Laboratory Medicine and Diagnostic

Pathology

Tim Hodgson, Barbara Carey, Emma Hayes, Richeal Ni Riordain,

Priya Thakrar, Sarah Viggor, and Paula Farthing

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Hematology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Full Blood Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bleeding Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Clinical Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Liver Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Renal Biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Bone Profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Hematinics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Fasting Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acute-Phase Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Fecal Calprotectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Serum Angiotensin-Converting Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Immunosuppressive Medication Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Short SynACTHen Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Glucose-6-phosphate Dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
C1 Esterase Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Human Leucocyte Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Amyloid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Indirect Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

T. Hodgson · B. Carey · E. Hayes · R. Ni Riordain ·

P. Thakrar · S. Viggor
Eastman Dental Hospital, London, UK
e-mail: Tim.Hodgson@ucl.ac.uk; barbara.carey@nhs.net;
Emma.Hayes1@nhs.net; r.niriordain@ucl.ac.uk;
Priya.thakrar@nhs.net; Sarah.Viggor@uclh.nhs.uk
P. Farthing (*)
School of Clinical Dentistry, University of Sheffield,
Sheffield, UK
e-mail: p.farthing@sheffield.ac.uk

# Springer International Publishing AG, part of Springer Nature 2018 1

C. S. Farah et al. (eds.), Contemporary Oral Medicine,
https://doi.org/10.1007/978-3-319-28100-1_4-1
2 T. Hodgson et al.

Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Autoantibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Microbiology and Serology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Mycoplasma pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Methicillin-Resistant Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Brucellosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Candida Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Pathological Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Laboratory Processing of Biopsy Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Histopathological Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Application of Molecular Biology Techniques in Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Interpretation of Biopsy Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Precision Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Abstract basis for accurate and meaningful diagnosis

Laboratory tests play an important role in and management strategies in oral medicine.
clinical diagnosis, and the results often direct
patient management. Tests should be Keywords
requested appropriately in order to refine the Diagnostic tests · Laboratory tests · Laboratory
differential diagnosis, and it is important to investigations · Oral disease · Hematology,
understand the theoretical basis of such tests Clinical chemistry · Histopathology ·
and to be able to interpret their findings. This Immunology · Microbiology · Anatomic
chapter covers the common laboratory tests pathology · Diagnostic pathology · In vitro
that may be requested in clinical oral medicine diagnostics · Molecular pathology · Precision
including hematology, clinical chemistry, medicine · Personalized medicine
immunology, and pathological investigations.
The reasoning behind the use of such tests in
diagnosis is explained, together with their Introduction
interpretation and relevance to diseases
affecting the oral and maxillofacial complex. Diagnostic tests are important aids to medical care
A more precision-based approach to clinical delivery, defined as investigations performed on
care is being mirrored in oral medicine by samples taken from the body used in a broad range
tailored laboratory investigations relying on of applications. These tests may be referred to as
molecular pathology approaches such as next- “in vitro diagnostics.” Results can be used to
generation sequencing. These will soon make assist the patient, physician, and caregiver in
their way into clinical practice and transform decision-making. Depending on the test and
patient care, so it is imperative that clinicians the methods used, testing can be performed in a
keep up to date with advances in technology, laboratory, at the hospital bedside, in the clini-
techniques, and their underlying scientific cian’s office, in the clinic, in the workplace, and
even at home. Diagnostic tests may be the least
Laboratory Medicine and Diagnostic Pathology
expensive component of the care pathway, but
they influence more than 70% of healthcare deci-
sions. All tests should be requested by the physi-
cian to answer a specific question. The requesting
clinician should make sure the test is fit for pur-
pose, they receive the result in a timely manner,
and they are able to interpret the results in relation
to the clinical presentation of the patient. From the
perspective of health economics, all tests should
be considered as positive additions to the diagnos-
tic process. It is not appropriate to order a test as a
“general health screen” in an oral medicine clinic.
Diagnostic tests provide objective information
about a person’s health. Some tests are used for
risk assessment to determine the likelihood a med-
ical condition is or will become present. Other
tests are used to monitor the course of a disease
and assess patient response to therapy or guide the
selection of further intervention and treatment.
Every clinical encounter begins with an initial
clinical impression and a subjective pretest prob-
ability of disease. The ultimate goal of diagnostic
testing is to refine this pretest probability to the
point where the physician can confidently make
a treat or no-treat decision. Each diagnostic test
results in a change in the physician’s probability
of disease, the posttest probability.
Most commonly, test results provide informa-
tion that, with the patient’s history and other med-
ical information, helps the clinician work with the
patient to decide the appropriate actions for addi-
tional testing or treatment. On some occasions, the
information from a single test is sufficient to pre-
vent a cascade of sophisticated medical interven-
tions; and sometimes it is what is needed to end
them. More often, diagnostic tests provide infor-
mation, along with other tests and observations,
which help determine whether or not a disease is
present, has progressed, or has changed its course.
This allows the physician to make a judgment on
what treatment regimen might be most appropri-
ate for a particular patient at a given time.
Different diagnostic tests for the same disease
often trade sensitivity for specificity or vice
versa. In general, the more sensitive a test is for
a disease, the higher its false-positive rate, lower-
ing its specificity. A test with a higher specificity
will usually sacrifice sensitivity by increasing its
3
false-negative rate. This makes a highly sensitive
test ideal for a screening examination. Highly
specific tests are best utilized in a confirmatory
role. Test results are not always easily interpreted.
Problems arise interpreting the results in the con-
text of the patient or considering the risks or
consequences for the patient’s treatment.
Discoveries about the human genome have
opened the door to precision medicine approaches
that can tailor medical treatments to individual
patient needs, transforming modern medicine.
The information provided by diagnostic tests
informs decisions made throughout the healthcare
continuum. Tests can be used to screen for a
disease or to provide early disease identification,
to diagnose a disease, to provide prognostic infor-
mation by assessing the degree of disease progres-
sion or severity, to assist in selecting drugs or
targeting medical treatment, and to monitor the
course of a disease or condition (Table 1). Tests
suggested for confirmation of common clinical
diagnoses relevant to the practice of oral medicine
and initiation of systemic immunosuppression are
detailed in Table 2.
Hematology
Full Blood Count
Full Blood Count (Complete Blood Count) is
normally collected in a 5 ml tube anticoagulated
with dry potassium EDTA (ethylenediaminete-
traacetic acid) or citrate. Blood cells are usually
automatically analyzed, and blood films are pre-
pared from the same sample if indicated by the
history or blood count results.
A full blood count would normally be
requested for oral medicine patients presenting
with:
1. Clinical orofacial signs suggestive of anemia:
• Persistent or recurrent oral ulceration
• Evidence of angular cheilitis or chronic
fungal infection
• A persistent sore or burning sensation of the
mouth
2. Recalcitrant oral disease
4 T. Hodgson et al.

Table 1 The application of diagnostic tests

Test use Purpose Example
Screening, early To detect asymptomatic disease or a Fecal calprotectin inflammatory bowel disease
disease detection predisposition to disease in order to take Blood cholesterol tests/heart disease
action to prevent it by modifying a risk Fecal occult blood tests/colorectal cancer
factor or to treat it earlier
Diagnosis To make a diagnosis when symptoms, Brain natriuretic peptide (BNP) and NT pro-BNP/
abnormalities on physical examination, or heart failure
other evidence suggests, but does not prove, Incisional biopsy for lichen planus
that a disease may be present Direct immunofluorescence for MMP and
pemphigus vulgaris
Disease staging, To determine the extent of disease Testing for comorbidities (e.g., hypertension,
prognosis progression or severity and the likelihood of cardiovascular disease, acute respiratory infection)
recovery or risk of future adverse health Blood clotting tests for presurgical risk assessment
outcomes (e.g., cancer relapse) Cardiac marker testing (e.g., troponin T and I and
CK-MB, myoglobin)/rapid assessment of heart
injury, heart attack
Proteins in Sjógren’s syndrome
Drug selection, To allow accurate and targeted treatment HLA and carbamazepine prescription
treatment selection tailored to individual needs HIV, HBC, and HCV testing prior to
monitoring immunosuppressant prescription e.g., Azathioprine
prescription
Full blood picture liver biochemistry for
immunosuppressant treatment e.g., Azathioprine
monitoring
Disease or To understand the course of the disease or Pemphigus antibody titer for disease response
condition the effect of a therapy in order to evaluate Blood glucose tests and HbA1c tests for diabetes
monitoring and the success of treatment and the need for monitoring
management additional testing or treatment Viral load, CD4 count to assess treatment response
in HIV patients
Cholesterol tests to monitor effectiveness of lipid-
lowering drug therapy

3. Clinical signs suggestive of immunosuppres- • Hemorrhage (blood loss)

sion or underlying hematological malignancy
4. Increased oral bleeding or mucosal petechiae
(low platelets)
5. Prior to initiation and for monitoring of
immune-modulating therapy
The cause of anemia can be further elicited by
assessing the hematocrit, red cell indices (mean
corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), mean corpuscular hemoglo-
bin concentration (MCHC)), differential white
cell count, blood film, and hematinic levels. A
simple guide to interpretation of hematological
tests is shown in Table 3 (Longmore et al. 2014).
Anemia is usually caused by one or more of the
following:
• Failure of erythropoiesis (red cell production)
• Abnormally increased breakdown of red blood
cells (hemolysis)
• Increased plasma volume (pregnancy)
Deficiencies of iron, vitamin B12, and folate are
the most common cause of a low hemoglobin
level in North America and Europe. Iron defi-
ciency is typically microcytic and hypochromic
with poikilocytosis (low MCV, MCH, and
MCHC), whereas vitamin B12 and folate defi-
ciency lead to production of large red blood cells
with a high MCV (macrocytosis). Some red cells
will be primitive and nuclei may remain present
(megaloblastic).
Laboratory Medicine and Diagnostic Pathology 5

Table 2 Tests indicated for common oral medicine diag- Table 2 (continued)
noses and for commencing systemic immunosuppressive
Varicella zoster antibodies
therapy
Fasting blood glucose/HbA1c
Pemphigus Glucose 6 phosphate dehydrogenase (dapsone)
Incisional biopsy of lesional and perilesional tissue TB risk assessment for latent disease and if suspected
Indirect immunofluorescence Quantiferon test
Work-up for immunosuppressant therapy
Mucous membrane pemphigoid
Incisional biopsy of lesional and perilesional tissue Other causes of anemia include autoimmune
Indirect immunofluorescence disease such as rheumatoid arthritis, drugs
Work-up for immunosuppressant therapy
(including dapsone), chronic alcohol misuse,
Recurrent mouth ulcers with no identified systemic
cause and renal failure. In sickle cell disease, a gene
Full blood count/complete blood count defect causes production of abnormal β globin
Vitamin B12 chains, resulting in the production of HbS rather
Ferritin/iron studies than HbA. Homozygosity for HbS (i.e., HbSS)
Red cell folate
In some cases: results in sickle cell anemia, while heterozygos-
Anti-tissue transglutaminase antibodies ity for HbS (i.e., HbAS) results in sickle-cell
Anti-endomysial antibodies trait. Patients with sickle-cell trait will only
Sjógren’s syndrome develop sickling in severe hypoxia. Sickle cell
Full blood count
disease patients are at risk of severe infections
Hematinic screen
Erythrocyte sedimentation rate (ESR) and aseptic bone necrosis. Thalassemia is caused
C-reactive protein (CRP) by reduced production of one or more of the
Antinuclear antibodies (ANA) hemoglobin chains leading to imbalanced hemo-
Extractable nuclear antigens (ENA)
globin synthesis. This leads to ineffective eryth-
Rheumatoid factor
Complement ropoiesis and hemolysis. There are two types: α
Lymphocyte subsets thalassemia and β thalassemia. Both produce a
Cryoglobulins hypochromic, microcytic anemia and are associ-
Serum immunoglobulins +/
subclasses
ated with recurrent infections and bony abnor-
Granulomatous disorders
malities. Analysis of a blood film may be
Incisional biopsy of lesional tissue
Full blood count requested to confirm the above diagnoses. Other
Hematinic screen inherited conditions causing anemia include
ESR, CRP hereditary spherocytosis, glucose 6 phosphate
Serum angiotensin-converting enzyme (ACE)
dehydrogenase deficiency, and pyruvate kinase
Fecal calprotectin
In some cases: deficiency.
Quantiferon test for TB Polycythemia is caused by a proliferation of
Oral dysesthesia red blood cells and may be primary (polycythemia
Full blood count rubra vera) or secondary to a number of disorders
Ferritin/iron studies
Vitamin B12
including renal disease and congenital heart
Red cell folate disease. There may be an associated bleeding
Fasting blood glucose tendency.
Commencing immunosuppressant therapy An abnormal white cell count may indicate
Full blood count infection or underlying immunosuppression. It
Renal function
Liver function is important to exclude immunosuppression in
Thiopurine methyltransferase (TPMT) (azathioprine/ patients due to commence immune-modulating
6-mercaptopurine) medication, such as azathioprine or myco-
HBV phenolate mofetil. A markedly increased white
HCV
HIV antibody cell count may indicate an underlying
(continued)
6 T. Hodgson et al.

Table 3 Simple guide to interpretation of full blood count (complete blood count)a
Measurement Reference range Increased Decreased Comments
Hemoglobin Males 13–18 g/dl Polycythemia Reduced erythropoiesis Can be affected by
(Hb) Females (e.g., hematinic age, gender,
11.5–16 g/dl deficiency, renal failure) pregnancy, altitude,
Hemorrhage and cigarette smoking
(menstruation,
gastrointestinal
pathology)
Hemolysis
(autoimmune disease,
drugs)
Hereditary conditions
(sickle cell anemia,
thalassemia)
Hematocrit Males 40–54% Dehydration Overhydration,
Females 38–47% Polycythemia pregnancy, hemorrhage,
Preeclampsia bone marrow failure,
nutritional deficiency
Red cell count 4.5–6.5  1012/L Polycythemia Hemorrhage, bone
(RBC) Congenital heart disease marrow failure,
Renal disease nutritional deficiency,
Dehydration overhydration
Mean 76–96 fl Vitamin B12 or folate Iron deficiency anemia, Classified into three
corpuscular deficiency, chronic lung thalassemia, pregnancy, types: microcytic (low
volume disease, liver disease, hemolytic anemia, bone MCV), normocytic
(MCV) bone marrow disorders, marrow disorders (normal MCV), and
alcoholism, drug effects, macrocytic (high
e.g., dapsone, MCV)
azathioprine,
metronidazole
Mean 27–32 pg Vitamin B12 and folate Iron deficiency
corpuscular deficiency
hemoglobin
(MCH)
Mean 30–36 g/dL Iron deficiency,
corpuscular hemorrhage, pregnancy,
hemoglobin anemia of chronic
concentration disease
(MCHC)
Red cell 11.6–14.6% Iron deficiency
distribution Hemolytic anemia
width (RDW)
White cell 4–10  109/l Stress, extreme cold or Drug therapy (e.g.,
count (WCC) heat, exercise, azathioprine),
pregnancy, infection, autoimmune disease,
inflammation, trauma, viral infection, bone
leukemia and other marrow disease
malignancy
(continued)
Laboratory Medicine and Diagnostic Pathology 7

Table 3 (continued)
Measurement Reference range Increased Decreased Comments
Platelets 150–400  109/l Essential Reduced production in
thrombocytosis, the bone marrow
inflammation, surgery, (aplastic anemia, acute
hemorrhage, leukemia, malignancy,
hyposplenism drugs, vitamin B12 or
folic acid deficiency)
Increased platelet
destruction (immune
thrombocytopenic
purpura (ITP))
Increased rate of
consumption
(disseminated
intravascular
coagulation (DIC),
drugs)
a
Laboratory reference ranges may vary. Clinicians should check with their local laboratory

hematological malignancy, such as leukemia. A
basic guide to the differential white cell count is
shown in Table 4 (Longmore et al. 2014).
Bleeding Disorders
Bleeding disorders are caused by either abnormal-
ities in platelet number or function or coagulation
defects. These can be hereditary or acquired.
An assessment of clotting function would
normally be carried out on oral medicine patients
presenting with the following:
1. Spontaneous gingival bleeding of unknown
cause
2. Oral mucosal purpura, petechiae, or ecchymoses
3. Excessive bruising of the skin or a history of
prolonged bleeding following tooth extraction
4. Patients taking anticoagulant medication or
those with suspected or confirmed liver disease
who are due to undergo oral biopsy
A full blood count will detect any abnormali-
ties in platelet count, a blood film will show any
alteration in the morphology, and platelet function
tests will detect abnormalities in platelet hemo-
static function.
Coagulation screening tests typically include
the following:
1. Prothrombin time (PT). This is the most sensi-
tive marker and assesses the extrinsic pathway
of the clotting cascade. It is usually expressed
as a ratio compared to control (international
normalized ratio or INR).
2. Activated partial thromboplastin time (APTT).
This assesses the intrinsic pathway of the
clotting cascade.
3. Thrombin time. This assesses conversion of
fibrinogen to fibrin.
Abnormalities will prompt onward referral to
a hematologist for investigation (Platt 2007).
Further specific tests are likely to be required to
accurately diagnose any bleeding disorder.
Clinical Chemistry
Glucose
The requirement for glucose by the human body is
continuous, and it is the preferred fuel source for
all tissue types. A water-soluble molecule crossing
the blood brain barrier makes it an unique energy
source for the brain. Sources of glucose include
diet, gluconeogenesis, and glycogenolysis. Phys-
iological concentrations of glucose range between
3.9 and 6.7 mmol/L. Diabetes mellitus is a meta-
bolic disease and is characterized by high blood
8 T. Hodgson et al.

Table 4 Basic guide to interpretation of the differential white cell counta

Cell type Reference range Increased Decreased
Neutrophils 2.0–7.5  109/l Bacterial infections Viral infections (HIV)
Inflammation (Myocardial Drugs (chemotherapy, sulfonamides)
infarction, rheumatoid arthritis) SLE
Myeloproliferative disorders Hemolytic anemia
Drugs (corticosteroids) Bone marrow failure
Disseminated malignancy Racial variant (Afro Caribbean)
Physiological (exercise, pregnancy) (If the neutrophil count falls below
Stress (trauma, burns, hemorrhage) 0.5 x109 risk of serious bacterial
infection)
Lymphocytes 1.5–4.5  109/l Acute viral infection SLE
Chronic bacterial infections HIV
(brucellosis, syphilis, Drug treatment (corticosteroids,
toxoplasmosis) azathioprine, mycophenolate mofetil)
Leukemia and lymphoma
Eosinophils 0.04–0.4  109/l Parasitic infections
Allergic disease (hay fever, asthma)
Drug reactions (Stevens-Johnson
syndrome)
Skin disease (pemphigus, psoriasis,
dermatitis herpetiformis)
Lymphoma and leukemia
Basophils 0–0.1  109/l Myeloproliferative disease
Viral infection
Hypersensitivity reactions
Inflammatory disorders
(rheumatoid arthritis, ulcerative
colitis)
Monocytes 0.2–0.8  109/l Chronic infection (TB, brucellosis)
Granulomatous disease
(sarcoidosis)
Malignancy
a
Laboratory reference ranges may vary. Clinicians should check with their local laboratory

glucose levels. This can be due to the inability to
produce enough insulin (type 1) or cellular resis-
tance to insulin (type 2). Biochemical tests used
to measure glycemic control include random glu-
cose, fasting glucose, post glucose challenge, and
glycosylated hemoglobin (HbA1c.) The fasting
glucose and post glucose challenge are commonly
used to help diagnose diabetes mellitus, while the
HbA1c gives a long-term view of glycemic
control.
The conditions that would require an oral med-
icine specialist to consider assessing glycemic
control include:
disease, a baseline HbA1c is required in order
to monitor the patient for development of dia-
betes. A random glucose is of little value as
glucose levels are variable and related to eating
and drinking.
• Suspected oral candidosis.
• Diabetic-related salivary hypofunction.
• In those patients presenting with fatigue as
part of their presenting complaints such as
Sjógren’s syndrome.
Liver Biochemistry

• Corticosteroid monitoring. For patients A typical liver biochemistry panel (LBP) test
who require long-term corticosteroid treatment includes bilirubin, alkaline phosphatase, alanine
such as those with active immunobullous transaminase, and albumin. International
Laboratory Medicine and Diagnostic Pathology
normalized ratio (INR) and gamma-glutamyl
transpeptidase (γGT or GGT) are also blood tests
used to assess liver function. Clotting factors II,
VII, IX, and X are produced in the liver, and hence
an elevated INR is indicative of potential liver
inflammation in the absence of other known
causes. INR, albumin, and bilirubin are the best
markers to assess liver function.
Alkaline phosphatase is found in both the cells
lining the bile duct as well as bone. Hence damage
to either of these sites releases the enzyme into
the blood and causes elevated levels. It is the
commonest marker of biliary obstruction. How-
ever natural elevations are noted in the 3rd trimes-
ter of pregnancy and in adolescence. γGT is also
found in the bile ducts, so in the presence of a
raised alkaline phosphatase, assessing the γGT
can help differentiate the cause of a raised alkaline
phosphatase between the bone and liver. Alkaline
phosphatase and γGT are markers of cholestasis,
and transaminases are markers for hepatocellular
disease.
Alanine transaminase (ALT) and aspartate
transaminase (AST) are both transaminases, and
levels of either/both may occur and be found on
an LBP dependent upon the laboratory used.
They are strong markers of liver disease as they
are found within the hepatic cell cytoplasm or cell
mitochondria. Elevated ALT or AST levels can be
secondary to viral infection, medications, chronic
alcohol use, and cirrhosis. An ALT greater than
1000 u/L is probably indicative of viral hepatitis.
Liver biochemistry should be requested by an
oral medicine specialist:
1. Before commencing systemic immuno-
suppression, for example, azathioprine/
mycophenolate mofetil
2. Monitoring of patients on particular systemic
medications, for example, azathioprine and
mycophenolate mofetil
3. Prior to the use of systemic azole antifungal
medications
4. For those patients with suspected liver disease
(persistent heavy alcohol intake requiring
mucosal biopsy)
9
The request for INR and γGT should be based
on clinical judgment and the result of liver
biochemistry.
Liver function abnormalities are a poor marker
for hepatitis C (HCV) infection as the function
may be unaffected for many years. While the
literature documents an association between oral
lichen planus (OLP) and HCV, if a clinician sus-
pects HCV in refractory OLP, then virology tests
should be carried out rather than a surrogate test of
liver function alone.
Albumin is a protein made by the liver; it is
a large molecule and found distributed in the
plasma. Persistently low albumin levels are usu-
ally a marker of either liver or kidney disease. It
is a test used in conjunction with other biochem-
istry tests. It is not necessarily required in every
day oral medicine practice unless clinically
justified.
Renal Biochemistry
A standard renal profile includes sodium, potas-
sium, creatinine, urea, and an eGFR (estimated
Glomerular Filtration Rate). This test provides
an overall view of kidney function. It may be
requested for those at risk of developing renal
disease (renal hypertension) or for monitoring
purposes for those with known kidney disease.
In oral medicine, a clinician may wish to
request renal biochemistry for:
1. Pre-prescription of systemic therapy, for exam-
ple, azathioprine and mycophenolate mofetil
2. Drug monitoring (e.g., immunosuppressant
therapy, colchicine, or carbamazepine)
3. Suspected systemic lupus erythematous (SLE)
especially prior to immunosuppression for
severe mucosal disease
Bone Profile
A blood bone profile can include the following:
calcium, corrected calcium, albumin, and alkaline
phosphatase.
10
The use of these tests within oral
medicine is normally disease specific and
are additional tests following the results of other
investigations such as radiographs. Examples
include metabolic bone disorders, Paget’s dis-
eases, and hyperparathyroidism.
Hematinics
Vitamin B12
Strictly speaking, the term “vitamin B12” should
be defined as cyanocobalamin. This form does not
occur in vivo. Cyanocobalamin releases a cyanide
group for every molecule of B12 that is used.
However, it is incorrect that hydroxocobalamin
is the active form of the vitamin. There are two
active forms of the B12 enzyme in the human cell.
First, methylcobalamin acts as a coenzyme for
the conversion of homocysteine to methionine.
Methionine subsequently acts as a methyl-donor
to a great number of reactions that need a methyl
group, including the synthesis of myelin,
serotonin, dopamine, noradrenalin, DNA, and
phospholipids. Secondly, adenosylcobalamin is a
coenzyme for the conversion of L-methylmalonyl-
CoA into succinyl-CoA that feeds into the citric
acid cycle.
Vitamin B12 has an essential role in red blood
cell production. Deficiency causes a macrocytic
anemia.
Vitamin B12 deficiency is commonly a result of
a poor diet or impaired absorption, including gas-
trectomy, ileal disease such as Crohn’s disease,
and coeliac disease.
Common causes of elevated vitamin B12
include hematological malignancies, liver dis-
ease, and polycythemia rubra vera.
In the practice of oral medicine, a clinician may
request vitamin B12 studies for:
1. Investigation of recurrent oral ulceration
2. Investigations of oral mucosal burning/
dysesthesia
3. Oral candidosis
4. Investigation of the impact of inflammatory
bowel disease
T. Hodgson et al.
5. Patients presenting with fatigue as part of
the wider clinical context, such as Sjógren’s
syndrome
Methylmalonic acid (MMA) testing is rarely
requested, sometimes along with homocysteine,
to help diagnose an early or mild B12 deficiency.
It may be requested as a follow-up to a vitamin
B12 test result that is in the lower end of the
normal range. Until more data supports its use
and consensus on its clinical utility and long-
term benefits are demonstrated, it will probably
not be routinely used by clinicians. It should
be noted, failing to correct folate levels prior to
treating vitamin B12 deficiency may lead to an
irreversible peripheral neuropathy.
Iron Studies
Investigation of iron levels and stores often involves
more than one biochemical test. Ferritin acts as an
intracellular iron store and serum ferritin is an indi-
rect marker of the bodies iron stores. It is the most
sensitive marker for iron deficiency; however, cli-
nicians must be aware that it is an acute-phase
protein and hence can give rise to false-negative
results. A reading below the lower reference level
can therefore be relied upon as a true deficiency.
Iron studies may also be requested and include
total iron-binding capacity (TIBC) that is high in
iron deficiency, transferrin saturation which is low
in iron deficiency and total iron.
Common causes of iron deficiency:
1. Poor diet
2. Blood loss. For example, heavy menstruation,
gastrointestinal blood loss: cancer, inflamma-
tory bowel disease, and gastric ulceration
3. Pregnancy
4. Malabsorption
5. Hook worm infestation (most common cause
in tropical regions)
Within the practice of oral medicine, a clinician
may request ferritin studies or iron studies for:
1. Recurrent oral ulceration
2. Oral candidosis
Laboratory Medicine and Diagnostic Pathology
3. Oral mucosal burning/dysesthesia
4. Investigation of inflammatory bowel disease
5. Fatigue
6. Patients presenting with fatigue as part of the
wider clinical context
Folate
There are two well-known laboratory tests for
measuring folate levels: serum folate and red
blood cell folate. Serum folate is closely matched
to recent folate intake, and therefore it cannot
differentiate between transient dietary changes as
a cause of deficiency or true chronic deficiency.
Red blood cell folate is a measure of long-term
deficiency. Erythrocytes take up folate at the
erythropoiesis stage. The life span of the cell is
120 days. Clinicians need to ensure they supple-
ment for at least 4 months prior to retesting to
observe any change.
The common causes of low folate levels
include dietary deficiency, malabsorption, drug
interaction, and alcohol excess.
Within oral medicine, a clinician may request
folate studies for:
1. Oral mucosal burning/soreness
2. Drug monitoring. For example, carbamazepine
3. Oral candidosis
4. Recurrent oral ulceration
Fasting Lipids
This test includes low-density lipoproteins (LDL),
high-density lipoproteins (HDL), total choles-
terol, and triglycerides. Within the practice of
oral medicine, clinicians have previously tested
lipids in patients requiring long-term corticoste-
roid treatment, at both initiation and monitoring
during therapy, as well as monitoring systemic
retinoids. Recent studies in rheumatoid arthritis
patients given corticosteroids for over 3 months
showed no significant difference in lipid profiles
when compared to those not prescribed cortico-
steroids suggesting that in this group of patients,
this test is not required (Schroeder et al. 2015).
Retinoids are associated with significant adverse
lipid values including high trigylcerides and
11
total cholesterol, and a lipid profile before and
after 2 months of therapy is indicated (Hansen
et al. 2016).
Acute-Phase Proteins
Acute-phase proteins are defined as those proteins
whose serum concentrations increase (positive
acute-phase proteins) or decease (negative acute-
phase proteins) by at least 25% during inflamma-
tory states (Gabay and Kushner 1999; Jain et al.
2011). Cytokines, primarily interleukin-6, are
considered to be the principal agents responsible
for stimulating the production of acute-phase pro-
teins (Tanaka and Kishimoto 2014; Gabay and
Kushner 1999). Positive acute-phase proteins
include C-reactive protein, ferritin, and elements
of the complement system (C3, C4, C9) and com-
ponents of the coagulation and fibrinolytic system
(fibrinogen, plasminogen, and protein S). Proteins
such as albumin, alpha-fetoprotein, and factor
XII are among those classified as negative
acute-phase proteins (Mizejewski 2015; Poudel-
Tandukar et al. 2017). These negative acute-phase
proteins will not be discussed further due to pre-
sent irrelevance to oral medicine practice.
C-Reactive Protein
C-reactive protein (CRP) is a positive acute-phase
protein that is produced by hepatocytes in
response to tissue injury, inflammation, or infec-
tion. It has multiple roles in response to inflam-
mation and infection including recognition of
foreign pathogens due to its ability to bind
phosphocholine in injured cells, complement acti-
vation, binding to phagocytic cells, induction
of tissue factor and cytokines in monocytes,
and prevention of neutrophil to endothelial cell
adhesion (Gabay and Kushner 1999). Secretion of
CRP begins 4–6 h after a stimulus (and peaks
at 36–50 h, with a reported half-life of 19 h)
(Vigushin et al. 1993).
Numerous factors may influence the baseline
level of CRP, including age and gender, with a
formula available reflecting the increases seen
with advancing age and female gender. The refer-
ence range commonly reported for CRP is less
12
than 5 mg/L; although in the absence of disease,
99% of patients will have a CRP of less than
10 mg/L (Shine et al. 1981).
CRP is not an investigation used for any spe-
cific condition managed in the scope of practice of
oral medicine. It is frequently used in allied med-
ical fields, especially in the assessment of patients
with connective tissue and rheumatological dis-
ease. For example, the measurement of CRP is
considered a key component in the monitoring of
disease activity in rheumatoid arthritis (RA). It is
recommended in the decision-making process
with regard to increasing the medication used in
the management of RA and can be incorporated
into the disease activity score based on the assess-
ment of 28 joints (DAS28) (National Institute for
Health and Care Excellence 2009). With regard to
systemic lupus erythematosus (SLE), studies have
demonstrated that almost two thirds of patients
have CRP levels greater than 10 mg/L (ter Borg
et al. 1990; Becker et al. 1980); however, these
clinically significant levels of CRP were not found
to correlate with disease activity (Dima et al.
2016). The rise in CRP in patients with SLE
is generally considered to be modest or muted
(Gaitonde et al. 2008) and therefore is not used
as a diagnostic test nor as a marker of disease
activity. Similarly with primary Sjógren’s syn-
drome (pSS), it has been long established that
CRP is not a good indicator of disease activity
(Moutsopoulos et al. 1983) as is it considered to
be only moderately raised in patient with pSS
(Pertovaara et al. 2009).
In gastroenterology, CRP is evaluated in
patients suspected to have inflammatory bowel
disease (IBD) or irritable bowel syndrome (IBS)
but is not considered diagnostic for either condi-
tion (National Institute for Health and Care Excel-
lence 2008). CRP is upregulated in both ulcerative
colitis (UC) and Crohn’s disease (CD); however,
there is no consistency with regard to the rise in
CRP. There is a more marked rise in CRP levels
seen in patients with CD than those with UC
with no sound evidence to support the discrep-
ancy in CRP between the conditions (Vermeire
et al. 2006).
T. Hodgson et al.
Erythrocyte Sedimentation Rate
Erythrocyte sedimentation rate (ESR) is
considered an acute-phase nonprotein or an indi-
rect acute-phase reactant. It is the coagulation
and fibrinolytic system acute-phase proteins, spe-
cifically fibrinogen that is directly correlated
with ESR. ESR is defined as the rate (expressed
in mm/hr) at which erythrocytes suspended
in plasma have fallen after 1 h in a vertical
column of anticoagulated blood under the influ-
ence of gravity. Although the reference range of
0–15 mm/h is commonly seen on laboratory
reports, age and gender can influence ESR.
A number of erythrocyte specific factors
can also influence ESR such as size, shape, and
number. ESR will be decreased in sickle cell
disease and iron deficiency anemia, while an
increased ESR will be seen in severe anemia and
macrocytosis. Conditions or diseases that result
in elevated levels of fibrinogen, including preg-
nancy, diabetes mellitus, end-stage renal failure,
and malignancy, will also cause a rise in ESR
(Brigden 1999).
As with CRP, ESR is not used in the diagnosis
or monitoring of any disease specific to oral med-
icine clinical practice but in allied medical spe-
cialties. ESR is a component of the diagnostic
criteria of polymyalgia rheumatica and giant cell
arteritis. Although neither of these conditions are
commonly managed in an oral medicine setting,
giant cell arteritis can present with headache and
jaw pain so may be included in the differential
diagnosis of these conditions. ESR is considered
the gold standard marker of inflammation in mon-
itoring both polymyalgia rheumatica and giant
cell arteritis with a marked decrease seen within
a few days of commencing systemic corticoste-
roids (Salvarani et al. 2005). Caution must be
taken in over dependence on ESR in patients
with polymyalgia rheumatica and giant cell arter-
itis, as there is a subgroup among this cohort of
patients in which ESR remains normal (Salvarani
et al. 2008).
The DAS28 used in the monitoring of disease
activity in patients with rheumatoid arthritis
commonly incorporates ESR, although CRP can
be used as mentioned previously. In patients with
SLE, ESR and CRP measured together can help to
Laboratory Medicine and Diagnostic Pathology
differentiate between an exacerbation of SLE and
a concurrent infection in patients with SLE. In an
exacerbation of SLE, the ESR will be raised but
not CRP (Fernando and Isenberg 2005).
Ferritin
The acute-phase protein ferritin is an iron storage
protein, and therefore serum ferritin can reflect the
body’s iron stores. A correlation between serum
ferritin and iron stores has shown that 1 μg/L of
serum ferritin corresponds to 8–10 mg of stored
iron (Walters et al. 1973). Low ferritin provides
evidence of iron deficiency and is recommended
to be included in the first-line investigations of
a microcytic anemia (WHO 2011). Its role as a
positive acute-phase reactant is due to the induc-
tion of the synthesis of ferritin by a number of
interleukins, including IL-1 and IL-6, tumor
necrosis factor (TNF)-α in hepatocytes. As ferritin
levels can be elevated in response to inflammation
or infection, a normal serum ferritin does not
exclude iron deficiency anemia. However, a ferri-
tin level >100 μg/L in a patient with a microcytic
anemia can be considered to exclude iron defi-
ciency anemia (Zhu et al. 2010).
Normal values of serum ferritin vary across
laboratories. Independent of iron overload and
inflammation, serum ferritin can be raised in
alcohol excess, viral hepatitis, nonalcoholic
steatohepatitis and renal failure (Adams
et al. 2005).
In an oral medicine setting, patients with oral
ulceration and oral burning symptoms are com-
monly investigated for anemia. Due to the recom-
mendation of the use of serum ferritin in the
investigation of microcytic anemia, this test will
be carried out and interpreted in these patients
(Scully and Porter 2008; Renton 2011).
Fecal Calprotectin
Calprotectin is a zinc- and calcium-binding protein
found in the cytosol of inflammatory cells (Smith
and Gaya 2012). It is derived mostly from neutro-
phils and monocytes and considered a valuable
marker of neutrophil activity (Muthas et al. 2017).
The function of calprotectin has not been clarified,
13
but it is thought to have bactericidal and fungicidal
properties (Steinbakk et al. 1990). It can remain in
stool samples at room temperature for 7 days and is
measured using ELISAwith as little as a 5 g sample
(Muthas et al. 2017; Yousefi et al. 2007).
The investigation is not carried out in oral
medicine but is recommended by the British
Society of Gastroenterology (BSG) in the diagno-
sis of IBD. Research has been carried out indicat-
ing the predictive value of fecal calprotectin
in determining relapse of both UC and CD (Mao
et al. 2012). It is not specific to IBD as it can
also be raised in gastric and colorectal cancer,
colorectal polyps, and with the used of nonsteroi-
dal anti-inflammatory medications (Khoshbaten
et al. 2014; Rendek et al. 2016).
It has been used in research regarding orofacial
granulomatosis (OFG) in oral medicine popula-
tion as a component of the diagnostic process in
determining those patients who may in fact have
Crohn’s disease (Gale et al. 2015).
Zinc
Zinc is an essential trace element found in tis-
sues including muscle, bone, and skin with
approximately 2 g of zinc distributed throughout
the human body (Thomas and Bishop 2007).
The biological functions of zinc can be broadly
categorized as catalytic, regulatory, and struc-
tural. It is considered a vital component of
numerous enzymes and proteins along with a
proposed role in the stimulation of bone growth
and mineralization (Yamaguchi et al. 1988;
Yamaguchi and Uchiyama 2004). It is an ele-
ment considered essential for growth and devel-
opment in humans (Bhattacharya et al. 2016). In
the oral cavity, zinc is present in plaque, saliva,
and enamel. During the development of the den-
tition, significant amounts of zinc are incorpo-
rated into enamel, while variable concentrations
of zinc have been found in plaque. It is proposed
that the oral mucosa is an important intraoral
reservoir of this essential trace element,
although no concrete evidence supports this
claim (Lynch 2011).
14
According to a recent review, up to 17% of the
population worldwide has insufficient dietary
intake of zinc. It is found in meat and fish, with
poor bioavailability in vegetables (Bhattacharya
et al. 2016). Zinc status of individuals is difficult
to assess. It is recommended to use of zinc con-
centrations in hair to determine dietary zinc, while
urinary zinc is a marker of plasma zinc concentra-
tions (Lowe 2016).
Although zinc is ubiquitous in the human body
and commonly found in the hard and soft tissues
of the oral cavity, it has a limited role in oral
medicine clinical practice. Deficiency in zinc has
historically been associated with taste distur-
bance, a symptom reported by patient that may
lead to a referral to oral medicine for further
investigation. This association was first proposed
following a 1972 US-based trial of the efficacy
of zinc supplementation in the management of
hypogeusia (Schechter et al. 1972). Subsequent
studies, in the late 1970s and early 1980s, have
demonstrated contradictory evidence regarding
the role of zinc supplementation in the manage-
ment of taste loss (Henkin et al. 1976; Mahajan
et al. 1980). A recent review, however, stated that
a deficiency in zinc is unlikely to be the cause of
hypogeusia (Cowart 2011).
Zinc is incorporated into oral health products,
as it is effective at controlling plaque and calculus
formation along with a noted reduction in oral
malodor. In an oral medicine setting, patients
may present with oral malodor, which may be
contributed to by odiferous compounds including
volatile sulfur compounds (VSCs). It is suggested
that zinc compounds, such as zinc chloride,
are efficient at removing VSC (Scully and
Greenman 2012).
Oral submucous fibrosis (OSF) is a long-
standing progressive condition leading to stiff-
ening of the oral mucosa and subsequent
limitation of mouth opening (Tilakaratne et al.
2016). Although this condition is the most com-
mon in south Asian, many patients with OSF are
managed in oral medicine units across the world.
Oral zinc supplementation has demonstrated a
positive effect on OSF (Warnakulasuriya and
Kerr 2016). This effect is thought to be due to
the role of zinc in chelating copper, a causative
T. Hodgson et al.
factor in the development of OSF (Kumar et al.
1991).
Serum Angiotensin-Converting
Enzyme
The primary source of serum angiotensin-
converting enzyme (ACE) is the endothelium of
the lung. The reference interval for pediatric
patients may be up to 50% higher than that of
adults. The serum ACE level is elevated in 75%
of untreated patients with sarcoidosis (Studdy and
Bird 1989). Other disease processes that have
been associated with an elevated serum ACE
include tuberculosis, silicosis, asbestosis, histo-
plasmosis, coccidiomycosis, diabetes mellitus,
Gaucher disease, Hodgkin disease, hypersensitiv-
ity pneumonitis, hyperthyroidism, leprosy, lung
cancer, and primary biliary cirrhosis. Approxi-
mately 5% of the healthy population has raised
levels. It has limited utility as a diagnostic test
for sarcoidosis due to poor sensitivity and
specificity with almost a 10% false-positive result
(Ungprasert et al. 2016). Nevertheless serum ACE
activity, which reflects the total amount of sarcoid
granulomas, has been proposed as a marker for the
activity of sarcoidosis. The ACE level may
decline in response to treatment or disease resolu-
tion with time. Corticosteroids independently sup-
press ACE levels, and reducing the dose may lead
to a rise in ACE level without a worsening of
disease activity.
Serologic markers such as serum amyloid A,
soluble interleukin-2 receptor, lysozyme, adeno-
sine deaminase, and the glycoprotein KL-6 have
been examined for potential roles in diagnosis
and monitoring disease activity in sarcoidosis
(Gungor et al. 2015). Serum amyloid A has
been found to be increased in sarcoidosis, but it
has not been shown to be clinically useful. Solu-
ble interleukin-2 receptor has been suggested as a
useful marker for determination of extra-
pulmonary involvement in sarcoidosis patients
(Grutters et al. 2003). Serum lysozyme is ele-
vated in the same way as ACE but in a smaller
number of patients with sarcoidosis. Elevated
adenosine deaminase levels may be found in
Laboratory Medicine and Diagnostic Pathology
serum and bronchoalveolar lavage fluid in sar-
coidosis patients. The clinical utilization of aden-
osine deaminase is limited given the low
sensitivity and specificity (Gungor et al. 2015).
Serum KL-6 levels are elevated in pneumonitis
of various causes including sarcoidosis. These
tests may eventually find a role in diagnosis and
monitoring.
Immunosuppressive Medication
Metabolism
Immunosuppression is a common method of
controlling mucosal inflammatory diseases.
These medications have multiple serious adverse
effects that may be mitigated by the measurement
of enzyme levels required for detoxification and
pathway end products.
Thiopurine Methyltransferase
Thiopurine methyltransferase (TPMT) is an impor-
tant enzyme-metabolizing immunosuppressive
drug such as azathioprine (AZA) and 6-mercapto-
purine (6-MP). TPMT is therefore measured prior
to commencing treatment with AZA/6-MP. High
levels of TPMT are found in erythrocytes. Varia-
tions in TPMT allow shunting of 6-MP/AZA into
6-methylmercaptopurine when levels are normal or
high. Trimodal population activity of TPMT has
been demonstrated (Lennard et al. 1989). Approx-
imately 89% of the population has wild-type
TPMT, which is associated with normal or high
TPMT enzyme activity. High metabolizers
(>55 nmol/g) may catabolize the intermediaries
so rapidly that they may remain refractory to con-
ventional doses of AZA/6-MP (Benmassaoud et al.
2016). Higher doses or alternative agents may be
necessary. Eleven percent are heterozygous with
intermediate TPMT enzyme activity and may be
subject to delayed bone marrow toxicity (Zur et al.
2016). Importantly, 0.3% of the population is
homozygous for mutations of TPMT and thus has
negligible activity. This causes AZA/6-MP to be
preferentially metabolized to produce high levels
of 6-TG, which then leads to bone marrow sup-
pression (Zur et al. 2016).
15
Assessment of TPMT activity may require
repeated measurements, while patients are taking
AZA/6-MP, since AZA and 6-MP can induce an
increase in TPMT activity. Similarly, it is impor-
tant to consider drug interactions that can affect
AZA/6-MP metabolism. 5-Amino salicylic acid
drugs, including sulfasalazine and mesalazine,
can reversibly inhibit TPMT activity and thus
lead to corresponding increases in 6-TG with
resultant leukopenia. Allopurinol inhibits the
enzyme xanthine oxidase and should normally
be avoided. In some specialist units, allopurinol
may be given with low-dose azathioprine to cause
shunting of 6-MMP metabolites with a resultant
shift in metabolism toward 6-TG (Sparrow et al.
2007; Hullah et al. 2015).
Thioguanine Nucleotides
Assessing 6-thioguanine nucleotides (6-TG)
and 6-methylmercaptopurine (6-MMP) concen-
trations, end products of AZA/6-MP metabolism,
has been shown to be clinically useful (Lee et al.
2015). Measurement of 6-TG levels, the active
end-product of AZA/6-MMP metabolism, corre-
lates with therapeutic efficacy and toxicity.
Several studies of AZA and 6-MMP have demon-
strated that therapeutic efficacy correlates with
concentrations of 6-TG levels between 230 and
400 pmol/8  108 red blood cells and bone mar-
row suppression is more likely with concentra-
tions of 6-TG greater than 400 pmol/8  108 red
blood cells (Osterman et al. 2006; Goldenberg
et al. 2004). While no correlation has been found
between therapeutic efficacy and 6-MMP levels,
hepatotoxicity correlates with concentrations
of 6-MMP greater than 5000 pmol/8  108 red
blood cells (Dubinsky et al. 2000). Thiopurine
metabolites are usually measured at 4 weeks to
confirm adherence, to optimize dosing, and to
identify early evidence of hypermethylation. The
metabolite level is then rechecked at 12–16 weeks
when it has reached a steady-state concentration.
Levels should be repeated 4–6 weeks after any
change in therapy.
Adherence to AZA/6-MMP can also be
assessed by monitoring the levels of 6-TG
and 6-MMP. Low or absent 6-TG levels in
non-responding patients may indicate non-
16
compliance, use of a sub-therapeutic dose of
AZA/6-MMP, or 6-MMP resistance.
There are limitations to using metabolite levels
to guide drug dosing. There may be substantial
variation in levels between measurements in indi-
vidual patients. Adverse reactions can also occur
unrelated to elevated levels. Therefore, measure-
ment of 6-TG and 6-MMP levels cannot replace
regular monitoring for toxicity (González-Lama
and Gisbert 2016).
Short SynACTHen Test
The use of potent topical corticosteroids such as
clobetasol applied to the oral mucosa, or injected
corticosteroids such as triamcinolone, may result
in adrenocortical suppression (Decani et al. 2014).
This is not clinically significant in most patients;
however, in those with compatible symptoms
such as fatigue, hypotension, and weight loss, it
should be referred for specialist endocrinology
review and investigated for iatrogenic suppression
of the hypothalamic-pituitary-adrenal axis with a
short synACTHen test. This involves checking
the baseline cortisol and rechecking it 30 min
after injection of a synthetic ACTH. A rise in
serum cortisol of greater than 200 nmol/L repre-
sents a normal response and therefore no adrenal
suppression.
A short synACTHen test, along with an ACTH
level, should be performed if Addison’s disease is
suspected as the cause of hyperpigmentation, in
which case there will be raised levels of ACTH
but an inadequate response to the synACTHen. A
random cortisol is not recommended to exclude
adrenal insufficiency, as the diurnal variation
and effects of stress make the results difficult to
interpret. However a midmorning cortisol in non-
stressed individuals may predict the short syn-
ACTHen outcome (Lee et al. 2003).
Glucose-6-phosphate Dehydrogenase
Glucose-6-phosphate Dehydrogenase (G6PD)
catalyzes the conversion of glucose-6-phosphate
to 6-phosphogluconate. Absence of functional
T. Hodgson et al.
G6PD (1–5% of normal activity) impairs the
reduction of NADP to NADPH via the pentose
phosphate pathway, which is the only method of
NADPH generation in erythrocytes. Dapsone is
used in an oral medicine setting for management
of mucous membrane pemphigoid. Dapsone has
long been recognized as a cause of oxidative
hemolysis and methemoglobinemia. Underlying
G6PD deficiency predisposes individuals to
severe hemolysis. Most prescribers measure
G6PD prior to commencing treatment.
G6PD deficiency is an X-linked (Xq28) condi-
tion. It is the most common enzymatic disorder of
red blood cells in humans, affecting 400 million
people worldwide. The World Health Organiza-
tion has classified the different G6PD variants
according to the level of enzyme deficiency and
the severity of hemolysis (WHO Working Group
1989). Class I variants have severe enzyme defi-
ciency (<10% of normal) and have chronic hemo-
lytic anemia. Class II variants also have severe
enzyme deficiency, but there are usually only
intermittent episodes of acute hemolysis associ-
ated with infection, drugs, or chemicals. Class III
variants have moderate enzyme deficiency
(10–60% of normal) with intermittent episodes
of acute hemolysis usually associated with infec-
tion, drugs, or chemicals. Class IV variants have
no enzyme deficiency. Class V variants have
increased enzyme activity. Absence of functional
G6PD (1–5% of normal activity) impairs the
NADPH system of oxidative metabolism. The
nitroblue-tetrazolium (NBT) test is diagnostic
and enzyme activity can be measured. Hemolytic
anemia is often present and can be triggered by
certain foods (fava beans) and drugs including
dapsone, primaquine, and salicylates. These
drugs interact with hemoglobin and oxygen, lead-
ing to the intracellular formation of H2O2 and
other oxidizing radicals. As these oxidants accu-
mulate within enzyme-deficient cells with low
glutathione levels, hemoglobin and other proteins
are oxidized, leading to erythrocyte death. Dap-
sone should be avoided in patients with G6PD
deficiency.
Laboratory Medicine and Diagnostic Pathology
Immunology
The number of immunological tests continues to
increase. There is potential use in oral medicine so
investigation choice, and the ability to interpret
test outcomes, remains extremely important.
C1 Esterase Inhibitor
C1-esterase inhibitor is a control protein of the
classical pathway. The key indication for testing
in oral medicine is angioedema occurring without
urticaria at any age. If urticaria is present, the
diagnosis is almost never C1-esterase inhibitor
deficiency. Normal immunochemical range in
adults is 0.18–0.54 g/L. The functional range,
reported as percentage activity compared with
normal, is 80–120%.
Two main types of hereditary angioedema
(HAE) are recognized. Both are inherited as auto-
somal dominant conditions.
• Type I (common, 80%): absence of immuno-
chemical C1-esterase inhibitor
• Type II (rare, 20%): presence of nonfunctional
C1-esterase inhibitor, due to point mutations
affecting enzyme active site
In type I, there is a low C1-inhibitor level
immunochemically, and this will become
undetectable in an acute attack. If immunochem-
ical C1-esterase inhibitor is low/absent, there is
no additional value in measuring functional
C1-esterase inhibitor. In type II, a normal or high
level of inhibitor will be measured immunochem-
ically, but function will be low or absent. C4 level
is a useful screen. A normal C4 level during an
attack excludes C1-esterase inhibitor deficiency.
If C4 level is low in a patient with angioedema and
immunochemical C1-esterase inhibitor is normal
or high, type II HAE should be suspected and the
C1-esterase inhibitor function should be assessed
(Bowen et al. 2010).
C1 inhibitor deficiency may be acquired
secondary to SLE, myeloma, and splenic villous
lymphoma. These may be distinguished from
17
HAE by a reduction in C1q, although this is not
always reliable (Zingale et al. 2006). Attacks are
triggered by similar stimuli to HAE. The develop-
ment of angioedema in an older patient should
lead to a search for a paraprotein (serum immuno-
globulins, serum and urine electrophoresis, serum
free light chains, and β-microglobulin). Detection
of autoantibodies (antinuclear antibody (ANA),
dsDNA, and extractable nuclear antigen (ENA)
antibodies) may be necessary to exclude connec-
tive tissue disease, although this will usually be
obvious. ACE level should be checked to exclude
ACE deficiency.
Complement
Complement was first identified as a heat-labile
component in blood that “complemented” the
ability of antibodies to destroy bacteria. Levels
of C3 and C4 are commonly used in clinical
practice. The measurement of C3 and/or C4 can
assist in the diagnosis of certain diseases, as well
as in monitoring the course of the disease. Both
C3 and C4 are acute-phase proteins, and levels
may remain within normal range even with rapid
consumption. Testing for C3 breakdown products
are more valuable as markers of complement
turnover.
Testing in oral medicine may be requested, but
other than for angioedema is not the first diagnos-
tic investigation of choice:
• Suspected SLE (low C3 and C4, raised C3d)
• Suspected Sjógren’s syndrome (low C4 and C3
or C3 alone)
• Suspected hereditary angioedema (C3, C4,
C1q, C1 esterase inhibitor, immunochemical,
and functional)
• Suspected anaphylaxis (anaphylatoxins C4a,
C5a)
Complement deficiency is common (especially
C4 and C2 deficiency) and predisposes to recur-
rent neisserial disease, bacterial infections, and
immune complex disease (Table 5). Patients with
low C4 complement levels at the time of diagnosis
18
Table 5 Interpretation of level of C3 and C4 testing
Low C4, normal C3 Normal C4, low C3
Genetic deficiency Streptococcal GN
SLE (active) Nephritic factor
Sjőgren’s syndrome Gram-negative sepsis
Hereditary angioedema
Type II cryoglobulins
of Sjógren’s syndrome display an increased risk of
developing lymphoproliferative disease (Table 5).
Human Leucocyte Antigen
The human leukocyte antigen (HLA) system is
synonymous with the human major histocompat-
ibility complex (MHC). These terms describe a
group of genes on chromosome six that encode a
variety of cell surface markers, antigen-presenting
molecules, and other proteins involved in immune
function. HLAs are proteins that assist the body’s
immune system to differentiate between its own
cells and harmful substances. Over 100 diseases
are associated with classical human leukocyte
antigen (HLA) I and II genes, as well as with
some of the non-HLA genes in the major histo-
compatibility complex (MHC) region (Shiina
et al. 2004).
Data regarding the relationship of human leu-
kocyte antigen (HLA) antigens to disease suscep-
tibility remain at the level of associations, not
disease mechanisms.
HLA region has been subdivided into three
regions: class I, class II, and class III.
• Class I region The class I region contains
the genes encoding the “classical” class I
HLA antigens: HLA-A, B, and C. Class I anti-
gens are expressed on almost all cells of the
body, except erythrocytes and trophoblasts.
• Class II region The class II region contains the
genes encoding the HLA class II molecules,
HLA-DR, DQ, and DP. Class II antigens are
constitutively expressed on B cells, dendritic
cells, and monocytes and can be induced dur-
ing inflammation on many other cell types that
normally have little or no expression.
T. Hodgson et al.
Low C4, low C3
Sepsis
SLE (active)
Subacute bacterial endocarditis
• Class III region The region in between class I
and class II is known as the class III region.
Although this region does not contain any of
the HLA genes, it does contain many genes of
importance in the immune response including
several complement components, tumor necro-
sis factor, and heat shock protein genes.
Many diseases relevant to oral medicine are
associated with classical HLA I and II genes, as
well as with some of the non-HLA genes in
the MHC region. HLA associations that are repro-
ducible and robust provide important clues about
the development of certain diseases.
Higher prevalence of HLA-B51 has been
found in Behçet’s disease patients in Italy,
Germany, and Middle Eastern and Far Eastern
countries along the Silk Road (63% vs. 9% in
controls) and of HLA-B52 (21% vs. 9%) in Israel
(Salvarani et al. 2001; Arber et al. 1991).
HLA-B5101 and, to a lesser extent, HLA-5108
alleles have been most closely linked in patients
along the Silk Road. Other HLA alleles
may increase (HLA-B15, HLA-B27, HLA-B57,
HLA-26) or decrease (HLA-B49, HLA-A03) the
risk for Behçet’s disease in various populations
(Hatemi et al. 2015).
Other well-established relationships include:
• HLA-B*15:02 with carbamazepine-induced
Stevens-Johnson syndrome/toxic epidermal
necrolysis (SJS/TEN) in South-East Asian
populations
• HLA-B27 with seronegative spondylo-
arthropathy
• HLA-DQ8 and HLA-DQ2 with celiac disease
• HLA-DQB1*0201 with vulvovaginal gingival
variant of lichen planus
Laboratory Medicine and Diagnostic Pathology
• HLA-DQB1*0301 with ocular mucous mem-
brane pemphigoid
HLA testing is not routinely performed in an
oral medicine clinical setting and the interpreta-
tion of results is complex.
Amyloid
Amyloid deposition is associated with a diverse
range of disorders that includes Alzheimer’s
disease, type II diabetes mellitus, and dialysis
arthropathy. Although less common, systemic
AA and AL amyloidosis remain important
because effective treatments have increasingly
become available. The pathology in all forms of
amyloidosis involves the extracellular deposition
of protein as characteristic fibrillar aggregates that
interfere with tissue structure and function. Amy-
loid fibrils are derived from different unrelated
proteins in the different forms of the disease but
share many common properties, including the
capacity to bind the normal plasma protein
serum amyloid P component. Thirty-one types of
extracellular, fibrillar proteins involved with
human amyloidosis have been identified (Sipe
et al. 2014).
Amyloid deposition is usually confirmed by
demonstration of apple-green birefringence on
Congo red staining on histological examination.
Measurement of serum immunoglobulins and
electrophoresis, serum free light chains, β -micro-
globulin, and CRP is essential if amyloid is
suspected. Head and neck amyloidosis is rarely
seen and is frequently associated with the immu-
noglobulin light chain-derived amyloid deposi-
tion (AL amyloidosis) secondary to plasma cell
dyscrasias (Penner and Műller 2006; Gouvêa et al.
2012); it may affect the larynx, sub-glottis, thy-
roid, and less frequently the oral cavity (Matsuo
et al. 2016). Other amyloidosis subtypes, like
the familial (AF) and (Apo) serum amyloid A
(AA) amyloidoses, are much rarer in the head
and neck, and data is limited to a very few case
reports. While the AF type is related to autosomal
dominant diseases, AA is associated with chronic
diseases, including rheumatoid arthritis and
19
chronic infections. Appropriate referral to hema-
tology should be arranged to diagnose the under-
lying disease.
Indirect Immunofluorescence
Indirect immunofluorescence (IIF) testing is used
to detect circulating antibodies. To perform this
test, the patient’s serum is overlaid on an epithelial
tissue substrate, incubated, and then stained for
fluorescent detection of antibodies. In oral medi-
cine, indirect immunofluorescence studies and
antigen-specific serologic testing may be utilized
to aid in the diagnosis and assessment of disease
activity in autoimmune blistering disease. If the
target of circulating antibodies is known, antigen-
specific testing can be used to detect the presence
of antibodies in serum.
Mucous Membrane Pemphigoid
Mucous membrane pemphigoid (MMP) may pre-
sent with IgG and IgA autoantibodies directed
against a variety of antigens, including BP180,
BP230, α6β4 integrin subunits in the hemides-
mosome, laminin 332, and collagen VII in the
adhesion complex. Early studies of MMP showed
IIF was positive in fewer than a third of patients.
Serum samples from MMP patients contain auto-
antibodies at low titers (usually 1:10–1:40) and
only in 50–80% of cases (Chan 2012). However,
the use of human basement membrane zone-split
skin and/or concentrated serum may increase
the sensitivity of this technique (Setterfield et al.
1998). IIF on a basement membrane zone-split
skin substrate may identify circulating autoanti-
bodies in up to 91% patients for IgG and 64%
patients with IgA (Setterfield et al. 1998). Dual
antibody response with IgG and IgA signifies
a more severe and persistent disease. There is a
significant relationship between the titer of IgG
and the presence of IgA with the severity of the
disease. In MMP, basement membrane zone-split
skin IIF is particularly useful for recognizing
patients at risk for malignancy-associated laminin
332 MMP; antibodies are found along the dermal
side. Antigen-specific testing for basement mem-
brane zone antibodies other than BP180 and
20
BP230 remains limited to research laboratories
and specialized centers (Amber et al. 2016). Fur-
ther research on the detection of salivary IgA and
IgG antibodies to BP180 NC16A by ELISA as a
useful diagnostic biomarker is necessary (Ali et al.
2016). Immunoprecipitation and immunoblotting
are additional serologic tests that have been uti-
lized in MMP. However, these tests are more labor
intensive to perform than IIF and ELISA and are
infrequently used in the clinical setting. Neverthe-
less, they may be the only way to definitively
identify laminin-332 pemphigoid.
Pemphigus Vulgaris
More than 80% of patients with pemphigus
vulgaris (PV) have circulating antibodies directed
against desmoglein 1 and desmoglein 3 detectable
by IIF (Di Zenzo et al. 2016). The substrate used
influences the test sensitivity. Monkey esophagus
is the preferred substrate for the diagnosis of pem-
phigus vulgaris. The transitional epithelium of
murine (rat) bladder is the preferred substrate in
patients with paraneoplastic pemphigus (PNP).
When performed on rat bladder epithelium, esti-
mates for the sensitivity and specificity of this test
have ranged from 74% to 86% and 83% to 100%,
respectively (Leger et al. 2012; Poot et al. 2013;
Joly et al. 2000). IIF is usually performed after
positive DIF studies are obtained, to help guide
prognosis and therapy (Ishii 2015). Serum dem-
onstrates a characteristic netlike intercellular
staining of IgG with an epithelial substrate.
ELISA for IgG antibodies to desmoglein 1 and
desmoglein 3 provides a simple and highly sensi-
tive approach to confirm the initial diagnosis of
PV. ELISA is more sensitive and specific than IIF
for the diagnosis of pemphigus vulgaris, with a
sensitivity exceeding 90%. ELISA may aid with
monitoring disease activity since desmoglein anti-
body levels often fall in response to treatment.
ELISA for antibodies against envoplakin and
periplakin has been utilized in studies of patients
with paraneoplastic pemphigus. In one study,
an ELISA based on the N-terminal fragment of
envoplakin had a sensitivity and specificity for
PNP of 81% and 99%, respectively; the ELISA
for periplakin had a sensitivity and specificity of
74% and 96% (Probst et al. 2009).
T. Hodgson et al.
Additional antigen-specific serological tests
may be used for the diagnosis of pemphigus,
including immunoblotting and immunoprecipita-
tion. Both tests are highly sensitive and specific
methods that have been used to detect autoanti-
bodies in PNP. The availability of such tests is
limited (Poot et al. 2013).
Immunoglobulins
Measurement of serum immunoglobulins does
not provide categorical diagnosis in any disease.
Normal serum immunoglobulins do not exclude
immunodeficiency. Measurement of serum immu-
noglobulin in an oral medicine setting is indicated
in the following diseases:
• Sarcoidosis (diagnosis)
• Sjögren’s syndrome (raised IgG1 with normal
or reduced IgG2, IgG3, and IgG4)
• Suspected immunodeficiency, 1 or 2
(diagnosis)
• Mycoplasma pneumoniae (raised IgM)
• Suspected myeloma (diagnosis)
• Lymphoma
• Liver disease (1 biliary cirrhosis, hepatitis,
cirrhosis)
There are no absolute indications for IgG sub-
class testing, as significant immunodeficiency can
occur in the presence of normal subclasses.
IgG4-related systemic disease (IgG4-RSD) is a
rare entity comprised of a collection of features
including tumor-like swelling of involved organs,
with a lymphoplasmocytic infiltrate enriched in
IgG4-positive plasma cells and elevated serum
levels of IgG4 (Kamisawa and Okazaki 2017).
All patients with recurrent infections should be
referred to an immunologist for further investiga-
tion. Any patient presenting with recurrent infec-
tions and reduced serum immunoglobulins has an
immunological problem until proven otherwise.
There are few indications for testing IgE
levels. Conditions associated with elevated
serum IgE include atopic disease, infectious dis-
ease (candidosis, CMV, EBV, HIV, tuberculosis,
parasitic infections), Hodgkin lymphoma,
Laboratory Medicine and Diagnostic Pathology
pemphigus vulgaris, bullous pemphigoid, some
primary immunodeficiencies, Churg-Strauss vas-
culitis, and hyper-IgE syndrome (Esposito et al.
2012). Therefore, elevated total serum IgE is not
specific to allergic disease.
Significant allergic disease is possible with low
levels of total IgE (including anaphylaxis). The
level does not correlate with severity of clinical
reactions. Patients with undetectable IgE are
unlikely to have allergic disease. It is important
to recognize that levels above the normal range
are compatible with no clinical allergic disease.
Levels of total IgE rarely provide information
about IgE to specific allergens, and the presence
of IgE to a specific allergen does not necessarily
equate to a clinical allergic response to that
substance. The results must be interpreted in
the context of the clinical history. Causes of
hypergammaglobulinemia and hypogammaglob-
ulinemia are outlined in Table 6.
Autoantibodies
Autoantibodies are generally divided into organ
specific and organ non-specific. Clinical testing
Table 6 Causes of hypergammaglobulinemia and hypogammaglobulinemia
Causes of hypergammaglobulinemia
Chronic infection: all immunoglobulins "
Osteomyelitis
Bacterial endocarditis
Tuberculosis
Sjögren’s syndrome: " IgG
SLE, rheumatoid arthritis: all immunoglobulins "
Sarcoidosis: " IgG and IgA
Liver disease:
Primary biliary cirrhosis (IgM may be very high)
Alcohol-related (" IgA).
Autoimmune hepatitis (" IgG, IgA)
Hodgkin’s disease: IgE " (also eosinophilia)
Viral infections:
Acute common viral infections: " IgM, $ IgG and IgA
Human immunodeficiency virus (HIV) – all
immunoglobulins " (IgG very high but polyclonal)
Epstein-Barr virus (EBV) – all "
21
infrequently follows this pattern. Autoantibodies
can be of any class but in most circumstances IgG
antibodies are usually sought. IgA autoantibodies
may also be diagnostically useful in celiac disease
where IgA endomysial antibodies have the
highest sensitivity and specificity. Autoantibodies
can also appear after infections, such as EBV
adenovirus, HIV, and acute and chronic bacterial
infections. They usually disappear after 6 months
and are not usually associated with clinical dis-
ease. Drugs may also induce autoantibodies.
These may cause disease and may persist after
the drug is withdrawn.
The most common autoantibodies associated
with infections are:
1. Rheumatoid factor (any infection)
2. Antinuclear antibodies – (adenovirus in chil-
dren), HIV, Gram-negative bacteria
3. Smooth muscle antibodies (adenovirus)
4. Liver-kidney microsomal antibodies (HCV)
5. Cardiolipin antibodies (EBV)
6. dsDNA antibodies – rare (HIV)
The most common autoantibodies associated
with drugs are:
Causes of hypogammaglobulinemia
Immunodeficiency:
X-linked agammaglobulinemia: all immunoglobulins
low/absent
Common variable immunodeficiency: low
immunoglobulins
Hyper-IgM syndrome: normal/raised IgM, low/absent
IgG, IgA
Selective IgA deficiency: absent IgA, $ IgG, IgM
Severe combined immunodeficiency: all
immunoglobulins low
Lymphoma: # IgM, IgA normal, IgG normal or low;
disease, chemotherapy, or radiotherapy
Infections:
Acute bacterial infections
Measles/rubella
Herpes viruses (rare)
Drugs: immunosuppressives, e.g., cyclophosphamide,
azathioprine, chemotherapy
Plasmapheresis
Renal loss: IgM $, IgG and IgA #
Gastrointestinal loss: IgM normal, IgG, and IgA #
22
1. Antinuclear antibodies (anti-histone) – (pro-
cainamide, hydralazine, ACE-inhibitors,
chlorpromazine, minocycline)
2. Non-M2 mitochondrial antibodies (alcohol)
Rheumatoid Factor
The test for rheumatoid factor (RhF) is widely
misused by clinicians. It is not a screening test
for rheumatoid arthritis and is positive in only
70–80% of patients. The normal ranges vary
with age. RhF is a non-specific test as it detects
immunoglobulins of any class reactive with the
Fc region of other immunoglobulins. The only
indication for this test is when patients have clin-
ical rheumatoid arthritis. A high titer in patients
with known rheumatoid arthritis is a risk factor
for extra-articular manifestations. There is little
value in serial monitoring of RhF as the titer
correlates poorly with disease activity. Raised
levels are also found in healthy elderly (asymp-
tomatic), chronic bacterial (SBE) and viral
infections (HIV, HCV, acute viral infections),
myeloma, lymphoma, and connective tissue dis-
eases (SLE, Sjögren’s syndrome, systemic sclero-
sis, polymyositis, undifferentiated connective
tissue disease). Requesting RhF is not helpful, as
positive results do not necessarily indicate disease
(Shmerling and Delbanco 1991). Anti-cyclic
citrullinated peptide antibodies are found in sera
of about 75% rheumatoid patients and are about
96% specific for rheumatoid arthritis. They appear
earlier than RhF, are unaffected by treatment, and
predict the development of erosions and localized
tissue damage (Gavrilă et al. 2016).
Antinuclear Antibodies
Antinuclear antibodies (ANA) is a general
term encompassing the group of antibodies to
intranuclear antigens. The ANA test is one of
the most unusual in medicine because a positive
result represents a classification criterion for the
diagnosis of autoimmune disease, while at the
same time, ANA positivity is present in a sub-
stantial proportion of the healthy population. It is
often thought of as a screening test for SLE or
other connective tissue diseases. Although ANA
is positive in 99% of cases of SLE, it is not
specific and can be positive in numerous
T. Hodgson et al.
situations including healthy individuals, hepatic
disease (primary biliary cirrhosis), pulmonary
disease (primary pulmonary hypertension),
chronic infections, malignancy, or may be drug-
induced. It should, therefore, be interpreted only
in the context of clinical symptoms (Pisetsky
2017). Most clinical laboratories report ANA as
a titer and the staining pattern produced by the
patient’s serum. The titer is determined by the
lowest dilution at which the fluorescence can still
be seen. A 1/40 titer, therefore, is less significant
than a 1/2560 titer. Nuclear staining patterns
include homogeneous, speckled, centromere,
and nucleolar. Antibodies in the serum of
Sjögren’s syndrome patients are likely to pro-
duce speckled or homogenous staining.
Low-titer positive ANAs in the elderly should
be interpreted in the context of relevant clinical
signs and symptoms. Positive ANA is useful for
the diagnosis of SLE and systemic sclerosis and
somewhat useful for the diagnosis of Sjögren’s
syndrome and polymyositis/dermatomyositis.
Detection of a strong positive ANA is an indica-
tor for confirmatory tests with ENA and dsDNA
to fully investigate antigenic specificities identi-
fied. Changes in ANA titer are not helpful
in disease monitoring in patients diagnosed
with an ANA-associated autoimmune disease.
Once a positive test has been obtained, repeat
measurements of ANA are not indicated
(Pisetsky 2017).
Antineutrophil Cytoplasmic Antibodies
Antineutrophil cytoplasmic autoantibodies
(ANCA) are antibodies directed against the lyso-
somal compartment of neutrophils and mono-
cytes. ANCA is often thought to be a screening
test for vasculitis (Bossuyt et al. 2017). In adults,
it is normally undetectable. The presence of
cytoplasmic-staining ANCA (cANCA) and pro-
teinase 3 (PR3) in combination has been reported
to be 99% specific (and ~55% sensitive) for
granulomatosis with polyangiitis. Similarly,
the presence of perinuclear-staining ANCA
(pANCA) and myeloperoxidase (MPO) together
is associated with microscopic polyangiitis
(approximately 90% specific) and Churg-Strauss
vasculitis. However, ANCA may be positive in
Laboratory Medicine and Diagnostic Pathology
many other situations including infection, scle-
rosing cholangitis, malignancy, and inflamma-
tory bowel disease. Therefore the full clinical
picture must be used in making treatment deci-
sions, and reliance should not be put on ANCA
alone. In granulomatosis with polyangiitis, serial
monitoring of cANCA is useful as a rising titer in
a patient in clinical remission heralds relapse
(Fussner et al. 2016). Whether there is any
value in serial monitoring of pANCA is less
certain.
Antibodies to Extractable Nuclear
Antigens
This test should always be carried out in patients
with suspected connective tissue disease. Moni-
toring at yearly intervals should be carried out in
diagnosed patients as the antibody pattern may
change with time, and this may correlate with
changes in the clinical profile. Normally laborato-
ries will carry out a six-antigen screen: Ro, La,
ribonucleoprotein (RNP), Sm, Jo-1, and Scl-70.
Results are reported qualitatively.
Antibodies to extractable nuclear antigens
(ENA) are listed below:
1. Anti-Ro/SSA – SLE, Sjögren’s, syndrome,
neonatal lupus, neonatal congenital complete
heart block
2. Anti-La/SSB – SLE, Sjögren’s syndrome, neo-
natal lupus, neonatal congenital complete heart
block
3. Anti-RNP – Undifferentiated connective tissue
disease (if present alone); SLE (if present with
dsDNA)
4. Anti-Sm – Highly specific marker for SLE
5. Anti- Jo-1 – Polymyositis, dermatomyositis
6. Anti-Scl-70 – Systemic sclerosis
7. Anti-Pm-Scl (PM1) – Scleroderma-myositis
overlap
8. Anti-Ku, Ki- Rare – SLE, UCTD, Sjögren’s
syndrome, polymyositis
9. Anti-Mi-2- Rare – Steroid-responsive
polymyositis
Anti-Ro (SSA) have been found in patients
with a range of autoimmune disorders, including
SLE, Sjögren’s syndrome, idiopathic
23
inflammatory myopathies, systemic sclerosis,
rheumatoid arthritis, as well as primary biliary
cholangitis and undefined connective tissue dis-
ease (UCTD). Sjögren’s syndrome (SS) is charac-
terized by the autoantibodies anti-Ro (SSA) and
anti-La (SSB) that play a key role in the classifi-
cation of the disease (Maślińska et al. 2017). Anti-
Ro (SSA) positivity is present in 50–70%, and
dual positivity for anti-Ro (SSA) and anti-La
(SSB) is found in approximately 30–60% of
cases of primary SS. Anti-Ro (SSA) antibodies
are found in approximately 50% of patients with
SLE. It is exceedingly rare to find patients with
single anti-La (SSB) antibodies positivity.
Seropositivity in primary SS is associated
with an earlier age of disease onset as well as
more frequent parotitis, and extraglandular fea-
tures, especially purpura and vasculitis. Sialogram
abnormalities are more frequently found in
patients with anti-Ro (SSA) positivity. Lip infil-
tration is more common in anti-La (SSB) positive
primary SS. Seropositivity and titers for anti-Ro
(SSA) and anti-La (SSB) remain relatively con-
stant throughout the course of the disease (Goules
and Tzioufas 2016).
Microbiology and Serology
Syphilis
Syphilis serology is used in both diagnosis and
treatment monitoring. Once a diagnosis of syphi-
lis is confirmed, care should be handed over to a
genitourinary specialist for further management
(Kingston et al. 2016).
Treponemal antibody tests can be classified
into:
1. Non-specific (cardiolipin, lipoidal, reagin,
or nontreponemal) tests: Venereal Disease
Research Laboratory (VDRL) carbon antigen
test/RPR test.
2. Specific (treponemal) tests: treponemal
enzyme immunoassay (EIA) or treponemal
chemiluminescent assay (CLIA); Treponema
pallidum hemagglutination assay (TPHA);
Treponema pallidum particle agglutination
24
assay (TPPA), fluorescent treponemal antibody
absorption test (FTA-abs), Treponema
pallidum immunoblot. Most of these tests
are now based on recombinant treponemal
antigens and detect treponemal IgG and
IgM antibody.
3. T. pallidum-specific IgM antibody tests: anti-
treponemal IgM EIA and immunoblot.
Demonstration of T. pallidum from infected
lymph nodes is by dark field microscopy and
should be performed by experienced observers
(Wheeler et al. 2004). It is less reliable in exam-
ining rectal and non-penile genital lesions and not
suitable for examining oral lesions due to the
presence of commensal treponemes. Polymerase
chain reaction can be used on oral or other lesions
where commensal treponemes may also be
present (Koek et al. 2006).
Mycobacterium tuberculosis
In the oral medicine clinic, the usual indication
for testing for tuberculosis (TB) is to prevent
reactivation of undiagnosed latent TB prior to
the patient commencing medications that will
cause immunosuppression. This can be done
with an interferon-γ release assay, in which there
is quantification of the interferon-γ released from
lymphocytes incubated with a protein from
M. tuberculosis (Doan et al. 2017). A patient
with an indeterminate or positive result should
be referred to an infectious diseases physician
for further assessment. The results of an inter-
feron-γ release assay are not influenced by BCG
vaccination status, unlike the Mantoux test.
Mycoplasma pneumoniae
IgM and IgG levels can be tested for if
Mycoplasma pneumoniae is suspected as a trigger
for erythema multiforme based on the clinical
history of a preceding respiratory illness. An
IgM over 770 U/ml is significantly positive,
although it may not start to rise until 7 days after
infection. It can stay raised for many months post-
T. Hodgson et al.
infection. It may not rise with reinfection, and
therefore IgG should be tested for simultaneously.
The normal range for IgG is less than 0.9 U/ml.
A fourfold increase in IgG indicates a current
infection (samples taken 2–3 weeks apart). Over
the past decade, there have been significant
advancements in the methods used for detecting
and characterizing M. pneumoniae, a common
cause of respiratory illness and community-
acquired pneumonia worldwide. The range of
available molecular diagnostics has expanded
from nucleic acid amplification techniques to
more sophisticated characterizing methods
including multi-locus variable-number tandem-
repeat analysis, multi-locus sequence typing,
matrix-assisted laser desorption ionization-time-
of-flight mass spectrometry, single nucleotide
polymorphism typing, and numerous macrolide
susceptibility profiling methods (Diaz and
Winchell 2016).
Toxoplasma gondii
The most usual presentation to an oral medicine
clinic would be cervical lymphadenopathy,
and the diagnosis is more likely to be obtained
via ultrasound guided FNA sample, which may
show tachyzoites. The diagnosis of toxoplasmosis
can be carried out by a number of methods,
including isolation of blood or body fluid
tachyzoites, parasite histological determination
in tissue, and protozoan DNA determination by
polymerase chain reaction and by antibody detec-
tion using serological tests. The latter is the most
currently used method, providing serological evi-
dence of immunoglobulin (IgG, IgM, IgA, and
IgE antibodies specific to T. gondii antigens).
However, these tests may display sensitivity and
specificity problems, leading to false-positive or
false-negative results (Zhang et al. 2016).
Methicillin-Resistant Staphylococcus
aureus
A swab for bacterial sensitivity and culture should
be taken from any lesion that does not respond to
Laboratory Medicine and Diagnostic Pathology
first-line therapy to guide future management.
A sterile swab is rubbed over the lesion, and this
is used to culture the bacteria present, which may
be pathogenic or commensal. The species are
subsequently identified as well as the antibiotics
they are sensitive to.
Brucellosis
This is not a commonly investigated condition in
oral medicine, but should be considered in
patients with erythema multiforme, particularly
if there is a history of contact with infected
animals such as cattle or consumption of
unpasteurized milk in countries where animals
are not vaccinated. In a systematic review, 51 of
72 patients (70.8%) achieved definitive diagnosis
based on isolation of bacteria of the genus
Brucella. Blood cultures were positive in 50%,
and pleural fluid culture was positive in 60.9%
in whom it was performed. Brucella spp. was
isolated in only 2 out of the 21 sputum cultures.
Diagnosis was based on indirect methods in the
remaining 21 patients. Standard tube agglutina-
tion was initially positive in 15 patients and was
positive during the course of the disease in another
4 patients. The other two patients were diagnosed
by PCR in one case and by detection of IgM and
IgG antibodies using enzyme-linked immunosor-
bent assay (ELISA) in the other case (Solera and
Solís García Del Pozo 2017).
Candida Culture
Candida culture and sensitivities can be requested
to ensure that treatment provided is appropriate.
This is not usually used at the initial assessment,
but rather if the patient has failed to respond to
first-line therapy such as amphotericin, nystatin,
miconazole, or fluconazole. Patients with immu-
nosuppression or mucocutaneous candidosis are
particularly more likely to have rare opportunistic
Candida present. A swab of the lesion, or a saliva
sample, is plated on Sabouraud’s dextrose agar to
allow Candida colonies to grow. These are subse-
quently identified and the sensitivity to antifungal
25
medication is assessed. Alternative methods
to identify candida include smears, which allows
identification of the blastospores and pseudo-
hyphae but does not provide sensitivities to
guide treatment. Candida may also be seen on
histological examination, but the species and sen-
sitivity will not be identified. The usefulness of
undertaking routine measurements of the presence
of oral yeast, and the level of amount of yeast
present in the oral cavity, during clinical apparent
disease is questionable (Manfredi et al. 2013).
However, these methods have been used in
research to shed light on the role of oral yeast in
oral mucosal disease and currently should not be
routinely requested (McCullough et al. 2002;
Alnuaimi et al. 2016).
Pathological Investigations
Biopsy
A biopsy of the patient’s tissues may be taken and
referred for histopathological examination either
to confirm a clinical diagnosis or to establish the
diagnosis where there is clinical doubt and treat-
ment critically depends on confirming the diagno-
sis. There are many mucosal lesions where
a biopsy is indicated. These include malignant
lesions such as squamous cell carcinoma, poten-
tially malignant disorders including leukoplakia
and erythroplakia, vesiculobullous disorders, and
ulceration of unknown cause. However, there may
be instances when a biopsy is contraindicated due
to an underlying medical condition. Examples
include patients with a bleeding disorder, such as
hemophilia, or patients taking anticoagulants,
such as warfarin. In these cases, if there is a degree
of clinical certainty about the diagnosis, for exam-
ple, in a patient with typical bilateral lesions of
oral lichen planus, then a biopsy may not be
necessary. In other patients with more sinister
lesions, then it may be necessary to adjust the
patient’s medication or arrange for the biopsy to
be taken under appropriate specialist care.
There are many different types of biopsy and it
is important that the correct biopsy is taken other-
wise diagnosis may be delayed.
26
Incisional Biopsy
An incisional biopsy involves taking a represen-
tative sample(s) of the patient’s lesion. This type
of biopsy is particularly suitable for large mucosal
lesions such as lichen planus and other types of
white patch, suspected potentially malignant
disorders or malignant lesions, ulceration, and
vesiculobullous disorders. From a pathological
point of view, an ellipse of tissue as large as
possible should be excised and should include
the underlying connective tissue and also the
edge of the lesion. If a very shallow, friable biopsy
is taken, it may curl and distort when placed
in fixative, and this may make it difficult for the
pathologist to orientate the specimen correctly. To
overcome this, it is useful if the clinician sutures
the specimen to a card to keep it flat. It is also
possible to indicate on the card the orientation of
the specimen if that is deemed clinically neces-
sary. However, in most cases an adequate-sized
specimen can be placed directly in fixative with-
out these additional measures. It is especially
important that the area to be biopsied is represen-
tative of the lesion and none more so than in oral
cancer and potentially malignant disorders where
failure to either initially recognize the disorders or
to biopsy the most appropriate representative part
can have adverse consequences for the patient.
Vital stains such as toluidine blue have been
used as an adjunct to identify oral cancer and
potentially aid diagnosis, but at the current time,
there is no evidence that its use significantly
increases detection rates (Su et al. 2010). A thor-
ough knowledge of the clinical features of
oral cancer and potentially malignant disorders
(Napier and Speight 2008) remains essential to
identify these lesions and to determine which area
to biopsy so the diagnosis is accurate and timely.
There are very few indications for using a
punch biopsy in the oral cavity as access is usually
good and there are major disadvantages from a
pathological point of view. The most important is
that the core of tissue produced by such biopsies
is very difficult to orientate, particularly if it is
a 4-mm punch biopsy or less. The resulting
difficulties mean that the sections taken from the
mucosal specimen are frequently not at right
angles to the surface and this can make it
T. Hodgson et al.
extremely difficult or even impossible to establish
the diagnosis. This may delay the diagnosis and
the patient may require a second biopsy.
Sometimes it is necessary to biopsy a lesion
deep to the mucosa in the underlying connective
tissues or salivary glands. Many such lesions
can be biopsied by raising a flap, but if the lesion
is very deep, such as in a salivary gland or a
lymph node, then a core biopsy under ultrasound
guidance may be indicated. Such biopsies are
usually carried out by radiologists, and, as the
cores are very small, it is important to ensure that
they are representative of the lesion. Lesions for
which a core biopsy may be indicated include
salivary gland neoplasms in the parotid and sub-
mandibular glands, other causes of salivary
swelling, and patients with enlarged cervical
lymph nodes.
Fine Needle Aspiration Biopsy
An aspiration or fine needle aspiration biopsy
(FNAB) is very rarely used for histopathological
diagnosis of mucosal disease in the oral cavity and
associated structures, as core biopsies are prefer-
able (Novoa et al. 2012). FNAB fails to provide
diagnostic material in 10–15% of cases compared
with 5% of core biopsies. In addition, a systematic
review has shown that core biopsies have a higher
specificity (99% compared with 96%), greater
accuracy (96% compared with 93%), and greater
negative predictive value (95% compared with
90%) than those of fine needle aspiration in
detecting malignancy. Core biopsies were also
shown to have a higher sensitivity in the diagnosis
of lymphoma than fine needle aspiration (92%
vs. 74%) (Novoa et al. 2012). Fine needle aspira-
tion biopsies are however useful to establish
whether a lesion is pus filled and infective or for
microbiological analysis but are not indicated for
pathological diagnosis.
Excisional Biopsy
Excisional biopsies are used to remove the lesion
in its entirety, and this may be an essential part of
the management of the lesion. In some cases, the
diagnosis has been established prior to the proce-
dure, for example, oral squamous cell carcinoma;
however, in other situations, such as fibrous
Laboratory Medicine and Diagnostic Pathology
hyperplasia and mucoceles, where the diagnosis is
made clinically, an excisional biopsy is used in an
attempt to provide both definitive treatments, as
well as to confirm the diagnosis. Excisional biop-
sies may also be used to remove small, nodular,
well-circumscribed lesions deep to the mucosa
without a firm clinical diagnosis. Examples of
such lesions include small neoplasms of the
minor salivary glands (common in the upper lip
and cheek) and neural tumors. Conversely it is
safer to perform an incisional biopsy on larger,
deep lesions with ill-defined borders in order to
establish the diagnosis before definitive treatment.
Excisional biopsy of salivary gland tumors of the
parotid and submandibular gland may also at
times be used to treat and confirm the diagnosis
without using an incisional biopsy to establish the
diagnosis prior to surgery. In these cases, detailed
radiological investigation is used together with
the clinical features to establish whether the lesion
is benign or malignant and to determine the
appropriate treatment. The lesion is removed in
its entirety and sent for histopathological
examination.
It is important that the pathologist knows
whether the biopsy is incisional or excisional as
the way the tissue sample is examined may differ.
For example, some excisional biopsies are used to
remove a sinister lesion in its entirety, and
a pathologist will make sure that all excision mar-
gins are sampled and examined to ensure com-
pleteness of excision. If the pathologist is sent
an incisional biopsy of a sinister lesion, it is not
necessary to examine the excision margins but
rather the body of the lesion to establish a
diagnosis.
Transport of Biopsies
Almost all biopsies are placed and fixed in 10%
formalin-buffered saline and sent with the appro-
priate clinical information and patient details to
the histopathology laboratory (Table 7). However,
occasionally it is necessary to send a fresh biopsy
so that specific tests such as immunofluorescence
can be carried out. This is particularly appropriate
for biopsies from patients with autoimmune
vesiculobullous disorders that normally require
27
Table 7 Information that should be supplied on a biopsy
request form
Patients name, address, date of birth, hospital number on
both request form and specimen jar
Requesting consultants name and hospital of origin
Date biopsy was taken
Clinical details including size, shape, color consistency,
distribution, duration of any symptoms, lesions
elsewhere
Site of biopsy
Relevant details from medical and social history
Indicate whether previous biopsy has been taken and
provide details
Provisional diagnosis or differential diagnosis
Indicate whether incisional or excisional biopsy
direct immunofluorescence for an accurate
diagnosis.
A fresh biopsy should be sent to the laboratory,
immediately after it has been taken in the clinic, so
it may be frozen promptly in liquid nitrogen. It
is good practice to let the laboratory know in
advance that such a biopsy has been planned so
there is no delay in processing the tissue once it
has arrived. Fresh tissue may be sent wrapped in a
gauze soaked in saline or placed in a drop of saline
in a sealed pot if the tissue is to be transported
immediately. If there is a delay of more than
30 min, then it is prudent to send the specimen
in a transport medium such as Michel’s medium
(Vaughan Jones et al. 1995). Transport of biopsies
from the clinic to the laboratory should always
follow established protocols. These may differ
between hospitals in the same country and also
between countries. It is important that the oral
medicine specialist familiarizes themselves with
these and it is good practice to liaise closely with
the laboratory to ensure accuracy appropriate pro-
cedures and safety.
The usual minimum requirement, if the speci-
men is to be transported locally without the use of
couriers or other forms of transport, is that the
biopsy specimen is placed in a screw-topped spec-
imen jar and then placed in a two-compartment,
sealable plastic bag (Fig. 1).
The specimen jar should be placed in one com-
partment and the request form in the other. If a
two-compartment bag is not available, then the
specimen jar should be placed in one bag, sealed,
28 T. Hodgson et al.

and then placed in another bag together with the
request form. It is essential that the two, the spec-
imen jar and the request form, are not separated
and are transported together. The bagged speci-
men should be placed in a suitable, sealed con-
tainer for transport to the laboratory.
The United Nations has produced guidelines
for the transport of biological specimens through
the post or by courier. The packaging must
comply with IATA 650 regulations (http://www.
un3373.com/info/regulations). The specimen jar
must be placed in a sealed bag as outlined above
together with the request form and then placed in a
leak-proof secondary container filled with suffi-
cient material to absorb the liquid from the spec-
imen jar. The secondary container should be
placed in a rigid strong out container and labeled
“Biological substance category B” (Fig. 2).

High-Risk Specimens
Biopsies from patients who are known to be HIV
positive or suffering from tuberculosis or other
infectious diseases should always be placed in
10% formalin-buffered saline and should never
be sent fresh as they present a hazard to the labo-
ratory staff. A “high-risk” sticker should be placed
on both the specimen pot and the request form to
alert the laboratory staff who will use special pro-
cedures to handle these specimens safely.

Fig. 1 Screw topped jar and a two-compartment sealable

Information Required by a Pathologist
plastic bag It is particularly important for an accurate diagno-
sis that all relevant clinical information is

Fig. 2 An outline of the Carriage of specimens in the post: must comply

IATA 650 guidelines for the with IATA 650 packing instructions.
transport of biological
specimens by post
UN3373 Biological Substance Category 3.

Absorbent
Senders name, material
Itemised contents

address and
telephone Leak proof
number secondary
container
Biological
substance
category B
Rigid strong
outer container
Laboratory Medicine and Diagnostic Pathology
provided to the pathologist. The patient’s details
must be on the accompanying request form as well
as on the biopsy specimen jar itself. It is also
essential that the name of the consultant or the
clinician sending the biopsy is provided as well as
the address or hospital and the date the biopsy was
taken (Table 7).
Clinical information is important to making an
accurate pathological diagnosis. A good example
is lichen planus and lichenoid reactions, that are
essentially a clinicopathological diagnosis. It is
not possible to reliably distinguish between lichen
planus and a lichenoid reaction to amalgam or a
lichenoid drug reaction on histopathological fea-
tures alone, although there are some pointers
(Thornhill et al. 2006). Thus, it is important for
the pathologist to know if there are bilateral or
unilateral lesions, which sites are affected, and
whether or not there are skin lesions. Furthermore,
whether the lesions are related to amalgam resto-
rations or whether the patient is taking any rele-
vant drugs. In rare cases patients who have had
bone marrow transplant may have graft versus
host disease that may resemble oral lichen planus.
Again, this information is essential to make an
accurate diagnosis.
Drug history is also important not only for the
accurate diagnosis of lichenoid reactions but in
the diagnosis of fungal infections such as chronic
hyperplastic candidosis. The diagnosis is made on
characteristic histopathological features including
pseudoepitheliomatous hyperplasia (deep exten-
sions of epithelium into the underlying connective
Fig. 3 Hematoxylin and eosin stained section of chronic hyperplastic candidosis showing pseudoepitheliomatous
hyperplasia (*) (a). The associated fungal hyphae are identified by Periodic-Acid Schiff (PAS) stain (b)
29
tissue) associated with the presence of fungal
hyphae in the superficial epithelial layers (Fig. 3).
If the patient had been prescribed antifungal
therapy prior to taking the biopsy, the fungal
hyphae may not be present although some
histopathological features including pseudo-
epitheliomatous hyperplasia may still be evident.
This causes diagnostic difficulties for the pathol-
ogist as pseudoepitheliomatous hyperplasia may
be seen in a number of other lesions affecting the
oral cavity that are not associated with Candida. It
is helpful for accurate diagnosis not to prescribe
antifungal therapy before taking the biopsy. If it is
necessary to do so, then that information should
be provided on the request form.
It could be argued that a course of antifungal
therapy should be prescribed before taking a
biopsy of a white patch so that any histopatholog-
ical changes caused by the candida, and which
may make the diagnosis of dysplasia difficult,
are minimized. An example of where candidal
infection is associated with dysplasia is shown
in Fig. 4.
When pseudoepitheliomatous hyperplasia is
not observed histopathologically, it will remain
unclear if the dysplasia is or is not the result of
the Candida infection. In an attempt to resolve
this issue, the pathologist may examine, if at all
possible, the degree of dysplasia away from the
Candida infection, to observe if it is still present
or is of a less extent. If it is not possible to
determine whether Candida is playing a role in
the dysplasia, then the clinician should consider
30
Fig. 4 Hematoxylin and eosin stained section of dysplasia
with Candida infection. No evidence of pseudo-
epitheliomatous hyperplasia is seen
prescribing antifungal therapy and re-biopsy to
determine whether the dysplasia has reduced in
grade or has resolved. This approach reduces the
necessity to prescribe antifungal therapy
irrespective of whether there is firm evidence of
Candida infection.
Another example of where clinical formation is
essential is in the accurate diagnosis of papilloma-
tous lesions. Simple, small papillomas are rela-
tively easy to diagnose; however, there are a group
of verruciform lesions that may be diagnostically
difficult to distinguish from each other, both clin-
ically and histologically. The differential diagno-
sis varies from well-differentiated squamous cell
carcinoma, verrucous carcinoma, proliferative
verrucous leukoplakia (a clinicopathological
diagnosis), and verruciform hyperplasia to reac-
tive lesions of both low- and high-risk human
papilloma virus (HPV). In these cases, it is essen-
tial that the size, site, and duration of the lesion is
given as well as a history of other lesions else-
where. Furthermore, if a previous biopsy of the
lesion has been taken, it is essential to note this so
a comparison can be made.
Clinical information about systemic disease is
essential even if it may not obviously be related
to the current lesion. An example is of a distant
malignancy arising in the kidney, breast, or pros-
tate. Such malignancies can metastasize to the
oral cavity, and if the pathologist knows in
T. Hodgson et al.
Fig. 5 Hematoxylin and eosin stained section of a “chev-
ron” pattern of hyperkeratosis associated with smoking
advance the detailed patient history, it will
speed diagnosis.
Information on the patient’s habits is also
important to the diagnosis. For example, smoking
and alcohol consumption are important risk
factors in the development of oral cancer, but
smoking is also associated with other specific
changes in the oral mucosa. These changes
include a chevron pattern of hyperkeratosis
(Fig. 5) as well as pigmentary incontinence
where melanin from the basal keratinocytes
drops out into the underlying connective tissue
and is taken up by macrophages (Fig. 6).
It is important to provide a provisional diag-
nosis or differential diagnosis on the request
form. This information may be used to deter-
mine the way the specimen is handled. For
example, if a potentially malignant lesion is
suspected, then the pathologist may order more
tissue sections to be cut than for a routine biopsy.
If this is done at the laboratory stage of the
specimen processing, it saves time and provides
a speedier result. Similarly, if the clinician sus-
pects a fungal lesion such as chronic hyperplas-
tic candidosis, then a Periodic acid Schiff (PAS)
stain may be ordered at an early stage to detect
the Candida hyphae.
Laboratory Processing of Biopsy
Specimens
Upon arrival in the laboratory, specimens are
logged and assigned an accession number by
Laboratory Medicine and Diagnostic Pathology
Fig. 6 Hematoxylin and eosin stained section of oral
lichen planus showing pigmentary incontinence (arrows).
Melanin from the basal keratinocytes has been engulfed by
macrophages (melanophages)
which they are subsequently identified. Particular
care is taken to check that the details on the
accompanying request form are the same as
those on the specimen jar. If there are discrepan-
cies, then such specimens are not accepted for
further processing until the differences have
been clarified by the clinician responsible for tak-
ing the biopsy. This is an essential step to ensure
that the correct biopsy specimen, and hence result
is attributed to the correct patient.
Sampling of Tissue Specimens
The pathologist examines each biopsy specimen
and decides whether all the tissue or only selected
parts will be processed by the laboratory. The
pathologist then cuts the tissue appropriately.
This is known as “cutting up” or “trimming” the
specimens.
For incisional biopsies, it is usual that all tis-
sues are processed. Mucosal specimens are rou-
tinely cut at right angles to the mucosal surface
and either bisected along their long axis or cut
transversely into multiple strips. Further discus-
sion on these differing approaches is outside the
scope of this chapter. The cut specimen is then
placed cut surface down in a labeled perforated
cassette and sent for processing (Fig. 7). Placing
31
mucosal tissue on the cut surface in this way and
maintaining that orientation throughout pro-
cessing mean that the tissue is sectioned at right
angles to the mucosal surface and the entire thick-
ness of the epithelium and underlying connective
tissue is available for the pathologist to assess on
the slides. This is essential for accurate diagnosis.
It has already been mentioned that trimming of
specimens, identification of the mucosal surface,
and maintaining that orientation are more difficult
if a small punch rather than an elliptical biopsy is
used.
Incisional biopsies of non-mucosal tissues
such as deep connective tissue, salivary glands,
and neural tumors are trimmed in the same way as
mucosal biopsies, but the orientation is not as
critical for diagnosis.
Excision biopsies may be trimmed or cut dif-
ferently. If the specimen is a benign lesion such as
a fibrous polyp and examination of the excision
margins is not essential for diagnosis and further
management, it may be bisected in the same way
as mucosal specimens. If however the specimen is
an excisional biopsy of a malignancy, then it is
essential that the excision margins are examined
to determine whether excision of the lesion is
complete. The pathologist must know the orienta-
tion of the specimen and be able to identify the
different excision margins. For mucosal resec-
tions of malignant tumors, accurate labeling of
the margins by the clinicians while the specimen
is being taken is essential. If, for whatever reason,
the specimen has either not been orientated or
there are discrepancies in the labeling, then
the pathologist may ask the responsible clinician
to confirm the orientation. However, once the
tissue is removed from the patient and placed in
fixative, its appearance changes significantly and
orientation by the clinician may be difficult, if not
impossible. In these cases, the pathologist will be
unable to give an accurate report on excision
margins to the detriment of the patient.
A pathologist will usually take a block of tissue
at right angles to the surgical margin, and the
number taken will depend on the nature and size
of the specimen. Particular care is taken to mark
the surgical margins with ink and an accurate
description of where the tissue block has been
32 T. Hodgson et al.

Fig. 7 A perforated
cassette and lid that will
house the biopsy specimen
during processing

taken is recorded. For most resections it is good

practice to take a photograph of the resection
specimen and mark where the tissue blocks have
been taken (Fig. 8). These photographs can also
be used to indicate where the margin is not clear if
appropriate and thus aid the surgeons. Further
discussion of the principles of trimming speci-
mens is outside the scope of this chapter.

Tissue Processing
Once the biopsy specimen has been examined,
cut, and placed in a cassette, it remains in formol
saline until it is processed. The aim of tissue
processing of fixed tissue is to infiltrate the tissue
with wax so it forms a solid block that can be cut
Fig. 8 An excisional biopsy specimen marked where
with a microtome to form tissue sections. For blocks of tissue will be taken for histopathological assess-
fresh tissues, a block is formed by freezing the ment of excision margins
tissue in liquid nitrogen and further processing is
not necessary. In most laboratories the cassettes specimen is still orientated correctly. The mold is
are processed in an automatic tissue processor then filled with molten wax and a cassette is placed
overnight that dehydrates the tissue using increas- on top and the whole thing is allowed to solidify on
ing concentrations of alcohol. After dehydration a cold plate. A tissue block is thus formed (Fig. 9).
the tissue is “cleared” with xylene to remove the
alcohol. Cutting and Staining Tissue Sections
Using an embedding center, a specialist labora- Very thin sections, approximately 4–5 μ in thick-
tory scientist will then open the cassette, place the ness, are cut from the paraffin blocks using
tissue in a mold, and carefully check if the a microtome. This is a skilled procedure and is
Laboratory Medicine and Diagnostic Pathology
Fig. 9 A tissue block from the excision margin of a
malignancy. The tissue is embedded in wax and the mar-
gins are marked with blue ink
carried out by a laboratory scientist who has to
ensure the sections transferred to the glass slides
are representative of the tissue sample. It is par-
ticularly important that the whole of the cut sur-
face of the specimen is present in the section.
Once the sections have been cut, they are gently
placed in a warm water bath to allow them to
“spread.” The sections are then transferred to a
glass slide and allowed to dry in an oven to
increase adherence of the tissue sections.
Before staining of the tissue sections can take
place, the wax must be removed using a solvent,
which is usually xylene. Once the staining is
completed, a coverslip is placed over the tissue
sections to make a permanent mount (Fig. 10).
Almost all tissue sections are examined initially
using a routine hematoxylin and eosin stain
(H&E). Hematoxylin is a basic dye that stains
nuclei blue/purple and eosin is an acidic dye
that stains the cytoplasm and extracellular connec-
tive tissues pink. Sometimes special stains are
used in diagnosis and these are described below.
33
Fig. 10 A hematoxylin and eosin stained section
Histopathological Diagnosis
A pathologist will initially read the request form
from the clinician and examine the appropriate
H&E stained slides provided by the laboratory.
In many cases they are able to make a firm diag-
nosis based on the histopathological features of
the sample and the clinical information provided.
In some cases, it is necessary to examine more
tissue to ensure the correct diagnosis. A request
is sent to the laboratory for “levels” – additional
sections usually taken at 100 μ apart.
The pathologist may also need additional
clinical information and often it is helpful to see
clinical images, especially if the clinical informa-
tion given is scant. If the lesion is intra-bony, it is
often essential for accurate diagnosis that the
pathologist can examine the appropriate radiolog-
ical imaging.
It is good practice for a pathologist to review
slides from previous biopsies if they are relevant
to the current biopsy. It is useful if the clinician
indicates whether previous biopsies have been
taken and also give the relevant accession
34 T. Hodgson et al.

Table 8 Common histochemical stains used in histopathological diagnosis

Purpose Stains used Indications
Detection of fungi such as Periodic-Acid Schiff Fungal infections of oral cavity and sinuses, e.g., chronic
Candida albicans Grocott hyperplastic candidosis, aspergillosis in antrum
Detection of Ziehl Neelsen Distinguishes tuberculosis from other causes of granulomatous
Mycobacterium Auramine inflammation
tuberculosis Rhodamine
To identify amyloid Congo red Distinguishes amyloid from other eosinophilic/hyaline deposits.
Does not distinguish between types
To detect melanin Masson Fontana Important in diagnosis of pigmented lesions: distinguishes
melanin from iron
To detect hemosiderin and Perls’ Prussian Blue Important in diagnosis of pigmented lesions: distinguishes iron
iron deposition from melanin
To detect glycogen and Periodic acid Schiff Important in the diagnosis of salivary gland tumors
mucins Periodic acid Schiff
with diastase
Alcian blue

numbers if possible. This may be essential to

monitor progression of a lesion over time.

Histochemical Stains
Almost all tissue sections are examined using a
routine H&E stain that stains nuclei blue/purple
and the cytoplasm of cells pink/red. However
sometimes other stains are required to detect addi-
tional histopathological features, such as fungal
hyphae and microorganisms (Table 8) (Figs. 11,
12, 13, and 14). It is not necessary for an oral
medicine specialist to request these, but it does
help the pathologist if adequate clinical informa-
tion and a provisional diagnosis indicating their Fig. 11 Periodic-Acid Schiff stain of superficial oral epi-
thelium in chronic hyperplastic candidosis. Fungal hyphae
potential presence are given.
are indicated by black arrows and glycogen in the epithelial
cells and neutrophils by asterisks (*)
Immunofluorescence
Immunofluorescence is used to detect autoanti-
bodies in the patient’s tissues and occasionally binds the antigen leaving the fixed tail region free.
the serum and is particularly relevant in the diag- The structure of the fixed tail region is constant
nosis of pemphigus and pemphigoid and other within any given antibody class so that all IgG
autoimmune vesiculobullous disorders. Tissue antibodies have the same fixed tail region as do all
should be sent fresh to the laboratory as fixative IgA antibodies and so forth. The immunofluores-
denatures the autoantibodies and renders their cence test uses antibodies raised in another spe-
detection impossible. It is good practice to take cies, such as mouse, rabbit, or rat, against the fixed
two biopsies, one from the lesion placed in for- tail region of human IgG, IgA, and IgM to detect
malin and a second for immunofluorescence from the autoantibodies bound in the patient’s tissues.
adjacent clinically unaffected mucosa, placed in These antihuman antibodies (mouse, rat, or rab-
Michel’s solution or sterile saline. bit) are linked to a fluorescent marker that can
All antibodies have a similar structure with a be visualized using a fluorescent microscope
variable and a fixed region. The variable region (Fig. 15). In addition to immunoglobulins, direct
Laboratory Medicine and Diagnostic Pathology 35

Fig. 12 Hematoxylin and eosin stained section of amyloid deposition in oral mucosa (a). The amyloid shows positive
staining with Congo red (b)

Fig. 13 Hematoxylin and eosin stained section showing hemosiderin deposition in hemangioma (a). The hemosiderin
stains positively for iron with Perls’ Prussian Blue (b)

immunofluorescence can also be used to assess
the presence of complement (C3) again using
antihuman antibodies raised in another species.
Indirect immunofluorescence assesses
whether autoantibodies are present in the serum
in vesiculobullous disorders. In this test the
patient’s serum is incubated with either normal
oral mucosa, skin, or monkey esophagus (Aoki
et al. 2010). The autoantibodies in the patient’s
serum will bind with the appropriate antigen in the
skin or mucosa. These autoantibodies are detected
using fluorescent-labeled antihuman antibodies in
the same manner as direct immunofluorescence.
The sensitivity of indirect immunofluorescence
using normal mucosal or skin is relatively low in
subepithelial blistering disorders, such as bullous
and mucosal pemphigoid, and may be improved
by the use of salt split skin (Woodley 1990). The
skin is incubated in a 1 M NaCl (sodium chloride)
solution that results in artificial separation of the
36 T. Hodgson et al.

Fig. 14 Hematoxylin and eosin stained section of a muco-epidermoid carcinoma showing cystic spaces (a). Staining
with Periodic-Acid Schiff with Diastase shows the cysts are filled with mucin (b)

Fig. 15 Diagram showing

the theoretical basis of
direct immunofluorescence. Fluorescent
The autoantibody is the labelled
patient’s antibody that is mouse/rat/rab
bit
directed towards specific
anti-human
self-antigens depending
antibody
upon the disease, for
example desmoglein 1 or
3 in pemphigus vulgaris.
The fluorescently labelled Autoantibody
anti-human antibody can be binding to
epithelial cells
raised in a number of
different animals and is
usually directed against a
specific antibody isotope, Epithelial
such as human IgG cells

skin through the basement membrane at the level
of the lamina lucida. This exposes the antigens
and increases the sensitivity (Kneisel and Hertl
2011). It also aids in diagnosis since the autoanti-
bodies may bind either on the epidermal or the
dermal side of the split. For example, the autoan-
tibodies bind to the epidermal side of the split in
bullous pemphigoid and to the dermal side of the
split in epidermolysis bullosa acquisita (Kneisel
and Hertl 2011).
The diagnosis of pemphigus, pemphigoid, and
other autoimmune blistering disorders is con-
firmed by the pattern of staining and the type of
antibody deposited (Kershenovich et al. 2014).
For example, in pemphigus vulgaris IgG (which
binds to the desmosomal component desmoglein
3) and C3 are deposited around the surface of the
keratinocytes in the prickle cell layer resulting in
a fish-scale appearance (Fig. 16). In pemphigus
foliaceus, IgG and C3 may be deposited higher up
in the prickle cell layer than seen in pemphigus
vulgaris. Pemphigus foliaceus is very rare in
the oral cavity as it is characterized by antibodies
against desmoglein 1 and their effects are
counteracted by high levels of desmoglein 3
(Mahoney et al. 1999).
Pemphigoid, whether bullous or mucous mem-
brane, is characterised by a linear deposition of
autoantibody at the basement membrane zone
(Fig. 17). Usually IgG and/or C3 is deposited,
but in addition occasionally IgA and/or IgM may
be seen (Chan et al. 2002). Linear IgA disease is
Laboratory Medicine and Diagnostic Pathology
Fig. 16 Direct immunofluorescent staining in pemphigus
vulgaris showing a “fish-scale” pattern of IgG around the
cells in the prickle cell layer
characterized by IgA and occasionally IgM depo-
sition (Betts et al. 2009). Other patterns of immu-
noglobulin deposition are seen in other blistering
disorders. For example, in dermatitis
herpetiformis, there may be granular deposition
of IgA in the connective tissue at the tips of the
connective tissue papillae.
Direct and indirect immunofluorescence
does not detect which antigens are targeted by
the autoantibodies merely where in the tissue the
autoantibodies are present. Thus, it is not possible
to distinguish between different pemphigoid
subtypes whose antibodies target different
components of the hemidesmosomes or basement
membrane zone. Other tests are required to
do this.
Enzyme-linked immunosorbent assay (ELISA)
is a method that can be used to detect either anti-
bodies or antigen. In vesiculobullous disorders,
ELISA is usually used to detect specific
37
Fig. 17 Direct immunofluorescent staining in pemphigoid
showing a linear deposition of IgG at the basement mem-
brane reproduced by kind permission of Jaypee Brothers
Medical Publishers (P) Ltd
autoantibodies (Heymann 2009). The basis of the
test is that a microtiter plate is coated with the
appropriate antigen, for example, BP180, and
then the patient’s serum is added. If antibodies
that recognize BP180 are present, they will bind
to the plate and may be detected by antihuman
antibodies conjugated to either alkaline phos-
phatase or horseradish peroxidase. These two
enzymes are detected by chemical means, and the
strength of the reaction gives an indication not only
which antibodies are present but also their titre.
ELISA may therefore be used as an adjunct to the
diagnosis of vesiculobullous disorders (Huang
et al. 2007) as well as to monitor the effect of
treatment and the progression of the disease
(Daneshpazhooh et al. 2007; Heymann 2009).
ELISA has been shown to be more sensitive than
indirect immunofluorescence in detecting serum
levels of autoantibodies in pemphigus vulgaris
(Ishii et al. 1997), although this does not appear
to be true in the diagnosis of pemphigoid where no
difference in either sensitivity or specificity was
found (Sárdy et al. 2013). Recent evidence sug-
gests that ELISA may be used to monitor salivary
levels of autoantibodies in mucosal pemphigus
vulgaris (Ali et al. 2016).
38
Immunoblotting (or western blotting) may also
be used in the diagnosis of vesiculobullous disor-
ders, although it is more often used in research.
Extracts of keratinocytes containing the putative
antigenic targets are separated on a SDS-poly-
acrylamide gel by electrophoresis. The proteins
are then transferred to a nitrocellulose membrane
where they are reacted with antibodies contained
within the patient’s serum. Binding of these anti-
bodies is detected using an antihuman antibody
conjugated to either horseradish peroxidase or
alkaline phosphatase that is visualized by enzy-
matic means.
In summary diagnosis of vesiculobullous dis-
orders is the result of careful clinical evaluation
and identification of autoantibodies in the tissues
and serum. This may be carried out by a combi-
nation of direct immunofluorescence, indirect
immunofluorescence, ELISA, and more infre-
quently immunoblotting.
Immunohistochemistry
Immunohistochemistry is used in the diagnosis of
certain pathological lesions when it is necessary
to reliably identify a tissue, cell, or organism. The
basis of the test is that antibodies that are either
directly or indirectly linked to a detection system,
such as hydrogen peroxidase or alkaline phospha-
tase, are used to detect antigens that are specific to
a cell or a restricted range of cell types (Table 9).
The immunostaining pattern is always interpreted
together with the H&E stained section and other
information. For example, S100 may be used to
identify nerves, Langerhans cells, myoepithelial
cells, melanocytes, melanocytic nevi, melanoma,
nerve sheath tumors, and granular cell tumors so
it has a broad staining pattern (Figs. 18, 19, 20,
21, 22, 23, and 24). The significance of its expres-
sion in a cell or group of cells is determined by
other histopathological diagnostic criteria of the
particular lesion. Immunohistochemistry is useful
in diagnosing malignant neoplasms of uncertain
origin or type and also in the diagnosis of lym-
phoma. Most antibodies work on fixed paraffin
embedded tissue and it is not necessary to send
tissue fresh.
T. Hodgson et al.
Molecular Diagnostic Pathology
Genetic material is carried on chromosomes
that are composed of double-stranded deoxyri-
bonucleic acid (DNA). Each strand is made up
of bases: adenine, cytosine, guanine, and thymine.
Adenine on one-strand binds with thymine on the
opposing strand and guanine binds with cytosine.
Portions of DNA that code for functionally impor-
tant ribonucleic acid (RNA) are known as genes.
When a gene, which codes for a polypeptide, is
transcribed, an enzyme called RNA polymerase
uses the base pair sequence on the DNA to make
complementary RNA. The resulting messenger
RNA (mRNA) travels from the nucleus into the
cytoplasm where it enters the ribosomes. The
mRNA is decoded to make the corresponding
polypeptide.
Polymerase chain reaction (PCR), real-time
PCR, and reverse transcriptase (RT)-PCR are
means of generating multiple copies of the gene
of interest. The basic principle is that the DNA
sequence of the gene is known and small primers
are generated that hybridize with the base
sequences either side of the gene. A heat stable
DNA polymerase enzyme catalyzes the formation
of multiple copies of the gene that can then be
visualized by electrophoresis on a gel. The advan-
tages of this technique are that it is quick and
sensitive, and it is robust in that it is able to
amplify badly degraded DNA. However, its use
in diagnostic pathology is limited as the tissue is
destroyed when the genetic material is removed
and it is only possible to indicate whether a par-
ticular gene is present, not where it is present in
the tissue, nor in what quantity.
Real-time PCR is a modification of the tech-
nique that allows the amount of DNA present in a
sample to be quantified. It does this by tagging
the sequence with a fluorescent dye and uses it to
measure the amount of DNA generated in the
early stages. The more copies of the gene that
are present in the tissues at the start of the reaction,
the greater the copy number will be early on in the
reaction.
Reverse transcriptase PCR (RT-PCR) is used
to detect mRNA in tissues or sample. A reverse
transcriptase enzyme is able to generate cloned
DNA (cDNA) complementary to the mRNA of
Laboratory Medicine and Diagnostic Pathology 39

Table 9 Commonly used antibodies in histopathological diagnosis

Antibody
against Identifies Usage
Cytokeratins Intermediate filaments in epithelial cells; Identification of epithelial cells. Identification of
epithelial cells of differing origins have anaplastic carcinoma; origin of metastatic tumors;
different cytokeratin profiles diagnosis of salivary gland tumors
CD45 Lymphocytes Diagnosis of lymphoma and cells of uncertain
origin
S100 S100 protein on nerves, melanocytes, Diagnosis of nerve sheath tumors, melanoma,
Langerhans dendritic cells, myoepithelial cells salivary gland tumors
Desmin Intermediate filaments in muscle Diagnosis of leiomyoma and rhabdomyosarcoma
Smooth Intermediate filament in muscle, Useful to differentiate smooth muscle from striated
muscle actin myofibroblasts, and myoepithelial cells muscle. Also identifies myoepithelial cells in
(SMA) salivary gland tumors
p63 (and p40) Nuclear transcription factors strongly Diagnosis of squamous origin in undifferentiated
expressed in myoepithelial cells and squamous carcinomas. Useful myoepithelial marker in
epithelium salivary gland tumors
Kappa and Heavy chains on plasma cells Distinguishes plasmacytoma (monoclonal for
Lambda either kappa or lambda) from reactive (polyclonal)
plasma cell proliferations including plasma cell
gingivostomatitis
P16 Cell cycle protein regulator that is Acts as a surrogate marker to identify
overexpressed in response to high-risk HPV HPV-positive oropharyngeal carcinomas
infection
Ki 67 Cell cycle protein that is only expressed in Indicates the proliferative fraction of a tissue.
mitotically active cells and not seen in resting Important in diagnosis of malignancy
cells

Fig. 18 Hematoxylin and eosin staining of a peripheral epithelium are highlighted by immunohistochemistry for
odontogenic fibroma (a). Islands of odontogenic epithe- cytokeratins (b)
lium in the connective tissue as well as the surface

interest. The cDNA is then amplified by PCR or is that it destroys the tissue sample and for patho-
real-time PCR. The advantages of this technique logical diagnosis the location of the mRNA is
are the same as those of PCR, but the disadvantage important.
40
Fig. 19 Nasopharyngeal carcinoma stained with hema-
toxylin and eosin: It is difficult to distinguish the carci-
noma from the infiltrating lymphocytes that are
characteristic of the malignancy (a). Immunohistochemical
In situ hybridization is used to detect mRNA in
tissue sections. A single-stranded RNA probe is
generated that is complementary to the mRNA of
interest. The probe hybridizes with high efficiency
to the mRNA and is detected by either fluores-
cence (FISH) or biochemical means. In situ
hybridization may also be used to detect DNA
sequences and gene rearrangements in tissue sec-
tions. The advantage of in situ hybridization is
that it identifies where in the tissue the mRNA or
DNA is expressed and it is used more frequently
than PCR or RT-PCR in pathological diagnosis.
T. Hodgson et al.
staining for cytokeratins identifies the carcinoma cells (b).
Immunohistochemical staining for CD45 identifies the
lymphocytes (c)
Application of Molecular Biology
Techniques in Diagnosis
Molecular techniques are beginning to play
an increasingly important role in the diagnosis of
malignancy and infectious disease affecting the
head and neck. At the current time, these include
salivary gland tumors, oropharyngeal cancers,
soft tissue neoplasms, and viral infections,
although it is likely the range of diseases will
increase in the future.
Laboratory Medicine and Diagnostic Pathology
Fig. 20 Hematoxylin and eosin staining of a nerve sheath tumor (schwannoma) (a). The schwannoma stains positively
for S100 protein by immunohistochemistry (b)
Molecular techniques have been used to refine
the classification of certain salivary gland tumors
and also to predict their behavior (Fonseca et al.
2016). For example, specific translocations
have been detected by molecular techniques
in mucoepidermoid carcinomas (Martins et al.
2004; Tonon et al. 2003), and the expression of
this CRTC1-MAML2 translocation is predictive
of a better prognosis. Similarly, an ETV6-NTRK3
gene translocation identified a subset of acinic
cell carcinomas that are now recognized as
a new entity, known as mammary analogue
secretory carcinoma of salivary glands (Skalova
et al. 2010).
Some viral infections may be detected by the
use of immunohistochemistry that identifies viral
proteins on tissue sections. However, it will only
detect viral infections that contain replicating
virus and are producing protein and does not
detect latent or transcriptionally active virus. In
situ hybridization is most commonly used to iden-
tify specific viral mRNA or DNA in tissue sam-
ples as the technique preserves tissue integrity.
Human Papilloma Virus
Human papilloma virus (HPV) is a ubiquitous
virus that has recently come to prominence in
diseases of the oral cavity and oropharynx. It is a
41
double-stranded DNA virus with up to 200 sub-
types identified to date (McBride 2017). The virus
infects mucosa and skin and has a propensity to
infect cells in the basal layer. It then replicates in
the suprabasal cells and is shed when the surface
epithelial cells desquamate. Human papilloma
virus causes a number of infections on the skin,
in the oral cavity, and in the cervix. Some of the
viruses are the so-called low-risk subtypes (e.g.,
types 6, 8, 13, 32) and are associated with squa-
mous papilloma, verruca vulgaris, condyloma
acuminatum, and multifocal epithelial hyperplasia
(Heck’s disease). These lesions have specific his-
tological features, and it is unusual to request in
situ hybridization to confirm the diagnosis.
However, the same is not true for oropharyn-
geal carcinoma and a small subset (less than 6%)
(Lingen et al. 2013) of oral cancers that are
associated with high-risk HPV subtypes 16 and
18 (but also 31 and 33). The incidence of oropha-
ryngeal HPV-associated cancers is rising, and in
the USA there has been a threefold increase
between 1988 and 2004 (Chaturvedi et al. 2011).
Similar increases have been reported in
Western Europe, and it has been estimated that
the number of HPV-positive oropharyngeal can-
cers will outnumber cervical cancers in the near
42
Fig. 21 Hematoxylin and eosin staining of a basal cell
adenoma (a). The myoepithelial cells surrounding the duc-
tal cells are highlighted by immunohistochemical staining
for SMA (b). The myoepithelial cells are also identified by
future (Chaturvedi et al. 2011; Berman and
Schiller 2017).
Two proteins produced during the infection
with high-risk HPV, E6 and E7 play a role in
driving the malignant transformation by interfer-
ing with the cell cycle pathway. E7 inactivates the
retinoblastoma gene protein that results in the
accumulation of the tumor suppressor protein
p16 (McBride 2017). HPV-positive tumors
have different histopathological features and
a better prognosis than HPV negative tumors,
in that they may show a better response to
chemo/radiotherapy (Wang et al. 2015; Stevens
and Bishop 2017). The WHO have recently
classified these lesions as HPV-associated
nonkeratinizing squamous cell carcinomas of
T. Hodgson et al.
immunohistochemical staining for P63 (c). The ductal cells
are identified by immunohistochemical stains for
cytokeratin (d)
the oropharynx (International Agency for
Research on Cancer 2017). For this reason, it is
necessary to confirm the HPV status of the cancer
so that the treatment may be optimized. The best
and most specific method for identification of
HPV is to use PCR or in situ hybridization, but
these are not widely available and may be expen-
sive. In routine practice therefore, staining for
p16 by immunohistochemistry in oropharyngeal
carcinoma is a surrogate marker, which is
interpreted together with the specific histopatho-
logical features (Westra 2014; Stevens and
Bishop 2017). In squamous cell carcinoma aris-
ing outside the oropharynx, p16 staining is not
necessarily indicative of HPV infection (Lingen
et al. 2013), and if HPV infection is suspected, it
Laboratory Medicine and Diagnostic Pathology 43

Fig. 22 Hematoxylin and

eosin staining section of an
HPV-associated
non-keratinizing squamous
cell carcinoma of the
oropharynx (a). The
carcinoma stains positively
for P16 by
immunohistochemistry (b)

Fig. 23 Hematoxylin and eosin stained section of amy- light chains indicative of a monoclonal proliferation (b).
loid in the oral mucosa associated with dense accumula- Very few plasma cells staining positively for lambda light
tions of plasma cells (a). Immunohistochemistry shows a chains are evident (c)
predominance of plasma cells staining positively for kappa
44
Fig. 24 A salivary neoplasm stained for Ki67 by immu-
nohistochemistry indicating the proliferative fraction of
epithelial cells
is necessary to confirm HPV infection by in situ
hybridization or PCR.
Related to HPV-positive oropharyngeal can-
cers, it is now becoming apparent there is a subset
of oral dysplastic lesions that are also associated
with high-risk HPV. These lesions have been
detected on the basis of specific histological fea-
tures with confirmation of high-risk HPV infec-
tion using P16 and in situ hybridization (McCord
et al. 2013). At the present time, it is not clear
whether they have a higher risk of transforming
into oral cancer.
Herpes Viruses
Herpes are a family of DNA viruses that are
associated with a number of diseases affecting
the oral cavity. Those viruses that cause oral
symptoms or lesions include herpes simplex, var-
icella zoster, cytomegalovirus, Epstein-Barr virus,
and human herpes eight (HHV8) virus. A biopsy
is very rarely used in the diagnosis, but there are
two pathologies where a biopsy is frequently
taken and it is necessary to determine the presence
of viral particles to confirm the diagnosis: hairy
leukoplakia and Kaposi’s sarcoma. Hairy leuko-
plakia is associated with Epstein-Barr viral infec-
tion, and this is detected by in situ hybridization
for the viral particle EBER (Fig. 25). Kaposi’s
sarcoma is associated with HHV8 that may be
detected by immunohistochemistry (Fig. 26).
Antibodies are also available to HSV1 and
T. Hodgson et al.
cytomegalovirus if tissue is taken as part of the
diagnostic procedure.
Interpretation of Biopsy Results
It is good practice to have a close working rela-
tionship with the pathologist who will report
the biopsies taken. This is important so the pathol-
ogist has confidence in the clinical information
and biopsy specimen provided, and the oral med-
icine specialist has confidence in and is able to
understand and interpret the pathologist’s report.
Dialogue between the two is essential in difficult
cases.
Histopathological Prognostic Factors
in Oral Potentially Malignant Disorders
and Oral Malignancy
Dysplasia
Oral potentially malignant disorders are lesions
that have an increased risk of progressing to oral
cancer (Warnakulasuriya and Ariyawardana
2016). Oral leukoplakia is the most common of
these lesions and is defined as a “white plaque of
questionable risk having excluded (other) known
diseases or disorders that carry no increased
risk for cancer” (Warnakulasuriya et al. 2007).
In order to confirm the diagnosis and exclude
other lesions, a biopsy is necessary. The pathol-
ogist will report on the histopathological features
to confirm whether the lesion is indeed a poten-
tially malignant disorder. The degree of dyspla-
sia is used as an indicator of the risk of malignant
transformation (Bouquot et al. 2006; Reibel et al.
2017).
Dysplasia is the term given to abnormalities in
both the appearance and differentiation of cells
(known as atypia) as well as the architecture of
the oral epithelium.
There are a number of ways to grade the degree
of dysplasia, but that recommended by the WHO
divides dysplasia into mild, moderate, and severe
(including carcinoma in situ) based on assessment
of defined architectural and cytological changes
(International Agency for Research on Cancer
2017) (Table 10).
Laboratory Medicine and Diagnostic Pathology
Fig. 25 Hematoxylin and eosin stained section of hairy
leukoplakia (a). Dense accumulations of fungal hyphae in
the superficial epithelial layers are identified by PAS
Fig. 26 Immunohistochemical staining for HHV8 in
Kaposi sarcoma
In general terms, mild dysplasia is diagnosed if
cytological atypia affects the epithelial cells in the
lower third of the epithelium, moderate dysplasia
if it affects the epithelial cells in the lower two
thirds, and severe dysplasia where more than two
45
staining (b). Clear evidence of Epstein Barr viral infection
identified by in-situ hybridization (c)
thirds of the epithelium are affected by cellular
atypia. In the diagnosis of dysplasia in the uterine
cervix where human papilloma virus (HPV) is
important in the etiology, this rule of grading
atypia depending on the extent or level of the
epithelium affected is more strictly applied. How-
ever, in the oral cavity, architectural changes also
play an important role in determining the degree
of dysplasia (Table 11) (Figs. 27, 28, and 29). The
grade of dysplasia may also change depending on
the severity of the cytological and the architectural
changes within a particular level. For instance,
marked cellular atypia and severe architectural
changes occurring within the lower third of the
epithelium may be sufficient to grade the dyspla-
sia as severe.
Diagnosis of the degree of dysplasia is to
a certain degree subjective, and there is both
intra- and interobserver variability with several
studies showing only poor to moderate agreement
(Abbey et al. 1995; Karabulut et al. 1995; Bosman
46 T. Hodgson et al.

Table 10 Architectural Cytological changes Architectural changes

and cytological changes in
the diagnosis of epithelial
dysplasia (International
Agency for Research on
Cancer 2017)
Abnormal variation in nuclear size
Abnormal variation in nuclear shape
Abnormal variation in cell size
Abnormal variation in cell shape
Increased nuclear to cytoplasmic ratio
Atypical mitotic figures
Increased number and size of nucleoli
Hyperchromasia
Irregular epithelial stratification
Loss of polarity of basal cells
Drop-shaped rete pegs
Increased numbers of mitotic figures
Abnormally superficial mitotic figures
Premature individual cell keratinization
Keratin pearls within rete ridges
Loss of epithelial cell cohesion

Table 11 Criteria for grading of oral epithelial dysplasia. (Adapted from Speight 2007)
Levels
Grade involved Cytological changes Architectural changes
Mild Lower Cell and nuclear pleomorphism Basal cell hyperplasia
third Nuclear hyperchromatism
Moderate Up to Cell and nuclear pleomorphism Disordered maturation from basal to
middle Anisocytosis and anisonucleosis squamous cells
third Nuclear hyperchromatism Loss of polarity Increased cellular density
Increased and abnormal mitotic Basal cell hyperplasia
figures Bulbous drop-shaped rete pegs
Severe (including Up to the Cell and nuclear pleomorphism Disordered maturation from basal to
carcinoma in situ) upper third Anisocytosis and anisonucleosis squamous cells
Nuclear hyperchromatism Increased cellular density
Increased and abnormal mitotic Basal cell hyperplasia
figures Dyskeratosis (premature keratinization
Enlarged nuclei and cells and keratin pearls deep in epithelium)
Hyperchromatic nuclei Increased Bulbous drop-shaped rete pegs
number and size of nucleoli Secondary extensions (nodules) on rete
Apoptotic bodies tips
Acantholysis

2001; Warnakulasuriya et al. 2008; Speight et al.
2015). However, there is a high level of agreement
between pathologists trained at the same institu-
tion, and a recent study has shown good agree-
ment on the grade of dysplasia between two
pathologists (agreed 69.6% of diagnoses) that
increased to 92.7% agreement after adjudication
by a third pathologist (agreement of two out of
three pathologists) (Speight et al. 2015). In an
attempt to ascertain which histopathological fea-
tures contributed most to the variability of diag-
nosis of dysplasia, one study demonstrated that
there was highest agreement between pathologists
on the number of mitotic figures, drop-shaped rete
ridges, increased nuclear size, and abnormal
variation in cell shape (Kujan et al. 2007).
There was least agreement on abnormal epithelial
stratification, abnormal mitoses and nuclear
hyperchromatism, loss of basal cell polarity, and
variation in nuclear size. These authors proposed
an alternative binary method of grading that
divides dysplasia into low and high grade (Kujan
et al. 2006). In this system, the pathologist
must decide whether the lesion has high-risk or
low-risk dysplasia. There is no intermediate cate-
gory. At present this grading system remains to
be fully validated, and it does not appear to
improve reliability of grading between patholo-
gists (Warnakulasuriya et al. 2008). The WHO
recommends the three-tier grading system should
continue to be used (International Agency for
Research on Cancer 2017).
Laboratory Medicine and Diagnostic Pathology 47

Fig. 27 Mild dysplasia.

Basal cell crowding,
nuclear hyperchromatism,
and enlargement as well as
anisonucleosis
(abnormality in shape) is
evident in the basal third of
the epithelium
(Hematoxylin and eosin
stain)

Fig. 28 Moderate
dysplasia. The epithelium
has a bulbous rete pattern,
and basal cell crowding,
nuclear enlargement,
anisonucleosis, and
individual cell
keratinization is seen in the
lower two thirds of the
epithelium (Hematoxylin
and eosin stain)

The risk overall of leukoplakia transforming
into malignancy is low, in the region of 0.1–2%
(Bouquot et al. 2006; Speight 2007), but this
increases when dysplasia is taken into account
(Mehanna et al. 2009). Approximately 50% of
leukoplakia show dysplasia, and the average
transformation rate for lesions with severe
dysplasia is 16% with a range of 7–50% (Bouquot
et al. 2006; Speight 2007). For lesions with mod-
erate dysplasia, the transformation rate is 3–15%,
and for those with mild dysplasia, a very low
transformation rate of less than 5% is seen
(Speight 2007). Thus it can be seen that the greater
degree of dysplasia, the greater the risk of
48
Fig. 29 Severe dysplasia.
The epithelium has a
proliferative bulbous rete
peg pattern, disordered
maturation, epithelial
crowding, and loss of
polarity; it shows marked
cellular atypia including
nuclear hyperchromatism,
anisonucleosis, and
anisocytosis. These changes
extend beyond the lower
two thirds of the epithelium
(Hematoxylin and eosin
stain)
malignant transformation. This correlates with the
finding that nonhomogeneous leukoplakia have a
higher risk of malignant transformation than
homogeneous leukoplakia and show a higher inci-
dence of dysplasia (Schepman et al. 1998;
Warnakulasuriya and Ariyawardana 2016). How-
ever, there have been several studies that show
dysplastic lesions do not necessarily progress but
may stay the same or even regress (Silverman
et al. 1976; Holmstrup et al. 2006). For an indi-
vidual patient, it is therefore difficult to predict the
risk of transformation based on dysplasia alone
(Schepman et al. 1998; Napier and Speight 2008;
Dost et al. 2014).
There has been a considerable amount of work
to identify biomarkers that might predict whether
or not a lesion will transform into squamous cell
carcinoma. These include aneuploidy that shows a
positive correlation with malignant transforma-
tion and severe dysplasia, oncogenes such as
EGFR (epidermal growth factor receptor) that
are responsible for cell growth and differentiation,
and tumor suppressor genes such as P53 that
effect cell signaling, genes controlling apoptosis,
and loss of heterozygosity (Pitiyage et al. 2009;
T. Hodgson et al.
Hunt et al. 2014). At the current time, although
these studies indicate the genetic changes that
are taking place in potentially malignant disor-
ders, they do not predict the risk of malignant
transformation. Thus dysplasia remains the best
current method to predict the risk of malignant
transformation.
Some lesions have marked architectural
changes and are considered high risk even though
the degree of cytological atypia is minimal.
Verruciform lesions, which may be present in
proliferative verrucous leukoplakia, are one
such example (Pentenero et al. 2014). It is there-
fore important for an oral medicine specialist to
have a close working relationship with the pathol-
ogist over such cases so the patient is treated
appropriately.
Oral Cancer
There have been many attempts to identify histo-
pathological features that will reliably predict the
prognosis of a carcinoma, in particular the risk of
recurrence and metastasis. Identification enables
a clinician to reliably stage the cancer and plan
management.
Laboratory Medicine and Diagnostic Pathology
The TNM system of clinical staging oral
cancer utilizes the surface diameter of the tumor
(T) and the presence of nodal (N) and distant
metastasis (M) as measured clinically and radio-
logically (International Agency for Research on
Cancer) (Table 12). This staging system correlates
well with prognosis. The pathological staging of a
tumor measures the same parameters but takes
place after the tumor has been excised. Thus the
surface diameter of the tumor and the presence of
lymph node metastasis are more accurately deter-
mined. This staging is known as the pTNM sys-
tem, and at the end of a report on the excision of
cancer, it is usual to state the pTNM stage.
In the latest 8th edition of the TNM classifica-
tion (Lydiatt et al. 2017; Brierley et al. 2016), the
depth of invasion of the tumor has been combined
with the surface diameter as the standard prognos-
tic indicator for oral cancer. Depth of invasion is
measured vertically from the level of the basement
membrane of the adjacent epithelium, and 5 mm
appears to be associated with a better prognosis
than those that are greater than 5 mm. A T1 tumor
has a surface diameter <2 cm and a depth of
invasion less than 5 mm. A T2 tumor has either a
surface diameter of less than 2 cm and a depth of
invasion between 5 and 10 mm or a surface diam-
eter between 2 and 4 cm and a depth of invasion of
<10 mm. A T3 tumor has a surface diameter
of >4 cm or a tumor of any size with a depth of
invasion of >10 mm.
Infiltration of a tumor from the regional
lymph nodes through the lymph node capsule
and into the surrounding tissues is known as extra-
nodal extension (ENE). Although this may be
detected by imaging or clinical examination, it is
best determined by pathological examination
(Fig. 30). ENE has now been added to the latest
pTNM staging as it is recognized that it has
an adverse effect on prognosis (Amin and
Edge 2017).
A pathologist may also report on other param-
eters that indicate prognosis. Guidelines on what
is considered “core” material and which should
be included in a report are produced in several
countries including Australia, the USA, and the
UK to ensure consistency in reporting
(Dahlstrom et al. 2012; Helliwell and Woolgar
49
2013; Richardson et al. 2012). In addition to the
parameters of surface diameter, depth of inva-
sion, and ENE outlined above, the degree of
differentiation and the pattern of invasion are
important. The degree of differentiation is
based on the original description of Broders in
1922 and adopted by the WHO and is defined as
well, moderately, or poorly differentiated (Inter-
national Agency for Research on Cancer 2017).
The most poorly differentiated part of the tumor
is used as the grade and is prognostically useful.
The pattern of invasion of the tumor is most
valuable in predicting metastasis particularly at
the invasive front (Helliwell and Woolgar 2013;
Woolgar and Triantafyllou 2009). The pattern of
invasion measures how dis-cohesive the tumor
islands are and is reported as “cohesive” or “non-
cohesive” (Fig. 31). A dis-cohesive pattern of
invasion is associated with a higher risk of
metastasis and recurrence (Helliwell and
Woolgar 2013). If this is assessed at the advanc-
ing front, there is a closer correlation with recur-
rence and metastasis (Woolgar and Scott 1995;
Odell et al. 1994).
Evidence of perineural extension ahead of the
advancing front of the tumor is a bad prognostic
sign and predicts local recurrence and nodal
metastases (Sethi et al. 2009; Rahima et al.
2004; Binmadi and Basile 2011; Woolgar 2006).
Evidence of invasion into lymphatic’s and blood
vessels should also be reported, although surpris-
ingly it is a weak predictor of nodal metastases
(Suzuki et al. 2007). Severe dysplasia adjacent to
a carcinoma and within 5 mm of the resection
margin is also one of the core items to be included.
Bone invasion is important in tumor staging.
These parameters may be commented upon in
both incisional and excisional biopsies, but the
accuracy of the assessment of some of these in
an incisional biopsy depends on how representa-
tive the incisional biopsy is of the lesions as
a whole.
A pathologist will also report on completeness
of excision for cancer resections and will measure
the distance of the cancer in millimeters from
the mucosal and deep margins. A tumor that is
greater than 5 mm from the margin is considered
completely excised, one that is between 1 and
50
Table 12 Staging of oral cancer using the TNM clinical classification (adapted from the 8th Edition) (Brierley 2016)
T – Primary Tumor
N – Regional Lymph
Nodes
M – Distant metastasis
pTNM Pathological Classification
The pT categories correspond to the clinical T categories
pN – Regional lymph
nodes
Histological examination
of a selective neck
dissection specimen will
ordinarily include 10 or
more lymph nodes.
Histological examination
of a radical or modified
radical neck dissection
specimen will ordinarily
include 15 or more lymph
nodes
Stage
Stage 0
Stage I
Stage II
Stage III
T. Hodgson et al.
TX Primary tumor cannot be assessed
T0 No evidence of primary tumor
Tis Carcinoma in situ
T1 Tumor 2 cm or less in greatest dimension and 5 mm or less depth of invasiona
T2 Tumor 2 cm or less in greatest dimension and more than 5 mm but no more than
10 mm depth of invasion or tumor more than 2 cm but not more than 4 cm in greatest
dimension and depth of invasion no more than 10 mm
T3 Tumor more than 4 cm in greatest dimension or more than 10 mm of invasion
T4a (Lip) Tumor invades through cortical bone, inferior alveolar nerve, floor of mouth,
or skin (of the chin or the nose)
T4a (Oral cavity) Tumor invades through the cortical bone of the mandible or maxillary
sinus, or invades the skin of the face
T4b (Lip and oral cavity) Tumor invades masticator space, pterygoid plates, or skull
base, or encases internal carotid artery
NX Regional lymph nodes cannot be assessed
N0 No regional lymph node metastasis
N1 Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension
without extranodal extension
N2 Metastasis described as:
N2a Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than
6 cm in greatest dimension without extranodal extension
N2b Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest
dimension, without extranodal extension
N2c Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in
greatest dimension, without extranodal extension
N3a Metastasis in a lymph node more than 6 cm in greatest dimension without
extranodal extension
N3b Metastasis in a single or multiple lymph nodes with clinical extranodal extensionb
M0 No distant metastasis
M1 Distant metastasis
pNX Regional lymph nodes cannot be assessed
pN0 No regional lymph node metastasis
pN1 Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension
without extranodal extension
pN2 Metastasis described as:
pN2a Metastasis in a single ipsilateral lymph node, less than 3 cm in greatest
dimension with extranodal extension or more than 3 cm but not more than 6 cm in
greatest dimension without extranodal extension
pN2b Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest
dimension, without extranodal extension
pN2c Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in
greatest dimension, without extranodal extension
pN3a Metastasis in a lymph node more than 6 cm in greatest dimension without
extranodal extension
pN3b Metastasis in a lymph node, more than 3 cm in greatest dimension with extranodal
extension or multiple ipsilateral or any contralateral or bilateral node(s) with extranodal
extension
Tis N0 M0
Tl N0 M0
T2 N0 M0
T3 N0 M0
T1, T2, T3 N1 M0
(continued)
Laboratory Medicine and Diagnostic Pathology
Table 12 (continued)
Stage IVA T4a
T1, T2, T3, T4a
Stage IVB Any T
T4b
Stage IVC Any T
Note:
a
Superficial erosion alone of bone/tooth socket by gingival primary is not sufficient to classify a tumor as T4a
b
The presence of skin involvement or soft tissue invasion with deep fixation/tethering to underlying muscle or adjacent
structures or clinical signs of nerve involvement is classified as clinical extranodal extension
Fig. 30 Extra-nodal extension of tumor. Hematoxylin and
eosin staining of a lymph node containing metastatic tumor
that has extended through the capsule and into the sur-
rounding tissues
5 mm from the margin is closely excised, and one
that is less than 1 mm from the margin is incom-
pletely excised (Woolgar 2006; Helliwell and
Woolgar 2013). A closely excised tumor, or one
with severe dysplasia at the margin, shows an
increased risk of local recurrence (Bradley et al.
2007; Langendijk et al. 2010). These features,
together with those outlined in the above para-
graphs, provide information for the multi-
disciplinary cancer team meetings that discuss
management of the patient.
A pathologist will write a report based on the
provided material as well as the results of previous
tests, the clinical history of the patient, and the
current clinical features. This information is
essential for the pathologist to examine the tissue
51
N0, N1 M0
N2 M0
N3 M0
Any N M0
Any N M1
in the appropriate manner, to order appropriate
tests, and to issue a meaningful report.
In addition, it is essential that there are good
lines of communication between the oral medi-
cine specialist and the pathologist. Failure at any
of these points may lead to an inaccurate report to
the detriment of the patient.
Precision Medicine
Recently, the term “precision medicine”
has become popular, fueled by scientific and
political perspectives. It has superseded the term
“personalized medicine,” which was defined syn-
onymously but then dismissed with the argument
physicians have always treated patients at a per-
sonalized level. Many common medicines pre-
scribed are ineffective in treating large numbers
of patients. Adverse drug reaction (ADR) rates
in the USA increased between 1999 and 2006,
with higher ADR death rates observed among
elderly individuals (Shepherd et al. 2012). In two
Australian hospitals, the proportion of over
65 year olds with potential ADR-related medical
admissions was 18.9%. Most (88.5%) ADR-
related admissions were considered preventable.
The most frequently implicated drug classes
were diuretics (23.9%), agents acting on the
renin angiotensin system (16.4%), β-blocking
agents (7.1%), antidepressants (6.9%), and
antithrombotic agents (6.9%) (Nair et al. 2017).
Moreover, healthcare costs are increasing with an
aging population alongside chronic disease that
becomes more prevalent.
Precision medicine starts with the patient.
However, rather than having a unique treatment
52
Fig. 31 Hematoxylin and eosin stained section of carcinoma of the lip with a cohesive invasion pattern (a). Carcinoma of
the tongue showing a dis-cohesive invasion pattern (b)
for each individual person, patients are subdivided
into groups based on their “molecular makeup,”
using biomarkers. Through stratification, medical
interventions can be tailored to be more effica-
cious in a particular group of patients than
under the currently dominant “one size fits all”
approach. In addition, clinical implementation of
genomic biomarkers may allow the prediction of
which patients are at high risk of serious adverse
reactions, in relation to genetic variants of metab-
olism enzymes, transporters, or genes active in
the immune responses underlying idiosyncratic
reactions. HLA allele B*1502 is a marker for
carbamazepine-induced Stevens-Johnson syn-
drome and toxic epidermal necrolysis in Han
Chinese. Genotyping all Asian individuals for
the allele has been recommended for patients
prescribed carbamazepine (Ferrell and McLeod
2008). Similarly TPMT levels aid systemic immu-
nosuppressant selection and dose of azathioprine
and decrease the risk of bone marrow suppression
(Hullah et al. 2015). Measuring glucose-6-phos-
phate dehydrogenase levels before prescribing
dapsone prevents hemolytic anemia.
The meaning of precision medicine and how
it is related to or different from other popular
terms such as “stratified medicine,” “targeted ther-
apy,” or “deep phenotyping” remains unclear.
Commonly used definitions of precision medicine
include the following aspects.
• Focus on result. Personalized treatment
strategies: some define precision medicine as
T. Hodgson et al.
“treatments targeted to the needs of individual
patients on the basis of genetic, biomarker, phe-
notypic or psychosocial characteristics that distin-
guish a given patient from other patients with
similar clinical presentations” (Jameson and
Longo 2015).
• Focus on process and utilized data. Others
emphasize the data by describing precision med-
icine as a model that integrates clinical and other
data to stratify patients into novel subgroups; it is
hoped that these have a common basis of disease
susceptibility and manifestation and thus poten-
tially allow for more precise therapeutic solu-
tions (McGrath and Ghersi 2016). Some
potential advantages offered by this new
approach include:
• Ability to make more informed medical
decisions
• Higher probability of desired outcomes thanks
to better-targeted therapies
• Reduced probability of adverse reactions to
medicines
• Focus on prevention and prediction of disease
rather than reaction to it
• Earlier disease intervention than has been pos-
sible in the past
• Improved healthcare cost containment
The move toward precision medicine can be
seen as an evolutionary rather than revolution-
ary process. Although some precision medicine
Laboratory Medicine and Diagnostic Pathology
approaches have been introduced, we remain at
an early stage of implementation. The imple-
mentation of precision medicine will result in a
steep increase in the number of new screening or
diagnostic tests administered in clinical
practice.
Conclusions and Future Directions
Evidence supports the transition of next-
generation sequencing (NGS) technology from
research to clinical practice. While our knowledge
of the relationship between disease pathogenesis
and genetic variations continues to expand, the
cost to NGS equipment and operation continues
to fall. Ultimately this will enable relatively cheap
exploration of specific nucleic acid regions, large
gene panels, whole exomes, genomes, trans-
criptomes, and the methylome.
Although more than 90% of head and neck
tumors are squamous cell carcinoma, recent stud-
ies have revealed this tumor is very heteroge-
neous. NGS studies of head and neck squamous
carcinoma (HNSCC) help better understand the
genetic aspects of a tumor traditionally considered
environmental. An extensive literature detailing
the molecular changes involved in the develop-
ment, prognosis, and management of HNSCC
now exists (Jessri and Farah 2014; Foy et al.
2017; Li et al. 2018).
Numerous genetic mutations have been asso-
ciated with susceptibility to chronic mucocutane-
ous candidosis. It has been 5 years since
heterozygous gain-of-function mutations of
STAT1 (STAT1-GOF) were first shown to cause
an intriguing familial susceptibility to severe
superficial fungal infection alongside autoim-
mune disease. These mutations lead to defective
responses in type 1 and type 17 helper T cells (Th1
and Th17), which, depending on the mutation,
also predispose to infection with Staphylococci,
Mycobacteria, and Herpesviridae. STAT1-GOF
mutations are the most common genetic etiology
for chronic mucocutaneous candidosis (CMC),
and mutation analysis should be considered in
these patients. Identifying specific genetic defects
53
in patients with CMC confirms the diagnosis,
facilitates genetic counseling, and allows
improved risk stratification and improved thera-
peutic management strategies (Toubiana et al.
2016).
We have no validated screening tests advo-
cated for use in oral medicine practice. While it
is widely acknowledged, oral disease may be the
presenting feature of a generalized disease pro-
cess, investigations should be appropriately
selected to answer the diagnostic question pro-
posed and not used as a “fishing exercise” for
the presence of other diseases unrelated to the
presenting symptoms and signs. The smallest
number of tests should be ordered to provide the
greatest diagnostic yield. For example, if pemphi-
gus vulgaris is suspected, an incisional biopsy
with direct immunofluorescence is the first-line
investigation and will provide a diagnosis. If the
presentation is severe and systemic immunosup-
pression is needed, a further group of appropriate
tests should be requested. These should include
a baseline indirect antibody titer to monitor
response to therapy. Investigations are expensive
and resources can be wasted by indiscriminate test
use. Investigations should only be requested when
they have impact on diagnosis, they contribute
directly to disease management, and the outcomes
are reviewed and actioned. Clinicians requesting
any investigation need to understand the limita-
tions of the test and be able to interpret the result.
Cross-References
▶ Clinical Evaluation of Oral Disease
▶ Clinical Immunology in Diagnoses of Maxillo-
facial Disease
▶ Diagnostic Imaging Principles and Applica-
tions in Head and Neck Pathology
▶ Gingival Pathology
▶ Head and Neck Tumors
▶ Interface Between Oral and Systemic Disease
▶ Non-odontogenic Bacterial Infections
▶ Oral and Maxillofacial Fungal Infections
▶ Oral and Maxillofacial Viral Infections
54
▶ Oral Manifestations of Systemic Diseases and
Their Treatments
▶ Oral Mucosal Malignancies
▶ Oral Vesicular and Bullous Lesions
▶ Pigmented Lesions of the Oral Mucosa
▶ Soft and Hard Tissue Operative Techniques in
the Diagnosis and Treatment of Oral Disease
▶ White and Red Lesions of the Oral Mucosa
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