalline powder, odorless or microstrip acetic acid Drug introduction
odor, slightly soluble in water, soluble in ethanol, soluble in It is a kind of coumarin anticoagulant and has anti-vitamin
ether, chloroform, and acidic in aqueous solution. K effect in the body. It can inhibit the synthesis of coagulation
2. It has proved to be effective in relieving mild or moderate factors II, VII, IX, and X involved in vitamin K in the liver. It
pain, such as toothache, headache, neuralgia, muscle is mainly used to prevent thromboembolic diseases.
aches, and dysmenorrhea. It is also used for colds, flu, Gene profile
antipyretic fever, treatment of rheumatic pain, and so on.
In recent years, it has been found that aspirin has an inhib- 1. The genes affecting drug therapy and evaluating drug
itory effect on platelet aggregation and can prevent throm- dosage are CYP2C9 and VKORC1.
bosis. It is clinically used to prevent transient ischemic 2. CYP2C9 is a p450 enzyme. The main enzymes of metab-
attack, myocardial infarction, artificial heart valve and olites in the human body are Cytochrome P450 proteins
venous fistula, or other postoperative thrombosis. (CYP), which are monooxygenases mainly found in the
liver and intestines. They are located on the endoplasmic
Gene profile reticulum of the cell and catalyze a variety of metabolism
The genes affecting drug treatment and evaluating drug of foreign substances (including most clinical drugs).
dosage are GP1BA and LTC4S.
876 Appendixes
3. CYP has multiple subfamilies, of which CYP2C9

50–1658T>G
(Cytochrome P450 2C9) is an important member of the Detection method
second subfamily, accounting for 20% of the total amount Direct sequencing technique is used to detect the geno-
of liver microsomal P450 protein. CYP2C9 is capable of type of the SNP locus of CACNA1C gene.
hydroxylating and metabolizing many different drugs,
mainly acidic substrates. D.2.3 Angiotensin II Receptor Inhibitor
Gene profile
Common polymorphic loci The genes that influence drug treatment are CYP2C9 and
CYP2C9: 1075A>C AGTR1.
VKORC1: 1639G>A Common polymorphic loci
Detection method and result interpretation CYP2C9: 1075A>C
The CYP2C9 gene and VKORC1 gene polymorphism AGTR1: 1166A>C
detection methods mostly use real-time quantitative PCR Detection method and result interpretation
TaqMan technology. Samples covering the CYP2C9 gene The CYP2C9 gene polymorphism detection method
homozygous wild type, heterozygous mutant, VKORC1- mostly uses real-time fluorescent quantitative PCR TaqMan
1639G>A homozygous mutant and VKORC1-1639G>A technology. A homozygous wild type, heterozygous mutant
heterozygous mutant were selected, and the sample DNA sample covering the CYP2C9 gene was selected. The sample
was extracted for real-time quantitative PCR amplification. DNA was extracted and subjected to real-time fluorescent
quantitative PCR amplification detection.
D.2 Antihypertensive Therapy
D.2.1 β1 Receptor Antagonist D.2.4 Angiotensin-Converting Enzyme Inhibitor
Drug introduction Drug introduction
The beta receptor antagonist is a drug that is contraindi- Angiotensin-converting enzyme inhibitor (ACEI) is a
cated in patients with severe left ventricular dysfunction, compound that inhibits angiotensin-converting enzyme
sinus bradycardia, severe atrioventricular block, and bron- activity. Angiotensin-converting enzyme catalyzes the pro-
chial asthma. Patients with myocardial infarction and liver duction of angiotensin II by angiotensin I, which is a potent
dysfunction should be used with caution. vasoconstrictor and activator of adrenocortical aldosterone
Gene profile release.
Gene profile
1. The genes that affect drug therapy and evaluate drug use The gene that affects drug treatment is ACE.
doses are ADRB1 and CYP2D6. Detection method and result interpretation
2. CYP2D6 is an enzyme encoded by the human CYP2D6 The ACE gene polymorphism detection method has the
gene which is mainly expressed in the liver and is also traditional Rigat method and the advanced three primer
highly expressed in the central nervous system, including methods commonly used in China. The results of agarose gel
the substantia nigra. electrophoresis or polyacrylamide gel electrophoresis are
often used as the interpretation criteria, stained with ethid-
Common polymorphic loci ium bromide, and observed under ultraviolet light.
ADRB1: 1165G>C
CYP2D6: 100C>T D.2.5 Thiazine Diuretic
Detection method and result interpretation Drug introduction
The CYP2D6*10 gene detection method mostly uses the
ASA PCR method. Two primers complementary to the 1. It is an antihypertensive drug that can be used alone to
unique intrinsic sequence of the CYP2D6*10 gene were treat early hypertension or to treat moderate to severe
designed to generate a 790 bp fragment as a control. The hypertension with other antihypertensive drugs.
experiment was carried out in two tubes, and the wild type 2. Thiazide can enhance the excretion of NaCl and water,
and CYP2D6*10 type were, respectively, verified by two producing a mild and long-lasting diuretic effect, but
specific primers, and the results were based on agarose gel long-term use can lead to hypokalemia.
electrophoresis bands.
Gene profile
D.2.2 Calcium Channel Blocker of Dihydropyridine The genes that influence drug treatment are ADD1 and
Gene profile PPKCA.
The gene that affects drug therapy is CACNA1C. Common polymorphic loci
Common polymorphic loci ADD1: 1378G>T
Appendixes 877
PPKCA: 1854+3730G>A TaqMan − MGB Needle Ct value − Type A

Detection method TaqMan − MGB probe Ct value) Determine genotype.
ADD1 gene polymorphism detection mostly uses PCR-
SSCP and DNA sequencing technology. Primers were D .5 Anti-inflammatory Treatment
designed based on different base variations. D.5.1 Celecoxib
Drug introduction
D.2.6 Furosemide It is used to relieve the symptoms and signs of osteoarthri-
Gene profile tis, relieve the symptoms and signs of rheumatoid arthritis,
The gene that affects drug treatment is ADD1. and treat acute pain in adults.
Detection method Gene profile
ADD1 gene polymorphism detection mostly uses PCR- The gene that affects the therapeutic effect of the drug and
SSCP and DNA sequencing technology. Primers were evaluates the dose of the drug is CYP2C9.
designed based on different base variations. Common polymorphic loci
1075A>C
D.3 Anticardiac Insufficiency Detection method
D.3.1 Digoxin Real-time fluorescent quantitative PCR TaqMan
Drug introduction technology.
Digoxin is a medium-effect cardiac glycoside drug, which
is white crystal or crystalline powder, odorless with bitter D.6 Antigout Treatment
taste. At the time of treatment, the effect on the heart is a D.6.1 Allopurinol
positive inotropic effect, slowing heart rate and inhibiting Drug introduction
cardiac conduction. Suitable for low-output congestive heart Allopurinol and its metabolites can inhibit xanthine oxi-
failure, atrial fibrillation, atrial flutter, paroxysmal supraven- dase, so that hypoxanthine and xanthine cannot be converted
tricular tachycardia. into uric acid, that is, uric acid synthesis is reduced. It reduces
Gene profile blood uric acid concentration and urate in bones and joints.
The gene for evaluating the dose of the drug is ABCB1. Gene profile
Common polymorphic loci The genes that affect the therapeutic effect of the drug and
3435C>T evaluate the dose of the drug are HLA-B*58:01 and ABCG2.
Detection method Common polymorphic loci
The ABCB1 gene was detected using Sanger HLA-B*58: 01:52A>T
sequencing. ABCG2: 421C>A
Detection method and result interpretation
D.4 Antiangina Pectoris Treatment
D.4.1 Glonoine 1. HLA-B*58:01 gene detection can be performed by fluo-
Drug introduction rescence in situ hybridization.
Nitroglycerin can directly relax vascular smooth muscle, 2. The in situ hybridization fluorescence staining analysis
especially small vascular smooth muscle, relax peripheral system automatically interprets the fluorescence signal
vasodilation, reduce peripheral resistance, reduce blood flow, value to obtain the fluorescence curve for HLA-B*5801
reduce cardiac output, reduce cardiac load, reduce myocar- genotyping and positive control. When one of the two
dial oxygen consumption, and relieve angina. fluorescent signals is higher than the internal reference
Gene profile signal, the site is positive, so the results of “+/−” and
The gene that affects the therapeutic effect of drugs is “+/+” indicate HLA-B*.
ALDH2.
Common polymorphic loci D.7 Antipeptic Ulcer Treatment
1510G>A D.7.1 Lansoprazole
1. Real-time fluorescent quantitative PCR is often used for 1. Lansoprazole, alias Dakpron, Langsonaze, is a white

ALDH2 gene polymorphism detection. crystalline powder of chemicals.
2. The TaqMan-MGB double probe was used to establish an 2. The benzimidazole compound is orally absorbed and

allelic typing method based on the difference ΔCt of the transferred to the gastric mucosa, and is converted into an
cycle threshold (Ct value) of the fluorescence signal of the active metabolite under acidic conditions, and the activa-
allele G and type A TaqMan-MGB probes (ΔCt = G type tor specifically inhibits the final step of gastric acid cell
878 Appendixes
secretion by blocking the H+/K+-ATPase system of the Detection method

gastric parietal cells. Real-time fluorescent quantitative PCR.
Gene profile D.8.2 Citalopram

The gene that affects the therapeutic effect of drugs is Drug introduction
CYP2C19. Citalopram is a selective serotonin reuptake inhibitor
Detection method (SSRI) that is a racemate. It can selectively inhibit the 5-HT
Real-time fluorescent quantitative PCR. transporter, block the reuptake of 5-HT by the presynaptic
D.7.2 Omeprazole membrane, prolong and increase the effect of 5-HT, thereby
Drug introduction producing an antidepressant effect.
Gene profile
1. Omeprazole, mainly used for duodenal ulcer and Zhuo- The genes that influence the therapeutic effect of drugs
Eye syndrome, can also be used for gastric ulcer and are CYP2C19 and FKBP5.
reflux esophagitis; intravenous injection can be used for Detection method
the treatment of acute bleeding of peptic ulcer. Real-time fluorescent quantitative PCR.
2. Combined with amoxicillin and clindamycin or with met-
ronidazole and clarithromycin to kill Helicobacter pylori. D.8.3 Escitalopram
As a proton pump inhibitor, it is a fat-soluble weakly Drug introduction
basic drug. Aspirin, its English name is escitalopram, the molecular
formula is C20H21FN2O, the CAS accession number is
Gene profile 128196-01-0, and the ATC code is N06AB10.
The gene that affects the therapeutic effect of drugs is Gene profile
CYP2C19.
Detection method 1. CYP2C19 is an important member of the second subfam-
Real-time fluorescent quantitative PCR. ily of CYP450 enzymes and is an important drug metabo-
D.7.3 Pantoprazole lizing enzyme in the human body.
Drug introduction 2. It is expressed in the liver. The CYP2C19 locus is located
on chromosome 10q24.2 and consists of 9 exons.
1. Clinically, it is a proton pump inhibitor drug that inhibits
gastric acid secretion. For the treatment of active peptic Detection method
ulcer reflux esophagitis and Zhuoyi’s syndrome. Real-time fluorescent quantitative PCR.
2. Pantoprazole is a third-generation proton pump inhibitor
that selectively acts on gastric mucosal cells, inhibits the D.9 Antipsychotic Treatment
activity of H, K-ATPase in parietal cells, and prevents H D.9.1 Risperidone
in parietal cells from being transported into the stomach. Drug introduction
Inhibition of gastric acid secretion.
1. For the treatment of acute and chronic schizophrenia.
Gene profile 2. It has a high affinity with the 5-HT2 receptor and the
The gene that affects the therapeutic effect of drugs is dopamine D2 receptor.
CYP2C19. 3. The oral absorption of this product is rapid and complete,
Detection method and its absorption is not affected by food. The blood drug
Real-time fluorescent quantitative PCR. peak concentration is reached after 1 h of administration,
and the elimination half-life is about 3 h. Most patients
D.8 Antidepressant Therapy reach steady state within 1 day.
D.8.1 Amitriptyline
Drug introduction Gene profile
Amitriptyline is used to treat various types of depression The gene that affects the efficacy is DRD2.
or depression. It can relieve chronic pain and it is also used Detection method and result interpretation
to treat children with enuresis and children with ADHD.
Gene profile 1. The real-time fluorescent quantitative PCR method is com-
CYP2C19 is an important member of the second subfam- monly used for the detection of DRD2 gene polymorphism.
ily of CYP450 enzyme and is an important drug metaboliz- 2. The DRD2 gene polymorphism site was amplified by
ing enzyme in the human body. gene amplification, and the obtained PCR product was
sequenced using a pyrosequencing apparatus.
Appendixes 879
D.10 Antiepileptic Treatment Gene profile

D.10.1 Carbamazepine The gene for evaluating the dose of the drug is
Drug introduction HLA-B*15:02.
The anticonvulsant mechanism of carbamazepine is Detection method
unclear and may be associated with its ability to increase Specimen DNA was extracted and subjected to routine
sodium channel inactivation, limit post-synaptic neurons, detection of HLA high-resolution typing by polymerase
and block presynaptic Na+ channels, thereby limiting pre- chain reaction-specific oligonucleotide probe hybridization
and post-synaptic neurons. (PCR-SSOP).
Gene profile
The gene for carbamazepine to evaluate the drug dose is D.11Antileukemia Treatment
HLA-B*15:02 or HLA-A*31:01, located on the short arm of D.11.1 Methotrexate
chromosome 6. Drug introduction
Detection method Methotrexate is an anti-folate anti-tumor drug. This prod-
Extracting specimen DNA and performing routine detec- uct is an orange-yellow crystalline powder. It inhibits the
tion of HLA high-resolution typing by polymerase chain synthesis of tumor cells mainly by inhibiting the synthesis of
reaction-specific oligonucleotide probe hybridization dihydrofolate reductase, and inhibits the growth and repro-
(PCR-SSOP). duction of tumor cells. Tetrahydrofolate is an important
coenzyme for the synthesis of purine nucleotides and pyrimi-
D.10.2 Dilantin dine deoxynucleotides in vivo. As a folate reductase inhibi-
Drug introduction tor, this product mainly inhibits dihydrofolate reductase and
prevents dihydrofolate from being reduced to physiological
1 . Phenytoin is an antiepileptic drug. activity. The tetrahydrofolate, which hinders the transfer of a
2. This product belongs to class IB anti-arrhythmia drug, mono-carbon group during the biosynthesis of purine nucle-
which has membrane stability and inhibits fast sodium otides and pyrimidine nucleotides, results in inhibition of
ion influx. DNA biosynthesis.
Gene profile
Gene profile
1. The genes evaluating its dose were SLCO1B1, MTRR,
1. The gene used to evaluate the dose of the drug is HLA-B and MTHFR.
or CYP2C9. 2. The SLCO1B1 gene is a key factor in the adverse reac-
2. CYP has multiple subfamilies, of which CYP2C9
tions caused by statins. The mutant SLCO1B1 gene can
(Cytochrome P450 2C9) is an important member of the cause a decrease in the ability of the liver to ingest statins,
second subfamily, accounting for 20% of the total amount causing an increase in blood concentration and an increase
of liver microsomal P450 protein. in the risk of rhabdomyolysis or myopathy. Methionine
3. The CYP2C9 gene is located on the chromosomal region synthase reductase (MTRR). The mutation of the 66th site
10q24.2 and has a full length of about 55 kb and is com- of the MTRR gene in children increases the risk of con-
posed of 9 exons. CYP2C9 shares 92% sequence homol- genital heart disease (CHD) in the offspring. The muta-
ogy with another P450 enzyme, CYP2C19, but these two tion of the 66th site of the mother MTRR gene significantly
enzymes have completely different substrate specificities. increases the risk of CHD in the offspring; the mother has
MTRR and MTR. In combination with variants, the risk
Detection method of CHD in their offspring increases.
Specimen DNA was extracted and subjected to routine 3. The genes closely related to folate metabolism are

detection of HLA high-resolution typing by polymerase MTHFR and MTRR. When these genes are mutated, the
chain reaction-specific oligonucleotide probe hybridization use of folic acid in pregnant women is inefficient, thereby
(PCR-SSOP) increasing the risk of neonatal birth defects or spontane-
ous abortion. The folate metabolism gene detection is
D.10.3 Oxcarbazepine mainly to detect the three sites of human metabolism folic
Drug introduction acid related genes MTHFR and MTRR genes
(MTHFR677C>T, MTHFR1298A>C, MTRR66A>G), to
1. Oxcarbazepine, a neurological drug, can be used for
evaluate the individual’s folate metabolism ability, to
localized and systemic seizures. achieve personalized supplementation of folic acid.
2. Oxcarbazepine is mainly used for clinically allergic reac- Objective: To scientifically guide folic acid supplementa-
tions to carbamazepine, and can be used as an alternative tion and reduce the risk of neonatal birth defects.
medicine for carbamazepine.
880 Appendixes
Detection method and result interpretation 2. It is a S phase specific anti-tumor drug with a retarding
PCR-fluorescent probe method is often used for SLCO1B1 effect on the S/G2 boundary. It is a commonly used spu-
gene detection. Real-time PCR detection The SLCO1B1 tum metabolic antagonist for inhibiting the synthesis of
A388G&T521C assay was performed according to the purines. It is a cell cycle-specific drug. It is most sensitive
instructions of the human SLCO1B1 gene detection kit, and to cells in S phase. In addition to inhibiting the synthesis
a positive/negative control (provided with the kit) was per- of cellular DNA, it also has a slight inhibitory effect on
formed. Fluorescence quantitative PCR reactions were per- RNA synthesis. This product is an analogue of guanine,
formed using the FQD96A real-time PCR instrument and the which must be converted from a phosphoribosyltransfer-
PCR 9600 PLUS software. PCR cycle parameters: 37 °C for ase to a 6-TG ribonucleotide in the human body.
10 min (UNG enzyme treatment decontamination); 95 °C
5 min; 95 °C 15 s, 60 °C 60 s, 40 cycles. Set the FAM, VIC, Gene profile
and ROX 3 channels to collect fluorescence signals. The genes for evaluating its dose were TPMT*3C and
Interpretation of the results: The end of the PCR amplifica- NUDT15.
tion, the SLCO1B1 genotype was determined based on the Detection method
amplification of the fluorescence curve of each sample. PCR-RFLP.
D.11.2 6-Mercaptopurine, Imuran D.12 Antineoplaston

Drug introduction D.12.1 Capecitabine
It is an anti-tumor drug, which is effective for acute leuke- Drug introduction
mia and is also effective for chronic myeloid leukemia; it is
used for chorionic epithelial cancer and malignant mole. 1. Tegafur digestive cancer has a certain effect on gastric
Mechanism of action of 6-mercaptopurine: 6-MP competi- cancer, colon cancer, rectal cancer, pancreatic cancer. It is
tively inhibits hypoxanthine-guanine phosphoribosyltrans- also effective for breast cancer and liver cancer.
ferase, which prevents the phosphoribosyl ribose in the 2. Capecitabine is rapidly absorbed by the intestinal mucosa
PRPP molecule from transferring to guanine and hypoxan- after oral administration, and then converted into an inac-
thine, and blocks the remedial synthesis pathway of purine tive intermediate 5′-deoxy-5-fluorocytidine in the liver by
nucleotides. carboxylesterase, followed by cytidine removal from liver
Gene profile and Common polymorphic loci and tumor tissues. The action of the aminoase is con-
verted to 5′-deoxy-5-fluorouridine and finally catalyzed
1. The genes for evaluating its dosage are TPMT*3C and by thymidine phosphorylase to fluorouracil (5-FU) in
NUDT15. tumor tissues.
2. TMTP*3C is one of the three common genes that cause a
decrease in TMTPase activity. Common polymorphism is Gene profile
719 A to G. The gene that affects the efficacy of drugs is DPYD.
Detection method
Detection method Sequencing can be used for the detection of the polymor-
The NUDT15 415C>T and TPMT*3C genotypes are phism of the DPYD gene.
determined by PCR-RFLP.
D.12.2 Carboplatin
D.11.3 Thioguanine Drug introduction
Drug introduction
1. Carboplatin is a broad-spectrum anti-tumor drug, has no
1. The chemical name of thioguanine is 2-aminoindole- cross-resistance with other anti-tumor drugs, and has
6(H)thione, which is white or yellowish powder, has a cross-resistance with DDP. The drug is easy to dissolve,
umami taste, is easily soluble in water, and is insoluble does not require hydration, diuresis, diuresis, and is con-
in organic solvents such as ethanol and acetone. It is venient to use. It mainly used for small cell lung cancer,
mainly used to treat acute leukemia. It also has a certain ovarian cancer, testicular tumor, head and neck squamous
effect on chronic myeloid leukemia. Its action is similar cell carcinoma.
to that of thiopurine, and it is active after being con- 2. In small cell lung cancer, the remission rate of this prod-
verted into thioguanosine monophosphate (6-TGRP) in uct alone was 60%; the total remission rate of treatment
the body. Finally, it is converted into deoxyguanine with Vp-16 and IFO was 78%; the remission rate of ovar-
nucleotides, which interfere with DNA function and ian epithelial cancer and head and neck squamous cell
produce anticancer effects. carcinoma was 65% and 29%, respectively. It can be used
for non-small cell lung cancer, bladder cancer, cervical
Appendixes 881
cancer, pleural mesothelioma, melanoma and endometrial idase or phosphatase present in the liver or tumor in the
cancer. It can also be used for digestive system tumors, human body and becomes an activated phosphoamido
liver cancer, etc. and radiotherapy. mustard. It has the broad anti-tumor spectrum and is the
first so-called latent broad-spectrum anti-tumor drug,
Gene profile which is effective for leukemia and solid tumors.
The gene that affects its efficacy is MTHFR. 2. This product is inactive in vitro, mainly through the

Detection method hydrolysis of liver P450 enzyme into aldehyde phos-
MTHFR gene polymorphism detection is commonly used phoramide and then run into the tissue to form phos-
in PCR-fluorescence probe method. phoramide mustard. Cyclophosphamide can be inactivated
by the conversion of dehydrogenase to carboxy-
D.12.3 Cisplatin phosphoramide or in the form of acrolein, resulting in
Drug introduction urinary tract toxicity. It is a periodic non-specific drug
with the same mechanism of action as nitrogen mustard.
1. Cisplatin is an orange-yellow or yellow crystalline pow-
der. Slightly soluble in water, soluble in dimethylfor- Gene profile
mamide. It can be gradually converted into trans and
hydrolyzed in aqueous solution. Clinically used for ovar- 1. The genes affecting the efficacy of the drug and evaluat-
ian cancer, prostate cancer, testicular cancer, lung cancer, ing its dosage are MTHFR, GSTP1, and SOD2.
nasopharyngeal cancer, esophageal cancer, malignant 2. The gene GSTP1 encoding GSTπ is located on chromo-
lymphoma, head and neck squamous cell carcinoma, thy- some 11q13, contains 7 exons and 6 introns, and there are
roid cancer and osteosarcoma can show efficacy. gene polymorphisms in exons 5 and 6. The A→G base
2. The role of tumors is of great significance. But only cis is occurs at the 81st position of exon 5 Substituting, can lead
meaningful, and trans is invalid. It can cross-link with to the 105th amino acid of the protein peptide chain
DNA strands and shows cytotoxic effects. After dissolving, changed from ATC isoleucine (Ile) to GTC proline (Va1),
it can pass through the charged cell membrane without car- which is recorded as GSTP1-I105V, GSTP1-I105V
rier transport in the body. Due to the low concentration of polymorphism, which can produce 105Ile/Ile, 105Ile/Val,
intracellular chloride ions (4 mmol/L), chloride ions are and 105Val/Val three genotypes.
replaced by water, and the charge is positive. It has the 3. The presence of GSTs gene polymorphisms can cause
function of a bifunctional group similar to an alkylating different activities of the corresponding enzymes that are
agent. It can bind to the base of DNA in the nucleus to form expressed, leading to changes in detoxification function,
three forms. Cross-linking causes DNA damage, disrupts thereby increasing the risk of specific tumors. Studies
DNA replication and transcription, and inhibits RNA and have shown that the frequency of distribution of different
protein synthesis at high concentrations. Cisplatin has the genotypes of GSTP1 has significant ethnic and regional
advantage of wide anticancer spectrum. differences. SOD2 contains manganese, 4p15.3-p15.1
located on chromosome 4.
Gene profile
The genes affecting the efficacy of the drug and evaluating Detection method and result interpretation
its dosage are XPC, TP53, GSTP1, and ERCC1. XPC provides Gene detection was performed by establishing artificially
instructions for making a protein involved in repairing damaged modified biallelic specific primers in combination with
DNA. Ultraviolet rays from the sun, toxic chemicals, radiation, SYBR Green I fluorescent PCR.
and unstable molecules called free radicals destroy DNA.
Detection method and result interpretation D.12.5 Fluorouracil
Gene detection was performed by establishing artificially Drug introduction
modified biallelic specific primers in combination with
SYBR Green I fluorescent PCR. The genotype is determined 1. Fluorouracil acts as an antimetabolite and, after being
based on the PCR product melting curve analysis. When the converted into an effective fluorouracil deoxynucleotide
melting peak appeared at (90.0 ± 0.5) °C and (90.5 ± 0.5) °C, in the cell, interferes with DNA by blocking the conver-
respectively. sion of deoxyribouroperidic acid to thymidylate by intra-
cellular thymidylate synthase. synthesis. Fluorouracil can
D.12.4 Cyclophosphamide also interfere with the synthesis of RNA. After intrave-
Drug introduction nous administration, fluorouracil is widely distributed in
body fluids and disappears from the blood within 4 h. It is
1. Cyclophosphamide (CTX) is a nitrogen mustard deriva- converted to nucleotides. It is preferentially taken up by
tive that acts upon activation of an excess of phosphoram- actively dividing tissues and tumors, and fluorouracil eas-
882 Appendixes
ily enters the cerebrospinal fluid. About 20% of the proto- kinase and metabolized by cytidine deaminase. This prod-
type is excreted from the urine, and most of the rest is uct is a pyrimidine anti-tumor drug, the mechanism of
metabolized in the liver by a mechanism that is generally action is the same as that of cytarabine, and its main
metabolized by uracil. metabolite is incorporated into DNA in cells, mainly in the
2. Pentafluorouracil and 6-mercaptopurine were the earliest G1/S phase. However, the difference is that in addition to
anticancer drugs, which were all extracted from sea cucum- the incorporation of DNA, difluorodeoxycytidine inhibits
ber. This product needs to be converted into the ribonucleotide reductase, resulting in a decrease in
5-fluorodeoxyuridine nucleotide by enzyme to have anti- intracellular deoxynucleoside triphosphate; another differ-
tumor activity. 5-FU inhibits DNA synthesis by inhibiting ence from cytarabine is that it inhibits deoxygenation.
thymidine nucleotide synthetase. The action of this enzyme Pyrimidine deaminase reduces the degradation of intracel-
may transfer one carbon unit of formyltetrahydrofolate to lular metabolites and has a self-enhancing effect.
deoxyuridine-phosphate to synthesize thymidine monoacid. 2 . Clinically, this product and cytarabine have different anti-
5-FU also has a certain inhibitory effect on the synthesis of tumor profiles and are effective against a variety of solid
RNA fluorouracil. 5-FU can be injected intravenously and tumors. For patients with advanced pancreatic cancer, as
intraluminally. 5 Fluorouracil was continuously adminis- a second-line medication after fluorouracil failure, it can
tered to patients with bladder cancer at a constant rate. improve the quality of life of patients; secondly, it is a
3. The concentration of the drug was the lowest at 10
first-line application for locally advanced (stage III) and
o’clock, and the highest concentration was between 22 already metastatic (stage IV) non-small cell lung cancer.
and 3 o’clock. When the constant velocity drop is not Recent data indicate that this product has palliative effects
used, the peak flow rate is set at 4 am, which allows the on ovarian cancer, breast cancer, bladder cancer, cervical
dose to be greatly increased and the toxicity is extremely cancer, liver cancer, biliary tract cancer, nasopharyngeal
low, and the therapeutic effect is enhanced. cancer, testicular tumor, lymphoma, mesothelioma, and
4. In addition, 5-FU metabolites can also enter the RNA and head and neck cancer.
DNA in the form of pseudo-metabolites, affecting cell func-
tion and producing cytotoxicity. 5-FU is an atypical cell Gene profile
cycle-specific drug that, in addition to its primary action on The gene associated with this drug is NT5C2. This gene
S phase, also has an effect on cells in other phases. encodes a hydrolase that acts primarily on inosine 5′-mono-
phosphate and other purine nucleotides and plays an important
Gene profile role in cell raft metabolism. It may play a key role in maintain-
ing a constant composition of the intracellular purine/pyrimi-
1. The genes affecting this therapeutic effect and the dose dine nucleotides in synergy with other nucleotidase enzymes.
for evaluation are DPYD and TP53. Preference is given to the hydrolysis of inosine 5′-monophos-
2. TP53 is a gene located on chromosome 17pl3.1, 20 kb phate (IMP) and other purine nucleotides. The location of the
long, containing 11 exons, encoding a protein of 393 amino cell: 10q24.32-q24.33, is the long (q) arm of chromosome 10
acids. P53 protein is a transcription factor involved in cell between 24.32 and 24.33. Molecular position: base pair 103,
cycle regulation, DNA repair, cell differentiation, and cell 088, 017 to 103, 193, 306 on chromosome 10 (Homophone
regulation. It mainly performs the DNA check “check- Update Note Version 109.20190607, GRCh38.p13).
point” function. If the DNA is damaged, the P53 protein Detection method
level rises rapidly and activates its downstream p2I/WAFI/ Gene detection was performed by establishing artificially
CIP/gene expression, which is a group of cyclin-dependent modified biallelic specific primers in combination with
protein kinases (cyclin). It arrests cells in the G phase and SYBR Green I fluorescent PCR.
performs DNA repair. If the repair fails, TP53 induces
death by activating the BAX gene pathway. Approximately D.12.7 Oxaliplatin
50% of human tumors are associated with allelic inactiva- Drug introduction
tion or mutation of the TP53 gene. Oxaliplatin is a third-generation platinum anticancer
drug, a platinum compound of diaminocyclohexane, in
Detection method which a 1,2-diaminocyclohexane group is substituted for the
Sequencing can be used for the detection of the polymor- amino group of cisplatin. It has the same action as other plat-
phism of the DPYD gene. inum drugs, that is, DNA is the target site, and platinum
atoms form a cross-link with DNA to antagonize its replica-
D.12.6 Gemcitabine tion and transcription. Patients with colorectal cancer who
Drug introduction are ineffective in 5-FU therapy are still effective against
other platinum-resistant patients.
1. Gemcitabine is a new cytosine derivative. Like cytarabine,
it enters the human body and is activated by deoxycytidine Gene profile
Appendixes 883
1. The gene that affects the efficacy of this drug is GSTP1. expressed at high levels in both the gonads and the brain.”
The gene encoding GSTπ, GSTP1, is located on chromo- The gene is located at the 15q21.2 region of the chromo-
some 11q13, contains 7 exons and 6 introns, and there are some and is about 123 kb in length, including a 30 kb
gene polymorphisms in exons 5 and 6. coding region and a 93 kb regulatory region. A large num-
2. The A→G base occurs at the 81st position of exon 5. ber of related studies in recent years have shown that the
Alternatively, the 105th amino acid of the protein peptide genetic polymorphism of CYP19A1 is associated with
chain can be changed from ATC isoleucine (Ile) to GTC AD. Mutations in some of the CYP19A1 loci increase the
proline (Va1), which is recorded as GSTP1-I105V, risk of AD.
GSTP1-I105V polymorphism, which can produce 105Ile/
Ile, 105Ile/Val, and 105Val/Val genotypes. The presence Detection method and result interpretation
of GSTs gene polymorphisms can cause different activi- The CYP2D6*10 gene detection method mostly uses the
ties of the corresponding enzymes that are expressed, ASA PCR method. Two primers complementary to the
leading to changes in detoxification function, thereby unique intrinsic sequence of the CYP2D6*10 gene were
increasing the risk of specific tumors. Studies have shown designed to generate a 790 bp fragment as a control. The
that the frequency of distribution of different genotypes experiment was carried out in two tubes, and the wild type
of GSTP1 has significant ethnic and regional and CYP2D6*10 type were, respectively, verified by two
differences. specific primers, and the results were based on agarose gel
electrophoresis bands.
313A>G D.12.9 Vincristine
Detection method Drug introduction
GSTP gene detection was performed by establishing arti-
ficially modified biallelic specific primers in combination 1. Vincristine (Oncovin, VCR) is an alkaloid extracted from
with SYBR Green I fluorescent PCR. the vinca flower of the oleander family. It has a good anti-
tumor effect and is currently used as a clinical anti-tumor
D.12.8 Tamoxifen drug. It is found in the periwinkle plant, periwinkle. When
Drug introduction recrystallization in methanol, it is needle crystal. The
melting point is 211–216 °C.
1. Tamoxifen is used to treat advanced breast and ovarian 2. It also has effects on mouse Ridgeway osteosarcoma,
cancer. In the clinical treatment of breast cancer, the Mecca lymphosarcoma, X-5563 myeloma and the like.
effective rate is generally 30%, the estrogen receptor- Vincristine has a significant anticancer effect on cultured
positive patients have better efficacy (49%), and the nega- human hepatoma cell line (SMMC-7721). When vincris-
tive patients have poor efficacy (7%). Both premenopausal tine is 100 and 50 ng/mL, most of the cancer cells can be
and postmenopausal patients can be used, and postmeno- rounded. The envelope becomes thick and falls off into a
pausal and 60 years of age or older are better than pre- suspended state.
menopausal and younger patients. 3. At the concentration of 100 and 50 ng/mL, most of the
2. From the point of view of the lesion, the skin, lymph tumor cells were rounded, the membrane became thick,
nodes, and soft tissues are effective, and the effect of bone and the shedding was suspended. When vincristine was
and visceral metastasis is poor. For the synthesis of anti- applied at a concentration of 100 ng/mL on the next day
estrogen drugs. and 50 ng/mL on the 6th day, the inhibition rate of cell
proliferation was very significant {inhibition rate 50%},
Gene profile and the concentration of vincristine 100 ng/mL on the 8th
day after the action. The proliferation inhibition rate was
1. Effect of drug efficacy of gene CYP2D6 group gene and 99.64%. At the same time, as the concentration of VCR
CYP19A1 genes. CYP2D6 (English: Cytochrome increased, the inhibition rate of protein content increased
P4502D6) is an enzyme encoded by the human CYP2D6 to 75.21% (25 ng/mL). Vincristine has a strong inhibitory
gene. CYP2D6 is mainly expressed in the liver and is also effect on human retinoblastoma cell line HOX-Rb44. The
highly expressed in the central nervous system, including half-inhibitory concentration IC50 is 0.31 μg/mL, and the
the substantia nigra. vincristine concentration is 0.01 μg/mL, which can sig-
2. The CYP19A1 gene encodes a cytochrome P450 aroma- nificantly induce the apoptosis of K562 cells.
tase (P450arom), and the aromatase is the last step-
limiting enzyme in hormone synthesis. Under its action, Gene profile
androgen androstenedione and testosterone are converted The gene associated with this pharmaceutical dose is
to estrogen and estrone, respectively. Glycol, which is CEP72.
884 Appendixes
Detection method is good. Because of its good curative effect, low toxicity,
Gene detection was performed by establishing artificially low cost, and convenient oral administration, it is listed as
modified biallelic specific primers in combination with the preferred anti-tuberculosis drug. The oral absorption
SYBR Green I fluorescent PCR. rate of isoniazid was 90%; the serum drug concentration
reached the peak 1–2 h after administration; Vd was
D.13 Antifungal Therapy 0.61 ± 0.11 L/kg, and the protein binding rate was very
D.13.1 Voriconazole low.
Drug introduction 2. The product is mainly metabolized by acetylation in the
body and partially hydrolyzed. Due to genetic differ-
1. The mechanism of action of voriconazole is to inhibit ences, the population can be divided into fast acetylated
cytochrome P450-mediated 14α-sterol demethylation in and slow acetylated. Their half-life was significantly
fungi, thereby inhibiting the biosynthesis of ergosterol. different, and the average t1/2 of fast acetylators was
2. In vitro tests have shown that voriconazole has a broad- 1.1 h. Slow acetylation is 3 h. The product easily passes
spectrum antifungal effect. This product has an antibacte- through the blood–brain barrier. Mainly used for the pro-
rial effect on Candida (including fluconazole-resistant gression of various types of tuberculosis, dissolution and
Candida krusei, Candida glabrata, and Candida albicans), dissemination period, absorption and improvement
and has a bactericidal effect on all tested Aspergillus fungi. period, can still be used for tuberculous meningitis and
3. In addition, voriconazole also has a bactericidal effect on other extrapulmonary tuberculosis.
other pathogenic fungi in vitro, including strains that are
less sensitive to existing antifungal agents, such as the Gene profile
genus Actinomyces and Fusarium.
1. The gene that affects the efficacy of the drug and evalu-
Gene profile ates its dose is NAT2.
The gene affecting the efficacy of the drug and evaluating 2. NAT2 is N-acetyltransferase 2 (aromatic amine N-
its dosage is CYP2C19. acetyltransferase). This gene encodes an enzyme that acti-
Detection method vates and inactivates aromatic amines and terpenoids and
Real-time PCR. carcinogens. The polymorphism of this gene is associated
with n-acetylated polymorphisms. In the n- acetylated
D.14 Anti-hyperthyroidism polymorphism, the human population is divided into fast,
D.14.1 Methimazole moderate, and slow acetylated phenotypes. Polymorphisms
Drug introduction in this gene are also associated with a high incidence of
Methimazole tablets are anti-thyroid drugs. They are suit- cancer and drug toxicity. The second aromatic amine
able for all types of hyperthyroidism. They are especially n-acetyltransferase gene (NAT1) is located near this gene
suitable for patients with milder disease, mild to moderate (NAT2).
thyroid enlargement; adolescents and children, and elderly 3. The location of the cell: 8p22 is the short arm (p) of posi-
patients with recurrence after thyroid surgery. It is not suit- tion 22 on chromosome 8, molecular positioning: base 8
able for patients treated with radioactive iodine 131I. of chromosome 8, 18, 386, 585~18, 401, 219.
Gene profile
The gene affecting its dosage is mainly HLA-B*38:02:01. Detection method
The gene is located on chromosome 6. Methods: The polymorphism of the NAT2rs1495741
Common polymorphic loci locus in tuberculosis patients was detected by modified mul-
g.2465195A>G tiplex ASe assay.
Detection method
Specimen DNA was extracted and subjected to routine D.15.2 Rifampicin
detection of HLA high-resolution typing by polymerase Drug introduction
chain reaction-specific oligonucleotide probe hybridization
(PCR-SSOP). 1. Rifampicin (also: rifamycin, meperidol, meptomycin,

weifuxian, xianduolun, lifuping or limidine, INN:
D.15 Anti-tuberculosis Treatment Rifampicin) is a kind of benefit A broad-spectrum antibi-
D.15.1 Isoniazid otic drug of the fumycin family has a strong antibacterial
Drug introduction effect against Mycobacterium tuberculosis, and is also
effective against Gram-positive or negative bacteria and
1.
Isoniazid has inhibitory and killing effect on viruses.
Mycobacterium tuberculosis, and its biofilm permeability
Appendixes 885
2. It is a red or dark red crystalline powder and is insoluble phosphatase PP2A. Assembly of signaling complexes
in water. Oral drugs, usually in capsules or tablets, work provides a mechanism to ensure specific and rapid signal-
synergistically with other anti-tuberculosis drugs and ing of this G protein coupled receptor. This gene is intron
delay the production of resistant strains. Mainly used to free. Different polymorphic forms, point mutations, and/
treat tuberculosis, meningitis, and Staphylococcus aureus or down-regulation of this gene are associated with noc-
infections. External use can treat trachoma. turnal asthma, obesity, and type 2 diabetes.
3. The location of the cell: 5q32 is the long (q) arm of chro-
Gene profile mosome 5 at position 32. Molecular localization: The
The gene affecting the efficacy and dosage of rifampicin base pairs on chromosome 5 are 148,826,593 pairs to
and pyrazinamide is NAT2. 148,828,634 pairs.
Detection method
The modified multiplex ASee assay. Detection method
Gene detection was performed by establishing artificially
D.16 Antiasthma treatment modified biallelic specific primers in combination with
D.16.1 Salbutamol SYBR Green I fluorescent PCR.
Drug introduction
.17 Anesthetic Treatment of Paroxysmal Pain
D
1. Salbutamol, a short-acting β2 adrenergic receptor agonist, D.17.1 Fentanyl
is used as an antiasthmatic agent to effectively inhibit the Drug introduction
release of allergic substances such as histamine and pre-
vent bronchospasm. For bronchial asthma, asthmatic 1. Fentanyl, suitable for all kinds of pain and analgesia after
bronchitis, bronchospasm, emphysema, and other surgery and surgery, surgery, gynecology, etc.; also used
symptoms. to prevent or reduce the occurrence of paralysis after sur-
2. Salmeterol is a novel selective long-acting beta 2 agonist gery; can also be combined with anesthetics, as an anes-
that lasts for 12 h at a single dose. There is a strong inhibi- thesia auxiliary; Pelidogine is combined into a “deep
tory effect on the release of allergic mediators from lung analgesic” for a large area dressing and minor analgesia.
mast cells, which can inhibit the early and late phase reac- It is an opioid receptor agonist and is a potent narcotic
tions induced by inhaled antigens and reduce airway analgesic with pharmacological effects similar to mor-
hyperresponsiveness. For asthma (including nocturnal phine. Animal experiments have shown that the analgesic
asthma and exercise asthma), asthmatic bronchitis, and effect is about 80 times that of morphine.
reversible airway obstruction. This product is a long- 2. The analgesic effect is rapid, but the duration is shorter,
acting selective β2 adrenergic receptor agonist with obvi- 1 min after the intravenous injection, and the peak is
ous bronchodilating effect. reached in 4 min, and the effect is maintained for 30 min.
3. Inhalation of this product 50 and 100 μg, the average time It takes about 7 min after intramuscular injection and is
to increase the forced inspiratory volume (FEV) per min- maintained for about 1–2 h. The respiratory inhibition of
ute by 15% is 17 min and 13 min, respectively, while this product is weaker than that of morphine, and the
inhaling salbutamol 200 μg, the same effect requires adverse reaction is smaller than that of morphine.
14 min. When the asthmatic patients inhaled the product
for 2 weeks, no rapid immunological effects on the lung Gene profile
effect of the product were observed. This product can
strongly and long-term inhibition of histamine, leukotri- 1. The genes affecting the dose and efficacy of these classes
enes, prostaglandins, and other inflammatory reactions, of drugs are CREB1 and OPRM1. The CREB1 gene
the effect lasts 12 h. encodes a transcription factor that is a member of the leu-
cine zipper family of DNA-binding proteins. This protein
Gene profile is combined with the camp reaction element, the octamer
palindrome, in the form of a homodimer. This protein is
1. The gene that affects the efficacy of salbutamol and sal- phosphorylated by multiple protein kinases and induces
meterol is ADRB2. This gene encodes a-2 adrenergic gene transcription when the hormone stimulates the
receptor and is a member of the G protein coupled recep- cAMP pathway. Alternate splicing of this gene can result
tor superfamily. in multiple transcript variants encoding different
2. This receptor is directly related to one of its final effec- subtypes.
tors, class C, calcium channel Ca(V)1.2. This receptor– 2. It is located at the location of the cell: 2q33.3, which is the
channel complex also contains a G protein, adenylate long (q) arm at position33.3 of chromosome 2. Molecular
cyclase, a camp-dependent kinase, and an equilibrium
886 Appendixes
localization: pairs 207, 529, 892 pairs on chromosome 2 Gene profile

to 207, 605, 989 pairs. The gene that affects the therapeutic effect of drugs is
3. The OPRM1 gene provides an indicator protein called μμ MTHFR.
opioid receptor. Opioid receptors are part of the endoge- Common polymorphic loci
nous opioid system, an internal system of the body that 677C>T
regulates pain, reward, and addictive behavior. Opioid 1298A>C
receptors are present in the nervous system and they are Detection method and result interpretation
located in the outer membrane of nerve cells (neurons). MTHFR gene polymorphism detection is commonly used
When an opioid binding to a receptor, this interaction in PCR-fluorescence probe method. Based on the amplifica-
triggers a series of chemical changes between the neuron tion curve, appropriate baseline and fluorescence thresholds
and the neuron, creating a feeling of pleasure and pain. are specified. The Ct values of different channels were
obtained to determine the genotype of the sample.
CREB1: g.208494234 D.19 Erectile Dysfunction
OPRM1: 118A>G D.19.1 Sildenafil
Polymerase chain reaction-restriction fragment length Sildenafil tablets are a drug that is used primarily to treat
polymorphism (PCR-RFLP) and polymerase chain reaction- penile erectile dysfunction (ED).
sequence specific primer (PCR-SSP) analysis techniques Gene profile
were used. After separation by agarose gel electrophoresis, The gene affecting the efficacy of the drug and evaluating
the gel imager system was processed for genotype the dose of the drug is GNB3. A heterotrimeric guanine
interpretation. nucleotide binding protein (G protein) is a protein that inte-
D.17.2 Tramadol grates signals between a receptor and an effector protein, and
Drug introduction consists of one, one, and one subunit. These subunits are
Tramadol is a non-opioid central analgesic but has a weak encoded by a family of related genes. This gene encodes a
affinity with opioid receptors. By inhibiting the reuptake of beta subunit belonging to the WD repeat G protein beta fam-
norepinephrine by neuronal synapses and increasing the con- ily. Subunits are important regulators of subunits and are
centration of serotonin outside the neurons, it affects the pain regulators of certain signal transduction receptors and effec-
transmission and produces an analgesic effect. Its intensity tors. The single-nucleotide polymorphism (C825T) of this
of action is 1/10 to 1/8 of morphine. No inhibition of respira- gene is associated with essential hypertension and obesity.
tion, low dependence, and significant analgesic effect. It has This polymorphism is also associated with the development
antitussive effect and the strength is 50% of codeine. Does of the splice variant GNB3-s, which appears to be more
not affect the release of histamine, does not cause smooth active. GNB3-s is an example of alternative splicing caused
muscle spasm. Oral, injection and absorption are good, the by nucleotide changes outside the splice donor and acceptor
analgesic effect is the same. Metabolized in the liver, 80% of sites. Alternative splicing results in multiple transcript vari-
the original form and metabolites are excreted from the urine ants. Other alternative splicing transcript variants of this
within 24 h. gene have been described, but their full-length properties are
Gene profile unclear. The position of the gene is located at the location of
The gene affecting the amount and efficacy of this phar- the cell: 12p13.31, which is the short arm (p) at position
maceutical agent is OPRM1. 13.31 of chromosome 12. Molecular position: base pairs on
Common polymorphic loci chromosome 12, 6,840,922 pairs to 6,847,393 pairs.
118A>G Common polymorphic loci
Detection method and result interpretation 825C>T
PCR-RFLP and PCR-SSP. Detection method
Gene detection was performed by establishing artificially
D.18 Disease Prevention modified biallelic specific primers in combination with
D.18.1 Folic Acid SYBR Green I fluorescent PCR.
Drug introduction
One of the folic acid vitamin B complexes, which is D.20 Hypoglycemic Therapy
equivalent to pteroylglutamic acid (PGA), is extracted and D.20.1 Melbine
purified from spinach leaves by M. K. Mitchell (1941). It has Drug introduction
the effect of promoting the maturation of young cells in the Metformin tablets are preferred for type 2 diabetes, which
bone marrow. The lack of folic acid in humans can cause is effective in diet control and physical exercise, especially
macrocytic anemia and leukopenia, which is especially obese type 2 diabetes. This product can be combined with
important for pregnant women. insulin to reduce the amount of insulin and prevent hypogly-
Appendixes 887
cemia. It can be combined with sulfonylurea hypoglycemic cal structure is a 23-membered macrolide antibiotic. It is a
agents and has a synergistic effect. powerful new type of immunosuppressive agent, which
Gene profile inhibits the release of interleukin-2 (IL-2) and inhibits the
The genes that influence the therapeutic effect of drugs action of T lymphocytes.
are C11orf65 and TCF7L2. Gene profile
Common polymorphic loci The gene for evaluating the dose of the drug is CYP3A5.
C11orf65: 108412434C>A Common polymorphic loci
TCF7L2: 53341C>T 219–237A>G
Detection method Detection method
The PCR-RFLP method is commonly used for the detec- Real-time quantitative PCR.
tion of C11otf65 gene polymorphism. The PCR product was
digested with restriction endonucleases. D.21.3 Tumor Necrosis Factor Inhibitor
Gene profile
D.20.2 Sulfonylurea The gene that affects the therapeutic effect of drugs is
Drug Introduction TNF.
Sulfonylureas (SU) are the earliest, most widely used, and Common polymorphic loci
most widely used oral hypoglycemic agents. In recent years, −308G>A
glimepiride has been developed. It is called the third-generation Detection method and result interpretation
SU class drug because of its small dosage, certain improve-
ment in insulin resistance and reduction of insulin dosage. 1. Detection of TNF gene polymorphism can be detected by
Gene profile real-time fluorescent quantitative PCR combined with
The genes that affect the therapeutic effect of drugs and high-resolution melting curve method.
evaluate the dose of drugs used are CYP2C9 and ABCC8. 2. Data were collected and genotypes were determined

Common polymorphism detection site according to the order of melting.
CYP2C9: 1075A>C
ABCC8: 16-3C>T D.22 Lipid Control Therapy
Detection method D.22.1 Fluvastatin
The CYP2C9 gene polymorphism detection method Drug introduction
mostly uses real-time fluorescent quantitative PCR TaqMan Fluvastatin is the first fully chemically synthesized
technology. Samples covering the wild type and heterozy- cholesterol-lowering drug. Compared with the listed natural
gous mutants of CYP2C9 gene were selected, and the sam- or semi-synthetic HMG-CoA reductase inhibitors lovastatin,
ple DNA was extracted for real-time quantitative PCR simvastatin, and pravastatin, fluvastatin has the advantages
amplification. of relatively simple structure, selective action, and low inci-
dence of adverse reactions. It is an excellent hypolipidemic
D.21 Immunosuppressive Therapy drug.
D.21.1 Ciclosporin A Gene profile
Drug introduction The genes affecting drug treatment and evaluating the
Cyclosporin A is composed of a cyclic polypeptide con- dose of drugs used are APOE and COQ2.
sisting of 11 amino acids and is a potent immunosuppressive Common polymorphic loci
agent. Clinically, it is mainly used for anti-rejection reaction APOE: 526C>T
of liver, kidney, and heart transplantation. It can also be used COQ2: 779–1022C>G
together with adrenocortical hormone to treat immune Detection method
diseases. PCR-fluorescence probe method.
Gene profile
The gene for evaluating the drug use dose is CYP3A5. D.22.2 Rosuvastatin
Common polymorphic loci Drug introduction
219–237A>G Rosuvastatin is a selective HMG-CoA reductase inhibitor.
Detection method The main site of action of rosuvastatin is the liver-targeting
Real-time quantitative PCR. organ that lowers cholesterol. Rosuvastatin increases the
number of surface receptors in liver LDL cells, promotes
D.21.2 Tacrolimus LDL uptake and catabolism, inhibits liver synthesis of
Drug introduction VLDL, thereby reducing the total number of VLDL and
Tacrolimus, also known as FK506, is a fermentation prod- LDL particles.
uct isolated from Streptomyces tsukubaensis and its chemi- Gene profile
888 Appendixes
The genes affecting drug therapy and evaluating drug use increased serum cholesterol clearance, lower levels of
doses are ABCG2, APOE, SLCO1B1, and COQ2. clinical use mainly to lower cholesterol, especially low-
Common polymorphic loci density lipoprotein cholesterol (LDL-C), for the treat-
APOE: 526C>T ment of atherosclerosis.
ABCG2: 421C>A
SLCO1B1: 521T>C Gene profile
COQ2: 779–1022C>G
Detection method 1. Genes affecting drug therapy and evaluating drug use
PCR-fluorescence probe method. doses are ApoE, COQ2, and SLCO1B1.
2. The COQ2 gene mainly encodes a para-hydroxybenzoate-
D.22.3 Pravastatin polyprenyl transferase, which is an important enzyme in
Drug introduction the process of synthesizing coenzyme Q10. When the
Pravastatin is a white crystalline powder of chemicals, human COQ2 gene is mutated, it will affect the biosyn-
often in the form of a sodium salt. Pravastatin is a lipid- thesis of coenzyme Q10, which will affect the electron
lowering drug and is mainly used in patients with primary transport chain during mitochondrial oxidation. Studies
hypercholesterolemia or hypertriglyceridemia (types IIa and have shown that human COQ2 gene can significantly
IIb) with uncontrolled dietary restriction. Atorvastatin increase the risk of systemic atrophy after functionally
Lipitor (English name Lipitor, commonly known as atorvas- impaired variants, suggesting that this gene mutation is
tatin calcium tablets) is a lipid-lowering drug developed by closely related to the pathogenesis of systemic atrophy.
Pfizer. It is applicable to reduce the risk of non-fatal myocar-
dial infarction, reduce the risk of fatal and non-fatal stroke, Common polymorphic loci
reduce the risk of revascularization, reduce the risk of hospi- APOE: 526C>T
talization due to congestive heart failure, and reduce the risk COQ2: 779–1022C>G
of angina. SLCO1B1: 521T>C
Gene profile Detection method
The genes affecting drug therapy and evaluating drug APOE gene and SLCO1B1 gene detection are mostly
dosage are APOE, KIF6, SLCO1B1, and COQ2. detected by PCR-fluorescence probe method. Real-time
Common polymorphic loci PCR detection was performed according to the instructions
APOE: 526C>T of the human SLCO1B1 and APOE gene detection kit.
KIF6: 2104T>C
SLCO1B1: 521T>C D.23 Risk Profile
COQ2: 779–1022C>G D.23.1 Alcohol Metabolism Assessment
Detection method Gene profile
PCR-fluorescence probe method. The gene for evaluating the efficacy of this drug is
ALDH2.
D.22.4 Statins Detection method and result interpretation
Drug introduction Real-time fluorescent quantitative PCR is often used for
ALDH2 gene polymorphism detection. The TaqMan-MGB
1. The statin, 3-hydroxy-3methylglutaryl coenzyme A
double probe was used to establish an allelic typing method
(HMG-CoA) reductase inhibitor, is currently the most based on the difference ΔCt of the cycle threshold (Ct value)
effective lipid-lowering drug, not only potently reducing of the fluorescence signal of the allele G and type A TaqMan-
total cholesterol (TC) and low-density lipoprotein (LDL), MGB probes (ΔCt = G type TaqMan − MGB Needle Ct
and can reduce triacylglycerol (TG) to a certain extent, value − Type A TaqMan − MGB probe Ct value) Determine
can also increase high-density lipoprotein (HDL), so genotype.
statins can also be called more comprehensive lipid-
lowering drugs. D.23.2 Children’s Asthma Risk
2. The mechanism of action of statins is to competitively Gene profile
inhibit the endogenous cholesterol synthesis rate-limiting The gene for evaluating the risk of this drug is ORMDL3.
enzyme HMG-CoA reductase, block the intracellular Detection method
hydroxyvalerate metabolic pathway, and reduce intracel- Primers were designed for the SNP site of the ORMDL3
lular cholesterol synthesis, thereby feedback-stimulated gene to perform multiplex PCR reactions. Detection by SNP
low cell membrane surface. Increased number and activ- typing technology.
ity of density lipoprotein (LDL) receptors resulting in
Appendixes 889
D.23.3 Risk of Venous Thrombosis D.23.8 Risk of Cardiovascular and Cerebrovascular

Gene profile Diseases
The gene for evaluating the risk of drug use is the PAI-1 Gene profile
gene. The genes evaluating the risk of drug use are MTHFR and
MTRR.
D.23.4 Risk of Alzheimer’s Disease
Gene profile D.23.9 Thrombosis Risk Susceptibility Gene Screening
The gene for evaluating the risk of drug use is APOE. for Thrombosis Risk
Gene profile
D.23.5 Risk of Habitual abortion The gene for evaluating the risk of drug use is the PAI-1
Gene profile gene.
The gene for evaluating the risk of drug use is MTHFR.
D.23.10 Thrombosis Risk of High Homocysteine
D.23.6 Risk of Postpartum Depression Susceptibility Gene Screening
Gene profile Gene profile
The gene for evaluating the risk of drug use is MTHFR. The genes evaluating the risk of drug use are MTHFR and
MTRR.
D.23.7 Risk of Birth Defects in Newborns and
Individualized Supplementation of Folic Acid D.23.11 Anti-thrombotic Protein Deficiency
Gene profile Susceptibility Gene Screening for Thrombosis Risk
The genes evaluating the risk of drug use are MTHFR and Gene profile
MTRR. The genes evaluating the risk of drug use are PROC and
THBD.

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Shiyang Pan

Clinical Molecular Diagnostics

ISBN 978-981-16-1036-3 ISBN 978-981-16-1037-0 (eBook)

© People’s Medical Publishing House Co. Ltd. 2021

Chinese Academy of Engineering Hongbing Shen

Xuan Cao Huanyu Liang Li Wang Shuxian Yang

Nanjing, China Shiyang Pan

Part I Principles of Clinical Molecular Diagnostics

1 Molecules of Disease and Their Detection Methods ����������������������������������������������� 3

Part II Molecular Biomarkers and Signals from Diseased Functional Organ

10 Molecules in Body Systems ��������������������������������������������������������������������������������������� 123

16 Coagulation and Fibrinolysis������������������������������������������������������������������������������������� 207

Part III Advanced Molecular Diagnostic Techniques

23 Next-Generation Sequencing (NGS)������������������������������������������������������������������������� 305

Part IV Disease Presentation and Clinical Laboratory Procedures

About the Editors

Jinhai Tang Prof. Tang is Full Professor of General Surgery, the

Hongbing Shen Prof. Shen is an academician of the Chinese

Yongtong Cao Department of Clinical Laboratory, China-Japan Friendship Hospital, Beijing,

Ming Guan Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical

Wenen Liu Department of Clinical Laboratory, Xiangya Hospital of Central South

Aiguo Tang Department of Laboratory Medicine, The Second Xiangya Hospital, Central

Zhenzhen Cai Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Huanhuan Chen Department of Laboratory Medicine, The First Affiliated Hospital of

Ye Jiang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Yuan Mu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Jia Wang Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

Yuqiao Xu Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing

The birth and development of molecular biology is an 1.1 Overview

© People’s Medical Publishing House Co. Ltd. 2021 3

Pre-rRNA m7GPPPN PolyA

m7GPPPN PolyA m7GPPPN PolyA m7GPPPN PolyA

Fig. 1.1 Protein production process

Table 1.1 Characteristics and applications of each sequencing technology

1.3.3 Nucleic Acid Hybridization Technology 1.3.4 Chip Technology

1.3.5 Biosensing Technology 1.4.1 Spectrum Technology

1.4.2 Protein Chip Technology 1.4.4 Mass Spectrometric Technique

© People’s Medical Publishing House Co. Ltd. 2021 11

Need for Precision Evaluation 2.1.4.1 Experimental Preparation

Select and Optimize Study Protocol Experimental Sample

2.1.4.2 Experimental Method

• Trueness The complete expression is the measurement

are available from the National Institute of Standards and

Matrix Effects Analyte Range

References 4. Clinical and Laboratory Standards Institute. EP09-A2 document:

3.1 Biological Reference

© People’s Medical Publishing House Co. Ltd. 2021 27

• Reference population A group of all reference to individu- 3.1.2 Clarifications

Table 3.1 Examples of possible exclusion criteria and partitioning

groups in the subject needs to be considered. Only on the     

4. Solberg HE, Stamm D. International Federation of Clinical

© People’s Medical Publishing House Co. Ltd. 2021 39

Table 4.1 Informed consent form for molecular genetic testing

INFORMED CONSENT FORM FOR MOLECULAR GENETIC TESTING

I have explained DNA testing to the patient/parent/guardian.

Clinician Signature Date Print name Date

3. Using IT technology and security to protect patient

5.1 Overview 5.1.2 Research Categories

European Institute of Bioinformatics It is the DNA and

National Institute of Genetics, Japan It is a Japanese

© People’s Medical Publishing House Co. Ltd. 2021 45

Fig. 5.1 FASTA format

Fig. 5.2 NCBI Home Page

Analysis of Nucleic Acid Sequence Characteristics In the

Chinese Academy of Engineering Hongbing Shen

Nanjing, China Shiyang Pan

Part I Principles of Clinical Molecular Diagnostics

1 Molecules of Disease and Their Detection Methods �� 3

Part II Molecular Biomarkers and Signals from Diseased Functional Organ

10 Molecules in Body Systems �� 123

16 Coagulation and Fibrinolysis�� 207

Part III Advanced Molecular Diagnostic Techniques

23 Next-Generation Sequencing (NGS)�� 305

Part IV Disease Presentation and Clinical Laboratory Procedures

The birth and development of molecular biology is an 1.1 Overview

1.3.3 Nucleic Acid Hybridization Technology 1.3.4 Chip Technology

1.3.5 Biosensing Technology 1.4.1 Spectrum Technology

1.4.2 Protein Chip Technology 1.4.4 Mass Spectrometric Technique

Need for Precision Evaluation 2.1.4.1 Experimental Preparation

2.1.4.2 Experimental Method

3.1 Biological Reference

• Reference population A group of all reference to individu- 3.1.2 Clarifications

groups in the subject needs to be considered. Only on the

5.1 Overview 5.1.2 Research Categories

5.2.2 Multiple Sequence Analysis Progressive alignment of multiple sequences

6.1 Overview fundamental source of biodiversity. Nongenetic variation is

Table 6.2 Table of nucleotide information in RNA 6.3.3.2 Silent Changes

specimens should be sent in a specified way to the labora- 7.3.1.1 Neonatal

Table 8.3 (continued) 8.4.3 Auditing Program

10.2.1 Sugars Provide an Energy Source