AABB Technical Manual 18th Ed 2014

1. AABB Technical Manual 18th Ed 2014

 AABB Technical Manual 18th Ed 2014
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Manual
1 8 T H EDITION
Other related publications available from the AABB:
Transfusion Therapy: Clinical Principles and Practice, 3rd edition Edited by Paul Mintz, MD
Transfusion Medicine: Self-Assessment and Review, 2nd edition By Douglas P. Blackall, MD; Jeffrey L.
Winters, MD; and Priscilla I. Figueroa, MD
Blood Transfusion Therapy: A Physician’s Handbook, 10th edition Edited by Karen King, MD
Judd’s Methods in Immunohematology, 3rd edition By John W. Judd, FIBMS; Susan T. Johnson, MSTM,
MT(ASCP)SBB; and Jill Storry, PhD, FIBMS
Antibody Identification: Art or Science? A Case Study Approach By Janis R. Hamilton, MS,
MT(ASCP)SBB; Susan T. Johnson, MSTM, MT(ASCP)SBB; and Sally V. Rudmann, PhD, MT(ASCP)SBB
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Technical
Manual
1 8 T H EDITION
Edited by
Mark K. Fung, MD, PhD
Fletcher Allen Health Care Burlington, VT
Brenda J. Grossman, MD, MPH
Washington University School of Medicine St. Louis, MO
Christopher D. Hillyer, MD
New York Blood Center New York, NY
Connie M. Westhoff, PhD, SBB
New York Blood Center New York, NY
 Mention of specific products or equipment by contributors to this AABB publication does not represent an
endorsement of such products by the AABB Press nor does it indicate a preference for those products over other
similar competitive products. Product listings, descriptions, and references are not intended to be
comprehensive. Any forms and/or procedures in this book are examples. AABB does not imply or guarantee
that the materials meet federal, state, or other applicable requirements. It is incumbent on the reader who intends
to use any information, forms, policies, or procedures contained in this publication to evaluate such materials
for use in light of particular circumstances associated with his or her institution.
AABB authors are requested to comply with a conflict of interest policy that includes disclosure of
relationships with commercial firms. A copy of the policy is located at http://www.aabb.org.
Efforts are made to have publications of the AABB consistent in regard to acceptable practices. However,
for several reasons, they may not be. First, as new developments in the practice of blood banking occur, changes
may be recommended to the Standards for Blood Banks and Transfusion Services. It is not possible, however, to
revise each publication at the time such a change is adopted. Thus, it is essential that the most recent edition of
the Standards be consulted as a reference in regard to current acceptable practices. Second, the views expressed
in this publication represent the opinions of authors. The publication of this book does not constitute an
endorsement by the AABB of any view expressed herein, and the AABB expressly disclaims any liability
arising from any inaccuracy or misstatement.
Copyright © 2014 by AABB. All rights reserved. No part of this book may be reproduced or transmitted in
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storage and retrieval system, without permission in writing from the Publisher.
The Publisher has made every effort to trace the copyright holders for borrowed material. If any such
material has been inadvertently overlooked, the Publisher will be pleased to make the necessary arrangements at
the first opportunity.
AABB ISBN No. 978-1-56395-888-5
8101 Glenbrook Road Printed in the United States
Bethesda, Maryland 20814-2749
Cataloging-in-Publication Data
Technical manual / editor, Mark K. Fung—18th ed. p.; cm.
Including bibliographic references and index.
ISBN 978-1-56395-888-5
1. Blood Banks—Handbooks, manuals, etc. I. Fung, Mark K. II. AABB. [DNLM: 1. Blood Banks-
laboratory manuals. 2. Blood Transfusionlaboratory manuals. WH 25 T2548 2014]
RM172.T43 2014 615’.39—dc23 DNLM/DLC

Technical Manual Authors

Colleen A. Aronson, MT(ASCP)SBB Aleksandar M. Babic, MD, PhD Robert A. Bray, PhD Laura Cooling,
MD, MS Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB
 Robertson D. Davenport, MD Meghan Delaney, DO, MPH Gregory A. Denomme, PhD, FCSMLS(D)
Katharine A. Downes, MD Larry J. Dumont, MBA, PhD Deborah F. Dumont, MT(ASCP)SBB Nancy M.
Dunbar, MD Anne F. Eder, MD, PhD William FitzGerald, LTC USA (Ret)
Susan A. Galel, MD Howard M. Gebel, PhD Mary Ghiglione, RN, MSN, MHA Janis R. Hamilton, MS,
MT(ASCP)SBB Jeanne E. Hendrickson, MD Eapen K. Jacob, MD Shweta Jain, MD Yameena Jawed, MD
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE Brian Johnstone, PhD Cassandra D. Josephson, MD
Melanie S. Kennedy, MD Scott A. Koepsell, MD, PhD Mickey B. C.Koh, MD, PhD Patricia M. Kopko, MD
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/ OE(ASQ)
Christine Lomas-Francis, MSc, FIBMS Keith March, MD, PhD Kim Maynard, BSN, RN, OCN
Catherine A. Mazzei, MD David H. McKenna Jr, MD Erin Meyer, DO, MPH Tania L. Motschman, MS,
MT(ASCP)SBB, CQA(ASQ)
Maria D.L.A. Muniz, MD Theresa Nester, MD Mona Papari, MD Jessica Poisson, MD Marilyn S. Pollack,
PhD Mark A. Popovsky, MD Kathleen E. Puca, MD, MT(ASCP)SBB Glenn Ramsey, MD Donna M. Regan,
MT(ASCP)SBB Rita A. Reik, MD Edward R. Samuel, PhD, MSc Scott Scrape, MD Ira A. Shulman, MD James
W. Smith, MD, PhD Steven L. Spitalnik, MD Simon Stanworth, FRCP, FRCPath, DPhil Jill R. Storry, PhD,
FIBMS Garnet Suck, PhD, MSc Ruth D Sylvester, MS, MT(ASCP)SBB Sreedhar Thirumala, PhD Alan
Tinmouth, MD, FRCPC, MSc Christopher A. Tormey, MD Lance D. Trainor, MD Wendy Trivisonno Phyllis S.
Walker, MS, MT(ASCP)SBB Connie M. Westhoff, PhD, SBB Theresa Wiegmann, JD Susan L. Wilkinson,
EdD, MS, MT(ASCP)SBB James C. Zimring, MD, PhD
 Acknowledgments
THE 18TH EDITION OF the Technical Manual was the work of many dedicated individuals. In addition to
the chapter authors, I would like to thank my three associate editors: Brenda Grossman, Chris Hillyer, and
Connie Westhoff. Their efforts and long hours in revising and rewriting chapters during the review process
made my job immeasurably easier.
We would also like to acknowledge the members of the following committees and program units for their
expert review of chapters, methods, and appendices for the 18th edition of the Technical Manual.
REVIEWING GROUPS
AABB Representative to ASFA AATB Representative Circular of Information Task Force Clinical
Transfusion Medicine Committee Cord Blood Subsection of the Cellular Therapies Section
Donor Center Accreditation Program Unit Donor History Task Force Immunohematology Reference
Laboratories Accreditation Program Unit Immunohematology Reference Laboratories Standards Program Unit
Information Systems Committee Molecular Testing Laboratories Accreditation Program Unit
Molecular Testing Laboratories Standards Program Unit
Novel Therapies and CT Product Development Subsection of the Cellular Therapies Section Patient Blood
Management Advisory Group Perioperative Accreditation Program Unit Perioperative Standards Program Unit
Product Collection and Clinical Practices Subsection of the Cellular Therapies Section Product Manufacturing
and Testing Subsection of the Cellular Therapies Section Quality Operations Subsection of the Cellular
Therapies Section
Quality Systems Accreditation Subcommittee Regulatory Affairs Subsection of the Cellular Therapies
Section
Relationship Testing Accreditation Program Unit
Relationship Testing Standards Program Unit Transfusion-Transmitted Disease Committee
Finally, we would like to thank the editors, authors, and program unit members of the 17th and earlier
editions of the Technical Manual for selected tables, figures, methods, and written sections of the chapters that
are valuable inclusions in the new edition.
Mark K. Fung, MD, PhD Editor in Chief
Preface
gg
IT is with tremendous pleasure that we introduce you to the 18th edition of the AABB Technical Manual.
As with all previous editions, this revision is based on the solid foundation of knowledge gathered by past
contributors to whom we are indebted. I would like to especially acknowledge the tremendous contributions of
Drs. Hillyer and Grossman who have helped guide previous editions. With the 18th edition they both conclude
their tenures as Associate Editors. Along the same lines, I want to welcome Dr. Westhoff who has joined me in
this new challenge of providing an up-to-date comprehensive resource of information in the field of transfusion
medicine and cellular therapies.
This edition of the Technical Manual will be most notable for several innovations. First, the cellular therapy
content has been expanded and reorganized to include many novel therapies that are moving from the research
setting into the clinical realm. In addition to updates on the sources of stem cells and the transfusion support of
stem cell transplantation, chapters focus on the quality and regulatory issues associated with cord banking,
novel stem cell therapies using nonhematopoietic stem cells, and tissue engineering. The new scope will be of
great value to the increasing number of professionals who now include some aspect of cellular therapy in their
daily responsibilities.
In a similar manner, the content on patient blood management (PBM) reflects the growing scope of what is
considered PBM. The traditional topics covered as part of discussions on blood utilization review and
perioperative blood recovery are augmented by detailed content on anemia management, optimization of
coagulation, and a host of
adjunctive therapies that can reduce the need for transfusion. As health-care economics join better patient
care in prompting continued adoption of PBM, readers will find that the new emphasis is highly relevant to their
needs.
Other content receiving special attention in this edition is that involving molecular testing. An increasing
number of organizations seek the more detailed test results possible through investments in molecular
technology. Those in the workforce today need (or soon will need) a solid understanding of the both the theory
and practice of molecular testing systems, which is found in this volume.
 Perhaps the most obvious upgrade in the new edition is the relocation of the methods from the printed pages
to electronic storage medium found inside the back cover. By moving the methods into the digital format, we
are able for the first time to give Technical Manual users methods that are already set up as standard operating
procedures (SOPs)—in a template that reflects how procedures would actually be used in real-life. Users are
invited to upload the methods to their facility networks and customize them for integration into their existing
SOPs.
In addition to these particular innovations, all chapters have been revised. Some of the chapter authors have
added substantial updates in great detail to assist the reader, whether working at the bench or the bedside. They
have embraced the task of explaining the issues that face all of us in the ever-changing world of transfusion
medicine and cellular therapies. We welcome your comments and feedback on the effectiveness of our labors.
Mark K. Fung, MD, PhD Editor in Chief
ix
Contents
si
Preface ix
QUALITY ISSUES
1. Quality Management Systems: Theory and Practice 1
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ);
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; and Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
Concepts in Quality 2
Practical Application of Quality Management Principles 4
Key Points 29
References 30
Appendix 1-1. Glossary of Commonly Used Quality Terms 33
Appendix 1-2. Code of Federal Regulations Quality-Related References ... 35 Appendix 1-3. Suggested
Quality Control Performance Intervals for
Equipment and Reagents 36
2. Facilities, Work Environment, and Safety 39
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and Tania L. Motschman, MS, MT(ASCP)SBB,
CQA(ASQ)
Facilities 40
Safety Program 42
Fire Prevention 45
Electrical Safety 46
Biosafety 47
Chemical Safety 55
Radiation Safety 60
Shipping Hazardous Materials 63
General Waste Management 63
Key Points 64
References 64
xi
Appendix 2-1. Safety Regulations and Recommendations Applicable to
Health-Care Settings 68
Appendix 2-2. General Guidelines for Safe Work Practices, Personal
Protective Equipment, and Engineering Controls 70
Appendix 2-3. Biosafety Level 2 Precautions 73
Appendix 2-4. Sample List of Hazardous Chemicals that May Be
Encountered in a Blood Bank 74
Appendix 2-5. Chemical Categories and Howto Work Safely with Them ... 76
Appendix 2-6. Incidental Spill Response 78
Appendix 2-7. Managing Hazardous Chemical Spills 81
3. Regulatory Issues in Blood Banking 83
 Glenn Ramsey, MD
Biological Products 84
Licensure and Registration 84
FDA Inspections 86
Blood-Related Devices 87
Hematopoietic Progenitor Cells as Tissues 87
Managing Recalls and Withdrawals 89
Medical Laboratory Laws and Regulations 89
Hospital Regulations and Accreditation 91
Conclusion 91
Key Points 91
References 92
4. Disaster Management 97
Ruth D. Sylvester, MS, MT(ASCP)SBB; William FitzGerald, LTC USA (Ret); Wendy Trivisonno; and
Theresa Wiegmann, JD
Background 98
Business Operations Planning 102
Regulatory Considerations in Emergencies 109
Testing the Disaster Plan Ill
Summary of Lessons Learned from Recent Disasters 112
Conclusion 112
Key Points 112
References 113
BLOOD DONATION AND COLLECTION
5 . Allogeneic and Autologous Blood Donor Selection 117
AnneF. Eder, MD, PhD, and Maria D.L.A. Muniz, MD
Overview of Blood Donor Screening 117
Selection of Allogeneic Blood Donors 118
Blood-Center-Defined Donor Eligibility Criteria 127
xiii
Abbreviated DHQ for Frequent Donors 130
Recipient-Specific “Designated” or “Directed” Blood Donation 130
Key Points 132
References 132
6. Whole-Blood Collection and Component Processing 135
Larry J. Dumont, MBA, PhD; Mona Papari, MD;
Colleen A. Aronson, MT(ASCP)SBB; and Deborah F. Dumont, MT(ASCP)SBB
WB Collection 135
Blood Component Preparation and Processing 146
Descriptions of Major Blood Components 148
Blood Component Modification 154
Quarantine 157
Labeling 158
QC of Blood Components 159
Key Points 160
References 161
7. Blood Component Collection by Apheresis 167
James W. Smith, MD, PhD
Component Collection 167
Instruments and Systems for Donor Apheresis Collections 173
Key Points 176
References 176
8. Infectious Disease Screening 179
Susan A. Galel, MD
Historical Overview of Blood Donor Screening 179
Donor Screening Tests 182
Residual Infectious Risks of Transfusion 192
Screening for Specific Agents 194
Pathogen Reduction Technology 204
Summary 205
Key Points 205
References 206
9. Hospital Storage, Monitoring, Pretransfusion Processing,
Distribution, and Inventory Management of
Blood Components 213
Nancy M. Dunbar, MD
Blood and Blood Component Storage and Monitoring 213
Pretransfusion Processing 221
Distribution 224
Inventory Management 227
Key Points 228
References 229
BLOOD GROUPS
10. Molecular Biology and Immunology in Transfusion
Medicine 231
James C. Zimring, MD, PhD, and Steven L. Spitalnilc, MD
Nucleic Acid Analysis 231
Protein Analysis 240
Basic Immunology 246
Key Points 252
References 253
11. Blood Group Genetics 255
Christine Lomas-Francis, MSc, FIBMS
Basic Principles of Genetics 256
Inheritance of Genetic Traits 265
Population Genetics 274
Relationship Testing 276
Blood Group Gene Mapping 277
Chimerism 278
Blood Group Terminology 278
Blood Group Genomics 279
Key Points 287
References 288
12. ABO, H, and Lewis Blood Groups and Structurally
Related Antigens 291
Laura Cooling, MD, MS
ABO System 291
The H System 301
The Lewis System 304
I and i Antigens 306
P Blood Groups/GLOB Collection 309
Key Points 313
References 313
13. The Rh System 317
Gregory A. Denomme, PhD, FCSMLS(D), and Connie M. Westhojf, PhD, SBB
Characterization of Rh 317
Terminology 319
xv
Rh Genes and Rh Proteins 319
Antigens 321
RhGenotyping 330
Rhnull Syndrome and RhAG Blood Group System 331
Rh Antibodies 331
Technical Considerations for Rh Typing 331
Key Points 333
References 334
Other Blood Group Systems and Antigens 337
Jill R. Storry, PhD, FIBMS
The MNS System 337
M (MNS1), N (MNS2), S (MNS3), and s (MNS4) 341
The Lutheran System 344
The Kell and Kx Systems 345
The Duffy System 349
The Kidd System 351
The Diego System 352
The Yt System 354
The Xg System 354
The Scianna System 354
The Dombrock System 354
The Colton System 355
The Landsteiner-Wiener System 355
The Chido/Rodgers System 356
The Gerbich System 357
The Cromer System 357
The Knops System 358
The Indian System 358
The Ok System 359
The Raph System 359
The John Milton Hagen System 359
The GILL System 359
The RHAG System 360
The FORS System 360
The Jr System 360
The Lan System 360
The Vel System 361
Antigens That Do Not Belong to a Blood Group System 361
Erythroid Phenotypes Caused by Mutations in Transcription
Factor Genes 362
Key Points 363
References 363
ANTIGEN AND ANTIBODY TESTING
15. Pretransfusion Testing 367
Katharine A. Downes, MD, and Ira A. Shulman, MD
Requests for Transfusion 367
Identification of Recipients and Labeling of Blood Specimens 368
Specimen Requirements 370
Serologic Testing Principles 371
Pretransfusion Tests 372
Tubeless Methods for Pretransfusion Testing 377
Comparison of Current Testing Results with Previous Records 378
Donor RBC Unit Testing 378
Donor RBC Unit Selection 378
Compatibility Testing or Crossmatch (Serologic or Computer/
Electronic) 379
Interpretation of Antibody Screening and Crossmatch Results 381
Pretransfusion Orders 381
Availability of Compatible Blood 384
Labeling of Blood and Blood Components with the Recipient’s
Information 384
Special Clinical Situations 385
Key Points 387
References 387
16. Identification of Antibodies to Red Cell Antigens 391
Phyllis S. Walker, MS, MT(ASCP)SBB, and Janis R. Hamilton, MS, MT(ASCP)SBB
Significance of Alloantibodies 392
Preanalytical Considerations 392
Analytical Phase of Antibody Identification 393
Postanalytical Considerations: Selecting Blood for Transfusion 419
Key Points 421
References 422
Suggested Readings 424
17. The Positive Direct Antiglobulin Test and Immune
Mediated Hemolysis 425
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
The DAT 425
Autoimmune Hemolytic Anemia 430
Drug-Induced Immune Hemolytic Anemia 440
Key Points 444
References 445
Appendix 17-1. Drugs Associated with Immune Hemolytic Anemia 448
xvii
18. Platelet and Granulocyte Antigens and Antibodies 453
Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB
Platelet Antigens and Antibodies 453
Granulocyte Antigens and Antibodies 466
Key Points 469
References 470
19. The HLA System 475
Robert A. Bray, PhD; Marilyn S. Pollack, PhD; and Howard M. Gebel, PhD
Genetics of the MHC 476
Biochemistry, Tissue Distribution, and Structure 480
Identification of HLA Antigens and Alleles 484
Crossmatching and Detection of HLA Antibodies 487
The HLA System and Transfusion 488
HLA Testing and Transplantation 491
Other Clinically Significant Aspects of HLA 493
Summary 494
Key Points 494
References 495
CLINICAL CONSIDERATIONS IN TRANSFUSION PRACTICE
20. Hemotherapy Decisions and Their Outcomes 499
Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD
Red Cell Transfusion 499
Platelet Transfusion 507
Plasma Transfusion 517
Cryoprecipitate Transfusion 522
Granulocyte Transfusion 523
Plasma-Derivative Transfusion 526
Key Points 532
References 533
21. Administration of Blood Components 545
Kim Maynard, BSN, RN, OCN
Events and Considerations Before Dispensing Components 545
Blood Component Transportation and Dispensing 549
Events and Considerations Before Component Administration 550
Manual Administration 552
Unique Transfusion Settings 555
Conclusions 556
Key Points 556
References 557
22. Perinatal Issues in Transfusion Practice 561
Melanie S. Kennedy, MD; Meghan Delaney, DO, MPH; and Scott Scrape, MD
HDFN 561
RhIG 565
ABO Hemolytic Disease 566
Immune Thrombocytopenia 567
Key Points 568
References 569
23. Neonatal and Pediatric Transfusion Practice 571
Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH
Transfusion in Infants Younger Than 4 Months 571
Transfusion in Infants Older Than 4 Months and Children 586
Prevention of Adverse Effects of Transfusion in Neonates,
Older Infants, and Children 589
Key Points 591
References 592
24. Patient Blood Management 599
Mary Ghiglione, RN, MSN, MHA, and Kathleen E. Puca, MD, MT(ASCP)SBB
Definition and Scope of Patient Blood Management 599
The Rationale for Patient Blood Management 600
Basic Elements of a PBM Program 603
Postoperative Strategies 608
Blood Utilization Review and Changing Physician Behavior 610
Program Development 612
Key Points 613
References 613
Appendix 24-1. Pharmacologic Therapies for Supporting Patient
Blood Management 620
Appendix 24-2. Responsibilities for Activity Levels 1, 2, and 3 PBM
Programs 629
25. Transfusion Support for Hematopoietic Stem Cell
Transplant Recipients 631
Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD
Implications of ABO- and Non-ABO-Antigen-Incompatible
Red Blood Cell Transplantation for Transfusion 632
Blood Component Considerations 633
Patients with Neutropenia and Infection that Is Unresponsive to
Antimicrobial Therapy 638
xix
Special Processing of Blood Components for HSCT Recipients 638
Special Considerations for Transfusions in Pediatric HSCT Recipients ... 639
Information Portability for HSCT Recipients 640
Key Points 640
References 641
26. Therapeutic Apheresis 645
Robertson D. Davenport, MD
Principles and Modalities 645
Indications 647
Anticoagulation 658
Adverse Effects 658
Vascular Access 660
Patient Evaluation 660
Key Points 661
References 662
27. Noninfectious Complications of Blood Transfusion 665
Catherine A. Mazzei, MD; Mark A. Popovsky, MD; and Patricia M. Kopko, MD
Hemovigilance 665
Recognition and Evaluation of a Suspected Transfusion Reaction 666
Delayed Transfusion Reactions 686
Fatality Reporting Requirements 691
Key Points 691
References 692
28. Approaches to Blood Utilization Auditing 697
Alan Tinmouth, MD, FRCPC, MSc, and Simon Stanworth, FRCP, FRCPath, DPhil
Rationale for Monitoring Blood Utilization 698
Types of Transfusion Audits 699
Interventions to Change Transfusion Practice 704
Effectiveness of Monitoring and Interventions to Change
Transfusion Practice 704
Selecting an Audit Process to Monitor Transfusions 705
Conclusions 705
Key Points 707
References 708
Appendix 28-1. Transfusion Order Form in Use at St. Vincent
Indianapolis Hospital Since 2001 711
xx AABB Technical Manual
TRANSPLANTATION
29. The Collection and Processing of Hematopoietic
Stem Cells 713
Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and David H. McKenna Jr, MD
Clinical Utility 713
Determination of Graft Source 717
Collection/Sources of HSCs 718
Processing of HSCs 720
Specialized Cell-Processing Methods 721
Cryopreservation 722
Shipping and Transport of HSC Cellular Products 723
Patient Care 724
Other Regulatory Considerations 725
Conclusion 725
Key Points 725
References 726
30. Umbilical Cord Blood Banking 729
Aleksandar M. Babic, MD, PhD, and Donna M. Regan, MT(ASCP)SBB
 Background 729
Donor-Related Issues 730
UCB Collection 733
UCB Processing 734
Shipment 737
Receipt of UCB for Transplantation 738
Thawing and Washing of UCB 740
Infusion of UCB 741
Economic Issues 742
Regulations and Standards 743
Key Points 747
References 747
31. Tissue-Derived Non-Hematopoietic Stem Cell Sources
for Use in Cell-Based Therapies 753
Yameena Jawed, MD; Brian Johnstone, PhD;
Sreedhar Thirumala, PhD; and Keith March, MD, PhD
MSC Sources 754
Properties of Clinical Relevance 758
Isolation and Expansion 758
Standardization of Methods for Isolation and Expansion of
Clinical Product 760
Cell Product Banking and Management of Supply Chain and End Use ... 761 Therapeutic Applications of
MSCs 762
xxi
Current Research and Development: Focus on Cell Culture and
Handling 764
Conclusions and Future Directions 765
Key Points 766
References 766
32. Human Allografts and the Hospital Transfusion Service 773
Lance D. Trainor, MD, and Rita A. Reik, MD
Tissue Transplantation 773
Regulations and Standards Ill
Hospital Tissue Services 778
Transfusion Service Support for Organ Transplantation 783
Key Points 783
References 784
33. Blood and Marrow-Derived Nonhematopoietic Stem Cell
Sources and Immune Cells for Clinical Applications 785
Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc; and Garnet Suck, PhD, MSc
Immune Cells for Clinical Therapy 786
Induced Pluripotent Stem Cells 795
Regulatory and Oversight Activities 796
Conclusion 797
Key Points 797
References 798
Index 803
METHODS
Methods Introduction
1. General Laboratory Methods—Introduction
Method 1-1. Shipping Hazardous Materials
Method 1-2. Monitoring Temperature During Shipment of Blood
Method 1-3. Treating Incompletely Clotted Specimens
Method 1-4. Solution Preparation Procedure
 Method 1-5. Serum Dilution Procedure
Method 1-6. Dilution of Percentage Solutions Procedure
Method 1-7. Preparing a 3% Red Cell Suspension
xxii
AABB Technical Manual
Method 1-8. Preparing and Using Phosphate Buffer Method 1-9. Reading and Grading Tube Agglutination
2. Red Cell Typing Methods—Introduction
Method 2-1. Determining ABO Group of Red Cells—Slide Test Method 2-2. Determining ABO Group of
Red Cells and Serum—Tube Test Method 2-3. Determining ABO Group of Red Cells and Serum—Microplate
Test Method 2-4. Initial Investigation of ABO Grouping Discrepancies Procedure Method 2-5. Detecting Weak
A and B Antigens and Antibodies by Cold Temperature Enhancement
Method 2-6. Confirming Weak A and B Antigens Using Enzyme-Treated Red Cells
Method 2-7. Confirming Weak A or B Subgroup by Adsorption and Elution Method 2-8. Testing Saliva for
A, B, H, Lea, and Leb Antigens Method 2-9. Confirming Anti-A: in an A, or Weak A Subgroup Method 2-10.
Resolving ABO Discrepancies Caused by Unexpected Alloantibodies
Method 2-11. Determining Serum Group Without Centrifugation Method 2-12. Determining Rh (D) Type—
Slide Test Method 2-13. Determining Rh (D) Type—Tube Test Method 2-14. Determining Rh (D) Type—
Microplate Test Method 2-15. Testing for Weak D Method 2-16. Preparing and Using Lectins Method 2-17.
Removing Autoantibody by Warm Saline Washes Method 2-18. Using Sulfhydryl Reagents to Disperse
Autoagglutination Method 2-19. Using Gentle Heat Elution to Test Red Cells with a Positive DAT Method 2-
20. Dissociating IgG by Chloroquine for Antigen Testing of Red Cells with a Positive DAT
Method 2-21. Using Acid Glycine /EDTA to Remove Antibodies from Red Cells Method 2-22. Separating
Transfused from Autologous Red Cells by Simple Centrifugation
Method 2-23. Separating Transfused from Autologous Red Cells in Patients with Hemoglobin S Disease
3. Antibody Detection, Identification, and Compatibility Testing—
Introduction
Method 3-1. Using Immediate-Spin Compatibility Testing to Demonstrate ABO Incompatibility
Method 3-2. Saline Indirect Antiglobulin Test Procedure
Method 3-3. Albumin or LISS-Additive Indirect Antiglobulin Test Procedure
Method 3-4. LISS-Suspension Indirect Antiglobulin Test Procedure
Method 3-5. PEG Indirect Antiglobulin Test Procedure
Method 3-6. Prewarming Procedure
Method 3-7. Detecting Antibodies in the Presence of Rouleaux—Saline Replacement
Method 3-8. Preparing Ficin Enzyme Stock, 1% w/v Method 3-9. Preparing Papain Enzyme Stock, 1% w/v
Method 3-10. Standardizing Enzyme Procedures
Table of Contents
xxiii
Method 3-11. Evaluating Enzyme-Treated Red Cells Method 3-12. One-Stage Enzyme Procedure Method 3-
13. Two-Stage Enzyme Procedure Method 3-14. Performing a Direct Antiglobulin Test Method 3-15. Antibody
Titration Procedure
Method 3-16. Using Sulfhydryl Reagents to Distinguish IgM from IgG Antibodies Method 3-17. Using
Plasma Inhibition to Distinguish Anti-Ch and -Rg from Other Antibodies with Similar Characteristics Method
3-18. Treating Red Cells Using DTT or AET Method 3-19. Neutralizing Anti-Sda with Urine Method 3-20.
Adsorption Procedure Method 3-21. Using the American Rare Donor Program
4. Investigation of a Positive DAT Result—Introduction
Method 4-1. Cold-Acid Elution Procedure Method 4-2. Glycine-HCl/EDTA Elution Procedure Method 4-3.
Heat Elution Procedure Method 4-4. Lui Freeze-Thaw Elution Procedure Method 4-5. Cold Autoadsorption
Procedure
Method 4-6. Determining the Specificity of Cold-Reactive Autoagglutinins Method 4-7. Cold Agglutinin
Titer Procedure
Method 4-8. Adsorbing Warm-Reactive Autoantibodies Using Autologous Red Cells
Method 4-9. Adsorbing Warm-Reactive Autoantibodies Using Allogeneic Red Cells
Method 4-10. Polyethylene Glycol Adsorption Procedure
Method 4-11. Performing the Donath-Landsteiner Test
 Method 4-12. Detecting Drug Antibodies by Testing Drug-Treated Red Cells
Method 4-13. Detecting Drug Antibodies by Testing in the Presence of Drug
5. Hemolytic Disease of the Fetus and Newborn—Introduction
Method 5-1. Testing for Fetomaternal Hemorrhage—The Rosette Test Method 5-2. Testing for Fetomaternal
Hemorrhage—Modified Kleihauer-Betke Test
Method 5-3. Using Antibody Titration Studies to Assist in Early Detection of Hemolytic Disease of the
Fetus and Newborn
6. Blood Collection, Component Preparation, and Storage—
Introduction
Method 6-1. Screening Donors for Anemia—Copper Sulfate Method Method 6-2. Preparing the Donor’s
Arm for Blood Collection Method 6-3. Collecting Blood and Samples for Processing and Compatibility Tests
Method 6-4. Preparing Red Blood Cells from Whole Blood Method 6-5. Preparing Prestorage Red Blood
Cells Leukocytes Reduced from Whole Blood
Method 6-6. Using High-Concentration Glycerol to Cryopreserve Red Cells— Meryman Method
AABB Technical Manual
Method 6-7. Using High-Concentration Glycerol to Cryopreserve Red Cells— Valeri Method
Method 6-8. Checking the Adequacy of Deglycerolization of Red Blood Cells
Method 6-9. Preparing Fresh Frozen Plasma from Whole Blood
Method 6-10. Preparing Cryoprecipitated AHF from Whole Blood
Method 6-11. Thawing and Pooling Cryoprecipitated AHF
Method 6-12. Preparing Platelets from Whole Blood
Method 6-13. Removing Plasma from Platelets (Volume Reduction)
Transplantation of Cells and Tissue—Introduction
Method 7-1. Infusing Cryopreserved Hematopoietic Cells Method 7-2. Processing Umbilical Cord Blood
Method 7-3. Investigating Adverse Events and Infections Following Tissue Allograft Use
Quality Control Methods—Introduction
Method 8-1. Validating Copper Sulfate Solution
Method 8-2. Calibrating Liquid-in-Glass Laboratory Thermometers
Method 8-3. Calibrating Electronic Oral Thermometers
Method 8-4. Testing Refrigerator Alarms
Method 8-5. Testing Freezer Alarms
Method 8-6. Calibrating Centrifuges for Platelet Separation Method 8-7. Calibrating a Serologic Centrifuge
for Immediate Agglutination Method 8-8. Calibrating a Serologic Centrifuge for Washing and Antiglobulin
Testing
Method 8-9. Testing Automatic Cell Washers Method 8-10. Monitoring Cell Counts of Apheresis
Components Method 8-11. Counting Residual White Cells in Leukocyte-Reduced Blood and Components—
Manual Method
APPENDICES
Appendix 1. Normal Values in Adults Appendix 2. Selected Normal Values in Children
Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation (Adults)
Appendix 4. Coagulation Factor Values in Platelet Concentrates Appendix 5. Approximate Normal Values
for Red Cell, Plasma, and Blood Volumes
Appendix 6. Blood Group Antigens Assigned to Systems Appendix 7. Examples of Gene, Antigen, and
Phenotype Symbols in
Conventional and International Society of Blood Transfusion Terminology Appendix 8. Examples of
Correct and Incorrect Terminology Appendix 9. Distribution of ABO/Rh Phenotypes by Race or Ethnicity
Appendix 10. Example of a Maximum Surgical Blood Order Schedule Appendix 11. Directory of Organizations
Abbreviations
complementary
AATB American Association of Tissue Banks cDNA
deoxyribonucleic acid
Center for Devices and
ACD acid-citrate-dextrose CDRH
Radiological
ACE angiotensin-converting enzyme Health
ACOG American College of Obstetricians and CFR Code of Federal Regulations
 Gynecologists CFU colony-forming unit
chronic granulomatous
ADP adenosine diphosphate CGD
disease
current good manufacturing
AET 2-aminoethylisothiouronium cGMP
practice
AHF antihemophilic factor cGTP current good tissue practice
AHG antihuman globulin cGy centiGray
AHTR acute hemolytic transfusion reaction Cl confidence interval
chronic inflammatory
AIDS acquired immune deficiency syndrome CIDP
demyelinating
AIHA autoimmune hemolytic anemia polyneuropathy
ALDH aldehyde dehydrogenase CJD Creutzfeldt-Jakob disease
Clinical Laboratory
ALT alanine aminotransferase CLIA
Improvement
AML acute myelogenous leukemia Amendments
Clinical and Laboratory
CLSI
Standards
AMR antibody-mediated rejection Institute
chronic myelogenous
ANH acute normovolemic hemodilution CML
leukemia
Centers for Medicare and
AORN Association of periOperative Registered CMS
Medicaid
Nurses Services
APC antigen-presenting cell CMV cytomegalovirus
aPTT activated partial thromboplastin time CNS central nervous system
citrate-phosphate-dextrose-
ARDP American Rare Donor Program CP2D
dextrose
AS additive solution CPD citrate-phosphate-dextrose
CPDA- citrate-phosphate-dextrose-
ASFA American Society for Apheresis
1 adenine-1
American Society for Histocompatibility and
ASHI CR complement receptor
Immunogenetics
ATP adenosine triphosphate CREG cross-reactive group
cryoprecipitate
BCR B-cell receptor CRYO
(Cryoprecipitated AHF)
C/T crossmatch/transfusion
BLA biologies license application r\/
BPD biological product deviation uv coemcieni 01 Vdiidiiori
BSA bovine serum albumin or body surface DAF decay-accelerating factor
area DAT direct antiglobulin test
deamino-D-arginine
BSC biological safety cabinet DDAVP
vasopressin
BSL-1 Biosafety Level 1 DHQ donor history questionnaire
delayed hemolytic
CAP College of American Pathologists DHTR
transfusion reaction
disseminated intravascular
CAS cold agglutinin syndrome DIC
coagulation
CBER Center for Biologies Evaluation and DMSO dimethylsulfoxide
 Research DNA deoxyribonucleic acid
(US) Department of
CCI corrected count increment DOT
Transportation
2,3-
CD clusters of differentiation 2,3-diphosphoglycerate
DPG
CDC Centers for Disease Control and DRG diagnosis-related group
Prevention
DSTR delayed serologic transfusion reaction
DTT dithiothreitol
EACA epsilon aminocaproic acid
EBAA Eye Bank Association of America
ECMO extracorporeal membrane oxygenation
ECV extracorporeal volume
EDTA ethylenediaminetetraacetic acid
EIA enzyme immunoassay
ELBW extremely low birthweight
ELISA enzyme-linked immunosorbent assay
EMAs emergency management agencies
EPO erythropoietin
FACT Foundation for the Accreditation of Cellular Therapy
FcR Fc gamma receptor
FDA Food and Drug Administration
FFP Fresh Frozen Plasma
FMH fetomaternal hemorrhage
FNAIT fetal/neonatal alloimmune thrombocytopenia
FNHTR febrile nonhemolytic transfusion reaction
FTA-ABS fluorescent treponemal antibody absorption test
G-CSF granulocyte colony-stimulating factor
GalNAc N-acetylgalactosamine
GM-CSF granulocyte-macrophage colonystimulating factor
GMP good manufacturing practice
GPIa glycoprotein la
GPA glycophorin A
GPB glycophorin B
GPC glycophorin C
GPD glycophorin D
GTP good tissue practice
GVHD graft-vs-host disease
Gy Gray
HAV hepatitis A virus
HAZMAT hazardous material
Hb hemoglobin
HBc hepatitis B core antigen
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
Hct hematocrit
HCT/Ps human cells, tissues, and cellular and tissue-based products
HCV hepatitis C virus
HDFN hemolytic disease of the fetus and newborn
HES hydroxyethyl starch
HHS (US) Department of Health and Human Services
HIT heparin-induced thrombocytopenia
HIV human immunodeficiency virus
HNA human neutrophil antigen
HPA human platelet antigen
HPC hematopoietic progenitor cell
HPG(A) HPCs from apheresis (HPC, Apheresis)
HPC(C) HPCs from cord blood (HPC, Cord Blood)
HPG(M) HPCs from marrow (HPC, Marrow)
HSC hematopoietic stem cell
HSCT hematopoietic stem cell transplantation
HTLV-I human T-cell lymphotropic virus, type 1
HTR hemolytic transfusion reaction
HUS hemolytic uremic syndrome
IAT indirect antiglobulin test
IATA International Air Transport Association
ICAM-1 intercellular adhesion molecule-1
ID indentification or individual donation
ig immunoglobulin
IL-1 a interleukin-1 alpha
IL-1P interleukin-1 beta
IL-2 interleukin-2
IM intramuscular
IND investigational new drug
INR international normalized ratio
iPSCs induced pluripotent stem cells
IRL immunohematology reference laboratory
IS immediate spin
ISBT International Society of Blood Transfusion
ISO International Organization for Standardization
ITP immune thrombocytopenia
IU international unit
IV intravenous
IVIG intravenous immune globulin
LDH lactate dehydrogenase
LDL low-density lipoprotein
LISS low-ionic-strength saline
ln2 liquid nitrogen
LR leukocyte-reduced
MAC membrane attack complex
2-ME 2-mercaptoethanol
MF mixed field
MHC major histocompatibility complex
 MNC mononuclear cell
MoAb monoclonal antibody
MPHA mixed passive hemagglutination assay
mRNA messenger ribonucleic acid
MSC mesenchymal stem cell
MSDS material safety data sheet
MSM male who has sex with another male
NAIT neonatal alloimmune thrombocytopenia
NAN neonatal alloimmune neutropenia
NAT nucleic acid testing
NHLBI National Heart, Lung, and Blood Institute
NIH National Institutes of Health
NIPA nonimmunologic protein adsorption
NK natural killer
NMDP National Marrow Donor Program
NRC Nuclear Regulatory Commission
NRF National Response Framework
OSHA Occupational Safety and Health Administration
P probability
PAD preoperative autologous (blood) donation
PBM patient blood management
PBS phosphate-buffered saline
PCH paroxysmal cold hemoglobinuria
PCR polymerase chain reaction
PEG polyethylene glycol
PF24 Plasma Frozen Within 24 Hours After Phlebotomy
PF24 Plasma Frozen Within 24 Hours After Phlebotomy Held at Room Temperature Up to 24
RT24 Hours after Phlebotomy
PPE personal protective equipment
PRA panel-reactive antibody
PRCA pure red cell aplasia
PRP platelet-rich plasma
PRT pathogen reduction technology
PT prothrombin time or proficiency testing
PTP posttransfusion purpura
PTT partial thromboplastin time
PVC polyvinyl chloride
QA quality assessment or quality assurance
QC quality control
QSE Quality System Essential
RBCs Red Blood Cells (blood donor unit)
RFLP restriction fragment length polymorphism
rFVIIa recombinant Factor Vila
Rh Rhesus factor
RHAG Rh-associated glycoprotein
RhIG Rh Immune Globulin
RIBA recombinant immunoblot assay
 RIPA radioimmunoprecipitation assay
RNA ribonucleic acid
RPR rapid plasma reagin (serologic test for syphilis)
RT room temperature or reverse transcriptase
SCF stem cell factor
SD standard deviation or solvent/detergent
SNP single nucleotide polymorphism
SOP standard operating procedure
SPRCA solid-phase red cell adherence
TA transfusion-associated
TACO transfusion-associated circulatory overload
TCR T-cell receptor
TMA transcription-mediated amplification
TNCs total nucleated cells
TNF-a tumor necrosis factor alpha
TPE therapeutic plasma exchange
TPO thrombopoietin
TRALI transfusion-related acute lung injury
TSE transmissible spongiform encephalopathy
TTP thrombotic thrombocytopenic purpura
UCB umbilical cord blood
UDP uridine diphosphate
UNOS United Network for Organ Sharing
use United States Code
vCJD variant Creutzfeldt-Jakob disease
VLBW very low birthweight
vWD von Willebrand disease
vWF von Willebrand factor
WAIHA warm autoimmune hemolytic anemia
WB whole blood or Western blot
WBC white blood cell
WHO World Health Organization
WNV West Nile virus
Chapter 1
Quality Management Systems: Theory and Practice
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ); Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;
and Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
gnA primary goal of transfusion medicine, cellular therapies, and clinical diagnostics is to promote high
standards of quality in all aspects of patient care and related products and services. This commitment to quality
is reflected in standards of practice set forth by the AABB. AABB standards use a quality management system
as the framework for quality. A quality management system includes the organizational structure,
responsibilities, policies, processes, procedures, and resources established by executive management to achieve
and maintain quality. (A glossary of quality terms used in this chapter is included in Appendix 1 -1.)
The establishment of a formal quality assurance program is required under the Centers for Medicare and
Medicaid Services (CMS) Clinical Laboratory Improvement Amendments (CLIA)1 and the Food and Drug
Admin
istration (FDA), especially in the current good manufacturing practice (cGMP) and current good tissue
practice (cGTP) regulations.2'5 The FDA regulations in the Code of Federal Regulations (CFR) Title 21, Part
211.22 require an independent quality control (QC) or quality assurance unit that has responsibility for the
overall quality of the facility’s finished product and authority to control the processes that may affect this
product.3 (See frequently used CFR quality-related citations in Appendix 1-2.) Professional and accrediting
organizations such as the AABB,6,7 College of American Pathologists (CAP),8 The Joint Commission,910
Clinical and Laboratory Standards Institute (CLSI),11 and Foundation for the Accreditation of Cellular Therapy
(FACT),12 have also established requirements and guidelines to address quality issues. The International
Organization for Standardization (ISO) quality manage
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality Director, Esoteric Business Unit,
Laboratory Corporation of America, Burlington, North Carolina; Betsy W. Jett, MT(ASCP),
CQA(ASQ)CQM/OE, Vice President for Quality and Regulatory Affairs, New York Blood Center, New York,
New York; and Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB, Interim Department Head, Analytical and
Diagnostic Sciences, College of Allied Health Sciences, and Associate Professor Emerita, University of
Cincinnati, Cincinnati, Ohio The authors have disclosed no conflicts of interest.
1

2
AABB TECHNICAL MANUAL
ment standards (ISO 9001) are generic to any industry and describe the important minimum elements of a
quality management system.13 The ISO 15189 standards are specific to laboratory medicine.14 In addition, the
Health Care Criteria for Performance Excellence published by the Baldrige Performance Excellence Program15
provides an excellent framework for implementing quality on an organizational level.
The AABB has defined the minimum elements that must be addressed in its quality system essentials
(QSEs).16 The AABB QSEs were developed to be compatible with ISO 9001 standards, the FDA Guideline for
Quality Assurance in Blood Establishments,5 and other FDA quality system approaches.17,18
CONCEPTS IN QUALITY
Quality Control, Quality Assurance, and Quality Management
The purpose of QC is to provide feedback to operational staff about the state of a process that is in progress.
QC tells staff whether to continue (everything is acceptable) or to stop until a problem has been resolved
(something is found to be out of control).
Historically, transfusion services and donor centers have used many QC measures as standard practices in
their operations. Examples include reagent QC; product QC; clerical checks; visual inspections; and
measurements, such as temperature readings on refrigerators and volume or cell counts on finished blood
components.
Quality assurance activities are not tied to the actual performance of a process. Rather, they include
activities, such as the development of documents like standard operating procedures (SOPs), to ensure
consistent and correct performance of processes, training of personnel, and qualification of materials and
equipment. Quality assurance activities also include retrospective reviews and analyses of operational
performance data to determine whether the overall process is in a state of control and to detect shifts or trends
that require attention. Quality assurance provides infor
mation to process managers regarding levels of performance that can be used in setting priorities for process
improvement. Examples of quality assurance activities in transfusion medicine and cellular therapies include
record reviews, monitoring of quality indicators, and internal assessments.
Quality management considers interrelated processes in the context of the organization and its relations with
customers and suppliers. It addresses the leadership role of executive management in creating a commitment to
quality throughout the organization, the understanding of suppliers and customers as partners in quality, the
management of human and other resources, and quality planning.
The quality systems approach described in this chapter encompasses all of these activities. It ensures
application of quality principles throughout the organization and reflects the changing focus of quality efforts
from detection to prevention.
Juran’s Quality Trilogy
Juran’s Quality Trilogy is one example of a quality management approach. This model centers around three
fundamental processes for managing quality in any organization: planning, control, and improvement.19(p25)
 The planning process for a new product or service includes activities to identify requirements, develop
product and process specifications that meet those requirements, and design the process. During the planning
phase, the facility must perform the following steps:
1. Establish quality goals for the project.
2. Identify the customers.
3. Determine customer needs and expectations.
4. Develop product and service specifications to meet customer, operational, regulatory, and accreditation
requirements.
5. Develop operational processes for production and delivery, including written procedures and resources
requirements.
6. Develop process controls and validate the process in the operational setting.
3
The results of the planning process are referred to as “design output.”13
Once the plan is implemented, the control process provides a feedback loop for operations that includes the
following:
1. Evaluation of performance.
2. Comparison of performance to goals.
3. Action to correct any discrepancy between the two.
The control process addresses inputs, production, and delivery of products and services to meet
specifications. Process controls should allow staff to recognize when things are going wrong and to either make
appropriate adjustments to ensure a product’s quality or stop the process.
An important goal in quality management is to establish a set of controls that ensure process and product
quality but are not excessive. Controls that do not add value should be eliminated to conserve limited resources
and allow staff to focus attention on those controls that are critical to the operation.
Statistical tools, such as process capability measurement and control charts, allow a facility to evaluate
process performance during the planning stage and in operations. These tools help determine if a process is
stable (ie, in statistical control) and if it is capable of meeting product and service specifications.19(p2219)
Quality improvement is intended to enable an organization to attain higher levels of performance by
creating new or better features that add value or by removing deficiencies in the process, product, or service.
Opportunities to improve may be related to deficiencies in the initial planning process; unforeseen factors
discovered on implementation; shifts in customer needs; or changes in starting materials, environmental factors,
or other variables that affect the process. Improvements must be based on data-driven analysis; an ongoing
measurement and assessment program is fundamental to that process.
Process Approach
In its most generic form, a process includes all of the resources and activities that transform an input into an
output. An understanding of how to manage and control processes in transfusion medicine, cellular therapies,
and clinical diagnostic activities is based on this simple equation:
INPUT ^ PROCESS OUTPUT
For example, a key process for donor centers is donor selection. The “input” includes the individual who
presents for donation and all of the resources required for that donor’s health screening. Through a series of
activities (a process), including the verification of the donor’s identity, a deferral status review, a mini-physical
exam, and a health history questionnaire, an individual is deemed an “eligible donor.” The “output” is either an
eligible donor who can continue to the next process (blood collection) or an ineligible donor who is deferred.
When the selection process results in a deferred donor, the resources (inputs) associated with that process do not
continue through the process but contribute to the cost of quality. One way that donor centers attempt to
minimize this cost is to educate potential donors before screening so that those who are not eligible do not enter
the selection process.
Strategies for managing a process should address all of its components, including its interrelated activities,
inputs, outputs, and resources. Supplier qualification, formal agreements, supply verification, and inventory
control are strategies for ensuring that the inputs to a process meet specifications. Personnel training and
competence assessment, equipment maintenance and control, management of documents and records, and
implementation of appropriate in-process controls provide assurance that the process will operate as intended.
End-product testing and inspection, customer feedback, and outcome measurement provide data to evaluate
product quality and improve the process. These output measurements and quality
 4
AABB TECHNICAL MANUAL
indicators are used to evaluate the effectiveness of the process and process controls.
To manage a system of processes effectively, the facility must understand how its processes interact and
what cause-and-effect relationships exist between them. In the donor selection scenario, the consequences of
accepting a donor who is not eligible reach into almost every other process in the facility. For example, if a
donor with a history of high-risk behavior is not identified as such during the selection process, the donated
unit(s) may return positive test results for one of the viral marker assays, triggering follow-up testing, look-back
investigations, and donor deferral and notification procedures. Components must be quarantined and their
discard documented. Personnel involved in collecting and processing the unit(s) are at risk of exposure to
infectious agents. Part of quality planning is to identify these relationships so that quick and appropriate
corrective action can be taken when process controls fail.
It is important to remember that operational processes include not only product manufacture or service
creation, but also the delivery of a product or service. Delivery generally involves interaction with the customer.
The quality of that transaction is critical to customer satisfaction and should not be overlooked in the design and
ongoing assessment of the quality management system.
Service vs Production
Quality management principles apply equally to a broad spectrum of activities, from those related to
processing and production to those involving interactions between individuals in delivering a service. However,
different strategies may be appropriate when there are differing expectations related to customer satisfaction.
Although the emphasis in a production process is on minimizing variation to create a product that consistently
meets specifications, service processes require a certain degree of flexibility to address customer needs and
circumstances at the time of the transaction. In production, personnel need to know how to maintain uniformity
in day-to-day operations.
In service, personnel need to be able to adapt a service in a way that meets customer expectations but does
not compromise quality. To do this, personnel must have sufficient knowledge and understanding of interrelated
processes to use independent judgment appropriately, or they must have ready access to higher-level decision
makers.
When one designs quality management systems for production processes, it is useful to think of the process
as the driver, with people providing the oversight and support needed to keep it running smoothly and
effectively. In service, people are the focus; the underlying process provides a foundation that enables staff to
deliver safe and effective services that meet customers’ needs in almost any situation.
Quality Management as an Evolving Science
The principles and tools used in quality management today will change as research provides new knowledge
of organizational behavior, technology provides new solutions, and the transfusion medicine and cellular
therapies fields present new challenges. Periodic assessments of the quality management system will help
identify practices that are no longer effective or that could be improved through the use of new technology or
new tools.
PRACTICAL APPLICATION OF QUALITY MANAGEMENT PRINCIPLES
The remainder of this chapter addresses the elements of a quality management system and practical
application of quality management principles to the transfusion medicine, cellular therapies, and clinical
diagnostics environments. These basic elements include the following:
■ Organization and leadership.
■ Customer focus.
■ Facilities, work environment, and safety.
■ Human resources.
■ Suppliers and materials management.
5
■ Equipment management.
■ Process management.
■ Documents and records.
■ Information management.
■ Management of nonconforming events.
■ Monitoring and assessment.
 ■ Process improvement.
Organization and Leadership
The facility should be organized in a manner that promotes effective implementation and management of its
operational and quality management system. The structure of the organization must be documented, and the
roles and responsibilities for the provision of tests, products, and services must be clearly defined. These
provisions should include a description of the relationships and avenues of communication between
organizational units and those responsible for key quality functions. Each facility may define its structure in any
format that suits its operations. Organizational trees or charts that show the structure and relationships are
helpful.
The facility should define in writing the authority and responsibilities of executive management to establish
and maintain the quality management system. These responsibilities include the following:
■ Establishing a quality policy and associated quality goals and quality objectives.
■ Providing adequate facilities as well as human, equipment, and material resources to carry out the
operations of the facility and the quality management system.
■ Ensuring appropriate design and effective implementation of new or modified processes and procedures.
■ Participating in the review and approval of quality and technical policies, processes, and procedures.
■ Enforcing adherence to operational and quality policies, processes, and procedures.
■ Overseeing operations and regulatory and accreditation compliance.
■ Periodically reviewing and assessing quality management system effectiveness.
■ Identifying designees and defining their responsibilities when assisting executive management in carrying
out these duties.
Executive management support for the quality management system goals, objectives, and policies is critical
to the program’s success. Executive management needs to clearly communicate its commitment to quality goals
and create an organizational culture that embraces quality principles.
The individual designated to oversee the facility’s quality functions should report directly to executive
management. In addition to having the responsibility to coordinate, monitor, and facilitate quality system
activities, this person should have the authority to recommend and initiate corrective action when appropriate.5
The designated individual need not perform all of the quality functions personally. Ideally, this person should be
independent of the facility’s operational functions. In small facilities, this independence may not always be
possible and carrying out this function may require some creativity. Depending on the organization’s size and
scope, the designated oversight person may work in the transfusion service, have laboratory-wide
responsibilities, supervise other workers (eg, a quality unit), or be part of an organization-wide quality unit (eg,
hospital quality or risk management). Individuals with dual quality and operational responsibilities should not
provide quality oversight for operational work that they have performed.
Quality oversight functions may include the following5:
■ Review and approval of SOPs and training plans.
■ Review and approval of validation plans and results.
■ Review and approval of document control and record-keeping systems.
■ Audit of operational functions.
■ Development of criteria for evaluating systems.
■ Review and approval of suppliers.
■ Review and approval of product and service specifications (eg, the requirements to be
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AABB TECHNICAL MANUAL
met in the manufacture, distribution, or administration of blood components, cellular therapy products,
tissues, and derivatives).
■ Review of reports of adverse reactions, deviations in the manufacturing process, nonconforming products
and services, and customer complaints.
■ Participation in decisions to determine whether blood components, cellular therapy products, tissues,
derivatives, and services are suitable for use, distribution, or recall.
■ Review and approval of corrective action plans.
■ Surveillance of problems (eg, event or incident reports, Form FDA 483 observations, or customer
complaints) and the effectiveness of corrective actions implemented to solve those problems.
 ■ Use of data resources to identify trends and potential problems before a situation worsens and patients
and/or products are affected.
■ Preparation of periodic (as specified by the organization) reports of quality issues, trends, findings, and
corrective and preventive actions.
Quality oversight functions may be shared among existing staff, departments, and facilities or, in some
instances, may be performed by an outside firm under a contract. The goal is to provide an independent
evaluation of the facility’s quality activities to the extent possible. Policies, processes, and procedures should
exist to define the roles and responsibilities of all individuals in the development and maintenance of these
quality goals. Quality management system policies and processes should be applicable across the entire facility.
A blood bank, tissue bank, transfusion service, or cellular therapy product service need not develop its own
quality policies if it is part of a larger entity whose quality management system addresses all of the minimum
requirements. The quality management system should address all matters related to compliance with federal,
state, and local regulations and accreditation standards that are applicable to the organization.
Customer Focus
A primary focus for any organization interested in quality is serving the needs of its customers. Customers
have a variety of needs and expectations. The most appropriate way to ensure that these needs and expectations
are met is for the facility and its customers to define them in an agreement, a contract, or another document.
Additional information on agreements can be found in the “Suppliers and Materials Management” section.
When planning for new or changed products or services, the facility should take the customer’s needs and
expectations into account. If these changes are determined to be critical to the quality or effectiveness of the
products and services provided by the facility, they should be incorporated into the product or service
specifications as customer requirements. The facility must have a process to manage needs and expectations that
are not met. For example, for a facility that has agreed to deliver leukocyte-reduced components daily to one of
its customers, processing components in a manner that ensures adequate leukocyte removal is critical to this
product’s quality. Such an expectation should be incorporated into the product specifications. Daily delivery of
products is a customer need and expectation, but it is not critical to the quality of the manufactured product. The
facility should have a process to manage this agreedon expectation and ensure that the product delivery
mechanism meets this customer need. If this need cannot be met, the facility should have a process to address
this failure.
Once agreements have been made between the facility and its customers, there should be a means to obtain
feedback from the customer to ensure that the facility is meeting the customer’s expectations. Mechanisms for
obtaining such feedback proactively include satisfaction surveys and periodic reviews of agreements. Reactive
feedback is obtained through customer complaints. A review of event data may also indicate failures to meet
customer needs and expectations. Data obtained through these mechanisms should be evaluated, and
appropriate follow-up actions
7
must be taken. One such action could be to change the agreement. Inadequately addressing customer
concerns or failing to meet expectations may result in loss of the customer.
Facilities, Work Environment, and Safety
The facility should provide a safe workplace with adequate environmental controls and emergency
procedures to ensure the safety of patients, donors, staff, and visitors. Space allocation, building utilities, and
the communication infrastructure should adequately support the facility’s activities. The facility should be kept
clean and well maintained so that the products and services provided are not compromised. Procedures should
be in place to address the following:
■ General safety.
■ Disaster preparedness, response, and recovery.
■ Biological safety (eg, protection from bloodborne pathogens).
■ Chemical safety.
■ Fire safety.
■ Radiation safety, if applicable.
■ Discard of biologic and other hazardous substances.
cGMP regulations require quality planning and control of the physical work environment, including the
following:
■ Adequate space and ventilation.
 ■ Sanitation and trash disposal.
■ Equipment for controlling air quality and pressure, humidity, and temperature.
■ Water systems.
■ Toilet and hand-washing facilities.
An evaluation of the infrastructure and its limitations before implementation of procedures or installation of
equipment will help ensure maximum efficiency and safety. A more thorough discussion of facilities and safety
can be found in Chapter 2.
Human Resources
This element of the quality management system is aimed at management of personnel, including selection,
orientation, training, competence assessment, and staffing.
Selection
Each facility should have a process to provide adequate numbers of qualified personnel to perform, verify,
and manage all activities in the facility. Qualification requirements for personnel are determined on the basis of
job responsibilities. The selection process should consider the applicant’s qualifications for a particular position
as determined by his or her education, training, experience, certifications, and licensure. For laboratory testing
staff, the standards for personnel qualifications should be compatible with the regulatory requirements
established under CLIA.1 Job descriptions are required for all personnel involved in processes and procedures
that affect the quality of tests, products, and services. Effective job descriptions clearly define the qualifications
and responsibilities of the positions as well as their reporting relationships.
Orientation, Training, and Competence Assessment
Once hired, employees should be oriented to their position and the organization’s policies and procedures
and be trained in their new duties. The orientation program should cover facility-specific requirements and
policies that address issues such as safety, quality, information systems, security, and confidentiality. The job-
related portion of the orientation program should cover operational issues specific to the work area. Training
should be provided for each procedure for which employees are responsible. The ultimate result of the
orientation and training program is to deem new employees competent to independently perform the duties and
responsibilities defined in their job descriptions. Time frames should be established to accomplish this goal.
Before the introduction of a new test or service, existing personnel should be trained
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to perform their newly assigned duties and must be deemed competent in these duties. During orientation
and training, employees should be given the opportunity to ask questions and seek additional help or
clarification. All aspects of the training should be documented, and the facility trainer or designated facility
management representative and employees should mutually agree on how employees’ competence will be
determined.
FDA cGMP training is required for staff involved in the manufacture of blood and blood products, including
staff involved in collection, testing, processing, preservation, storage, distribution, and transport.3 Likewise,
cGTP training is required for personnel involved in similar activities for human cells, tissues, and cellular and
tissue-based products (HCT/Ps).4 Such training should provide staff with an understanding of the regulatory
basis for the facility’s policies and procedures as well as the specific application of the cGMP and cGTP
requirements as described in the facility’s own written operating procedures. This training should be provided
periodically to ensure that personnel remain familiar with regulatory requirements.
To ensure that skills are maintained, the facility should have regularly scheduled competence evaluations of
all staff members whose activities affect the quality of laboratory testing, manufacture of products, or provision
of products or services.1,5'10 Competence assessment of management personnel should also be considered.
Depending on the nature of the job duties, competence assessments may include written evaluations; direct
observations of activities; reviews of work records or reports, information system records, and QC records;
testing of unknown samples; and evaluations of employees’ problem-solving skills.5 For all testing personnel,
CMS requires that each of the following methods be used, when applicable, for each test system annually1:
■ Direct observations of
- Routine patient test performance (including patient preparation, if applica
ble), specimen handling, processing, and testing.
- Performance of instrument maintenance and function checks.
■ Monitoring of the recording and reporting of test results.
 ■ Review of intermediate test results or worksheets, QC records, proficiency testing results, and preventive
maintenance records.
■ Assessment of
- Test performance by testing previously analyzed specimens, internal blind testing samples, or external
proficiency testing samples.
- Problem-solving skills.
A formal competency plan that includes a schedule of assessments, a defined minimum for acceptable
performance, and remedial measures is one way to ensure appropriate and consistent competence assessments.
Assessments need not be targeted to each individual test or procedure performed by the employee; instead, they
can be grouped together to assess similar techniques or methods. However, any test with unique aspects,
problems, or procedures should be assessed separately to ensure that staff maintain their competency to report
test results promptly, accurately, and proficiently.1 Written tests can be used effectively to evaluate problem-
solving skills and cover many topics by asking one or more questions for each area to be assessed. CMS
requires that employees who perform testing be assessed semiannually during the first year that patient
specimens are tested and annually thereafter.1 Initial training verification activities may serve as the first of
these competence assessments.
The quality oversight personnel should assist in the development, review, and approval of training
programs, including the criteria for retraining.5 Quality oversight personnel also monitor the effectiveness of
training programs and competence evaluations, and they recommend changes as needed. In addition, The Joint
Commission requires analyses of aggregate competence assessment data to identify staff learning needs.9
9
Staffing
Management should have a staffing plan that describes the number and qualifications of personnel needed to
perform the facility’s functions safely and effectively. There should be an adequate number of staff to perform
the operational activities and support quality management system activities. Organizations should assess
staffing effectiveness by evaluating human resource indicators (eg, overtime, staff injuries, and staff
satisfaction) in conjunction with operational performance indicators (eg, adverse events and patient complaints).
The results of this evaluation should feed into the facility’s human resource planning process along with
projections based on new or changing operational needs.
Suppliers and Materials Management
Materials, supplies, and services used as inputs to a process are considered critical if they affect the quality
of products and services being produced. Examples of critical supplies are blood components, blood bags, test
kits, and reagents. Examples of critical services are infectious disease testing, blood component irradiation,
transportation, equipment calibration, and preventive maintenance. The suppliers of these materials and services
may be internal (eg, other departments within the same organization) or external (outside vendors). Supplies
and services used in the collection, testing, processing, preservation, storage, distribution, transport, and
administration of blood components, cellular therapy products, tissues, and derivatives that have the potential to
affect quality should be qualified before use and obtained from suppliers who can meet the facility’s
requirements. Three important elements of supplier and materials management are 1) supplier qualification; 2)
agreements; and 3) receipt, inspection, and testing of incoming supplies.
Supplier Qualification
Critical supplies and services must be qualified on the basis of defined requirements. Similarly, suppliers
should be qualified to ensure
that they are reliable sources of materials. The facility should clearly define requirements or expectations for
its suppliers and share this information with staff and suppliers. Suppliers’ ability to consistently meet
specifications for a supply or service should be evaluated along with performance relative to availability,
delivery, and support. The following are examples of factors that could be considered to qualify suppliers:
■ Licensure, certification, or accreditation.
■ Supply or product requirements.
■ Supplier-relevant quality documents.
■ Results of audits or inspections.
■ Quality summary reports.
■ Customer complaints.
■ Experience with that supplier.
 ■ Cost of materials or services.
■ Delivery arrangements.
■ Financial security, market position, and customer satisfaction.
■ Support after sales.
A list of approved suppliers should be maintained that includes both primary suppliers and suitable
alternatives for contingency planning. Critical supplies and services should be purchased only from suppliers
that have been qualified. Once suppliers are qualified, periodic evaluations of supplier performance help ensure
suppliers’ continued ability to meet requirements. Tracking suppliers’ ability to meet expectations gives the
facility valuable information about the stability of each supplier’s processes and commitment to quality.
Documented failures of supplies or suppliers to meet defined requirements should result in immediate action by
the facility, including notification of the supplier, quality oversight personnel, and management with contracting
authority, if applicable. Supplies may need to be replaced or quarantined until all quality issues are resolved.
Agreements
Contracts and other agreements define expectations and reflect the concurrence of the parties involved.
Periodic reviews of agreements
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ensure that the expectations of all parties continue to be met. Changes should be mutually agreed upon and
incorporated as needed.
Transfusion and cellular therapy services should maintain written contracts or agreements with outside
suppliers of critical materials and services, such as blood components, cellular therapy products, irradiation,
compatibility testing, or infectious disease marker testing. An outside supplier may be another department
within the same facility that is managed independently, or it may be another facility (eg, contract manufacturer).
The contracting facility assumes responsibility for the manufacture of the product; ensuring the safety, purity,
and potency of the product; and ensuring that the contract manufacturer complies with all applicable product
standards and regulations. Both the contracting facility and the contractor are legally responsible for the work
performed by the contractor.
It is important for the transfusion or cellular therapy service to participate in the evaluation and selection of
suppliers. The service should review contracts and agreements to ensure that all important aspects for their
critical materials and services are addressed. Examples of issues that could be addressed in an agreement or
contract include the responsibility for a product or blood sample during shipment; the supplier’s responsibility
to promptly notify the facility when changes have been made that could affect the safety of blood components,
cellular therapy products, tissues, derivatives, or services for patients; and the supplier’s responsibility to notify
the facility when information is discovered indicating that a product may not be considered safe, such as during
look-back procedures.
Receipt, Inspection, and Testing of Incoming Supplies
Before acceptance and use, critical materials such as reagents, blood components, cellular therapy products,
tissues, and derivatives should be inspected and tested (if necessary) to ensure that they meet specifications for
their intended use. It is essential that supplies used in the collection, processing, preserva
tion, testing, storage, distribution, transport, and administration of blood, components, tissues, and cellular
therapy products also meet FDA requirements.
The facility must define acceptance criteria for critical supplies (see 21 CFR 210.3)3 and must develop
procedures to control and prevent the inadvertent use of materials that do not meet specifications. Corrective
action may include returning the material to the vendor or destroying it. Receipt and inspection records enable
the facility to trace materials that have been used in a particular process and provide information for ongoing
supplier qualification.
Equipment Management
Equipment that must operate within defined specifications to ensure the quality of blood components,
cellular therapy products, tissues, derivatives, and services is referred to as “critical equipment” in the quality
management system. Critical equipment may include instruments, measuring devices, and computer systems
(hardware and software).
Activities designed to ensure that equipment performs as intended include qualification, calibration,
maintenance, and monitoring. Calibration, functional and safety checks, and preventive maintenance should be
scheduled and performed according to the manufacturer’s recommendations and regulatory requirements of the
FDA2-4 and CMS.1 Written procedures for equipment use and control should comply with the manufacturer’s
recommendations unless an alternative method has been validated by the facility and, in some instances,
approved by the appropriate regulatory and accrediting agencies.
When one selects new equipment, it is important to consider not only the performance of the equipment as it
will be used in the facility but also any issues regarding ongoing service and support by the supplier. There
should be a written plan for installation, operational, and performance qualifications.6 The plan should provide
for 1) installation according to the manufacturer’s specifications, 2) verification of the equipment’s functionality
11
before use by ensuring that the criteria established by the manufacturer for its intended use are met, and 3)
assurance that the equipment performs as expected in the facility’s processes. After the equipment is installed,
any problems and follow-up actions taken should be documented. Recalibration and requalification may be
necessary if repairs are made that affect the equipment’s critical operating functions. Recalibration and
requalification should also be considered when existing equipment is relocated.
The facility must develop a mechanism to uniquely identify and track all critical equipment, including
equipment software versions, if applicable. The unique identifier assigned by the manufacturer may be used, or
a unique identification code may be applied by the transfusion or cellular therapy service or assigned through a
laboratory-wide or organization-wide identification system. Maintaining a list of all critical equipment helps in
the control function of scheduling and performing functional and safety checks, calibrations, preventive
maintenance, and repair. The equipment list can be used to ensure that all appropriate actions have been
performed and recorded. Evaluating and trending equipment calibration, maintenance, and repair data help the
facility assess equipment functionality, manage defective equipment, and identify equipment needing
replacement. When equipment is found to be operating outside acceptable parameters, the potential effects on
the quality of products or test results must be evaluated and documented.
Process Management
Written and approved policies, processes, and procedures must exist for all critical functions performed in
the facility, and these functions must be carried out under controlled conditions. Each facility should have a
systematic approach for identifying, planning, and implementing new (and making changes to existing)
policies, processes, and procedures that affect the quality of the facility’s tests, products, or services. Such
activities should include a review of at least the following:
■ Customer needs and expectations.
■ Accreditation and regulatory requirements.
■ Specifications to be met.
■ Risk assessment.
■ Performance measures.
■ Nonconformance analyses.
■ Current knowledge (eg, of other successful practices).
■ Resource needs (eg, financial, facility, human, materials, and equipment).
■ Interrelationships of the new or changed process(es) with other processes.
■ Documents needed for the new or changed process(es).
The documents developed should be reviewed by management personnel with direct authority over the
process and by quality oversight personnel before implementation. Changes in policies, processes, and
procedures should be documented, validated, reviewed, and approved. Additional information on policies,
processes, and procedures can be found in the “Documents and Records” section later in this chapter.
Once a process has been implemented, the facility should have a mechanism to ensure that procedures are
performed as defined and that critical equipment, reagents, and supplies are used in conformance with
manufacturers’ written instructions and facility requirements. Table 1-1 lists elements that constitute sound
process control (among other elements of a quality management system). A facility using critical equipment,
reagents, or supplies in a manner that is different from the manufacturer’s directions should validate such use. If
the activity is covered under regulations for blood and blood components or HCT/Ps, the facility may be
required to request FDA approval to operate at variance to requirements (see 21 CFR 640.1202 or 21 CFR
1271.1554). If a facility believes that changes to the manufacturer’s directions would be appropriate, it should
encourage the manufacturer to make such changes in the labeling (ie, the package insert or user manual).
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 TABLE 1-1. Components of a Quality Management System
Quality System Component
procedures
Organization and leadership
Customer focus
Facilities, work environment, anc
safety
relationships
Human resources
use, and safety
Suppliers and materials
management
products
Quality Functions and Responsibilities
■ Organization structure and function
■ Leadership roles and responsibilities, authority, and relationships
■ Establishment of a quality management system
■ Customer needs
■ Planned products and services
■ Documented, followed, and improved policies, processes, and
■ Quality representative
■ Management reviews
■ Provision of adequate resources
■ Adequate design and effective implementation
■ Conformance with requirements
■ Effective communication
■ Effective process improvement
■ Customer requirements
■ Agreements
■ Customer feedback
■ Minimal health and safety risks
■ Design and space allocations
■ Clean work environment
■ Controlled environment
■ Communication and information management systems
■ Storage facilities
■ Health and safety programs
■ Hazard discards
■ Emergency preparedness
■ Adequate and qualified staff
■ Job descriptions and qualifications
■ Defined roles and responsibilities for all staff and their reporting
■ Staff selection
■ New hire orientation
■ Training on the quality system, job-related activities, computer
■ Staff competence
■ Continuing education
■ Staff identifying information
■ End-of-employment activities
■ Supplier qualification
■ Qualifying materials
■ Agreement reviews
■ Inventory management
■ Adequate storage conditions
■ Receipt, inspection, and testing of incoming materials and
■ Acceptance and rejection of materials and products
■ Tracing critical supplies and services
 13
TABLE 1-1. Components of a Quality Management System (Continued)
Quality Quality
System Functions and
Component Responsibilities
■
Selection and acquisition
■
Unique identification code
■
Equipment Verification of performance
■
management Installation, operational, and performance qualification
■
Calibration
■
Preventive maintenance and repairs Retirement
■
■
■
■
Process development Change control Process validation Process
■
implementation
Process ■
Adherence to policies, processes, and procedures Quality control program
management ■
Inspection of products and services Concurrent creation of records
■
Requirements for critical activities Traceability
■
■
■
■
■
■
Standardized formats Document creation Unique identification code
Documents ■
Review and approval process Document use and maintenance Change
and records ■
control Record archiving and storage Record retention and destruction
■
■
■
■
■ Confidentiality
Information
■ Prevention of unauthorized access Data integrity Data backup Alternative
management
■ system
■
■ Detection of deviations and nonconformances
Management
■ Complaint file
of
■ Adverse event reporting
nonconforming
■ Investigations
events
■ Immediate actions
■ Monitoring and assessment of specified requirements
■ Quality indicators
Monitoring
■ Internal and external assessments
and assessment
■ Laboratory proficiency testing
■ Data analyses
Process ■ Identifying opportunities for improvement
improvement ■ Systems approach to continual improvement
■ Root cause evaluation
■ Corrective action plans
■ Preventive action plans
■ Monitoring for effectiveness

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Process Validation
Validation is used to demonstrate that a process is capable of consistently and reliably achieving planned
results.13 It is critical to validate processes in situations where it is not feasible to measure or inspect each
finished product or service to fully verify conformance with specifications. However, even when effective end-
product testing can be achieved, it is advisable to validate important processes to generate information that can
be used to optimize performance. Prospective validation is used for new or revised processes. Retrospective
validation may be used for processes that are already in operation but were not adequately validated before
implementation. Concurrent validation is used when required data cannot be obtained without performance of a
“live” process. If concurrent validation is used, data are reviewed at predefined periodic intervals before full
implementation receives final approval. Modifications to a validated process may warrant revalidation,
depending on the nature and extent of the change. It is up to the facility to determine the need for revalidation
on the basis of its understanding of how the proposed changes may affect the process.
Test Method Validation
When the laboratory wishes to implement a nonwaived test using an FDA-approved or -cleared test system,
CLIA requires that the performance specifications established by the manufacturer be verified by the laboratory
before it reports patient results.1 At a minimum, the laboratory must demonstrate that it can obtain performance
specifications comparable to those of the manufacturer for accuracy, precision, reportable range, and reference
intervals (normal values).
If the laboratory develops its own method, introduces a test system not subject to FDA approval or
clearance, or makes modifications to an FDA-approved or -cleared test system, or if the manufacturer does not
provide performance specifications, then the laboratory must establish the test system performance
specifications before reporting patient results.1 At a minimum, the following must be established for the test
system:
■ Accuracy.
■ Precision.
■ Reportable range of test results for the test system.
■ Reference intervals (normal values).
■ Analytical sensitivity.
■ Analytical specificity, including interfering substances.
■ Any other performance characteristic required for test performance (eg, specimen or reagent stability).
Based on performance specifications, the laboratory must also establish calibration and control procedures
and document all activities for test method validation. (See 42 CFR 493.1253.1)
Validation Plan
Validation should be planned if it is to be effective. Development of a validation plan is best accomplished
after one obtains an adequate understanding of the system or framework within which the process will occur.
The plan should include conducting the process as designed. Additionally, a significant amount of effort should
be targeted at attempts to “break” the process to identify weaknesses and limitations. Many facilities develop a
template for the written validation plan to ensure that all aspects are adequately addressed. Although no single
format for a validation plan is required, most plans include the following common elements:
■ System description.
■ Purpose or objectives.
■ Risk assessment.
■ Responsibilities.
■ Validation procedures.
■ Acceptance criteria.
■ Approval signatures.
■ Supporting documentation.
15
The validation plan should be reviewed and approved by quality oversight personnel before the validation
activities are carried out.
Staff responsible for carrying out the validation activities should be trained in the process before the plan is
executed. The results and conclusions of these activities may be appended to the approved validation plan or
may be recorded in a separate document. This documentation typically contains the following elements:
■ Expected and observed results.
■ Interpretation of results as acceptable or unacceptable.
■ Corrective action for and resolution of unexpected results.
■ Explanation of and rationale for any deviations from the validation plan.
■ Conclusions and limitations.
■ Approval signatures.
■ Supporting documentation.
■ Implementation timeline.
When a validation process does not produce the expected outcome, its data and corrective actions must be
documented as well. The responsible quality oversight personnel should provide final review and approval of
the validation plan, results, and corrective actions and determine whether new or modified processes and
equipment may be implemented as planned or implemented with specified limitations.
Equipment Validation
Validation of new equipment used in a process should include installation, operational, and performance
qualifications, as follows20:
■ Installation qualification demonstrates that the instrument is properly installed in environmental
conditions that meet the manufacturer’s specifications.
 ■ Operational qualification demonstrates that the installed equipment operates as intended. It focuses on the
equipment’s capability to operate within the established lim
its and specifications supplied by the manufacturer.
■ Performance qualification demonstrates that the equipment performs as expected for its intended use in
the processes established by the facility and that the output meets the facility’s specifications. It evaluates the
adequacy of equipment for use in a specific process that uses the facility’s own personnel, procedures, and
supplies in a normal working environment.
Computer System Validation
The FDA considers computerized systems to include “hardware, software, peripheral devices, networks,
personnel, and documentation.”21 End-user validations of computer systems and the interfaces between
systems should be conducted in the environment in which they will be used. Testing by the computer software
vendor or supplier is not a substitute for computer validation at the facility. End-user acceptance testing may
repeat some of the validation performed by the developer, such as load or stress testing and verification of
security, safety, and control features, to evaluate performance under actual operating conditions. In addition, the
end user should evaluate the ability of personnel to use the computer system as intended within the context of
actual work processes. The hardware and software interface should be designed so that staff can navigate
successfully and respond appropriately to messages, warnings, and other functions. If changes to the computer
system result in changes to the way a process is performed, process revalidation should also be performed. As
with process validation, quality oversight personnel should review and approve validation plans, results, and
corrective actions and should determine whether implementation may proceed with or without limitations.
For additional information, facilities should refer to FDA guidance on computer system validation in the
user’s facility.21 Those who develop their own software should consult Title 21 CFR Part 880 and FDA
guidance
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regarding general software validation principles.22
Quality Control
QC testing is performed to ensure the proper functioning of materials, equipment, and methods during
operations. QC performance expectations and acceptable ranges should be defined and be made readily
available to staff so they will recognize, and respond appropriately to, unacceptable results and trends. The
frequency for QC testing is determined by the facility in accordance with the applicable CMS, FDA, AABB,
state, and manufacturer requirements. QC results should be documented concurrently with performance.2
Records of QC testing should include the following:
■ Identification of personnel performing the test.
■ Identification of reagents (including lot numbers and expiration dates).
■ Identification of equipment.
■ Testing date and, when applicable, time.
■ Results.
■ Interpretation (eg, meets or fails to meet established criteria).
■ Reviews.
Unacceptable QC results must be investigated and corrective action must be implemented, if indicated,
before the QC procedure is repeated or the operational process is continued. If products or services have been
provided since the last acceptable QC results were obtained, it may be necessary to evaluate the conformance of
these products or services. Examples of QC performance intervals for equipment and reagents are included in
Appendix 1-3.
Documents and Records
Documentation provides a framework for understanding and communicating about processes throughout the
organization. Documents provide a description of or instructions regarding what is supposed to happen.
Documents describe how processes are intended to work, how they interact, where
they must be controlled, what their requirements are, and how to implement them. Records provide
evidence of what did happen (ie, that a process was performed as intended), and provide information needed to
assess product and service quality. Together, documents and records are used by quality oversight personnel to
evaluate the effectiveness of a facility’s policies, processes, and procedures. ISO 9001 provides an example of
quality system documentation that includes the following items13:
 ■ The quality policy and objectives.
■ A description of the interactions between processes.
■ Documented procedures for the control of documents, records, and nonconforming products and for
corrective action, preventive action, and internal quality audits.
■ Records related to the quality management system, operational performance, and product or service
conformance.
■ All other “documents needed by the organization to ensure the effective planning, operation, and control
of its processes.”
Written policies, process descriptions, procedures, work instructions, job aids, labels, forms, and records are
all part of the facility’s document management system. They may be paper based or electronic. A document
management system provides assurance that documents are comprehensive, current, and available and that
records are accurate and complete. A well-structured document management system links policies, process
descriptions, procedures, forms, and records together in an organized and workable system.
Documents
Documents should be developed in a format that conveys information clearly and provides staff with the
necessary instructions and templates for recording data. The CLSI offers guidance regarding general levels of
documentation11 as well as detailed instructions on how to write procedures.23 General types of documentation
are described below.
17
POLICIES. Policies communicate the organization’s highest-level goals, objectives, and intent. The rest of
the organization’s documentation interprets, and provides instructions regarding implementation of, these
policies.
PROCESSES. Process documents describe a sequence of actions and identify responsibilities, decision
points, requirements, and acceptance criteria. Process diagrams or flowcharts are often used for this level of
documentation. It is helpful to show process control points on a diagram as well as the flow of information and
handoffs between departments or work groups.
PROCEDURES, WORK INSTRUCTIONS, AND JOB AIDS. These documents provide step-bystep
directions on performing job tasks and procedures. Procedures and work instructions should include enough
detail to perform a task correctly but not so much as to be difficult to read. The use of standardized formats
helps staff know where to find specific elements and facilitates implementation and control. Job aids are
excerpted from an approved document and condense information into a shorter, more readily viewable format.
External documents (eg, from a manufacturer’s manual or package insert) may also be incorporated into the
facility’s procedures manual by reference. Relevant procedures should be available to the staff in each area in
which the corresponding job tasks are performed.2,5,8
FORMS. Forms provide templates for capturing data on paper or electronically. These documents specify
the data requirements called for in SOPs and processes. Forms should be carefully designed to be easy to use,
minimize the likelihood of errors, facilitate data and information retrieval, effectively capture outcomes, and
support process traceability When it is not immediately evident what data should be recorded or how to record
them, forms should include instructions for their use. Forms should indicate units of measure for recording
quantitative data.
LABELS. Product labels, such as blood component or HCT/P labels, are subject to many of
the requirements in a document management system. Many facilities maintain a master set of labels that can
be used as a reference to verify that only currently approved stock is in use. The accuracy of new stock labels
should be verified before this stock is put into inventory; comparison against a master label provides a
mechanism for this verification. Change control procedures should be established for the use of on-demand
label printers to prevent nonconforming modifications of label format or content.
Each facility should have a defined process for developing and maintaining documents. This process should
identify basic elements required for documents; procedures for review and approval of new or revised
documents; a method for keeping documents current; a process for control of document distribution; and a
process for archiving, protecting, and retrieving obsolete documents. Training should be provided to the staff
responsible for the content of new or revised documents. Document management systems include these
established processes:
■ Verifying the adequacy of the document before its approval and issuance.
■ Periodically reviewing, modifying, and reapproving documents as needed to keep them current.
 ■ Identifying changes and revision status.
■ Ensuring that documents are legible, identifiable, and readily available in the locations in which they will
be used.
■ Retaining and retrieving previous versions for the required retention period.
■ Preventing unintended use of outdated or obsolete documents.
■ Protecting documents from alteration, damage, or unintended destruction.
External documents that are incorporated by reference become part of the document management system
and should be identified and controlled. The facility should have a mechanism to detect changes to external
documents in its system, such as manufacturers’ package inserts or user manuals, so that corre
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sponding changes to procedures and forms can be made.
When new or revised policies, process descriptions, procedures, or forms are added to or replaced in the
facility’s manual, these documents should be marked with the date on which they were first put into use (ie,
effective date).
One copy of retired documents should be retained as defined by existing and applicable standards and
regulations.
A master list of all current policies, process descriptions, procedures, forms, and labels is useful for
maintaining document control. It should include document titles, individuals or work groups responsible for
maintaining each document, revision dates, unique document identifiers, and the areas in which each document
is used. It should also identify the number and locations of controlled copies in circulation. Copies of
documents that are used in the workplace should be identified and controlled to ensure that none are overlooked
when changes are implemented.
Records
Records provide evidence that critical steps in a procedure have been performed appropriately and that
products and services conform to specified requirements. Records should be created concurrently with the
performance of each significant step and should clearly indicate the identity of the individuals who performed
each step and when each step was completed.2,6,7 Data should be recorded in a format that is clear and
consistent. When forms are used for capturing or recording data, steps, or test results, the forms become records.
The process for managing records should address the following items:
■ Creation and identification of records.
■ Protection from accidental or unauthorized modification or destruction.
■ Verification of completeness, accuracy, and legibility.
■ Storage and retrieval.
■ Creation of copies or backups.
■ Retention periods.
■ Confidentiality.
Records review is an important tool to help evaluate the effectiveness of the quality management system.
The facility should define a process and time frames for records review to ensure accuracy, completeness, and
appropriate follow-up. It should determine how reports and records are to be archived and how to define their
retention period. Specific requirements for records to be maintained by AABB-accredited facilities are included
in the relevant AABB standards.
Record-keeping systems should allow for ready retrieval of records within time frames established by the
facility and permit traceability of blood components, cellular therapy products, tissues, and derivatives as
required by federal regulations.2,4 When copies of records are retained, the facility should verify that each copy
contains the complete, legible, and accessible content of the original record before the original is destroyed.
If records are maintained electronically, adequate backups should exist in case of system failure. Electronic
records should be readable for the entire duration of their retention period. Obsolete computer software that is
necessary to reconstruct or trace records should also be archived appropriately. If the equipment or software
used to access archived data cannot be maintained, the records should be converted to another format or copied
to another medium to permit continued access. Converted data should be verified against the original to ensure
completeness and accuracy. Electronic media such as magnetic tapes, optical disks, or online remote servers are
widely used for archiving documents. Records kept in this manner must meet FDA requirements for electronic
record-keeping.24 Microfilm or microfiche may also be used to archive records. The medium selected should
be appropriate to comply with the retention requirements.
Each facility must have a policy for altering or correcting records.6 The date of the changes and the identity
of the individual making each change must be documented. In
19
some instances, it may also be important to indicate the reason for the change. The original wording must
not be obliterated in written records; the original may be crossed out with a single line, but it should remain
legible. Writeovers and scratch-outs should not be used. Electronic records must permit tracking of both original
and corrected data and must include the date and identity of the person who made the change. There should be a
process for controlling changes. A method for referencing changes to records that is linked to the original record
and a system for reviewing changes for completeness and accuracy are essential. Audit trails for changed data in
computerized systems are required by the FDA.24
The following issues might be considered when planning record storage:
■ Storage of records in a manner that protects them from damage and accidental or unauthorized destruction
or modification.
■ Degree of accessibility of records in proportion to frequency of their use.
■ Method and location of record storage related to the volume of records and the amount of available
storage space.
■ Availability of properly functioning equipment, such as computer hardware, and software to view
archived records.
■ Documentation that all records copied, transferred to microfiche, or converted to digital files legitimately
replace originals that are stored elsewhere or destroyed.
■ Retention of original color-coded records when only black-and-white reproductions are available.
Considerations for electronic records include the following:
■ A method of verifying the accuracy of data entry.
■ Prevention of unintended deletion of data or access by unauthorized persons.
■ Adequate protection against inadvertent data loss (eg, when a storage device is full).
■ Validated safeguards to ensure that a record can be edited by only one person at a time.
■ Security of and access to confidential data.
The facility should maintain a record of names; inclusive dates of employment; and corresponding
signatures, identifying initials, or identification codes of personnel authorized to create, sign, initial, or review
reports and records. Magnetically coded employee badges and other computer-related identifying methods are
generally accepted in lieu of written signatures, provided that the badges or other methods meet electronic
record-keeping requirements.
Information Management
The quality management system should ensure the confidentiality and appropriate use of data and
information in both oral and written communications. Privacy of patient and donor records should be addressed
to maintain the security and confidentiality of such records.
The system should prevent unauthorized access, modification, or destruction of the data and information.
Individuals who are authorized to make changes to data should be defined by name, code, or job responsibility.
Information systems should be designed with security features to prevent unauthorized access and use. Systems
may include levels of security defined by job responsibility and require the use of security codes and passwords
or, for paper-based systems, locked cabinets and keys.
The integrity of data should be maintained so that data are retrievable and usable. Periodic integrity checks
should be conducted to ensure that critical data have not been inadvertently modified, been lost, or become
inaccessible. When data are sent manually or electronically from one point to another, a process should be in
place to ensure that the data accurately and reliably reach their final destination in a timely manner.
Backup versions (eg, disks, tapes, or duplicate hard copies) of critical data should be maintained in the event
of an unexpected loss from the storage medium. It is advisable to store backup or archived computer records
and databases off-site at a sufficient distance away to ensure that disasters will not affect both the originals and
the backups. The
20
AABB TECHNICAL MANUAL
 backup storage facility should be secure. Environmental conditions in the backup storage facility should be
maintained in a way that protects and preserves the equipment and media for the duration of their storage.
Temperature and humidity should be monitored and controlled. Archival copies of computer operating systems
and applications software required to view original records should be stored in the same manner.
The facility should develop and maintain alternative systems to ensure access to critical data and
information in the event that computerized data or primary sources of information are not available. The backup
and recovery procedures for computer downtime should be defined, and validation documentation should show
that the backup system works properly. The associated processes should be tested periodically to ensure that the
backup system remains effective. Special consideration should be given to staff competence and readiness to
use the backup system.
Management of Nonconforming Events
The quality management system should include a process for detecting, investigating, and responding to
events that result in deviations from accepted policies, processes, and procedures or in failures to meet
requirements, as defined by the facility, AABB standards, or applicable regulations.2'4 This process should be
implemented after, for example, the discovery of nonconforming products and services or of adverse reactions
to donation, blood components, cellular therapy products, tissues, or derivatives. The facility should define how
to perform the following for nonconforming events:
■ Document and classify occurrences.
■ Determine the effect, if any, on the quality of products or services.
■ Evaluate the effect on interrelated activities.
■ Analyze the event to understand root causes.
■ Implement corrective action as appropriate, including notification and recall, on the
basis of investigations and root cause analyses.
■ Implement preventive actions as appropriate on the basis of analyses of aggregate data about events and
their causes.
■ Report to external agencies when required.
■ Evaluate effectiveness of the corrective actions and preventive actions taken.
The CLSI has published a consensus standard on occurrence management that explores event management
in more detail.25
Facility personnel should be trained to recognize and report such occurrences. Depending on the severity of
an event and risk to patients, donors, and products as well as the likelihood of recurrence, investigation of
contributing factors and underlying causes may be warranted. The cGMP regulations require investigation and
documentation of results if a specific event could adversely affect patient safety or the safety, purity, or potency
of blood or components.2,3 The cGTP regulations require similar activities for deviations and possible product
contamination or communicable disease transmission.4 Tools and approaches for performing root cause
analysis and implementing corrective action are discussed in the section on “Process Improvement.” A
summary of each event, investigation, and any follow-up activities must be prepared. Table 1-2 outlines
suggested components of an internal event report.
Fatalities related to blood collection or transfusion or to HCT/Ps must be reported as soon as possible to the
FDA Center for Biologies Evaluation and Research (CBER). [See 21 CFR 606.170(b) and 1271.350(a) (i),
respectively.] Instructions for reporting to CBER are available in published guidance27 and on the FDA
website.28 A written follow-up report must be submitted within 7 days of the fatality and should include a
description of any new procedures implemented to avoid recurrence. AABB Association Bulletin #04-06
provides additional information on these reporting requirements, including a form for reporting donor
fatalities.29
Regardless of their licensure and registration status with the FDA, all donor centers,
21
TABLE 1-2. Components of an Internal Event Report26
Component Examples
Who ■ Reporting individual(s)
■ Individual(s) involved (by job title) in committing, compounding, discovering, investigating, and
initiating any immediate action
■ Patient or donor identification code
■ Reviewer(s) of report
 What ■ Brief description of event
■ Effects on and outcomes for patient, donor, blood component, or tissue
■ Name of component and unit identification number
■ Manufacturer, lot number, and expiration dates of applicable reagents and supplies
■ Immediate actions taken
When ■ Date of report
■ Date and time event occurred
■ Date and time of discovery
■ Date (and time, if applicable) that immediate action was taken
And as applicable, date and time of:
■ Blood component collection, processing steps, and shipping
■ Order for blood component
■ Order for patient testing
■ Patient sample collection, transport, and receipt
■ Test performance and reporting
Where ■ Physical location of event
■ Where in process event was detected
■ Where in process event was initiated
Why and How ■ How event occurred
■ Contributing factors to event
■ Root cause(s)
Follow-up ■ External reports or notifications (eg, regulatory* or accreditation agencies, manufac
turer, or patient’s physician)
■ Corrective actions
■ Implementation dates
■ Effectiveness of actions taken
■ Linkage to preventive action if appropriate
*AII blood establishments (including licensed, registered but unlicensed, and unregistered transfusion
services)2 (21 CFR 606.121) are required to notify the FDA of deviations from cGMP, applicable standards, or
established specifications that may affect the safety, purity, or potency of biological products or otherwise cause
the biological products to be in violation of the Food, Drug, and Cosmetic Act or the Public Health Service Act
(21 CFR 600.14).2 The FDA has identified the following examples as reportable events if components or
products are released for distribution:
■ Arm preparation not performed or performed incorrectly.
■ Units released from donors who are (or should have been) either temporarily or permanently deferred
because of their medical history or a history of repeatedly reactive viral marker tests.
■ Shipment of a unit with repeatedly reactive viral markers.
■ ABO/Rh or infectious disease testing not performed according to the manufacturer’s package insert.
■ Units released from donors for whom test results were improperly interpreted because of testing errors
related to improper use of equipment.
■ Units released before completion of all tests (except as an emergency release).
■ Sample used for compatibility testing that contains incorrect identification.
■ Testing error that results in the release of an incorrect unit.
■ Incorrectly labeled blood components (eg, ABO group or expiration date).
(Continued)
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AABB TECHNICAL MANUAL
TABLE 1-2. Components of an Internal Event Report26 (Continued)
■ Incorrect crossmatch label or tag.
■ Storage of biological products at an incorrect temperature.
■ Microbial contamination of blood components when the contamination is attributed to an error in
manufacturing. Deviations involving distributed HCT/Ps and relating to core cGTP requirements must also be
reported to the FDA if they
occurred in the facility or in a facility that performed a manufacturing step for the facility under contract,
agreement, or other arrangement.4 Each report must contain a description of the HCT/P deviation, information
relevant to the event and the manufacture of the HCT/Ps involved, and information on all follow-up actions that
have been or will be taken in response to the deviation.
CFR = Code of Federal Regulations, FDA = Food and Drug Administration; cGMP = current good
manufacturing practice; HCT/Ps = human cells, tissues, and cellular and tissue-based products; cGTP = current
good tissue practice.
blood banks, and transfusion services must promptly report biological product deviations—and information
relevant to these events—to the FDA2,30 using Form FDA-3486 when the event 1) is associated with
manufacturing (ie, collecting, testing, processing, packing, labeling, storing, holding, or distributing); 2)
represents a deviation from cGMP, established specifications, or applicable regulations or standards or that is
unexpected or unforeseen; 3) may affect the product’s safety, purity, or potency; 4) occurs while the facility had
control of, or was responsible for, the product; and 5) involves a product that has left the facility’s control (ie,
has been distributed).
Using the same form, facilities must also promptly report biological product deviations associated with a
distributed HCT/P if the event represents a deviation from applicable regulations, standards, or established
specifications that relate to the prevention of communicable disease transmission or HCT/P contamination. This
requirement pertains to events that are unexpected or unforeseeable but may relate to the transmission or
potential transmission of a communicable disease or may lead to HCT/P contamination.4 More information
concerning biological product deviation reporting can be found on the FDA website.31
There must also be a mechanism to report medical device adverse events to the FDA and the device
manufacturer.8,32 The Joint Commission encourages reporting of sentinel events, including hemolytic
transfusion reactions involving the administration of blood or
components having major blood group incompatibilities.9,10
Hemovigilance processes also provide the opportunity to detect, investigate, and respond to adverse
transfusion reactions and events that result in deviations from safe blood transfusion and collection practices.
Adverse transfusion reactions and events (or incidents) can be reported voluntarily to the Centers for Disease
Control and Prevention (CDC) National Healthcare Safety Network (NHSN) Hemovigilance Module. This
system was developed through a public-private collaboration that included the Department of Health and
Human Services and its agencies, and the private sector, including AABB, America’s Blood Centers, and the
American Red Cross. The AABB Center for Patient Safety, a licensed Patient Safety Organization, works with
hospitals to provide additional analysis and benchmarking of hospital transfusion event reports, while protecting
data confidentiality. AABB also administers the AABB United States Donor Hemovigilance Program where
blood collectors can report, analyze, and benchmark adverse donor reactions.
Each facility should track reported events and look for trends. The use of classification schemes may
facilitate trend analysis and typically involves one or more of the following categories: the nature of the event,
the process (or procedure) in which the event occurred, the outcome and severity of the event, and the
contributing factors and underlying causes. If several events within a relatively short period involve a particular
process or procedure, that process or procedure should be further inves

23
tigated. The most useful schemes involve the use of multiple categories for each event, which allows data to
be sorted in a variety of ways so that patterns that were previously not obvious can emerge. (See example in
Table 13.) Such sorting or stratification can result in identification of situations that require closer monitoring or
problems needing corrective or preventive action. For smaller facilities that may not have sufficient data to
identify trends, pooling data with a larger entity (eg, the laboratory or all transfusion services in a healthcare
system) or following national trends from data provided by organizations such as the AABB, CAP, or The Joint
Commission, may also prove helpful. The extent of monitoring and the length of time to monitor processes
depends on the frequency and critical aspects of the occurrences. Reporting and monitoring of events are
essential problem identification methods for process improvement activities in a quality management system.
Occasionally, there may be a need for a facility to deviate from approved procedures to meet the unique
medical needs of a particular patient. When this situation arises, a medically indicated exception should be
planned and approved in advance by the facility’s medical director. The rationale and nature of the planned
exception should be documented. Careful consideration should be given to maintaining a controlled process and
verifying the safety and quality of the resulting product or service. Any additional risk to the patient must be
disclosed.
Monitoring and Assessment
The quality management system should describe how the facility monitors and evaluates its processes.
Assessments are systematic examinations to determine whether actual activities comply with planned activities,
are implemented effectively, and achieve objectives. Depending on the focus, assessments can
TABLE 1-3. Example of an Event Classification
Event: A unit of Red Blood Cells from a directed donor was issued to the wrong oncology patient. The unit
was not transfused.
Event Classification
■ Type of event: patient
■ Procedure involved: issuing products
■ Process involved: blood administration
■ Product involved: Red Blood Cells
■ Location: transfusion service
■ Other factors: directed donor
■ Other factors: oncology patient
Underlying Causes
■ Proximate cause: two patients with similar names had crossmatched blood available
■ Root cause: inadequate procedure for verification of patient identification during issue
Outcome
■ Severity: serious, FDA reportable
■ Patient: no harm, correct product was obtained and transfused
 ■ Product: no harm, product returned to inventory
■ Donor: not applicable
Successful Barriers
■ Problem detected during the patient identification verification step of blood administration
FDA = Food and Drug Administration.

24
AABB TECHNICAL MANUAL
include: 1) evaluation of process outputs (ie, results); 2) the activities that make up a process as well as its
outputs; or 3) a group of related processes and outputs (ie, the system).
Assessments can be internal or external and can include quality assessments, peer reviews, self-assessments,
and proficiency testing. Evaluations typically include comparisons of actual to expected results.
Internal Assessments
Internal assessments may include evaluations of quality indicator data, targeted audits of a single process, or
system audits that are broader in scope and may cover a set of interrelated processes. These assessments should
be planned and scheduled. The details of who performs the assessments and how they are performed should be
addressed. Assessments should cover the quality system and the major operating systems in the donor center
and transfusion or cellular therapy service.
In addition, there should be a process for responding to the issues raised as a result of the assessment,
including review processes and time frames. The results should be documented and submitted to management
personnel who have authority over the process assessed as well as to executive management. Management
should develop corrective action plans with input from operational staff and quality oversight personnel for any
deficiencies noted in the assessment. Quality oversight personnel should track progress toward implementation
of corrective actions and monitor them for effectiveness.
To make the best use of these assessments, there should be a process to track, monitor trends in, and analyze
the problems identified so that opportunities for improvement can be recognized. Early detection of trends
makes it possible to develop preventive actions before patient safety, blood, components, tissues, or derivatives
are adversely affected. Evaluation summaries provide information that is useful for addressing individual or
group performance problems and ensuring the adequacy of test methods and equipment. Any corrective or
preventive action associated
with assessment results should be reviewed by executive management.
Quality Indicators
Quality indicators are performance measures designed to monitor one or more processes during a defined
time and are useful for evaluating service demands, production, personnel, inventory control, and process
stability. These indicators can be process based or outcome based. Process-based indicators measure the degree
to which a process can be consistently performed. An example of a process-based indicator is turnaround time
from blood product ordering until transfusion. Outcome-based indicators are often used to measure what does
or does not happen after a process is or is not performed. The number of incorrect test result reports is an
example of such an indicator. For each indicator, thresholds are set that represent warning limits, action limits,
or both. These thresholds can be determined from regulatory or accreditation requirements, benchmarking, or
internal facility data.
Tools frequently used for displaying quality indicator data are run charts and control charts. In a run chart,
time is plotted on the x-axis and values on the y-axis. In control charts, the mean of the data and the upper and
lower control limits, which have been calculated from the data, are added to the chart. Single points outside the
upper and lower control limits result from special causes. Statistical rules for interpreting consecutive points
outside 1 standard deviation (SD), 2 SDs, and 3 SDs should be used to recognize a process that is out of control.
The root cause should be determined, and corrective action should be initiated, if indicated.
Blood Utilization Assessment
The activities of blood usage review committees in the transfusion setting are an example of internal
assessment. Guidelines are available from the AABB for both adult and pediatric utilization review.34'36 Peer
review of transfusion practices, required by the AABB, is also required by The Joint Commission9 for hospital
accreditation, by CMS1 for hospitals to
25
qualify for Medicare reimbursement, and by some states for Medicaid reimbursement.
Transfusion audits provide reviews of policies and practices to ensure safe and appropriate transfusions and
are based on measurable, predetermined performance criteria. (See Chapter 28.) Transfusion services should
investigate an adequate sample of cases (eg, 5% of cases within a defined time frame or 30 cases, whichever is
larger). Audits assess a facility’s performance and effectiveness in the following6:
■ Ordering practices.
■ Patient identification.
■ Sample collection and labeling.
■ Infectious and noninfectious adverse events.
■ Near-miss events.
■ Usage and discard.
■ Appropriateness of use.
■ Blood administration policies.
■ Ability of services to meet patient needs.
■ Compliance with peer-review recommendations.
■ Critical laboratory results before and after transfusion.
One method of assessing the blood administration process is to observe a predetermined number of
transfusions by following a unit of blood as it is issued for transfusion and is then transfused.34
Assessments of transfusion safety policy and practice may include reviews of transfusion reactions and
transfusion-transmitted diseases. The review committee may monitor policies and practices for notifying
recipients or recipients’ physicians of recalled products and notifying donors of abnormal test results. Other
assessments important in transfusion practice include reviews of policies for informed consent, indications for
transfusion, releases of directed donor units, and outpatient or home transfusions. Additional assessments
should include, where appropriate, 1) therapeutic apheresis; 2) use of blood recovery devices; 3) procurement
and storage of hematopoietic progenitor cells; 4) perioperative autologous blood collection; 5) procurement and
storage of tissue; and 6) evaluation of
evolving technologies and products, such as growth factors and cytokines.
External Assessments
External assessments include inspections, surveys, audits, and assessments performed by those not affiliated
with the organization, such as the AABB, CAR CMS, COLA, FACT, the FDA, The Joint Commission, or state
and regional health departments. Participation in an external assessment program provides an independent
objective view of the facility’s performance. External assessors often bring broad-based experience and
knowledge of best practices that can be shared. Such assessments are increasingly being performed
unannounced or with minimal notification.
In the preparation phase, there is typically some data gathering and information to submit to the
organization performing the assessment. To prepare, facilities can perform internal audits and conduct drills to
ensure that staff can answer questions. For most external assessments, there is an increased emphasis on
observations of the processes and dialogue with nonmanagement staff, so preparation is key. During the
assessment phase, it is important to know who is responsible for the assessors or inspectors while they are in the
facility. Clear descriptions of what information can be given to these individuals—and in what form— help the
facility through the assessment or inspection process. After the assessment, identified issues should be
addressed. Usually, a written response is submitted.
Proficiency Testing for Laboratories
Proficiency testing (PT) is one means for determining that test systems (including methods, supplies, and
equipment) are performing as expected. As a condition for certification, CMS requires laboratories to
participate successfully in an approved PT program for CLIAregulated testing. When no approved PT program
exists for a particular analyte, the laboratory must have another means to verify the accuracy of the test
procedure at least twice annually.1 Some accrediting agencies may require more frequent verification of
accuracy.
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AABB TECHNICAL MANUAL
PT must be performed using routine work processes and conditions if it is to provide meaningful
information. PT samples should generally be handled and tested in the same way as patient or donor specimens.
However, a CLIA-certified laboratory is prohibited from discussing the PT or sending the samples to a
laboratory with a different CLIA number during the active survey period, even if the two laboratories are within
the same organization and that would be the routine manner for handling patient or donor specimens.
Supervisory review of the summary evaluation report should be documented along with investigation and
corrective action for unacceptable results. Quality oversight personnel should monitor the PT program and
verify that test systems are maintained in a state of control and appropriate corrective action is taken when
indicated.
Process Improvement
Continual improvement is a fundamental goal in any quality management system. In transfusion and cellular
therapies and clinical diagnostics, this goal is tied to patient safety goals and expectations for the highest quality
health care. The importance of identifying, investigating, correcting, and preventing problems cannot be
overstated. The process of developing corrective and preventive action plans involves identification of problems
and their causes as well as identification and evaluation of solutions to prevent future problems. This process
should also include evaluation of near-miss events and a mechanism for data collection and analysis as well as
follow-up to evaluate the effectiveness of the actions taken. Statistical tools and their applications may be found
in publications from the AABB and the
American Society for Quality.37-39 The Joint Commission standards for performance improvement are
outlined in Table 1-4.910
Corrective action is defined as action taken to address the root causes of an existing nonconformance or
other undesirable situation to reduce or eliminate the risk of recurrence. Preventive action is defined as action
taken to reduce or eliminate the potential for a nonconformance or other undesirable situation to prevent
occurrence. Corrective action can be thought of as a reactive approach to address the root causes of actual
nonconformances, deviations, complaints, and process failures, whereas preventive action can be thought of as a
proactive approach to address the underlying causes of anticipated problems identified through the analysis of
data and information.40 In contrast, remedial action is defined as action taken to alleviate the symptoms of
existing nonconformances or any other undesirable situation.41 Remedial action, sometimes called correction,
addresses only a problem’s visible indicator and not the actual cause. (See comparisons in Table 1-5.) Effective
corrective and preventive action cannot be implemented until the underlying cause is determined and the
process is evaluated in relationship to other processes. Pending such evaluation, it may be desirable to
implement interim remedial action.
Identification of Problems and Their Causes
Sources of information for process improvement activities include process deviations, nonconforming
products and services, customer complaints, QC records, PT, internal audits, quality indicators, and external
assessments. Active monitoring programs may be set
TABLE 1-4. Applicable Joint Commission Performance Improvement Standards940
■ The organization collects data to monitor its performance, including the following:
- Blood and blood component use.
- All confirmed transfusion reactions.
■ The organization compiles and analyzes data.
■ The organization improves performance on an ongoing basis.

27
TABLE 1-5. Comparison of Remedial, Corrective, and Preventive Actions40
Action Problem Approach Outcome
Remedial Existent Reactive Alleviates symptoms
Corrective Existent Reactive Prevents recurrence
Preventive Nonexistent Proactive Prevents occurrence
up to help identify problem areas. These programs should be representative of the facility processes and
consistent with organizational goals, and they should reflect customer needs. Preparation of a facility quality
report at least annually in which data from all these sources are aggregated and analyzed can be valuable to
identify issues for performance improvement.
 Once identified, problems should be analyzed to determine their scope, potential effects on quality
management and operational systems, relative frequency, and extent of their variation. Such an analysis is
important to avoid tampering with processes that are merely showing normal variations or problems with little
effect.
The underlying causes of an undesirable condition or problem can be identified by an individual or group.
The more complex the problem and the more involved the process, the greater the need to enlist a team and to
formalize the analysis. Three commonly used tools for identifying underlying causes in an objective manner are
process flowcharting, use of the “repetitive why,” and the cause-andeffect diagram.
PROCESS FLOWCHART. A process flowchart gives a detailed picture of the multiple activities and
important decision points within that process. By examining this picture, one may identify problem-prone areas.
REPETITIVE WHY. The “repetitive why” is used to work backward through the process. One repeatedly
asks “Why did this happen?” until 1) no new information can be gleaned; 2) the causal path cannot be followed
because of missing information; or 3) further investiga
tion is impractical, impossible, or outside the boundaries of the organization. Use of the “repetitive why”
prevents the mistake of interpreting an effect as a cause.
CAUSE-AND-EFFECT DIAGRAM. The causeand-effect diagram, also known as the Ishikawa or fish-bone
diagram, uses a specialized form of brainstorming that breaks down problems into “bite-sized” pieces. (An
example of a cause-and-effect diagram is shown in Fig 1-1.) The method used in the diagram is designed to
focus ideas around the component parts of a process as well as to give a pictorial representation of the ideas that
are generated and their interactions. When using the cause-and-effect diagram, one looks at equipment,
materials, methods, environment, and human factors.
These three tools identify both active and latent failures. Active failures are those that have an immediate
adverse effect. Latent failures are more global actions and decisions with potential for damage that may lie
dormant and become evident only when triggered by the presence of localized factors. The key to successfully
determining root causes is to avoid stopping too soon or getting caught in the trap of placing blame on an
individual.
Most problems, particularly those that are complex, have several root causes. A method that can be of use
when such problems occur is the Pareto analysis. A chart of causes, laid out in order of decreasing frequency, is
prepared. Those that occur most frequently are considered the “vital few”; the rest are considered the “trivial
many.” This method offers direction on where to dedicate resources for maximal effect. An example of a Pareto
chart is shown in Fig 1-2.

28
AABB TECHNICAL MANUAL
Root Cause Analysis of Failed Test Runs

FIGURE 1-1. Example of a cause-and-effect diagram. SOP = standard operating procedure.

Identification and Evaluation of Solutions
 Potential solutions to problems are identified during the creative phase of process improvement.
Brainstorming and process flowcharting can be particularly helpful in this phase. Benchmarking with other
organizations can also be helpful. Possible solutions should be evaluated relative to organizational constraints
and should be narrowed down to those that are most reasonable. Individuals who implement the process are
usually the most knowledgeable about what will work. They should be consulted when possible solutions are
being considered. Individuals with knowledge of the interrelationships of processes who have a more “global”
view of the organization should also be involved in this step. Solutions may fail if representatives with those
perspectives are not involved.
Potential solutions should be tested before full implementation and a clear plan should be created regarding
methods, objectives, timelines, decision points, and algorithms for all possible results of a trial. Large
scale solutions can be tried on a limited basis and can be expanded if successful; small-scale solutions can
be implemented pending an effectiveness evaluation. Data should be collected to evaluate the effectiveness of
the proposed change. Data can be collected using the methods employed initially to identify the problems or
methods specially designed for the trial. Once solutions have been successfully tested, full implementation can
occur. After implementation, data should be collected at least periodically to ensure the continuing effectiveness
and control of the changed process.
Other Process Improvement Methods
Failure modes and effects analysis is a systematic stepwise approach for identifying all possible failures
within a process, product, or service; studying and prioritizing the consequences, or effects, of those failures;
and eliminating or reducing the failures, starting with those of highest priority. Despite their relative complexity,
LEAN and Six Sigma process improvement methods from the manufacturing
29
Reasons for Rejecting Crossmatch Specimens Ordered by Frequency

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i i Number rejected Cumulative percent rejected
FIGURE 1-2. Example of a Pareto chart.
industry are finding increasing use in the tion of these principles and techniques can
 health-care setting. LEAN emphasizes speed improve performance, reduce costs and waste,
and efficiency. Six Sigma emphasizes precision cut time, and eliminate non-value-added ac
and accuracy. Six Sigma uses the data-driven tions. Additional information about both
approach to problem solving of define, mea- methods can be found on the website of the
sure, analyze, improve, and control. Applica- American Society for Quality.42
KEY POINTS
1. Organization and Leadership. A defined organizational structure in addition to top management’s support
and commitment to the quality policy, goals, and objectives are key to ensuring the success of the quality
management system.

30
AABB TECHNICAL MANUAL
 2. Customer Focus. Quality organizations should understand and meet or exceed customer needs and
expectations. These needs and expectations should be defined in a contract, agreement, or other document
developed with feedback from the customer.
3. Facilities, Work Environment, and Safety. Procedures related to general safety; biological, chemical, and
radiation safety; fire safety; and disaster preparedness are required. Space allocation, building utilities,
ventilation, sanitation, trash, and hazardous substance disposal must support the organization’s operations.
4. Human Resources. Quality management of all personnel addresses adequate staffing levels and staff
selection, orientation, training, and competence assessment as well as specific regulatory requirements.
5. Suppliers and Materials Management. Suppliers of critical materials and services (ie, those affecting
quality) should be qualified, and these requirements should be defined in contracts or agreements. All critical
materials should be qualified and then inspected and tested upon receipt to ensure that specifications are met.
6. Equipment Management. Critical equipment may include instruments, measuring devices, and computer
hardware and software. This equipment must be uniquely identified and operate within defined specifications,
as ensured by qualification, calibration, maintenance, and monitoring.
7. Process Management. A systematic approach to develop new, and control changes to, policies, processes,
and procedures includes process validation, test method validation, computer system validation, equipment
validation, and QC. Validation must be planned and results reviewed and accepted.
8. Documents and Records. Documents include policies, process descriptions, procedures, work
instructions, job aids, forms, and labels. Records provide evidence that the process was performed as intended
and allow assessment of product and service quality.
9. Information Management. Unauthorized access, modification, or destruction of data and information
must be prevented and confidentiality of patient and donor records maintained. Data integrity should be
assessed periodically and backup devices, alternative systems, and archived documents maintained.
10. Management of Nonconforming Events. Deviations from facility-defined requirements, standards, and
regulations must be addressed by documenting and classifying occurrences, assessing effects on quality,
implementing remedial actions, and reporting to external agencies as required.
11. Monitoring and Assessment. Evaluation of facility processes includes internal and external assessments,
monitoring of quality indicators, blood utilization assessment, proficiency testing, and analysis of data.
12. Process Improvement. Opportunities for improvement may be identified from deviation reports,
nonconforming products and services, customer complaints, QC records, proficiency testing results, internal
audits, quality indicator monitoring, and external assessments. Process improvement includes determination of
root causes, implementation of corrective and preventive actions, and evaluation of the effectiveness of these
actions.
REFERENCES
1. Code of federal regulations. Title 42, CFR Part 493. Washington, DC: US Government Printing Office,
2013 (revised annually).
2. Code of federal regulations. Title 21, CFR Parts 606, 610, 630, and 640. Washington, DC: US
Government Printing Office, 2014 (revised annually).
3. Code of federal regulations. Title 21, CFR Parts 210 and 211. Washington, DC: US Government Printing
Office, 2014 (revised annually).
 31
4. Code of federal regulations. Title 21, CFR Parts 1270 and 1271. Washington, DC: US Government
Printing Office, 2014 (revised annually).
5. Food and Drug Administration. Guideline for quality assurance in blood establishments (July 11,1995).
Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 1995.
6. Judith Levitt, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD:AABB,
2014.
7. Fontaine M, ed. Standards for cellular therapy services. 6th ed. Bethesda, MD: AABB, 2013.
8. College of American Pathologists. Laboratory Accreditation Program checklists. Chicago: CAR 2013.
 9. The Joint Commission. Hospital accreditation standards. Oakbrook Terrace, IL: Joint Commission
Resources, 2014.
10. The Joint Commission. Laboratory accreditation standards. Oakbrook Terrace, IL: Joint Commission
Resources, 2014.
11. Clinical and Laboratory Standards Institute. Quality management system: A model for laboratory
services; approved guideline (GP26A4/QMS 01-A4). 4th ed. Wayne, PA: CLSI, 2011.
12. Foundation for the Accreditation of Cellular Therapy and the Joint Accreditation Committee of ISCT
and EBMT. FACT-JACIE international standards for cellular therapy product collection, processing, and
administration. 5th ed. Omaha, NE: FACT-JACIE, 2012.
13. ANSI/ISO/ASQ Q9001-2008 series—quality management standards. Milwaukee, WI: ASQ Quality
Press, 2008.
14. International Organization for Standardization. ISO 15189:2012. Medical laboratories— Requirements
for quality and competence. Geneva, Switzerland: ISO, 2012. [Available at
http://www.iso.org/iso/catalogue_detaibcsnumber=56115 (accessed November 6,2013).]
15. Baldrige Performance Excellence Program. Health care criteria for performance excellence.
Gaithersburg, MD: National Institute of Standards and Technology, 2013-2014 (revised biannually).
16. Quality program implementation. Association bulletin #97-4. Bethesda, MD: AABB, 1997.
17. Food and Drug Administration. Guidance for industry: Quality systems approach to pharmaceutical
cGMP regulations (September, 2006). Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2006.
18. Code of federal regulations. Title 21, CFR Part 820. Washington, DC: US Government Printing Office,
2014 (revised annually).
19. Juran JM, Godfrey AB. Juran’s quality handbook. 5th ed. New York: McGraw-Hill, 1999.
20. Food and Drug Administration. Guidance for industry: Process validations: General principles and
practices (January, 2011). Silver Spring, MD: CBER Office of Communication, Outreach, and Development,
2011.
21. Food and Drug Administration. Guidance for industry: Blood establishment computer system validation
in the user’s facility (April, 2013). Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2013.
22. Food and Drug Administration. General principles of software validation: Final guidance for industry
and FDA staff (January, 2002). Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2002.
23. Clinical and Laboratory Standards Institute. Quality Management System: Development and
management of laboratory documents; approved guideline. 6th ed. (GP02-A6/QMS 02-A6). Wayne, PA: CLSI,
2013.
24. Code of federal regulations. Title 21, CFR Part 11. Washington, DC: US Government Printing Office,
2014 (revised annually).
25. Clinical and Laboratory Standards Institute. Management of nonconforming laboratory events; approved
guideline (GP32-A/QMS 11A). Wayne, PA: CLSI, 2007.
26. Motschman TL, Santrach PJ, Moore SB. Error/ incident management and its practical application. In:
Duckett JB, Woods LL, Santrach PJ, eds. Quality in action. Bethesda, MD: AABB, 1996:37-67.
27. Food and Drug Administration. Guidance for industry: Notifying FDA of fatalities related to blood
collection or transfusion (September, 2003). Silver Spring, MD: CBER Office of Communication, Outreach,
and Development, 2003.
28. Food and Drug Administration. Transfusion/ donation fatalities: Notification process for transfusion
related fatalities and donation related deaths. Silver Spring, MD: Center for Biologies Evaluation and Research,
2007. [Available at http://www.fda.gov/BiologicsBloodVac cines/SafetyAvailability/ReportaProblem/
TransfusionDonationFatalities/default.htm (accessed August 23,2013).]
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AABB TECHNICAL MANUAL
29. AABB. Reporting donor fatalities. Association bulletin #04-06. Bethesda, MD: AABB, 2004.
30. Food and Drug Administration. Guidance for industry: Biological product deviation reporting for blood
and plasma establishments (October, 2006). Silver Spring, MD: CBER Office of Communication, Outreach,
and Development, 2006.
 31. Food and Drug Administration. Biological product deviations: Includes human tissue and cellular and
tissue-based product (HCT/P) reporting (BPDR). Silver Spring, MD: Center for Biologies Evaluation and
Research, 2010. [Available at http://www.fda.gov/Biologics BloodVaccines/SafetyAvailability/Reporta
Problem/BiologicalProductDeviations/de fault.htm (accessed August 23,2013).]
32. Code of federal regulations. Title 21, CFR Part 803. Washington, DC: US Government Printing Office,
2014 (revised annually).
33. Strong DM, AuBuchon J, Whitaker B, Kuehnert MJ. Biovigilance initiatives. ISBT Sci Ser 2008; 3:77-
84.
34. Shulman LA, Lohr K, Derdiarian AK, Picukaric JM. Monitoring transfusionist practices: A strategy for
improving transfusion safety. Transfusion 1994;34:11-15.
35. Roback J, Waxman D, for the Clinical Transfusion Medicine Committee and the Transfusion
Medicine Section Coordinating Committee. Guidelines for patient blood management and blood utilization.
Bethesda, MD: AABB, 2011.
36. Strauss RG, Blanchette VS, Hume H. National acceptability of American Association of Blood Banks
Hemotherapy Committee guidelines for auditing pediatric transfusion practices. Transfusion 1993;33:168-71.
37. Vaichekauskas L. You need the tools to do the job. In: Walters L, ed. Introducing the big Q: A practical
quality primer. Bethesda, MD: AABB Press, 2004:181-206.
38. Walters L. So many tools, so little understanding. In: Walters L, Carpenter-Badley J, eds. S3: Simple Six
Sigma for blood banking, transfusion, and cellular therapy. Bethesda, MD: AABB Press, 2007:9-24.
39. Tague NR. The quality toolbox. 2nd ed. Milwaukee, WI: ASQ Quality Press, 2005.
40. Motschman TL. Corrective versus preventive action. AABB News 1999;21(8):5,33.
41. Russell JR Regel T. After the quality audit: Closing the loop on the audit process. 2nd ed. Milwaukee,
WI: ASQ Quality Press, 2000.
42. American Society for Quality. Learn about quality. Milwaukee, WI: ASQ, 2013. [Available at
http://asq.org/learn-about-quality/ (accessed August 23,2013).]
33
■ APPENDIX 1-1
Glossary of Commonly Used Quality Terms
Collection and analysis of adverse event data for the purpose of improving outcomes in
Biovigilance
the use of blood products, organs, tissues, and cellular therapies.
Comparison of measurements performed with an instrument to those made with a more
Calibration accurate instrument or standard for the purpose of detecting, reporting, and eliminating errors
in measurement.
Established procedures for planning, documenting, communicating, and executing
changes to infrastructure, processes, products, or services. Such procedures include the
Change
submission, analysis, approval, implementation, and postimplementation review of the
control
change and decisions made about the change. Formal change control provides a measure of
stability and safety and avoids arbitrary changes that might affect quality.
A graphic tool used to determine whether the distribution of data values generated by a
Control process is stable over time. A control chart plots a statistic vs time and helps to determine
chart whether a process is in control or out of control according to defined criteria (eg, a shift from
a central line or a trend toward upper or lower acceptance limits).
Documents, records, and evidence in any other format used to verify that design goals
have been met. Design output should identify characteristics of a product or service that are
Design crucial to safety and function and to meeting regulatory requirements. It should contain or
output make reference to acceptance criteria. Examples of design output include standard operating
procedures; specifications for supplies, reagents, and equipment; identification of quality
control requirements; and results of verification and validation activities.
End-product
Verification through observation, examination, or testing (or a combination) that the
test and
finished product or service conforms to specified requirements.
inspection
Near-miss An unexpected occurrence that did not adversely affect the outcome but could have
event resulted in a serious adverse event.
 Process Ability of a controlled process to produce a service or product that fulfills requirements or
capability a statistical measure of the inherent process variability for a given characteristic relative to
design specifications. The most widely accepted formula for process capability is Six Sigma.
Process Activities intended to minimize variation within a process to produce a predictable output
control that meets specifications.
Demonstration that an entity is capable of fulfilling specified requirements and
verification of attributes that must be met or complied with for a person or thing to be
considered fit to perform a particular function. For example, equipment may be qualified for
Qualification
an intended use by verifying performance characteristics, such as linearity, sensitivity, or ease
of use. An employee may be qualified on the basis of technical, academic, and practical
knowledge and skills developed through training, education, and on-the-job performance.
(Continued)

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AABB TECHNICAL MANUAL
■ APPENDIX 1-1
Glossary of Commonly Used Quality Terms (Continued)
Activities involving quality planning, control, assessment, reporting, and improvement
Quality
necessary to ensure that a product or service meets defined quality standards and
assurance
requirements.
Quality Operational techniques and activities used to monitor and eliminate causes of
control unsatisfactory performance at any stage of a process.
Measurable aspects of processes or outcomes that provide an indication of the condition
Quality
or direction of performance over time. Quality indicators are used to monitor progress toward
indicators
stated quality goals and objectives.
The organizational structure, processes, and procedures necessary to ensure that the
overall intentions and direction of an organization’s quality program are met and that the
Quality
quality of the product or service is ensured. Quality management includes strategic planning,
management
allocation of resources, and other systematic activities, such as quality planning,
implementation, and evaluation.
A stated or obligatory need or expectation that can be measured or observed and that is
necessary to ensure quality, safety, effectiveness, or customer satisfaction. Requirements can
Requirement
include things that the system or product must do, characteristics that it must have, and levels
of performance that it must attain.
Description of a set of requirements to be satisfied by a product, material, or process
indicating, if appropriate, the procedures to be used to determine whether the requirements
Specification
are satisfied. Specifications are often in the form of written descriptions, drawings,
professional standards, and other descriptive references.
 Validation Demonstration through objective evidence that the requirements for a particular
application or intended use have been met. Validation provides assurance that new or changed
processes and procedures are capable of consistently meeting specified requirements before
implementation.
Confirmation, by examination of objective evidence, that specified requirements have
Verification
been met.

35
■ APPENDIX 1-2
Code of Federal Regulations Quality-Related References
Code of Federal
Regulations, Title 21
Topic Biologies, Blood Drugs Tissues, HCT/Ps
Personnel 600.10,606.20 211.25,211.28 1271.170
Facilities 600.11, 606.40 211.42-58 1271.190
Environmental control
211.42 1271.195
and monitoring
211.63-72,211.105,
Equipment 606.60 1271.200
211.182
Supplies and reagents 606.65 211.80 1271.210
Standard operating
606.100 211.100-101 1270.31,1271.180
procedures
Process changes and
211.100-101 1271.225,1271.230
validation
Quality
assurance/quality control 211.22
unit
610.60-64,
Label controls 211.122-130 1271.250,1271.370
606.120-122
Laboratory controls 606.140 211.160
 Records and record 600.12,606.160 211.192,211.194, 1270.33,1271.55
reviews 211.196 1271.270
Receipt, predistribution,
606.165 211.142,211.150 1271.265
and distribution
Adverse reactions 606.170 211.198 1271.350
Tracking 211.188 1271.290
Complaints 606.170-171 211.198 1271.320
Reporting deviations 600.14, 606.171 1271.350
640.2,640.11, 640.25,
Storage 211.142 1271.260
640.34, 640.54, 640.69
HCT/Ps = human cells, tissues, and cellular and tissue-based products.

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AABB TECHNICAL MANUAL
■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
Equipment and Reagent Frequency of Quality Control
Refrigerators/freezers/platelet storage
Refrigerators
■ Recorder Daily
■ Manual temperature Daily
■ Alarm system board (if applicable) Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
Freezers
■ Recorder Daily
■ Manual temperature Daily
■ Alarm system board (if applicable) Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
Platelet incubators
■ Recorder Daily
■ Manual temperature Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
■ Ambient platelet storage Every 4 hours
Laboratory equipment
Gentrifuges/cell washers
■ Speed Quarterly
■ Timer Quarterly
■ Function Yearly
■ Tube fill level (serologic) Day of use
■ Saline fill volume (serologic) Weekly
■ Volume of antihuman globulin dispensed (if applicable) Monthly
■ Temperature check (refrigerated centrifuge) Day of use
■ Temperature verification (refrigerated centrifuge) Monthly
CHAPTER 1 Quality Management Systems: Theory and Practice
37
■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents* (Continued)
Equipment and Reagent Frequency of Quality Control
Heating blocks/waterbaths/view boxes
■ Temperature Day of use
■ Quadrant/area checks Periodically
Component thawing devices Day of use
pH meters Day of use
Blood irradiators
■ Calibration Yearly
■ Turntable (visual check each time of use) Yearly
■ Timer Monthly/quarterly
■ Source decay Dependent on source type
■ Leak test Twice yearly
■ Dose delivery check (with indicator) Each irradiator use
■ Dose delivery verification
- Cesium-137 Yearly
- Cobalt-60 Twice yearly
- Other source As specified by manufacturer
Thermometers (vs NIST-certified or traceable thermometer)
■ Liquid-in-glass Yearly
■ Electronic As specified by manufacturer
Timers/clocks Twice yearly
Pipette recalibration Quarterly
Sterile connecting device
■ Weld check Each use
■ Function Yearly
Blood warmers
■ Effluent temperature Quarterly
■ Heater temperature Quarterly
■ Alarm activation Quarterly
(Continued)
 38
AABB TECHNICAL MANUAL
■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents* (Continued)
Frequency of Quality
Equipment and Reagent
Control
Blood collection equipment
Whole blood equipment
■ Agitators Day of use
■ Balances/scales Day of use
■ Gram weight (vs NIST-certified) Yearly
Microhematocrit centrifuge
■ Timer check Quarterly
■ Calibration Quarterly
■ Packed cell volume Yearly
Cell counters/hemoglobinometers Day of use
Blood pressure cuffs Twice yearly
Apheresis equipment
As specified by
■ Checklist requirements
manufacturer
Reagents
Red cells Day of use
Antisera Day of use
Antiglobulin serum Day of use
Transfusion-transmissible disease marker testing Each test run
Miscellaneous
Copper sulfate Day of use
Shipping containers for blood and component transport (usually at
Twice yearly
temperature extremes)
‘The frequencies listed above are suggested intervals, not requirements. For any new piece of equipment,
installation, operational, and performance qualifications must be performed. After the equipment has been
suitably qualified for use, ongoing QC testing should be performed. Depending on the operational and
performance qualification methodology, the ongoing QC may initially be performed more often than the
ultimately desired frequency. Once a record of appropriate in-range QC results has been established (during
either equipment qualification or the ongoing QC), the frequency of testing can be reduced. At a minimum, the
frequency must comply with the manufacturer’s suggested intervals; if no such guidance is provided by the
manufacturer, the intervals given in this table are appropriate to use.
NIST = National Institute of Standards and Technology, QC = quality control.

Chapter 2
Facilities, Work Environment, and Safety

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ)
^RjTHE physical work environment Mean have a significant impact on the safety, efficiency, and
effectiveness of work processes and on the quality of work process outcomes. It should be designed and
managed in a way that meets operational needs and provides for the safety of staff and visitors. The layout of
the physical space; management of utilities, such as water and air ventilation; flow of personnel, materials, and
waste; and ergonomic factors should all be considered in the facility management plan.
In addition to providing adequate facilities, the organization should develop and implement a safety
program that defines policies and procedures for safe work practices and emergency responses. Such a program
also includes requirements for training, hazard communication, use of engineering controls, and protective
equipment. All employees are re
sponsible for protecting their own safety and the safety of others by adhering to policies and procedures set
forth in the facility safety program.
The AABB requires its accredited facilities to plan, implement, and maintain a program to minimize risks to
the health and safety of donors, patients, volunteers, and employees from biological, chemical, and radiological
hazards.1,2 Other professional and accrediting organizations, including the College of American Pathologists
(CAP), the Clinical and Laboratory Standards Institute, and The Joint Commission, have similar or more
detailed safety program requirements.3'6
US federal regulations and recommendations intended to protect the safety of workers and the public in
health-care settings are listed in Appendix 2-1. Appendix 2-1 also lists relevant safety recommendations of
trade and
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice President for Quality and Regulatory Affairs, New
York Blood Center, New York, New York; Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB, Interim Department
Head, Analytical and Diagnostic Sciences, College of Allied Health Sciences and Associate Professor Emerita,
University of Cincinnati, Cincinnati, Ohio; and Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ),
Quality Director, Esoteric Business Unit, Laboratory Corporation of America, Burlington, North Carolina The
authors have disclosed no conflicts of interest.
39

40
AABB TECHNICAL MANUAL
professional associations. The contents of these regulations and guidelines are discussed in more detail in
each section of this chapter. US state and local government regulations may have additional safety
requirements, including architectural and construction safety considerations.
FACILITIES
Facility Design and Workflow
Effective design and maintenance of facilities along with the physical organization of work activities can
help reduce or eliminate many potential hazards. Facility design, workflow, and maintenance also affect process
efficiency, productivity, error rates, employee and customer satisfaction, and the quality of products and
services.
During the design phase for a new or renovated space, the location and flow of personnel, materials, and
equipment should be considered in the context of the processes to be performed. Adequate space must be
allotted for personnel movement, location of supplies and large equipment, and private or distraction-free zones
for certain manufacturing tasks (eg, donor interviewing, record review, and blood component labeling). The
facility must offer designated “clean” and “dirty” spaces and provide for controlled movement of materials and
waste in and out of these areas to avoid contamination. Chemical fume hoods and biological safety cabinets
(BSCs) should be located away from drafts and high-traffic areas. The number and location of eyewash stations
and emergency showers must also be considered in planning. Staff handling hazardous materials must have
ready access to handwashing sinks. In some cases, additional special water sources for reagent preparation must
be provided. The location of very heavy equipment, such as irradiators, should be taken into account to ensure
that the flooring has sufficient load-bearing capacity.
Laboratories must be designed with adequate illumination, electrical power, and conveniently located
electrical outlets. Emergency backup power sources, such as uninterrupt
ible power supplies and backup generators, should be considered to ensure that blood components, cellular
therapy products, and critical reagents are not compromised during power failures. The National Electrical Code
is routinely used as a national guideline for the design of essential electrical distribution systems, with
modifications approved by the local building authority that has jurisdiction.7
Heating, ventilation, and air conditioning must be adequate for the needs of the facility. Environmental
monitoring systems should be considered for laboratories that require positive or negative air pressure
differentials or where air filtration systems are used to control particle levels. The nationally accepted
specifications for ventilation are published by the American Society of Heating, Refrigerating, and Air-
Conditioning Engineers.8
Housekeeping
The workplace should be kept clean and free of clutter. Work surfaces and equipment should be regularly
cleaned and disinfected. Items that may accumulate dust and debris should not be stored above clean supplies or
work surfaces. Exits and fire safety equipment must not be blocked or obstructed in any way. Receptacles and
disposal guidelines for nonhazardous solid waste and biohazardous, chemical, and radiation waste should be
clearly delineated. Housekeeping responsibilities, methods, and schedules should be defined for every work
area. Written procedures, initial training, continuing education of personnel, and ongoing monitoring of
housekeeping effectiveness are essential to safe operations.
Clean Rooms
Clean-room facilities should be considered for open processing activities that cannot be accommodated in a
BSC. Laboratories that process cellular therapy products may choose to adopt clean-room specifications and
maintenance practices to meet the requirements of the Food and Drug Administration (FDA) current good tissue
practice regulations.9
International standards for clean rooms are published by the International Organiza
41
 tion for Standardization and provide specifications for general manufacturing applications to limit airborne
particulates, contaminants, and pollutants.10 These standards also provide guidance for pharmaceutical and
biotechnology applications that include methods to assess, monitor, and control biocontamination.11 Aspects of
a biocontamination-control system include 1) developing air-sampling plans using validated equipment; 2)
assessing biocontamination of surfaces, textiles, and liquids; 3) evaluating laundering processes; and 4)
maintaining personnel training and work practices.
Restricted Areas
Hazardous areas should be clearly and uniformly identified with warning signs in accordance with federal
Occupational Safety and Health Administration (OSHA) and Nuclear Regulatory Commission (NRC) standards
so that personnel entering or working around them are aware of existing biological, chemical, or radiation
dangers.12'15 Staff members not normally assigned to these areas should receive adequate training to avoid
endangering themselves. Risk areas can be stratified. For example, high-risk areas might include those that
contain chemical fume hoods, BSCs, and storage areas for volatile chemicals or radioisotopes. Technical work
areas might be considered moderate risk and restricted to laboratory personnel. Administrative and clerical
areas are generally considered low risk and not restricted. Guidelines for restricted access based on biosafety
levels are published by the US Department of Health and Human Services (DHHS).16 Where possible,
functions not requiring special precautions should be separated from those performed in restricted areas.
Organizations should consider establishing specific safety guidelines for visitors with business in restricted
areas and verifying that safety guidelines have been reviewed before the visitors enter the area. Casual visitors
should not be allowed in restricted areas. Children should not be allowed in areas where they could be exposed
to hazards and should be closely supervised in those areas where their presence is permitted.
Mobile Sites
Mobile blood collection operations can present special challenges. An advance safety survey of the
proposed collection site helps ensure that hazards are minimized.
Responsibility for site safety should be assigned to an individual with adequate knowledge to recognize
safety concerns and the authority to address them in a timely manner. All mobile personnel should be trained to
recognize unsafe conditions and understand how to effectively implement infection-control policies and
procedures in a variety of settings.
Hand-washing access is essential at collection sites. Carpeted or difficult-to-clean surfaces may be covered
using an absorbent overlay with waterproof backing to protect them from possible blood spills. Portable screens
and crowd-control ropes are helpful in directing traffic flow to maintain safe work areas. Food service areas
should be physically separated from areas for blood collection and storage. Blood-contaminated waste must be
either returned to a central location for disposal or packaged and decontaminated in accordance with local
regulations for medical waste.
Ergonomics
Consideration in physical design should be given to ergonomics and to accommodations for individuals
covered under the Americans with Disabilities Act [42 United States Code (USC) Sections 2101-12213,1990],
Several factors may contribute to employee fatigue, musculoskeletal disorder syndromes, or injury, including
the following17:
■ Awkward postures—positions that place stress on the body, such as reaching overhead, twisting, bending,
kneeling, or squatting.
■ Repetition—performance of the same motions continuously or frequently.
■ Force—the amount of physical effort used to perform work.
■ Pressure points—pressing of the body against hard or sharp surfaces.
■ Vibration—continuous or high-intensity hand/arm or whole-body vibration.
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AABB TECHNICAL MANUAL
■ Other environmental factors—extreme high or low temperatures or lighting that is too dark or too bright.
Both the total time per work shift and the length of uninterrupted work periods can make significant
contributions to physical problems. Actions to correct problems associated with ergonomics may include the
following:
■ Engineering improvements to reduce or eliminate the underlying cause, such as making changes to
equipment, workstations, or materials.
 ■ Administrative improvements, such as providing variety in tasks; adjusting work schedules and work
pace; providing recovery or relaxation time; modifying work practices; ensuring regular housekeeping and
maintenance of work spaces, tools, and equipment; and encouraging exercise.
■ Provision of personal protective equipment (PPE), such as gloves, knee and elbow pads, protective
footwear, and other items that employees wear to protect themselves against injury.
SAFETY PROGRAM
An effective safety program starts with a wellthought-out safety plan. This plan identifies the applicable
regulatory requirements and describes how they will be met. A safety plan includes procedures to:
■ Provide a workplace free of recognized hazards.
■ Evaluate all procedures for potential exposure risks.
■ Evaluate each employment position for potential exposure risks.
■ Identify hazardous areas or materials with appropriate labels and signs.
■ Educate staff, document training, and monitor compliance.
■ Apply standard precautions (including universal and blood and body fluid precautions) to the handling of
blood, body fluids, and tissues.
■ Dispose of hazardous waste appropriately.
■ Report incidents and accidents, and provide treatment and follow-up.
■ Provide ongoing review of safety policies, procedures, operations, and equipment.
■ Develop facility-specific plans for disaster preparedness and response, and test these plans at defined
intervals (Chapter 4).
Safety programs should consider the needs of all persons affected by the work environment. Most obvious is
the safety of technical staff members, but potential risks for blood donors, ancillary personnel, volunteers,
visitors, housekeeping staff, and maintenance and repair workers must also be evaluated. Appropriate
provisions must be applied if these individuals cannot be excluded from risk areas.
Laboratories should consider appointing a safety officer who can provide general guidance and expertise.4
Typical duties of a safety officer are to develop the safety program, oversee orientation and training, perform
safety audits, survey work sites, recommend changes, and serve on or direct the activities of safety committees.
Facilities using hazardous chemicals and radioactive materials often assign specially trained individuals to
oversee chemical and radiation protection programs as needed.12,15 Five basic elements must be addressed for
each type of hazard covered in the safety program:
■ Training.
■ Hazard identification and communication.
■ Engineering controls and PPE.
■ Safe work practices, including waste disposal.
■ Emergency response plan.
Management controls should be established to ensure that these elements are implemented and maintained
and that they are effective. Management is responsible for the following:
■ Developing and communicating the written plan.
43
■ Ensuring implementation and providing adequate resources.
■ Providing access to employee health services related to prevention strategies and treatment of exposures.
■ Monitoring compliance and effectiveness.
■ Evaluating and improving the safety plan.
Basic Elements of a Safety Program
Training
Employees must be trained to recognize the hazards in their workplace and take appropriate precautions.
Supervisors are responsible for assessing and documenting each employee’s understanding of and ability to
apply safety precautions before independent work is permitted. Safety training must precede even
temporary work assignments if significant potential for exposure exists. Staff members who do not
demonstrate the requisite understanding and skills must receive additional training. Training should be provided
not only to laboratory staff, but also to housekeeping and other personnel who may come into contact with
hazardous substances or waste. Table 2-1 lists topics to cover in work safety training programs.
Hazard Identification and Communication
 Employers are required to provide information about workplace hazards to their staff to help reduce the risk
of occupational illnesses and injuries. Staff need to know what hazardous
TABLE 2-1. Topics to Cover in a Work Safety Training Program
Work safety
training programs
should ensure that all
personnel:
Have access to a copy of pertinent regulatory texts and an explanation of the
■
contents.
Understand the employer's exposure control plan and how to obtain a copy of the
■
written plan.
Understand how hepatitis and human immunodeficiency virus (HIV) are
■ transmitted and how often; be familiar with the symptoms and consequences of
hepatitis B virus (HBV), hepatitis C virus, and HIV infection.
■ Know that they are offered vaccination against HBV.
■ Recognize tasks that pose infectious risks, and distinguish them from other duties.
Know what protective clothing and equipment are appropriate for the procedures
■
they will perform and how to use them.
Know and understand the limitations of protective clothing and equipment (eg,
■ different types of gloves are recommended according to the permeability of the
hazardous material to be used).
■ Know where protective clothing and equipment are kept.
Become familiar with and understand all requirements for work practices specified
■ in standard operating procedures for the tasks they perform, including the meaning of
signs and labels.
Know how to remove, handle, decontaminate, and dispose of contaminated
■
material.
Know the appropriate actions to take and the personnel to contact if exposed to
■
blood or other biologic, chemical, or radiologic hazards.
Know the corrective actions to take in the event of spills or personal exposure to
■ fluids, tissues, and contaminated sharp objects; appropriate reporting procedures; and
medical monitoring recommended when parenteral exposure may have occurred.
■ Know their right to access to medical treatment and medical records.
■ Know fire safety procedures and evacuation plans.
 44
AABB TECHNICAL MANUAL
substances they are working with and where those materials are located in the facility. This communication
is achieved by means of signage, labels on containers, written information, and training programs.
Engineering Controls and PPE
If the physical workspace cannot be designed to eliminate the potential for exposure to hazards, appropriate
protective gear must be provided. Engineering controls are physical plant controls or equipment, such as
sprinkler systems, chemical fume hoods, and needleless systems, that isolate or remove the hazard from the
workplace.
PPE is specialized clothing or equipment, such as gloves, masks, and laboratory coats, worn by employees
for protection against a hazard. Employees should remove their PPE, such as gloves and laboratory coats, and
should wash their hands with soap and water when leaving a laboratory area. General guidance on the use of
engineering controls and PPE is included in Appendix 2-2.
Safe Work Practices
Employees must be trained in how to work with hazardous materials in ways that protect themselves, their
coworkers, and the environment. Safe work practices are defined as tasks performed in a manner that reduces
the likelihood of exposure to workplace hazards. General recommendations for safe work practices are included
in Appendix 2-2.
Emergency Response Plan
When engineering and work practice controls fail, employees must know how to respond promptly and
appropriately. The purpose of advance planning is to control a hazardous situation as quickly and safely as
possible. Regular testing of the emergency response plan identifies areas for improvement and builds staff
confidence in their ability to respond effectively in a real emergency. OSHA requires facilities with more than
10 employees to have a written emergency response plan. Verbal
communication of the plan is acceptable for facilities with 10 or fewer employees.18
Management Controls
Supervisory personnel must monitor safety practices in their areas of responsibility. Continuing attention to
safety issues should be addressed in routine staff meetings and training sessions. Periodic audits performed by a
safety professional increase safety awareness. Management should seek staff input on the design and
improvement of the facility’s safety plan.
The safety program policies, procedures, guidelines, and supporting references should be documented in
writing and made available to all personnel at risk. These documents should be reviewed on a regular basis and
updated as technology evolves and new information becomes available. Work sites and safety equipment should
be inspected regularly to ensure compliance and response readiness. Checklists may be helpful for documenting
safety inspections and assessing safety preparedness.3,4,19
Employee Health Services
Hepatitis Prophylaxis
All employees routinely exposed to blood must be offered hepatitis B virus (HBV) vaccine if they do not
already have HBV-protective antibodies (ie, anti-HBs). OSHA requires that the vaccine be offered at no cost to
all employees and, if any employee refuses the vaccine, that the refusal be documented.14
 Mon itoring Programs
Employers must provide a system for monitoring exposure to certain substances as defined in the OSHA
standard if there is reason to believe that exposure levels routinely exceed the recommended action level.20
Medical First Aid and Follow-Up
When requested by a worker who has sustained known or suspected blood exposure, monitoring for HBV,
hepatitis C virus (HCV), and human immunodeficiency virus (HIV) in
45
fection should be provided with appropriate counseling. In some states, consent is required for this
voluntary testing; rejection of offered testing must be documented. The usual schedule includes immediate tests
of the worker and the source of the potentially infectious material, with follow-up testing of the worker at
intervals after exposure.13,14 All aspects of accident follow-up should be appropriately documented.
The Centers for Disease Control and Prevention (CDC) has published recommendations for both pre-
exposure and post-exposure prophylaxis if the contaminating material is HBV-positive or if this information is
unknown.21 HBV immune globulin is usually given concurrently with HBV vaccine in cases of penetrating
injuries. When administered in accordance with the manufacturer’s directions, both products are very safe and
carry no documented risk of infection with HBV, HCV, or HIV.21 Post-exposure prophylaxis for HIV is
continually evolving; policies are generally based on Public Health Service recommendations and current
standards of practice.
Reporting Accidents and Injuries
When an injury occurs, relevant information should be documented, including the date, time, and place
where the injury occurred; the nature of the injury; descriptions of what happened from the injured person and
any witnesses; and first aid or medical attention provided. The supervisor should complete any accident reports
and investigation forms required by the institution’s insurer and worker’s compensation agencies. Employers
must report fatalities and injuries resulting in the hospitalization of three or more employees to OSHA within 8
hours of the accident.22
OSHA requires health service employers with 11 or more workers to maintain records of occupational
injuries and illnesses requiring a level of care that exceeds the capabilities of a person trained in first aid.23
Initial documentation must be completed within 6 days of the incident. Records of first aid provided by a
nonphysician for minor injuries, such as cuts or burns, do not need to be retained. All logs,
summaries, and supplemental records must be preserved for at least 5 years beyond the calendar year of
occurrence. Medical records of employees should be preserved for the duration of employment plus 30 years,
with few exceptions.24
Latex Allergies
Adverse reactions associated with latex, powdered gloves, or both include contact dermatitis, allergic
dermatitis, urticaria, and anaphylaxis. Medical devices that contain latex must bear a caution label. The National
Institute for Occupational Safety and Health (NIOSH) offers the following recommendations to prevent these
allergic reactions25:
■ Make latex-free gloves available as an alternative to latex, and encourage the use of latex-free gloves for
activities and work environments where there is minimal risk of exposure to infectious materials.
■ If latex gloves are used, consider providing reduced-protein and powder-free gloves.
■ Use good housekeeping practices to remove latex-containing dust from the workplace.
■ Use work practices that reduce the chance of reaction, such as hand washing and avoiding oil-based hand
lotions.
■ Educate workers about latex allergy.
■ Evaluate current prevention strategies.
■ Periodically screen high-risk workers for latex allergy symptoms.
■ If symptoms develop, have workers avoid direct contact with latex and consult a physician about allergy
precautions.
FIRE PREVENTION
Fire prevention relies on a combination of facility design that is based on the National Fire Protection
Association (NFPA) Life Safety Code, which identifies processes to maintain fire protection systems in good
working order, and fire safe-work practices.26 The Life Safety Code includes both active and passive
fireprotection systems (eg, alarms, smoke detectors, sprinklers, exit lights in corridors, and fire-rated barriers).
46
 AABB TECHNICAL MANUAL
Training
Fire safety training is recommended at the start of employment and at least annually thereafter. Training
should emphasize prevention and an employee’s awareness of the work environment, including how to
recognize and report unsafe conditions, how to report fires, where the nearest alarm and fire-containment
equipment are located and how to use it, and what the evacuation policies and routes are.
All staff members in facilities accredited by CAP or The Joint Commission are required to participate in fire
drills at least annually.3,5 In areas where patients are housed or treated, The Joint Commission requires
quarterly drills on each shift. Staff participation and understanding should be documented.
Hazard Identification and Communication
Emergency exits must be clearly marked with an exit sign. Additional signage must be posted along the exit
route to show the direction of travel if it is not immediately apparent. All flammable materials should be labeled
with appropriate hazard warnings, and flammable storage cabinets should be clearly marked.
Engineering Controls and PPE
Laboratories storing large volumes of flammable chemicals are usually built with 2-hour fire separation
walls, or with 1-hour separation walls if there is an automatic fire-extinguishing system.4 Fire detection and
alarm systems should be provided in accordance with federal, state, and local regulations. All fire equipment
should be inspected on a regular basis to ensure that it is in good working order. Fire extinguishers should be
readily available, and the staff should be trained to use them properly. Housekeeping and inventory management
plans should be designed to control the accumulations of flammable and combustible materials stored in the
facility. In areas where sprinkler systems are installed, all items should be stored at least 18 inches below the
sprinkler heads. Facilities should consult local
fire codes, which may require greater clearance.
Safe Work Practices
Emergency exit routes must be clear of anything that would obstruct evacuation efforts. Exit doors must not
be locked in such a way as to impede egress. Permanent exit routes must be designed to allow free and
unobstructed exit from all parts of the facility to an area of safety. Secondary exits may be required for areas
larger than 1000 square feet; facilities should consult local safety authorities with jurisdiction, such as the local
fire marshal and NFPA, for guidance on secondary exits.
Emergency Response Plan
The fire emergency response plan should encompass both facility-wide and area-specific situations. It
should describe reporting and alarm systems; location and use of emergency equipment; roles and
responsibilities of the staff during the response; “defend-in-place” strategies; and conditions for evacuation,
evacuation procedures, and exit routes.5,18
When a fire occurs, the general sequence for immediate response should be to 1) rescue anyone in
immediate danger; 2) activate the fire alarm system and alert others in the area; 3) confine the fire by closing
doors and shutting off fans or other oxygen sources if possible; and 4) extinguish the fire with a portable
extinguisher if the fire is small, or evacuate if it is too large to manage.
ELECTRICAL SAFETY
Electrical hazards, including fire and shock, may arise from the use of faulty electrical equipment; damaged
receptacles, connectors, or cords; or unsafe work practices. Proper use of electrical equipment, periodic
inspection and maintenance, and hazard recognition training are essential to help prevent accidents that may
result in electric shock or electrocution. The severity of shock depends on the path that the electrical current
takes through the body, the amount of current flowing
47
through the body, and the length of time that current is flowing through the body. Even lowvoltage
exposures can lead to serious injury.27
Training
Safety training should be designed to make employees aware of electrical hazards associated with
receptacles and connectors. This training should also help them recognize potential problems, such as broken
receptacles and connectors, improper electrical connections, damaged cords, and inadequate grounding.
Hazard Identification and Communication
The safety plan should address the proper use of receptacles and connectors. Equipment that does not meet
safety standards should be marked to prevent accidental use.
 Engineering Controls and PPE
OSHA requires that electrical systems and equipment be constructed and installed in a way that minimizes
the potential for workplace hazards. When purchasing equipment, the facility should verify that it bears the
mark of an OSHA-approved independent testing laboratory, such as Underwriters Laboratories.28 Adequate
working space should be provided around equipment to allow easy access for safe operation and maintenance.
Groundfault circuit interrupters should be installed in damp or wet areas.
Safe Work Practices
Electrical safety practices focus on two factors: 1) proper use of electrical equipment and 2) proper
maintenance and repair of this equipment. Staff should not plug or unplug equipment from an electrical source
with wet hands. Overloading circuits with too many devices may cause the current to heat the wires to a very
high temperature and generate a fire. Damaged receptacles and faulty electrical equipment must be tagged and
removed from service until they have been repaired and
checked for safety. Flexible cords should be secured to prevent tripping and should be protected from
damage from heavy or sharp objects. Flexible cords should be kept slackened to prevent tension on electrical
terminals, and cords should be checked regularly for cut, broken, or cracked insulation. Extension cords should
not be used in lieu of permanent wiring.
Emergency Response Plan
In case of an emergency in which it is not possible to decrease the power or disconnect equipment, the
power supply should be shut off from the circuit breaker. If it is not possible to interrupt the power supply, a
nonconductive material, such as dry wood, should be used to pry a victim from the source of current.27 Victims
must not be touched directly. Emergency first aid for victims of electrical shock must be sought. Water-based
fire extinguishers should not be used on electrical fires.
BIOSAFETY
The facility must define and enforce measures to minimize the risk of exposure to biohazardous materials in
the workplace. Requirements published by OSHA (Blood-borne Pathogens Standard) and recommendations
published by the US DHHS provide the basis for an effective biosafety plan.13,14,16
Blood-Borne Pathogens Standard
The OSHA Blood-Borne Pathogens Standard is intended to protect employees in all occupations where
there is a risk of exposure to blood and other potentially infectious materials. It requires the facility to develop
an exposure control plan and describes appropriate engineering controls, PPE, and work practice controls to
minimize the risk of exposure. The standard also requires employers to provide HBV vaccinations to any staff
members with occupational exposure, provide medical follow-up care in case of accidental exposure, and keep
records related to accidents and exposures.
48
AABB TECHNICAL MANUAL
Standard Precautions
Standard precautions represent the most current recommendations by the CDC to reduce the risk of
transmission of blood-borne pathogens and other pathogens in hospitals. Originally published in 1996 in the
Guidelines for Isolation Precautions in Hospitals, standard precautions build on earlier recommendations,
including body substance isolation (1987), universal precautions (1986), and blood and body fluid precautions
(1983).13 Standard precautions apply to all patient care activities, regardless of diagnosis, in which there is a
risk of exposure to 1) blood; 2) all body fluids, secretions, and excretions, except sweat; 3) nonintact skin; or 4)
mucous membranes.
The OSHA Blood-Borne Pathogens Standard refers to the use of universal precautions. However, OSHA
recognizes the more recent guidelines from the CDC and, in Directive CPL 02-02-069, allows hospitals to use
acceptable alternatives, including standard precautions, as long as all other requirements in the standard are
met.29
Biosafety Levels
Recommendations for biosafety in laboratories are based on the potential hazards pertaining to specific
infectious agents and the activities performed.16 Biosafety recommendations include guidance on both
engineering controls and safe work practices. The four biosafety levels are designated in ascending order, with
increasing protection for personnel, the environment, and the community:
■ Biosafety Level 1 (BSL-1) involves work with agents of no known or of minimal potential hazard to
laboratory personnel and the environment. Activities are usually conducted on open surfaces, and no
containment equipment is needed.
■ BSL-2 work involves agents of moderate potential hazard to personnel and the environment, usually from
contact-associated exposure. Most blood bank laboratory activities are considered BSL-2.
■ BSL-3 includes work with indigenous or exotic agents that may cause serious or potentially lethal disease
as a result of exposure to aerosols (eg, Mycobacterium tuberculosis) or by other routes (eg, HIV) that would
result in grave consequences to the infected host. Recommendations for BSL-3 work are designed to contain
biohazardous aerosols and minimize the risk of surface contamination.
■ BSL-4 applies to work with dangerous or exotic agents that pose high individual risk of life-threatening
disease from aerosols (eg, agents of hemorrhagic fevers or filoviruses). BSL-4 is not applicable to routine
blood-bank-related activities.
The precautions described in this section focus on BSL-2 requirements. Laboratories should consult the
CDC or National Institutes of Health guidelines for precautions appropriate for higher levels of containment.
Training
OSHA requires annual training for all employees whose tasks increase their risk of infectious
exposure.14,29 Training programs must be tailored to the target group both in level and content. General
background knowledge of biohazards, understanding of control procedures, or work experience cannot meet the
requirement for specific training, although an assessment of such knowledge is a first step in planning program
content. Workplace volunteers require at least as much safety training as paid staff members who perform
similar functions.
Hazard Identification and Communication
The facility’s exposure control plan communicates the risks present in the workplace and describes controls
to minimize exposure. BSL2 through BSL-4 facilities must have a biohazard sign posted at the entrance when
infectious agents are in use. The sign notifies personnel and visitors of the presence of infectious agents,
provides a point of contact for
49
the area, and indicates any special protective equipment or work practices required.
Biohazard warning labels must be placed on containers of regulated waste; refrigerators and freezers
containing blood or other potentially infectious material; and other containers used to store, transport, or ship
blood or other potentially infectious materials. Blood components that are labeled to identify their contents and
have been released for transfusion or other clinical use are exempted.
Engineering Controls and PPE
OSHA requires that hazards be controlled by engineering or work practices whenever possible.14
Engineering controls for BSL-2 laboratories include limited access to the laboratory when work is in progress
and BSCs or other containment equipment for work that may involve infectious aerosols or splashes,
Handwashing sinks and eyewash stations must be available. The work space should be designed so that it can
be easily cleaned, and bench tops should be impervious to water and resistant to chemicals and solvents.
To help prevent exposure or cross-contamination, work area telephones can be equipped with speakers to
eliminate the need to pick up the receiver. Computer keyboards and telephones can be covered with plastic.
Such equipment should be cleaned on a regular basis and when visibly soiled.
BSCs are primary containment devices for handling moderate-risk and high-risk organisms. There are three
types—Classes I, II, and III—with Class III providing the highest protection to workers. In addition to
protecting personnel during the handling of biohazardous materials, a BSC may be used to prevent
contamination of blood and cellular therapy products during open processing steps. A comparison of the
features and applications of the three classes of cabinets is provided in Table 2-2.
BSCs are not required by standard precautions, but centrifugation of open blood samples or manipulation of
units known to be positive for HBV surface antigen or HIV are ex
amples of blood bank procedures for which a BSC could be useful. The effectiveness of the BSC is a
function of directional airflow inward and downward through a high-efficiency filter. Efficacy is reduced by
anything that disrupts the airflow pattern (eg, arms moving rapidly in and out of the BSC, rapid movements
behind an employee using the BSC, downdrafts from ventilation systems, or open laboratory doors). Care
should be taken not to block the front intake and rear exhaust grills. Performance of the BSC should be certified
annually.31
Injuries from contaminated needles and other sharp objects (sometimes called “sharps”) continued to be a
major concern in health-care settings even after the BloodBorne Pathogens Standard went into effect. In 2001,
OSHA revised the standard to refer to engineered sharps injury protections and needleless systems.14 The
revised standard requires that employers implement appropriate new control technologies and safer medical
devices in exposure control plans and that employers solicit input from their employees to identify, evaluate,
and select engineering and work practice controls. Examples of safer devices are needleless systems and self-
sheathing needles in which the sheath is an integral part of the device.
Decon tam ination
Reusable equipment and work surfaces that may be contaminated with blood require daily cleaning and
decontamination. Obvious spills on equipment or work surfaces should be cleaned up immediately; routine
wipe-downs with disinfectant should occur at the end of each shift or a different frequency that provides
equivalent safety. Equipment that is exposed to blood or other potentially infectious material must be
decontaminated before it is serviced or shipped. When decontamination of all or a portion of the equipment is
not feasible, a biohazard label stating which portions remain contaminated should be attached before the
equipment is serviced or shipped.
50
AABB TECHNICAL MANUAL
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AABB TECHNICAL MANUAL
Choice of Disinfectants
 The Environmental Protection Agency (EPA) maintains a list of chemical products that have been shown to
be effective hospital antimicrobial disinfectants.32 The Association for Professionals in Infection Control and
Epidemiology also publishes a guideline to assist health-care professionals with decisions involving judicious
selection and proper use of specific disinfectants.33 For facilities covered under the Blood-Borne Pathogens
Standard, OSHA allows the use of EPA-registered tuberculocidal disinfectants, EPA-registered disinfectants
that are effective against both HIV and HBY a diluted bleach solution to decontaminate work surfaces, or a
combination of these.29
Before selecting a disinfectant product, workers should consider several factors. Among them are the type
of material or surface to be treated; the hazardous properties of the chemical, such as corrosiveness; and the
level of disinfection required. After a product has been selected, procedures need to be written to ensure
effective and consistent cleaning and treatment of work surfaces. Some factors to consider for effective
decontamination include contact time, type of microorganisms, presence of organic matter, and concentration of
the chemical agent. Workers should review the basic information on decontamination and follow the
manufacturer’s instructions.
Storage
Hazardous materials must be segregated, and areas for different types of storage must be clearly
demarcated. Blood must be protected from unnecessary exposure to other materials and vice versa. If
transfusion products cannot be stored in a separate refrigerator from reagents, specimens, and unrelated
materials, areas within the refrigerator must be clearly labeled, and extra care must be taken to reduce the
likelihood of spills and other accidents. Storage areas must be kept clean and orderly; food or drink is never
allowed where biohazardous materials are stored.
PPE
When hazards cannot be eliminated, OSHA requires employers to provide appropriate PPE and clothing and
to clean, launder, or dispose of PPE at no cost to their employees.14 Standard PPE and clothing include
uniforms, laboratory coats, gloves, face shields, masks, and safety goggles. Indications and guidelines for their
use are discussed in Appendix 2-2.
Safe Work Practices
Safe work practices appropriate for standard precautions include the following:
■ Wash hands after touching blood, body fluids, secretions, excretions, and contaminated items, whether or
not gloves are worn.
■ Wear gloves when touching blood, body fluids, secretions, excretions, and contaminated items, and
change gloves between tasks.
■ Wear a mask and eye protection or a face shield during activities that are likely to generate splashes or
sprays of blood, body fluids, secretions, and excretions.
■ Wear a gown during activities that are likely to generate splashes or sprays of blood, body fluids,
secretions, or excretions.
■ Handle soiled patient-care equipment in a manner that prevents exposure; ensure that reusable equipment
is not used for another patient until it has been cleaned and reprocessed appropriately; and ensure that single-use
items are discarded properly.
■ Ensure that adequate procedures are defined and followed for the routine care, cleaning, and disinfection
of environmental surfaces and equipment.
■ Handle soiled linen in a manner that prevents exposure.
■ Handle needles, scalpels, and other sharp instruments or devices in a manner that minimizes the risk of
exposure.
■ Use mouthpieces, resuscitation bags, or other ventilation devices as an alternative to mouth-to-mouth
resuscitation methods.
■ Place in a private room patients who are at risk of contaminating the environment or
53
are not able to maintain appropriate hygiene (eg, patients with tuberculosis).
Laboratory Biosafety Precautions
Several factors need to be considered when assessing the risk of blood exposures among laboratory
personnel. Some of these factors include the number of specimens processed, personnel behaviors, laboratory
techniques, and type of equipment.34 The laboratory director may wish to institute BSL-3 practices for
procedures that are considered to be higher risk than BSL-2. When there is doubt whether an activity is BSL-2
or BSL-3, the safety precautions for BSL-3 should be followed. BSL-2 precautions that are applicable to the
laboratory setting are summarized in Appendix 2-3.
Considerations for the Donor Room
The Blood-Borne Pathogens Standard acknowledges a difference between hospital patients and healthy
donors, in whom the prevalence of infectious disease markers is significantly lower. The employer in a
volunteer blood donation facility may determine that routine use of gloves is not required for phlebotomy as
long as the following conditions exist14:
■ The policy is periodically reevaluated.
■ Gloves are made available to those who want to use them, and their use is not discouraged.
■ Gloves are required when an employee has cuts, scratches, or breaks in skin; when there is a likelihood
that contamination will occur; while an employee is drawing autologous units; while an employee is performing
therapeutic procedures; and during training in phlebotomy.
Procedures should be assessed for risks of biohazardous exposures and risks inherent in working with a
donor or patient during the screening and donation processes. Some techniques or procedures are more likely to
cause injury than others, such as using lancets for finger puncture, handling capillary tubes, crushing vials for
arm cleaning, handling any
unsheathed needles, cleaning scissors, and giving cardiopulmonary resuscitation.
In some instances, it may be necessary to collect blood from donors known to pose a high risk of infectivity
(eg, collection of autologous blood or source plasma for the production of other products, such as vaccines).
The FDA provides guidance on collecting blood from such “high-risk” donors.35,36 The most recent
regulations and guidelines should be consulted for changes or additions.
Emergency Response Plan
Table 2-3 lists steps to take when a spill occurs. Facilities should be prepared to handle both small and large
blood spills. Good preparation for spill cleanup includes several elements:
■ Work areas designed so that cleanup is relatively simple.
■ A spill kit or cart containing all necessary supplies and equipment with instructions for their use. It should
be placed near areas where spills are anticipated.
■ Responsibility assigned for kit or cart maintenance, spill handling, record-keeping, and review of
significant incidents.
■ Personnel trained in cleanup procedures and procedures for reporting significant incidents.
Biohazardous Waste
Medical waste is defined as any waste (solid, semisolid, or liquid) generated in the diagnosis, treatment, or
immunization of human beings or animals in related research, production, or testing of biologies. Infectious
waste includes disposable equipment, articles, or substances that may harbor or transmit pathogenic organisms
or their toxins. In general, infectious waste should be either incinerated or decontaminated before disposal in a
sanitary landfill.
If state law allows, blood and components, suctioned fluids, excretions, and secretions may be carefully
poured down a drain connected to a sanitary sewer. Sanitary sewers may also be used to dispose of other
potentially infectious wastes that can be ground and
54
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TABLE 2-3. Blood Spill Cleanup Steps
■ Evaluate the spill.
■ Wear appropriate protective clothing and gloves. If sharp objects are involved, gloves must be puncture
resistant, and a broom or other instrument should be used during cleanup to avoid injury.
■ Remove clothing if it is contaminated.
■ Post warnings to keep the area clear.
■ Evacuate the area for 30 minutes if an aerosol has been created.
■ Contain the spill if possible.
■ If the spill occurs in the centrifuge, turn the power off immediately and leave the cover on for 30 minutes.
The use of overwraps helps prevent aerosolization and contain the spill.
■ Use absorbent material to mop up most of the liquid contents.
■ Clean the spill area with detergent.
 ■ Flood the area with disinfectant and use it as described in the manufacturer’s instructions. Allow adequate
contact time with the disinfectant.
■ Wipe up residual disinfectant if necessary.
■ Dispose of all materials safely in accordance with biohazard guidelines. All blood-contaminated items
must be autoclaved or incinerated.
flushed into the sewer. State and local health departments should be consulted about laws and regulations
pertaining to disposal of biologic waste into the sewer.
Laboratories should clearly define what will be considered hazardous waste. For example, in the blood
bank, all items contaminated with liquid or semiliquid blood are biohazardous. Items contaminated with dried
blood are considered hazardous if there is potential for the dried material to flake off during handling.
Contaminated sharp objects are always considered hazardous because of the risk for percutaneous injury.
However, items such as used gloves, swabs, plastic pipettes with excess liquid removed, or gauze contaminated
with small droplets of blood maybe considered nonhazardous if the material is dried and will not be released
into the environment during handling.
Guidelines for Biohazardous Waste Disposal
Employees must be trained before handling or disposing of biohazardous waste, even if it is
packaged. The following disposal guidelines are recommended37:
■ Identify biohazardous waste consistently; red seamless plastic bags (at least 2 mm thick) or containers
carrying the biohazard symbol are recommended.
■ Place bags in a protective container with closure upward to avoid breakage and leakage during storage or
transport.
■ Prepare and ship waste transported over public roads according to US Department of Transportation
(DOT) regulations.
■ Discard sharps (eg, needles, broken glass, glass slides, and wafers from sterile connecting devices) in
rigid, puncture-proof, leakproof containers.
■ Put liquids in leakproof, unbreakable containers only.
■ Do not compact waste materials.
Storage areas for infectious material must be secured to reduce accident risk. Infectious waste must never be
placed in the public trash collection system. Most facilities hire private
55
carriers to decontaminate and dispose of infectious or hazardous waste. The facility should disclose all risks
associated with the waste in their contracts with private companies. The carrier is responsible for complying
with all federal, state, and local laws for biohazardous (medical) waste transport, treatment, and disposal.
Treating Infectious or Medical Waste
Facilities that incinerate hazardous waste must comply with EPA standards of performance for new
stationary sources and emission guidelines for existing sources.38 In this regulation, a
hospital/medical/infectious waste incinerator is any device that combusts any amount of hospital waste or
medical/infectious waste.
Decontamination of biohazardous waste by autoclaving is another common method for decontamination or
inactivation of blood samples and blood components. The following elements are considered in determining
processing time for autoclaving:
■ Size of load being autoclaved.
■ Type of packaging of item(s) being autoclaved.
■ Density of items being autoclaved.
■ Number of items in a single autoclave load.
■ Placement of items in the autoclave to allow for steam penetration.
It is useful to place a biologic indicator in the center of loads that vary in size and contents to evaluate
optimal steam penetration times. The EPA provides detailed information about choosing and operating such
equipment.37
For decontamination, material should be autoclaved for a minimum of 1 hour. For sterilization, longer
treatment times are needed. A general rule for decontamination is to process for 1 hour for every 10 pounds of
waste. Usually, decontaminated laboratory wastes can be disposed of as nonhazardous solid wastes. The staff
should check with the local solid waste authority to ensure that the facility is in compliance with regulations for
the area. Waste
 containing broken glass or other sharp items should be disposed of using a method consistent with policies
for the disposal of other sharp or potentially dangerous materials.
CHEMICAL SAFETY
One of the most effective preventive measures that a facility can take to reduce hazardous chemical
exposure is to choose alternative nonhazardous chemicals whenever possible. When the use of hazardous
chemicals is required, purchasing these supplies in small quantities reduces the risks associated with storing
excess chemicals and then dealing with their disposal.
OSHA requires that facilities using hazardous chemicals develop a written chemical hygiene plan (CHP)
and that the plan be accessible to all employees. The CHP should outline procedures, equipment, PPE, and work
practices that are capable of protecting employees from hazardous chemicals used in the facility.15,20 The CHP
must also provide assurance that equipment and protective devices are functioning properly and that criteria to
determine implementation and maintenance of all aspects of the plan are in place. Employees must be informed
of all chemical hazards in the workplace and be trained to recognize chemical hazards, protect themselves when
working with these chemicals, and know where to find information about particular hazardous chemicals.
Safety audits and annual reviews of the CHP are important control steps to help ensure that safety practices
comply with the policies set forth in the CHP and that the CHP is up to date.
Establishing a clear definition of what constitutes hazardous chemicals is sometimes difficult. Generally,
hazardous chemicals pose a significant health risk if an employee is exposed to them or a significant physical
risk, such as fire or explosion, if they are handled or stored improperly. Categories of health and physical
hazards are listed in Tables 2-4 and 2-5. The NIOSHPocket Guide to Chemical Hazards provides a quick
reference for many common chemicals.39
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TABLE 2-4. Categories of Health Hazards
Hazard Definition
Carcinogens Cancer-producing substances
Agents causing irritation (eg, edema or burning) to skin or mucous membranes upon
Irritants
contact
Corrosives Agents causing destruction of human tissue at the site of contact
Toxic or
Substances causing serious biologic effects following inhalation, ingestion, or skin
highly toxic
contact with relatively small amounts
agents
Reproductive Chemicals that affect reproductive capabilities, including chromosomal damages and
toxins effects on fetuses
Hepatotoxins; nephrotoxins; neurotoxins; agents that act on the hematopoietic system;
Other toxins
and agents that damage the lungs, skin, eyes, or mucous membranes
The facility should identify a qualified chemical hygiene officer to be responsible for developing guidelines
for hazardous materials.20 The chemical hygiene officer is also accountable for monitoring and documenting
accidents and for initiating process change as needed.
Training
Employees who may be exposed to hazardous chemicals must be trained before they begin work in an area
in which hazards exist. If a new employee has received prior training, it may not be necessary to retrain the
individual, depending on the employer’s evaluation of the new employee’s level of knowledge. New employee
training is likely to be necessary regarding such specifics as the location of each relevant material safety data
sheet (MSDS), details on chemical labeling, PPE to be used, and sitespecific emergency procedures.
Training must be provided for each new physical or health hazard when it is introduced into the workplace
but not for each new chemical that falls within a particular hazard class.15 For example, if a new solvent is
brought into the workplace and the solvent has hazards similar to existing chemicals for which training has
already been conducted, then the employer need only make employees aware of the
new solvent’s hazard category (eg, corrosive or irritant). However, if the newly introduced solvent is a
suspected carcinogen and carcinogenic hazard training has not been provided, then new training must be
conducted for employees with potential exposure. Retraining is advisable as often as necessary to ensure that
employees understand the hazards linked to the materials with which they work, particularly any chronic and
specific target-organ health hazards.
Hazard Identification and Communication
Hazard Communication
Employers must prepare a comprehensive hazard communication program for all areas in which hazardous
chemicals are used to complement the CHP and “ensure that the hazards of all chemicals produced or imported
are classified, and that information concerning the classified hazard is transmitted to employers and
employees.”15 The program should include labeling of hazardous chemicals, instructions on when and how to
post warning labels for chemicals, directions for managing MSDS reports for hazardous chemicals in the
facilities, and employee training. Safety materials made available to employees should include the following:

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TABLE 2-5. Categories of Physical Hazards
Hazard Definition
Combustible or Chemicals that can burn (including combustible and flammable liquids,
flammable chemicals solids, aerosols, and gases)
Compressed gases Gases or mixtures of gases in a container under pressure
Unstable or reactive chemicals that undergo violent chemical changes at
Explosives
normal temperatures and pressure
Unstable (reactive) Chemicals that could be self-reactive under certain conditions (shocks,
chemicals pressure, or temperature)
Chemicals that react with water to release a gas that either is flammable or
Water-reactive chemicals
presents a health hazard
■ The facility’s written CHP.
■ The facility’s written program for hazard communication.
■ Identification of work areas where hazardous chemicals are located.
■ Required list of hazardous chemicals and the relevant MSDS. (It is the responsibility of the facility to
determine which chemicals may present a hazard to employees. This determination should be based on the
quantity of chemical used; physical properties, potency, and toxicity of the chemical; manner in which the
chemical is used; and means available to control the release of, or exposure to, the chemical.)
Hazardous Chemical Labeling and Signs
The Hazard Communication Standard requires manufacturers of chemicals and hazardous materials to
provide the user with basic information about the hazards of these materials through product labeling and the
MSDS.15 Employers are required to provide employees who are expected to work with these hazardous
materials with information about what the hazards of the materials are, how to read the labeling, how to
interpret symbols and signs on the labels, and how to read and use the MSDS.
MSDS forms typically include the following:
■ Identification.
■ Hazard(s) identification.
■ Composition/information on ingredients.
 ■ First-aid measures.
■ Fire-fighting measures.
■ Accidental release measures.
■ Handling and storage considerations.
■ Exposure controls/personal protection information.
■ Physical and chemical properties.
■ Stability and reactivity.
■ Toxicologic information.
■ Ecologic information.
■ Disposal considerations.
■ Transport information.
■ Regulatory information.
■ Other information.
At a minimum, hazardous chemical container labels must include the name of the chemical, name and
address of the manufacturer, hazard warnings, symbols, designs, and other forms of warning to provide visual
reminders of specific hazards. The label may refer to any MSDS for additional information. Labels applied by
the manufacturer must remain on containers. The user may add storage requirements and dates of receipt,
opening, and expiration. If chemicals are aliquotted into

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secondary containers, the secondary container must be labeled with the name of the chemical and
appropriate hazard warnings. Additional information, such as precautionary measures, concentration if
applicable, and date of preparation, are helpful but not mandatory.
It is a safe practice to label all containers with their content, even water. Transfer containers used for
temporary storage need not be labeled if the person performing the transfer retains control and intends the
containers to be used immediately. Information regarding acceptable standards for hazard communication
labeling is provided by the NFPA40 and the National Paint and Coatings Association.41
Signs meeting OSHA requirements must be posted in areas where hazardous chemicals are used. Decisions
about where to post warning signs are based on the manufacturer’s recommendations regarding the chemical
hazards, the quantity of the chemical in the room or laboratory, and the potency and toxicity of the chemical.
MSDS
The MSDS identifies the physical and chemical properties of a hazardous chemical (eg, flash point or vapor
pressure), its physical and health hazards (eg, potential for fire, explosion, and signs and symptoms of
exposure), and precautions for the chemical’s safe handling and use. Specific instructions in an individual
MSDS take precedence over generic information in the hazardous materials program.
Employers must maintain copies of each required MSDS in the workplace for each hazardous chemical and
ensure that MSDS copies are readily accessible during each work shift to employees when they are in their
work areas. When household consumer products are used in the workplace in the same manner that a consumer
would use them (ie, when the duration and frequency of use, and therefore exposure, are not greater than those
that the typical consumer would experience), OSHA does not require that an MSDS be provided to purchasers.
However, if exposure to such prod
ucts exceeds that normally found in consumer applications, employees have a right to know about the
properties of such hazardous chemicals. OSHA does not require or encourage employers to maintain an MSDS
for nonhazardous chemicals.
Engineering Controls and PPE
Guidelines for laboratory areas in which hazardous chemicals are used or stored must be established.
Physical facilities, and especially ventilation, must be adequate for the nature and volume of work conducted.
Chemicals must be stored according to chemical compatibility (eg, corrosives, flammables, and oxidizers) and
in minimal volumes. Bulk chemicals should be kept outside work areas. NFPA standards and others provide
guidelines for proper storage.4,40,42
Chemical fume hoods are recommended for use with organic solvents, volatile liquids, and dry chemicals
with a significant inhalation hazard.4 Although constructed with safety glass, most fume hood sashes are not
designed to serve as safety shields. Hoods should be positioned in an area where there is minimal foot traffic to
avoid disrupting the airflow and compromising the containment field.
PPE that may be provided, depending on the hazardous chemicals used, includes chemical-resistant gloves
and aprons, shatterproof safety goggles, and respirators.
Emergency showers should be accessible to areas where caustic, corrosive, toxic, flammable, or
combustible chemicals are used.4,43 There should be unobstructed access, within 10 seconds, to the showers
from the areas where hazardous chemicals are used. Safety showers should be periodically flushed and tested
for function, and associated floor drains should be checked to ensure that drain traps remain filled with water.
Safe Work Practices
Hazardous material should not be stored or transported in open containers. Containers and their lids or seals
should be designed to prevent spills or leakage in all reasonably anticipated conditions. Containers should be
59
able to safely store the maximum anticipated volume and be easy to clean. Surfaces should be kept clean
and dry at all times.
When an employee is working with a chemical fume hood, all materials should be kept at least 6 inches
behind the face opening. The vertical sliding sash should be positioned at the height specified on the
certification sticker. The airfoil, baffles, and rear ventilation slot must not be blocked. Appendix 2-5 lists
suggestions for working safely with specific chemicals
Emergency Response Plan
The time to prepare for a chemical spill is before it occurs. A comprehensive employee training program
should provide each employee with all tools necessary to act responsibly at the time of a chemical spill. The
employee should know response procedures, be able to assess the severity of a chemical spill, know or be able
to quickly look up the basic physical characteristics of the chemicals, and know where to find emergency
response telephone numbers. The employee should be able to assess, stop, and confine the spill; either clean up
the spill or call for a spill cleanup team; and follow procedures for reporting the spill. The employee must know
when to ask for assistance, when to isolate the area, and where to find cleanup materials.
Chemical spills in the workplace can be categorized as follows44:
■ Incidental releases are limited in quantity and toxicity and pose no significant safety or health hazard to
employees. They may be safely cleaned up by employees familiar with the hazards of the chemical involved in
the spill. Waste from the cleanup may be classified as hazardous and must be disposed of in the proper fashion.
Appendix 26 describes appropriate responses to incidental spills.
■ Releases that may be incidental or may require an emergency response may pose an exposure risk to
employees depending on the circumstances. Considerations such as the hazardous substance properties, cir
cumstances of release, and mitigating factors play a role in determining the appropriate response. The
facility’s emergency response plan should provide guidance on how to determine whether a spill is incidental or
requires an emergency response.
■ Emergency response releases pose a threat to health and safety regardless of the circumstances
surrounding their release. These spills may require evacuation of the immediate area. The response typically
comes from outside the immediate release area by personnel trained as emergency responders. These spills
include those that involve immediate danger to life or health, serious threat of fire or explosion, and high levels
of toxic substances.
Appendix 2-7 addresses the management of hazardous chemical spills. Spill cleanup kits or carts tailored to
the specific hazards present should be available in each area. The kits or carts may contain rubber gloves and
aprons, shoe covers, goggles, suitable aspirators, general absorbents, neutralizing agents, a broom, a dust pan,
appropriate trash bags or cans for waste disposal, and cleanup directions. Chemical absorbents, such as clay
absorbents or spill blankets, can be used for cleaning up a number of chemicals and thus may be easier for
employees to use in spill situations.
With any spill of a hazardous chemical, but especially of a carcinogenic agent, it is essential to refer to the
MSDS and contact a designated supervisor or designee trained to handle these spills and hazardous waste
disposal.4 Facility environmental health and safety personnel can also offer assistance. The employer must
assess the extent of the employee’s exposure. After an exposure, the employee must be given an opportunity for
medical consultation to determine the need for a medical examination.
Another source of workplace hazards is the unexpected release of hazardous vapors into the environment.
OSHA has set limits for exposure to hazardous vapors from toxic and hazardous substances.45 The potential
risk associated with a chemical is determined by the manufacturer and listed on the MSDS.
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Chemical Waste Disposal
Most laboratory chemical waste is considered hazardous and is regulated by the EPA through the Resource
Conservation and Recovery Act (42 USC §6901 et seq, 1976). This regulation specifies that hazardous waste
can be legally disposed of only at an EPA-approved disposal facility. Disposal of chemical waste into a sanitary
sewer is regulated by the Clean Water Act (33 USC §1251 et seq, 1977), and most US states have strict
regulations concerning disposal of chemicals in the water system. Federal and applicable state regulations
should be consulted when a facility is setting up and reviewing its waste disposal policies.
RADIATION SAFETY
Radiation can be defined as energy in the form of waves or particles emitted and propagated through space
or a material medium. Gamma rays are electromagnetic radiation, whereas alpha and beta emitters are examples
of particulate radiation. The presence of radiation in the blood bank, from either radioisotopes used in
laboratory testing or self-contained blood irradiators, requires additional precautions and training.4,46
Radiation Measurement Units
The measurement unit quantifying the amount of energy absorbed per unit mass of tissue is the gray (Gy) or
radiation absorbed dose (rad); 1 Gy= 100 rad.
Dose equivalency measurements are more useful than simple energy measurements because dose
equivalency measurements take into account the ability of different types of radiation to cause biologic effects.
The ability of radiation to cause damage is assigned a number called a quality factor (QF). For example,
exposure to a given amount of alpha particles (QF = 20) is far more damaging than exposure to an equivalent
amount of gamma rays (QF = 1). The common unit of measurement for dose equivalency is the roentgen or rad
equivalent man (rem). Rem is the dose from any type of radiation that pro
duces biologic effects in humans equivalent to 1 rad of x-rays, gamma rays, or beta rays. To obtain the dose
from a particular type of radiation in rem, the number of rad should be multiplied by the QF (rad x QF = rem).
Because the QF for gamma rays, x-rays, and most beta particles is 1, the dose in rad is equal to the dose in rem
for these types of radiation.
Biologic Effects of Radiation
Any harm to tissue begins with the absorption of radiation energy and subsequent disruption of chemical
bonds. Molecules and atoms become ionized or excited (or both) by absorbing this energy. The direct action
path leads to radiolysis or formation of free radicals that, in turn, alter the structure and function of molecules in
the cell.
Molecular alterations can cause cellular or chromosomal changes, depending on the amount and type of
radiation energy absorbed. Cellular changes can be manifested as a visible somatic effect (eg, erythema).
Changes at the chromosome level may be manifested as leukemia or other cancers or possibly as germ-cell
defects that are transmitted to future generations.
Several factors influence the level of biologic damage from exposure, including the type of radiation, part of
the body exposed, total absorbed dose, and dose rate. The total absorbed dose is the cumulative amount of
radiation absorbed in the tissue. The greater the dose, the greater the potential for biologic damage. Exposure
can be acute or chronic. The low levels of ionizing radiation likely to occur in blood banks should not pose any
detrimental risk.47'50
Regulations
The NRC controls the use of radioactive materials by establishing licensure requirements. States and
municipalities may also have requirements for inspection, licensure, or both. The type of license for using
radioisotopes or irradiators depends on the scope and magnitude of the use of radioactivity. US facilities should
contact the NRC and appropriate state agencies for information on license require
61
ments and applications as soon as such activities are proposed.
Each NRC-licensed establishment must have a qualified radiation safety officer who is responsible for
establishing personnel protection requirements and for proper disposal and handling of radioactive materials.
Specific radiation safety policies and procedures should address dose limits, employee training, warning signs
and labels, shipping and handling guidelines, radiation monitoring, and exposure management. Emergency
procedures must be clearly defined and readily available to the staff.
 In 2005, the NRC imposed additional security requirements for high-risk radioactive sources, including
those used in blood irradiators. The purpose of the increased controls is to reduce the risk of unauthorized use of
radioactive materials that may pose a threat to public health and safety. These 2005 measures include controlled
access, approval in writing of individuals deemed trustworthy and reliable to have unescorted access, a system
of monitoring to immediately detect and respond to unauthorized access, and documentation of authorized
personnel and monitoring activities.51 In 2007, a requirement for fingerprinting was added.52
Exposure Limits
The NRC sets standards for protection against radiation hazards arising from licensed activities, including
dose limits.12 Such limits, or maximal permissible dose equivalents, are a measure of the radiation risk over
time and serve as standards for exposure. The occupational total effective-dose-equivalent limit is 5 rem/year,
the shallow dose equivalent limit (skin) is 50 rem/year, the extremity dose equivalent limit is 50 rem/year, and
the eye dose equivalent limit is 15 rem/year.12,47 Dose limits for an embryo or fetus must not exceed 0.5 rem
during pregnancy.12,47,53 Employers are expected not only to maintain radiation exposure below allowable
limits, but also to keep exposure levels as far below these limits as can reasonably be achieved.
Radiation Monitoring
Monitoring is essential for early detection and prevention of problems resulting from radiation exposure.
Monitoring is used to evaluate the facility’s environment, work practices, and procedures and to comply with
regulations and NRC licensing requirements. Monitoring is accomplished with the use of dosimeters, bioassays,
survey meters, and wipe tests.4
Dosimeters, such as film or thermoluminescent badges, rings, or both, measure personnel radiation doses.
The need for dosimeters depends on the amount and type of radioactive materials in use; the facility radiation
safety officer determines individual dosimeter needs. Film badges must be changed at least quarterly and in
some instances monthly, be protected from high temperature and humidity, and be stored at work away from
sources of radiation.
Bioassays, such as thyroid and whole body counting or urinalysis, may be used to determine whether there
is radioactivity inside the body and if so, how much. If necessary, bioassays are usually performed quarterly and
after an incident where accidental intake may have occurred.
Survey meters are sensitive to low levels of gamma or particulate radiation and provide a quantitative
assessment of radiation hazard. Survey meters can be used to monitor storage areas for radioactive materials or
wastes, testing areas during or after completion of a procedure, and packages or containers of radioactive
materials. Survey meters must be calibrated annually by an authorized NRC licensee. Selection of appropriate
meters should be discussed with the radiation safety officer.
In areas where radioactive materials are handled, all work surfaces, equipment, and floors that may be
contaminated should be checked regularly with a wipe test. In the wipe test, a moistened absorbent material (the
wipe) is passed over the surface and then measured for radiation.
Training
Personnel who handle radioactive materials or work with blood irradiators must receive radi
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ation safety training before beginning work. This training should address the presence and potential hazards
of radioactive materials in the employee’s work area, general health protection issues, emergency procedures,
and radiation warning signs and labels in use. Instruction in the following areas is also suggested:
■ NRC regulations and license conditions.
■ The importance of observing license conditions and regulations and of reporting violations or conditions
of unnecessary exposure.
■ Precautions to minimize exposure.
■ Interpretation of results of monitoring devices.
■ Requirements for pregnant workers.
■ Employees’ rights.
■ Documentation and record-keeping requirements.
The need for refresher training is determined by the license agreement between the NRC and the facility.
Engineering Controls and PPE
Although self-contained blood irradiators present little risk to laboratory staff and film badges are not
required for routine operation, blood establishments with irradiation programs must be licensed by the NRC.48
 The manufacturer of the blood irradiator usually accepts responsibility for radiation safety requirements
during transportation, installation, and validation of the unit as part of the purchase contract. The radiation
safety officer can help oversee the installation and validation processes and should confirm that appropriate
training, monitoring systems, procedures, and maintenance protocols are in place before use and that they
reflect the manufacturer’s recommendations. Suspected malfunctions must be reported immediately so that
appropriate actions can be initiated.
Blood irradiators should be located in secure areas so that only trained individuals have access. Fire
protection for the unit must also be considered. Automatic fire detection
and control systems should be readily available in the immediate area. Blood components that have been
irradiated are not radioactive and pose no threat to the staff or the general public.
Safe Work Practices
Each laboratory should establish policies and procedures for the safe use of radioactive materials. These
policies and procedures should include requirements for following general laboratory safety principles,
appropriate storage of radioactive solutions, and proper disposal of radioactive wastes. Radiation safety can be
improved with the following procedures:
■ Minimizing the time of exposure by working as efficiently as possible.
■ Maximizing the distance from the source of the radiation by staying as far from the source as possible.
■ Maximizing shielding (eg, by using a selfshielded irradiator or wearing a lead apron) when working with
certain radioactive materials. These requirements are usually stipulated in the license conditions.
■ Using good housekeeping practices to minimize the spread of radioactivity to uncontrolled areas.
Emergency Response Plan
Radioactive contamination is the dispersal of radioactive material into or onto areas in which it is not
intended—for example, the floor, work areas, equipment, personnel clothing, or personnel skin. The NRC
regulations state that gamma or beta radioactive contamination cannot exceed 2200 disintegrations per minute
(dpm) per 100 cm2 in the posted (restricted) area or 220 dpm/100 cm2 in an unrestricted area, such as a
corridor. For alpha emitters, these values are 220 dpm/100 cm2 and 22 dpm/100 cm2, respectively.54
If a spill occurs, employees’ contaminated skin surfaces must be washed several times, and the radiation
safety officer must be notified immediately to provide further guidance. Others must not be allowed to enter the
area until emergency response personnel arrive.
63
Radioactive Waste Management
Policies for the disposal of radioactive waste, whether liquid or solid, should be established with input from
the radiation safety officer.
Liquid radioactive waste may be collected into large sturdy bottles labeled with an appropriate radiation
waste tag. The rules for separation by chemical compatibility apply. Bottles must be carefully stored to protect
against spillage or breakage. Dry or solid waste may be sealed in a plastic bag and tagged as radiation waste.
The isotope, its activity, and the date on which the activity was measured should be recorded on the bag.
Radioactive waste must never be discharged into the facility’s drain system without prior approval by the
radiation safety officer.
SHIPPING HAZARDOUS MATERIALS
Hazardous materials commonly shipped by transfusion medicine, cellular therapy, and clinical diagnostic
services include infectious substances, biologic substances, liquid nitrogen, and dry ice.
The US DOT regulations for transportation of hazardous materials are harmonized with the international
standards published annually by the International Air Transport Association (IATA).55,56 These regulations
provide instructions for identifying, classifying, packaging, marking, labeling, and documenting hazardous
materials to be offered for shipment on public roadways or by air.
Specimens are classified as Category A if they are known or likely to contain infectious substances in a
form that is capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy
humans or animals when an exposure occurs. The proper shipping name for Category A specimens is
“infectious substances, affecting humans” (UN2814) or “infectious substances, affecting animals only”
(UN2900).
Specimens that may contain infectious substances but do not have the level of risk described above are
classified as Category B, and the proper shipping name is “biological
 substance, Category B” (UN3373). HIV or HBV in culture are classified as Category A infectious
substances, but these viruses present in a patient blood specimen are classified as Category B.
Patient specimens with minimal likelihood of containing pathogens are exempt from hazardous materials
regulations if the specimens are properly packaged and marked. Blood components, cellular therapy products,
and tissue for transfusion or transplantation are not subject to hazardous material regulations. Method 1-1
provides additional shipping instructions for safe transport of these materials. However, the most recent revision
of the IATA or US DOT regulations should be consulted for the most current classification, packaging, and
labeling requirements as well as for limitations on the volumes of hazardous materials that can be packaged
together in one container.
GENERAL WASTE MANAGEMENT
Those responsible for safety at a facility must be concerned with protecting the environment as well as all
staff members. Every effort should be made to establish facility-wide programs to reduce solid wastes,
including nonhazardous and, especially, hazardous wastes (ie, biohazardous, chemical, and radioactive wastes).
A hazardous waste-reduction program instituted at the point of use of the material achieves several goals. It
reduces the institutional risk for occupational exposures to hazardous agents, reduces “cradle-to-grave” liability
for disposal, and enhances compliance with environmental requirements to reduce pollution generated from
daily operations of the laboratory.37,57,58
Facilities can minimize pollution of the environment by practicing the “three R’s”: reduce, reuse, and
recycle. Seeking suitable alternatives to materials that create hazardous waste and separating hazardous waste
from nonhazardous waste can reduce the volume of hazardous waste and decrease costs for its disposal.
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Changes in techniques or materials to reduce the volume of infectious waste or render it less hazardous
should be carefully considered, and employees should be encouraged to identify safer alternatives wherever
possible.
Facilities should check with state and local health and environmental authorities about current requirements
for storage and disposal of a particular multihazardous waste before creating that waste. If creating the
multihazardous waste cannot be avoided, the volume of waste generated should be minimized.
In some states, copper sulfate contaminated with blood is considered a multihazardous waste. The disposal
of this waste poses several problems with transportation from draw sites to a central facility for disposal of the
final containers. State and local health departments must be involved in reviewing transportation and disposal
practices where this is an issue, and procedures must be developed in accordance with state and local
regulations as well as those of the US DOT.
KEY POINTS
1. Facilities should be designed and maintained in a way that supports the work being done in the physical
space. Designing the space to accommodate planned work flow, the need to restrict certain areas, the movement
of materials and waste, equipment location, special airhandling requirements, and other critical aspects of the
operation help ensure safety for staff and visitors as well as the quality of products and services.
2. The facilities safety program should 1) strive to reduce hazards in the workplace, 2) ensure that staff are
trained to handle known hazards and potential risks, 3) ensure that known hazards are clearly identified and
marked, and 4) describe policies and procedures for workplace safety and emergency response.
3. Safety programs should address fire, electrical, biological, chemical, and radioactive hazards that may be
found in the facility.
4. For each type of hazard, five basic elements that must be covered are 1) training; 2) hazard identification
and communication; 3) engineering controls and PPE; 4) safe work practices, including waste disposal; and 5)
an emergency response plan.
5. Management controls ensure that the safety program is implemented, maintained, and effective.
Management is responsible for 1) developing and communicating the written plan, 2) ensuring implementation
of the plan and providing adequate resources for this implementation, 3) providing access to employee health
services for prevention strategies and treatment of exposures, 4) monitoring compliance and effectiveness, and
5) evaluating and improving the safety plan.
REFERENCES
1. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
2. Fontaine M, ed. Standards for cellular therapy product services. 6th ed. Bethesda, MD: AABB, 2013.
 3. Laboratory Accreditation Program laboratory general checklist. Chicago: College of American
Pathologists, 2014.
4. Clinical laboratory safety: Approved guideline. 3rd ed. NCCLS Document GP17-A3. Wayne,
PA: Clinical and Laboratory Standards Institute, 2012.
5. Hospital accreditation standards. Oakbrook Terrace, IL: The Joint Commission, 2014.
6. Laboratory accreditation standards. Oakbrook Terrace, IL: The Joint Commission, 2014.
7. NFPA 70—National electrical code. Quincy, MA: National Fire Protection Association, 2014.
8. ANSI/ASHRAE Standard 62.1-2013. Ventilation for acceptable indoor air quality. Atlanta, GA:

65
American Society of Heating, Refrigerating, and Air-Conditioning Engineers, Inc., 2013.
9. Code of federal regulations. Title 21, Part 1271.190. Washington, DC: US Government Printing Office,
2014 (revised annually).
10. ISO-14644: Cleanrooms and associated controlled environments, Parts 1-9. ISO/TC 209. Geneva,
Switzerland: International Organization for Standardization, 1999-2012.
11. ISO-14698: Cleanrooms and associated controlled environments—bio-contamination control, Part 1:
General principles and methods. ISO/TC 209. Geneva, Switzerland: International Organization for
Standardization, 2003.
12. Code of federal regulations. Title 10, Part 20. Washington, DC: US Government Printing Office, 2014
(revised annually).
13. Siegel JD, Rhinehart E, Jackson M, et al. 2007 Guideline for isolation precautions: Preventing
transmission of infectious agents in healthcare settings. Atlanta, GA: Centers for Disease Control and
Prevention (Healthcare Infection Control Practices Advisory Committee), 2007. [Available at
http://www.cdc.gov/ hicpac/pdf/isolation/Isolation2007.pdf (accessed January 6, 2013).]
14. Code of federal regulations. Title 29, CFR Part 1910.1030. Washington, DC: US Government Printing
Office, 2013 (revised annually).
15. Code of federal regulations. Title 29, CFR Part 1910.1200. Washington, DC: US Government Printing
Office, 2013 (revised annually).
16. US Department of Health and Human Services. Biosafety in microbiological and biomedical
laboratories. 5th ed. Washington, DC: US Government Printing Office, 2009.
 17. Bernard B, ed. Musculoskeletal disorders and workplace factors: A critical review of epidemiologic
evidence for work-related musculoskeletal disorders of the neck, upper extremity, and low back. NIOSH
publication no. 97-141. Washington, DC: National Institute for Occupational Safety and Health, 1997.
18. Code of federal regulations. Title 29, CFR Part 1910.38. Washington, DC: US Government Printing
Office, 2013 (revised annually).
19. Wagner KD, ed. Environmental management in healthcare facilities. Philadelphia: WB Saunders, 1998.
20. Code of federal regulations. Title 29, CFR Part 1910.1450. Washington, DC: US Government Printing
Office, 2013 (revised annually).
21. Centers for Disease Control and Prevention. Public Health Service guidelines for the management of
occupational exposures to HBV, HCV and HIV and recommendations for postexposure prophylaxis. MMWR
Morb Mortal WklyRep 2001;50:1-52.
22. Code of federal regulations. Title 29, CFR Part 1904.39. Washington, DC: US Government Printing
Office, 2013 (revised annually).
23. Code of federal regulations. Title 29, CFR Part 1904.1, Part 1904.7. Washington, DC: US Government
Printing Office, 2013 (revised annually)
24. Code of federal regulations. Title 29, CFR Part 1910.1020. Washington, DC: US Government Printing
Office, 2013 (revised annually).
25. NIOSH Alert: Preventing allergic reactions to natural rubber latex in the workplace. (June 1997) NIOSH
Publication No. 97-135. Washington, DC: National Institute for Occupational Safety and Health, 1997.
[Available at http:// www.cdc.gov/niosh/docs/97-135/ (accessed January 6,2013).]
26. NFPA 101: Fife safety code. Quincy, MA: National Fire Protection Association, 2012.
27. Fowler TW, Miles KK. Electrical safety: Safety and health for electrical trades student manual. (January
2002) NIOSH Publication No. 2002-123. Washington, DC: National Institute for Occupational Safety and
Health, 2002.
28. OSHA technical manual: TED 1-0.15A. Washington, DC: US Department of Labor, 1999.
29. Enforcement procedures for the occupational exposure to bloodborne pathogens. Directive CPL 02-02-
069. Washington, DC: US Department of Labor, 2001.
30. US Department of Health and Human Services. Primary containment for biohazards: Selection,
installation, and use of biological safety cabinets. Washington, DC: US Government Printing Office, 2009.
[Available at http:// www.cdc.gov/biosafety/publications (accessed January 6,2013).]
31. Richmond JY. Safe practices and procedures for working with human specimens in biomedical research
laboratories. J Clin Immunoassay 1988;11:115-19.
32. ATP— tested actively registered hospital disinfectant products. (March 2010) Washington, DC: US
Environmental Protection Agency, 2012. [Available at http://www.epa.gov/ oppad001/chemregindex.htm
(accessed January 6, 2013).]
66
AABB TECHNICAL MANUAL
33. Rutala WA. APIC guideline for selection and use of disinfectants. Am J Infect Control 1996; 24:313-42.
34. Evans MR, Henderson DK, Bennett JE. Potential for laboratory exposures to biohazardous agents found
in blood. Am J Public Health 1990;80:423-7.
35. Memorandum: Guideline for collection of blood products from donors with positive tests for infectious
disease markers (“high risk” donors). (October 26, 1989) Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 1989.
36. Memorandum: Revision to 26 October 1989 guidelines for collection of blood or blood products from
donors with positive tests for infectious disease markers (“high-risk” donors). Silver Spring, MD: CBER Office
of Communication, Outreach, and Development, 1991. [Available at http://www.fda.gov/biolog
icsbloodvaccines/guidancecomplianceregula toryinformation/other recommendations for
manufacturers/memorandumtobloodestab lishments.default.htm.]
37. US Environmental Protection Agency. EPA guide for infectious waste management. EPA/ 530-SW-86-
014. NTIS #PB86-199130. Washington, DC: National Technical Information Service, 1986.
38. Code of federal regulations. Title 40, CFR Part 264. Washington, DC: US Government Printing Office,
2013 (revised annually).
39. NIOSH pocket guide to chemical hazards. Washington, DC: National Institute for Occupational Safety
and Health, 2010. [Available at http://www.cdc.gov/niosh/npg (accessed November 8, 2010).]
 40. NFPA 704—Standard for the identification of the hazards of materials for emergency response. Quincy,
MA: National Fire Protection Association, 2012.
41. Hazardous Materials Identification System. HMIS implementation manual. 3rd ed. Neenah, WI: JJ
Keller and Associates, Inc., 2001.
42. Lisella FS, Thomasston SW. Chemical safety in the microbiology laboratory. In: Fleming DO,
Richardson JH, Tubs JJ, Vesley D, eds. Laboratory safety, principles, and practices. 2nd ed. Washington, DC:
American Society for Microbiology Press, 1995:247-54.
43. American national standards for emergency eyewash and shower equipment. ANSI Z358.12009. New
York: American National Standards Institute, 2009.
44. Inspection procedures for 29 CFR 1910.120 and 1926.65, paragraph (q): Emergency response to
hazardous substance releases. OSHA Directive CPL 02-02-073. Washington, DC: Occupational Safety and
Health Administration, 2007.
45. Code of federal regulations. Title 29, CFR Part 1910.1000. Washington, DC: US Government Printing
Office, 2013 (revised annually).
46. Cook SS. Selection and installation of selfcontained irradiators. In: Butch S, Tiehen A, eds. Blood
irradiation: A user’s guide. Bethesda, MD: AABB Press, 1996:19-40.
47. Beir V. Health effects of exposure to low levels of ionizing radiation. Washington, DC: National
Academy Press, 1990:1-8.
48. Regulatory Guide 8.29: Instruction concerning risks from occupational radiation exposure. Washington,
DC: Nuclear Regulatory Commission, 1996.
49. NCRP Report No. 115: Risk estimates for radiation protection: Recommendations of the National
Council on Radiation Protection and Measurements. Bethesda, MD: National Council on Radiation Protection
and Measurements, 1993.
50. NCRP Report No. 105: Radiation protection for medical and allied health personnel: Recommendations
of the National Council on Radiation Protection and Measurements. Bethesda, MD: National Council on
Radiation Protection and Measurements, 1989.
51. EA-05 090. Enforcement action: Order imposing increased controls (licensees authorized to possess
radioactive material quantities of concern). (November 14, 2005) Rockville, MD: US Nuclear Regulatory
Commission, 2005.
52. RIS 2007-14. Fingerprinting requirements for licensees implementing the increased control order. (June
5, 2007) Rockville, MD: US Nuclear Regulatory Commission, 2007.
53. US Nuclear Regulatory Commission Regulatory Guide 8.13: Instruction concerning prenatal radiation
exposure. Washington, DC: NRC, 1999.
54. Nuclear Regulatory Commission regulatory guide 8.23: Radiation surveys at medical institutions.
Washington, DC: NRC, 1981.
55. Code of federal regulations. Title 49, CFR Parts 171.22. Washington, DC: US Government Printing
Office, 2014 (revised annually).
56. Dangerous goods regulations manual. 54th ed. Montreal, PQ, Canada: International Air Transport
Association, 2014 (revised annually).
67
57. United States Code. Pollution prevention act. Clinical and Laboratory Standards Institute,
42 U.S.C. §§13101 and 13102 etseq. 2011.
58. Clinical laboratory waste management. Approved guideline. 3rd ed. GP05-A3. Wayne, PA:
68
AABB TECHNICAL MANUAL
■ APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
Agency/Organization Reference Title
Federal Regulations and
Recommendations
Nuclear Regulatory
10CFR 20 Standards for Protection Against Radiation
Gommission
Licenses and Radiation Safety Requirements for
10CFR 36
Irradiators
 Guide 8.29 Instructions Concerning Risks from Occupational
Radiation Exposure
Occupational Safety and Health 29CFR
Occupational Exposure to Bloodborne Pathogens
Administration 1910.1030
29CFR
Access to Employee Exposure and Medical Records
1910.1020
29CFR
Ionizing Radiation
1910.1096
29CFR
Hazard Communication Standard
1910.1200
29 CFR Occupational Exposure to Hazardous Chemicals in
1910.1450 Laboratories
49CFR 171-
Department of Transportation Hazardous Materials Regulations
180
Environmental Protection
EPA Guide for Infectious Waste Management
Agency (EPA)
Centers for Disease Control
Guideline for Isolation Precautions in Hospitals
and Prevention
21 CFR Current Good Manufacturing Practice for Blood and
Food and Drug Administration
606.3-606.171 Blood Components
General Requirements for Blood, Blood
21 CFR 630.6
Components, and Blood Derivatives
21 CFR Additional Standards for Human Blood and Blood
640.1-640.120 Products
21 CFR 211.1- Current Good Manufacturing Practice for Finished
211.208 Pharmaceuticals
21 CFR 1270 Human Tissue Intended for Transplantation
Human Cells, Tissues, and Cellular and Tissue-
21 CFR 1271
Based Products

69
■ APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings (Continued)
Agency/Organization Reference Title
 Trade and Professional
Organizations
National Fire
Protection Association NFPA 70 National Electrical Code
(NFPA)
Electrical Safety Requirements
NFPA 70E
for Employee Workplaces
NFPA 101 Life Safety Code
NFPA 99 Standards for Health Care Facilities
Standard for Identification of the Hazards
NFPA 704
of Materials for Emergency Response
National Paint and Hazardous Materials Identification System
Coatings Association Implementation Manual
International Air
Dangerous Goods Regulations
Transport Association
CFR = Code of federal regulations.

70
AABB TECHNICAL MANUAL
■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and Engineering Controls
UNIFORMS AND LABORATORY COATS
Personnel should wear closed laboratory coats or full aprons over long-sleeved uniforms or gowns when
they are exposed to blood, corrosive chemicals, or carcinogens. The material of required coverings should be
appropriate for the type and amount of hazard exposure. Plastic disposable aprons may be worn over cotton
coats when there is a high probability of large spills or splashing of blood and body fluids; nitrile rubber aprons
may be preferred when caustic chemicals are poured.
Protective coverings should be removed before the employee leaves the work area and should be discarded
or stored away from heat sources and clean clothing. Contaminated clothing should be removed promptly,
placed in a suitable container, and laundered or discarded as potentially infectious. Home laundering of
garments worn in Biosafety Level 2 areas is not permitted because unpredictable methods of transportation and
handling can spread contamination and home laundering techniques may not be effective.1
GLOVES
Gloves or equivalent
barriers should be used
whenever tasks are likely
to involve exposure to
hazardous materials.
TYPES OF GLOVES
Glove type varies
with the task:
Sterile gloves: for procedures involving contact with normally sterile areas of
■
the body.
Examination gloves: for procedures involving contact with mucous membranes,
■ unless otherwise indicated, and for other patient care or diagnostic procedures that
do not require the use of sterile gloves.
Rubber utility gloves: for housekeeping chores involving potential blood
contact, instrument cleaning and decontamination procedures, and handling
■ concentrated acids and organic solvents. Utility gloves may be decontaminated and
reused but should be discarded if they show signs of deterioration (eg, peeling,
cracks, or discoloration) or if they develop punctures or tears.
■ Insulated gloves: for handling hot or frozen material.
GUIDELINES FOR
USE
The following
guidelines should be used
to determine when gloves
are necessary1:
For donor phlebotomy when the health-care worker has cuts, scratches, or other
■
breaks in his or her skin.
For phlebotomy of autologous donors or patients (eg, therapeutic apheresis
■
procedures or intraoperative red cell collection).
■ For persons who are receiving training in phlebotomy.
■ When handling open blood containers or specimens.
When collecting or handling blood or specimens from patients or donors known
■
to be infected with a bloodborne pathogen.
■ When examining mucous membranes or open skin lesions.
■ When handling corrosive chemicals and radioactive materials.
■ When cleaning up spills or handling waste materials.
When the likelihood of exposure cannot be assessed because of lack of
■
experience with a procedure or situation.
 71
■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and Engineering Controls
(Continued)
The Occupational Safety and Health Administration (OSHA) does not require the routine use of gloves by
phlebotomists working with healthy prescreened donors or the changing of unsoiled gloves between donors if
gloves are worn.12 Experience has shown that the phlebotomy process is low risk because donors typically
have low rates of infectious disease markers. Also, exposure to blood is rare during routine phlebotomy, and
other alternatives can be used to provide barrier protection, such as using a folded gauze pad to control any
blood flow when the needle is removed from the donor’s arm.
Employers whose policies and procedures do not require routine gloving should periodically reevaluate the
potential need for gloves. Employees should never be discouraged from using gloves, and gloves should always
be available.
GUIDELINES FOR
USE
Guidelines for the safe
use of gloves by
employees include the
following3'4:
Securely bandage or cover open skin lesions on hands and arms before putting
■
on gloves.
Change gloves immediately if they are torn, punctured, or contaminated; after
■ handling high-risk samples; or after performing a physical examination (eg, on an
apheresis donor).
Remove gloves by keeping their outside surfaces in contact only with outside
■
and by turning the glove inside out while taking it off.
Use gloves only when needed, and avoid touching clean surfaces such as
■
telephones, doorknobs, or computer terminals with gloves.
Change gloves between patient contacts. Unsoiled gloves need not be changed
■
between donors.
■ Wash hands with soap or other suitable disinfectant after removing gloves.
Do not wash or disinfect surgical or examination gloves for reuse. Washing with
surfactants may cause “wicking” (ie, enhanced penetration of liquids through
■
undetected holes in the glove). Disinfecting agents may cause deterioration of
gloves.
Use only water-based hand lotions with gloves, if needed; oil-based products
■
cause minute cracks in latex.
FACE SHIELDS,
MASKS, AND SAFETY
GOGGLES
Where there is a risk of blood or chemical splashes, the eyes and mucous membranes of the mouth and nose
should be protected.5 Permanent shields fixed as a part of equipment or bench design are preferred (eg, splash
barriers attached to tubing sealers or centrifuge cabinets). All barriers should be cleaned and disinfected on a
regular basis.
Safety glasses alone provide impact protection from projectiles but do not adequately protect eyes from
biohazardous or chemical splashes. Full-face shields or masks and safety goggles are recommended when
permanent shields cannot be used. Many designs are commercially available; eliciting staff input on comfort
and selection can increase use.
 Masks should be worn whenever there is danger from inhalation. Simple, disposable dust masks are
adequate for handling dry chemicals, but respirators with organic vapor filters are preferred for areas where
noxious fumes are produced (eg, for cleaning up spills of noxious materials). Respirators should be fitted to
their wearers and checked annually.
(Continued)

72
AABB TECHNICAL MANUAL
■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and Engineering Controls
(Continued)
HANDWASHING
Frequent, thorough hand washing is the first line of defense in infection control. Blood-borne pathogens
generally do not penetrate intact skin, so immediate removal reduces the likelihood of transfer to a mucous
membrane or broken skin area or of transmission to others. Thorough washing of hands (and arms) also reduces
the risks from exposure to hazardous chemicals and radioactive materials.
Employees should always wash their hands before leaving a restricted work area or using a biosafety
cabinet, between medical examinations, immediately after becoming soiled with blood or hazardous materials,
after removing gloves, or after using the toilet. Washing hands thoroughly before touching contact lenses or
applying cosmetics is essential.
OSHA allows the use of waterless antiseptic solutions for hand washing as an interim method.2 These
solutions are useful for mobile donor collections or in areas where water is not readily available for cleanup
purposes. If such methods are used, however, hands must be washed with soap and running water as soon as
possible thereafter. Because there is no listing or registration of acceptable hand-wipe products similar to the
one that the Environmental Protection Agency maintains for surface disinfectants, consumers should request
data from the manufacturer to support advertising claims.
EYEWASHES
Laboratory areas that contain hazardous chemicals must be equipped with eyewash stations.36 Unobstructed
access within a 1 - second walk from the location of chemical use must be provided for these stations.
Eyewashes must operate so that both of the user’s hands are free to hold open the eyes. Procedures and
indications for use must be posted, and routine function checks must be performed. Testing eyewash fountains
weekly helps ensure proper function and flushes out stagnant water. Portable eyewash systems are allowed only
if they can deliver flushing fluid to the eyes at a rate of at least 1.5 liters per minute for 15 minutes. They should
be monitored routinely to ensure the purity of their contents.
Employees should be trained in the proper use of eyewash devices, although prevention—through consistent
and appropriate use of safety glasses or shields—is preferred. If a splash occurs, the employee should be
directed to keep his or her eyelids open and to use the eyewash according to procedures, or the employee should
go to the nearest sink and direct a steady, tepid stream of water into his or her eyes. Solutions other than water
should be used only in accordance with a physician’s direction.
 After eyes are adequately flushed (many facilities recommend 15 minutes), follow-up medical care should
be sought, especially if pain or redness develops. Whether washing the eyes is effective in preventing infection
has not been demonstrated, but it is considered desirable when accidents occur.
1. Code of federal regulations. Title 29, CFR Part 1910.1030. Fed Regist 1991;56:64175-82.
2. Occupational Safety and Health Administration. Enforcement procedures for the occupational exposure to
bloodborne pathogens. OSHA Instruction CPL 02-02-069. Washington, DC: US Government Printing Office,
2001.
3. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory
Standards Institute, 2012.
4. Medical glove powder report. (September 1997) Rockville, MD: Food and Drug Administration, 2009.
[Available at http://www.fda. gov/MedicalDevices/DeviceRegulationand Guidance/GuidanceDocuments/ucml
13316.htm (accessed January 6,2013).]
5. Inspection checklist: General laboratory. Chicago: College of American Pathologists, 2012.
6. American national standards for emergency eyewash and shower equipment. ANSI Z358.1 -2009. New
York: American National Standards Institute, 2009.

73
■ APPENDIX 2-3
Biosafety Level 2 Precautions
Biosafety Level 2 precautions as applied in the blood establishment setting include at least the following1'2:
■ High-risk activities are appropriately segregated from lower-risk activities, and the boundaries are clearly
defined.
■ Bench tops are easily cleaned and are decontaminated daily with a hospital disinfectant approved by the
Environmental Protection Agency.
■ Laboratory rooms have closable doors and sinks. An air system with no recirculation is preferred but not
required.
■ Workers are required to perform procedures that create aerosols (eg, opening evacuated tubes,
centrifuging, mixing, or sonicating) in a biological safety cabinet or equivalent or to wear masks and goggles in
addition to gloves and gowns during such procedures. (Note: Open tubes of blood should not be centrifuged. If
whole units of blood or plasma are centrifuged, overwrapping is recommended to contain leaks.)
 ■ Gowns and gloves are used routinely and in accordance with general safety guidelines. Face shields or
their equivalents are used where there is a risk from splashing.
■ Mouth pipetting is prohibited.
■ No eating, drinking, smoking, applying cosmetics, or manipulating contact lenses occurs in the work area.
All food and drink are stored outside the restricted area, and laboratory glassware is never used for food or
drink. Personnel are instructed to avoid touching their face, ears, mouth, eyes, or nose with their hands or other
objects, such as pencils and telephones.
■ Needles and syringes are used and disposed of in a safe manner. Needles must never be bent, broken,
sheared, replaced in a sheath, or detached from a syringe before being placed in puncture-proof, leakproof
containers for controlled disposal. Procedures are designed to minimize exposure to sharp objects.
■ All blood specimens are placed in well-constructed containers with secure lids to prevent leaking during
transport. Blood is packaged for shipment in accordance with regulatory agency requirements for etiologic
agents or clinical specimens, as appropriate.
■ Infectious waste is not compacted and is decontaminated before its disposal in leakproof containers.
Proper packaging includes double, seamless, tear-resistant, orange or red bags that are enclosed in protective
cartons. Both the cartons and the bags inside display the biohazard symbol. Throughout delivery to an
incinerator or autoclave, waste is handled only by suitably trained persons. If a waste management contractor is
used, the agreement should clearly define the respective responsibilities of the staff and the contractor.
■ Equipment to be repaired or submitted for preventive maintenance, if potentially contaminated with
blood, must be decontaminated before its release to a repair technician.
■ Accidental exposure to suspected or actual hazardous material is reported to the laboratory director or
responsible person immediately.
1. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory
Standards Institute, 2012.
2. Fleming DO. Laboratory biosafety practices. In: Fleming DO, Richardson JH, Tulis JJ, Vesley DD, eds.
Laboratory safety, principles, and practices. 2nd ed. Washington, DC: American Society for Microbiology
Press, 1995:203-18.

74
AABB TECHNICAL MANUAL
■ APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
Chemical Hazard
Ammonium chloride Irritant
Bromelin Irritant, sensitizer
Calcium chloride Irritant
Carbon dioxide, frozen (dry ice) Corrosive
Carbonyl iron powder Oxidizer
Chloroform Toxic, suspected carcinogen
Chloroquine Irritant, corrosive
Chromium-111 chloride hexahydrate Toxic, irritant, sensitizer
Citric acid Irritant
Copper sulfate (cupric sulfate) Toxic, irritant
Dichloromethane Toxic, irritant
Digitonin Toxic
Dimethyl sulfoxide Irritant
Dry ice (carbon dioxide, frozen) Corrosive
Ethidium bromide Carcinogen, irritant
Ethylenediaminetetraacetic acid Irritant
Ethyl ether Highly flammable and explosive, toxic, irritant
Ficin (powder) Irritant, sensitizer
Formaldehyde solution (34.9%) Suspected carcinogen, combustible, toxic
Glycerol Irritant
Hydrochloric acid Highly toxic, corrosive
Imidazole Irritant
Isopropyl (rubbing) alcohol Flammable, irritant
Liquid nitrogen Corrosive
Lyphogel Corrosive
2-Mercaptoethanol Toxic, stench
Mercury Toxic
Mineral oil Irritant, carcinogen, combustible
Papain Irritant, sensitizer
Polybrene Toxic
Sodium azide Toxic, irritant, explosive when heated
Sodium ethylmercurithiosalicylate (thimerosal) Highly toxic, irritant
75
■ APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank (Continued)
Chemical
Sodium hydrosulfite
Sodium hydroxide
Sodium hypochlorite (bleach)
Sodium phosphate
Sulfosalicylic acid
Trichloroacetic acid
Trypsin
Xylene
Hazard
Toxic, irritant
Corrosive, toxic
Corrosive
Irritant, hygroscopic
Toxic, corrosive
Corrosive, toxic
Irritant, sensitizer
Highly flammable, toxic, irritant

76
AABB TECHNICAL
■ APPENDIX 2-5
Chemical Categories and How to Work
Chemical Category Hazard
Acids, alkalis, and corro- Irritation, severe burns, sive compounds tissue damage
Acrylamide Neurotoxic, carcino
genic, absorbed through the skin
Compressed gases
Explosive
UAL

Precautions Special Treatment

 During transport, protect Store concentrated acids large containers with in acid safety cabinets, plastic or
rubber bucket Limit volumes of concarriers. centrated acids to 1
During pouring, wear eye liter per container, protection and chemi- Post cautions for materical-resistant-
rated als in the area,
gloves and gowns as Report changes in
recommended. appearance (perchloric
Always add acid to water, acid may be explosive if
never add water to acid. it becomes yellowish or
When working with large brown) to chemical
jugs, have one hand on safety officer,
the neck and the other hand at the base, and position them away from the face.
Wear chemically rated gloves.
Wash hands immediately after exposure.
Label contents.
Leave valve safety covers on until use.
Open valves slowly for use.
Label empty tanks.
Store in a chemical cabinet.
Transport using hand trucks or dollies.
Place cylinders in a stand or secure them to prevent tipping over.
Store in well-ventilated separate rooms.
Do not store oxygen close to combustible gas or solvents.
Check connections for leaks using soapy water.

77
■ APPENDIX 2-5
Chemical Categories and How to Work Safely with Them (Continued)
Chemical Category Hazard Precautions Special Treatment
Flammable solvents Classified according to Use extreme caution Make every attempt to
flash point—see mate- when handling. replace hazardous
rial safety data sheet, Post “No Smoking” signs materials with less hazclassified according to in working
area. ardous materials
volatility Keep a fire extinguisher Store containers larger
and solvent cleanup kit than 1 gallon in a flam
in the room. mable solvent storage
Pour volatile solvents room or a fire safety under a suitable hood. cabinet.
Use eye protection and Ground metal containers chemical-resistant neo- by connecting the can
prene gloves when to a water pipe or
pouring. ground connection. If
No flame or other source the recipient container
of possible ignition is also metal, it should
should be in or near be electrically conareas where flammable nected to the delivery
solvents are being container during pour
poured. ing.
Label as “flammable.”
Liquid nitrogen Freeze injury, severe Use heavy insulated The tanks should be
burns to skin or eyes gloves and goggles securely supported to when working with liq- avoid being tipped
uid nitrogen. over.
 The final container of liquid nitrogen (freezing unit) must be securely supported to avoid
being tipped over.

78
AABB TECHNICAL MANUAL
■ APPENDIX 2-6
Incidental Spill Response*
Chemicals Hazards PPE Control Materials
If inhaled, causes
severe irritation.
Contact causes
burns to skin and
Acids
eyes.
Acetic
Spills are
Hydrochloric
corrosive. Acid-resistant gloves Acid neutralizers or absorbent
Nitric
Fire or contact Apron and coveralls Goggles material Absorbent boom Leakproof
Perchloric
with metal may and face shield Acid- containers Absorbent pillow Mat (cover
Sulfuric
produce irritating or resistant foot covers drain) Shovel or paddle
Photographic
poisonous gas.
chemicals
Nitric,
(acidic)
perchloric, and
sulfuric acids are
water-reactive
oxidizers.
Bases and Spills are Gloves, impervious
Base control/neutralizer
caustics corrosive. apron
Potassium Fire may produce
or coveralls Absorbent pillow
hydroxide irritat
Sodium ing or poisonous
Goggles or face shield, Absorbent boom
hydroxide gas.
Photographic
Drain mat
chemicals impervious foot covers
Leakproof container Shovel or paddle
(basic)
Inhalation can
Chlorine Gloves (double set of 4H Chlorine control powder
cause
respiratory
Bleach undergloves and butyl Absorbent pillow
irritation.
Sodium Liquid contact
or nitrile overgloves), Absorbent material
hypochlorite can pro
duce irritation of
impervious apron or Absorbent boom
the
eyes or skin. coveralls Drain mat
Toxicity is
Goggles or face shield Vapor barrier
caused by
alkalinity,
Impervious foot covers Leakproof container
possible
 chlorine gas (neoprene boots for Shovel or paddle
generation, and emergency response
oxidant properties. releases)
Self-contained breathing
apparatus (emergency
response releases)
Cryogenic Contact with
Full face shield or gog Hand truck (to transport
gases liquid nitro
Carbon gen can produce
gles, neoprene boots, cylinder outdoors if
dioxide frost
Nitrous
bite. gloves (insulated to necessary)
oxide
Liquid Release can
provide protection from Soap solution (to check
nitrogen create an
oxygen-deficient
for leaks)
atmosphere.
the cold) Putty (to stop minor pipe and line
Nitrous oxide has
leaks)
anesthetic effects.

79
■ APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials
Flammable gases Simple Face shield and goggles, Hand truck (to
Acetylene Oxygen gases asphyxiant neoprene boots, double set of transport cylinder outdoors
Butane Propane (displaces air). gloves, coveralls with hood and feet if needed)
Inhaled vapors Soap solution (to check
have an anesthetic for leaks)
potential.
 Flammable
gases pose an
extreme fire and
explosion hazard.
Release can
create an oxygen-
deficient
atmosphere.
Vapors are
harmful if inhaled
(central nervous
system
depressants).
Absorbent material
Flammable liquids Liquid is Gloves (double set of 4H
Absorbent boom
Acetone Xylene harmful if undergloves and butyl or nitrile
Absorbent pillow Shovel
Methyl alcohol absorbed through overgloves), impervious apron or
or paddle (nonmetal,
toluene Ethyl alcohol the skin. coveralls, goggles or face shield,
nonsparking) Drain mat
Other alcohols Substances are impervious foot covers
Leakproof container
extremely
flammable.
Liquid
evaporates to form
flammable vapors.
Vapors are
harmful if inhaled;
liquids are harmful
if absorbed
through skin.
Substances are
Aldehyde neutralizer
Formaldehyde and irritants to skin,
Gloves (double set of 4H or absorbent Absorbent
glutaraldehyde 4% eyes, and
undergloves and butyl or nitrile boom Absorbent pillow
formaldehyde 37% respiratory tract.
overgloves), impervious apron or Shovel or paddle
formaldehyde 10% Formaldehyde
coveralls, goggles, impervious foot (nonsparking)
formalin 2% is a suspected
covers Drain mat
glutaraldehyde human carcinogen.
Leakproof container
37%
formaldehyde
should be kept
away from heat,
sparks, and
flames.
(Continued)

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■ APPENDIX 2-6
Incidental Spill Response* (Continued)
Chemicals Hazards PPE Control Materials
 Mercury Cantor tubes Thermometers Barometers Sphygmomanometers Mercuric chloride
Mercury and mercury vapors are rapidly absorbed in respiratory tract, gastrointestinal (Gl) tract, or skin.
Short-term exposure may cause erosion of respiratory or Gl tracts, nausea, vomiting, bloody diarrhea, shock,
headache, or metallic taste.
Inhalation of high concentrations can cause pneumonitis, chest pain, dyspnea, coughing, stomatitis,
gingivitis, and salivation.
Avoid evaporation of mercury from tiny globules by quick and thorough cleaning.
Gloves (double set of 4H undergloves and butyl or nitrile overgloves), impervious apron or coveralls,
goggles, impervious foot covers
Mercury vacuum or spill kit
Scoop
Aspirator
Hazardous waste containers
Mercury indicator powder Absorbent material Spatula
Disposable towels Sponge with amalgam Vapor suppressor
‘This list of physical and health hazards in not intended as a substitute for the material safety data sheet
(MSDS) information. In case of a spill or if any questions arise, always refer to the chemical-specific MSDS for
more complete information.

CHAPTER 2 Facilities, Work Environment, and Safety

81
■ APPENDIX 2-7
Managing Hazardous Chemical Spills
Actions Instructions for Hazardous Liquids, Gases, and Mercury
Liquids: For 37% formaldehyde, deenergize and remove all sources of ignition within 10
feet of spilled hazardous material. For flammable liquids, remove all sources of ignition.
Deenergize.
Gases: Remove all sources of heat and ignition within 50 feet for flammable gases.
Remove all sources of heat and ignition for nitrous oxide release.
Isolate, Isolate the spill area and evacuate everyone from the area surrounding the spill except
evacuate, and those responsible for cleaning up the spill. (For mercury, evacuate within 10 feet for small
secure the area. spills or 20 feet for large spills.) Secure the area.
Have the See Appendix 2-2 for recommended PPE.
appropriate
personal
protective
equipment
(PPE).
Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release (quantity, location, and
Contain the
ventilation). If circumstances indicate that it is an emergency response release, make
spill.
appropriate notifications; if the release is determined to be incidental, contact the supplier for
assistance.
Liquids: Confine the spill to the initial spill area using appropriate control equipment and
material. For flammable liquids, dike off all drains.
Confine the
Gases: Follow the supplier’s suggestions or request outside assistance. Mercury: Use
spill.
appropriate materials to confine the spill (see Appendix 2-6). Expel mercury from the aspirator
bulb into a leakproof container, if applicable.
Liquids: Apply appropriate control materials to neutralize the chemical (see Appendix 2-
Neutralize
6).
the spill
Mercury: Use a mercury spill kit if needed.
Liquids: Scoop up solidified materials, booms, pillows, and any other materials. Put used
materials into a leakproof container. Label the container with the name of the hazardous
materials. Wipe up residual material. Wipe the spill area surface three times with a detergent
solution. Rinse the areas with clean water. Collect the supplies used (eg, goggles or shovels)
and remove gross contamination; place equipment to be washed and decontaminated into a
Clean up separate container.
the spill. Gases: Follow the supplier’s suggestions or request outside assistance. Mercury: Vacuum
up the spill using a mercury vacuum, or scoop up mercury paste after neutralization and collect
the paste in a designated container. Use a sponge and detergent to wipe and clean the spill
surface three times to remove absorbent. Collect all contaminated disposal equipment and put
it into a hazardous waste container. Collect supplies and remove gross contamination; place
equipment that will be thoroughly washed and decontaminated into a separate container.
(Continued)

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■ APPENDIX 2-7
Managing
Hazardous Chemical
Spills (Continued)
Actions Instructions for Hazardous Liquids, Gases, and Mercury
 Dispose. Liquids: Dispose of material that was neutralized as solid waste. Follow the
facility’s procedures for disposal. For flammable liquids, check with the facility safety
officer for appropriate waste determination.
Gases: The manufacturer or supplier will instruct the facility about disposal if
applicable.
Mercury: Label with appropriate hazardous waste label and Department of
Transportation diamond label.
Follow appropriate spill documentation and reporting procedures. Investigate the
Report.
spill; perform a root cause analysis if needed. Act on opportunities for improving safety.

Chapter 3
Regulatory Issues in Blood Banking
Glenn Ramsey, MD

^K|IN THE UNITED STATES, when fedfij3l eral laws are enacted, they are published chronologically as
statutes and placed into the appropriate 1 of 50 subject areas (titles) of the United States Code (USC).1 The
government agency responsible for the law—for bloodrelated laws, the Food and Drug Administration (FDA)
—then writes regulations (rules) to enforce the law. Proposed rules for public comment and final rules, along
with background and interpretive information, are published chronologically in the daily Federal Register.2 The
current edition of all rules is collated annually in the appropriate title of the Code of Federal Regulations (CFR);
Title 21 addresses food and drugs, and Title 42 focuses on health care.3
Some rules have associated guidance documents to provide current thinking about regulatory topics.
Guidance documents contain recommendations, not requirements, but they are usually followed by industry.
Memoranda to blood establishments were issued before 1998, but some are still active references. These
publications can be accessed from the FDA blood and blood products webpage.4
The FDA regulates drugs and medical devices under the Food, Drug, and Cosmetic Act (USC Title 21,
Sections 301-399d).5 Blood products are defined as drugs because they are intended to cure, mitigate, treat, or
prevent disease (21 USC 321).6,7 The law requires blood product manufacturers to register with the FDA,
obtain biologies licenses, and follow current good manufacturing practice (cGMP) regulations. It also prohibits
adulteration and misbranding, authorizes inspections, and defines civil and criminal penalties for violations. The
act controls the use of unapproved drugs and devices during their investigational phases and public health
emergencies.
Medical devices include instruments, invitro reagents, and their parts and accessories for the diagnosis and
treatment of disease. The FDA classifies devices as Class I, II, or III, in ascending order of associated risk [21
USC 360(c)].8,9 Class I devices have no potential unreasonable risk. Class II devices must meet performance
standards beyond basic FDA regulations. Some Class I devices and most Class II devices must be cleared by the
FDA based on substantial equivalence with another device
Glenn Ramsey, MD, Medical Director, Blood Banks, Northwestern Memorial Hospital and Ann & Robert
H. Lurie Children’s Hospital of Chicago, and Department of Pathology, Feinberg School of Medicine,
Northwestern University, Chicago, Illinois The author has disclosed no conflicts of interest.
 83

84
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already on the market. This process is called 510(k) clearance, referring to the applicable section of the act
describing the submission of applications to the FDA for such devices [21 USC 360(k)].8 Class III devices pose
potential unreasonable risk and require specific premarket approval from the FDA, as do unprecedented new
devices before their class is determined.
BIOLOGICAL PRODUCTS
Biological products come from living sources and include blood, blood components, derivatives, therapeutic
serum, and vaccines applicable to the prevention or treatment of disease. They are regulated by Section 351 (42
USC 262) of the Public Health Service Act, which addresses licensure, labeling, inspections, recalls, and
penalties for violations in producing biological drugs. This set of regulations provides the core of federal law
that is most specific to blood products.10 Section 361, Regulations to Control Communicable Diseases (42 USC
264), grants the Surgeon General inspection and quarantine powers to prevent the spread of infectious diseases
and is invoked in the regulation of tissues (see below).11
The FDA’s key regulations enforcing the Food and Drug Act and the Public Health Service Act are in Title
21 of the CFR, Food and Drugs, and in particular, Parts 200 through 299 for drugs, 600 through 680 for
biologicals, 800 through 898 for devices, and 1270 through 1271 for tissues (Table 3-1).12 The FDA website
has links to pertinent laws in the USC and regulations in the CFR.13
Within the FDA, the Center for Biologies Evaluation and Research (CBER) regulates blood products and
most other biological therapies.14 The Center for Devices and Radiological Health (CDRH) regulates most
medical devices, but CBER retains primary jurisdiction over medical devices used with blood and cellular
products.15 The FDA’s Office of Regulatory Affairs (ORA) has responsibility for all field operations, which
include inspections and investigations.
As part of the development process for FDA regulations and guidance documents,
several forums are offered for input from the public and regulated groups.16 Proposed rules and draft
guidance documents are published with an invitation for written comments, which are filed in public dockets.17
When final rules are published in the Federal Register, the accompanying commentaries offer response to key
questions. The FDA also receives petitions to write or change regulations. Expert opinions on current issues are
sought from several advisory committees, including the FDA’s committees on blood products and on cellular,
tissue, and gene therapies, and the Department of Health and Human Services (DHHS) Committee on Blood
Safety and Availability. Public meetings hosted by the FDA on selected topics provide an additional opportunity
for input.
Facilities may apply to CBER for approval of exceptions to the regulations for blood products or alternative
procedures. These approvals are periodically published, although the circumstances for these approvals may not
apply to other facilities.18
LICENSURE AND REGISTRATION
Blood establishments are classified into three categories by the FDA: 1) licensure and registration for
interstate commerce, 2) registration for manufacturers not involved in interstate commerce, and 3) exemption
from registration for transfusion services as described in the regulations.19 Licensed and/or registered facilities
are inspected by the FDA. For transfusion services that perform minimal manufacturing and are certified for
reimbursement by the Centers for Medicare and Medicaid Services (CMS), the FDA accepts CMS-sanctioned
laboratory inspections and approval.20 However, the FDA still has jurisdiction and may conduct its own
inspections if warranted. All military blood facilities, including transfusion services, register with and are
inspected by the FDA.21
Facilities use the Biologies License Application (BLA, Form 356h) to apply for their establishment and
product licenses for interstate commerce.22 Subsequent license amendments are based on the potential for
85
TABLE 3-1. Regulations of Interest in Title 21 of the CFR (Food and Drugs)
Topic Section Topic Section
Donor
FDA general 630.6
notification
 Enforcement 1-19 Blood product standards 640
Research and development 50-58 Donor eligibility 640.3
Labeling 201 Blood collection 640.4
cGMP for drugs 210-211 Blood testing 640.5
Biological products 600-680 Red Blood Cells 640.10-.17
General 600 Platelets 640.20-.27
Licensing 601 Plasma 640.30-.34
cGMP for blood components 606 Cryoprecipitated AHF 640.50-.56
Personnel, 606.20-.65 Exceptions, alternatives 640.120
resources
Standard operating
606.100 Medical devices 800-898
procedures
Labeling 606.120-.122 Adverse events 803
Compatibility testing 606.151 Hematology and pathology 864
Records 606.160-.165 Tissues
Adverse reactions 606.170 Tissues for transplantation 1270
Cellular and tissue-based
Product deviations 606.171 1271*
products
Establishment
607 General provisions 1271.1-.20
registration
General standards 610 Registration and products 1271.21-.37
Donor testing 610.40 Donor eligibility 1271.45-.90
Donor deferral 610.41 cGTP 1271.145-.320
Look-back 610.46-.48 Section 361 provisions* 1271.330-.440
Dating periods 610.53
*The following citations represent Subparts A, B, C, D, and E-F, respectively. fAutologous and related
donors (see text).
CFR = Code of Federal Regulations, FDA = Food and Drug Administration, cGMP = current good
manufacturing practice; cGTP = current good tissue practice.

86
AABB TECHNICAL MANUAL
adverse effects of the changes.23 Unlicensed blood products, whether manufactured by a licensed or an
unlicensed facility, may be shipped across state lines for a medical emergency if necessary, but this shipping
should be unscheduled and infrequent.24 The shipping facility must retain documentation of the emergency for
FDA review.
Facilities must register annually with the FDA if they collect, manufacture, prepare, or process blood
products (Form FDA 2830).25,26 Transfusion services are exempt from this requirement if they are approved
for reimbursement by CMS and their preparation activities are basic, such as preparing Red Blood Cells from
whole blood, converting unused plasma to recovered plasma, pooling components, reducing leukocytes with
bedside filters, or collecting blood only in emergencies. However, facilities that routinely collect blood
(including autologous units) or perform such procedures as irradiation, washing, laboratory leukocyte reduction,
freezing, deglycerolization, and rejuvenation must register as community or hospital blood banks. Transfusion
services acting as depots for forwarding products to other hospitals must register as distribution centers. Clinical
laboratories performing infectious disease testing required for blood donors must register unless they are CMS
approved. If blood irradiation is performed outside the blood bank or transfusion service, such as in a nuclear
medicine department, that facility must register as well.
FDA INSPECTIONS
New facilities are generally inspected within 1 year of establishment by a team from CBER and ORA.
Subsequent routine inspections are generally performed by ORA every 2 years or earlier depending on the
facility’s compliance history.
ORA publishes online manuals and instructions for FDA investigators for inspections in general and for
inspections of licensed and unlicensed blood banks in particular.25,26 The blueprints for blood bank inspections
are the general regulations for cGMP and drugs (21 CFR 210-211) and the specific require
ments for blood components (21 CFR 606).27 Routine inspections address the FDA’s five required layers of
blood safety—donor screening, donor testing, product testing, quarantining, and monitoring and investigating
problems. Investigators review the following operational systems that create these safety layers: quality
assurance, donor eligibility, product testing, quarantine/inventory management, and production and processing.
Full inspections of all systems are designated Level I. After a favorable inspection profile, facilities with
four or five systems sometimes have streamlined Level II inspections of three systems. (Focused inspections for
problems or fatalities need not follow these formats.) Within each system, the investigators review standard
operating procedures, personnel and training, facilities, equipment calibration and maintenance, and records.
Specific requirements for individual systems and processes are discussed in detail in their respective chapters of
this book.
If the investigator judges that significant objectionable practices, violations, or conditions are present under
which a drug or device is or could be adulterated or injurious to health, these observations are written and
presented to the facility (Form 483). Investigators are instructed to seek and record the management’s intentions
to make corrections.25 However, most facilities also respond in writing immediately with questions or
corrective actions. Final determination that a violation has occurred is made by ORA.
The FDA can take a number of steps in response to a violation, including no action at all. For significant
violations, the FDA may issue a warning letter, which is an advisory action to provide the facility with the
opportunity for voluntary compliance. Enforcement actions are categorized as administrative, judicial, or recall.
Administrative actions include formal citations of violation and—for licensed facilities—suspension or
revocation of a license. Judicial actions range from seizures of products to court injunctions, civil monetary
penalties, and criminal prosecution. The FDA may also conduct recalls if necessary. ORA’s
87
Regulatory Procedures Manual provides details on available sanctions.28
BLOOD-RELATED DEVICES
CBER has lead responsibility in collaboration with CDRH for equipment marketed for transfusion and the
collection and processing of blood products, hematopoietic stem cells, and autotransfusions.29'30 These devices
include diagnostic tests for infectious diseases in blood donors and pretransfusion testing in patients. Blood
bank computer software programs are CBER-approved devices.
Most blood-related devices are in Class II and cleared by 510(k) equivalence. Examples of Class I devices
are copper sulfate solutions for hemoglobin screening, blood grouping view boxes, and heat sealers. Human
immunodeficiency virus (HIV) and hepatitis B and C virus donor tests are regulated as Class II or III devices.
Screening tests for blood donations are regulated as BLAs. BLAs and Class III devices require specific
premarket approval. Each category of device is assigned a code, and all cleared or approved vendors and
products for that code are searchable in the Establishment Registration and Device Listing database on the
CDRH website.31
Serious adverse events related to medical devices must be reported (21 CFR 803).32 User facilities must
report deaths or serious injuries in which a device was or may have been a factor. Serious injury is defined as
being life threatening, causing permanent impairment or damage, or needing medical or surgical intervention.
Reports of serious injuries are sent to the device maker using FDA Medwatch Form 3500A within 10 working
days of the event, and deaths must also be reported to the FDA. In years when a Form 3500A report is
submitted, an annual summary must be sent to the FDA by January 1 of the following year (Form 3419) ,22
Users may voluntarily report other device-related adverse events to the FDA (Form 3500).22 All possible
adverse events, whether reported or not, must be investigated and their records must be kept on file for 2 years.
HEMATOPOIETIC PROGENITOR CELLS AS TISSUES
Unmanipulated marrow cell transplants are regulated by the Health Resources and Services Administration
of DHHS, which also oversees marrow and cord blood donations and transplant procedures coordinated by the
National Marrow Donor Program. Other hematopoietic progenitor cells (HPCs) collected before May 25, 2005
are regulated by FDA rules for tissue transplants in 21 CFR 1270.12. The FDA’s regulations in 21 CFR 1271 for
human cells, tissues, and cellular and tissue-based products (HCT/Ps) apply to HPCs collected on or after May
25,2005.12'33'34
HCT/P manufacturers must comply with rules for 1) facility registration and product listing; 2) donor
eligibility, including testing for communicable diseases; and 3) production according to current good tissue
practice (cGTP) regulations (Table 3-1). Registrants using FDA Form 3356 report each type of HPC they
manufacture in three categories: 1) HCT/Ps as described in 21 CFR 1271.10(a), 2) biologies, and 3) drugs.
Table 3-2 outlines how the FDA defines, regulates, and (when applicable) approves each type of HPC. Basic
peripheral blood stem cells (PBSCs) from autologous or first- or second-degree related donors are in the first
category; these products are regulated mainly with regard to preventing transmission of communicable diseases
or contamination (Public Health Service Act, Section 361,42 USC 264).11 Typical PBSCs from unrelated
donors are biologies, and the FDA is considering licensure requirements for them. Since October 20, 2011,
unrelated umbilical cord cells must be licensed or used under an investigational new drug application (see
Chapter 30). Any HPCs that are more than minimally manipulated (eg, by expansion, property alteration, or
gene therapy) or used for a different function (eg, tissue engineering) are categorized as a drug and must have
premarket approval.
The FDA specifies required infectious disease tests for tissue donors and posts lists of tests that have been
licensed or cleared for this indication.45 The elements of cGTP are analogous to those of cGMP for blood
products. The
TABLE 3-2. US Regulations for Manufacturers of Hematopoietic Progenitor Cells:
AABB TECHNICAL MANUAL
k<°
c\j i "55 o o S
00“
1— o
O '-d CM
O 00 0 ^0
C/3 qj) d
03 o O
>- = c
Q £ -O
i 15
E5 s i
O E -g •— r~) 03
co co
ctS q co
in autologous transfusion or in allogeneic transfusion of donations from a first- or second-degree relative.
HPC = hematopoietic progenitor cell, CFR = Code of Federal Regulations, FDA = Food and Drug
Administration, PHS = Public Health Service, HCT/Ps = human cells, tissues, and cellular and tissue-based
products, IND = investigational new drug, CBER = Center for Biologies Evaluation and Research, CDRH =
Center for Devices and Radiological Health, BLA = biologies license application.
 89
Circular of Information for the Use of Cellular Therapy Products is jointly written by the AABB and other
organizations for users of these products.46 The AABB and the Foundation for Accreditation of Cellular
Therapy set voluntary standards and accredit facilities for collection and processing of HPCs (see Chapters 29
and 30 for information on these standards and accreditation procedures).
The FDA tissue regulations do not apply to facilities that receive, store, and administer tissues but do not
perform any manufacturing steps. However, The Joint Commission (TJC) has hospital standards for handling
and tracing tissues and investigating adverse events (TS.03.01.01 to 03.03.01) (see Chapter 32 for information
on these standards).47
MANAGING RECALLS AND WITHDRAWALS
The FDA’s requirements for monitoring and investigating problems with drugs extend to the time after a
product’s release. When blood banks discover after distribution that a blood product was in violation of rules,
standards, specifications, or cGMP regulations, they must report the problem to the FDA and their consignee.
These problems comprise a subset of biological product deviations (BPDs)—events in which the safety, purity,
or potency of a distributed blood product may be affected—all of which must be reported to the FDA.48
A recall is defined as the removal or correction of a marketed product that is in violation of the law (21 CFR
7.3).49 The FDA classifies recalls by severity. Most blood component recalls are in Class III, not likely to cause
adverse health consequences. Class II recalls are for products that may cause temporary adverse effects or
remotely possible serious problems. Class I recalls involve a reasonable probability of serious or fatal adverse
effects. Ah recalls are published by the FDA and involve about 1 in 5800 blood components issued in the
United States.50
Market withdrawals are required for products found to be in minor violation of the law. Problems beyond
the control of the manufacturer, such as postdonation donor infor
mation, are often in this category. Withdrawals are much more common than recalls but are not published.
Recommendations for managing common notices have been published in the United States and
Canada.50,51 Transfusion services need to have procedures and training for rapid responses as advised when
recalls and withdrawals are received along with records of actions, reviews, and follow-up actions, as indicated.
Accreditation requirements of the AABB (Standard 7.1) and the College of American Pathologists (CAP)
(Checklists TRM.42120 and TRM.42170) address aspects of this topic.52,53
Most of these blood components are transfused before a notice is received of their nonconformance. In some
recent blood guidance documents on infectious diseases, the FDA has included recommendations on whether or
not to notify the recipient’s physician about transfused units. In cases of possible recent infectious disease
exposure in donors or transfusion recipients, the seroconversion window periods for tests should be consulted
for scheduling prospective testing or reviewing retrospective results, such as for a donor who has been retested
since an exposure.50
The most common reasons for BPDs and blood component recalls are given in Table 33. Look-backs
investigations on units from donors found after donation to have HIV or hepatitis C virus are discussed in
Chapter 5.
MEDICAL LABORATORY LAWS AND REGULATIONS
CMS regulates all US medical laboratories under the Clinical Laboratory Improvement Amendments
[CLIA, 42 USC 263(a) and 42 CFR 493] to Section 353 of the Public Health Service
Act.54,55
The law and regulations establish the requirements and procedures for laboratories to be certified under
CLIA as both a general requirement and a prerequisite for receiving Medicare and Medicaid payments.
To be certified, laboratories must have adequate facilities and equipment, supervisory and technical
personnel with training and
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AABB TECHNICAL MANUAL
TABLE 3-3. Most Common Reasons for Biological Product Deviations and Blood Component Recalls
Biological Product Deviations (Ranked by number Blood Component Recalls (Ranked by number of
of events) units)
Malaria travel/residence Collection sterility and arm preparation
Variant Creutzfeldt-Jakob disease travel/residence Storage temperature
Production according to current good
Postdonation illness
manufacturing practice
Tattoo Donor suitability
Donor deferral missed via incorrect donor
Product quality control
identification
Biological product deviations shown are from licensed establishments.48 Blood component recalls are
compiled from Food and Drug Administration Enforcement Reports.50 Market withdrawals are not included in
public recalls data.50
experience appropriate to the complexity of testing, a quality management system (see Chapter 1), and
successful ongoing performance in CMS-approved proficiency testing (PT) ,56 All laboratories must register
with CMS, submit to inspection by CMS or its designees, and obtain recertification every 2 years.
All laboratory tests are rated for complexity by the FDA for CMS as waived, moderate complexity, and high
complexity. Waived tests are simple and easily performed with limited technical training. Examples include
over-thecounter tests, urinalysis dipsticks, copper sulfate specific-gravity hemoglobin screens, spun
microhematocrits, and some simple devices for measuring hemoglobin. Laboratories that perform only waived
tests register with CMS for a certificate of waiver. The Centers for Disease Control and Prevention provides
technical and advisory support to CMS for laboratory regulation and has published practice recommendations
for waived testing sites.57
Nonwaived tests are classified as being of moderate or high complexity based on a scoring system of needs
for training, preparation, interpretive judgment, and other factors (42 CFR 493.17).54 The CDRH website
provides the searchable CLIA database on the complexity levels of specific tests.31
Blood banks and transfusion services have three pathways to obtain a CLIA certifi
cate to perform testing: 1) certificate of compliance: approval via state health department inspections using
CMS requirements; 2) certificate of accreditation: approval via a CMSapproved accrediting organization; and 3)
CMS-exempt status: licensure programs for nonwaived laboratories in New York and Washington States that
are accepted by CMS.58
 The CLIA regulations delineate general requirements for facilities; quality systems, including quality
assurance and quality control systems; and management and technical personnel qualifications. High-
complexity tests require more stringent personnel qualifications. Immunohematology laboratories have
standards for blood supply agreements, compatibility testing, blood storage and alarms, sample retention,
positive identification of blood product recipients, investigation of transfusion reactions, and documentation (42
CFR 493.1103 and 493.1271).54 Viral and syphilis serologic tests are part of the immunology requirements.
CMS has published guidelines for conducting surveys (inspections).59
CMS has approved six laboratory accreditation organizations with requirements that meet CMS regulations:
the AABB, American Osteopathic Association, American Society for Histocompatibility and Immunogenetics
(ASHI), CAP, COLA (formerly the Commission on Office Laboratory Accreditation), and The

91
Joint Commission.60 The Joint Commission has cooperative agreements with ASHI, CAP, and COLA to
accept their laboratory accreditations in facility surveys.61
AABB and CAP can coordinate joint surveys by facilities seeking both types of accreditation. CMS may
perform its own follow-up surveys to validate those of the accreditation organizations.
CMS requires successful PT for ongoing laboratory certification of nonwaived testing. Within each
laboratory section, CMS regulations specify tests and procedures (regulated analytes) that must pass approved
PT if the laboratory performs them. The CMS website has a list of approved PT providers.62 PT is discussed in
Chapter 1.
HOSPITAL REGULATIONS AND ACCREDITATION
CMS approves hospitals for Medicare reimbursement through state surveys or accreditation programs from
The Joint Commission, the American Osteopathic Association, and DNV Healthcare. These inspections cover
CMS regulations for blood administration and the evaluation of transfusion reactions [42 CFR 482.23(c)].63
The Joint Commission has standards for preventing misidentification of
laboratory specimens and transfusion recipients (NPSG.01.01.01, .01.03.01), checking blood products in the
Universal Protocol preprocedure verification process (“time-out”) (UP.01.01.01), and assessing transfusion
appropriateness (MS.05.01.01).47The Joint Commission includes hemolytic transfusion reactions in its Sentinel
Events reporting program.64
Facilities also should be familiar with all relevant state and local laws and regulations, including
professional licensure requirements for medical and laboratory personnel. In some situations, facilities
providing products or services in other states must comply with local regulations in the customer’s location.
CONCLUSION
Blood components and other blood products are regulated as pharmaceutical agents, and blood banks and
transfusion services are also regulated as medical laboratories. These many requirements make compliance
complex. However, close regulation underscores the recognized importance to public health of safe, effective
blood products provided via good laboratory practices using the quality management systems described in
Chapter 1 and the procedures discussed throughout this manual.
KEY POINTS
1. The FDA regulates the manufacture and use of drugs and medical devices, including blood components,
blood-derived biological products, and their ancillary testing and transfusion equipment. The FDA website
provides links to blood-related regulations in Title 21 CFR and to explanatory guidance documents.
 2. The FDA requires licensure and registration for blood manufacturers engaged in interstate commerce and
registration alone for manufacturing activities, such as irradiation or washing. Transfusion services performing
only basic component preparation need not register but must have laboratory approval from CMS.
3. Licensed and registered blood facilities are inspected by the FDA every 2 years, with a focus on current
cGMP (21 CFR 210-211) and blood component requirements (21 CFR 606). Observations of significant
noncompliance or hazards are reported to the facility for its response and correction and may result in a
spectrum of sanctions, possibly including license revocation and criminal prosecution.
4. HPCs for transplantation are regulated as tissues by the FDA, with an emphasis on preventing
transmissions of infections and contaminations in autologous or closely related allogeneic transplants. The FDA
has a licensure process for unrelated umbilical cord blood HPCs.

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5. The FDA requires drug (and blood) manufacturers to conduct recalls or market withdrawals when
noncompliance is found after products are issued, such as for donor malaria risk or potentially compromised
component sterility. Transfusion services should have procedures for evaluating and managing the potential
impact of these recalls and withdrawals on patients transfused with such products.
6. All US medical laboratories must be approved by CMS every 2 years. Laboratory regulations emphasize
the need for adequate facilities and qualified personnel commensurate with the complexity of testing, and they
require ongoing successful performance in proficiency testing by CMS-approved vendors. Laboratory approval
by CMS is granted via inspections performed by CMS-approved accrediting agencies or state health
departments.
 7. Health-care facilities also have CMS regulations for their activities, and The Joint Commission and other
organizations accredit many hospitals for CMS compliance. CMS and The Joint Commission have requirements
for monitoring transfusion practices, evaluating adverse transfusion reactions, and preventing mistransfusions.
REFERENCES
1. Office of the Law Revision Council. Search the United States Code. Washington, DC: US House of
Representatives, 2013. [Available at http://uscode.house.gov/search/criteria.shtml (accessed November
15,2013).]
2. Federal register. Washington, DC: National Archives and Records Administration and US Government
Printing Office, (updated daily). [Available at https://www.federalregister.gov (accessed November 15,2013).]
3. Electronic code of federal regulations (e-CFR). Washington, DC: Office of the Federal Register, National
Archives and Records Administration, 2013. [Available at http://www.ecfr.gov (accessed November 15,2013).]
4. Blood and blood products. Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2013. [Available at http:// www.fda.gov/BiologicsBloodVaccines/Blood
BloodProducts/default.htm (accessed November 15, 2013).]
5. Federal Food, Drug, and Cosmetic Act (FD&C Act). Silver Spring, MD: FDA, 2011. [Available at
(http://www.fda.gov/RegulatoryInforma tion/Legislation/FederalFoodDrugandCos
meticActFDCAct/default.htm (accessed November 16,2013).]
6. United States code. Title 21, USC Part 321.
7. Compliance policy guides. CPG 230.120— Human blood and blood products as drugs. Silver Spring,
MD: Food and Drug Administration, 2011. [Available at http://www.fda.gov/ ICECI/ ComplianceManuals /
CompliancePoli cyGuidanceManualZucm073863.htm (accessed March 30,2014).]
8. FD&C Act Chapter V: Drugs and devices. Silver Spring, MD: Food and Drug Administration, 2012.
[Available at http://www.fda.gov/Regu latorylnformation/Legislation/FederalFood
DrugandCosmeticActFDCAct/FDCActChap terVDrugsandDevices / default.htm (accessed November
16,2013).]
9. General and special controls. Silver Spring, MD: Food and Drug Administration, 2013. [Available at
http://www.fda.gov/MedicalDe vices/DeviceRegulationandGuidance/Over view/GeneralandSpecialControls/ de
fault.htm (accessed November 16,2012).]
10. United States code. Title 42, USC Part 262.
11. United States code. Title 42, USC Part 264.
12. Code of federal regulations. Title 21, CFR Parts 200-299, 600-680, and 1270-1271. Washington, DC:
US Government Printing Office, 2014 (revised annually).
13. Food and Drug Administration. Washington, DC: FDA, 2013. [Available at http://www.fda. gov
(accessed November 8,2013).]
14. Food and Drug Administration. Vaccines, blood & biologies. Washington, DC: CBER Office of
Communication, Outreach, and Development, 2013. [Available at http://www.fda. gov/BiologicsBloodVaccines
(accessed Novembers, 2013).]
15. Medical devices. Washington, DC: Food and Drug Administration, 2013. [Available at http:/ /
www.fda.gov/MedicalDevices/default.htm (accessed November 8,2013).]
16. Comment on regulations. Silver Spring, MD: Food and Drug Administration, 2009. [Available at
http://www.fda.gov/AboutFDA/Con
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tactFDA/CommentonRegulations/default, htm (accessed November 16,2012).]
17. Regulations.gov. Washington, DC: US Government, 2013. [Available at http://www.regula tions.gov
(accessed November 8,2013).]
18. Exceptions and alternative procedures approved under 21 CFR 640.120. Silver Spring, MD: CBER
Office of Communication, Outreach, and Development, 2013. [Available at
http://www.fda.gov/BiologicsBloodVaccines/ BloodBloodProducts/RegulationoftheBlood
Supply/ExceptionsandAlternativeProcedures/ default.htm (accessed November 8,2013).]
19. Code of federal regulations. Title 21, CFR Part 607.65(f). Washington, DC: US Government Printing
Office, 2013 (revised annually).
20. MOU 225-80-4000. Memorandum of understanding between the Health Care Financing Administration
and the Food and Drug Administration. (June 6,1983) Silver Spring, MD: Food and Drug Administration, 1983.
[Available at http://www.fda.gov/AboutFDA/Part nershipsCollaborations/MemorandaofUnder
standingMOUs/DomesticMOUs/ucml 16313. htm (accessedNovember 16,2013).]
21. MOU 225-74-1017. Memorandum of understanding between the Department of Defense and the Food
and Drug Administration. (June 19, 1974) Silver Spring, MD: Food and Drug Administration, 1974. [Available
at http:// www.fda.gov/AboutFDA/PartnershipsCollab orations/MemorandaofUnderstandingMOUs/
DomesticMOUs/ucml 15877.htm (accessed November 16, 2013).]
22. FDA forms. Silver Spring, MD: Food and Drug Administration, 2010. [Available at http://
www.fda.gov/AboutFDA/ReportsManuals Forms/Forms/default.htm (accessed November 16, 2013).]
23. Guidance for industry. Changes to an approved application: Biological products: Human blood and
blood components intended for transfusion or for further manufacture. (July 2001) Silver Spring, MD: CBER
Office of Communication, Outreach, and Development, 2001. [Available at http://www.fda.gov/
BiologicsBloodVaccines/GuidanceComplian ceRegulatorylnformation/Guidances/Blood/ ucm076729.htm
(accessed November 21, 2013).]
24. Compliance policy guides. CPC Sec. 220.100. IS shipment biologicals for medical emergency. Silver
Spring, MD: Food and Drug Administration, 2011. [Available at http://www.fda.
gov/ICECI/ComplianceManuals/Complian cePolicyGuidanceManual/ucm073860.htm (accessed November
8,2013).]
25. Investigations operations manual 2012. Silver Spring, MD: Food and Drug Administration, 2012.
[Available athttp://www.fda.gov/ICECI/ Inspections/IOM/default.htm (accessed November 16,2013).]
26. 7342.001. Inspection of licensed and unlicensed blood banks, brokers, reference laboratories, and
contractors. (December 2010) Silver Spring, MD: Food and Drug Administration, 2013. [Available at
http://www.fda.gov/ BiologicsBloodVaccines / GuidanceComplian ceRegulatorylnformation/ComplianceActivi
ties/Enforcement/CompliancePrograms/ ucm095226.htm (accessed November 8,
2013) .]
27. Code of federal regulations. Title 21, CFR Parts 210, 211, and 606. Washington, DC: US Government
Printing Office, 2014 (revised annually)
28. Regulatory procedures manual—2012. Silver Spring, MD: Food and Drug Administration, 2011.
[Available athttp://www.fda.gov/ICECI/ ComplianceManuals/RegulatoryProcedures Manual/default.htm
(accessed March 30,
2014) .]
29. Intercenter agreement between the Center for Biologies Evaluation and Research and the Center for
Devices and Radiological Health. (October 31,1991) Bethesda and Silver Spring, MD: Food and Drug
Administration, 1991. [Available at http://www.fda.gov/Combina tionProducts/Jurisdictionallnformation/
ucml21175.htm (accessed November 16, 2013).]
 30. Devices regulated by the Center for Biologies Evaluation and Research. Silver Spring, MD: CBER
Office of Communication, Outreach, and Development, 2013. [Available at http://
www.fda.gov/biologicsbloodvaccines/devel opmentapprovalprocess/510kprocess/ ucml33429.htm (accessed
November 8, 2013).]
31. Medical device databases. Silver Spring, MD: Food and Drug Administration, 2013 (revised monthly).
[Available at http://www.fda.gov/ MedicalDevices/DeviceRegulationandGuid ance/Databases/default.htm
(accessed Novembers, 2013).]
32. Code of federal regulations. Title 21, CFR Part 803. Washington, DC: US Government Printing Office,
2014 (revised annually).
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33. Tissues and tissue products. Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2010. [Available at http:// www.fda.gov/BiologicsBloodVaccines/Tissue
TissueProducts/default.htm (accessed November 16, 2013).]
34. Halme DG, Kessler DA. Regulation of stemcell-based therapies. N Engl J Med 2006;355: 1730-5.
35. Guidance for industry: Regulation of human cells, tissues, and cellular and tissue-based products
(HCT/Ps)—small entity compliance guide. (August 2007) Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 2007. [Available at http://www.fda. gov/BiologicsBloodVaccines/GuidanceCom
plianceRegulatorylnformation / Guidances / Tissue/ucm073366.htm (accessed November 16,2013).]
36. Guidance for industry: Current good tissue practice (CGTP) and additional requirements for
manufacturers of human cells, tissues, and cellular and tissue-based products (HCT/Ps). (December 2011)
Silver Spring, MD: CBER Office of Communication, Outreach and Development, 2011. [Available at
http://www.fda. gov/downloads/BiologicsBloodVaccines/ GuidanceComplianceRegulatorylnforma
tion/Guidances/Tissue/UCM285223.pdf (accessed November 16,2013).]
37. 7341.002—Inspection of human cells, tissues, and cellular and tissue-based products (HCT/ Ps). Silver
Spring, MD: CBER Office of Communication, Outreach, and Development, 2012. [Available at
http://www.fda.gov/Biolog icsBloodVaccines/GuidanceComplianceRegu latorylnformation /
ComplianceActivities / En forcement/CompliancePrograms/ucm 095207.htm (accessed November 21,2013).]
38. Compliance programs (CBER). Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2012. [Available at http://www.fda.gov/BiologicsBloodVaccines/
GuidanceComplianceRegulatorylnformation/ ComplianceActivities/Enforcement/Compli
ancePrograms/default.htm (accessed November 16, 2013).]
39. 7341.002A—Inspection of tissue establishments. (December 1, 2007) Silver Spring, MD: CBER Office
of Communication, Outreach, and Development, 2007. [Available at http://
www.fda.gov/BiologicsBloodVaccines/Guid anceComplianceRegulatorylnformation/
ComplianceActivities/Enforcement/Compli
ancePrograms/ucm095218.htm (accessed November 16,2013).]
40. 7342.007 Addendum—Imported human cells, tissues, and cellular and tissue-based products (HCT/Ps).
(January 1, 2010) Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 2010.
[Available at http:// www.fda.gov/BiologicsBloodVaccines/Guid anceComplianceRegulatorylnformation/
ComplianceActivities/Enforcement/Compli ancePrograms/ucm095250.htm (accessed November 16,2013).]
41. Guidance for industry: BLA for minimally manipulated, unrelated allogeneic placental/umbilical cord
blood intended for hematopoietic and immunologic reconstitution in patients with disorders affecting the
hematopoietic system. (March 2014) Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2009. [Available at http://www. fda.gov/downloads/BiologicsBloodVaccines/
GuidanceComplianceRegulatorylnforma tion/ Guidances / CellularandGeneTherapy / de fault.htm (accessed
March 30,2014).]
42. Guidance for industry and FDA staff: IND applications for minimally manipulated, unrelated allogeneic
placental/umbilical cord blood intended for hematopoietic and immunologic reconstitution in patients with
disorders affecting the hematopoietic system. (March 2014) Silver Spring, MD: CBER Office of
Communication, Outreach, and Development, 2009. [Available at http://www.fda.gov/down
loads/BiologicsBloodVaccines/Guidance ComplianceRegulatorylnformation/Guidanc
es/CellularandGeneTherapy/default.htm (accessed March 30,2014).]
 43. 7345.848—Inspection of biological drug products. (October 1, 2010) Silver Spring, MD: CBER Office
of Communication, Outreach, and Development, 2010. [Available at http://
www.fda.gov/BiologicsBloodVaccines/Guid anceComplianceRegulatorylnformation/
ComplianceActivities/Enforcement/Compli ancePrograms/ucm095393.htm (accessed November 16,2013).]
44. Inspection of medical device manufacturers. Program 7382.845. (February 2, 2011) Silver Spring, MD:
Food and Drug Administration, 2011. [Available at http://www.fda.gov/Medi
calDevices/DeviceRegulationandGuidance/ GuidanceDocuments/ucm072753.htm (accessed November 16,
2013).]
CHAPTER 3 Regulatory Issues In Blood Banking
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45. Testing HCT/P donors for relevant communicable disease agents and diseases. Silver Spring, MD:
CBER Office of Communication, Outreach, and Development, 2013. [Available at
http://www.fda.gov/BiologicsBloodVac cines/SafetyAvailability/Tissue Safety/ucm 095440.htm (accessed
November 8, 2013).]
46. AABB, America’s Blood Centers, American Association of Tissue Banks, American Red Cross,
American Society for Apheresis, American Society for Blood and Marrow Transplantation, College of
American Pathologists, Foundation for the Accreditation of Cellular Therapy, ICCBBA, International Society
for Cellular Therapy, Joint Accreditation Committee, National Marrow Donor Program, and Netcord. Circular
of information for the use of cellular therapy products. Bethesda, MD: AABB, 2013. [Available at
http://www.aabb. org/ (accessed November 8,2013).]
47. 2012 Comprehensive accreditation manual for hospitals: The official handbook (CAMH). Oakbrook
Terrace, IL: The Joint Commission, 2012.
48. Biological product deviations. Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2012. [Available at http://www.fda.gov/BiologicsBloodVaccines/
SafetyAvailability/ReportaProblem/Biologi calProductDeviations/default.htm (accessed November 16, 2013).]
49. Code of federal regulations. Title 21, CFR Part 7.3. Washington, DC: US Government Printing Office,
2014 (revised annually).
50. Ramsey G. Blood component recalls and market withdrawals: Frequency, reasons, and management in
the United States. Transfus Med Rev 2013:7:82-90.
51. Recommendations for the notification of recipients of a blood component recall. (March 21, 2012) St.
John’s, NT and Ottawa, ON: National Advisory Committee on Blood and Blood Products and Canadian Blood
Services, 2012 [Available at http://www.nacblood.ca/re sources/guidelines/recall-recipient-notifica tion.html
(accessed January 14,2014).]
52. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
53. College of American Pathologists Laboratory Accreditation Program checklists. Northfield, IL: College
of American Pathologists, 2013 (revised annually).
54. Code of federal regulations. Title 42, CFR Part 493. Washington, DC: US Government Printing Office,
2013 (revised annually).
55. United States code. Title 42, USC Part 263a.
56. Rauch CA, Nichols JH. Laboratory accreditation and inspection. Clin Lab Med 2007;27: 845-58.
57. Howerton D, Anderson N, Bosse D, et al. Good laboratory practices for waived testing sites: Survey
findings from testing sites holding a certificate of waiver under the Clinical Laboratory Improvement
Amendments of 1988 and recommendations for promoting quality testing. MMWR Recomm Rep 2005;54(RR-
13):132.
58. Clinical Laboratory Improvement Amendments (CLIA): How to obtain a CLIA certificate. (March
2006) Baltimore, MD: Centers for Medicare and Medicaid Services, 2006. [Available at
http://www.cms.hhs.gov/CLIA/down loads/HowObtainCLIACertificate.pdf (accessed November 18, 2012).]
59. Interpretive guidelines for laboratories. Appendix C. Survey procedures and interpretive guidelines for
laboratories and laboratory services. Baltimore, MD: Centers for Medicare and Medicaid Services, 2013.
[Available at http://www.cms.hhs.gov/CLIA/03_Interpre tive_Guidelines_for_Laboratories.asp (accessed
November 8,2013).]
60. List of approved accreditation organizations under CLIA. Baltimore, MD: Centers for Medicare and
Medicaid Services, 2013. [Available at http://www.cms.gov/Regulations-and-Guid
ance/Legislation/CLIA/Downloads/AOList. pdf (accessed November 8,2013).]
 61. Laboratory services. Facts about the cooperative accreditation initiative. Oakbrook Terrace, IL: The
Joint Commission, 2013. [Available at http://www.jointcommission.Org/assets/l/
18/cooperative_accreditation_initiative_6_ 15_ll.pdf (accessed November 8,2013).]
62. CLIA-approved proficiency testing programs—2013. Baltimore, MD: Centers for Medicare and
Medicaid Services, 2012. [Available at http://www.cms.hhs.gov/CLIA/down loads/ptlist.pdf (accessed
November 8,2013).]
63. Code of federal regulations. Title 42, CFR Part 482.23(c). Washington, DC: US Government Printing
Office, 2013 (revised annually).
64. Sentinel event. Oakbrook Terrace, IL: The Joint Commission, 2012. [Available at http://www.
jointcommission.org/sentinel_event.aspx (accessed November 18, 2013).]

Chapter 4
Disaster Management
Ruth D. Sylvester; MS, MT(ASCP)SBB; William FitzGerald, LTC USA (Ret); Wendy Trivisonno; and
Theresa Wiegmann, JD
much has been learned over the B81 past decade about preparing for and responding to disasters. A disaster
is defined by the United Nations International Strategy for Disaster Reduction as a serious event that disrupts
the normal functioning of a community or society and that exceeds the affected area’s ability to cope.1 Disasters
can be small, only affecting a single business, or large, encompassing an entire region. Whether a disaster
involves natural forces (eg, hurricanes, earthquakes, floods, or pandemic influenza) or is the result of human
activity (eg, terrorism or industrial accidents), organizations around the world have put forth tremendous
resources to counteract and mitigate the threats related to these events.
The goal of this chapter is to provide an overview of disaster management, which encompasses much more
than just preparing for a disaster. This chapter addresses the entire cycle of disaster management, including
elements
often overlooked in traditional disaster plans. This chapter is more strategic than procedural. Many of the
specific and tactical information and procedures mentioned in this chapter are described in detail in the AABB
Disaster Operations Handbook, which should be considered a complementary text to this chapter.2
Even though this chapter focuses primarily on emergency management strategies used in the United States,
many of the methods can also be adapted for use in other countries. Hospital-based blood banks and transfusion
services should note that many of the activities discussed in this chapter are intended for stand-alone
organizations (eg, blood collection centers) and may already be addressed in a hospital’s overall disaster plan.
However, hospital staff members should review both their hospital and departmental disaster plans to ensure
that blood-related issues (including those pertaining to cellular and related biologic therapies) are sufficiently
covered.

Ruth D Sylvester, MS, MT(ASCP)SBB, Director, Regulatory Services, America’s Blood Centers,
Washington, District of Columbia; William FitzGerald, LTC USA (Ret), Senior Advisor, Biomedical
Preparedness, American Red Cross, Washington, District of Columbia; Wendy Trivisonno, Director, Strategic
Biologic Sourcing and Logistics, Blood Centers of America, West Warwick, Rhode Island; and Theresa
Wiegmann, JD, Director of Public Policy, AABB, Bethesda, Maryland The authors have disclosed no conflicts
of interest.
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BACKGROUND
Disaster Management Cycle
Disaster management includes plans for, responses to, and recovery from disasters. Planning by its nature is
cyclic and, as with many activities, future responses to disasters can be improved by learning from past events.
The disaster management cycle includes four functional areas: mitigation, preparedness, response, and
recovery.3 Organizations should consider each of these areas when creating an effective disaster management
program.
■ Mitigation efforts focus on making permanent changes to plants and property to reduce the overall
exposure to various known hazards. Mitigation strategies typically involve the “where” and “how” of building
physical structures and protecting employees and assets (eg, building code requirements, flood plain analysis,
and storm shelters). Mitigation techniques also involve taking relatively minor steps to reduce loss or injury (eg,
fastening bookshelves or file cabinets to a wall).
■ Preparedness focuses on areas of risk that cannot be addressed through mitigation efforts alone.
Emergencies are dynamic and complex events, and careful preparation efforts are needed to reduce the loss of
life and property while sustaining business operations. Preparation efforts begin with a thorough risk analysis of
all known hazards (both natural and human) that could affect an organization (eg, fires, severe storms,
earthquakes, pandemics, and terrorism). Based on the risk assessment, planning efforts should focus on the
people and organizational systems that are most likely to be affected. Effective preparation involves a continual
improvement process whereby the disaster plan is routinely tested by a series of real or simulated events (drills)
through which gaps are identified and remedied.
■ Response takes place during an emergency and typically involves time-sensitive action steps taken by the
staff to protect life and property and to stabilize the organization (eg, communication with staff, evacuation
procedures, and responses to customers and the media). Effective response strategies center on clearly
outlined processes or procedures that can be practiced during drills and be implemented at any time of the day
or night (eg, during night and weekend shifts). These strategies are also based on well-defined lines of authority.
■ Recovery efforts begin once the initial response actions have taken place. These efforts focus on restoring
critical systems (eg, communications, water, power, and sewage) to resume and maintain business operations.
Recovery efforts complete the cycle of disaster management by providing insights into additional mitigation
strategies that can be deployed for future emergencies.
Risk Assessment
The cornerstone of an effective disaster management program is a thorough assessment of the known
hazards that can affect the organization. Risk can be defined as the potential losses (physical, economic, and
social) associated with a hazard and can be described in terms of expected probability and frequency, causative
factors, and locations or areas affected.4 The goal of risk assessment is to identify and reduce the number of
potential risks associated with a hazard given its probability of occurrence and scope. Potential hazards can be
grouped in different ways, such as natural vs man-made or internal vs external to an organization. Using the
latter categorization, hazards that are generated externally include natural events, such as earthquakes, floods,
wildfires, pandemics, and hurricanes, as well as human events, such as terrorism or industrial accidents,
including chemical, hazardous material, and nuclear power plant emergencies.
Another class of potential hazards to consider involves events that may occur internally at the organization.
Examples of internal hazards include internal flooding caused by burst water pipes or fire sprinkler
malfunctions, fires, natural gas leaks, workplace violence, and hazardous spills. Each of these hazards may
involve service disruptions and evacuations of staff, donors, and patients. An
99
example of a question to answer in a risk assessment of internal disasters is, what is the potential risk to
stored blood components and patients if the hospital’s blood bank becomes flooded or must be relocated
because of smoke damage from a fire?
There are many approaches to conducting an effective risk assessment. This text does not provide guidance
on a specific risk assessment process. However, a sample risk assessment chart is provided in Table 4-1, which
highlights the major functional areas and hazards to consider. For instance, a facility located in an area of
seismic activity would list earthquakes as a potential external hazard and would need to determine the
immediate effect of an earthquake on its operational status (to humans, property, and business) and the
resources needed to recover and restore operations. Conversely, an organization located far from hurricane and
flood zones would reflect that situation in its risk assessment chart. The AABB Disaster Operations Handbook
contains a list of common hazards, including impact factors as well as preparedness and response strategies.2
Emergency Operations in the United States
It is important for staff members to understand the relationships between emergency
operations at different levels of government and an organization’s disaster management program. An
understanding of the entire spectrum of emergency operations allows an organization to communicate
effectively when working with emergency management professionals during a disaster event or a drill.
Disasters are inherently local events requiring citizens, local organizations, and first responders to work
together. Key to effective emergency response is the integrated planning for disasters that starts at the national
level and extends to the local emergency management agency (EMA). State and national support systems are
available to assist if a local municipality is overwhelmed by a disaster and with the development of local
response plans and training. These national and local systems include the following:
■ National Incident Management System (NIMS). The NIMS is a nationally accepted system for helping
responders from all levels of government, various jurisdictions, and the private sector to communicate and
coordinate their response efforts.5 The NIMS uses a unified approach for incident management that offers
standard command and management structures and emphasizes preparedness, mutual aid, and resource
management. Organizations should
TABLE 4-1. Risk Assessment Chart with Sample Data4
Recovery
Human Property Business
Probability of Occurrence Resources Total
Effect Effect Effect
Needed
High Low High Low High Low High Low High Low
5 <->1 5 <->1 5 <->1 5 <-> 1 5 <->1
External Hazards
Earthquake 5 3 4 5 4 21
Hurricane 1 1 1 2 2 7
Internal Hazards
 Flooding 4 1 4 3 2 14
Workplace violence 2 5 2 4 1 14

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be familiar with NIMS protocols so that they are prepared to communicate effectively with responding
organizations [eg, police and fire departments and the Federal Emergency Management Agency (FEMA)].
Online training courses are available on the FEMA website.6
■ National Response Framework (NRF). The NRF is a federally operated all-hazards response plan that is
activated in response to a presidentially declared disaster under the Robert T. Stafford Disaster Relief and
Emergency Assistance Act (amended June 2006).7,8 Under this act, the federal government, coordinated by the
Department of Homeland Security (DHS), provides personnel, assets, and financial support to state and local
governments that are overwhelmed during a major disaster. The need for blood is covered under the medical
and public health section of the NRF (Emergency Support Function No. 8).8(p33)'9 Blood collection and
hospital facilities should be familiar with the NRF to request and receive assistance during major emergencies.
■ National Terrorism Advisory System (NTAS). The DHS assesses threats posed by terrorists and
communicates those threats to the US public, government agencies, and the private sector through the NTAS
alerts.10 This system replaces the color-coded Homeland Security Advisory System (HSAS) used in the past.
The NTAS uses only two tiers of alerts:
- Imminent Threat Alerts warn of “credible, specific, and impending terrorist threads] against the United
States.”
- Elevated Threat Alerts warn of credible, but not specific, threats against the United States.
NTAS alerts are brief, clearly articulating what can be done to prevent and even, if possible, mitigate a
threat’s impact and respond if necessary. Unlike the previously used HSAS that issued ongoing warnings,
NTAS alerts are issued for a specific period and expire once that period has ended.
■ National Disaster Medical System (NDMS). The NDMS is a cooperative asset-sharing program directed
by the Department of
Health and Human Services (DHHS). The NDMS augments local medical care when an emergency exceeds
the scope of a community’s health-care system. The NDMS consists of more than 9000 medical and support
personnel from federal, state, and local governments and the private sector as well as civilian volunteers.11
■ AABB Interorganizational Task Force on Domestic Disasters and Acts of Terrorism (AABB Disaster Task
Force). The AABB Disaster Task Force provides support to blood collection and transfusion facilities during
major emergencies, including during NRF activation under the Stafford Act. Facilities should review the AABB
Disaster Task Force response plan and integrate its response processes into their organizational disaster plans.2
■ EMAs. EMAs are local, tribal, state, and federal agencies tasked with helping individuals and
organizations deal with all cycles of disaster management. These agencies are staffed by both full-time and
volunteer personnel (emergency managers) and use standard methods when responding to a disaster.11
Major Disaster Management Factors for Blood and Transfusion
In conjunction with comprehensive disaster planning strategies, blood collection and hospital facilities
should consider the disaster management factors, described in this section, that are key for blood and blood
components during all phases from collection to transfusion.12'16
Most of the blood and blood components used for transfusion in the United States are collected, processed,
tested, and stored at regional blood centers and must be delivered to hospitals before transfusion. During an
emergency, this “just-in-time” delivery system results in the need for close coordination between blood centers
and the hospitals they serve. This involves robust communication and information systems, transportation and
logistics support, and critical utilities, such as fuel and electrical power, to ensure that blood
101
can be transported and stored at required temperatures.
Emergency management personnel are often unaware of issues related to the collection, processing, storage,
and distribution of blood and blood components and may assume that blood is collected and stored directly in
hospitals. As a result, blood-related issues (eg, the need for transportation and logistics support) are often
overlooked during real and simulated disasters. A continuing process of education for emergency managers at
both the local and national levels is imperative to increase their awareness of issues related to blood. Blood
centers should pursue active participation in EMA planning activities and participate in activities of the
emergency operations center to ensure continuous representation of blood-related issues.
Historically, an average of about 200 units of blood are needed by victims of most disasters. However, the
public response to disasters has traditionally resulted in increases in blood donations regardless of the true
medical need for transfusions. A sudden increase in unneeded blood donations can disrupt both local and
national blood supplies. Efforts should be made to establish the medical need for blood during a disaster and
communicate this need to national blood organizations and the AABB Disaster Task Force, blood donors, and
the public through a clear and consistent messaging strategy.12'16
In disasters resulting in injuries that do require blood transfusions to treat victims, the available local blood
inventory is the primary source for the initial treatments. Because blood collected in response to a disaster takes
24 to 48 hours to process, it is critical for the local blood supply to remain at ample levels to treat victims of
potential hazards as defined in the organizational risk assessment. The AABB Disaster Task Force recommends
maintaining a 5- to 7-day supply [combined inventory at the blood collector and hospital(s) it serves] to address
potential disasters.2 Efforts are made to resupply the affected facilities if needed from neighboring blood
collection centers or through national support by the AABB Disaster Task Force. However, delivering blood
from
outside the immediate vicinity to the affected hospital might take 12 to 24 hours.
Disasters resulting from the use of biologic, chemical, radiologic, or nuclear agents may result in
widespread donor deferrals and the potential quarantine of blood components already in the manufacturing
process or storage until the nature and effect of the agent can be determined. Radiologic-nuclear attacks also
would dramatically increase the demand for platelets, other blood components, and stem cells for several weeks
following an event. In addition, in the event of an influenza pandemic, blood supplies may be severely strained
for weeks due to high staff absenteeism and donor shortages as these individuals become ill, need to stay home
to take care of loved ones, or fear exposure to influenza in public places. An influenza pandemic could also
severely affect the availability of needed supplies as manufacturers face similar absenteeism and transportation
difficulties.17,18
Hospitals and transfusion services should develop blood shortage management plans to maximize the
probability of optimal blood distribution and use during a severe shortage. It is critical that detailed blood triage
plans developed by hospital transfusion services, management staff, and physicians who make transfusion
decisions be available and be regularly reviewed in advance of a severe disaster. Blood centers should work
with their hospital customers to ensure that they have triage plans in place. The Minnesota Department of
Health’s Minnesota Healthcare System Preparedness Program has developed an extensive set of materials to
help hospitals prepare to respond to disasters including “When Healthcare Resources are Scarce,” a set of
guidelines to assist health-care facilities in planning for events that would overwhelm the system’s resources,
including blood and blood components.19,20
Finally, certain disasters may affect a blood collector’s ability to collect and process blood during the entire
event, thus straining the local blood supply for days or weeks. For instance, as a hurricane approaches, a blood
collector may suspend collections for a few days before and after the storm has passed,
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resulting in a significant loss of expected collections. Elective surgeries typically are postponed during this
period, helping alleviate the strain on supply. However, lessons learned from previous disasters indicate that
blood collection and transfusion facilities should prepare for the potential acute shortage of blood components
in the days following a hurricane or disaster of similar magnitude, paying special attention to the supply of
platelets. Facilities should augment supplies before a predictable hazard (eg, a hurricane or severe winter storm)
and contact the AABB Disaster Task Force for support if routine supply channels are inadequate to bolster
supplies before or after the event.
BUSINESS OPERATIONS PLANNING
Business operations planning is generally a three-step process: assembling a planning team; analyzing the
current situation, including hazards and capabilities; and developing a plan to continue operations in an
emergency. Once planning has been completed, implementation of the plan (which includes training, testing,
evaluating, and revising the plan) can proceed.21,22
Disaster Planning Team
The first step in disaster planning is to identify the person or team of people in an organization who are
responsible for emergency operations planning. The number of people involved will depend on the size and
complexity of the operation. Assistance will be needed from key people, such as the organization’s accountant,
attorney, human resources staff, and insurance agent. In addition, establishing a relationship with local EMAs
can be vital to the success of a disaster plan in an actual emergency.
Once the responsible person has been identified or the team has been formed, a project plan should be
developed to identify the key steps and a timeline for completing the disaster plan. Business continuity planning
has proliferated over the past 5 years as organiza
tions have seen human and natural disasters wreak havoc in the United States and abroad. References,
training materials, and toolkits with step-by-step instructions and templates are widely available on the Internet
and should be acquired and used when developing a disaster plan.21'25
Analysis of Current Situation
The success of disaster planning depends on a risk analysis of the current situation (see section on “Risk
Assessment”). This assessment includes determining the hazards to which an organization is most vulnerable,
the probability of each hazard, and the anticipated effect of each event on human and physical resources and on
business operations.4,21 Effective prevention strategies can be used for risks such as fires, power interruptions,
facility security breaches, and workplace violence. What cannot be prevented should be mitigated. For example,
conducting essential operations away from floodplains, having multiple sources (but not necessarily multiple
suppliers) of critical supplies, and relocating vital records (including critical information technology resources)
to upper floors in flood-prone areas are all mitigation strategies. Plans should be developed for events that have
a high likelihood of occurring as well as low-probability events with potentially catastrophic consequences (eg,
Category 5 hurricanes or pandemic influenza).
Continuity of Operations Plan
A Continuity of Operations Plan (COOP) is designed to ensure continued operation of essential functions in
the event of an emergency or disaster.21,25 A COOP should cover the emergencies that are most likely to occur
or that could have a significant effect. A COOP should address at least the areas identified in Table 4-2. These
elements are discussed in this section.
Hospital-based blood banks and transfusion services may be covered by the overall hospital or laboratory
COOP; however, the various COOPs should be reviewed to ensure that issues specific to blood components and
transfusion are sufficiently covered. Such
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TABLE 4-2. Essential Elements of a Continuity of Operations Plan19,20'23
1. Identify essential processes and functions required for continued operations.
2. Develop decision trees for implementing the Continuity of Operations Plan.
3. Consider alternate facility options.
4. Establish safety and security plans for staff facilities and fleet vehicles.
5. Identify and ensure protection and survivability of vital records and databases.
6. Review insurance coverage and ensure that it is adequate for potential risks.
7. Establish minimum cash reserves necessary to continue operations for 90 days.
8. Develop an emergency communications plan.
9. Establish a chain of command and an order of succession for decision-making authority.
10. Develop a plan for dealing with the news media.
11. Identify designated spokespersons, and train them to handle risk and emergency communications.
12. Plan to maintain supplies and logistical support for operations.
13. Evaluate utility needs and develop contracts or memoranda of agreement to ensure replenishment and
restoration of essential utilities.
14. Review information technology systems and develop redundancies to ensure that vital systems and their
supporting subsystems remain operational during an emergency.
15. Identify staffing issues, including essential personnel and key contacts necessary to carry out essential
functions as well as employee compensation and benefits during and after the disaster.
16. Develop procedures for transitioning back to normal operations after a disaster.
plans are required by the AABB in its Standards for Blood Banks and Transfusion Services as well as by
The Joint Commission.26,27
Essential Functions
Essential functions are those that must be carried out to ensure business continuity, such as the storage of
blood and blood components and their transportation and delivery to hospitals. Even though blood collections,
component preparation, and testing may be interrupted, the continued viability of a blood center depends on its
ability to provide hospital customers with blood. Blood can be imported from outside the affected area to ensure
an adequate inventory until collections can resume. Likewise, hospitals must be certain that they can receive,
store, and transfuse blood to patients in need.
COOP Decision Trees
COOP operations and decisions are best thought out in advance during normal operations. Flowcharts and
decision trees that list options can be developed to guide leaders during emergency operations. Such flow charts
and decision trees can be developed by groups and refined using lessons learned from exercises and real-world
events. An example of a decision tree developed to support blood bank operations by the California Blood Bank
Society is available on the Internet.28
Alternate Facilities
Alternate facilities that can be used to continue operations should be identified during the planning phase of
a COOP. Thought should be given to the need for equipment, supplies, information technology support, and
other
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items at the alternate location. Consideration should also be given to practical locations for alternate
facilities. For example, an alternate facility across the street will most likely be similarly affected by an external
disaster. If funding is not available to pay for an alternate facility, the organization should consider establishing
formal agreements with other organizations in the area to provide alternate space if one facility is incapacitated.
Security and Safety
In a disaster, security of critical resources— ranging from the facility and fleet to staff and information
systems—can become a major concern.22 For example, crowd control may become an issue if large numbers of
donors gather at collection locations or in the event of special screening requirements during a pandemic
influenza outbreak. In natural disasters in which fuel shortages occur, the security of fuel supplies can become a
major challenge. A COOP should include the measures that will be necessary to ensure the security of all
critical resources.
Planners should review their normal operations security plan and determine whether the plan adequately
addresses potential threats and hazards to the local area. Coordination with EMA officials can provide
information on threat or risk assessments. Planners should consider the implementation of measures to increase
the security of their facilities, staff, volunteers, and donors during changes intheNTAS.10
Lastly, planners should ensure that the facility complies with all local building codes and applicable
Occupational Safety and Health Administration (OSHA) regulations. The actions implemented should be
discussed with the local EMA and the insurance company, and any suggestions that they provide should be
addressed.22
Vital Records
The protection of vital records is especially critical in an industry that depends on records
to document the safety and availability of blood components. In addition to donor records, the
organizational human resource files, legal records, payroll records, and financial records (such as accounts
receivable and accounts payable) are critical to a business’s survivability during a disaster.22,29 Records of
insurance policies, bank account numbers, and supplies of blank checks must be available during emergency
operations to ensure continuity of operations. Corporation records, strategic plans, and research-related records
must also be maintained. Redundancy of computerized records should be established by periodically by backing
records up on a duplicate server. Geographic considerations must be taken into account in identifying off-site
storage locations. Not all records are maintained electronically, so provisions to safely keep paper copies of
records must be considered, especially if alternate facility operations are implemented.
Insurance
Adequate insurance is essential to the survivability of any business. The two major types of insurance
coverage for blood collection and transfusion facilities are property and liability. Key provisions of these
policies include valuations of property; perils covered, such as flooding, wind, and power interruptions;
deductibles; and how to file claims. Business interruption insurance can cover loss of income. Additional
background information on relevant insurance policies is available from a variety of resources, including the
Small Business Administration and insurance companies.24,30
Cash Reserves
 The single most crucial element for the survivability of a business during a disaster is access to cash that can
provide support until normal operations can be resumed.31,32 Cash reserves equal to several months’ worth of
operational expenses should be accrued during normal operations.
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Emergency Communications Plan
Clear, quick, and effective communication is vital to protecting life and property during a disaster, and
communication systems are often the first assets to be strained during an emergency. Telecommunication
systems can quickly become overloaded or jammed; computer systems may be disrupted because of a loss of
power; and the staff may become separated, resulting in general confusion and the inability to make rapid
decisions during and after a disaster. Organizations should invest heavily in ensuring that the staff can
communicate both internally and externally and should develop an Emergency Communications Plan (ECP).
No single definition or all-encompassing set of procedures exists for an ECR but organizations should consider
the following areas when designing one.
IDENTIFYING INTERNAL AND EXTERNAL AUDIENCES FOR THE ECP. Blood collection and
hospital facilities should identify and map the essential internal departments and personnel that need to
communicate both during a disaster (response) and after a disaster (recovery). All employees need to be
included in the ECP because all employees will need to receive information regarding the response procedures
and the operational status of the facility (eg, when to report back to work).
External audiences need to be identified and assessed for their ability to communicate with an organization
(eg, vendors must have an ECP for communications with an organization). For instance, a hospital blood bank
or transfusion service should identify all essential departments and personnel at its blood supplier(s).
Conversely, blood centers should identify the key departments and personnel at hospitals or the testing
providers that need to be contacted during and after an emergency.
KEY CONTACTS. Key lists should be maintained and periodically updated of all of the critical internal and
external personnel, departments, and organizations that need to disseminate or receive information during an
emergency. The contact lists should be readily available both electronically (eg, through
smart phone applications) and in print (eg, on laminated cards) for emergency response personnel to use
during a disaster.
Internal contact lists should include essential personnel and key leaders. External contact lists should
include customers, suppliers, and vendors of critical supplies; the AABB Disaster Task Force; personnel at state
and local EMAs; and other key business resources, such as insurance agents, utility service representatives,
legal representatives, and banking personnel. Facilities should consider collecting redundant contact
information for each person in their contact lists (ie, business, mobile, and home telephone numbers; e-mail
addresses; and text messaging number). For organizational contacts (eg, blood bag suppliers, delivery services,
and electrical utility providers), facilities should obtain alternate direct (“back door”) contact information to
bypass standard automated answering systems during an emergency.
Human resources staff and legal counsel should be consulted to ensure that they support the collection of
staff contact information. In some cases, the staff may be unwilling to provide personal contact information due
to privacy concerns.
CHAIN OF COMMAND. Leadership is critical for maintaining control and direction during a disaster.
Organizations should clearly define and communicate to all personnel and external partners who will be (or who
are) in charge of a given event.4(p6) In addition to a primary authority, clearly defined lines of decisionmaking
authority, delegation, and communication should be established among the leadership team and employees.
STRATEGIES FOR COMMUNICATION REDUNDANCY AND COMPATIBILITY. A critical challenge
for organizations during real or simulated disasters is the ability (or inability) to communicate using routine
methods. These methods quickly become overloaded, requiring organizations to use alternate communication
methods. It is vital to identify and test these redundant communication channels before a disaster because
significant
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delays can occur in switching to alternate methods during an event, resulting in the potential loss of life and
property.
No single method is used by organizations to design communications redundancy. Local and state
jurisdictions use different types of emergency communications equipment (eg, fire and police personnel use
radios) and have different local telecommunications infrastructures (eg, cell towers) and capacities.
Organizations should acquire multiple redundant communications technologies and identify the order in which
they should be used during an emergency. Alternative communication technologies are described in the AABB
Disaster Handbook.2
COMMUNICATION WITH LOCAL, STATE, AND NATIONAL EMERGENCY RESPONSE
AGENCIES. In addition to communicating with routine suppliers and vendors, facilities should establish and
maintain relationships with local, state, tribal, and national emergency response organizations as shown in Fig
4-1 (see also section on Participating in Disaster Drills with EMAs).
In particular, blood collection and transfusion facilities should identify agencies that
can assist with obtaining 1) transportation support for components, critical supplies, and staff; 2) power
restoration; 3) fuel for backup generators; 4) fuel for fleet and essential staff vehicles; 5) communication
support; 6) security if staff or property is threatened; and 7) other utilities support (eg, telecommunications and
Internet). Facilities should educate these agencies on how the blood supply operates both locally and nationally
(eg, the use of “just-in-time” delivery methods and the need to deliver blood from regional blood centers to
hospitals) and should request that they be deemed a critical health entity or critical infrastructure entity in the
emergency response structure.8
Blood centers and hospitals should work with the local EMA to ensure their inclusion in local emergency
communications systems. These systems may include ongoing videoconferences or conference calls, blast e-
mail lists, and 800-MHz radio networks. Furthermore, blood centers should seek admission to any priority
networks established by local hospitals to improve communications during an incident.
Periodically, management should provide informational briefings to members of the EMA and the public
health department about

FIGURE 4-1. Communication with external agencies. EMAs = emergency management agencies.
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the mission of the organization, the scope of its operations, and the effect on local healthcare facilities if the
blood center or hospital is not operational. Management should also become familiar with the NIMS and NRF
documents and should consider enrolling in FEMA online educational courses30 or disaster training courses
provided by the state or local community. Management should request that the EMA provide informational
briefings to key staff of the blood center or hospital. During exchanges with the EMA, management should
discuss resource issues (eg, transportation assistance, fuel support, storage, security, inclusion in EMA
notification systems, and assignment of a blood center or hospital liaison to the EMA). Management should
invite the EMA to participate in its disaster exercises and actively encourage the EMA to include the blood
center or hospital in its disaster exercise programs.
National assistance during disasters (eg, coordination of national media messages and resupply of blood
components to affected facilities) is provided through the AABB Disaster Task Force in coordination with other
national and federal organizations. Along with local planning efforts, blood collection and transfusion facilities
are encouraged to integrate the task force response system into their disaster plans. The response system is
described in the AABB Disaster Operations Handbook on the AABB website.2
working WITH PUBLIC MEDIA. Creating and disseminating a clear and consistent message about the
status of the local and national blood supply is a critical component of an ECR12'16 Unnecessary donor surges
can be prevented, or donations of specific blood components (eg, group 0 red cells or platelets) can be requested
by working with public media outlets to effectively convey messages about the status of the blood supply
following a disaster. The AABB Disaster Task Force has developed media-related resources for facilities,
including boilerplate press releases that are available in the AABB Disaster Operations Handbook.2
Only individuals who have received training in risk and emergency communication
 should speak with members of the media. Specific activities should take place before, during, and after each
interview.33 For instance, written background information should be provided to a reporter before an interview,
and a spokesperson should focus the most salient points of the message into “sound bites” that can be quoted in
a story. Another useful technique when communicating with the media is message mapping.34 Message maps
help communicators develop and synthesize complex information by identifying three key messages to be
delivered in three short sentences with a total of 27 words. This process accommodates both broadcast and print
media. Most important, a spokesperson should never speak off the record with a reporter. Many other tips and
techniques can be used to ensure that the correct messages are conveyed. One resource for such tips is the
Northwest Center for Public Health Practice in collaboration with Public Health—Seattle and King County.35
Logistics
During the development of a COOP, facility planners must place heavy emphasis on the management of
daily operations, including making available the supplies and equipment required to perform essential functions
related to 1) recruiting of donors, 2) collecting donated blood, 3) manufacturing and testing blood components,
and 4) delivering the components to the hospital for transfusion to patients. During a disaster, the normal
logistical systems may not be available or capable of providing the necessary support. Key logistical issues to
address in a COOP are described below.
TRANSPORTATION. Past disaster and terrorist events have clearly demonstrated the vulnerability of
transportation systems. Blood centers should coordinate with their local EMAs to ensure that vehicles engaged
in collecting or distributing blood are duly authorized as emergency support vehicles and are granted the
appropriate clearances to operate on the roads. In addition, memoranda of understanding, agreements, or
statements of understanding should be established with the
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appropriate EMAs regarding access to the roads and use of law enforcement, National Guard, and state
militia personnel or vehicles to deliver blood. Under the provisions of the NRF, DHHS can ask the Department
of Transportation to assist with the movement of blood by air, rail, water, and motor vehicle.8 Such requests
should be directed to the AABB Disaster Task Force.2
“JUST-IN-TIME” SYSTEMS. Many blood centers and hospitals have adopted just-intime supply systems to
minimize expenses associated with the storage of large quantities of supplies, maintenance of storage facilities,
staffing, and outdated products. Blood centers and hospitals should determine the critical items required for
their business operations, the vendor’s resupply capability (including manufacturing schedule and transport
timelines), the shelf life of each supply item, and any storage requirements (eg, special environmental
factors).36
STORAGE. Planners should consider the storage capacity of their customers and identify local storage
companies that could provide an emergency storage site. Contingency contracts or contingency clauses in
existing contracts with customers and ice and dry-ice suppliers should be considered. Temporary storage space
may be provided by commercial containers, including prevalidated refrigerated containers that could be placed
at the blood center, depending on space and electrical power. The local EMA may permit storage of
consumables at municipal disaster supply facilities at no cost.
CONTRACTS. All vendor contracts should identify the process for emergency deliveries of supplies,
equipment, and services. The vendor or supplier should have a disaster plan that demonstrates the ability to
meet a facility’s supply requirements in a disaster. It is advisable to require vendors to have disaster plans for
their operations. Facilities might also develop contracts containing noncompete agreements with local
competitor organizations for implementation during disasters.
BIOHAZARDS. In the aftermath of a disaster, the disposal of biohazardous material may become a serious
problem for a blood center or hospital. Planners should incorporate appropriate language in their biohazard
material disposal contracts that will ensure that vendors establish contingency plans to meet the facility’s
requirements. The blood center or hospital should coordinate with the EMA and fire or safety agencies and
should provide information on the types of biohazardous wastes that it generates. Organizations that have
irradiators should provide local fire or safety agencies with a detailed building plan that includes photographs of
the ingress routes to the device and of the device as well as the manufacturer’s specifications and contact
information. Affected facilities should contact the AABB Disaster Task Force2 if local and state EMA
authorities cannot provide this assistance.
Utilities
 Blood centers and hospitals rely on utility services (eg, electricity, water, fuel, sewage disposal,
telecommunications, and Internet) from a variety of sources, such as municipalities or publicly or privately
owned companies. Planners should establish contingency support plans with their service providers. These
plans should identify emergency points of contact at the utility service provider and include their telephone
numbers and e-mail addresses. Most utility services have priority lists for service restoration during
emergencies. During the planning process (and not during or after an emergency), planners should solicit the
support of the local EMA and hospitals to ensure that their organization will be designated to receive priority
restoration of services.
Staffing
Blood centers and hospitals depend on their highly trained, competent, and motivated staff and volunteers to
be successful in their mission of recruiting blood donors and collecting, manufacturing, testing, and distributing
blood components. Management should promote disaster preparedness to staff and volunteers
109
and should encourage them to complete family preparedness plans.8 22
ESSENTIAL PERSONNEL. During the emergency planning process, the management team should identify
staff members and volunteers (if applicable) who are responsible for the vital services required to maintain
business operations. Each department should develop a “smart book” that describes its essential functions,
essential personnel who accomplish them, vital information and records, contract information, and customer
information.
Employees who are deemed essential because of their responsibilities must receive extensive training in the
facility’s emergency plans and participate in disaster training exercises to refine operational procedures under
emergency conditions. Consideration should be given to establishing alternate work sites and/or implementing
telework programs to mitigate the effects of the loss of the blood center or hospital on operations. The human
resources department should work with management to ensure that employee-training plans provide for the
succession of personnel into positions held by designated essential personnel should the incumbent become
unavailable. Management should also plan for housing and feeding essential personnel and providing them with
fuel to help them travel to and from work when the local infrastructure is not operational.23
CROSS-FUNCTIONAL TRAINING. Planners, in close coordination with the human resources department
and, if applicable, any collective bargaining units, should develop a crossfunctional training program that
identifies personnel whose routine duties do not support the facility’s essential functions during a disaster
response. These reassigned personnel may serve as drivers, public affairs representatives, and recruiters or
perform other nonregulated duties. The human resources department and management should make the
necessary modifications to the job descriptions of the affected employees or volunteers, develop training plans,
and conduct initial and
refresher training as required. Management may need to negotiate with collective bargaining units to modify
existing contracts.
HUMAN resources ISSUES. Of importance to employees—beyond the safety of loved ones and availability
of food and shelter—are issues such as salary continuation, flexible work schedules, implementation of
telework procedures, and benefits.8,23 Because these issues are influenced by many factors, having a
predeveloped decision matrix can be particularly helpful during times of crisis.
REGULATORY CONSIDERATIONS IN EMERGENCIES
The blood collection and transfusion community is highly regulated, and even slight changes to processes
and procedures can have farreaching effects on facilities and patients, especially during an emergency. Blood
collection and transfusion facilities should carefully consider regulatory issues in their disaster management
plans.
Federal Agencies Involved
The blood community is regulated by numerous agencies. The Center for Biologies Evaluation and
Research (CBER) of the Food and Drug Administration (FDA) has primary responsibility for ensuring the
safety, purity, and potency of all biologic products, including blood and blood components. The Center for
Devices and Radiological ffealth (CDRH), also part of the FDA, regulates medical devices, including radiation-
emitting devices. The Department of Transportation regulates the movement of cargo, including blood, blood
components, and progenitor cells, through the transportation network. OSHA ensures the safety of the US
workforce. Likewise, the mission of the Environmental Protection Agency is to protect human health and the
environment. The Centers for Medicare and Medicaid Services under the Clinical Laboratory Improvement
Amendments of 1988 oversees patient (as distinct from donor) testing at blood
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centers, hospital blood banks, and transfusion services.
Consideration must be given to the regulations and requirements of each of these agencies when developing
emergency response plans. Such regulatory considerations generally fall into three categories: effect on
available blood components, effect on donors, and potential consequences for operations.
Effects on Components in Available Inventory
All blood centers and hospitals must have written procedures to follow in an emergency. These procedures
should address the provision of emergency electrical power and continuous temperature monitoring during
storage. These procedures should address the potential effects of unsuitable storage conditions, such as extreme
(high or low) temperatures or exposure to smoke or water.37 Consideration should also be given to
nontraditional emergencies that may affect blood, such as a radiologic event or exposure to a biologic or
chemical agent.38
Blood components that have potentially been exposed in an event should be quarantined, and a
determination of component suitability should be made before the components are returned to inventory. When
the quality, safety, purity, or potency of a component is in question, CBER should be consulted. An exception or
variance may be needed to use components in such situations.39 In addition, the AABB Disaster Task Force is
available to assist facilities with questions about a component’s safety, purity, or potency during a disaster.2
All blood released for use during a disaster should be fully tested, including for infectious diseases. An
exception to full testing should be made in the event that blood supplies are exhausted, resupply is not possible,
and blood is needed immediately to save lives. Blood-testing samples should be retained for retrospective
testing after a disaster. Thorough documentation of the circumstances of the disaster is essential, and physicians
should
be notified regarding what tests have and have not been completed on the blood.39 In very extreme
circumstances, the FDA may issue emergency guidance to help ensure an adequate blood supply. For example,
on September 11, 2001, the FDA issued the Policy Statement on Urgent Collection, Shipment, and Use of
Whole Blood and Blood Components Intended for Transfusion to Address Blood Supply Needs in the Current
Disaster Situation.40 Blood collection facilities should closely follow all developments regarding donor
deferrals and all emergency FDA guidance during disasters.
Effects on Donors
Disasters, particularly emergencies involving potential infectious diseases or hazardous chemicals, can
affect donor eligibility. Infectious and chemical agents should be evaluated for 1) transmissibility by blood
transfusion, 2) potential to harm recipients, and 3) potential for an asymptomatic interval of infectivity or
toxicity after exposure but before or after any donor illness. If an agent is determined to be transmissible, a
deferral period that ensures an adequate period of safety from infectivity or adverse effect must be estimated,
and donors must be deferred appropriately. As with component suitability, CBER and the AABB Disaster Task
Force should be consulted about potential donor deferral issues.
Potential Consequences on Operations
Emergencies involving power outages, floods, or structural damage to facilities disrupt normal operations.
Disasters that do not directly affect the blood center’s or hospital’s physical infrastructure can still disrupt
operations. For example, a pandemic influenza outbreak may cause severe staffing shortages with up to a 40%
absenteeism rate.17 Collections should be performed only by facilities that routinely collect blood. Facilities
that collect only autologous blood should not attempt to collect allogeneic blood during a disaster.
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Adequate staffing can be challenging immediately before, during, and after an emergency while staff
members tend to their families and personal needs. Only staff trained in regulated functions should perform
these functions. The FDA recommends that blood establishments have adequate backup personnel in the event
of a disaster and that more than one backup person be trained for each critical function. This training should be
documented.41 Volunteers can be used to perform nonregulated functions, such as predonation education and
maintenance of the canteen. Likewise, appropriate licensure is required to safely operate fleet vehicles.
Emergency plans should include lists of staff trained in multiple functions. For example, staff members who
have transferred from one area to another (and who have maintained competency in the prior area) could
perform the initial function with appropriate certification and any necessary commercial vehicle licenses.
Equipment and supplies should also be assessed for exposure to water, humidity, and temperature extremes.
CDRH has published an information guide about the effects of disasters on medical devices; it can be useful in
planning as well as responding to disasters.42
Records Management
Vital records must be secured and maintained. These documents include records of donors, donations,
manufacturing, testing, quality assurance, and product disposition. If these records are damaged or lost during a
disaster, blood in inventory at that time may require quarantine and recall. Efforts should be made to retrieve
and preserve any damaged records. CBER should be consulted to determine the disposition of inventory when
records are lost.
TESTING THE DISASTER PLAN
Continual improvement of a disaster plan is critical for maintaining a high state of readiness for future
emergencies, and participation in disaster drills is one way to achieve this goal. By simulating the conditions of
a real disaster,
a facility can identify and correct deficiencies and gaps in its disaster plan.
Internal and External Disaster Drills
Drills can be conducted internally (eg, a fire drill or simulated hazardous spill cleanup) or in conjunction
with other organizations. Multiple-organization disaster drills are typically conducted by local or state EMAs.
Unfortunately, blood-related issues are often overlooked in the exercise scenarios developed by these agencies
because many of the planners are unaware of how blood is collected, stored, and distributed to hospitals. Blood
collection facilities should develop relationships with EMAs, as described in the “Major Disaster Management
Factors for Blood and Transfusion” section.
Participating in Disaster Drills with EMAs
The National Strategy for Homeland Security directed the establishment of a national exercise strategy.
Homeland Security Directive 8 directed the DHS to establish a National Exercise Program (NEP).43 In addition
to full-scale, integrated, national-level exercises, the NEP provides tailored exercise activities that serve as the
primary DHS vehicle for training national leaders and staff. The NEP enhances collaborations among partners
at all levels of government for assigned homeland security missions. National-level exercises provide the means
to conduct “full-scale, full-system tests” of collective preparedness, interoperability, and collaboration across all
levels of government and the private sector.44 The program also incorporates elements to allow identification of
any implications of changes to homeland security strategies, plans, technologies, policies, and procedures.
Blood centers and hospitals should seek inclusion in disaster drills and exercises by contacting their local
EMAs. Participation in local, state, and federal exercises increases a facility’s preparedness. This participation
also increases awareness by the emergency preparedness officials at all levels of the critical role that blood
centers play in providing the blood compo
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nents required by victims of a disaster and the resources that are required to do so.
Including Blood Issues in Drills
Disaster drills range from informal discussions about improving response methods to fullscale, real-time
exercises that involve hundreds of organizations. Each method can be used by a single organization or in
conjunction with multiple organizations. When an organization participates in drills with other organizations (ie,
full-scale exercises), it is important for blood collection and transfusion facilities to “inject” blood-related
scenarios into the drills during the planning stages. For example, drills should address 1) the need to transport
blood from a blood center to a hospital (or hospitals) to treat victims when local roads have been damaged or
destroyed and 2) the effect of an unknown biologic agent release on the local blood supply, donors (eg, need to
defer donors), or both. Additional information on the basic types of drills can be found on the FEMA
Emergency Management Institute website.45
After-Action Review
Perhaps the most important aspect of a wellexecuted drill or actual disaster is the after-action review
process. During this process, participants evaluate what worked well and what did not work well, with the aim
of identifying lessons learned and implementing corrective actions to improve future responses.
SUMMARY OF LESSONS LEARNED FROM RECENT DISASTERS
Blood collection facilities and hospitals around the world have dealt with significant
 disasters in recent years. Hurricanes, tsunamis, earthquakes, tornados, fires, industrial accidents, and
terrorism have tested emergency response systems. Organizations have used the learning points from these
events to further sharpen their disaster plans. Many of these lessons can be found in the AABB Disaster
Operations Handbook.2 Blood collection and transfusion facilities are encouraged to share the major learning
points from real or simulated disasters that they have experienced with AABB through the AABB National
Blood Exchange at 1.800.458.9388 or by e-mail to nbe@aabb.org. AABB is collecting such lessons and sharing
them with facilities around the world.
CONCLUSION
In the wake of disasters—such as the September 11, 2001, attacks on the World Trade Center and Pentagon;
the rail transit bombings in Great Britain, Spain, and Boston; and the devastating natural disasters that occur
every year—organizations around the world have focused significant attention and resources on strengthening
their disaster plans. Given the heightened awareness of the need for organizations to be prepared, both the
public and members of the media are holding organizations to the highest standards of excellence for ensuring
that all necessary planning has been done to protect both life and property from known threats. Blood collection
and transfusion facilities are not immune to this reality and should therefore strive to maintain the highest
standards of disaster preparedness. Above all else, the most important benefit of careful and continual planning
is assurance that blood and blood components are available to patients when and where needed.
KEY POINTS
1. Planning is the key to success during a disaster. Disaster planning is not a one-time event; it requires
continual review, exercises and practice, and learning and changing.
2. Continuity of operations planning provides a framework for facilities to maintain or restore the critical
functions necessary to collect, manufacture, store, and distribute blood components.

CHAPTER 4 Disaster Management

113
3. The cornerstone of an effective disaster plan is a thorough risk assessment of the probable hazards and
their impact on critical operations. Risks can include internal events, such as a broken pipe that floods a
laboratory, or external events, such as tornadoes and earthquakes.
4. Facilities should use the resources of the AABB Interorganizational Task Force on Domestic Disasters
and Acts of Terrorism in disaster management planning (eg, Disaster Operations Handbook) and incorporate the
task force’s national response procedures into local plans.
5. The ability to communicate during a disaster determines much of the success of a response; facilities need
well-developed and routinely tested communication plans.
6. Hospitals should have emergency plans for triage of blood components in disasters in which demand
exceeds supply (eg, a severe pandemic or nuclear or biologic attack). Blood centers should work with their
hospital customers to ensure that such plans are in place.
7. Adequate cash reserves and insurance are essential for a blood center to survive a major disaster.
8. Coordination with local emergency management officials before a disaster is critical to success during
that disaster.
9. Regulatory considerations during a disaster include the effects on the blood components on the shelf, the
donors in the affected area, and operations.
10. Facilities should routinely reinforce their disaster plan by conducting information sessions with staff,
volunteers and their families, suppliers, and hospital customers. The disaster plan should be tested through
participation in local or regional disaster drills.
REFERENCES
1. UNISDR terminology on disaster risk reduction 2009. Geneva, Switzerland: United Nations International
Strategy for Disaster Reduction, 2009:10. [Available at: http://www.
unisdr.org/files/7817_UNISDRTerminology English.pdf (accessed November 12,2012).]
2. Disaster operations handbook: Coordinating the nation’s blood supply during disasters and biological
events. Version 2.0. Bethesda, MD: AABB, 2008. [Available at: http://www.aabb. org/ (accessed November
12,2012).]
3. Comprehensive preparedness guide (CPG) 101: Developing and maintaining emergency operations plans.
Washington, DC: Federal Emergency Management Agency, 2010:1.4-1.6. [Available at:
http://www.fema.gov/library/ viewRecord.do?action=back&id=5697 (accessed May 27,2013).]
4. Multi-hazard identification and risk assessment: A cornerstone of the national mitigation strategy.
Washington, DC: Federal Emergency Management Agency, 1997:314-5. [Available at
http://www.fema.gov/library/viewRecord. do?id=2214 (accessed May 25,2013).]
5. National incident management system. Washington, DC. Department of Homeland Security, 2008:4-8.
[Available at http://www.fema.
gov/pdf/emergency/nims/NIMS_core.pdf (accessed May 25,2013).]
6. Training. Washington, DC: Federal Emergency Management Agency, 2013. [Available at:
http://www.fema.gov/training (accessed May 28,2013).]
7. Robert T Stafford Disaster Relief and Emergency Assistance Act (Public Law 93-288) as amended, and
related authorities. (June 2007) Washington, DC. US Government Printing Office, 2007. [Available at:
http://www.fema.gov/ pdf/about/stafford_act.pdf (accessed November 8,2013).]
8. National response framework. Washington, DC: Department of Homeland Security, 2013. [Available at:
http://www.fema.gov/library/ viewRecord.do?id=7371 (accessed May 27, 2013).]
9. Emergency support function #8: Public health and medical services annex. Washington, DC: Department
of Health and Human Services, 2008. [Available at http://www.fema.gov/pdf/ emergency/nrf/nrf-esf-08.pdf
(accessed 27 May 2013).]
10. NTAS public guide. Washington, DC: Department of Homeland Security, 2013. [Available at:
http://www.dhs.gov/ntas-public-guide (accessed May 27, 2013).]
 114
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11. Public health emergency: National disaster medical system. Washington, DC: Department of Health and
Human Services, 2013. [Available at: http://www.phe.gov/emergency/pag es/default.aspx (accessed May
27,2013).]
12. Hess JR, Thomas MJG. Blood use in war and disaster: Lessons from the past century. Transfusion
2003;43:1622-33.
13. Linden JV) Davey RJ, Burch JW. The September 11,2001, disaster and the New York blood supply.
Transfusion 2002;42:1385-7.
14. Schmidt PJ. Blood and disaster—supply and demand (editorial). N Engl J Med 2002;346: 617-20.
15. Klein HG. Earthquake in America. Transfusion 2001;41:1179-80.
16. Glascow SM, Allard S, Doughty H, et al. Blood and bombs: The demand and use of blood following the
London bombing of 7 July 2005—a retrospective review. Transfus Med 2012;22: 244-50.
17. Pandemic influenza update. Association Bulletin #09-07. Bethesda, MD: AABB, 2009.
18. Pandemic influenza preparedness and response guidance for healthcare workers and healthcare
employers. Washington, DC: Occupational Safety and Health Administration, 2009. [Available at
http://www.osha.gov/Pub lications/OSHA_pandemic_health.pdf (accessed May 27,2013).]
19. Healthcare emergency preparedness. St. Paul, MN: Minnesota Department of Public Health, 2013.
[Available at http://www.health.state, mn.us/oep/healthcare/in dex.html (accessed November 8,2013).]
20. When healthcare resources are scarce. St Paul, MN: Minnesota Department of Health, 2012. [Available
at http://www.health.state.mn.us/ oep/healthcare/scarce/index.html (accessed November 8,2013).]
21. Open for business: A disaster planning toolkit for the small to mid-sized business owner. Tampa, FL:
Institute for Business and Home Safety, 2007. [Available at http://www.disaster safety.org/wp-
content/uploads/open-forbusiness-english.pdf (accessed November 8, 2013).]
22. Ready business: Preparedness planning for your business. Washington, DC: Federal Emergency
Management Agency, 2013. [Available at http://www.ready.gov/business (accessed Novembers, 2013)]
 23. Federal Continuity Guidance Circular 1 (CGC1). Continuity guidance for non-federal entities
(states, territories, tribal and local government jurisdictions and private sector organization). Washington,
DC: Federal Emergency Management Agency, 2009. [Available at http://
www.fema.gov/pdf/about/org/ncp/coop/ continuity_guidance_circular.pdf (accessed May 27,2013).]
24. 10 things to do to prepare: A step by step guide to building your disaster preparedness plan. Hartford,
CT: The Hartford Financial Services, 2013. [Available at http://thehartford.com/ business/disaster-preparedness-
planningguide (accessed November 8,2013).]
25. Emergency preparedness. Washington, DC: Small Business Administration, 2013. [Available at
http://www.sba.gov/prepare (accessed May 27,2013).]
26. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
27. Comprehensive accreditation manual for hospitals (CAMH). Oak Brook Terrace, IL: The Joint
Commission, 2012.
28. Disaster response plan. Sacramento, CA: California Blood Bank Society, 2012. [Available at http: / /
www.cbbsweb.org/attachmnts/cbbsdi sasterplan.pdf (accessed May 27,2013).]
29. Myers KN. Manager’s guide to contingency planning for disasters. New York: John Wiley and Sons,
1999.
30. Business insurance. Washington, DC: Small Business Administration, 2013. [Available at http: / /
www.sba.gov/ content/business-insur ance (accessed May 27,2013).]
31. The conference on emergency response planning for your business. Mission, KS: SkillPath Seminars,
2002.
32. Gannon B. The best laid plans. Presentation at America’s Blood Centers 2006 Annual Meeting.
Houston, TX, March 4-6,2006.
33. Reynolds B, Seeger M. Crisis and emergency risk communication (CERC). Atlanta, GA: Centers for
Disease Control and Prevention, 2012: 2-16,157-9.
34. Covello VT. Invited paper presented at the 2002 World Health Organization conference on bio-terrorism
and risk communication. Geneva, Switzerland, October 1,2002.
35. Li-Vollmer M. Emergency risk communication: An online course. Seattle, WA: Northwest Center for
Public Health Practice, University of Washington School of Public Health, 2013. [Available at
http://www.nwcphp.org/train ing/opportunities/online-courses/emergen
CHAPTER 4
Disaster Management
115
cy-risk-communication-for-public-healthprofessionals (accessed May 27,2013).]
36. Ready.gov business continuity planning. Washington, DC: Federal Emergency Management Agency,
2013. [Available at http://www. ready.gov/sites/default/files/documents/ files/BusinessContinuityPlan.pdf
(accessed May 29,2003).]
37. Impact of severe weather conditions on biological products. Silver Spring, MD: CBER Office of
Communication, Outreach and Development, 2013. [Available at http://www.fda.
gov/BiologicsBloodVaccines/SafetyAvailabili ty/ProductSecurity/ucml47243.htm (accessed May 27,2013).]
38. Hamburg M. FDA emergency operations plan. Rockville, MD; Food and Drug Administration, 2010.
[Available at http://www.fda.gov/down loads/EmergencyPreparedness/Emergency
Preparedness/UCM230973.pdf (accessed May 27,2013).]
39. Food and Drug Administration. Exceptions and alternative procedures approved under 21 CFR 640.120.
Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 2014. [Available at
http://www.fda.gov/ BiologicsBloodVaccines/BloodProducts/Reg ulationoftheBloodSupply/ExceptionsandAl
ternativeProcedures / default.htm.]
40. Policy statement on urgent collection, shipment, and use of whole blood and blood components intended
for transfusion to address blood supply needs in the current disaster situation. Silver Spring, MD: CBER Office
of
Communication, Outreach and Development, 2011.
41. Guidance for industry: Recommendations for blood establishments: Training of back-up personnel,
assessment of blood donor suitability and reporting certain changes to an approved application. Silver Spring,
MD: CBER Office of Communication, Outreach and Development, 2010. [Available at http://www.fda.
gov/BiologicsBloodVaccines/GuidanceCom plianceRegulatorylnformation / Guidances / Blood/default.htm.]
 42. FDA offers tips about medical devices and hurricane disasters. Washington, DC: Food and Drug
Administration, 2013. [Available at http://www.fda.gov/MedicalDevices/Safety/
EmergencySituations/ucm055987.htm (accessed May 27,2013).]
43. Homeland Security Presidential Directive 8: National preparedness. Washington, DC: Department of
Homeland Security, 2011. [Available at http://www.dhs.gov/xlibrary/assets/ presidential-policy-directive-8-
national-pre paredness.pdf (accessed May 27,2013).]
44. Exercise. Washington, DC: Federal Emergency Management Agency, 2013. [Available at http://
www.fema.gov/exercise (accessed May 27, 2013).]
45. IS-120A. An introduction to exercises (FEMA independent study). Emmitsburg, MD: Federal
Emergency Management Agency Emergency Management Institute, 2008. [Available at
http://training.fema.gov/EMIWeb/IS/course Overview.aspx?code=is-120.a (accessed May 27,2013).]

Chapter 5
Allogeneic and Autologous Blood Donor Selection
AnneF.Eder, MD, PhD, and Maria D.L.A. Muniz, MD
HR THE FOREMOST RESPONSIBILITY Off of blood centers is to maintain a safe and adequate blood
supply. The selection of appropriate blood donors is essential to protect their health during and following
donation, and to ensure the safety, quality, identity, purity, and potency of the donated blood components to
protect the transfusion recipient. Key elements of the selection process, as part of the overall approach to blood
safety, are education, the donor health history questionnaire, a focused physical examination, infectious disease
testing (see Chapter 8), and management of postdonation information.
This chapter describes the current federal regulations, accreditation requirements, and medical
considerations related to screening blood donors before their blood is collected and tested for various blood-
borne diseases.1'3
OVERVIEW OF BLOOD DONOR SCREENING

Blood centers provide prospective blood donors with information on the donation process and potential
donation-related adverse effects and instruct them not to donate if they may be infected with blood-borne
pathogens. The screening process includes a focused physical examination and direct questioning about specific
risk behaviors, medications, travel, and other factors that potentially affect recipient or donor safety. The
questions address risks related to infectious diseases for which sensitive tests are currently performed [eg,
hepatitis B and C viruses and human immunodeficiency virus (HIV)], for which tests are not universally used
(eg, Chagas disease), and for which licensed screening tests are not yet available (eg, babesiosis and malaria).
Anne F. Eder, MD, PhD, Executive Medical Officer, American Red Cross, National Headquarters,
Biomedical Services Medical Office, Holland Laboratory, Rockville, Maryland; and Maria D.L.A. Muniz, MD,
Clinical Pathologist, Allegheny Health Network, Pittsburgh, Pennsylvania The authors have disclosed no
conflicts of interest.
117

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AABB TECHNICAL MANUAL
If individuals are instructed not to donate blood for others because of their health history, reactive test
results, behavioral risks, or medical reasons, they may be added to a confidential deferral list to prevent future
donations. Deferrals maybe for a defined interval of time or an indefinite period, or they may be permanent with
no potential for reinstatement as a blood donor in the future.1 In addition, blood centers are required to manage
information received after the donation that could affect the safety, quality or potency of the donated blood
components and the future eligibility of the donor, and keep records of deferred individuals.2
The criteria to evaluate individuals who are donating blood for their own use (ie, autologous donation) may
be less stringent than those for allogeneic donation. However, the focus remains on providing the safest possible
blood for transfusion to the donor-patient and on evaluating the risks that the collection procedure poses to his
or her health.
The blood center must determine donor eligibility prior to collection and on the day of collection, in
accordance with federal and state regulations and voluntary accreditation standards. Specific criteria used to
select donors are established by the Food and Drug Administration (FDA) through the Code of Federal
Regulations (CFR), guidance documents, and memoranda to industry.2 The AABB also develops professional
standards for donor selection with which accredited blood establishments must comply.1
An industry-wide effort led by AABB to streamline and standardize donor screening culminated with the
release by the FDA, in October 2006, of a final guidance document recognizing the donor history questionnaire
(DHQ) as an effective donor screening tool that provides licensed and unlicensed facilities with a way to meet
all FDA requirements.4 The DHQ is currently used by most blood centers in the United States.5,6 The
references in this chapter are to DHQ Version 1.3 (May 2008), which the FDA officially recognized as
acceptable DHQ documentation in May 2010.4,7
Medical directors of blood collection facilities are responsible for determining donor
eligibility policies on issues that are not covered by regulations or standards.3,8'11 Consequently, medical
decisions regarding the same issue may differ among blood centers or even among physicians at the same blood
center. Considerable variability exists in national and international practices for determining donor eligibility,
which reveals the inherent uncertainty in risk assessment.8
A precautionary approach attempts to eliminate the risk of known or potential transfusion-transmitted
diseases from the blood supply but also results in the disqualification of large segments of the healthy
population. Prospective donors who are deferred from blood donation may be disappointed, angry, or confused
about the relevance that certain questions have to their health or ability to donate blood for transfusion to others.
Blood center staff should be able to explain the intended purpose of AABB and FDA requirements as well as
their center-specific eligibility screening practices.
The most frequently asked questions about federal regulations defining blood donor eligibility are available
to the public in the “Questions about Blood” section of the FDA website.12 Questions about the interpretation
or underlying rationale of AABB Standards for Blood Banks and Transfusion Services should be directed to the
AABB Standards Department (standards@aabb.org); responses and discussion of selected issues are posted on
the AABB website.
SELECTION OF ALLOGENEIC BLOOD DONORS
Registration and Donor Identification
In the United States, blood components for allogeneic transfusion are typically collected from volunteer,
nonremunerated donors; otherwise, they must be labeled as being from paid donors.2
Prospective blood donors should provide an acceptable form of identification, and each donor must be
properly identified by the collection staff before each donation. Acceptable forms of identification include
government
119
issued documents, such as a driver’s license or passport, or a blood-center-issued donor card with a unique
alphanumeric code. Many blood collectors no longer use social security numbers for donor identification
because of regulations restricting their use and privacy concerns.
Accurate records are essential to identify all prior donations from any given donor, including whether the
donor has ever given blood under a different name, so that the link with all prior donations within the blood
system is maintained. Accurate records are also essential to ensure that the donor can be contacted in the weeks
following the donation and informed of test results or other relevant information from the current donation, if
necessary. Blood centers must make reasonable attempts to notify the donor within 8 weeks of the donation if
any test results disqualify the individual from continued donation.2 Blood centers often require donors to
provide an address and telephone number where they can be reached during this interval for counseling or other
follow-up, if necessary. Accurate donation records must be kept by the donor center for the requisite amount of
time, according to current regulations and standards.1,2
Notably, there is currently no national registry of deferred blood donors in the United States, and individuals
who are deferred by one blood center may be eligible in another blood system. The available evidence suggests
that such donor deferral registries are not necessary because they do not contribute to blood safety and do not
prevent the release of unsuitable components.13
Educational Materials and Donor Consent
US blood centers provide all prospective blood donors with information about blood donation during the
informed consent process. The DHQ educational materials incorporate all necessary elements required by
federal regulations and AABB Standards, and blood centers have added elements to protect both blood donors
and transfusion recipients.1(ppl5'16,60‘63) 4 At each encounter, the blood center staff
should explain the collection procedure to the donor in terms that the donor understands and document the
donor’s consent, which indicates that the donor has considered all the educational materials and has had an
opportunity to ask questions.ltpl6) The donor should be informed about possible adverse reactions to the
collection procedure and the infectious disease tests that will be performed on his or her donated blood
components. The donor should also be informed of the notification process for positive test results, any
reporting requirements to federal or state health authorities, and the possibility of inclusion in the donor center’s
deferral registry and subsequent deferral from future donation. The individual should agree not to donate if his
or her blood could pose a risk to the blood supply. Donors should also be informed if investigational tests or
other research may be performed on samples or information collected during the blood donation. Finally, the
limitations of the tests to detect early infections and the possibility that a test may not be performed if samples
are not adequate should be explained to the donor.
No one should donate blood for the purpose of receiving free infectious disease testing. Information about
alternative HIV test sites or public health options to obtain HIV tests should be provided to all prospective
donors.
All prospective donors must be able to give informed consent and provide an accurate health history. They
should be given an opportunity to ask questions and they have the right to withdraw from the process at any
time. Blood donation centers should document donors’ consent to have their blood collected and tested.
 Blood centers must comply with applicable state laws to obtain permission from parents or guardians for
minors (ie, 16- or 17-year olds) or legally incompetent adults.1(pl6) Moreover, AABB Standard 5.2.2 requires
that collection facilities have a process to provide information about the donation process to parents or legally
authorized representatives of donors when parental permission is required.llpl6)
Donor centers should also establish policies on accommodating individuals who are
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AABB TECHNICAL MANUAL
not fluent in English or are illiterate, are vision or hearing impaired, or have other physical disabilities.
Many blood centers try to make reasonable accommodations for donors’ special needs. However, centers must
also ensure that the collection procedure does not pose undue risk to donors or staff members and that the
informed consent process is not compromised. The final authority for such decisions rests with the donor
center’s physician who supervises donor qualification and phlebotomy.1(pl)
Donor Qualification by Focused Physical Examination and Hemoglobin or Hematocrit Measurement
Qualification procedures for blood donation include a focused physical examination (eg, taking the donor’s
temperature and inspecting his or her arm) and a hemoglobin or hemato
crit measurement. These tests have potential implications for the potency or safety of the collected
component (Table 5-1). Additional requirements apply to apheresis donors, who must meet the weight and
hemoglobin or hematocrit requirements approved by the FDA for the automated collection device.
Prior to phlebotomy, the collection staff inspect the donor’s antecubital skin, which must be free of lesions
and evidence of injection drug use, such as multiple needle punctures (eg, small scars lined up in “tracks”) or
sclerotic veins. Scars or pitting on the forearm associated with frequent blood donation should not be mistaken
for evidence of injection drug use. Common and mild skin disorders (eg, poison ivy rash) are not a cause for
deferral unless there are signs of localized bacterial superinfection or the condition interferes with proper skin
disinfection in the antecubital site before phlebotomy.
TABLE 5-1. Physical Examination and Requirements for Allogeneic and Autologous Whole-Blood
Donation
Allogeneic Autologous
AABB Reference Standard 5.4.1A; Title 21, CFR Part AABB Standard 5.4.4;
640.3 Title 21, CFR Part 640.3
Alternate requirements
Age Conform to applicable state law or >16 years
defined by
blood center’s medical
Blood No requirement in AABB standards, systolic and diastolic
director (AABB Standard
pressure blood pressure “within normal limits” [Title 21, CFR Part 3(2)].
5.4.4).
Pulse No requirement in AABB standards or CFR.
Whole
Maximum of 10.5 mL/kg of donor weight, including
blood volume
samples.
collected
8 weeks after whole blood donation; 16 weeks after 2-unit
Donation
red cell collection; 4 weeks after infrequent plasmapheresis;
interval
and >2 days after plasma-, platelet-, or leukapheresis.
Deferral for conditions
<37.5 C (99.5 F) if measured orally or equivalent if
Temperature presenting risk of bacteremia
measured by another method.
(AABB Standard 5.4.4.4).
Hemoglobin
>12.5 g/dL (>38%). >11 g/dL (>33%).
(hematocrit)
CFR = Code of Federal Regulations.
 121
The FDA defines the minimum acceptable hemoglobin concentration for both men and women as 12.5
g/dL, and the essentially equivalent hematocrit requirement of 38%.2 This requirement is controversial and is
periodically debated because normal hemoglobin values are influenced by gender, race, nutritional status, and
other factors.1415 In particular, a single acceptance criterion for men and women is contentious because normal
hemoglobin values for women are lower than those for men. However, this requirement has remained
unchanged for more than 30 years. Hemoglobin or hematocrit screening may prevent collection of blood from a
donor with significant anemia, which could have implications for the health of the donor as well as the potency
of the collected component.
A hemoglobin level lower than the 12.5 g/dL cutoff is the most common reason for disqualification of
potential blood donors. This single deferral criterion can be problematic for both male and female donors. Some
female donors with hemoglobin levels below the 12.5 g/dL cutoff are actually in the normal range. In contrast,
some male donors with a hemoglobin level above 12.5 g/dL may, in fact, be anemic. Furthermore, hemoglobin
screening does not ensure that the donor has an adequate store of iron.1617 Debate continues on the minimum
hemoglobin requirement for blood donation, frequent donors’ iron status, and allowable donation intervals.18
Donor hemoglobin screening may ensure a minimum content of hemoglobin in a unit of Red Blood Cells
(RBCs), but currently neither the FDA nor the AABB define potency standards for RBC units prepared from
wholeblood collection. If a donor’s hemoglobin level is 12.5 g/dL, a 500-mL whole-blood collection is expected
to yield about 62.5 g of hemoglobin per unit of RBCs, but determining the final content of hemoglobin in an
RBC unit prepared from whole blood is not required. AABB Standards require apheresis RBC units to be
prepared using a method known to ensure a final component containing a mean hemoglobin level of >60 g of
hemoglobin, with 95% of the units sampled containing more than 50 g of hemoglobin.1(p27)
 In general, neither the FDA nor the AABB specifies the test method, specimen type (capillary, venous
blood), or acceptable performance characteristics for tests used for hemoglobin/hematocrit screening. One
exception is that a capillary sample collected from an earlobe puncture is not an acceptable specimen for
hemoglobin/hematocrit screening for allogeneic or autologous donors.1(ppl9,60) Most US blood centers use
fingerstick capillary samples for hemoglobin determination. These samples tend to give slightly higher
hemoglobin values than venous samples.
The methods to measure hemoglobin or hematocrit are generally selected for their ease of use in the mobile
blood collection setting. The copper sulfate density method (Method 61) is still an acceptable screening tool in
blood centers in the United States but has been largely replaced by methods such as spectra photometric
measurement of hemoglobin with portable devices (eg, HemoCue Donor Hb Checker, HemoCue AB,
Angelholm, Sweden; DiaSpect Hemoglobin, DiaSpect Medical AB, Uppsala, Sweden) or hematology
analyzers. The point-of-care methods that use portable devices yield quantitative hemoglobin results, with a
coefficient of variation (CV) of 1.5%.15 A typical automated analyzer measures hemoglobin levels in a venous
sample with a CV <1.2%.15 Most quantitative methods currently in use reliably measure hemoglobin levels
within approximately 0.2 to 0.5 g/dL, and the vast majority of deferred donors have hemoglobin or hematocrit
values near the cutoff value. For capillary-sample-based methods, the most likely source of preanalytical error
is the sampling technique, and testing must be performed in compliance with the manufacturer’s instructions.
Health History Assessment—DHQ
The AABB DHQ is now used by most blood centers in the United States, although its use is not currently
mandated by the FDA (Table 5-2).19 Alternative procedures for collecting required information from blood
donors are subject to FDA review and acceptance. In addition, the FDA acknowledges that the DHQ
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TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
Donor History Questionnaire Brief Description of Eligibility Criteria
The prospective donor shall appear to be in good health and shall be
1. Are you feeling healthy and free of major organ disease (eg, heart, liver, lungs), cancer, or abnormal
well today? bleeding tendency, unless determined eligible by the medical director.
Variable, temporary deferral criteria as defined by the medical director.
Variable, temporary deferral if treated for active infections, as defined
2. Are you currently taking an by the medical director.
antibiotic? No deferral for prophylactic (preventive) use of antibiotics (eg,
tetracycline for acne).
3. Are you currently taking any
Variable, temporary deferral if treated for active infections.
other medication for an infection?
Examples:
Potent teratogens (potential for harm to unborn children):
■ Finasteride (Proscar, Propecia), isotretinoin (Absorica, Accutane,
4. Are you now taking or have
Amnesteem, Claravis, Myorisan, Sotret, Zenatane): Defer for 1 month
you ever taken any medications on
after last dose.
the Medication Deferral List?
■ Dutasteride (Avodart, Jalyn): Defer for 6 months after last dose.
■ Acitretin (Soriatane): Defer for 3 years after last dose.
■ Etretinate (Tegison): Defer indefinitely.
Potential vCJD risk (but no documented cases of transmission):
■ Bovine insulin manufactured in the United Kingdom: Defer
indefinitely.
5. Have you read the
N/A
educational materials?
Medications that irreversibly inhibit platelet function preclude use of
6. In the past 48 hours have you the donor as sole source of platelets:
taken aspirin or anything that has ■ Defer for at least 36 hours after ingestion of aspirin.
aspirin in it? ■ Defer for use of other medications as defined by the facility's
medical director.
 7. Female donors: Have you Defer if donor has been pregnant within the last 6 weeks.
been pregnant or are you pregnant
now? (Males: Check “1 am male.”)
Donation interval requirements:
■ Defer for 8 weeks after whole blood donation.
8. In the past 8 weeks have you ■ Defer for 16 weeks after 2-unit red cell collection.
donated blood platelets or plasma? ■ Defer for 4 weeks after infrequent plasmapheresis.
■ Defer >2 days after plasmapheresis, plateletpheresis, or
leukapheresis.

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TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History Questionnaire Brief Description of Eligibility Criteria
No deferral for receipt of toxoids or synthetic or killed viral, bacterial, or
rickettsial vaccines listed below if the donor is symptom free and afebrile.
9. In the past 8 weeks have
■ Anthrax, cholera, diphtheria, hepatitis A, hepatitis B, influenza, Lyme
you had any vaccinations or
disease, paratyphoid, pertussis, plague, pneumococcal polysaccharide, polio
other shots?
(Salk/injection), Rocky Mountain spotted fever, tetanus, or typhoid (by
injection).
No deferral for receipt of recombinant vaccines (eg. human
papillomavirus vaccine).
No deferral for receipt of intranasal live attenuated flu vaccine.
Defer for 2 weeks for receipt of the following live attenuated viral and
bacterial vaccines:
■ Measles (rubeola), mumps, polio (Sabin/oral), typhoid (oral), or yellow
fever.
Defer for 4 weeks for receipt of the following live attenuated viral and
bacterial vaccines:
■ German measles (rubella) or chicken pox (varicella zoster).
Defer for 12 months unless otherwise indicated by medical director for
receipt of other vaccines, including unlicensed vaccines.
 10. In the past 8 weeks have Refer to current FDA guidance.
you had contact with someone
who had a smallpox
vaccination?
11. In the past 16 weeks have
you donated a double unit of red
Defer for 16 weeks after 2-unit red cell collection.
cells using an apheresis
machine?
12. In the past 12 months
have you had a blood Defer for 12 months for receipt of blood, components,
transfusion?
13. In the past 12 months
have you had a transplant such human tissue, or plasma-derived clotting factor concentrates.
as organ, tissue, or bone Defer indefinitely for receipt of human (cadaveric) dura
marrow?
14. In the past 12 months
have you had a graft such as mater.
bone or skin?
(Continued)

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AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History Questionnaire
Brief Description of Eligibility Criteria
15. In the past 12 months have you come into contact with someone else’s blood?
16. In the past 12 months have you had an accidental needle-stick?
Defer for 12 months
■ From the time of mucous membrane exposure to blood.
■ For nonsterile skin penetration with instruments or equipment contaminated with blood or body fluids
other than the donor’s own.
17. In the past 12 months have you had sexual contact with anyone who has HIV/AIDS or has had a positive
test for the HIV/AIDS virus?
18. In the past 12 months have you had sexual contact with a prostitute or anyone else who takes money or
drugs or other payment for sex?
19. In the past 12 months have you had sexual contact with anyone who has ever used needles to take drugs
or steroids, or anything not prescribed by their doctor?
Defer for 12 months from the time of sexual contact with an individual with HIV infection or at high risk of
HIV infection.
20. In the past 12 months have you had sexual contact with anyone who has hemophilia or has used clotting
factor concentrates?
21. Female donors: In the past 12 months have you had sexual contact with a male who has ever had sexual
contact with another male? (Males: check “I am male.”)
22. In the past 12 months have you had sexual Defer for 12 months for sexual contact or for having lived
contact with a person who has hepatitis? with an individual who meets any of the following criteria:
23. In the past 12 months have you lived with a person who has hepatitis?
Has acute or chronic hepatitis B (positive hepatitis B surface antigen test).
 Has symptomatic hepatitis C.
Is symptomatic for any other viral hepatitis.
24. In the past 12 months have you had a tattoo?
25. In the past 12 months have you had ear or body piercing?
Defer for 12 months from the time of nonsterile skin penetration with instruments or equipment
contaminated with blood or bodily fluids other than the donor’s own, including for tattoos or permanent makeup
unless, applied by a stage-regulated entity with sterile needles and ink that is not reused.
26. In the past 12 months have you had or Defer for 12 months or for longest applicable period for the been
treated for syphilis or gonorrhea? following situations:
■ Following the completion of treatment for syphilis or gonorrhea.
■ For a reactive screening test for syphilis where no confirmatory testing was performed.
■ For a confirmed positive test for syphilis (FDA reentry protocol after 1 year applies).

125
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History
Brief Description of Eligibility Criteria
Questionnaire
27. In the past 12
months have you been in
juvenile detention, lockup, Defer for 12 months.
jail, or prison for more than
72 hours?
Malaria:
■ Defer for 3 years after departure from malaria-
endemic area(s) for individuals(s) who have lived for
28. In the past three longer than 5 consecutive years in a country(ies) with
years have you been outside areas considered endemic by the Malarial Branch,
the United States or Centers for Disease Control and Prevention, US
Canada? Department of Health and Human Services.
■ Defer for 12 months after departure from
malariaendemic area(s) for other individuals who have
traveled to an area where malaria is endemic.
29. From 1980 through
1996, did you spend time
that adds up to three (3)
months or more in the
United Kingdom? (Review
list of countries in the UK.)
30. From 1980 through
1996, were you a member of
the US military, a civilian Defer indefinitely for risk of vCJD, as defined in
military employee, or a most recent
dependent of a member of
the U.S. military?
31. From 1980 to the
present, did you spend time
that adds up to five (5) years FDA guidance.
or more in Europe? (Review
list of countries in Europe.)
32. From 1980 to the
present, did you receive a
blood transfusion in the
United Kingdom? (Review
list of countries in the UK.)
33. From 1977 to the
present, have you received
Defer indefinitely donors:
money, drugs, or other
payment for sex?
Excluded by current
FDA regulations and
recommendations for the
prevention of HIV
transmission by blood
34. Male donor: From
and components.
1977 to the present, have
With present or past
you had sexual contact with ■
clinical or laboratory
another male, even once? ■
evidence of infection
(Female donors: Check “1
with HCV, HTLV, or
am female.”)
HIV or as excluded by
current FDA regulations.
For use of a needle to
administer
nonprescription drugs.
35. Have you EVER had
a positive test for the ■
HIV/AIDS virus?
36. Have you EVER
used needles to take drugs,
steroids, or anything not
prescribed by your doctor?
(Continued)
 126
AABB TECHNICAL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History Questionnaire Brief Description of Eligibility Criteria
■ Defer as directed by the medical director donors with abnormal
37. Have you EVER used bleeding tendency.
clotting factor concentrates? ■ Defer for 12 months for receipt of blood, components, human tissues,
or plasma-derived clotting factor concentrates.
38. Have you EVER had
Defer indefinitely for history of viral hepatitis after 11th birthday.
hepatitis?
39. Have you EVER had Defer for 3 years after becoming asymptomatic prospective donors who
malaria? have had a diagnosis of malaria.
40. Have you EVER had
Defer indefinitely for a history of babesiosis or Chagas ■ disease.
Chagas disease?
41. Have you EVER had
babesiosis?
42. Have you EVER received
a dura mater (or brain covering) Defer indefinitely for receipt of human (cadaveric) dura mater.
graft?
43. Have you EVER had any Variable deferral criteria, as defined by medical director. Examples:
type of cancer, including ■ Nonhematologic cancer: Defer for 5 years after completion of
leukemia? treatment.
 ■ Local skin cancer, in situ cancer: No deferral if treated completely
with excision and healed.
■ Hematologic cancer (eg, leukemia): Defer indefinitely.
44. Have you EVER had any
problems with your heart or Variable deferral criteria, as defined by medical director.
lungs?
45. Have you EVER had a
bleeding condition or a blood Variable deferral criteria, as defined by medical director.
disease?
Defer indefinitely, as excluded by current FDA regulations and
recommendations for the prevention of HIV transmis■ sion by blood and
46. Have you EVER had
blood components.
sexual contact with anyone who
The questions for risk of HIV group 0 can be deleted from the DHQ if
was born in or lived in Africa?
the blood center is using an HIV antibody test that has been approved by
FDA to include a claim for detection of HIV group 0.
47. Have you EVER been in
Africa?
48. Have any of your relatives
Defer indefinitely for family history of Creutzfeldt-Jakob disease.
had CreutzfeldtJakob disease?
vCJD = variant Creutzfeldt-Jakob disease; N/A = not applicable; HIV = human immunodeficiency virus;
AIDS = acquired immunodeficiency syndrome; FDA = Food and Drug Administration; UK = United Kingdom;
HCV = hepatitis C virus; HTLV = human T-cell lymphotropic virus.

127
documents contain questions related to the following issues not addressed by any FDA requirement or
recommendation: cancer; organ, tissue, or marrow transplants; bone or skin grafts; and pregnancy. However,
AABB recommends that blood centers implement the DHQ materials, including the following documents, in
their entirety:
■ Blood donor educational materials.
■ Full-length DHQ.
■ User brochure, including glossary and references.
■ Medication Deferral List.
The use of the DHQ flow charts is optional. All DHQ documents that the FDA has recognized as acceptable
are available on the FDA website.20 The most current version is also available on the AABB website.19
The wording, order, and text of the DHQ questions should not be changed. A collection facility may make
minor changes to the time frame of a question on the DHQ to make the question more restrictive so that its use
results in the deferral of more donors. Blood centers may choose to include additional questions as long as they
place these questions at the end of the DHQ. The DHQ documents are intended to be self-administered by the
donor, but blood centers may choose to use direct oral questioning, self-administration, or a combination of both
methods to administer the DHQ.
If AABB standards or FDA regulations do not address specific medical conditions that a blood center has
chosen to include in the DHQ, the blood center must develop standard operating procedures (SOPs) for
determining the criteria for acceptance or deferral of a donor. A rational approach to donor health history
assessment attempts to balance the need to take appropriate precautions to protect the blood supply with
avoiding unnecessarily restrictive policies that disqualify large segments of the population without contributing
to either recipient or donor safety.3 Decisions about donor eligibility should be based on available evidence
regarding the risk that the
medical condition or history poses to the blood donor and transfusion recipients.
If a potential exists for risk to the recipient or donor, the effectiveness and incremental benefit of screening
donors by questioning should be evaluated, especially in light of other safeguards that protect the donor or other
transfusion practices that mitigate potential risks to the recipient. If the blood center receives information after
the donation that would have been cause for deferral had it been reported at the time of donation, then any
subsequent actions, such as product retrieval, market withdrawal, or consignee notification, should be
commensurate with the potential hazard and likelihood of possible harm to the recipient. The center’s approach
to developing donor deferral criteria should take into account evidence as it becomes available to modify those
decisions. Some of these issues that allow for medical judgment and for which questions exist in the DHQ can
be explored further with the donor, but each donor center must develop and follow its own SOPs.3
BLOOD-CENTER-DEFINED DONOR ELIGIBILITY CRITERIA
Unlike questions about potential risks to transfusion recipients, selection criteria directed primarily at
protecting donor safety are left to the discretion of the blood center’s medical director. Consequently, practice
varies at different blood centers.3,8 AABB Standards requires that prospective donors appear to be in good
health and be free of major organ disease (eg, diseases of the heart, liver, or lungs), cancer, or abnormal
bleeding tendency, unless determined eligible by the medical director.1(p61) The rationale for each deferral for
medical conditions should be carefully considered because even temporary deferrals adversely affect the
likelihood that individuals will return to donate blood.21 Donor centers have successfully eliminated some
health-related questions and removed screening requirements, such as assessment of pulse, without a deleterious
effect on donor health or donation-related reactions.4,22 These findings support the conclusion that some
arbitrary and
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AABB TECHNICAL MANUAL
rigid selection criteria, ostensibly intended to protect donors, are unnecessary and can be safely eliminated.
Cancer
Each year in the United States, blood centers receive hundreds of reports of cancer in individuals who had
donated blood.
Direct transmission of cancer through blood transfusion—although biologically plausible—has not yet been
documented to occur even though people with cancer frequently donate blood before discovering their
diagnosis.23 A retrospective study examined the incidence of cancer among patients in Denmark and Sweden
who received blood from donors with subclinical cancer at the time of donation.24 Of the 354,094 transfusion
recipients, 12,012 (3%) were exposed to blood components from precancerous donors, yet there was no excess
risk of cancer among these recipients compared with recipients of blood from donors without cancer.24 These
data indicate that cancer transmission by blood collected from blood donors with incident cancer, if it occurs at
all, is so rare that it could not be detected in a large cohort of transfusion recipients that included the total blood
experience of two countries over several years.
In considering the future eligibility of donors with cancer, some degree of caution is warranted to allow
sufficient time for donors to recover after chemotherapy or other treatment. There are currently no US federal
regulations or professional standards regarding the criteria that should be used to evaluate donors with a history
of cancer. For this reason, a blood center’s medical director has considerable flexibility in determining donor
eligibility policies.
Almost all licensed blood centers currently accept donors who report localized cancers after treatment, with
no deferral period. These cancers include skin cancer (eg, basal cell or squamous cell carcinoma) and carcinoma
in situ (eg, cervical) that have been fully excised and are considered cured. Most blood centers defer individuals
with a history of a solid organ or nonhematologic malignancy for a defined
period after completion of treatment provided that the donor remains symptom free without relapse. The
deferral period following completion of treatment for nonhematologic cancer ranges from 1 to 5 years.24
Hematologic malignancy typically results in permanent deferral from allogeneic blood donation, although some
US blood centers reportedly accept adults if they have been successfully treated for childhood leukemia or
lymphoma and have had a long (eg, 10- to 20-year) cancer-free period following completion of treatment. These
various deferral policies are currently defensible but should be reevaluated if new information becomes
available about the potential for cancer transmission through blood transfusion.
Bleeding Conditions or Blood Diseases
Bleeding conditions and blood diseases have the potential to affect donor safety as well as product potency,
and blood centers must define their procedures for handling donors with hematologic disorders. In general,
prospective donors should be evaluated for bleeding conditions or blood diseases that 1) place the donors at risk
of bleeding or thrombosis as a result of the collection procedure or 2) may affect the hemostatic efficacy of their
blood and its suitability for transfusion to others.3
Plasma components and Cryoprecipitated Antihemophilic Factor should contain adequate amounts of
functional coagulation factors and should not contain significant inhibitory or prothrombotic factors. Similarly,
platelet components intended as the sole source for patients should contain platelets that have adequate function
and are not irreversibly impaired by the presence of inhibitors.
Individuals with a history of significant bleeding complications are usually counseled to avoid blood
donation. However, screening donors for such a history does not prevent the rare but serious thrombotic or
hemorrhagic complications in otherwise healthy blood donors.
Individuals with hemophilia, clotting factor deficiencies, or clinically significant inhibitors—all of which
are manifested by variable
129
bleeding tendencies—require deferral for both donor safety and product potency considerations. The
exception is Factor XII deficiency, which is not associated with either bleeding or thrombosis.
Carriers of autosomal-recessive or sexlinked recessive mutations in clotting factors usually are not at risk of
bleeding. They typically have decreased factor levels but are accepted by most blood centers because of the
normal, wide variability in clotting factor activity levels (50%-150%) compared to the much lower relative
activity that is necessary to maintain hemostasis (5%-30%).3 Individuals with von Willebrand disease are
typically deferred by most blood centers, although some centers may allow individuals with mild disease and no
history of bleeding to donate red cells. Individuals taking anticoagulants to treat or prevent venous
thromboembolism are deferred for both donor safety and product potency considerations.
Heart and Lung Conditions
Cardiovascular disease is common in the United States, affecting an estimated 80 million (1 in 3) adults.25
Prospective blood donors are asked if they have ever had problems with their heart or lungs as a donor safety
measure, but the criteria for accepting donors with a history of heart or lung disease are defined by each blood
center.
The collective, published experience with autologous donation by patients scheduled for cardiac procedures
has demonstrated that adverse effects are not more frequent than in donors without a history of cardiac
disease.26'30 Despite the relative frequency of cardiovascular disease in the adult population, vasovagal
reactions occur in only about 2% to 5% of whole-blood donations by healthy donors and are actually more
likely to occur among young, healthy adolescents than older adults.31
 A rational approach to screening donors with a history of cardiac disease allows the acceptance of donors
who are asymptomatic on the day of donation, have been medically evaluated, and report no functional
impairment or limitations on daily activity after being diag
nosed or treated for cardiac disease. Some donor centers advise individuals to wait at least 6 months after a
cardiac event, procedure, or diagnosis. These centers then allow these individuals to donate if they have been
asymptomatic and able to perform their usual daily activities during this interval.
Causes for deferral may include recent symptoms, limitations on activity or functional impairment resulting
from unstable angina, recent myocardial infarction, left main coronary disease, ongoing congestive heart failure,
or severe aortic stenosis.3
Medications
The DHQ and Medication Deferral List contain the requirements for deferrals for specific medications as
stipulated by the AABB and the FDA. These requisite medication deferrals fall into five broad categories:
1. Potent teratogens that pose potential harm to unborn children (although there have been no documented
cases of adverse fetal outcomes related to transfusions from donors taking these medications).
2. Anticoagulants and antiplatelet agents that affect component potency (plasma or platelet components
only).
3. Potential variant Creutzfeldt-Jakob disease risk from bovine insulin manufactured in the United Kingdom
(although there have been no documented cases of transfusion transmission from donors taking bovine insulin).
4. Human-pituitary-derived growth hormone, which theoretically increases the risk of Creutzfeldt-Jakob
disease (although there have been no documented cases of transfusion transmission from donors taking these
growth hormones).
5. Antibiotics or antimicrobials to treat an infection that could be transmitted through blood transfusion
(excluding preventive antibiotics for acne, rosacea, and other chronic conditions).
Although blood centers may add medications whose use requires donor deferral to the
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AABB TECHNICAL MANUAL
Medication Deferral List, many have chosen to use the list as developed by the FDA and the AABB or have
added only a few drugs. The DHQ task force has encouraged blood centers to fully consider the reasons behind
each local deferral and avoid unnecessary deferral practices.3
FDA’s pregnancy risk categories, which are designed to assess the benefit vs risk ratio if drugs are taken
during pregnancy, are often inappropriately used for blood donor selection. For example, categories D and X
include some commonly used drugs (eg, oral contraceptives and anticholesterol agents) that may be
contraindicated in pregnancy but pose negligible, if any risk, to any transfusion recipient.
Local medication deferrals are often based on concerns about the reason for the potential donor’s use of the
medication and his or her underlying medical condition rather than on any inherent threat posed by residual
medication in the collected blood component. Most drugs used by donors pose no harm to recipients, and many
factors should be considered when evaluating the potential risk of a drug’s use by a donor (eg, the medication’s
half-life, mean and peak plasma concentration, residual concentration in blood component, and dilution when
transfused to a recipient).
ABBREVIATED DHQ FOR FREQUENT DONORS
Blood centers have recognized for years that frequent and repeat donors must answer the same questions at
every donation about remote risk factors that are not likely to change—a situation that leaves many dedicated
donors dissatisfied with the donation experience. A small number of US blood centers have received approval
from the FDA and implemented an abbreviated DHQ (aDHQ) in 2003 for donors who have successfully
completed the full-length DHQ on at least two separate occasions and given one or more donations within the
past 6 months.32
The AABB aDHQ, which was developed and validated by the same task force as the full-length DHQ, has
been officially recog
nized by the FDA as “acceptable” and can be implemented by blood establishments using the corresponding
full-length DHQ.33 Two “capture questions” about new diagnoses or treatments since the last donation replace
17 previous questions about remote risks (eg, blood transfusion, Chagas disease, and babesiosis). Although the
aDHQ is only somewhat shorter than the DHQ and its use is restricted to frequent donors, it may improve
donors’ experience.
RECIPIENT-SPECIFIC “DESIGNATED” OR “DIRECTED” BLOOD DONATION
 Exceptional Medical Need
In certain clinical circumstances, a patient may benefit from blood components collected from a specific
donor, such as 1) a patient with multiple antibodies or with antibodies to highincidence antigens who requires
units from donors whose red cells are negative for the corresponding antigens or 2) an infant with neonatal
alloimmune thrombocytopenia who requires antigen-negative platelets from his or her mother.
Frequent donation by a specific donor for a specific patient with a medical need requires that the donor
center have a procedure that typically calls for both a request from the patient’s physician and approval by the
donor center’s physician. The donor must meet all allogeneic donor selection requirements, with the exception
of donation frequency, provided that they are examined and certified by a physician [21 CFR 640.3(f)]. For
frequent blood donors, the CFR allows centers to qualify a donor by testing the first donation in each 30-day
period.2 In emergency medical situations, blood components can be released before infectious disease test
results are available provided that the units are labeled and managed in accordance with the CFR. Testing on the
units must be completed as soon as possible after release or shipment, and results must be promptly reported to
the hospital or transfusion service.2
131
Directed Blood Donations
The use of directed donations has decreased over the last 10 years, but the ongoing demand from patients
for transfusions from specific donors during scheduled surgeries likely still reflects the skewed perception
among the general public of the risk for HIV associated with blood transfusion. Most blood centers and
hospitals surmount the associated collection, storage, and tracking logistical difficulties to provide this service.
Directed donations have higher viral marker rates than volunteer donations, mostly but not entirely
reflecting the higher prevalence of first-time donors among the former group.34 There is no evidence that
directed donations are safer to use than donations from volunteer community donors. On the contrary, some
concerns persist that directed donors may feel unduly pressured to give blood, which could compromise blood
safety.
Directed donors must meet the same criteria as voluntary donors, and their blood can be used for other
patients if not needed by the individual for whom the donations were initially intended. If collection of whole
blood is required from a directed donor more than once in an 8-week period, the CFR requires that the donor be
examined and certified to be in good health by a physician on the day of donation [21 CFR 640.3(f)],
The donor center should clearly communicate its directed-donation procedures so that the expectations
regarding availability of directed-donor units are known to the ordering physician and patient. The
communication required includes defining the mandated interval between collection of the blood and its
availability to the patient, mentioning the possibility that the patient will identify donors who are not ABO
compatible or not otherwise acceptable blood donors, and defining the policy for release of donor-directed units
for transfusion to other patients.
Autologous Blood Donations
Autologous donations have declined dramatically in the United States since the 1990s. Wan
ing interest in autologous donations may reflect the decline in viral risk associated with allogeneic blood
transfusion and, consequently, the minimal medical benefit and increased cost of autologous blood.35,36
In general, the use of preoperative autologous blood donation alone provides only a relatively small benefit
in reducing the probability of allogeneic transfusion and may actually increase the risk of lower postoperative
hematocrits, with enhanced risk of ischemic complications after surgery. Preoperative autologous donations
may still be used in conjunction with other blood conservation methods, such as acute normovolemic
hemodilution, perioperative blood recovery, and pharmacologic strategies. (See further discussion in Chapter
24.)
Patients identified as candidates for autologous donation are evaluated by the donor center. The following
criteria for autologous donations are specified by the FDA, AABB, or both:
■ A prescription or order from the patient’s physician.
■ Minimum hemoglobin concentration of 11 g/dL or hematocrit of 33%.
■ Collection at least 72 hours before the anticipated surgery or transfusion.
■ Deferral for conditions presenting a risk of bacteremia.
■ Use only for the donor-patient if labeled “autologous use only.”
Contraindications to autologous blood donation should be defined by the blood center and may include
medical conditions associated with the greatest risk from blood donation, such as 1) unstable angina, 2) recent
myocardial infarction or cerebrovascular accident, 3) significant cardiac or pulmonary disease with ongoing
symptoms but without an evaluation by the treating physician, or 4) untreated aortic stenosis.37 Both the
ordering physician and the donor center physician need to carefully balance the risks of the collection procedure
against any perceived benefit to the patient-donor.
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AABB TECHNICAL MANUAL
KEY POINTS
1. The DHQ and associated documents were developed by an AABB task force, and their use is recognized
by the FDA as an adequate process to determine the eligibility of volunteers for allogeneic blood donation.
2. The most current versions of the DHQ and associated documents are available on the AABB website, and
all DHQ documents that the FDA has recognized as acceptable are available on the FDA website.19,20
3. Prospective blood donors are informed of the risks of blood donation, clinical signs and symptoms
associated with HIV infection, behavioral risk factors for transmission of bloodborne pathogens, and
importance of refraining from blood donation if they are at an increased risk of being infected.
4. To be accepted for allogeneic blood donation, individuals must feel healthy and well on the day of
donation and must meet all AABB and FDA requirements as well as medical criteria defined by the donor
center.
5. In certain clinical circumstances (eg, rare phenotype or antigen-negative Red Blood Cell units needed), a
patient may benefit from blood components collected from a specific donor who may not otherwise meet all the
requirements for a volunteer donor. The blood center must have procedures and follow federal regulations for
frequent donation by a specific donor for a designated patient with a medical need.
6. The ongoing demand from patients to choose specific donors to provide blood for their transfusions
during scheduled surgeries in the absence of a defined medical need has dramatically decreased in the last 10
years but persists despite the lack of evidence of improved safety with directed donations.
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2. Code of federal regulations. Title 21, CFR Parts 600 to 799. Washington, DC: US Government Printing
Office, 2014 (revised annually).
3. Eder A, Bianco C, eds. Screening blood donors: Science, reason, and the donor history questionnaire.
Bethesda, MD: AABB Press, 2007.
4. US Food and Drug Administration, Center for Biological Evaluation and Research. Guidance for
industry: Implementation of acceptable full-length donor history questionnaire and accompanying materials for
use in screening donors of blood and blood components. (October 2006) Silver Spring, MD: CBER Office of
Communication, Outreach, and Development, 2006. [Available at http://www.fda.gov/ biologicsbloodvaccines /
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6. Fridey JL, Townsend M, Kessler D, Gregory K. A question of clarity: Redesigning the AABB blood
donor history questionnaire—a chronology and model for donor screening. Transfus Med Rev 2007;21:181-
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by blood and blood products, (May 2010) Silver Spring, MD: CBER Office of Communication, Outreach, and
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11. Reik RA, Burch JW, Vassallo RR, Trainor L. Unique donor suitability issues. Vox Sang 2006;90:255-64.
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11,2013).]
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registries for transfusiontransmitted disease markers. Transfusion 2008;48:34-42.
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hemoglobin concentration? Blood 2006; 107: 1747-50.
15. Cable RG. Hemoglobin determination in blood donors. Transfus Med Rev 1995;9:13144.
16. Simon TL, Garry PJ, Hooper EM. Iron stores in blood donors. JAMA 1981;245:2038-43.
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Evaluation (RISE) study. Transfusion2012;52:702-ll.
18. Triulzi DJ, Shoos KL. AABB Association Bulletin #12-03: Strategies to monitor, limit or prevent iron
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October23,2012).]
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(accessed November 11,2013).]
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after temporary deferral. Transfusion 2011;51:1188-96.
22. Germain M, Delage G, Gregoire Y, Robillard R Donation by donors with an atypical pulse rate does not
increase the risk of cardiac ischaemic events. Vox Sang 2013;104:319-16.
23. Eder AF. Blood donors with a history of cancer. In: Eder AF, Bianco C, eds. Screening blood donors:
Science, reason, and the donor history questionnaire. Bethesda, MD: AABB Press, 2007:77-92.
24. Edgren G, Hjalgrim H, Reilly M, et al. Risk of cancer after blood transfusion from donors with
subclinical cancer: A retrospective cohort study. Lancet 2007;369:1724-30.
25. AHA Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics—
2007 Update. Circulation 2007; 115: e69-171.
 26. Kasper SM, Ellering J, Stachwitz R et al. All adverse events in autologous blood donors with cardiac
disease are not necessarily caused by blood donation. Transfusion 1998;38:669-73.
27. Mann M, Sacks HJ, Goldfinger D. Safety of autologous blood donation prior to elective surgery for a
variety of potentially high risk patients. Transfusion 1983;23:229-32.
28. Klapper E, Pepkowitz SH, Czer L, et al. Confirmation of the safety of autologous blood donation by
patients awaiting heart or lung transplantation. A controlled study using hemodynamic monitoring. J Thorac
Cardiovasc Surg 1995;110:1594-9.
29. Dzik WH, Fleisher AG, Ciavarella D, et al. Safety and efficacy of autologous blood donation before
elective aortic valve operation. Ann Thorac Surg 1992;54:1177-80.
30. Popovsky MA, Whitaker B, Arnold NL. Severe outcomes of allogeneic and autologous blood donation:
Frequency and characterization. Transfusion 1995;35:734-7.
31. Eder AF, Dy BA, Kennedy J, Notari E, et al. The American Red Cross donor hemovigilance program:
Complications of blood donation reported in 2006. Transfusion 2008;48:1809-19.
32. Kamel HT, Bassett MB, Custer B, et al. Safety and donor acceptance of an abbreviated do
134
AABB TECHNICAL MANUAL
nor history questionnaire. Transfusion 2006; 46:1745-55.
33. Food and Drug Administration. Guidance for industry: Implementation of acceptable abbreviated donor
history questionnaire and accompanying materials for use in screening frequent donors of blood and blood
components. (May, 2013) Silver Spring, MD: CBER Office of Communication, Outreach, and Development,
2013. [Available at http://www.fda.gov/ BiologicsBloodVaccines/GuidanceComplian
ceRegulatorylnformation/Guidances/Blood/ UCM351107.htm (accessed January 7,2014).]
34. Dorsey KA, Moritz ED, Steele WR, et al. A comparison of human immunodeficiency virus,
hepatitis C virus, hepatitis B virus and human T-lymphotropic virus marker rates for directed versus
volunteer blood donations to the American Red Cross during 2005 to 2010. Transfusion 2013;53:1250-6.
35. Brecher ME, Goodnough LT. The rise and fall of preoperative autologous blood donation. Transfusion
2002;42:1618-22.
36. Schved JF. Preoperative autologous blood donation: A therapy that needs to be scientifically evaluated.
Transfus Clin Biol 2005;12:365-9.
37. Goodnough LT. Autologous blood donation. Anesthesiol Clin North Am 2005;23:263-70.
Chapter 6
Whole-Blood Collection and Component Processing
Larry J. Dumont, MBA, PhD; Mona Papari, MD; Colleen A. Aronson, MT(ASCP)SBB; and Deborah F.
Dumont, MT(ASCP)SBB
HR THE COLLECTION OF whole blood B181 (WB) from the donor and subsequent processing of the
donation into components requires careful attention to proper techniques to ensure the optimal care of both the
donor and the recipient. The classical, manually intense component-preparation methods are undergoing
significant changes as more automation is incorporated by developers, from hands-off expression of
components to completely hands-off methods for the entire process. Differences are also recognized in methods
for platelet preparation by the “huffy coat” intermediary process and the “plateletrich plasma” (PRP)
intermediary process.
This chapter describes WB collection and component preparation and processing that may take place at the
blood center or the hospital-based collection facility.
WB COLLECTION
The phlebotomy staff involved in WB collection must be trained in proper collection techniques to
minimize contamination of donated units and phlebotomy-related local complications, such as hematoma or
nerve injury, using locally approved standard operating procedures. Phlebotomy must be performed only after
the donor has been found to be eligible for blood donation and the following items have been properly labeled
using a unique donation identification number (DIN) label: the blood donor record, primary and satellite
containers, and sample tubes. A final check of appropriate labeling before phlebotomy helps ensure that the
donor history data, laboratory data, and other manufacturing data are associated with the correct blood
components. The
 Larry J. Dumont, MBA, PhD, Associate Professor, Geisel School of Medicine at Dartmouth, Lebanon, New
Hampshire; Colleen A. Aronson, MT(ASCP)SBB, Regional Director, Transfusion Services, ACL Laboratories,
Rosemont, Illinois; Mona Papari, MD, Medical Director, LifeSource, Rosemont, Illinois; and Deborah F.
Dumont, MT(ASCP)SBB, Research Technologist, Center for Transfusion Medicine Research, Dartmouth
Hitchcock, Lebanon, New Hampshire
L. Dumont has disclosed financial relationships with Advanced Preservation Technologies, New Health
Sciences, Fenwal, Terumo BCT, Haemonetics/Hemerus, Advanced Cell Technology, BioMed/CitraLab, and
EryDel. M. Papani, C. Aronson, and D. Dumont have disclosed no conflicts of interest.
135

136
AABB TECHNICAL MANUAL
final check may be documented by having the staff initial the donor record.
Blood Containers
Desirable Properties
WB collection containers must be approved by the appropriate regulatory authority [eg, the Food and Drug
Administration (FDA); Japanese Ministry of Health, Labor and Welfare; or Health Canada] and must be CE
marked for use in Europe. The containers should be sterile, pyrogen free, and identified by a lot number. They
should also list an expiry date and include other label data required by regulatory authorities.
The desired properties of storage containers include being easily formed and processed; flexible; kink
resistant; transparent; and resistant to damage throughout the anticipated use, including component processing,
storage, and transfusion. In addition, the container’s material must be compatible with sterilization by gamma
irradiation, ethylene oxide, electron beam, and/or steam.
The gas (oxygen, carbon dioxide, and water) permeability properties should be appropriate for the intended
use. Gas exchange and water loss before use is often limited by an overwrap or pouch. After removal from a
pouch, the collection set should be used within the time prescribed by the manufacturer. Pouches should be
labeled with the date and time that they are opened, and unused containers should be stored within closed
pouches.
Blood containers are usually made of thermoplastics, such as polyvinylchloride (PVC), ethylvinylacetate,
polyolefins (polyethylene or polypropylene), or fluoropolymers. The material formulation determines the
physical properties that are to be matched to the use requirements, such as high gas permeability for platelet
storage or low glass transition temperature for freezing.
The glass transition temperature of PVC bags is about -25 C to -30 C. At and below these temperatures, the
container is brittle and should be treated as if it were made of glass and fragile enough to break during trans
port and handling. PVC is rigid at room temperature and requires the addition of a plasticizer such as di-(2-
ethylhexyl) phthalate (DEHP). DEHP not only imparts flexibility to the PVC but also has been shown to protect
red cells against hemolysis during storage. Because of concerns over possible toxicity of DEHP, alternative
plasticizers, such as butyryltrihexyl-citrate, have been made available in some collection sets. Because of the
protective effect of DEHP on red cells, finding an equally effective substitute for DEHP has been challenging.
This has recently been reviewed by Simmchen.1
Blood collection and processing sets are generally latex free. The manufacturer’s label should be consulted
to verify the absence of latex before use in patients with a latex allergy.
Configurations, Anticoagulants, and Additive Solutions
Collection and storage systems come in different configurations based on the intended processing methods
(manual or automated) and the number and types of final components to be prepared from a unit of WB. Blood
collection systems may be intended for final processing with a classical combination of manual and mechanical
methods (eg, centrifugation, manual bag transfers, and manual expression of contents) or may be designed for
use in a fully automated system of blood component preparation. There will be differences in the configurations
based on the method of choice for preparing platelets (huffy coat or PRP processes). Blood collection systems
may also include in-line filters for removal of leukocytes from WB, Red Blood Cell (RBC) units, and/or
platelets. Approved anticoagulants include anticoagulant citrate dextrose solution Formula A (ACD-A) or
Formula B (ACD-B), anticoagulant citrate phosphate dextrose solution (CPD), anticoagulant citrate phosphate
double-dextrose solution (CP2D), and citrate phosphate dextrose adenine solution (CPDA1) (see Tables 6-1 and
6-2). RBCs anticoagulated with CPDA-1 have a shelf life of 35 days in the United States. The use of additive
solutions (ASs) enables extension of RBC shelf life to
137
TABLE 6-1. Anticoagulant-Preservative Solutions for Collection of 450-mL Whole Blood*
CPDA-
Variable CPD CP2D
1
5.3-
pH 5.0-6.0 5.0-6.0
5.9
Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 63 mL)
Sodium citrate, dehydrate 1660 1660 1660
Citric acid, anhydrous 188 206 188
Dextrose, monohydrate 1610 3220 2010
Monobasic sodium phosphate, monohydrate 140 140 140
Adenine 0 0 17.3
*Data were supplied and verified by manufacturers.
= citrate-
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-
phosphate-
dextrose-dextrose; CPDA-1 adenine; FDA = Food and Drug
dextrose
Administration.
42 days in the United States and to 56 days in tween bags. This
tubing is marked with repeat
some other jurisdictions. Four ASs are current- ing serial numbers
and may be sealed at
ly approved in the United States, and others several locations with
either a dielectric (heat)
are approved in other regions (Table 6-3).2 sealer or a metal clip
(grommet) to prepare ap
Containers have integrally attached tub- proximately 13 to 15
segments. These seg
ing that permits aseptic/closed transfers be- ments may be used
later for ABO/Rh typing,
TABLE 6-2. Anticoagulant-Preservative Solutions for Collection of
500-mL Whole Blood*
CPDA-
Variable CPD CP2D
1
5.3-
pH 5.0-6.0 5.0-6.0
5.9
Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 70 mL)
Sodium citrate, dehydrate 1840 1840 1840
Citric acid, anhydrous 209 229 300
Dextrose, monohydrate 1780 3570 2230
Monobasic sodium phosphate, phosphate, monohydrate 155 155 155
Adenine 0 0 19.3
*Data were supplied and verified by manufacturers.
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-dextrose-dextrose; CPDA-1 = citrate-
phosphate-dextroseadenine; FDA = Food and Drug Administration.
 138
AABB TECHNICAL MANUAL
TABLE 6-3. RBC Additive Solutions Currently in Routine Use around the World*1"
AS-1
Adsoi AS-3 AS-5 AS-7
Fresensius Nutricel Optisol SOLX
Constituents Kabi Haemon Terumo Haemon PAGGSM
(mM) SAGM (Fenwal) etics BCT etics MAP MacoPharma
NaCI 150 154 70 150 85 72
NaHC03 26
Na2HP04 12 16
NaH2P04 23 6 8
Citric acid 2 1
Na3-citrate 23 5
Adenine 1.25 2 2 2.2 2 1.5 1.4
Guanosine 1.4
Dextrose
45 Ill 55 45 80 40 47
(glucose)
Mannitol 30 41 45.5 55 80 55
pH 5.7 4.6-7.2 5.8 5.5 8.5 5.7 5.7
Density
1.02 1.012 1.005 t 1.01 f 1.02
(g/mL)
Osmolality 347 -
462 418 393 237 f 270 - 310
(mOsm) 383
Anticoagulant CPD CPD CP2D CPD CPD ACD CPD
FDA-licensed No Yes Yes Yes Yes No No
Countries
Europe United United United United Japan Germany
used
United States States States States
Kingdom Canada Europe
Australia
 Canada
New
Zealand
Adapted from Sparrow.2
‘Data were supplied and verified by manufacturers.
tBrand names and primary sources are listed for each solution. Formulations may be available through other
sources.
*Not reported.
SAGM = saline, adenine, glucose, and mannitol; AS = additive solution; MAP = mitogen-activated protein;
PAGGSM = phosphate-adenine-glucose-guanosine-saline-mannitol; NaCI = sodium chloride; NaHC03 =
sodium bicarbonate; Na2HP04 = sodium hydrogen phosphate; NaH2P04 = monosodium phosphate; l\la3-citrate
= trisodium citrate; FDA = Food and Drug Administration.
139
crossmatching, antigen typing, investigation of adverse events of transfusion, or other appropriate laboratory
tests. A unique number is imprinted on each segment. A printed DIN is wrapped around the segment and is
archived for investigative purposes in case any transfusion reactions occur.
Containers also contain integral access ports for open connection of infusion sets or other spike entry. Such
open access limits the outdate from the time of entry to reduce the risk of septic transfusion reactions caused by
bacteria. This period may range from 24 hours for RBCs held at 1 to 6 C to 4 hours for RBCs held at room
temperature, cryoprecipitate, plasma, and platelets.
Base labels and any additional labels that are placed directly on the container must use approved adhesives.
In accordance with the FDA guideline issued in 1985 for the uniform labeling of blood and blood components,
only those substances that are FDA approved as “indirect food additives” may be used in adhesives and coating
components for labels placed over the base label.3 The FDA has additional standards for labels that are applied
directly on plastic blood containers. Because of a lack of sufficient space for attaching labels directly to the
container, tie tags may be used as an appropriate alternative, particularly for items that do not have to be directly
affixed to the container. National regulatory requirements should be verified when selecting labels and
establishing labeling policies.
Diversion Pouch
Collection systems intended for the preparation of platelets must include a diversion pouch immediately
after the access needle.4(p22) Diversion of the first few milliliters of blood into a pouch has been shown to
reduce the risk of bacterial contamination of the blood unit by capturing bacteria from the skin (also known as a
skin plug).5 The diversion pouch should be isolated by a heat seal or metal clip prior to any collection into the
primary collection container. After the pouch is isolated, blood in the pouch may be used for donor testing.
Container Modification by Sterile Connecting Device
Blood containers may be modified by using a sterile connecting device cleared or approved by the
appropriate regulatory body for that purpose.6 FDA-approved indications for the use of a sterile connecting
device are presented in Table 6-4. Use of such a device is increasing, primarily to obtain a sample from
apheresis platelets for bacterial contamination testing, attaching a leukocyte reduction filter to prepare
prestorage leukocyte-reduced (LR) RBCs and platelets, and preparing prestorage pooled platelets and pooled
cryoprecipitate. Recently available computer software permits tracking of lot numbers (eg, for wafers, bags, and
filters) and product codes, as well as other steps that involve the scanning of bar codes.
Donor and Blood Component Identification
Identification of blood components and test results to maintain association and linkage to the blood donor is
critical to ensure the transfusion recipient’s safety by permitting lookback investigations and product
withdrawals if indicated. Before phlebotomy, the donor is asked to present appropriate identification. Donor-
identifying information commonly includes the donor’s first name, middle name or initial, last name, and birth
date. A donor’s Social Security number is less commonly used as an identifier because of concerns about
identity theft. Contact information is necessary for future recruitment and notification of the donor about
abnormal test results.
The blood component identity (ID) process uniformly uses both a bar-coded and an eye-readable, unique
DIN that is assigned to each sample tube and each component prepared from the donation. The donor history
form and blood sample tubes are similarly labeled with the unique number for each donation. Electronic records
of the donation are also assigned the same number. The DIN should be verified on the donation record,
collection set primary and secondary containers, and sample tubes before blood collection can
140
AABB TECHNICAL MANUAL
TABLE 6-4. Use of Sterile Connecting Devices for Modification of Collection Containers6
Use Comment
To add a new or smaller needle to If a needle is added during the procedure, an approved device to
a blood collection set weld liquid-filled tubing should be used.
Examples include adding a fourth bag to make cryoprecipitate,
To prepare components
adding solution to the RBC unit, or adding an in-line filter.
Appropriate use of a sterile connecting device to pool platelets
To pool blood products prepared from whole blood collection may obviate potential
contamination from the spike and port entries commonly used.
To prepare an aliquot for pediatric FDA provides specific guidance if this activity is considered to
use and divided units involve manufacturing of new products.
To connect additional saline or
SOPs are required, although prior approval from the FDA is not
anticoagulant lines during an
required.
automated plasmapheresis procedure
To attach processing solutions Examples include washing and freezing RBCs.
To add an FDA-cleared leukocyte
The purpose is to prepare prestorage leukocytereduced RBCs.
reduction filter
To remove samples from blood
The label may need revision if the product’s cell count is affected.
product containers fortesting
RBC = Red Blood Cell; FDA = Food and Drug Administration; SOPs = standard operating procedures.
proceed as well as during and after the collection.
Phlebotomy
Vein Selection
The phlebotomist selects one arm for phlebotomy after inspecting both of the donor’s arms. The
phlebotomist checks for the presence of a prominent, large, firm vein in the antecubital fossa to permit a single,
readily accessible phlebotomy site that is devoid of scarring or skin lesions. A blood pressure cuff inflated at a
pressure of between 40 and 60 mm Hg or a tourniquet is applied to enlarge the vein. Palpating the vein is also
recommended before the phlebotomy site is prepared.
The first choice for the phlebotomy site is the well-anchored median vein, which is centrally located in the
antecubital fossa. The second choice is the cephalic vein that lies laterally
(shoulder side) and is often superficial. The third choice is the basilic vein, which lies on the underside of
the antecubital fossa; the basilic vein is not well anchored and may roll during phlebotomy. Excessive probing
with the needle should be avoided to prevent nerve injury.
Although the Occupational Safety and Health Administration provides an exemption from the requirements
to wear gloves during phlebotomy of volunteer blood donors, some blood collection centers require their
employees to wear gloves for phlebotomy of all allogeneic and autologous donors.
Disinfection Methods for Venipuncture Site
Specific instructions in the product insert for the use of approved agents should be followed for arm and
phlebotomy site preparation (Method 6-2). These methods provide surgical
 141
cleanliness, but none of the methods can achieve an absolutely aseptic site.
Approximately 50% of donors have no bacterial colonies on culture of the venipuncture site after
disinfection with povidone iodine or isopropyl alcohol plus iodine tincture. Bacterial growth with low colony
numbers (1 to 100) occurs in about half of the donors. More than 100 colonies is rare (1%) in such cultures.7
Bacteria residing deep within skin layers are not accessible to disinfectants. Therefore, if skin tissue enters the
collection bag, it can result in contamination. In one study, pigskin epidermal cells were detectable in the lavage
fluid in 1 out of 150 punctures.8 Whether human skin fragments or epidermal cells are carried into the bag
during routine blood donation has not been studied in detail. Nonetheless, AABB Standards for Blood Banks
and Transfusion Services (Standards)4^ requires collection containers that divert the first few milliliters of
blood into a special pouch that retains skin plugs to prevent contamination when platelet components will be
prepared from the collection. Diversion of the first aliquot of blood passing through the needle has been shown
to reduce the proportion of blood products containing viable bacteria.9
Blood Collection Process
Method 6-3 describes steps for blood collection and sample collection for processing and compatibility
tests. The average time to collect 500 mL of blood is less than 10 minutes. A draw time longer than 15 to 20
minutes may not be suitable for collecting platelets or plasma for transfusions.
The collection bag should be periodically mixed during the collection to ensure uniform distribution of
anticoagulant. Devices are available to automatically mix the unit during collection.
Samples for donor testing may be collected from the diversion pouch after the pouch is isolated either by
clamping or sealing the entry line. Samples for testing may also be aseptically obtained from the collection bag
with either an integrally attached sample container or a
sampler that is sterile and connected to the bag. If insufficient volume is available for test samples, a second
phlebotomy may be performed immediately after the blood collection. Care should be taken to avoid diluting
sample tubes.
Blood collection containers now include safety devices, such as a sliding sheath needle guard, to prevent
accidental needle-stick injuries. These devices allow retraction of the needle into a safety guard at the
completion of blood collection. Capping of needles should be avoided to prevent injuries.
Volume of Blood Collected
AABB Standards permits collection of 10.5 mL of blood per kilogram of the donor’s weight for each
donation, including the blood unit and all samples for testing.4(p60) In North America and Europe, the volume
of blood collected during routine phlebotomy is typically either 450 ± 10% (405-495 mL) or 500 mL ± 10%
(450550 mL). The volume may be different in other regions and may be as low as 200 to 250 mL. For general
blood banking applications, the volume of blood collected can be determined from the net weight in grams
collected divided by the density of WB (1.053 g/mL).
Empirical estimates may be used for the density of RBC components as reported by Burstain et al.10 The
theoretical density may also be calculated from the densities of the red cells (DPJiC), density of the suspending
medium (Ds), and measured hematocrit (H, L/L): density of RBC component = (DRBC - Ds) x H + Ds x (l-II).
Collection volumes must be within the manufacturer’s specified range to ensure the correct anticoagulant-
to-WB ratio. High-volume allogeneic collections should be discarded. Low-volume allogeneic collections
should be relabeled as “RBCs Low Volume” (Table 6-5). Plasma and platelets from low-volume units should be
discarded.
Low-volume autologous collections may be retained if they are approved by a physician. Plasma from low-
volume units has a higher citrate concentration than plasma from high-volume units and should be discarded.
142
AABB TECHNICAL MANUAL
TABLE 6-5. Weight and Volumes of Routine-Volume, Low-Volume, and Overweight Whole Blood
Collections*
450 mL Collection 500 mL Collection
Bag Bag
333-449
Low-Volume Collection Volume 300-404 mL Volume
mL
Weight 316-425 g Weight 351-473 g
Routine-Volume 450-550
Volume 405-495 mL Volume
Collection mL
Weight 427-521 g Weight 474-579 g
Overweight Collection Volume >495 mL Volume >550 mL
Weight >521 g Weight >579 g
*A density of 1.053 g/mL was used for conversion calculations. The volume and weight of anticoagulant
and bag(s) are not accounted for in this table. Low-volume Red Blood Cell products must be labeled as low-
volume products.
The evidence indicates that the volume collected does not affect in-vivo red cell survival. Red cell recovery
(average) after 21 days of storage in CPD was reported to be acceptable (approximately 80%) when only 300 g
(285 mL) of blood was collected.11 Recovery was more than 90% when 400 g (380 mL) of blood was collected
and stored for 21 days. Similarly, as much as 600 g (570 mL) of blood collected in a standard 450-mL CPD
container and stored for 21 days had normal in-vivo red cell recovery. Undercollected units (275 mL or 290 g)
in CPDA-1 stored for 35 days had a mean 24-hour survival rate of 88%.12 These data show that for
undercollected and overcollected units, variability in the amount collected does not adversely affect red cell
storage for 21 to 35 days.
Volumes of other components may also be determined gravimetrically by dividing the net weight in grams
by the appropriate density in g/mL. Table 6-6 provides generally accepted densities and, for some, the
applicable regulatory documents provide a specific value.13,16 Often, the specific gravity (ratio of density
component to density water) is used for this purpose, assuming that the density of water is 1.00 g/mL.
Donor Care After Phlebotomy
Immediately after collection, the needle is withdrawn into a protective sleeve to prevent accidental injuries.
Local pressure is applied
by hand to the gauze placed directly over the venipuncture site while the donor’s arm is kept elevated.
Pressure is applied until hemostasis is achieved, which may require more time for donors who are taking
anticoagulants or antiplatelet drugs. Once hemostasis is evident, a bandage or tape may be applied.
Postphlebotomy care includes observing the donor for signs or symptoms of reactions. If the donor tolerates
a sitting position without problems, he or she may proceed to the canteen area and is encouraged to drink fluids
and wait until being released. Local or state laws may specify the amount of time that the donor must wait after
the donation and before leaving the collection area. The donor is encouraged to drink more fluids and refrain
from heavy exercise for the next 4 hours, avoid alcohol ingestion for several hours, and refrain from smoking
for 30 minutes. The donor is also instructed to apply local pressure to the phlebotomy site if any bleeding recurs
and to call the blood center if the bleeding does not stop with pressure. A telephone number is provided so that
the donor can report if he or she feels that the donated unit should not be used, has any reactions, or experiences
any signs or symptoms of infection.
Adverse Donor Reactions
In donor follow-up surveys, minor reactions, such as dime-sized hematomas at the phlebotomy site, are
reported by as many as one-third
 143
TABLE 6-6. Density of Principal Blood Cells and Components
Specific
Blood Cell or Component Source*
Gravity
Cell Council of Europe13
Platelet 1.058
Monocyte 1.062
Lymphocyte 1.070
Neutrophil 1.082
Red cell 1.100
Components
Red cells collected by automated methods
1.06* FDA guidance14
with additive solution
Red cells collected by automated methods
1.08* FDA guidance14
without additive solution
Apheresis Platelets 1.03 FDA guidance15
Dependent on manufacturer of plasma or
Plasma
plasma derivatives
Octapharma AG* 1.026
CSL Plasma5 1.027
Fenwal (Amicus Cell Separator)" 1.027
FDA guide for inspection of blood
Whole blood 1.053*
banks16
‘Specific gravity (SG) is the density relative to water. Assuming a density of water of 1000 g/mL, the values
of SG and density are equal.
fSee text for alternate methods to determine density of whole blood and Red Blood Cell components.
♦Octapharma AG, Lachen, Switzerland.
§CSL (previously ZLB) Plasma, Boca Raton, FL.
"Fenwal, Lake Zurich, IL.
FDA = Food and Drug Administration.
of all donors.17 Adverse reactions occur at the time of donation or are reported later in about 3.5% of
donations. The American Red Cross observed a similar rate of 4.35% (435 per 10,000 donations) in 4.3 million
donations in 2007 (see Table 6-7).18 Reactions that need medical care after the donor has left the donation site
occur in 1 in 3400 donors. A population-based European study found the rate of complications leading to long-
term morbidity or disablement to be 5/100,000 donations and 2.3/100,000, respectively.19 Donor
hemovigilance programs instituted by the American
Red Cross also recorded a lower rate of complication rates for WB collections than with apheresis methods
for platelet and 2-unit RBC collection, with minor presyncopal reactions and small hematomas representing
most of the reactions. Major reactions were slightly more common for WB collection (7.4/10,000) compared
with plateletpheresis (5.2/10,000) and 2-unit red cell apheresis (3.3/10,OOO).20
Systemic Reactions
Vasovagal reaction complex includes dizziness, sweating, nausea, vomiting, weakness,
144
AABB TECHNICAL MANUAL
TABLE 6-7. Postcollection Adverse Event Rates in Calendar Year 2007 Based on 4,348,686 Whole Blood
Collections in the American Red Cross System*
Adverse Event Number Rate per 10,000 Collections
Minor reactions
Presyncope 120,561 277.2
Hematoma (small) 61,029 140.3
Citrate (minor)1 31 0.1
Other (minor) 165 0.4
Allergic (minor) 30 0.1
Subtotal 181,816 418.1
LOC and major reactions
LOC (<1 minute) 4,269 9.8
LOC (>1 minute) 591 1.4
Prolonged recovery 842 1.9
LOC with injury 438 1.0
Other (major) 82 0.2
Citrate (major)1 3 0.0
Allergic (major) 2 0.0
Subtotal 6,227 14.3
Major phlebotomy-related reactions
Hematoma (large) 387 0.9
Nerve irritation 314 0.7
Arterial puncture 449 1.0
Subtotal 1,150 2.6
All adverse events 189,193 435.1
Outside medical care 1,814 4.2
Adapted with permission from Benjamin et al.18
‘Citrate reactions after WB donations represent errors in classification.
*95% confidence interval includes 1.0.
LOC = loss of consciousness; ND = not determined.
apprehension, pallor, hypotension, and bradycardia. In severe cases, syncope and convulsions may be
observed. The pulse rate is often low during vasovagal reactions, but the rate is often high during volume
depletion. Some donors with severe reactions or with prolonged recovery times may need short-term observa
tion, intravenous fluid administration in the emergency room, or both. A protocol for telephone follow-up of
donors who have experienced severe reactions is helpful to assess the donors for any residual symptoms. Donor
reactions after WB donation do not predict accurately the possibility of recurrent syncope in
 145
returning donors, although they reduce the likelihood of future donations.21
Approximately 60% of systemic reactions occur in the canteen area.17 The main predictors of immediate
and delayed vasovagal and presyncopal reactions are young age, low estimated blood volume, and first-time
donation status.22,23 Ingestion of antihypertensive medications does not appear to be a risk factor.24
Approximately 15% of reactions occur away from the donation site, usually within 1 hour of donation.17 In
donors experiencing a reaction, an injury to head, face, or extremity may occur. The staff should be vigilant to
detect reactions early and prevent injuries as much as possible. Deferral strategies for young donors with
estimated low blood volume (<3.5 liters) and physiologic strategies to minimize donor reactions in young
donors are aimed at improving donor safety.21 Donor education, environmental controls, instructions to donors
to drink water right before donation, dietary interventions, and muscle tension are all effective in reducing the
rate of adverse reactions, especially in young donors, in whom a 20% reduction has been observed.25,26
In case of a vasovagal reaction, phlebotomy should be stopped, and the donor should be placed in a
recumbent position as soon as the reaction is suspected. Applying cold wet towels to the donor’s neck and
shoulder area and loosening the donor’s clothes can assist in symptom management.
Oral fluid intake before and soon after nonautomated WB donation appears to reduce the frequency of
systemic reactions.17 Coffee ingestion can reduce the frequency of reactions, but it may also reduce blood flow
during collection because of its vasoconstrictive effect.
Bruise or Hematoma
Both symptoms are common after phlebotomy but generally do not prevent donors from donating again.
Fatigue
First-time donors and females are more likely to report fatigue after donation. Donor return
is reduced by one-third among donors experiencing this symptom.17
Local Nerve Injury
Nerve injuries may be unavoidable because nerves cannot be palpated. In 40% of cases of nerve injuries,
phlebotomy is performed without difficulty.17 Donors may complain of sensory changes away from the
phlebotomy site, such as in the forearm, wrist, hand, upper arm, or shoulder. These injuries are usually transient,
and recovery almost always occurs; however, in 7% of injured donors, recovery may take 3 to 9 months.17 In
severe cases, referral to a neurologist may be indicated.
Arterial Puncture
Presence of bright red blood, rapid collection (within 4 minutes), and a pulsating needle suggest arterial
puncture. The hematoma rate is higher with arterial puncture. When puncture is recognized early, the needle
should be pulled out immediately, and local pressure should be applied for an extended period. Most donors
recover quickly and completely, but some might present with waxing and waning hematomas and should be
evaluated for pseudoaneurysm by ultrasound studies.
Upper-Extremity Deep Vein Thrombosis
Only one case of this thrombosis has been reported in the literature.17 Symptoms include pain; antecubital
fossa tenderness; swelling of the arm; and a prominent, palpable, cord-like thickening of the thrombosed vein.
Medical referral of donors who are experiencing deep vein thrombosis should not be delayed so that treatment
can begin promptly.
Postdonation Mortality
The FDA requires blood establishments to report deaths caused by blood donation. For fiscal years 2008-
2012, the FDA received 50 reports of postdonation fatalities for automated and manual collections, of which
only 11 followed WB donation. After medical review, one case was ruled out, and in the remaining
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10 cases, there was no evidence of a causal relationship between blood donation and the donors’ demise.27
Most deaths that take place after blood donation are coincidentally related to the donation rather than caused by
it.
BLOOD COMPONENT PREPARATION AND PROCESSING
Improvements and innovations continue to be made to WB collection and its processing into components.
Apheresis is growing in importance for collection of all major components, as discussed in Chapter 7. Single
WB units are collected in plastic containers containing anticoagulant; typically CDP, CP2D, or CPDA-1
(Methods 6-3,6-4, and 6-5). Innovations in this area include scales for monitoring the collection volume plus
automatic mixing and devices to add anticoagulant at a fixed ratio as the blood is withdrawn from the vein.
Methods and devices are available to process WB in a variety of manners for WB leukocyte reduction,
separation of plasma, and separation of red cells and platelets. Important secondary processing includes
leukocyte reduction, irradiation to prevent graft-vs-host disease (GVHD), and pathogen reduction.
For preparation of platelets from WB, two primary methods are available (Method 6-12): preparation from
huffy coats and preparation from PRP. The huffy coat method is employed in many regions but is not currently
cleared in the United States. In short, non-leukocytereduced WB units are first centrifuged under a high g-force
(ie, hard or heavy spin) and plasma, and red cells and huffy coats are removed for further processing. Buffy
coats from 4 or 5 units are pooled with 1 unit of plasma and then centrifuged under a low g-force (ie, soft or
light spin) to separate the platelet concentrate for additional processing, such as leukocyte reduction. These
processes are often associated with extended holding of the WB and buffy coats for 8 to 24 hours at 20 to 24
C.28
Preparation of platelets from PRP begins with a soft spin of the WB, followed by separation and hard spin
of the PRP. Plasma for fur
ther processing is removed from the platelet pellet, which is held undisturbed for 30 to 60 minutes before
being resuspended.29 Leukocyte reduction may occur at the PRP or final platelet concentrate stage, depending
on the device system used. Platelet concentrates are labeled and stored as individual units or, if cleared by
regulatory authorities, pooled and stored as transfusable doses.30
Compared to the PRP preparation method, the buffy coat method yields more plasma, greater red cell loss,
better initial white blood cell (WBC) reduction before filtration, and moderate reduction in viable bacteria in the
platelets.
Plastic collection containers, satellite bags, and integrally attached tubing that are hermetically sealed allow
component manufacturing to take place in a closed system. The blood container should not be entered before
issue except for the purposes of blood collection or transfer of components to a different container. The
container material should not have any adverse effect on the safety, purity, or potency of the blood. Components
prepared with an open system require a reduction in their expiration time from the time that the system was
opened. The use of approved sterile connection devices maintains a functionally closed system when various
connections are performed, such as pooling or sampling, thereby maintaining the product’s original expiry date.
WB Handling Before Component Processing—Timing and Temperatures
The temperature conditioning and handling of WB immediately after collection is determined by
downstream processing requirements for component preparation. In some cases, this may mean that collections
at mobile blood drives or fixed collection sites should be transported as soon as possible to the central
component preparation laboratory. With other processing methods, transportation may not be as urgent.
Requirements for cooling and transportation methods are quite variable, and the specifications of the device
manufacturer should be
147
carefully followed. By the time phlebotomy is completed, the blood has air cooled to about 30 C.31 If such
units are left at the ambient temperature, the cooling rate is quite slow and about 6 hours more are needed for
units to reach 25 C.31 To cool blood more rapidly, units are typically placed in specific storage environments.
Some centers use cooling plates that provide rate-controlled cooling toward 20 C. These cooling plates contain
1,4-butanediol that has a melting temperature of 20 C and serves as a heat absorber. With the cooling plates,
about 2 hours are needed for the collected blood to reach 20 C.31
Many WB leukocyte reduction systems allow filtration at ambient temperature beginning soon after
collection and lasting up to 24 hours. WB filtration may also be started and/ or completed at refrigerator
temperatures. WB destined for platelet preparation should not be cooled to less than 20 C. Platelets may need to
be separated from WB within 8 hours in some regions or with some collection systems. Other methods are
approved or are under consideration for up to a 24-hour hold of WB at 20 to 24 C before separation of plasma
and platelets. All processes for preparing platelets by the huffy coat methods have an extended hold of WB at
20 to 24 C for approximately 2 to 24 hours following collection and before the separation of the huffy coat and
plasma.13 The pooled huffy coat is often held undisturbed for approximately 2 to 18 hours at 20 to 24 C before
separation of the platelet concentrate.
The details of these hold times are generally adjusted to ease the operational logistics for the blood center.
For example, morning collections are held until the afternoon when plasma is separated and huffy coats
prepared. The huffy coats are held overnight as either individual units or pools, and the platelets are separated
the following morning. Likewise, afternoon collections may be held overnight, the huffy coats are prepared the
next morning, and platelets are prepared in the afternoon.
WB that will not be used to prepare platelets should be cooled to refrigerator temperature as soon as
possible; this is often accomplished by placing the unit on wet ice or other appropriate cooling media. The
allowable time
window for separation of plasma from WB is quite variable from region to region. FFP should be separated
from the WB and frozen within the time frame specified in the directions for use of the blood collection,
processing, and storage system. In the United States, plasma that is separated from WB collection and placed in
the freezer within 8 hours can be labeled “FFP”; if it is within 8 to 24 hours after collection of WB (manual or
apheresis method), it can be labeled as “Plasma Frozen Within 24 Hours After Phlebotomy.” In Europe, if the
WB is refrigerated, plasma may be separated within 18 hours; if the WB is held at 20 to 24 C for platelet
production, plasma can be separated within 24 hours, frozen, and labeled as FFP.
Shipping containers and cooling methods used to transport and hold WB and components must be
sufficiently validated according to local and national regulations and guidelines. Validation is generally
performed with various quantities of units in the transport container and a fixed amount of ice or gelpack to
determine the number of units that can be stored and transported while maintaining the desired temperature.
Portable temperature monitors are available to record temperature continuously.
Separation by Centrifugation
All current methods for separation and preparation of the three major blood components— RBCs, platelets,
and plasma—rely on one or more centrifugation steps. As mentioned above, the first step may be a high g-force
centrifugation followed by a low g-force step for the huffy coat platelet preparation method (the opposite of the
PRP platelet separation method) or an automated sequence of steps in recendy developed systems. Centrifuges
should be properly validated, maintained, and calibrated in a way that is consistent with local and regional
guidelines. Centrifuge methods for blood component preparation should be calibrated or checked in a
systematic manner to verify the processing conditions.
The major variables that affect the recovery of cells from WB by differential centrifugation are rotor size,
centrifuge speed, duration
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of centrifugation, and acceleration/deceleration protocol. Published papers often refer to relative centrifugal
force (g-force) that is derived from the radius of the centrifuge rotor and its revolutions. For a given centrifuge,
the rotor size is generally not variable. Therefore, the other two variables (centrifuge speed and duration) can be
altered in a stepwise fashion in a simplex strategy to determine the optimal conditions for preparing PRR32 The
simplex strategy can also be used to identify the optimal conditions of centrifugation for platelet concentrates
when quality control (QC) data show that the platelet counts in the platelet concentrates are not satisfactory.
Method 8-4 describes a process of functional calibration of the centrifuge to maximize platelet yield. The device
manufacturer should be consulted for recommendations on validating the performance of automated systems.
 On the day of preparation, some platelet units may contain clumps composed of platelet aggregates.33 In
routine practice, visual inspection is adequate to determine the degree of clumping subjectively and ensure that
units with excessive clumping are not released for labeling. Most of the clumps seen on day 0 disappear on day
1 of storage with continuous agitation, particularly those showing light to moderate clumping.33 The
temperature at which platelets are prepared may influence clumping; platelets prepared at 24 C appear to show
the least amount of clumping compared to those prepared at less than 24 C.33 Visual inspections after the
platelet concentrates are prepared have shown an absence of visible red cells in the vast majority of units, which
implies that the units contain fewer than 0.4 x 109 red cells. Generally, the number of red cells in a unit of
platelets does not exceed 1.0 x 109.34
Blood Component Division
Following separation by centrifugation, components must be carefully divided into separate containers for
further processing. Many laboratories use manual expressers for this purpose. Automated and semiautomated
devices, available in some regions, control the rate of expression, monitor component
weights, add storage solutions, clamp and seal tubing, and perform other useful functions that assist in the
consistent preparation of blood components. Some systems also combine nearly all of the functions, including
centrifugation, component expression, filtration, sealing, and additive addition, without relying on operator
interventions. The systems and methods used for these steps, whether predominantly manual or fully automated,
should be validated by the user.
Blood component extractors are available for making components in a semiautomated manner. After
primary centrifugation, WB is placed in the extractor, and a pressure plate creates an outflow of components
from the container. Outflow can occur from the top or bottom of the container, depending on the device.
When a cellular interface is detected by an optical sensing device, outflow tubing is automatically clamped
at an appropriate level. Such devices may improve the standardization of components, but they are not widely
used in the United States. An automated system is available in Europe that performs multiple functions to
prepare pooled platelet concentrates derived from WB by the buffy-coat method. Steps that are automated
include pooling, rinsing, centrifugation, transfer, filtration, and sealing.
DESCRIPTIONS OF MAJOR BLOOD COMPONENTS
WB
The minimum hematocrit of WB units in anticoagulant-preservative, such as CPD, is usually approximately
33%. WB is most often separated into components and is rarely used for transfusion directly. Severe
hemorrhage cases, such as those resulting from trauma, may benefit from fresh WB transfusions when platelets
are not available.35 Labile coagulation factors diminish and platelets may activate and develop a storage lesion
as the storage interval increases.
WB in ACD, CPD, or CP2D has an expiration date of 21 days when stored at 1 to 6 C; the
149
maximum storage time for units in CPDA-1 is 35 days. WB may be reconstituted by combining RBCs with
thawed plasma to achieve a desired hematocrit level—for example, when used for neonatal exchange
transfusions. Additional information about the preparation of reconstituted WB is provided in Chapter 9.
RBCs
RBCs in anticoagulant-preservative CPD or CPD2 have a shelf life of 21 days at 1 to 6 C with a hematocrit
of 65% to 85%, or 35 days in CPDA-1 with a hematocrit of <80%. The addition of additive solution (Table 6-3)
reduces the hematocrit to approximately 55% to 65%. Additive solutions maintain the red cells during storage
(for example, reduce hemolysis) and extend the expiry date to 42 days or 56 days, depending on regulatory
approvals. Hemolysis at the end of storage should be less than 1% in the United States or less than 0.8% in the
European Union (EU).
Hemoglobin content per unit varies because of differences between donors and processing specifics. For
example, more hemoglobin is generally lost when huffy coat preparation methods are used than with PRPtype
methods.
Hemoglobin content per unit may be more precisely controlled with automated collections. Total
hemoglobin content is not directly regulated in the United States but has a lower limit of 45 g per unit or 40 g
per unit for leukoreduced RBCs in the EU.36 Some experts have advocated for standardizing the amount of
hemoglobin per RBC unit at 50 g.37 Depending on local practice, RBCs stored for less than 7 to 10 days are
issued for neonatal or pediatric transfusions, and some neonatologists may prefer RBCs without additive
solutions (see Chapter 23).
 RBCs that are labeled as low-volume units are made available for transfusion when 300 to 404 mL of WB is
collected into an anticoagulant volume calculated for 450 ± 45 mL, or when 333 to 449 mL of WB is collected
into an anticoagulant volume calculated for 500 ± 50 mL. Although the resulting RBCs may be trans
fused, other components, such as platelets, plasma, and cryoprecipitate, should not be prepared from low-
volume units.
RBCs may be subject to several secondary processing steps, for example, leukocyte reduction, gamma or x-
ray irradiation to prevent GVHD, and pathogen reduction. The FDA requires leukoreduced units to contain <5 x
106 WBCs per unit and retain >85% of the preleukoreduced RBC content. The EU regulations call for <1 x 106
WBCs per unit.
Retention segments are held at the blood center for 7 days after the expiration date of the unit containing red
cells and at the hospital transfusion service for 7 days after the transfusion. Hemolysis identified in a segment
by visual inspection often does not correlate with the presence of hemolysis in the unit. In one study,
approximately threefourths of the visual assessments did not agree with the hemoglobin levels measured by
chemical methods, indicating a high falsepositive rate with visual assessment.38
Visual inspection of RBC units can detect white particulate matter, abnormal color caused by bacterial
contamination, hemolysis, and clots. Abnormal color caused by bacterial contamination may be observed in the
bag, where some segments appear to be darker than others because of oxygen consumption in the bag. The bag
content may look purple with or without hemolysis, and large clots may be evident. The unit can be centrifuged
to facilitate inspection of the supernatant in the case of suspected bacterial contamination, and visual inspection
of the supernatant may reveal murky, brown, or red fluid.39 However, visual inspection will not detect all
contaminated units.
Blood clots in RBC units are often too small to be detected by visual inspection. Clots are sometimes
revealed during transfusion when they clog the filter or in the component laboratory when the units are filtered
through a leukocyte reduction filter. Units known to have clots should not be released for transfusion.
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AABB TECHNICAL MANUAL
Platelets
The two major methods used in preparing platelets from WB, huffy coat, and PRP, are described above and
in Method 6-12. Single units of platelets are normally suspended in 40 mL to 70 mL of plasma, although studies
have shown good recovery and survival rates when platelets are stored in plasma volumes of 35 to 40 mL.40,41
Buffy-coat-derived platelets are nearly always pooled with the addition of 1 unit of plasma from one of the
donor units or the addition of a platelet additive solution. In the United States, no platelet additive solutions are
approved for use with platelets prepared from WB. Platelets prepared by the PRP or the buffy-coat methods can
be further processed to reduce leukocytes by using a leukocyte reduction filter as described in Method 612.
Platelets have demonstrated superior in-vivo recovery when stored between 20 and 24 C.42 However, at this
storage temperature, contaminating bacteria can proliferate during storage and result in septic transfusion
reactions, some of which are fatal. Therefore, platelet shelf life is limited to 5 days in the United States, 3 days
in Japan, 4 days in Germany, and up to 7 days in most EU and other countries. These limits are primarily
determined by regulatory risk assessments of the likelihood of septic transfusion reactions.
Practices to prevent and detect bacterial contamination have been effectively implemented for apheresis
platelets and prestorage pooled platelets from WB. However, developing effective and affordable detection
methods for single-unit platelets from WB has proved challenging, although several manufacturers are working
to develop an effective testing solution. Pathogen-reduction technologies are anticipated by many to be the best
general protection but have been slow to gain regulatory approval and be implemented in the field.
Platelets are metabolically active throughout the storage period. They use both glycolysis and oxidative
phosphorylation to produce adenosine triphosphate (ATP), a requirement to maintain cell integrity and function.
The product of glycolysis in platelets, lac
tic acid, is buffered by bicarbonate and exogenous buffers in additive solutions (eg, phosphate). These
buffer systems are limited, and lactic acid can cause the pH of the stored platelets to decline to less than 6.2 at
22 C, a level that results in unacceptably low in-vivo recoveries.43 Use of oxidative phosphorylation does not
result in acid production, and it is a much more efficient pathway for ATP production that, in turn, reduces the
need for glycolysis to run at a high rate—again limiting acid production. Modern platelet storage containers
permit oxygen to enter the storage bag to support oxidative phosphorylation and carbon dioxide escape; the
latter maintains the bicarbonate buffer function. This advance in storage containers has allowed the extended
storage of platelets beyond 3 days. The use of acetate, a component of many platelet additive solutions, as a
metabolic fuel also contributes positively to pH control because it consumes protons and is metabolized through
oxidative phosphorylation.44
Because of their metabolic requirements, platelets must be continuously agitated during storage. This action
keeps platelets suspended in the storage media, ensuring effective exchange of oxygen, carbon dioxide, and
lactic acid between the platelets and the suspending media. Long periods of static storage of platelets interrupts
this dynamic and can result in lactic acid production with a decrease in pH.45 During transport to hospitals from
the blood center or long-distance air transport, when components are exchanged between blood centers,
platelets are not agitated. Instead, they are double wrapped and secured in a shipping system. In-vitro studies
have shown that platelets are not damaged when they are stored without agitation for 24 hours.
Platelets must be stored and shipped at 20 to 24 C. Shipping, temporary equipment failures, and power
outages present challenges to meeting this requirement. Platelets maintained in-vitro function when they are
stored at 37 C for 6 hours followed by room-temperature storage without agitation for an additional 18 hours.46
Several studies have demonstrated negative effects on in-vivo platelet recovery and survival when platelets are
stored at tem
151
peratures <20 C.47,48 Thus, proper steps should be taken to maintain the required range of temperatures
during storage at the blood center and during transport.
Plasma
Plasma preparations are defined and regulated through a dizzyingly broad combination of collection
methods, storage temperatures, freezing methods, secondary processing, timing, and storage after thawing.
These specifications are covered in an array of standards, rules, and guidelines overlaid with various
requirements of the country where the plasma is prepared and/or used. Major, although not exhaustive, sources
of this information include the US Code of Federal Regulations, FDA guidance documents, the US Circular of
Information,49 the AABB Standards, and EU directives. The definitions and requirements of the country where
the plasma is prepared should always be consulted.
Plasma is prepared from WB collections and by apheresis. Plasma is generally frozen to maintain factor
activity and provide an extended shelf life. Frozen plasma is thawed for clinical use and may be maintained at 1
to 6 C for some time prior to use. Frozen plasma is also the source of cryoprecipitate and cryoprecipitate-
reduced plasma. There are several methods available for pathogen reduction of plasma that may be applied
depending on national regulatory approvals. Plasma may be used for preparation of specific plasma protein
products through fractionation processes. The descriptions below are based on the prevailing guidance
documents and regulations of the FDA and Council of Europe.
FFP
In the United States, FFP is plasma collected either from a single unit WB collection or by apheresis. FFP
must be placed in the freezer within 8 hours of collection; within 6 hours if anticoagulated with ACD; or as
directed by the manufacturer’s instructions for use of the blood collection, processing, and storage system. FFP
has a shelf life of 12 months when
stored at -18 C or colder. FFP that is stored at -65 C may be stored for longer than 12 months, but such
storage requires FDA approval.4(pp56'57) Rapid freezing of plasma can be accomplished using a blast freezer,
dry ice, or a mixture of dry ice with either ethanol or antifreeze. Plasma should be thawed at 30 to 37 C in a
waterbath or by using an FDA-cleared device. When a waterbath is used, the component should be placed in a
protective plastic overwrap. Thawing of larger units of FFP collected by apheresis may require more time. FFR
once thawed, has a shelf life of 24 hours at 1 to 6 C. Thawed plasma held longer than 24 hours must be
relabeled as Thawed Plasma, and it can be stored for an additional 4 days at 1 to 6 C.
In the past several years, many blood centers in the United States have increased the amount of WB
collected from 450 mL to 500 mL, resulting in a larger amount of plasma per unit. A unit can contain 500 mL to
800 mL of plasma when collection is performed by automated (single-donor) plasmapheresis.
The Council of Europe defines “plasma, fresh frozen” as prepared from either WB or collected by apheresis.
Plasma freezing must be initiated within 6 hours of collection, within 18 hours if the WB is held at 1 to 6 C, or
within 24 hours if WB or apheresis plasma is rapidly conditioned to 20 to 22 C following collection. Freezing
must be completed within 1 hour to less than -30 C. Plasma, fresh frozen has an expiry time of 36 months if
held at less than -25 C or 3 months if held at -18 C to -25 C.13 The Council of Europe does not address specific
thawing methods or treatment of the plasma following thawing, including expiry dating.
 AABB requires4tpl7) interventions to minimize the preparation of high-plasma volume components
(apheresis platelets, plasma products, and WB) for transfusion from donors who are at risk of developing HLA
antibodies.50 These products should be collected from males, never-pregnant females, or parous female donors
who test negative for HLA antibodies.
FFP contains normal amounts of all coagulation factors, antithrombin, and ADAMTS13.
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Quarantine FFP
Quarantine plasma was introduced to increase the viral safety of plasma. The Council of Europe notes that
quarantine FFP can be released from quarantine after the donor returns to the blood center and has repeatedly
negative test results for, at a minimum, hepatitis B and C viruses, HIV-1, and HIV-2 beyond a minimum
quarantine period that is greater than the diagnostic window period for viral infection, typically 6 months. With
the use of nucleic acid tests for viral screening, this window period for quarantine FFP may be reduced.51
Plasma Frozen within 24 Hours After Phlebotomy
The FDA defines plasma that is frozen within 24 hours of collection as Plasma Frozen within 24 Hours
After Phlebotomy (PF24). Once thawed, PF24 has a shelf life of 24 hours at 1 to 6 C. Thawed plasma held
longer than 24 hours must be relabeled as Thawed Plasma, which can be stored for an additional 4 days at 1 to 6
C.
Plasma Frozen within 24 Hours After Phlebotomy Held at Room Temperature up to 24 Hours After
Phlebotomy
The FDA defines Plasma Frozen within 24 Hours After Phlebotomy Held at Room Temperature up to 24
Hours After Phlebotomy (PF24RT24) as apheresis plasma that is held for up to 24 hours after collection at room
temperature and then stored at less than -18 C. Once thawed, PF24 and PF24RT24 have a shelf life of 24 hours
at 1 to 6 C. Thawed plasma held for longer than 24 hours must be relabeled as Thawed Plasma and can be
stored for an additional 4 days at 1 to 6 C. A similar product prepared from WB held at room temperature for
more than 8 hours (ie, overnight hold) may be defined in the future if FDA approves an overnight-hold WB
process.
Thawed Plasma
The FDA defines Thawed Plasma (which is not licensed by the FDA) as FFR PF24, or
PF24RT24 that has been thawed and held at 1 to 6 C for >24 hours. Thawed Plasma may be held at 1 to 6 C
for up to 5 days after thawing. Stable Factor II, fibrinogen, and reduced amounts of other factors have been
observed in Thawed Plasma. Thawed Plasma prepared from FFP and stored for 5 days contains reduced levels
of Factor V (>60%) and Factor VIII (>40%). AD AMTS 13 levels are well maintained for 5 days at 1 to 6 C in
Thawed Plasma.
Plasma Cryoprecipitate Reduced
Plasma cryoprecipitate reduced (United States) or plasma, fresh frozen cryoprecipitate depleted (Europe) is
a byproduct of cryoprecipitate preparation. In the United States, plasma cryoprecipitate reduced must be
refrozen within 24 hours at less than -18 C. The storage temperatures and expiration that apply to FFP apply to
this component in both the United States and Europe. The product contains a normal level of Factor V (85%).
Even after the removal of cryoprecipitate, the product has a fibrinogen level of about 200 mg/dL.52 The levels
of the following coagulation factors have been demonstrated to be normal in plasma cryoprecipitate reduced:
Factor I, Factor VII, Factor X, antiplasmin, antithrombin, protein C, and protein S. Levels of Factor VIII, the
von Willebrand factor (vWF) antigen, vWF activity, fibrinogen, and Factor XIII are decreased.53
Liquid Plasma
In the United States, liquid plasma for transfusion can be separated from WB at any time during storage and
stored at 1 to 6 C for up to 5 days after the WB’s expiration date.
Recovered Plasma
Blood centers often convert plasma and liquid plasma to an unlicensed component, “recovered plasma
(plasma for manufacture),” which is usually shipped to a fractionator and processed into derivatives, such as
albumin and/ or immune globulins. To ship recovered plasma, the collecting facility must have a “short supply
agreement" with the manufacturer. Because recovered plasma has no expiration
153
date, records for this component should be retained indefinitely. Storage conditions for recovered plasma are
established by the fractionator. FFP used as human plasma for fractionation in Europe must comply with the
applicable European Pharmacopoeia guidelines.
Pathogen-Reduced Plasma
Plasma can be treated to inactivate microbial agents for pathogen reduction. Four such methods are
available and in use in Europe: the methylene blue, psoralen (amotosalen), riboflavin, and solvent/detergent
treatments.
Methylene blue (approximately 0.085 mg/ unit of plasma) can be added to thawed FFP, followed by
activation using white light. After removal of methylene blue with a filter (residual concentration: 0.3 pmol),
plasma can be frozen. Methylene-blue-treated plasma contains approximately 15% to 20% less Factor VIII and
fibrinogen than untreated plasma.
Plasma prepared from WB or by automated methods can be treated with 15 mL of amotosalen per 250 mL
of plasma, followed by illumination with ultraviolet A light (320-400 nm) with 3.0 J/cm2. After amotosalen is
removed by exposing treated plasma to an adsorption device, the unit is frozen for storage at -18 C. Average
activity values for coagulation and antithrombotic factors are reported to be within reference ranges for
untreated plasma.
Plasma prepared by apheresis or from single WB units (volume range 170-360 mL) can be treated with the
Mirasol system by adding 35 mL of riboflavin (vitamin B2) followed by illumination for 6 to 10 minutes.
Immediately after illumination, the plasma can be released or frozen below -30 C for 2 years. Residual RBC
levels up to 15 x 109 per liter have been qualified to result in successful pathogen reduction and leukocyte
inactivation. Coagulation and anticoagulation proteins are well preserved in plasma treated with the Mirasol
system.54,55
Solvent/detergent-treated plasma (SD plasma) is prepared from a pool of plasma from many donors (no
more than 2500) that undergoes treatment with 1% tri-n-butyl
phosphate and 1% Triton X-100 for pathogen reduction. This treatment has been shown to significantly
inactivate lipid-enveloped viruses. SD plasma is manufactured in facilities that can manage large-scale
production rather than in blood centers. Each unit contains 200 mL of plasma that is stored frozen at -18 C with
an expiration date of 12 months.56 All coagulation factors are reduced by 10% in SD plasma, except for Factor
VIII, which is reduced by 20%.57 Also, SD plasma contains 50% less functional protein S in comparison to the
nontreated FFR58 The product is labeled with the ABO blood group and, once thawed, should be used within
24 hours. SD plasma is available in Europe and has been recently approved in the United States.59
Cryoprecipitated Antihemophilic Factor
Cryoprecipitated antihemophilic factor (AHF), or simply “cryoprecipitate” in Europe, is prepared from FFP.
Cold-insoluble protein that precipitates when FFP is thawed to 1 to 6 C is collected by centrifugation;
supernatant plasma is transferred into a satellite container; the precipitate is resuspended in a small amount of
residual plasma, generally 15 mL; and the precipitate is refrozen as described in Method 6-11. FFP can be
thawed to prepare cryoprecipitated AHF by placing the FFP in a refrigerator (at 1 to 6 C) overnight or in a
circulating waterbath at 1 to 6 C. An alternate method that uses microwaves for thawing has been described.
The cryoprecipitated AHF is placed in a freezer within an hour of removal from the refrigerated centrifuge and
can be stored at -18 C for 12 months from the original collection date. In Europe, thawing is to be performed at
2 to 6 C, and the product can be stored for up to 36 months below -25 C and for 3 months at -18 to -25 C.
AABB Standards4(pp28-29) requires that cryoprecipitated AHF contain at least 80 international units (IU)
of Factor VIII and 150 mg of fibrinogen per unit, although the average fibrinogen content is generally 250
mg.60 European standards are at least 70 IU/unit of Factor VIII, 140 mg/unit of fibrinogen, and 100 IU/unit
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of vWF. More current preparations are reported to have much higher amounts of fibrinogen (median, 388
mg/unit).61 Cryoprecipitated AHF also contains the vWF ristocetin cofactor activity (approximately 170
units/bag), Factor XIII (approximately 60 units/bag), and fibronectin. Rapid freezing of FFP is found to increase
the Factor VIII yield in cryoprecipitated AHF.62 ADAMTS13 levels are normal in cryoprecipitated AHF.63
Anti-A and anti-B are known to be present in cryoprecipitated AHF, but the combined amount of these
antibodies from the unit of plasma is only 1.15% of the total.64
Thawed cryoprecipitated AHF should be used as soon as possible but may be held at room temperature (20-
24 C) for 6 hours as single units or a pool prepared as a closed system using an approved sterile connecting
device or for 4 hours if pooling was with an open system. Pooling may be accomplished with the aid of a
diluent, such as 0.9% sodium chloride (USP), to facilitate removal of material from individual bags.
 At room temperature, the mean declines of Factor VIII levels at 2,4, and 6 hours are approximately 10%,
20%, and 30%, respectively.65 Cryoprecipitated AHF from blood groups A and B has higher levels of Factor
VIII compared to that derived from blood group O donors (about 120 vs 80 IU per bag, respectively).66
Thawed cryoprecipitate should not be refrozen.
Granulocytes
Although it is possible to prepare granulocytes from fresh WB, current practice is to collect granulocytes by
apheresis. Refer to Chapter 7 for information on automated collections.
BLOOD COMPONENT MODIFICATION
Prestorage Leukocyte Reduction by Filtration
For RBCs that are leukocyte reduced, the FDA requires a residual number of <5.0 x 106 leukocytes per unit.
The Council of Europe requires that the residual number be <1 x 106 per unit. In the United States, leukocyte
reduction by filtration of RBCs should result in a compo
nent that contains at least 85% of the original red cell content. The Council of Europe’s standards require a
minimum of 40 g of hemoglobin to be present in each unit after leukocyte reduction.13
For WB-derived platelets, AABB Standards requires that the leukocyte reduction process ensures that 95%
of the platelet units sampled contain <8.3 x 105 leukocytes per unit, at least 75% of the units sampled contain
5.5 x 1010 platelets, and at least 90% of the units sampled have a pH of 6.2 at the end of the allowable storage
period.4(p29) The number in the Council of Europe’s standards is <0.2 x 106 residual leukocytes per unit of
platelets from WB.13
In practice, approximately 1% of RBC components do not achieve the levels of <1 x 106 residual
leukocytes in the component. Sickle cell trait of red cells is the most common cause of filter failure.
Approximately 50% of the RBC units with sickle cell trait fail to filter. Although the other 50% pass through
the filter, the residual leukocyte content may be higher than the allowable limits.67
Prestorage leukocyte reduction is generally performed soon after WB collection and is always performed
within 5 days of collection. In-line WB filters are available in collection sets that permit preparation of LR
RBCs and FFP WB filters that spare platelets are now available as well. If WB is collected without the in-line
leukocyte reduction filter, a filter can be attached to the tubing by an FDA-cleared sterile connecting device.
The number of platelets in LR platelet concentrates is generally lower than in non-LR platelet concentrates.
Methods that measure residual leukocytes include Nageotte hemocytometry and flow cytometry (see
Method 8-11). In a multicenter study, flow cytometry gave better results than Nageotte hemocytometry when
freshly prepared samples (within 24 hours) were tested. For instance, at a concentration of 5 white cells per pL
for RBC units, the intersite coefficient of variation was 4.9% for flow cytometry and 54% for Nageotte
hemocytometry. In general, Nageotte hemocytometry tends to underestimate the number of white cells
compared to flow cytometry.68 A new semiau
155
tomated methodology offers to reduce the technical burden associated with both Nageotte and flow
cytometry methods.69
Cryopreservation
RBCs
Glycerol is the most commonly used cryopreservative agent and is added in either high or low concentration
to RBCs within 6 days of collection. Two commonly used protocols for the high-glycerol method are described
in Methods 6-6 and 6-7.
Frozen RBCs must be stored at temperatures colder than -65 C and expire after 10 years. Rare frozen units
may be used beyond the expiration date, but only after medical review and approval based on the patient’s
needs and availability of other rare compatible units. The units should be handled with care because the
containers may crack if they are bumped or handled roughly. The containers can also crack during
transportation.
The units should be thawed at 37 C and generally take about 10 minutes to thaw completely. Glycerol must
be removed after thawing and before transfusion. This removal is generally accomplished by instruments that
allow the addition and removal of sodium chloride solutions. In most cases, addition of the glycerol and its
removal (deglycerolization) require the system to be opened; for this reason, thawed and deglycerolized units
can be stored only for 24 hours at 1 to 6 C. The final solution in which cells are suspended is 0.9% sodium
chloride and 0.2% dextrose. Dextrose provides nutrients and has been shown to support satisfactory
posttransfusion viability for 4 days of storage after deglycerolization.70 For QC, determining the volume of red
cells in the unit after deglycerolization and examining the last wash for hemolysis are recommended (see
Method 6-8).
Recently automated addition of glycerol to RBCs and removal from RBCs in a closed system has become
possible. With this system, glycerol is added within 6 days of WB collection. Postthaw red cells prepared in this
manner are suspended in additive solution 3 (AS-3) and can be stored for 14 days at 4 C.70
Postwash units have a hematocrit of 51% to 53% and contain a mean of about 9.0 x 106 leukocytes per
unit.71
Platelets
Cryopreservation of platelets is not widely available because the procedures for cryopreservation are
complex and not routinely practiced at most blood centers. Several cryoprotectants have been described for
platelet cryopreservation72,73 However, 5% or 6% dimethyl sulfoxide (DMSO) is most commonly used,
mainly for autologous platelet transfusions in patients who are refractory to allogeneic platelets. Cryopreserved
platelets can be stored for at least 2 years. After thawing, the platelet recovery rate in vitro is about 75%, which
may be reduced further if thawed platelets are centrifuged to remove the DMSO before transfusion. The in-vivo
recovery rate after transfusion of thawed, DMSO-reduced platelets is about 35% to 42%.74 In vivo, the platelets
that survive after transfusion are hemostatically effective.75
Irradiation
Cellular blood components can be irradiated for prevention of GVHD. Frozen plasma components, such as
FFP and cryoprecipitated AHF, are generally not irradiated because they are considered noncellular
components. In addition, the small number of T lymphocytes present in the components may not survive the
freeze-thaw cycle.
The irradiation sources in use include gamma rays—from either cesium-137 or cobalt-60 sources—and x-
rays produced by radiation therapy linear accelerators or standalone units. Both sources achieve satisfactory
results in rendering T lymphocytes inactive. Freestanding instruments that allow the use of each of these
sources are commercially available for blood bank use. The US Nuclear Regulatory Commission requires an
increased number of controls for the radioactive material license that is needed for a gamma irradiator and its
source onsite.76 The increased controls are designed to reduce the risk of unauthorized use of radioactive
materials. The licensee
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AABB TECHNICAL MANUAL
is required to secure any area where radioactive source is present, and only approved individuals may access
the site to perform their duties.
In the United States, the radiation dose to the center of the irradiation field must be at least 25 gray (Gy)
[2500 centigray (cGy)] and no more than 50 Gy (5000 cGy).77 During dosimetry, the delivered dose of 25 Gy
is targeted to the internal midplane of the container. Moreover, the minimum delivered dose to any portion of
the blood components must be at least 15 Gy in a fully loaded canister.4(p25) The European standard requires a
higher dose, with no part of the component receiving less than 25 Gy and a maximum dose to any part of the
component of 50 Gy.13
Each instrument must be routinely monitored to ensure that an adequate dose is delivered to the container
that houses the blood components during irradiation. Dose mapping is used to monitor the instrument’s
function. For this purpose, irradiation-sensitive films or badges that monitor the delivered dose are used for QC
of the irradiators. Several systems consisting of irradiation films or badges are commercially available.77
Verification of the delivered dose must be performed annually for the cesium-137 source and semiannually for
the cobalt-60 source. For x-ray irradiators, the dosimetry should be performed in accordance with the
manufacturer’s recommendations. Dose verification is also required after major repairs or relocation of the
irradiator. For gamma irradiators, the turntable operation, timing device, and lengthening of irradiation time
caused by source decay should also be monitored periodically.
Another important quality assurance step is the demonstration that the product that was irradiated received
the desired amount of irradiation. For that reason, irradiation-sensitive labels are used to demonstrate that
irradiation of each batch of units was accomplished; this is a requirement in Europe.13
In the United States, RBCs may be irradiated up to the end of their storage shelf life. The postirradiation
expiration date is 28 days or the original expiration date, whichever is earlier. In Europe, RBCs may be
irradiated only
 up to day 28 following collection. Irradiated cells may not be stored longer than the earlier of 14 days
postirradiation or 28 days postcollection. Platelets may be irradiated until their expiration date, and their
postirradiation expiration date is the same as the original expiration date.
Irradiation of RBCs followed by storage does result in a decrease in the percentage of recovery after
transfusion. In addition, an increased efflux of potassium from red cells causes the potassium levels to rise
approximately twofold compared to nonirradiated units. Platelets are not damaged by an irradiation dose as high
as 50 Gy.78
Pooling
Platelets that are pooled have an expiration time of 4 hours from when the system was opened for pooling.
A closed system for prestorage pooling of platelets has been licensed by the FDA. This system permits the
storage of pooled platelets for up to 5 days from the time of WB collection. Four to six LR or non-LR platelet
units that are ABO identical can be pooled using a set consisting of a multilead tubing manifold for sterile
connection. If non-LR units are pooled, they are then filtered as part of the pooling process. The shortest
expiration date of the pooled units determines the expiration date of the pool.
In the United States, each pool prepared from LR platelets must have <5.0 x 106 residual leukocytes. The
approved pooling set also allows sampling of the pool for detection of bacterial contamination. A record of the
unique DIN for each individual member of the pool must be available. The pool must be labeled with the
approximate total volume, ABO/Rh type of the units in the pool, and number of units in the pool.
Many countries in Europe prepare prestorage pools of buffy-coat platelets that are preserved in platelet
additive solution or in the plasma from one of the units from which platelets are prepared.79 Instruments that
automate the pooling process are increasingly used in Europe. In a few countries in Europe, systems for
prestorage pooling of buffy-coat
157
derived platelets followed by pathogen-reduction treatment have become available. The latter systems are
not yet licensed by the FDA.
Cryoprecipitated AHF units may be pooled immediately before transfusion in an “open” system; the pool
has an expiration time of 4 hours at 20 to 24 C storage. Prestorage pools can also be prepared in an “open”
system and stored for 12 months at -18 C as described in Method 6-11. After thawing, the product expires in 4
hours. Prestorage pools prepared with the use of an FDA-cleared sterile connecting device are stored for 12
months at -18 C; postthaw expiration time is 6 hours. The number of units pooled may vary and can consist of
4, 5, 6, 8, or 10 units. Prestorage pools must be placed in a freezer within 1 hour. The potency of the pool is
calculated by assuming that each unit in the pool contains 80 IU of coagulation Factor VIII and 150 mg of
fibrinogen multiplied by the number of units in the pool. If normal saline is used to rinse the bags during
preparation of the pool, the amount of saline in the pool must be stated on the label.
Volume Reduction (Platelets)
Volume-reduced platelets may be needed for patients in whom a reduced amount of plasma is desired to
prevent cardiac overload, to minimize ABO antibody infusion, or for intrauterine transfusion. Method 6-13,
describes volume reduction by centrifugation. Platelet concentrate volume can be reduced to 10 to 15 mL/unit.
In-vitro properties such as platelet morphology, mean platelet volume, hypotonic shock response, synergistic
aggregation, and platelet factor 3 activity, appear to be maintained in the volume-reduced platelets stored for 5
days.80 The in-vitro recovery rate of platelets is about 85% after the volume-reduction step. Platelets from WB
that are volume reduced by centrifugation (580 x g for 20 minutes) from an approximate volume of 60 mL to
between 35 and 40 mL yield a high platelet count (>2.3 x 109/L).81 Lowering the pFT of platelets prepared
from WB avoids platelet aggregates visible to the unaided eye (macroaggregates).82 The addition of 10% ACD-
A to
platelets to lower the pH before centrifugation can help with resuspension of high-concentration platelets
and avoid aggregation.
Apheresis platelets can also be volume reduced during the collection process. In early trials, collection of
apheresis platelets in approximately 60 mL showed good in-vitro platelet characteristics and function. In-vivo
autologous recovery was at least equivalent to that of control apheresis platelets at standard concentrations
when the storage time of 1, 2, or 5 days was adjusted based on the platelet concentration.83,84 For apheresis
platelets, the volume reduction from 250 mL to 90 mL by centrifugation has been shown to cause a mild
increase in platelet activation and an impaired aggregation response to adenosine diphosphate but not to
collagen. In these experiments, the platelet count before the volume reduction was 1.0 x 109/mL; after the
reduction, the count was 1.9 x 109/mL.85
Recent developments include refinements in collection protocols for apheresis instruments that result in the
collection of platelets in a high concentration that may, therefore, obviate the need for volume reduction.
Platelet collections were at concentrations as high as 3.0 to 4.0 x 109/L.83,86 Furthermore, the highly
concentrated platelets may be suspended in a platelet additive solution with the autologous plasma at a ratio of
5:1 to 3:1. Platelet units prepared in this manner contain much lower amounts of plasma compared to standard
apheresis platelets.80 Recently, the FDA has approved additive solutions for apheresis platelets.
Posttransfusion platelet increments have been satisfactory after transfusion of volumereduced platelets.80
However, the overall invivo survival data for volume-reduced platelets are limited. If an open system is used,
the maximum allowable storage time is 4 hours. The maximum allowable storage time has not been established
for closed systems.
QUARANTINE
All units of blood collected should be immediately placed in quarantine in a designated area until donor
information and donation records
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AABB TECHNICAL MANUAL
have been reviewed, the current donor information has been compared to the previous information, the
donor’s previous deferrals have been examined, and all laboratory testing has been completed.87 Because of the
limited amount of time after collection that is available for component separation, WB units may be separated
into components before all of the earlier processes have been completed. Separated components are quarantined
at the appropriate temperature until all of the suitability steps have been completed and reviewed. Often,
physical and electronic quarantine are used simultaneously.
Certain blood components from previous donations by donors whose more recent donations test positive for
infectious disease also require quarantine and appropriate disposition, as do units identified as unsuitable for
transfusion because of postdonation information. Other components may need to be quarantined so that the QC
samples can be taken and analyzed. For instance, if a sample is obtained for bacterial contamination testing, the
component is held in quarantine for some preset amount of time and then released if the test results are negative.
A thorough understanding of the quarantine process is needed to prevent erroneous release of unsuitable
blood components. Components may be removed from the quarantine area, labeled, and released for
distribution if all of the donor information, previous donor records, and current test results are satisfactory.
Some nonconforming autologous blood components may be released for autologous use only.
Some blood components require emergency release because they have a very short storage time. Such is the
case for granulocytes. Emergency release requires physician approval and a label or tie tag to indicate that
testing was incomplete at the time of release.
Despite the widespread use of software to control manufacturing processes, instances of failure of
quarantine and release of unsuitable products continue to be reported to the FDA. For instance, during fiscal
year 2011, the FDA received 54,947 reports of blood and plasma
product deviations. Of these reports, 4258 (7.7%) involved QC and distribution errors.88
LABELING
Blood component labeling is a highly regulated activity, and the documents that describe the requirements
are listed below. Readers are advised to consult these documents and specific national regulations for details.
The FDA requirements for labeling of blood and components are detailed in the Guidelines for Uniform
Labeling of Blood and Blood Components published in 1985.3 The FDA approved the International Society of
Blood Transfusion (ISBT) 128 symbology, Version 1.2.0, in 2000 and Version 2.0.0 in 2006.89 Detailed
requirements are described in the Code of Federal Regulations (Title 21, CFR Parts 606.120, 606.121, and
606.122). AABB Standards requires that accredited facilities use ISBT 128 labels.4(pl2)
The FDA rule that requires all blood components to be labeled with a bar-coded label became effective on
April 26,2006. The rule requires that, at a minimum, the label contain the following bar-coded information: 1)
the unique facility identifier (eg, registration number), 2) lot number relating to the donor, 3) product code, and
4) ABO group and Rh type of the donor. These pieces of information must be present in eye-readable and
machine-readable format. The rule applies to blood centers that collect and prepare blood components. The rule
also applies to hospital transfusion services that prepare pooled cryoprecipitate and/or prepare divided units or
aliquots of RBCs, platelets, and plasma for pediatric use.
 Another major part of labeling in the United States is the information circular. The circular must be made
available to everyone involved in the transfusion of blood components. The Circular of Information for the Use
of Human Blood and Blood Components is produced by AABB, America’s Blood Centers, the American Red
Cross, and the Armed Services Blood Program and is recognized as acceptable by the FDA.49 The circular
provides important information about each blood compo
159
nent and should be consulted for information not included in this chapter.
Special message labels may also be affixed to blood component containers. The labels may include one or
more of the following indications: 1) hold for further manufacturing, 2) for emergency use only, 3) for
autologous use only, 4) not for transfusion, 5) irradiated, 6) biohazard, 7) from a therapeutic phlebotomy, and 8)
screened for special factors [eg, HLA type or cytomegalovirus (CMV) antibody status]. ISBT 128 allows
incorporation of special attributes of the component, such as CMV antibody status.
Additional information on the container can be conveyed using a tie tag. Tie tags are especially useful for
autologous and directed donations. Tie tags include the patient’s identifying information, name of the hospital
where the patient will be admitted for surgery, date of surgery, and other information that may be helpful to the
hospital transfusion service.
Each component must also bear a unique DIN that can be traced back to the blood donor. If components are
pooled, a pool number must allow tracing to the individual units within a pool.
For groups planning to implement ISBT 128, an important source of information is ICCBBA (formerly
known as the International Council for Commonality in Blood Banking Automation). This organization’s
website features updates and a revised list of product codes.90
Immediate benefits of ISBT 128 include the following:
■ Uniform labels applied on blood components manufactured by different collection centers.
■ Better traceability of components.
■ Improved self-checking features per character.
■ Encoding of entire ASCII character set that includes alphanumeric and special characters.
■ Improved accuracy through reduction in the number of misreads during scanning of the bar-coded
information.
■ Double-density coding of numeric characters, which permits encoding of more information in a given
space.
■ Larger number of product codes, which allows more detailed descriptions of blood components.
■ Enhanced scanning, which permits more facile auditing of movements of blood components from one
location to another.
■ Ability to add information for autologous donations.
■ Ability to read more than one bar code with a single sweep (concatenation).
■ Less laborious importation of “foreign” inventory into “own” inventory via the availability of uniform
systems for DINs and product codes.
In the future, ISBT 128 is expected to permit information transfer by radiofrequency ID tags or other means
of electronic data transmission.
QC OF BLOOD COMPONENTS
A quality management system is critical for establishing a current good manufacturing practice-compliant
operation (see Chapter 1). Testing of blood components is necessary to ensure the safety, purity, and potency of
the product and verify compliance with national regulatory requirements, such as those of FDA. These
requirements are minimum standards, and an individual manufacturer may establish more stringent ones. QC
failures can serve as an indicator of unexpected suboptimal reagents or materials. Furthermore, QC data can
reveal previously unrecognized variations from validated procedures and processes. A timely detection system
provides a proactive approach to early identification and resolution of a manufacturing problem.
Equipment QC is necessary to ensure that blood components achieve desired properties in a consistent
manner. Suggested QC steps for critical equipment in component laboratories are described in Chapter 1 and are
listed in Appendix 1-3.
Limitations of QC are demonstrated, for example, in QC failures that are due to poor
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AABB TECHNICAL MANUAL
 sampling techniques and failures that may be ascribed to donor-related variables that cannot be controlled.
Examples of donor-associated variables include occult donor bacteremia or viremia and leukocyte reduction
filter failure resulting from the presence of sickled red cells.
National regulatory authorities provide specific minimum requirements for QC testing of blood products.
These may differ from country to country, and the most recent guidance documents and regulations should be
reviewed. For certain blood components, it may
be impractical to perform the QC steps. For example, the FDA and Council of Europe do not require QC of
bedside leukocyte reduction filters.
A statistical process control approach has been suggested for QC of blood components.15,91,92 Such an
approach is expected to provide a definition of product conformance to a standard with a given probability. This
approach also allows a limit to be established for nonconformance, facilitates implementation of corrective
actions, and permits QC to be individualized for different blood components.
KEY POINTS
1. Modern blood containers are composed of soft plastic and identified by a lot number. They should be
pyrogen-free and flexible yet tough and both kink- and scratch-resistant. During frozen storage, each plastic
container has a glass transition temperature below which it becomes brittle and susceptible to breakage during
transportation.
2. The initial 35 to 45 mL of blood drawn is allowed to collect into a diversion pouch at the collection
tubing. The pouch reduces bacterial contamination of collected blood by diverting bacteria from the skin that
may have otherwise entered the collection. Blood in this pouch may be used for laboratory tests.
3. The average rate of adverse donor reactions after donation is 3.5% to 4.5%. Most reactions are mild and
require no further medical care. These reactions can be systemic (eg, fainting) or local (eg, hematoma). About 1
in 3400 donors experience a reaction after leaving the donation site and may need medical care. Deferral of
low-blood-volume (less than 3.5 L) donors may be helpful in reducing the risk of reactions, especially in young
donors.
4. During centrifugation for component preparation, primary variables that affect cell separation and cell
recovery are rotor size, centrifuge speed, and duration. The platelet-rich plasma method is used in the United
States for platelet concentrate preparation, and the buffycoat method is more common in Canada and Europe.
5. LR RBCs and platelets are not to exceed 5.0 x 106 residual WBCs per transfusion dose in the United
States or 1.0 x 106 residual WBCs per transfusion dose in Europe.
6. In plasma prepared from WB (by manual or apheresis methods) and labeled as “Plasma Frozen Within 24
Hours After Phlebotomy,” the levels of all the coagulation factors are similar to those of FFP except for some
decrease in coagulation Factor VIII.
7. The radiation dose must be 25 to 50 Gy with a minimum delivered dose to any portion of the blood
components of 15 Gy in the United States. RBCs may be irradiated up until the end of their storage shelf life;
the postirradiation expiration date is 28 days after collection or the original expiration date, whichever is sooner.
The European standard requires that no part of the component receive less than 25 Gy, a maximum dose to any
part of the component of 50 Gy, and irradiation of RBCs only until day 28, with storage for no longer than 14
days after irradiation or 28 days after collection, whichever is earliest. Platelet shelf life is not reduced after
irradiation.
8. Bar-coded and eye-readable container labels are increasingly using the ISBT symbology (ISBT 128).
ISBT 128 offers a number of advantages over the Codabar symbology, including identification of the
manufacturer throughout the world, more product codes, better accu
 161
racy as a result of reduced misreads during scanning, and enhanced conveyance of other labeling
information.
9. Various approaches for QC of blood components have been promulgated by the AABB, Council of
Europe, and FDA.
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pooled filtered PLT concentrates in plasma. Transfusion 2004;44:202-9.
29. Levin E, Culibrk B, Gyongyossy-Issa MI, et al. Implementation of buffy coat platelet component
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red cell 2,3-DPG and the accumulation of lactate. Transfusion 1999;39:4927.
32. Reiss RR Katz AJ. Optimizing recovery of platelets in platelet-rich plasma by the Simplex strategy.
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33. Welch M, Champion AB. The effect of temperature and mode of agitation on the resuspension of
platelets during preparation of platelet concentrates. Transfusion 1985;25:283-5.
34. Berseus O, Hogman CF, Johansson A. Simple method of improving the quality of platelet concentrates
and the importance of production control. Transfusion 1978;18:333-8.
35. Nessen SC, Eastridge BJ, Cronk D, et al. Fresh whole blood use by forward surgical teams in
Afghanistan is associated with improved survival compared to component therapy without platelets.
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36. European Union. Commission Directive 2004/ 33/EC of 22 March 2004 implementing Directive
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and blood components 30.3.2004. EUR-Lex 2004;91:25-39. [Available at http://eur-lex.europa.eu/Lex
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37. Hogman CF, Meryman HT. Red blood cells intended for transfusion: Quality criteria revisited.
Transfusion 2006;46:137-42.
38. Janatpour KA, Paglieroni TG, Crocker VL, et al. Visual assessment of hemolysis in red blood
163
cell units and segments can be deceptive. Transfusion 2004;44:984-9.
39. Kim DM, Brecher ME, Bland LA, et al. Visual identification of bacterially contaminated red cells.
Transfusion 1992;32:221-5.
40. Holme S, Heaton WA, Moroff G. Evaluation of platelet concentrates stored for 5 days with reduced
plasma volume. Transfusion 1994; 34:39-43.
41. Ali AM, Warkentin TE, Bardossy L, et al. Platelet concentrates stored for 5 days in a reduced volume of
plasma maintain hemostatic function and viability. Transfusion 1994;34:447.
42. Murphy S, Gardner FH. Platelet preservation. Effect of storage temperature on maintenance of platelet
viability—Deleterious effect of refrigerated storage. N Engl J Med 1969;280: 1094-8.
43. Dumont LJ, AuBuchon JP, Gulliksson H, et al. In vitro pH effects on in vivo recovery and survival of
platelets: An analysis by the BEST Collaborative. Transfusion 2006;46:1300-5.
44. Bertolini F, Murphy S, Rebulla P, Sirchia G. Role of acetate during platelet storage in a synthetic
medium. Transfusion 1992;32:152-6.
45. Dumont LJ, Gulliksson H, van der Meer PF, et al. Interruption of agitation of platelet concentrates: A
multicenter in vitro study by the BEST Collaborative on the effects of shipping platelets. Transfusion
2007;47:1666-73.
46. MoroffG, George VM. The maintenance of platelet properties upon limited discontinuation of agitation
during storage. Transfusion 1990; 30:427-30.
47. Gottschall JL, Rzad L, Aster RH. Studies of the minimum temperature at which human platelets can be
stored with full maintenance of viability. Transfusion 1986;26:460-2.
48. Moroff G, Holme S, George VM, Heaton WA. Effect on platelet properties exposure to temperatures
below 20 degrees C for short periods during storage at 20 to 24 degrees C. Transfusion 1994;34:317-21.
49. Food and Drug Administration. Guidance for industry: An acceptable circular of information for the use
of human blood and blood components. (August 2013) Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 2013. [Available at http:// www.fda.gov/BiologicsBloodVaccines/Guid
anceComplianceRegulatorylnformation/ Guidances/Blood/ucm364565.htm (accessed August 16,2013).]
50. AABB. TRALI risk mitigation for plasma and whole blood for transfusion. Association Bulletin #14-02.
Bethesda, MD, AABB, 2014.
51. Roth WK. Quarantine Plasma: Quo vadis? Transfus Med Hemother 2010;37:118-22.
52. Smak Gregoor PJH, Harvey MS, Briet E, Brand A. Coagulation parameters of CPD fresh-frozen plasma
and CPD cryoprecipitate-poor plasma after storage at 4 C for 28 days. Transfusion 1993;33:735-8.
53. Yarraton H, Lawrie AS, Mackie IJ, et al. Coagulation factor levels in cryosupernatant prepared from
plasma treated with amotosalen hydrochloride (S-59) and ultraviolet A light. Transfusion 2005;45:1453-8.
54. Larrea L, Calabuig M, Roldan V et al. The influence of riboflavin photochemistry on plasma
coagulation factors. Transfus Apher Sci 2009; 41:199-204.
55. Rock G. A comparison of methods of pathogen inactivation of FFP Vox Sang 2011;100:169-78.
56. Hellstern P, Haubelt H. Manufacture and composition of fresh frozen plasma and virusinactivated
therapeutic plasma preparations: Correlation between composition and therapeutic efficacy. Thromb Res 2002;
107(Suppl l):S3-8.
57. Sharma AD, Sreeram G, Erb T, Grocott HE Solvent-detergent-treated fresh frozen plasma: A superior
alternative to standard fresh frozen plasma? J Cardiothorac Vase Anesth 2000;14:712-17.
58. Murphy K, O'Brien P, O’Donnell J. Acquired protein S deficiency in thrombotic thrombocytopenic
purpura patients receiving solvent/detergent plasma exchange. Br J Haematol 2003;122:518-19.
59. Food and Drug Administration. Octaplas. Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 2013. [Available at http://www.fda.gov/BiologicsBloodVac
cines/BloodBloodProducts/ApprovedProd ucts/LicensedProductsBLAs/ucm336140.htm (accessed August
12,2013).]
60. Ness PM, Perkins HA. Fibrinogen in cryoprecipitate and its relationship to factor VIII (AHF) levels.
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61. Callum JL, Karkouti K, Yulia L. Cryoprecipitate: The current state of knowledge. Transfus Med Rev
2009;23:177-88.
62. Farrugia A, Prowse C. Studies on the procurement of blood coagulation factor VIII: Effects of plasma
freezing rate and storage conditions on
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cryoprecipitate quality. J Clin Pathol 1985; 122:686-92.
63. Scott EA, Puca KE, Pietz BC, et al. Analysis of ADAMTS13 activity in plasma products using a
modified FRETS-VWF73 assay (abstract). Blood 2005; 106(Suppl):165a.
64. Smith JK, Bowell PJ, Bidwell E, Gunson HH. Anti-A haemagglutinins in factor VIII concentrates. J
Clin Pathol 1980;33:954-7.
65. Pesquera-Lepatan LM, Hernandez FG, Lim RD, Chua MN. Thawed cryoprecipitate stored for 6 h at
room temperature: A potential alternative to factor VIII concentrate for continuous infusion. Haemophilia
2004;10:6848.
66. Hoffman M, Koepke JA, Widmann FK. Fibrinogen content of low-volume cryoprecipitate. Transfusion
1987;27:356-8.
67. Schuetz AN, Hillyer KL, Roback JD, Hillyer CD. Leukoreduction filtration of blood with sickle cell
trait. Transfus Med Rev 2004;18:168-76.
68. Dzik S, Moroff G, Dumont L. A multicenter study evaluating three methods for counting residual WBCs
in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorimetry.
Transfusion 2000;40: 513-20.
69. Whitley PH, Wellington M, Sawyer S, et al. A simple, new technology for counting low levels of white
blood cells in blood components: Comparison to current methods. Transfusion 2012;52(Suppl):59A.
70. Valeri CR, Ragno G, Pivacek LE, et al. A multicenter study of in vitro and in vivo values in human
RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4°C in AS-3:
Assessment of RBC processing in the ACP 215. Transfusion 2001;41: 933-9.
71. Valeri CR, Pivacek LE, Cassidy GP Ragno G. The survival, function, and hemolysis of human RBCs
stored at 4°C in additive solution (AS1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen,
thawed, washed, and stored at 4°C in sodium chloride and glucose solution for 24 hours. Transfusion
2000;40:1341-5.
72. Alving BM, Reid TJ, Fratantoni JC, Finlayson JS. Frozen platelets and platelet substitutes in transfusion
medicine. Transfusion 1997;37: 866-76.
73. Lee DH, Blajchman MA. Novel platelet products and substitutes. Transfus Med Rev 1998; 12:175-87.
74. Dumont LJ, Cancelas JA, Dumont DF, et al. A randomized controlled trial evaluating recovery and
survival of 6% dimethyl sulfoxide-frozen autologous platelets in healthy volunteers. Transfusion 2013;53:128-
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75. Lelkens CC, Koning JG, de Kort B, et al. Experiences with frozen blood products in the Netherlands
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76. Nuclear Regulatory Commission. Holders of material licenses authorized to possess radioactive material
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77. Moroff G, Leitman SF, Luban NLC. Principles of blood irradiation, dose validation, and quality control.
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78. Voak D, Chapman J, Finney RD, et al. Guidelines on gamma irradiation of blood components for the
prevention of transfusion-associated graft-versus-host disease. Transfus Med 1996;6:261-71.
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80. Moroff G, Friedman A, Robkin-Kline L, et al. Reduction of the volume of stored platelet concentrates
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81. Pisciotto P, Snyder EL, Napychank PA, Hopper SM. In vitro characteristics of volumereduced platelet
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83. Dumont LJ, Krailadsiri R Seghatchian J, et al. Preparation and storage characteristics of white-cell-
reduced high-concentration platelet concentrates collected by an apheresis system for transfusion in utero.
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concentration platelet concentrates. Transfusion 2002;42:1333-9.
85. Schoenfeld H, Muhm M, Doepfmer UR, et al. The functional integrity of platelets in vol
CHAPTER 6 Whole-Blood Collection and Component Processing
165
ume-reduced platelet concentrates. Anesth Analg 2005;100:78-81.
86. Ringwald J, Walz S, Zimmerman R, et al. Hyperconcentrated platelets stored in additive solution:
Aspects of productivity and in vitro quality. Vox Sang 2005;89:11-18.
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Fiscal Year 2012. Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 2013.
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blood and blood components using ISBT 128. Version 2.0.0, November 2005. Silver Spring, MD: CBER Office
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of leukoreduced components: Report of the BEST working party of the ISBT. Transfusion 1996;36:11-20.
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and blood components intended for transfusion. Silver Spring, MD: CBER Office of Communication, Outreach,
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ceComplianceRegulatorylnformation/Guid ances/Blood/UCM320641.pdf (accessed November 12,2013).]

Chapter 7
Blood Component Collection by Apheresis
*
James W. Smith, MD, PhD
MR “APHERESIS,” “PHERESIS,” “hemaMipheresis,” and all of the various terms used to refer to
automated blood component collection procedures are derived from the Greek word “aphairos,” meaning “to
take from.” Specifically, whole blood is separated into components during collection, the desired component is
removed/modified, and the remaining components are returned to the donor or patient. Centrifugal and
membranebased apheresis techniques were under development in the late 19th and early 20th centuries. By the
1970s, multiple improved technologies had emerged and apheresis processes advanced rapidly.
Today, centrifugal technique is primarily used in the United States, whereas membrane filtration is used in
other parts of the world (primarily Europe and Japan) for donor apheresis.
Early versions of automated, computerized, and centrifugal techniques facilitated large-scale donations of
platelets, plasma, and granulocytes. As the technology continued to evolve, equipment, disposables, and
software became increasingly sophisticated, and now
the collection of various combinations of components is possible (see Table 7-1). This chapter provides a
discussion of the technology and instrumentation used during donor apheresis, with special consideration given
to the associated regulatory requirements.
COMPONENT COLLECTION
The collection of components by apheresis follows many of the same rules and guidelines that apply to
whole-blood donation. Like whole-blood donors, apheresis donors must be given sufficient information to
enable them to give informed consent to donate blood. Although the apheresis collection and preparation
processes differ from those used for whole-blood-derived components, the storage and transportation
requirements and several quality-control steps are the same for both processes. Another similarity is that the
facility must maintain written procedures and protocols for all types of collections used and must keep records
of each procedure as required by AABB Standards for Blood Banks and Transfu

James W. Smith, MD, PhD, Medical Director, Oklahoma Blood Institute, Oklahoma City, Oklahoma The
author has disclosed no conflicts of interest.
167

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AABB TECHNICAL MANUAL
TABLE 7-1. Components that Can Be Collected with Various Instruments
Instrument GRAN PLT cRBC 2-RBC PLASMA cPLASMA
Fenwal ALYX X X X
Fenwal Amicus X X X
Fenwal Autopheresis G X
Fresenius AS104 X
TerumoBCT (COBE) Spectra X X X
TerumoBCT Spectra Optia X
TerumoBCT Trima V-4 X X X X
TerumoBCT Trima Accel X X X X
Haemonetics Cymbal X
Haemonetics MCS+ LN9000 X X X
Haemonetics MCS+ LN8150 X X X
Haemonetics PCS-2 X
‘Concurrent collection refers to the ability to collect more than one type of product.
GRAN = granulocytes; PLT = plateletpheresis (single, double, triple); cRBC = concurrent* 1 unit of Red
Blood Cells (RBCs); 2-RBC = double unit of RBCs; PLASMA = 1 unit of plasma; cPLASMA = concurrent
plasma; V-4 = software version 4.
sion Services.'1^'2'67' The circumstances that are unique to apheresis collection are addressed in the sections
that follow.
Platelets
Apheresis is used to obtain platelets from volunteer donors, patients’ family members, or donors with HLA
or platelet-antigen-compatible phenotypes. By design, apheresis procedures are intended to collect large
numbers of platelets from an individual, thereby providing a more potent product with fewer donor exposures
for the patient. AABB Standards requires that an apheresis platelet component contain at least 3 x 1011 platelets
in 90% of sampled units.1(pp28'29)
 With newer technology and more efficient processes, higher yields of platelets may be obtained from one
donor, and the original apheresis unit may be split into multiple units,
each of which must meet minimum standards. Some instruments are programmed to calculate the platelet
yield based on the donor’s hematocrit, platelet count, height, and weight.
For alloimmunized patients who do not respond to random allogeneic platelets, transfusions of platelets
from an apheresis donor selected on the basis of a compatible platelet crossmatch or that are matched for HLA
antigens may be the only way to achieve a satisfactory posttransfusion platelet increment. In the United States,
the use of apheresis platelets has been steadily increasing over the past 25 years. It is estimated that 90% of
platelets transfused in the United States are apheresis platelets.2
Donor Selection and Monitoring
Plateletpheresis donors may donate more frequently than whole-blood donors but must

169
meet all of the other criteria for whole-blood donation. The interval between donations should be at least 2
days, and donors should not undergo plateletpheresis more than twice in a week or 24 times in a rolling 12-
month period.1(p201,3 If the donor donates a unit of whole blood or if it becomes impossible to return the
donor’s red cells during plateletpheresis, at least 8 weeks should elapse before a subsequent plateletpheresis
procedure unless the extracorporeal red cell volume (ECV) is less than 100 mL. Platelets may be collected from
donors who do not meet these requirements only if the component is expected to be of particular value to a
specific intended recipient and a physician certifies in writing that the donor’s health will not be compromised
by the donation. Donors who have taken antiplatelet medications that irreversibly inhibit platelet function are
deferred for specific intervals before donation (48 hours for aspirin/aspirincontaining medications and
piroxicam, 14 days for clopidogrel and ticlopidine) because apheresis platelets are often the sole source of
platelets given to a patient.1(p61)'3
A platelet count is not required before the first apheresis collection; however, triple collections of platelets
may not be drawn from first-time donors unless a qualifying platelet count is obtained on a sample collected
before the procedure.3 If the donation interval is less than 4 weeks, many facilities prefer that the donor’s
platelet count be above 150,000/pL before plateletpheresis occurs to prevent a postdonation count of less than
100,000/pL. AABB Standards permits qualification of a donor with a platelet count from a sample collected
immediately before the procedure or one obtained either before or after the previous procedure.1(p21)
Exceptions to these laboratory criteria should be approved in writing by the apheresis program physician based
on documented medical need.
The Food and Drug Administration (FDA) specifies that the total volume of plasma collected should be no
more than 500 mL (or 600 mL for donors weighing more than 175 lb) or the volume described in the labeling of
the automated blood cell separator device (which may be more or less than the 500-mL or 600
mL volume previously mentioned). The platelet count of each unit should be kept on record but need not be
written on the component label. Units containing less than 3.0 x 1011 platelets should be labeled with the actual
platelet count.3
 It is possible to collect plasma concurrendy with platelets. Such collection is discussed in more detail in the
“Plasma” section below.
Vasovagal and hypovolemic reactions are rare in apheresis donors but may occur. Paresthesias (tingling
sensations) and other reactions to citrate anticoagulant are not uncommon. (Similar citrate toxicity reactions in
recipients are discussed along with whole blood transfusions in Chapter 27.) Serious reactions occur less often
among apheresis donors than whole blood donors.4
Laboratory Testing
Tests for ABO group, Rh type, unexpected alloantibodies, and transfusion-transmitted diseases must be
performed by the collecting facility in the same manner as for other blood components. Each unit must be tested
unless the donor is undergoing repeated procedures to support a specific patient, in which case testing for
infectious disease markers needs to be repeated only at 30-day intervals.5
If red cells are visible in a product, the hematocrit should be determined. AABB Standards state that if the
component contains more than 2 mL of red cells, the red cells must be ABO compatible with the recipient’s
plasma and be crossmatched.1(pp37'38) In such cases, a sample of donor blood is attached to the container for
compatibility testing. In some instances, it may be desirable for the donor plasma to be ABO compatible with
the recipient’s red cells (eg, if the recipient is a child or an ABO-mismatched allogeneic progenitor cell
transplant recipient). In the United States, to be considered leukocyte reduced, apheresis platelets must contain
less than 5 x 106 leukocytes per unit and platelets must meet the specifications of the apheresis device
manufacturer.3 In Europe, the guideline for
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AABB TECHNICAL MANUAL
leukocyte-reduced components is fewer than 1 x 106 leukocytes per unit.
Record-Keeping
Complete records must be kept on each procedure. All adverse reactions occurring during collection
procedures (or transfusion) must be documented along with the results of thorough investigations. Records of
all laboratory findings and collection data must be periodically reviewed by a knowledgeable physician and
must be found to be within acceptable limits. FDA guidelines require a periodic review of donor records to
monitor platelet counts.3 Facilities must have policies and procedures in place to ensure that donor red cell loss
during each procedure does not exceed acceptable limits.1(pl8)
Plasma
Apheresis devices can be used to collect plasma for transfusion or as Source Plasma for subsequent
manufacturing. Recently, the FDA approved apheresis devices for the collection of plasma held at 1 to 6 C
within 8 hours and frozen within 24 hours after phlebotomy and plasma held at room temperature for up to 24
hours and frozen within 24 hours after phlebotomy.
The FDA has provided guidance with regard to the volume of plasma that may be collected using automated
devices. A distinction is made between infrequent plasmapheresis, in which the donor undergoes
plasmapheresis no more frequently than once every 4 weeks, and serial plasmapheresis (or Source Plasma
collection, the process to collect plasma for fractionation into plasma components), in which the donation is
more frequent than once every 4 weeks. For donors in infrequent plasmapheresis programs, donor selection and
monitoring requirements are the same as those for whole-blood donation. Plasma obtained by these processes is
intended for direct transfusion.
For serial plasma (Source Plasma) collection using either automated instruments or manual techniques, the
following principles apply6:
1. Donors must give consent for the procedure, and they must be observed closely during the procedure.
Emergency medical care must always be available.
2. Red-cell losses related to the procedure, including samples collected for testing, should be monitored so
that no more than 200 mL of red cells are removed in 8 weeks. If the donor’s red cells cannot be returned during
an apheresis procedure, hemapheresis or whole-blood donation should be deferred for 8 weeks.
3. For manual collection systems, a mechanism must exist to ensure safe reinfusion of the autologous red
cells.
4. In manual procedures for donors weighing 110 to 175 lb, no more than 500 mL of whole blood should be
removed at one time or no more than 1000 mL during a session or within a 48-hour period. The limits for
donors who weigh > 175 lb are 600 mL and 1200 mL, respectively. For automated procedures, the allowable
volume has been determined for each instrument by the FDA.
 5. At least 48 hours should elapse between successive procedures. Donors should not undergo more than
two procedures within a 7-day period.
6. At the time of initial plasmapheresis and at 4-month intervals for donors undergoing serial (large-volume)
plasmapheresis (donors undergoing plasmapheresis more often than once every 4 weeks), serum or plasma must
be tested for total protein and for serum protein electrophoresis or for quantitative immunoglobulins. Results
must be within normal limits.
7. A qualified licensed physician, knowledgeable about all aspects of hemapheresis, must be responsible for
the program.
For manual collection systems, a process that is rarely used in the United States at present, requirements are
outlined in the Code of Federal Regulations6 and have been summarized in previous editions of this Technical
Manual.
171
Red Cells and Multicomponent Donations
Both AABB Standards and FDA guidance documents address the removal of red cells by automated
apheresis methods. A guidance document issued in 2001 by the FDA finalized recommendations for the use of
automated apheresis equipment to collect the following7:
■ Single units of RBCs and plasma.
■ Single units of RBCs and platelets.
■ Single units of RBCs, platelets, and plasma.
■ Double units of RBCs only.
The guidance document includes FDA regulations requiring that equipment perform and be used in the
manner for which it was designed to collect or process blood and components. Standard operating procedures,
including device manufacturers’ instructions for use and maintenance of current records, are described. The
sections below summarize the information in the guidance document.
Donor Selection and Monitoring
The FDA requires that an adequate hemoglobin level be determined by a quantitative method for
predonation hemoglobin or the hematocrit of donors undergoing double-RBC collection. The procedure is
limited to persons who are larger and have a higher hematocrit than the minimum standards for whole-blood
donations. For males, the minimum weight is 130 lb, and the minimum height is 5' 1". For females, the
minimum weight is 150 lb, and the minimum height is 5' 5". The minimum hematocrit is 40% for both genders.
Donors who are shorter than the minimum height or who weigh less than the minimum weight, as established
by the FDA in device operator manuals, should be further evaluated. These donors must also meet all relevant
FDA criteria for allogeneic or autologous whole-blood donation.
Donors who have given a single unit of RBCs with platelets, plasma, or both should be deferred for at least
8 weeks. The exception is when a donor serves as a plateletpheresis donor or a donor of platelets with plasma
by
products within 8 weeks and the ECV of the procedure is <100 mL. Donors should be deferred for at least
16 weeks after a double-RBC donation. If an apheresis procedure is discontinued before completion and
absolute red cell loss is <200 mL, the donor may donate again within 8 weeks if he or she meets all donor
eligibility criteria.
If a donor has a second red cell loss of <100 mL during a subsequent donation within 8 weeks of a previous
donation, the donor should be deferred for 8 weeks. If the total absolute red cell loss within 8 weeks is >300
mL, the donor should be deferred for 16 weeks from the date of the last red cell loss. If an apheresis procedure
is discontinued and the absolute red cell loss is >200 mL but <300 mL, the donor should be deferred for 8
weeks. If an apheresis procedure is discontinued and total absolute red cell loss is >300 mL, the donor should be
deferred for 16 weeks.
Saline infusion is used to minimize volume depletion.
Quality-Control Issues
The FDA has promulgated quality-control (QC) programs for RBC unit collection by apheresis. There are
two phases:
■ In Phase I QC, 100 consecutive RBC units are tested to determine the expected or target red cell volume
in accordance with the specifications in the device operator’s manual. Target values are compared with the
actual values to determine product acceptability. If the QC results are satisfactory, which includes that at least
95% of the units meet product specifications, the establishment may proceed to Phase II.
 ■ Phase II QC consists of monthly testing of a representative sample of 50 units of the manufactured
product from each collection center. At least 1 unit from a single RBC protocol or both units from a doubleRBC
protocol device used at the center should be included in the testing. At least 95% of the products tested should
meet product specifications as described in the device operator’s manual.
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AABB TECHNICAL MANUAL
Record Requirements
US blood establishments must update their blood establishment registrations and product listing forms with
the FDA to collect RBCs using automated methods. Automated RBC collection has substantial potential to have
an adverse effect on the identity, strength, quality, purity, or potency of a product. Blood establishments that are
approved to manufacture RBCs with one manufacturer’s device and that want to use another manufacturer’s
device instead must submit a prior approval supplement and must receive FDA approval before distribution of
the product manufactured on the new device. The FDA requires these establishments to make available a
number of records and forms regarding RBC or multicomponent unit collection for FDA inspection. These
records and forms include documents addressing donor consent, donor eligibility, product collection, and
product QC.7
Granulocytes
The use of granulocyte transfusions has been controversial for a number of years. Analysis of randomized
controlled trials of granulocyte transfusions in adults has indicated that an acceptable minimum dose (>1 x 1010
granulocytes/day) and crossmatch compatibility (no recipient antibodies to granulocyte antigens have a major
impact on the effectiveness of such transfusions.8 Recently, there has been renewed interest in granulocyte
transfusion therapy because much larger cell doses may be obtained from donors who have received
recombinant colony-stimulating factors.
Agents Administered to Increase Yields
AABB Standards requires that 75% of granulocyte components contain at least 1 x 1010 granulocytes,
1(p30) although the optimal therapeutic dose in adult patients is unknown. For infants and children, a dose of 10
to 15 mL/kg may provide an adequate number of granulocytes per dose. To collect this number of cells in a unit
of granulocytes, one must administer drugs or sedimenting materials to the donor. Sedimenting agents enhance
granulocyte har
vest by increasing sedimentation of the RBCs, thereby enhancing the interface in the collection device and
resulting in minimal RBC content in the final product. The donor’s consent to the procedure must provide
permission for any of these drugs or sedimenting agents to be used.
HYDROXYETHYL STARCH. A common sedimenting agent, hydroxyethyl starch (HES) causes red cells
to aggregate, thereby sedimenting them more completely. Because HES can be detected in donors as long as a
year after infusion, AABB Standards requires facilities performing granulocyte collections to have a process to
control the maximum cumulative dose of any sedimenting agent administered to a donor within a given
interval.1(p23) HES is a colloid that acts as a volume expander. Donors who receive HES may experience
headaches or peripheral edema because of expanded circulatory volume. A boxed warning from the FDA exists
for its use due to increased mortality and severe renal injury in certain patient populations.9
CORTICOSTEROIDS. Corticosteroids can double the number of circulating granulocytes by mobilizing
granulocytes from the marginal pool. The common protocol is to use 60 mg of oral prednisone in a single or
divided dose before donation to collect large numbers of granulocytes with minimal systemic steroid activity.
Another protocol uses 8 mg of oral dexamethasone. Donors should be questioned about their relevant medical
history before they use systemic corticosteroids. Hypertension, diabetes, cataracts, or peptic ulcer can be
relative or absolute contraindications to corticosteroid use.
GROWTH FACTORS. Although not a licensed indication, granulocyte colony-stimulating factor (G-CSF)
can effectively increase granulocyte yields. Hematopoietic growth factors given alone can result in the
collection of up to 4 to 8 x 1010 granulocytes per apheresis procedure. Typical doses of G-CSF are 5 to 10
pg/kg given 8 to 12 hours before granulocyte collection. Preliminary evidence suggests that invivo recovery and
survival of these granulo
173
cytes are excellent and that growth factors are well tolerated by donors.
Laboratory Testing
 Testing for ABO and Rh group, red cell antibodies, and infectious disease markers is required on a sample
drawn at the time of phlebotomy. Red cell content in granulocyte products is inevitable; the red cells should be
ABO compatible with the recipient’s plasma. If >2 mL of red cells are present, the component should be
crossmatched for Rh compatibility and HLA compatibility, 1(pp37‘38)
Storage and Infusion
Granulocyte function deteriorates rapidly during storage, and concentrates should be transfused as soon as
possible after preparation. AABB Standards mandates a storage temperature of 20 to 24 C for no longer than 24
hours.1(p55) Agitation during storage is undesirable. Irradiation is required for products to be administered to
immunodeficient recipients and is indicated for nearly all recipients because their primary diseases are likely to
involve deficiencies in their immune systems. Use of a microaggregate or leukocyte reduction filter is
contraindicated because it removes the collected granulocytes.
INSTRUMENTS AND SYSTEMS FOR DONOR APHERESIS COLLECTIONS
This section provides descriptions of equipment that is available for use in the United States for the
collection of blood components by automated techniques. A brief description is given of each instrument; more
detailed information can be found in other resources.10,11 This section does not include the CS3000 and
CS3000+ (Fenwal, Lake Zurich, IL) because the company has discontinued the sale of these devices in the
United States (although disposables for the devices are still available at the time of this writing). These two
devices are capable of collecting RBCs, platelets, concurrent plasma, and granulocytes.
Plasma
Fenwal Autopheresis C
The Autopheresis C (Fenwal) is an instrument designed to collect plasma only.11 It uses a rotating
cylindrical filter to separate the plasma from the cellular elements of blood. Because of the high efficiency of
the rotating filter, the filter is small and the system’s ECV is approximately 200 mL. The Autopheresis C is a
single-access system, and saline replacement can be administered. It is considered an open system and can
collect several units of plasma. According to the FDA definition, an open system requires that plasma outdates
4 hours after thawing. Variances can be granted that allow 24-hour outdates, but these units cannot be relabeled
as Thawed Plasma.
Haemonetics PCS-2
The PCS-2 (Haemonetics, Braintree, MA), a simplified version of the MCS Plus, is designed for plasma
collection.12 The PCS-2 uses a blowmolded (grenade-shaped) centrifuge bowl to separate plasma from cellular
elements. Depending on the degree of cell reduction required, one of three versions of the PCS-2 bowl can be
used: standard, filter core, or high separation core. The standard bowl uses centrifugal force to remove the
plasma from the top of the bowl. To increase cell reduction, the filter core and high separation core bowls allow
plasma to pass through the core, which is covered with a filter membrane.1315 Use of this device results in the
greatest cell reduction, but it has not been released in the United States at the time of this writing. The ECV of
the PCS-2 is variable depending on the hematocrit of the donor and ranges from 491 mL (38% hematocrit) to
385 mL (50% hematocrit). The PCS-2 is a single-access system, and saline replacement can be administered. It
is considered an open system. As noted for the Auto-C, a variance can be granted allowing 24-hour outdates of
plasma processed with this device, but these units may not be relabeled as Thawed Plasma. The PCS-2 can
collect several units of plasma at a time.
174
AABB TECHNICAL MANUAL
Equipment for Concurrent Plasma Collection
Plasma can be collected as a concurrent product during collection of apheresis platelets or automated RBCs.
The amount that can be collected is determined by the volume of platelets, red cells, or both, that are being
collected and by the maximum volume that can be removed from the donor. Equipment capable of concurrent
plasma collection includes Haemonetics MCS+ LN9000, Haemonetics MCS+ LN8150, Fenwal Amicus,
Fenwal ALYX, TerumoBCT (COBE) Spectra (TerumoBCT, Lakewood, CO), TerumoBCT Trima, and
TerumoBCT Trima Accel.
Platelets
TerumoBCT (COBE) Spectra
The TerumoBCT (COBE) Spectra is capable of collecting single, double, or triple apheresis platelet units as
well as concurrent plasma, depending on the size and platelet count of the donor.10,16'19 This device uses a
dual-stage (different radius) channel to collect leukocytereduced platelets. Leukocyte reduction to <5 x 106
White Blood Cells (WBCs) can be obtained in approximately 85% of the collections for software versions that
are lower than 5.0.20Use of software versions 5.0 and 7.0 can more consistently result in the collection of
products containing <1.0 x 106 WBCs with the leukocytereduction system (LRS), which uses a cone in the
centrifuge and saturated, fluidized, particlebed filter technology to remove residual WBCs leaving the second
stage of the channel.10,16'19 The Spectra is capable of single- or doubleaccess procedures. The ECV of the
singleaccess kit is 361 mL and of the double-access kit is 272 mL. The Spectra is being replaced by the more
efficient Trima or Trima Accel.11
TerumoBCT Trima and Trima Accel
The TerumoBCT Trimas were designed as automated donor collection machines for platelets, plasma, and
RBCs only. The TerumoBCT Trima (Version 4) uses a smaller, mod
ified, dual-stage channel and LRS cone to consistently collect leukocyte-reduced platelets (<1.0 x
106WBCs). To increase platelet yields, the Trima Accel (Versions 5.0, 5.01, and 5.1) uses a single-stage,
doughnut-shaped channel and larger LRS cone to consistently collect leukocyte-reduced platelets.21 The
Trimas use single-access kits only. The ECV of the Trimas is 182 to 196 mL. They are capable of collecting
single, double, or triple units of apheresis platelets as well as concurrent plasma and RBCs, depending on donor
size, platelet count, and hematocrit.21'23
Fenwal Amicus
The Amicus is capable of collecting single, double, or triple apheresis platelets as well as concurrent plasma
and RBCs (single-access kit only), depending on the donor’s size, platelet count, and hematocrit.10,16,22,24
The Amicus uses centrifugal force and a double compartment belt wrapped around a spool to separate the
platelets. The platelets accumulate in the collection chamber and are transferred to the final collection bags at
the end of the procedure. The Amicus is capable of single- or double-access procedures. The ECV of the
double-access kit is 210 mL, and that of the single-access kit is 209 mL. The ECV of the whole-blood bag is
adjustable. Consistent leukocyte reduction is accomplished without external filtration, and this process is
approved by the FDA for submission of a prior approval supplement.
Haemonetics MCS+ LN9000
The Haemonetics MCS+ LN9000 system is capable of conducting several different apheresis procedures,
including apheresis platelet collection. It uses the Latham conical bowl, plasma-controlled hematocrit, and
plasma surge technique to build up the platelets and float them off the bowl with rapid plasma infusion.
Although this technique results in leukocytereduced platelets, the use of an inline leukocyte reduction filter
ensures consistency in leukocyte reduction.10,25'28 The LN9000 uses a single-access kit, and the ECV ranges
from 480 mL (38% hematocrit) to 359 mL (52% he
175
matocrit). The LN9000 is capable of collecting single, double, or triple units of apheresis platelets as well as
concurrent plasma, depending on donor size and platelet count.10,25-28
RBCs
TerumoBCT Trima and Trima Accel
As previously mentioned, the Trima can collect a single unit of RBCs concurrently with platelets.11,23,29 A
kit is available to collect a double unit of RBCs with or without concurrent plasma, depending on the donor’s
size and hematocrit. The Trima uses the singlestage channel for the double-RBC collection, and saline is
returned to the donor during the collection. The addition of the preservative solution and leukocyte reduction by
filtration are performed manually and off-line after the collection is complete for both the single and double
units of RBCs.
FenwalALYX
The Fenwal ALYX was developed as an automated, double-RBC collection system only. Recently, it has
been approved to collect concurrent plasma as well. The ALYX uses a rigid, cylinder-shaped chamber in the
centrifuge to separate the plasma from the cells. The plasma is collected in one bag, and the red cells are
collected in a separate bag. During reinfusion, the plasma and saline are returned to the donor. When the
collection is complete, the ALYX automatically adds the preservative solution and pumps the red cells through
an inline leukocyte reduction filter into the final storage bags. The ALYX uses a single-access kit only, and its
ECV is approximately 110 mL. The ALYX is capable of collecting 2 units of RBCs or 1 unit of RBCs and
concurrent plasma at a time, depending on donor size and hematocrit.11,28-30
Fenwal Amicus
 As mentioned previously, the Fenwal Amicus can collect a single unit of RBCs concurrently with
plateletpheresis, but only with the single
access kit.11,31 The addition of the preservative solution and leukocyte reduction by filtration are
performed manually and off-line after the collection is complete.
Haemonetics Cymbal
The Cymbal is a collection system that uses an expanding, variable-volume bowl. The system has a
relatively small ECV when compared to the Haemonetics MCS+ LN8150 and collects a double-RBC unit.32
Haemonetics MCS+ LN8150
The Haemonetics MCS+ LN8150 was designed as a donor instrument only for the collection of RBCs and
concurrent plasma. The LN8150 uses the blow-molded bowl that is also used for plasma collection. It uses a
single-access kit and its ECV varies, depending on the donor’s hematocrit, from 542 mL (38% hematocrit) to
391 mL (54% hematocrit). The LN8150 is capable of collecting a single or double unit of RBCs with or without
concurrent plasma, depending on the donor’s size and hematocrit.11,29,33 The addition of preservative and
leukocyte reduction by filtration are performed manually and off-line after the collection is complete.
Granulocytes
TerumoBCT (COBE) Spectra
The Spectra is capable of collecting granulocytes.11,34,35 It uses a doughnut-shaped, singlestage channel in
the centrifuge to isolate the granulocytes that are continuously collected in the final storage bag. It uses a
double-access kit only, and its ECV is 285 mL.
TerumoBCT Spectra Optia
The Spectra Optia system (not to be confused with the Spectra system above) used mainly for therapeutic
apheresis procedures has recently been approved for granulocyte collections.36 Its ECV is 191 mL, as it is
modified from the MNC protocol.
176
AABB TECHNICAL MANUAL
Fresenius AS 104
The Fresenius AS 104 is capable of collecting several components, including granulocytes.11,37 It uses a
doughnut-shaped, singlestage channel to separate the granulocytes. The granulocytes build up in the centrifuge
and are harvested into the final collection bag intermittently. The AS 104 uses a doubleaccess kit, and its ECV
is 175 mL.
Flaemonetics MCS+ LN9000
The LN9000 can also be used to collect granulocytes. It uses the conical Latham bowl for separation, and
the huffy coat is then transferred to one of two bags. The red cells settle to the bottom of the bag before being
returned to the donor. The LN9000 switches from one bag to another to return the red cells. A single- or double-
access kit can be used for granulocyte collection, and the ECV ranges, based on the hematocrit of the donor,
from 480 mL (38% hematocrit) to 359 mL (52% hematocrit).
KEY POINTS
1. Apheresis components must meet many of the same basic regulatory requirements (eg, donor consent,
storage conditions, and transportation requirements) as whole-blood-derived components, although more
specific requirements also apply to each type of apheresiscomponent collection.
2. The majority of platelets collected and transfused in the United States are apheresis-derived platelets.
3. Plasma can be collected by apheresis for transfusion or as Source Plasma for subsequent manufacturing.
The FDA provides guidance regarding the volume of plasma that may be collected using automated devices.
4. In multicomponent donations, a variety of components or combinations of components may be collected
with apheresis technology. Regulations specific to this practice apply to donor selection and monitoring, QC,
and records.
5. Red cells can be removed concurrently with other components, or a double RBC unit may be collected.
6. Several instruments and systems have been developed and/or adapted for apheresis collection of blood
components that use different technologies. Some are appropriate for the collection of only one type of
component, and others can collect multiple types of components.
7. Granulocyte collection differs from that of other components. Specific techniques should be used and
certain factors must be taken into consideration for the optimal collection of granulocytes by apheresis.
REFERENCES
1. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
 2. US Department of Health and Human Services. The 2011 national blood collection and utilization survey
report. Washington, DC: DHHS, 2013:19.
3. Food and Drug Administration. Guidance for industry and FDA review staff: Collection of
platelets by automated methods. (December 17, 2007) Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 2012. [Available at http://www.fda.gov/Biolog
icsBloodVaccines/GuidanceComplianceRegu latorylnformation/guidances/Blood/ucm 073382.htm (accessed
December 10,2013).]
4. Wiltbank TB, Giordano GE. The safety profile of automated collections: An analysis of more

CHAPTER 7 Blood Component Collection by Apheresis

177
than 1 million collections. Transfusion 2007; 47:1002-5.
5. Code of federal regulations. Title 21, CFR Part 610.40. Washington, DC: US Government Printing Office,
2014 (revised annually).
6. Code of federal regulations. Title 21, CFR Part 640, Subpart G. Washington, DC: US Government
Printing Office, 2014 (revised annually).
7. Food and Drug Administration. Guidance for industry: Recommendations for collecting red blood cells
by automated apheresis methods. (January 30,2001) Silver Spring, MD: CBER Office of Communication,
Outreach, and Development, 2001. [Available at http://www.fda. gov/downloads/BiologicsBloodVaccines/
GuidanceComplianceRegulatorylnformation/ Guidances/Blood/ucm080764.pdf (accessed December 10,2013).]
8. Massey E, Paulus U, Doree C, Stanworth S. Granulocyte transfusions for preventing infections in patients
with neutropenia or neutrophil dysfunction. Cochrane Database Syst Rev 2009;1:CD005341.
9. Food and Drug Administration. Safety communication: Boxed warning on increased mortality and severe
renal injury, and additional warning on risk of bleeding, for use of hydroxyethyl starch solutions in some
settings. Rockville, MD: Office of Community Outreach and Development, 2013. [Available at: http://
www.fda.gov/BiologicsBloodVaccines/Safety Availability/ucm358271.htm (accessed March 30, 2014).]
10. Burgstaler EA. Current instrumentation for apheresis. In: McLeod BC, Szczepiorkowski ZM, Weinstein
R, Winters JL, eds. Apheresis: Principles and practice. 3rd ed. Bethesda, MD: AABB Press, 2010:71-110.
11. Burgstaler EA. Blood component collection by apheresis. J Clin Apher 2006;21:142-51.
 12. Hood M, Mynderup N, Doxon L. Evaluation of Haemonetics PCS-2 and Fenwal Auto-C plasmapheresis
collection systems (abstract). J ClinApher 1996;11:99.
13. Burkhardt T, Kappelsberger C, Karl M. Evaluation of a new combined centrifugation/filtration method
for the collection of plasma via plasmapheresis (abstract). Transfusion 2001; 41(Suppl):50S.
14. Burnouf T, Kappelsberger C, Frank K, Burkhardt T. Protein composition and activation markers in
plasma collected by three apheresis procedures. Transfusion 2003;43:1223-30.
15. Burnouf T, Kappelsberger C, Frank K, Burkhardt T. Residual cell content in plasma produced by three
apheresis procedures. Transfusion 2003;43:1522-6.
16. Burgstaler EA, Pineda AA, Bryant SC. Prospective comparison of plateletapheresis using four apheresis
systems on the same donors. J ClinApher 1999;14:163-70.
17. Perseghin P, Mascaretti L, Riva M, et al. Comparison of plateletapheresis concentrates produced with
Spectra LRS version 5.1 and LRS Turbo version 7.0 cell separators. Transfusion 2000;40:789-93.
18. Zingsem J, Glaser A, Weisbach V. Evaluation of a platelet apheresis technique for the preparation of
leukocyte-reduced platelet concentrates. Vox Sang 1998;74:189-92.
19. Zingsem J, Zimmermann R, Weisbach V et al. Comparison of COBE white cell-reduction and standard
plateletapheresis protocols in the same donors. Transfusion 1997;37:1045-9.
20. Maresh S, Randels M, Strauss R, et al. Comparison of plateletapheresis with a standard and an improved
collection device. Transfusion 1993;33:835-7.
21. McAteer M, Kagen L, Graminske S, et al. Trima Accel improved platelet collection efficiency with the
merging of single stage separation technology with leukoreduction performance of the LRS chamber (abstract).
Transfusion 2002;42(Suppl) :37S.
22. Burgstaler EA, Winters JL, Pineda AA. Paired comparison of Gambro Trima Accel vs Baxter Amicus
single-needle plateletapheresis. Transfusion 2004;44:1612-20.
23. Elfath MD, Whitley P, Jacobson MS, et al. Evaluation of an automated system for the collection of
packed RBCs, platelets, and plasma. Transfusion 2000;40:1214-22.
24. Yockey C, Murphy S, Eggers L, et al. Evaluation of the Amicus separator in the collection of apheresis
platelets. Transfusion 1999;38:848
25. Valbonesi M, Florio G, Ruzzenenti MR, et al. Multicomponent collection (MCC) with the latest
hemapheresis apparatuses. Int J Artif Organs 1999;22:511-15.
26. Paciorek L, Holme S, Andres M, et al. Evaluation of the continuous filtration method with double
platelet products collected on the MCS+ (abstract). J ClinApher 1998; 13:87.
27. Ford K, Thompson C, McWhorter R, et al. Evaluation of the Haemonetics MCS+ LN9000 to produce
leukoreduced platelet products (abstract). J ClinApher 1996; 11:104.
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AABB TECHNICAL MANUAL
28. Rose C, Ragusa M, Andres M, et al. Evaluation of the MCS + LN9000 in-line leukoreduction filter
(abstract). Transfusion 1996;36(Suppl):85.
29. Picker SM, Radojska SM, Gathof BS. Prospective evaluation of double RBC collection using three
different apheresis systems. Transfus Apher Sci 2006;35:197-205.
30. Snyder EL, Elfath MD, Taylor H, et al. Collection of two units of leukoreduced RBCs from a single
donation with a portable multiple-component collection system. Transfusion 2003; 43:1695-705.
31. Moog R, Frank Y Muller N. Evaluation of a concurrent multicomponent collection system for the
collection and storage of WBCreduced RBC apheresis concentrates. Transfusion 2001;41:1159-64.
32. Nussbaumer W, Grabmer C, Maurer M, et al. Evaluation of a new mobile two unit red cell apheresis
system (abstract). J Clin Apher 2006; 21:20.
33. Smith JW. Automated donations: Plasma, red cells, and multicomponent donor procedures. In: McLeod
BC, Szczepiorkowski ZM, Weinstein R, Winters JL, eds. Apheresis: Principles and practice. 3rd ed. Bethesda,
MD: AABB Press, 2010:125-40.
34. Worel N, Kurz M, Peters C, Hocker P. Serial granulocyte apheresis under daily administra
tion of rHuG-CSF: Effects on peripheral blood counts, collection efficiency, and yield. Transfusion
2001;41:390-5.
35. Dale DC, Lises WC, Llewellyn C, et al. Neutrophil transfusions: Kinetics and functions of neutrophils
mobilized with granulocyte colony-stimulating factor and dexamethasone. Transfusion 1998;38:713-21.
 36. Food and Drug Administration. Substantially equivalent 510(k) device information, BK130065
summary. TerumoBCT Spectra Optia. Silver Spring, MD: FDA, 2013. [Available at://
http://www.fda.gov/BiologicsBloodVac cines/BloodBloodProducts/ApprovedProd ucts /
SubstantiallyEquivalent51 OkDeviceln formation/ucm390957.htm (accessed March 31,2014).]
37. Kretschmer V Biehl M, Coffe C, et al. New features of the Fresenius blood cell separator AS104. In:
Agishi T, Kawamura A, Mineshima M, eds. Therapeutic plasmapheresis (XII): Proceedings of the 4th
International Congress of Apheresis of the World Apheresis Association and the 12th Annual Symposium of the
Japanese Society for Apheresis, 3-5 June 1992, Sapporo, Japan. Utrecht, the Netherlands: VSP BY 1993:851-5.
Chapter 8
Infectious Disease Screening
Susan A. Galel, MD
^KjBLOOD COMPONENTS, LIKE all ISSi other medications in the United States, are regulated by the
Food and Drug Administration (FDA). The FDA requires medication manufacturers to verify the suitability of
every raw material in their products.1 For biologic pharmaceuticals, the donor is the key ingredient whose
suitability must be scrutinized.
Blood banks test a sample of blood from each donation to identify donors and donated components that
might harbor infectious agents. This screening process is critically important because many blood components
(eg, red cells, platelets, plasma, and cryoprecipitate) are administered intravenously to recipients without
pasteurization, sterilization, or other treatments to inactivate infectious agents. Thus, infectious agents in a
donor’s blood at the time of donation that are not detected by the screening process can be transmitted directly
to recipients.
HISTORICAL OVERVIEW OF BLOOD DONOR SCREENING
Table 8-1 shows the progression over time of donor testing for infectious diseases in the
United States. Initially, donors were screened only for syphilis. In the 1960s, studies showed that more than
30% of patients who received multiple transfusions developed posttransfusion hepatitis (PTH).2 Studies in the
early 1970s found that hepatitis B virus (HBV) accounted for only 25% of PTH cases.2 Both HBV and non-A,
non-B (NANB) hepatitis occurred more frequently in recipients of blood from commercial (paid) blood donors
than in recipients of blood from volunteer donors. By the mid-1970s, implementation of sensitive tests for
hepatitis B surface antigen (HBsAg) and a nearly universal changeover to a volunteer donor supply resulted in a
dramatic reduction in the incidence of both HBV and NANB PTH. Still, NANB PTH continued to occur in
approximately 6% to 10% of multitransfused patients.2,3
In the absence of a specific test for the causative agent of NANB PTH, investigators searched for surrogate
markers that could be used to identify donations associated with NANB hepatitis. The presence of antibody to
hepatitis B core antigen (anti-HBc) and/or the presence of elevated alanine aminotransferase in blood donors
were shown to be associ

Susan A. Galel, MD, Associate Professor of Pathology, Stanford University School of Medicine, and
Medical
Director of Clinical Services, Stanford Blood Center, Palo Alto, California
The author has disclosed a financial relationship with Roche Molecular Systems.
179

180
AABB TECHNICAL MANUAL
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182
AABB TECHNICAL MANUAL
ated with an increased risk of NANB PTH.4'7 However, concerns about the nonspecific nature of these tests
led to a delay in their implementation for donor screening.
The concept of surrogate testing was revisited in the early 1980s when concerns arose about the
transmission of AIDS by transfusions before the identification of its causative agent. In an effort to reduce the
potential transmission of AIDS by transfusion, some blood banks implemented donor testing for anti-HBc
(because this antibody was highly prevalent in populations at increased risk of AIDS) or donor screening for
inverted CD4/ CD8 T-cell ratio (an immune abnormality found in both AIDS patients and those with the pre-
AIDS lymphadenopathy syndrome).8 The leaders of most blood banks, however, believed that the risk of
transmitting AIDS by transfusion was too low to warrant surrogate interventions.9 After the human
immunodeficiency virus (HIV) was isolated and identified as the causative agent of AIDS, a donor screening
test for antibody to this agent was rapidly developed and implemented in 1985.
Once the HIV antibody test became available and cases of HIV were recognized in both prior donors and
transfusion recipients, it became clear that the risk of transmitting HIV via blood transfusion had been greatly
underestimated.10 The HIV experience highlighted the fact that an infectious agent associated with a lengthy
asymptomatic carrier state could be present in the blood supply for years without being recognized.
 In the wake of this realization, the approach to donor screening was extended beyond known agents. Current
donor history evaluations include screening for, and exclusion of, donors with, an increased risk of exposure to
blood-borne or sexually transmitted diseases. The intention is to reduce the likelihood that the blood supply will
contain other, as-yet-unidentified agents that are potentially transmissible by blood.
Rare transmissions of HIV persisted even after implementation of donor testing due to a delay of weeks or
months between the time a person is infected with HIV and the time the screening test for HIV antibody shows
positive
results.11 Blood donated during this “window period” can contain infectious HIV but is not detected by the
donor screening tests.
The most straightforward means of protecting the blood supply from window-period donations is to exclude
potential donors with an increased likelihood of exposure to HIV The FDA initially recommended that blood
banks provide informational materials to donors listing HIV risk activities and requesting that individuals not
donate if they had engaged in these activities. Later, in 1990, the FDA recommended asking each donor directly
about each risk activity. In 1992, the FDA issued comprehensive guidance describing this questioning process.
In the years since the discovery of HIV the risk of transfusion-transmitted disease has been progressively
reduced through a variety of measures:
1. Use of donor education and questioning to minimize window-period donations and to screen for
infections for which no tests are available.
2. Shortening of the window period for specific agents by improving and/or adding tests to detect earlier
stages of infection.
3. Use of questions and tests that exclude donors at increased risk of blood-borne or sexually transmitted
infections.
4. Surveillance for transfusion-transmissible diseases and implementation of new donor screening tests,
when available.
The approach used to screen potential donors for a specific agent depends on whether specific risk factors
are identifiable and whether donor screening tests are available. Table 8-2 lists the types of screening
approaches used for different infectious agents.
DONOR SCREENING TESTS
The donor infectious disease tests required by the FDA are specified in Title 21, Section 610.40, of the Code
of Federal Regulations (CFR).1 The process of amending the CFR is slow; therefore, the FDA initially
communi
183
TABLE 8-2. Approaches to Donor Screening
Approach Context for Use Example(s)
Infectious agents with defined risk factors and no
Questioning only Malaria, prions
sensitive and/or specific test
Donor test is available, but no question to distinguish
Testing only West Nile virus
individuals at risk of infection
Human
Questioning and Agents for which there are both identified risk factors immunodeficiency
testing and effective tests virus, hepatitis B and C
viruses
Use of blood
Agents with a high prevalence in donors but for which
components that test
an identifiable subset of recipients can benefit from blood Cytomegalovirus
negative for specific
components that test negative
recipients
Testing of blood
Infectious agent not detectable in donor samples Bacteria
components
cates changes in its recommendations by issuing guidance publications. Although FDA guidance documents
do not constitute legal requirements, they define the expected standard of practice in the United States. The
AABB also issues recommendations to the blood banking community, and these are communicated either by
Association Bulletins or by inclusion in AABB Standards for Blood Banks and Transfusion Services. AABB
recommendations and standards do not have the force of law, except that one US state (California) has
incorporated some AABB standards into state law.
AABB standards are often considered throughout the United States as defining a standard of practice in the
blood banking community and therefore are widely implemented. Since 1985, the FDA and AABB have issued
a series of recommendations and/or standards for additional screening tests in addition to the long-standing
donor screens for syphilis and HBsAg. Table 8-1 summarizes the chronology of changes in donor infectious
disease testing, and Table 8-3 lists the donor
screening tests that are currently performed by US blood banks.
Logistics of Testing
All infectious disease testing for donor qualification purposes is performed on samples collected at the time
of donation and sent to the donor testing laboratory. In addition, platelet components are tested for bacterial
contamination typically by the component manufacturing facility.
Laboratories that perform FDA-mandated donor testing must be registered with the FDA as biologies
manufacturers because this “qualification of raw materials” is considered part of the blood component
manufacturing process. The infectious disease tests and testing equipment used to screen donors must be
approved (licensed or cleared) for this purpose by the FDA Center for Biologies Evaluation and Research. The
tests must be performed exactly as specified in the manufacturers’ package inserts. Tests and test platforms that
are approved only for diagnostic use may not be used for screening whole blood donors.
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AABB TECHNICAL MANUAL
TABLE 8-3. Blood Donor Screening Tests Performed in the United States
Marker
Agent Screening Test Method Supplemental Assays*
Detected
■ Positive HBV DNA
Hepatitis B surface (FDA)*
HBV ■ ChLIA or EIA
antigen ■ Neutralization
(FDA)
IgM and IgG antibody
■ ChLIA or EIA
to hepatitis B core antigen
■ HBV DNA* TMA or PCR
■ Positive HCV RNA
IgG antibody to HCV
HCV ■ ChLIA or EIA (FDA)*
peptides
■ RIBA (FDA)§
■ HCV RNA* TMA or PCR
■ Positive HIV RNA
(FDA)*
IgM and IgG antibody
HIV-1/2 ■ ChLIA or EIA ■ HIV-1: IFA or
to HIV-1/2
Western blot (FDA)
■ HIV-2: EIA (FDA)
■ HIV-1 RNA* TMA or PCR
IFA, Western blot,
IgG antibody to HTLV-
HTLV-I/II ■ ChLIA or EIA RIPA, and line
I/II
immunoblot
T. pallidum antigen-
IgG or IgG + IgM
■ Microhemagglutination specific
Syphilis antibody to Treponema
or or EIA immunofluorescence or
pallidum antigens
agglutination assays
T. pallidum antigen-
Nontreponemal Solid-phase red cell
specific
■ serologic test for syphilis adherence or particle
immunofluorescence or
(eg, rapid plasma reagin) agglutination
agglutination assays
WNV ■ WNV RNA* TMA or PCR
 Trypanosoma ■ IgG antibody to T. ChLIA or EIA ESA (FDA) RIPA
cruzi cruzi (one time)11
‘Supplemental assays with “(FDA)” are FDA-approved supplemental assays. Other supplemental assays
listed are not required but may be useful for donor counseling.
tPositive results on some nucleic acid tests are approved by FDA as providing confirmation for reactive
HBsAg, HIV antibody, and HCV antibody serology tests. If a nucleic acid test result is negative, serologic
supplemental test(s) must be performed.
♦ Screening for DNA or RNA in the United States is usually performed on minipools of 6 to 16 donor
samples.
§As of 2013, RIBA is not available. FDA variance may be obtained to use a second licensed screening test
as an alternative.
11T. cruzi antibody testing may be limited to one-time testing of each donor.
HBV = hepatitis B virus; ChLIA = chemiluminescent immunoassay; EIA = enzyme immunoassay; DNA =
deoxyribonucleic acid, FDA = Food and Drug Administration; Ig = immune globulin; TMA = transcription-
mediated amplification; PCR = polymerase chain reaction; HCV = hepatitis C virus; RNA = ribonucleic acid;
RIBA = recombinant immunoblot assay; HIV-1/2 = human immunodeficiency virus types 1 and 2; IFA =
immunofluorescence assay; HTLV-I/II = human T-cell lymphotropic virus, types I and II; RIPA =
radioimmunoprecipitation assay, WNV = West Nile virus; ESA = enzyme strip assay.

CHAPTER 8 Infectious Disease Screening

185
Serologic Testing Process
Most of the serologic screening tests (assays for the detection of antibody or antigen) are enzyme
immunosorbent assays or chemiluminescent immunoassays. Typically, the process involves performing each
required screening test once on each donor sample. If the screening test is nonreactive, the test result is
considered negative (ie, there is no evidence of infection). If a test is reactive during the first round of testing
(“initially reactive”), the package insert for the test typically requires that the test be repeated in duplicate. If
both of the repeat results are nonreactive, the final interpretation is nonreactive or negative, and the unit may be
used. If one or both of the repeat results are reactive, the donor sample is characterized as “repeatedly reactive,”
and the blood unit is not permitted to be used for allogeneic transfusion. (In the case of cellular therapy
products, there are some circumstances in which repeatedly reactive donations may be used. See
“Considerations in Testing Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products” later in
this chapter.)
The infectious disease tests that are approved for donor screening have performance characteristics chosen
to make them highly sensitive. They are designed to detect almost all infected individuals and minimize
falsenegative results. However, to achieve this sensitivity, the assays also react with samples from some
individuals who are not infected (falsepositive results). Because the blood donor population is preselected by
questioning to be at low risk of infection, the vast majority of repeatedly reactive results in donors do not
represent true infections. To determine whether a repeatedly reactive screening result represents a true infection
rather than a false-positive result, additional, more specific testing may be performed on the donor sample.
FDA requires that repeatedly reactive donor specimens be further evaluated by FDAapproved
supplemental/confirmatory assays when such assays are available.1 FDA has approved confirmatory assays for
HBsAg, HIV type 1 (HIV-1) antibody, hepatitis C virus (HCV) antibody, and antibody to Trypanoso
ma cruzi. The HCV antibody confirmatory test (the recombinant immunoblot assay) recently became
unavailable, but blood centers can obtain FDA approval to use an alternative HCV supplemental testing
pathway.12 If no licensed confirmatory assay is available, unlicensed supplemental tests or retests of the donor
sample using a second licensed screening test may be helpful for donor counseling. Table 8-3 displays the
available confirmatory and supplemental assays.
A donation that is repeatedly reactive on a screening test is not permitted to be used for allogeneic
transfusion, regardless of the results of confirmatory or supplemental testing. Syphilis is the only agent for
which negative results on supplemental tests can, in some circumstances, enable use of a screening testreactive
unit. (This applies only to donations screened using a nontreponemal assay.) This situation is discussed more
fully in the “Syphilis” section.
Nucleic Acid Testing
Nucleic acid testing (NAT) was implemented to reduce the window periods described above. The process of
screening donor samples for viral nucleic acid [ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)] is
somewhat different from the serologic screening process. NAT requires the extraction of nucleic acid from
donor plasma followed by use of a nucleic acid amplification test to amplify and detect viral genetic sequences.
The test systems that were initially developed in 1999 to screen donors for HIV and HCV RNA were
semiautomated and had insufficient throughput to allow individual testing of each donor sample. Testing of
seroconversion panels showed little loss of sensitivity if donor plasma samples were tested in small pools
[minipools (MPs)] because levels of HIV and HCV RNA are typically high in the blood of infected individuals
and NAT assays are exquisitely sensitive.
Thus, in the initially approved NAT donor screening systems, MPs of 16 to 24 donor samples were prepared
and tested together. If a pool tested negative, all donations contribut
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AABB TECHNICAL MANUAL
ing to that pool were considered negative for HIV and HCV RNA. If a pool showed reactivity on the nucleic
acid test, further testing of smaller pools and, ultimately, individual samples was performed to determine which
donation was responsible for the reactive test result. Donations that were nonreactive on this additional testing
could be released for transfusion. Donations that were reactive at the individual sample level were considered
positive for viral nucleic acid and could not be released for transfusion.
In recent years, fully automated NAT systems have been developed. The two automated test platforms that
are approved by the FDA for donor screening use multiplex assays that detect HIV RNA, HCV RNA, and HBV
DNA in one reaction chamber. Reactive donations are subjected to discriminatory testing to identify which
virus is present. These systems are approved for testing of individual donations and pools of 6 to 16 donor
samples, depending on the platform. The availability of fully automated NAT platforms raises the possibility of
moving to routine screening of individual donor samples [individual donation (ID) screening], rather than
testing of pools (MP-NAT).
However, it has been estimated that IDNAT screening would minimally increase detection of infected
donors, whereas the associated testing cost would be significandy higher than with MP-NAT.13 Furthermore, it
is not clear that ID-NAT screening of the entire US blood supply would be logistically feasible using the
available platforms. An additional concern is that donors might be deferred for falsepositive results more
frequently with ID-NAT screening than pooled screening.
 In contrast to serologic testing policies, repeat testing is not permitted by the FDA for an individually
reactive NAT sample to determine whether the initially reactive result represents a true-positive result. If an
individual (unpooled) specimen is reactive on a NAT screen for HIV HCV] or HBV the FDA requires that the
corresponding blood component be discarded and the donor be deferred indefinitely.
ID-NAT screening rather than MP-NAT screening is recommended when West Nile vi
rus (WNV) activity is high in a specific geographic area. Circulating levels of viral RNA are often low
during WNV infection. When donor samples are combined into MPs, the RNA from a WNV-infected sample
may become diluted to below the detectable level. It has been estimated that MP-NAT screening for WNV RNA
may fail to detect 50% or more of infected donations.14'16 Therefore, both the FDA and AABB have
recommended ID-NAT screening for WNV RNA at times of high WNV activity in a region.18,17
Implications of Reactive Test Results
A repeatedly reactive result on a screening test (or individually reactive NAT result) typically results in
mandatory discarding of the reactive donation. Most blood bank computer systems prevent labeling and/or
release of products from donations with reactive test results. A reactive test result may also indicate that the
donor should be prohibited from making future donations, because many infections are persistent. Furthermore,
past donations may also be considered suspect because the exact date of onset of a donor’s infection cannot be
determined.
Both the FDA and AABB have issued recommendations regarding whether reactive test results affect a
donor’s eligibility for future donations, components from prior donations should be retrieved (and if so, how far
back in time), and patients who previously received components from that donor should be notified. These
recommendations are often guided by the results of supplemental or confirmatory testing performed on the
donor sample. Many of these recommendations have evolved over time.
Because these recommendations are complex, blood bank laboratories typically create checklists that list
each of the actions to be performed after a specific reactive test result is obtained. Staff use these checklists to
document completion of each action as they perform it.
Table 8-416'37 lists federal regulations, FDA guidance documents, AABB standards, and AABB
Association Bulletins with recommen
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following
Reactive Results
CHAPTER 8 Infectious Disease Screening
187
FDA guidance, October 200434 NAT for HIV-1 and HCV
 TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following
Reactive Results* (Continued)
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AABB TECHNICAL MANUAL
NAT = nucleic acid testing; HBsAg = hepatitis B surface antigen.
 189
dations regarding management of blood donors with reactive test results, retrieval of other components, and
notification of prior recipients. These regulations and recommendations are described briefly below.
Donor Eligibility
FDA regulation Title 21, CFR Part 610.41, addresses donors with reactive screening test results. FDA
guidance documents and AABB Association Bulletins contain more detailed recommendations regarding
additional testing, donor eligibility, and donor counseling for these and other tests. Donors should be notified of
any test results that affect their eligibility or that could have important implications for their health. Blood banks
should have systems that prevent future collections from ineligible donors and the release of any components
inadvertently collected from such individuals.
For donors deferred for reactive screening tests, Title 21, CFR Part 610.41, provides for reinstatement by
means of FDA-defined requalification algorithms. The FDA has issued guidance documents (listed in Table 8-
4) that define reentry pathways for donors deferred for reactivity on HIV HCV, HBsAg, anti-HBc, serologic
syphilis, and HIV/HCV/HBV NAT tests. Most of the pathways require that the donor have negative results on
specified tests after a defined waiting period. Blood banks that desire to reenter donors must follow the
FDAdefined algorithms explicitly.
Retrieval of Prior Donations and Notification of Prior Recipients (“Look-Back”)
The FDA and AABB offer guidance with regard to the appropriate management of previously collected
blood components from donors whose current donation is repeatedly reactive (or, in the case of NAT,
individually reactive) on an infectious disease screening test. These recommendations address the concern that
at the time of the previous donation, the donor could have been in the window period of an early in
fection even though the screening test results were negative.
For HIV and HCV tests, the algorithms for managing prior donations and recipients of prior donations are
spelled out in Title 21, CFR Parts 610.46 and 610.47. These requirements are replicated in the Centers for
Medicare and Medicaid Services regulations (Title 42, CFR Part 482.27) to ensure hospital transfusion service
compliance with recipient notification requirements. For other agents, recommendations for the management of
previously donated components may be found in the FDA guidance documents or AABB Association Bulletins
(or both) listed in Table 8-4.
In most cases, the FDA and AABB recommend retrieval and quarantine of any remaining components from
prior donations of that donor. It is essential that the retrieval of indate components be initiated immediately after
the repeatedly reactive result is obtained. This approach prevents transfusion of these components while
confirmatory testing is performed. The FDA requires initiation of retrieval within 3 calendar days of a reactive
HIV or HCV test and within 1 week of reactive HBsAg, anti-HBc, or anti-HTLV screening tests. If
confirmatory test results on the current donation are negative, the FDA, in some circumstances, permits
rerelease of the prior donations. In many cases, some or all of the components from prior donations will have
been transfused. For some infectious agents, the FDA and AABB recommend that the recipients of prior
donations from confirmed positive donors be notified of their possible exposure to the infectious agent.
Recommendations for notification of recipients of prior donations (“look-back”) are usually issued by the
AABB, FDA, or both at the time a new test is implemented, but these recommendations may evolve as
confirmatory tests become available or medical treatments are developed for the infection in question. Look-
back is required by law only for HIV and HCV tests (Title 21, CFR Parts 610.46 and 610.47). For an HIV look-
back investigation involving a deceased prior recipient, the next of kin must be notified. The CFR spells out
specific timelines for component retrieval and
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AABB TECHNICAL MANUAL
recipient notification. It also specifies how far back in time (ie, to which donations) the retrieval and
notification should extend. For other agents, such as WNV and T. cruzi, recommendations regarding retrieval
and recipient notification are included in FDA guidance documents and AABB Association Bulletins. Table 8-4
indicates which of these documents address product retrieval or recipient notification.
In the absence of published guidance, it is not always obvious whether or when it is appropriate to notify
prior recipients of their possible exposure to infection. If there is no confirmatory assay available, it may be
difficult to determine whether a repeatedly reactive screening test result for a donor represents a true infection.
Furthermore, if there is no effective treatment for that infection, there may be no obvious benefit to the recipient
of being told that he or she might have been exposed. There could, however, be a public health benefit from
such a notification. Specifically, a recipient who is alerted of a potential exposure can be tested and, if the
results are positive, take precautions to avoid further spread of the infection.
Cytomegalovirus Testing of Products for Immunocompromised Recipients
Some common infections cause relatively innocuous illnesses in immunocompetent individuals but can
cause severe disease in immunocompromised patients. Such is the case with cytomegalovirus (CMV).
CMV is a lipid-enveloped DNA virus in the Herpesviridae family. Like other herpesviruses, CMV causes
lifelong infection, typically in a latent state, with the potential for reactivation. Primary CMV infection in
immunologically competent individuals is mild, with symptoms ranging from none to an infectious
mononucleosis-type syndrome. In immunocompromised patients, however, both primary infection and
reactivation disease can be overwhelming and even fatal. CMV can be transmitted by blood transfusion,
primarily through intact white cells contained in cellular blood components. Frozen/thawed plasma
components do not appear to transmit CMV infection. Immunocompromised patients who are at increased
risk of transfusion-transmitted disease include fetuses, low-birthweight premature infants who are born to
CMV-seronegative mothers, and CMV-seronegative recipients of solid-organ or allogeneic hematopoietic cell
transplants from seronegative donors.38
The majority of blood donors have had prior exposure to CMV) indicated by the fact that they have CMV
antibodies. Therefore, it would not be possible to produce an adequate supply of blood if all CMV-antibody-
positive donations were discarded.
It is possible, however, to minimize CMV transmission to patients at risk of severe CMV disease, such as
those described above. These patients should be supported with cellular blood components that have a reduced
risk of transmitting CMV These reduced-risk options include using blood components from donors who are
CMV antibody negative or components that have been effectively leukocyte reduced. The literature suggests
that these two methods have similar but not identical efficacy, with an estimated transmission risk by
seronegative components of 1% to 2% vs a risk of 2% to 3% with leukocyte-reduced components.38'40 A
recent study, however, found no CMV transmissions among 100 carefully monitored allogeneic hematopoietic
cell transplant recipients who received CMV-untested, leukocyte-reduced components.41 Because many atrisk
patients receive leukocyte-reduced components and are monitored closely for CMV infection, treated early with
anti-CMV drugs, or both, it is difficult to measure a benefit from also providing CMV-seronegative components
to these patients.
Autologous Donations
The FDA requires infectious disease testing of autologous donations that are shipped from one facility to
another. If the receiving facility does not permit autologous donations to be crossed over to the general
inventory, the FDA requires testing of only the first donation in each 30-day period [Title 21, CFR Part
191
610.40(d)], The labeling of the unit must be consistent with its testing status. Units from donors with
repeatedly reactive tests must be labeled with biohazard labels. Some hospitals have policies that prohibit
acceptance of autologous units with positive results on some tests because there is a potential for an infectious
unit to be transfused to the wrong patient. The AABB has warned that refusal of test-positive units could be
interpreted as violating the Americans with Disabilities Act.42
Considerations in Testing Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products
Both the questions and tests required by FDA to screen donors of human cells, tissues, and cellular and
tissue-based product (HCT/Ps) differ from those for blood donors, and screen
ing requirements vary by type of tissue. These requirements are spelled out in Title 21, CFR Part 1271 and
an August 2007 FDA guidance document and are summarized in Table 85 43,44 yqe qme frames for testing
HCT/P donors are also specified in these documents.
In most cases, the samples for infectious disease testing must be obtained 7 days before or after the tissue
donation. However, samples of peripheral blood hematopoietic progenitor cells or marrow may be tested up to
30 days before donation. Autologous tissues and reproductive tissues from sexually intimate partners may be
exempt from some testing requirements.
Blood bank laboratories that test samples from HCT/P donors must take care to check package inserts for
HCT/P testing methods; a package insert may require a different testing method for HCT/P donors than for
blood
TABLE 8-5. FDA Testing Requirements for HCT/Ps (as of March 2014)
Tissue Type Agent Tests
Antibody to HIV-1
HIV
and HIV-2*
HIV-1 RNA*
Hepatitis B
HBV
surface antigen*
Antibody to
All tissues hepatitis B core
antigen*
Antibody to
HGV hepatitis C* HCV
RNA*
FDA-cleared
Treponema pallidum screening or
diagnostic test
For donors of viable leukocyte-rich HGT/Ps (eg,
Antibody to
hematopoietic progenitor cells or semen) also test for the HTLV-I/ll
HTLV-I/ll*
following in addition to the above:
FDA-cleared screening
CMV test for anti-CMV (total
IgG and IgM)
FDA-licensed,
For donors of reproductive tissues, test for the following
Chlamydia trachomatis approved, or cleared
in addition to the above:
diagnostic test
Neisseria gonorrhea FDA-licensed,
approved, or cleared
 diagnostic test
‘These tests must be FDA licensed for donor screening.
HCT/Ps = human cells, tissues, and cellular and tissue-based products; HIV = human immunodeficiency
virus; RNA = ribonucleic acid; HBV = hepatitis B virus; HCV = hepatitis C virus; FDA = Food and Drug
Administration; HTLV = human T-cell lymphotropic virus; CMV = cytomegalovirus; Ig = immune globulin.

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AABB TECHNICAL MANUAL
donors. For example, NAT for most types of HCT/P donors must be performed on individual donor samples,
and MP-NAT is not permitted for most HCT/P donor categories.
In some cases, FDA regulations permit the use of HCT/P donations that are reactive on infectious disease
screening tests. These exceptions are listed in Title 21, CFR Part 1271.65. FDA has issued specific labeling,
storage, and notification requirements for these tissues.
Testing of HCT/P donors for WNV RNA and antibody to T. cruzi is not required by FDA as of March 2014.
However, FDA has indicated that it considers WNV to be a “relevant communicable disease,” and draft
guidance documents indicate that FDA is considering a requirement to test at least some HCT/P donors for
these agents.44
International Variations in Donor Testing
Although this chapter focuses on infectious disease screening in the United States, the general approach to
donor screening is similar in other countries. However, the specific donor questions and tests vary from country
to country based on the regional epidemiology of infections and tests available. For example, most countries
where WNV is not endemic do not test for this agent, although they may question donors about travel to WNV-
affected countries. Countries where HBV is hyperendemic cannot exclude donations from individuals who test
positive for anti-HBc without adversely affecting the adequacy of their blood supply. The AABB Standards
Program Unit in conjunction with the AABB Subcommittee for the Evaluation of International Variances
considers variance applications from facilities in countries where national practices and available tests differ
from those in the United States.
RESIDUAL INFECTIOUS RISKS OF TRANSFUSION
Despite donor screening, blood components may still transmit infections. The residual risk of transmission
varies according to the inci
dence of the infection in the donor population and the nature of the donor screening processes in place.
Agents for Which Blood Is Tested
Transfusion transmission of HIV, HCV, and HBV is now so rare that the rate of transmission cannot be
measured by prospective clinical studies. The risk can only be estimated by theoretical modeling.
One theoretical source of risk is a virus strain that the current test kits do not detect. The Centers for Disease
Control and Prevention and the test manufacturers conduct surveillance for such emerging strains. Over time,
the FDA has required that test manufacturers expand their detection capabilities to include new strains. A
second potential cause of transmission is a quarantine failure (ie, a blood bank’s failure to quarantine a unit that
tests positive). Quarantine errors are thought to be rare in blood banks that use electronic systems to control
blood component labeling and release because these systems are designed to prevent the release of any unit with
incomplete testing or a reactive test result. Erroneous releases appear to occur more frequently in blood banks
that rely on manual records and quarantine processes.45
The primary cause of residual transmissions is thought to be donations from individuals in the window
period of early infection, before test results are positive. Figure 8-1 displays the sequence in which different
types of donor screening tests demonstrate reactivity. Over time, the window periods have been shortened by
implementation of donor screening tests that detect earlier infections. However, because no test gives a positive
result immediately after an individual acquires an infection, a window period remains. With NAT, the average
duration of the window period is estimated to be 9.0-9.1 days for HIV and 7.4 days for HCV.13,46 The window
period for HBV is longer, as discussed in the “HBV” section below.
The likelihood that a blood donation has been obtained from a donor in the window pe
193

FIGURE 8-1. Time sequence of the appearance of various markers of infection.

riod can be estimated mathematically using the incidence-window period model46:
Probability a donation was made during the window period = length of window period x incidence of
infections in the donor population
The incidence of infections in donors can be calculated from the observed number of donors with a negative
test result for one donation but a positive result for a subsequent donation (ie, seroconverting donors). This
method measures incidence rates only in repeat donors and does not permit assessment of the likelihood that
first-time donors might be in the window period.
Other methods permit measurement of new infection rates in both first-time and repeat donors using tests
that differentiate new from established infections. Such tests include nucleic acid tests (donor blood that
contains HIV or HCV RNA but not antibody most likely represents a very early infection) and “sensitive/less
sensitive” antibody testing.13,46-49 When these alternative methods have been used, new HIV and HCV
infections were two to four times more common among first-time donors than among repeat donors.46'48
Howev
er, both of these donor populations have significantly lower infection rates than the general population. The
continued importance of using donor questioning to select donors with a low incidence of infection is explored
in more detail in the “HIV” section below.
The current estimated risks of HIV, HCV, and HBV transmission in donors, based on window-period and
incidence calculations, are shown in Table 8-6.
Agents for Which No Donor Screening Tests Are Available
 Essentially any infectious agent that can circulate in the blood of an apparently healthy person could be
transmitted by transfusion. It is impossible to estimate the risk of transmission for each of the infectious agents
for which donors are not tested. However, transfusiontransmitted infections are rarely identified. The infections
that are most likely to be recognized as transmitted by transfusion are those that have a distinctive clinical
presentation and are otherwise rare in the United States. The likelihood that an infection will be recognized as
transmitted by transfusion is enhanced if the infection is usually associated with a behavioral risk that the
transfusion
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AABB TECHNICAL MANUAL
TABLE 8-6. Estimated Risks of Transfusion-Transmitted Infection in the United States Based on Window-
Period and Incidence Estimates*
Study Incidence per 105 Infectious Window Residual Risk per
Agent
Period Person/Years Period (days) Donated Unit
2007-
HIV 3.1 9.1 1:1,467,000
200846*
2007-
HCV 5.1 7.4 1:1,149,000
200846*
2009- 1:843,000 to
HBV 1.6 26.5-18.5
201150t 1:1,208,000
*HIV and HCV risk estimates are based on minipool nucleic acid testing in pools of 16.
4HBV risk estimates are based on minipool nucleic acid testing in pools of 16 using the Novartis Ultrio Plus
assay. The range indicated for the HBV window period reflects uncertainty regarding the minimum infectious
dose of HBV (1 copy in 20 mL plasma vs 10 copies in 20 mL).
HIV = human immunodeficiency virus; HCV = hepatitis C virus; HBV = hepatitis B virus.
recipient lacks (eg, when malaria develops in a transfusion recipient who has not traveled outside the United
States).
If a life-threatening agent is recognized as a potential threat to the blood supply, both the AABB and FDA
typically consider whether a donor screening question could be used to exclude potentially exposed donors in
the absence of a donor screening test. Donor questioning regarding travel to and residence in endemic areas is
currently the only means of protecting the US blood supply from malaria and variant Creutzfeldt-Jakob disease
(vCJD). Most infectious agents, however, do not have such clear geographic risk areas. In general, it is difficult
to design donor questions that are both sensitive (ie, detect most infected individuals) and specific (ie, exclude
only infected individuals).
An alternative method of protecting the blood supply from infectious agents is pathogen inactivation or
reduction. Heat inactivation, solvent/detergent treatment, nanofiltration, chromatography, cold ethanol
fractionation, and other approaches have been used with remarkable success to inactivate or remove residual
pathogens in plasma derivatives. Pathogen reduction systems for cellular blood components are not available in
the United States as of March 2014, although systems for platelet components are in use in
some other countries. Pathogen reduction systems are discussed in the “Pathogen Reduction Technology”
section later in this chapter.
The AABB Transfusion-Transmitted Diseases Committee published an extensive review of infectious
agents that are possible threats to the blood supply.51 Potential mitigation strategies were discussed for each
agent, including the documented or theoretical efficacy of pathogen reduction processes. AABB keeps these
materials up to date and adds materials for new potential threats as they are identified.52 The agents deemed to
pose the highest threat from either a scientific or public perspective are briefly discussed in this chapter. (See
the 2009 supplement to TRANSFUSION and updates on the AABB website for a more thorough review of
these potential infectious risks.51,52)
SCREENING FOR SPECIFIC AGENTS
HIV
HIV-1, a lipid-enveloped, single-stranded RNA virus, was identified in 1984 as the causative agent of AIDS,
and blood donor screening for antibodies to this virus was implemented in the United States in 1985. In 1992,
donor screening tests were modified to include de
 195
tection of antibodies to HIV-2, a closely related virus identified initially in West Africa.
HIV can be transmitted sexually, parenterally, and from infected mothers to their infants. Although
heterosexual and vertical spread of HIV predominate in some parts of the world, new HIV cases in the United
States continue to be concentrated in men who have sex with men (MSM) and individuals with high-risk
heterosexual contact (defined as contact with an individual who is HIV positive or in an identified risk group
for HIV, such as MSM or injection drug users).53
Current donor screening for HIV includes NAT testing for HIV-1 RNA and serologic testing for antibodies
to HIV. The antibody tests approved for donor screening detect both immune globulin M (IgM) and IgG
antibody to both HIV-1 and HIV-2. Some of the approved tests also detect antibody to the HIV-1 group 0, a
strain of HIV-1 found primarily in Central and West Africa. If the antibody test used by a donor center does not
have an HIV-1 group 0 detection claim, the donor center must use donor questioning to exclude individuals who
have resided in, received medical treatment in, or had sex partners from HIV-1 group 0 endemic areas.31
The average lag time from HIV-1 exposure to test detection (window period) is currently estimated to be 9-
9.1 days for MP-NAT.13,48 Based on window-period and incidence-rate calculations, the current risk of
acquiring HIV from transfusion is estimated to be approximately 1 in 1.5 million units (Table 8-6).
In the United States, blood donor screening questions exclude very broadly defined populations at increased
risk of HIV. Given the short delay of only days between exposure and detection of infection by NAT, experts
have questioned whether donor interviews and exclusion of donors with increased risk remain medically
necessary.54 The continued importance of a low-risk donor population becomes evident if different HIV
incidence figures are entered into the blood safety calculation. For example, HIV incidence rates as high as 1%
to 8% have been observed in some high-risk populations, such as young urban MSM.55,56 If an individual
from a population with a 1% inci
dence of HIV donates blood, the likelihood that this individual is in the window period and that the
component will transmit HIV can be calculated as follows:
Risk that the donation is in window period = length of window period x incidence of infection in donor
population = (9.0 days/365 days/ year) x (1/100 person-years) = 1/4100.
This is the likelihood that a unit from this high-risk donor would harbor HIV but be missed by the current
donor screening. This risk is clearly much higher than the estimated HIV transmission risk of 1 in 1.5 million
for a unit of blood obtained from the current donor population. Thus, despite the short window period with
current testing, inclusion of donors with a high risk of HIV would have a profoundly adverse impact on blood
safety. Accordingly, questioning of donors for risk and temporarily excluding those at increased risk to
minimize window-period donations continue to be critical for preserving blood safety.
Despite the efficacy of current donor questioning approaches, there has been great interest in developing a
more specific donorscreening algorithm for MSM that would exclude only individuals who are truly at
increased risk of HIV. Although more specific donor screening criteria for MSM are desirable, the FDA states
that no algorithm has yet been developed that reliably identifies MSM whose HIV risk is as low as that of
current blood donors.57
FDA’s rationale for permanently excluding MSM from donation has been less clear. Most other high-risk
populations are excluded only temporarily (for 1 year after the risk activity). The FDA’s rationale for excluding
MSM beyond 1 year appears to be based on two concerns. One concern is that there are other potentially
transfusion-transmissible infections that are more prevalent in MSM populations (such as human herpesvirus 8,
the causative agent of Kaposi sarcoma), and donors are not tested for all of these infections.54 The other
concern is “quarantine release errors,” or the risk that a blood center might inadvertently release a unit with a
positive test result.
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However, based on current electronic controls used by most US blood centers, release errors appear to be
extremely rare.
Mathematical modeling suggests that the deferral period for MSM could be shortened without a substantial
increase in risk to recipients.45 A US Department of Health and Human Services (DHHS) advisory committee
concluded in 2010 that available scientific data were inadequate to support any specific alternative policy.58
DHHS subsequently issued requests for proposals to evaluate alternative approaches to donor screening that
could maintain the current level of blood safety.
HBV
HBV is a lipid-enveloped, double-stranded DNA virus. Like HIV, HBV is transmitted parenterally, sexually,
and perinatally. Jaundice is noted in only 25% to 40% of adult cases and in a smaller proportion of childhood
cases. A large percentage of perinatally acquired cases result in chronic infection, but most HBV infections
acquired in adulthood are cleared. HBV is highly prevalent in certain parts of the world, such as Eastern Asia
and Africa, where perinatal transmission and resultant chronic infection have amplified infection rates in the
population. In the United States, the incidence of acute HBV infection has decreased dramatically with the
implementation of routine vaccination programs. Perinatal screening and newborn prophylaxis have also been
effective in reducing perinatal transmission.
During HBV infection, DNA and viral envelope material (HBsAg) are typically detectable in circulating
blood. Antibody to the core antigen is produced soon after the appearance of HBsAg, initially in the form of
IgM antibody, followed by IgG. As infected individuals produce antibody to the surface antigen (antiHBsAg),
the HBsAg is cleared.
The FDA requires donor screening for HBsAg, HBV DNA, and total anti-HBc (IgM and IgG antibody).
Measurements of HBV incidence in donors have been complicated by the transience of HBsAg and false-
positive results on the HBsAg test.48,50 Published estimates of the infectious window have varied because of
differences in the sensitivity of various HBV assays and lack of certainty regarding the level of virus in a
blood component that is required for infectivity.50,59 Recent publications provide window-period estimates for
different potential infectious doses of virus (eg, 10 copies/20 mL of plasma vs 1 copy/20 mL of plasma). The
infectious window prior to a positive result on the Abbott PRISM (Abbott Laboratories, Abbott Park, IL)
HBsAg test has been estimated to be 30 to 38 days.59 With the addition of HBV DNA testing in MPs of 16, the
window period is estimated to have been reduced to 18.5 to 26.5 days.50 Using these MP testing estimates, US
HBV transfusion-transmission risk has been estimated to be between 1 in 843,000 donations to 1 in 1.2 million
donations (Table 8-6).50
Donor screening for HBV DNA can be of value at a variety of points in infection. HBV DNA may be
detected during the infectious window period before HBsAg detection; however, DNA levels may be low and
could be below the limits of detection of MP-NAT assays.50,59 Later in infection, following the clearance of
HBsAg, HBV NAT may detect persistent (ie, “occult” HBV) infection.60 Such infections are interdicted in the
United States by the donor screening test for anti-HBc. HBV NAT testing can also detect acute HBV infections
in individuals who have previously been vaccinated.61,62 Such individuals may never develop detectable
HBsAg, but they may have detectable DNA. The infectivity of such donations is not known because these units
contain vaccine-induced antibodies to HBsAg in addition to the virus. Routine HBV DNA screening of US
blood donations detects at least some of these infections.
HCV
HCV is a lipid-enveloped, single-stranded RNA virus. HCV was shown to be the cause of up to 90% of
cases previously called NANB transfusion-related hepatitis.63 The majority of HCV infections are
asymptomatic. However, HCV infection is associated with a high risk of chronicity, which can result in liver
cirrhosis and hepatocellular carcinoma.
197
HCV is thought to be transmitted primarily through blood exposure. In the United States, about 55% of
HCV infections are associated with injection drug use or receipt of transfusion before 1992, but the risk factors
for the remainder of the infections are not clear.64 Sexual and vertical transmissions are uncommon, although
coinfection with HIV may increase transmission rates by these routes.
 Current donor screening for HCV includes NAT testing for HCV RNA and serologic testing for antibodies
to HCV. The average window period between exposure and detection of infection by MP-NAT is estimated to
be 7.4 days.13 The serologic test detects only IgG antibody, a relatively late marker of infection. Therefore,
there may be a significant lag (1.5 to 2 months) between detection of RNA and detection of antibody.65 Donor
questioning has limited potential to exclude individuals who may be harboring HCV infection because a large
proportion of infected individuals are asymptomatic and have no identifiable risk factors. Despite this
limitation, the current estimated US risk of HCV transmission by transfusion is extremely low—approximately
1 in 1.1 million (Table 8-6).46
Human T-Cell Lymphotropic Virus, Types I and II
Human T-cell lymphotropic virus type I (HTLV-I) is a lipid-enveloped RNA virus. It was the first human
retrovirus identified, isolated in 1978 from a patient with cutaneous T-cell lymphoma. A closely related virus,
HTLV-II, was later isolated from a patient with hairy cell leukemia. Both viruses are highly cell associated,
infect lymphocytes, and cause lifelong infections, but most of these infections are asymptomatic.
Approximately 2% to 5% of HTLV-I-infected individuals develop adult Tcell leukemia/lymphoma after a lag of
20 to 30 years. A smaller percentage develop a neurologic disease called HTLV-associated myelopathy or
tropical spastic paraparesis. HTLV-II disease associations remain unclear. Both in
fections are thought to be spread through blood, sexual contact, and breast feeding.
HTLV-I infection is endemic in certain parts of the world, including regions of Japan, South America, the
Caribbean, and Africa. In the United States, infections are found in immigrants from endemic areas, injection
drug users, and the sexual partners of these individuals. Approximately one-half of the HTLV infections in US
blood donors are from HTLV
jj 66,67
The only FDA-approved donor tests for HTLV infection are screening assays for IgG antibody to HTLV-I
and HTLV-II. Units that are reactive on the screening assay may not be released for transfusion. Because there
is no FDA-approved confirmatory assay and the majority of reactive donor samples are thought to represent
false-positive results, the FDA does not require permanent deferral of donors after one reactive donation.
Donors are permanently excluded only if their sample reacts again on a subsequent donation (Tide 21, CFR Part
610.41). Unlicensed supplemental tests, such as immunoblots or immunofluorescence assays, may be very
helpful for counseling donors about the likelihood that the screening test reactivity represents a true infection.
Some of these supplemental assays can also differentiate between HTLV-I and HTLV-II infection.66,67 Rather
than use unlicensed supplemental tests, some blood centers retest reactive samples on a different FDAlicensed
donor screening assay; samples with nonreactive results on the alternative screening assay are most likely false-
positive. Donors whose samples are reactive on the second screen, however, must be counseled and deferred.35
Risk estimates for transfusion-transmitted HTLV are somewhat uncertain, given the absence of well-defined
window periods for the current HTLV antibody tests and the absence of confirmatory assays to definitively
measure true case rates in donors.48 Like CMV HTLV is thought to be transmitted only by white-cell-
containing blood components and not by frozen/thawed plasma components.66,67
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Syphilis
Syphilis is caused by the spirochete bacterium Treponema pallidum. Donor screening for syphilis has been
performed for more than 60 years. Donors were initially screened by nontreponemal serologic tests that detect
antibody to cardiolipin (eg, rapid plasma reagin). In recent years, however, most blood banks have begun using
tests that detect specific antibodies to T. pallidum because these tests can be performed with automated testing
instruments.
The vast majority of reactive donor test results do not represent active cases of syphilis. Most reflect either
biologic false-positive results or persistent antibody in previously treated individuals (the latter are detected by
treponemal-specific antibody screening tests). The FDA has permitted use of additional, treponemal-specific,
confirmatory assays (eg, fluorescent treponemal antibody absorption test) to guide the management of both
donors and components. Specifically, the FDA has permitted release of units from donors who have reactive
nontreponemal screening test results and negative treponemal confirmatory results if the units are labeled with
both test results.1,37 If, however, the result of the treponemal-specific confirmatory test is positive or if no
additional testing is performed, the component may not be used and the donor must be deferred for at least 12
months.
 The current value of donor screening for syphilis is controversial.68'70 Although numerous cases of
transfusion-transmitted syphilis were reported before World War II, no cases have been reported in the United
States for more than 40 years. The low transmission risk is probably related to a declining incidence of syphilis
in donors as well as the limited survival of the T. pallidum spirochete during blood storage.
One issue that has been considered is whether the syphilis screen improves blood safety by serving as a
surrogate marker of highrisk sexual activity. However, studies have demonstrated that donor screening for
syphilis does not provide incremental value in detecting other blood-borne and sexually trans
mitted infections, such as HIV, HBY HCV or HTLV70
Other Bacteria
Bacterial contamination of blood components (mainly platelets) continues to cause some transfusion-related
fatalities.71 Bacteria are reportedly present in approximately 1 in 3000 cellular blood components.72 The
source of the bacteria can be either the donor’s skin or asymptomatic bacteremia in the donor.
The level of bacteria in components just after collection is generally too low to detect or to cause symptoms
in the recipient. However, bacteria can multiply during component storage, particularly in platelet components,
which are kept at room temperature. Bacteria proliferate to a lesser extent in refrigerated Red Blood Cells, and
septic reactions occur much less commonly with these components. To reduce the risk of septic transfusion
reactions associated with platelets, the AABB implemented a requirement for processes to limit and detect
bacterial contamination in all platelet components in 2004. (Since 2009 the requirement has been to “limit and
detect or inactivate” bacterial contamination in platelet components.18111111)
To limit blood component contamination by bacteria from donor skin, two elements of the blood collection
process are critical. Before venipuncture, the donor skin must be carefully disinfected using a method with
demonstrated efficacy. Most of these methods involve iodophors, chlorhexidine, or alcohol.73 Second,
diversion of the first 10 mL to 40 mL of donor blood, which can contain skin and appendages, away from the
collection container (eg, into a sample pouch) further reduces the likelihood that skin contaminants will enter
the component.73,74 Since 2008, the AABB has required that collection sets with diversion pouches be used for
all platelet collections, including whole-blood collections from which platelets are made.18(p22]
A variety of technologies are available for detection of bacteria in platelet components. AABB Standards
requires blood centers to use a bacteria detection method that is approved
199
by the FDA or validated to provide sensitivity equivalent to FDA-approved methods. None of these
methods is sensitive enough to detect bacteria immediately after collection. All methods require a waiting time
for bacteria contaminants to multiply before the component is sampled.
The process most commonly used in the United States to screen apheresis platelets is a culture-based system
that requires the platelet component to be stored for 24 hours before sampling. After that time, a sample is
withdrawn and inoculated into one or more culture bottles. The bottles are then incubated in the culture system.
Some blood centers continue to hold the component during the first 12 to 24 hours of culture and release it for
use only if the culture is negative at the end of that time. In all cases, the culture is continued for the shelf life of
the unit. If the culture becomes positive after the component is released, the blood center attempts to retrieve it.
If the component has not been transfused, resampling of the product for culture is very informative because
approximately two-thirds of the initially positive signals are determined to be caused by either contamination of
the bottle (and not the component) or false signals from the culture system.73,74All positive cultures should be
tested to determine the identity of the organism. If a true-positive result is related to an organism that is not a
skin contaminant, the donor should be notified and advised to seek medical consultation.19
Other methods approved in the United States for platelet quality control testing early in the storage period
include a culture-based system with a one-time-point readout and an optical scanning system. All of the
methods are approved for testing leukocyte-reduced apheresis platelets, and some are approved for testing pools
of leukocyte-reduced, wholeblood-derived platelets. These methods are not generally used for routine screening
of individual (unpooled) whole-blood-derived platelet concentrates. Low-technology methods for screening
platelets just before issuesuch as visually inspecting the platelets for swirling or testing them for low glucose or
pH—lack both sensitivity and specificity and
do not fulfill the AABB standard for bacteria detection.18(pll)'20'73 However, two FDA-cleared point-of-
issue assays can be used for bacteria detection testing of platelet concentrates that are pooled immediately
before issue.
 Since the implementation of routine bacterial screening of apheresis platelets, the frequency of FDA-
reported fatalities from contaminated apheresis platelets has declined.71 However, some contaminated apheresis
platelets escape detection by this early testing, presumably because bacterial concentrations are below limits of
detection at the time of sampling; thus, septic, and even fatal, reactions do still occur. AABB has recommended
consideration of policies to further reduce the risk of bacterially contaminated platelets.75 The point-of-issue
assays mentioned above are cleared by FDA as adjunct (time-of-issue) tests for apheresis platelets that have
been screened by another method. In a large clinical trial, one of these assays detected nine bacterially
contaminated components among 27,620 apheresis platelets (1 in 3069 components) previously screened by an
early-storage culture-based assay.76 There were also 142 false-positive results. As of March 2014, point-of-
issue retesting of apheresis platelets had not been widely implemented in the United States.
All of the above methods provide incomplete assurance of bacteria detection, and none is practical for
screening the red cell inventory. Pathogen-reduction methods, which impair proliferation of bacteria in the
blood component, could theoretically be used to reduce the risk of septic reactions from bacteria in blood
components. Indeed, in some regions outside the United States, pathogen reduction has replaced bacteria
detection testing for platelets, but these technologies are not approved for use in the United States as of March
2014.
Infections Transmitted by Insect Vectors
Until recently, malaria was the only vectortransmitted disease that was widely recognized as having the
potential for secondary transmission by transfusion in the United
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AABB TECHNICAL MANUAL
States. Malaria is rare in the United States, and the blood supply has been effectively protected by questions
that exclude donors who have recendy traveled to or resided in malariaendemic regions. In the past decade,
however, other vector-transmitted infections have been recognized as threats to the US blood supply, and these
infections are targeted by the newest blood donor screening assays.
WNV
WNV is a lipid-enveloped RNA vims in the Flaviviridae family. First detected in the United States in 1999,
it subsequently spread throughout North America, appearing in annual epidemics every summer and autumn.
Birds are thought to be the primary reservoir for WNV with infection spreading to humans through mosquito
bites. Approximately 80% of human cases are asymptomatic; 20% are associated with a self-limited febrile
illness; and less than 1% are associated with severe neuroinvasive disease, such as meningoencephalitis or acute
flaccid paralysis.
Transfusion transmission of WNV was first recognized in 2002 and traced to blood donations containing
viral RNA in the absence of antibody. Thus NAT, rather than serologic testing, was required to protect the blood
supply. Donor screening was widely implemented in 2003 using investigational NAT assays. Donor tests for
WNV RNA are now approved by FDA and required by both the FDA and AABB.17,18(pp31'32) The
predominant method of testing is in MPs.
However, as discussed in the “NAT” section above, circulating levels of viral RNA are frequently low
during WNV infection, and a donor sample containing a low concentration of RNA may escape detection if it is
diluted in a minipool. Therefore, both the AABB and FDA recommend testing of individual donor samples, not
minipools, when WNV activity is high in a particular collection region.16,17 Regional WNV activity is
monitored through active communications between neighboring blood collection agencies about viremic donors
detected as well as by public health reports of
clinical WNV cases and animal and mosquito surveillance in the area.
Two documented cases of WNV transmission by transfusion were traced to donations screened by MP-NAT
during periods when neighboring blood collection facilities were screening donors by ID-NAT.77 The most
recent reported transmission was traced to a donation containing WNV antibody and a low level of virus that
was not reproducibly detectable even by ID-NAT.78 It is possible that some transmissions have escaped
recognition because most WNV infections lack distinctive symptoms.
T. cruzi
T. cruzi, the protozoan parasite that causes Chagas disease, is endemic in parts of Mexico, Central America,
and South America. It is transmitted to humans by an insect vector, the reduviid bug. Acute infection is usually
selflimited but may be severe in immunocompromised patients. Most infections become chronic but
asymptomatic. Decades after the initial infection, 10% to 40% of infected individuals develop late-stage
manifestations, including intestinal dysfunction or cardiac disease, which can be fatal. Transfusion transmission
of T. cruzi from the blood of chronically infected, asymptomatic donors has been reported in endemic areas.
A blood donor screening enzyme immunoassay for antibodies to T. cruzi was approved by the FDA for US
use in December 2006. Although not initially required by the FDA, the test was widely implemented by US
blood centers during 2007. Supplemental testing of reactive specimens using an FDAlicensed enzyme strip
assay or unlicensed radioimmunoprecipitation assay (RIPA) is very helpful for guiding donor counseling. Based
on the results of the latter assay, about 25% of reactive US donors appear to be truly infected.79,80 All donors
whose samples are reactive on the screening assay, however, must be permanently deferred, regardless of the
results of the supplemental testing, pending FDA approval of a reentry algorithm.29
201
The vast majority of US donations with reactive I cruzi screening test results and positive supplemental
results are from donors born in T. crazi-endemic areas. Other confirmed-positive donors appear to have
congenitally acquired infections (ie, the donor’s mother is from a T. crazi-endemic area), and only a small
number of donor infections appear to have been acquired from vector exposure within the United States
(autochthonous cases). In the first 2 years of US donor screening, no donor seroconversions were
identified.79,80 In December 2010, the FDA issued guidance recommending one-time screening of every US
donor for T. cruzi.29
Before implementation of donor screening, seven cases of transfusion-transmitted T. cruzi had been
identified in the United States and Canada; all of the cases with available data were linked to platelet
transfusions. Since implementation of donor screening, RIPA-positive donors have been identified, and
recipients of their prior donations have been notified and tested. Thus far, only two prior recipients (of platelets
from one donor) appear to have positive test results. Thus, despite reported transmission rates of 10% to 20%
from components from infected donors in endemic areas, no T. cruzi transmissions by red cells in the United
States have been documented to date. The lower infectivity of US red cell components compared to those in
endemic areas may be at least partly attributable to more frequent use of fresh whole blood in endemic areas.
Babesia
Babesia are intraerythrocytic parasites. Cases of transfusion-transmitted babesiosis are being identified with
increasing frequency, and some have been fatal.71,81,82 There is no FDAapproved donor screening test for this
infection.
Human Babesia infections are zoonotic, usually acquired through the bite of an infected tick. In the
northeastern and Midwestern United States, the most common Babesia species is B. microti. The vector is
Ixodes scapularis, the same tick that transmits Lyme disease.
Reported human infections with B. microti are becoming more frequent. In the western United States,
Babesia infections appear to be less common, and a different species, B. duncani, predominates. The vector for
B. duncani has not been clearly defined.
Babesia infection is usually asymptomatic, even though parasites can circulate for months to years. In some
individuals, however, Babesia infection presents as a severe malarialike illness that can be fatal.
Immunocompromised, elderly, and asplenic patients are at increased risk of severe disease.
Babesia infection is diagnosed when the intraerythrocytic parasites are seen on a blood smear. If a patient is
suspected of having acquired the infection by transfusion, donors of the patient’s components can be recalled
and tested for antibodies to Babesia using an immunofluorescence assay; the presence of high-titer antibody in
the donor is suggestive of recent infection. Most of the donors implicated in transfusion-transmitted cases have
been residents of endemic areas, although some were residents of nonendemic areas who were apparently
exposed to Babesia during travel to endemic areas.82
Studies in Connecticut have found B. microti antibodies in approximately 1% of blood donors, suggesting
that B. microti infections are highly prevalent in the state’s population and grossly underrecognized.83
In the absence of FDA-approved tests for blood donor screening, there have been public discussions of
potential interventions to reduce transfusion risk in the most highly endemic regions.81,84 Clinical trials of
some investigational serologic and nucleic acid tests are currently under way.85
Malaria
Malaria is caused by an intraerythrocytic parasite of the genus Plasmodium. Infection is transmitted to
humans through a mosquito bite. Five species account for most human infections: P. falciparum, P. vivax, P.
malariae, P ovale, and P knowlesi.
No FDA-approved test is available to screen US blood donations for malaria infection.
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AABB TECHNICAL MANUAL
Screening is accomplished solely by donor questioning. Donors are excluded temporarily from donating
blood after traveling to malariaendemic areas, residing in malaria-endemic countries, or after recovery from
clinical malaria. Donor questioning has been remarkably effective at preventing transfusion-transmitted malaria
in the United States, with only six cases reported between 1999 and 2012. All six cases were linked to donors
with a history of residence in Africa; on re-interview, five of the six donors were verified to have met
acceptance criteria, but one had emigrated less than 3 years before donation.86,90
This level of transfusion safety has been achieved at substantial cost in terms of donor loss; malaria-related
questions have excluded hundreds of thousands of otherwise acceptable US donors annually. Although travel to
Mexico has accounted for the largest proportion of deferred donors, the risk of acquiring malaria during tourist
travel to Mexico is exceedingly low.91 The FDA recently issued guidance redefining malaria-endemic areas as
only those for which chemoprophylaxis is recommended.92 With this new definition, travel to many popular
tourist locations will no longer be considered a malarial risk. However, the new guidance adds a complex
algorithm for evaluating travel by donors who have lived for more than 5 years in malaria-endemic countries
because of a concern of partial immunity in such donors.
Some other countries that exclude donors after travel to malaria-endemic areas permit reinstatement of these
donors if they test negative for malaria antibodies 4 to 6 months after completion of travel. In the absence of an
approved assay, such a “test-in” reinstatement strategy has not been accepted by the FDA.
Other Vector-Transmitted Infections
There are many other vector-transmitted infections that could be secondarily transmitted by transfusion.
These agents and potential intervention strategies are reviewed in the publicly available AABB emerging
infectious disease resources.51,52 Two of these agents, dengue and chikungunya viruses, have received
recent attention because donations containing viral nucleic acid have been documented during epidemics
outside the continental United States. Dengue activity has also been documented within the southern United
States and Hawaii.93 The degree to which these agents threaten the US blood supply is unclear.94
Prions
Prions are proteinaceous infectious particles that induce disease by triggering conformational changes in
naturally occurring protein counterparts. These agents cause fatal infections of the nervous system called
“transmissible spongiform encephalopathies” (TSEs).
Classical CJD is a TSE with both a sporadic form and familial forms and an incidence of about 1 per
million. CJD has been transmitted by infusion or implantation of products from infected central nervous system
tissues. Blood components do not appear to transmit classical CJD. Nevertheless, blood donations are not
accepted from donors who are at increased risk of this disease.95
Another TSE, vCJD, does appear to be transmissible by blood transfusion. This TSE is caused by the same
prion that causes bovine spongiform encephalopathy (BSE), also known as “mad cow disease.” As of March
2014, four cases of vCJD transmission by transfusion had been reported in the United Kingdom, the area in
which BSE was most endemic. In addition, one latent vCJD infection was identified in a patient with
hemophilia in the United Kingdom who died of other causes. This patient had received UK-plasma-derived
Factor VIII, including material from a donor who later developed vCJD, suggesting that vCJD might have been
transmitted by clotting factor concentrates.51,52 vCJD infection is extremely rare in the United States. The few
reported cases have been in individuals who most likely acquired their infections elsewhere, and no US
transfusion-transmitted cases have been reported.
There are no FDA-approved donor screening tests for prion infections. Blood donors in the United States
are screened solely by questioning and are excluded if they have an
203
increased risk of either CJD or vCJD. CJD exclusions are based on family history of the disease, receipt of
human growth hormone derived from pituitary glands, or receipt of a dura mater tissue graft. vCJD exclusions
are for residence in the United Kingdom or Europe during specified times when BSE was endemic, receipt of a
transfusion in the United Kingdom or France, or receipt of UK bovine insulin.95 It is thought that plasma
derivative manufacturing processes remove substantial amounts of TSE infectivity.95
In-Process Screening for Plasma Derivatives
Commercial plasma derivatives are prepared from large pools of plasma derived from thousands of donors.
Before the incorporation of specific pathogen-reduction processes, contamination of these large pools with viral
agents was common. Today, plasma derivative manufacturing processes incorporate methods—such as
prolonged heat or solvent/detergent (SD) treatment—that remove or inactivate most known pathogens. SD
treatment inactivates lipid-enveloped agents, such as HIV HCV, and HBV. Pathogen infectivity may also be
reduced by nanofiltration, chromatography, or cold ethanol fractionation, which are used in the production of
certain products. Not all infectious agents, however, are removed or inactivated by these processes.
One agent that can persist in plasmaderivative products is human parvovirus B19. This small, nonenveloped
DNA virus is extremely resistant to physical inactivation. Acute infection is typically mild and selflimited;
clinical manifestations include “fifth disease” (erythema infectiosum) and polyarthropathy. Acute infection is
associated with transient red cell aplasia that may be clinically significant in immunodeficient individuals and
those with underlying hemolytic processes. The aplasia in immunodeficient individuals can be prolonged.
Intrauterine infection is associated with severe fetal anemia and hydrops fetalis.
Parvovirus B19 infection is very common; most adults have antibodies to this agent, indi
cating previous exposure. Levels of viral DNA during acute infection may exceed 1012 IU/mL, decreasing
over weeks to months in association with antibody production.
Viral DNA, mosdy at low concentrations, has been detected in approximately 1% of blood donations and in
essentially all lots of pooled plasma derivatives. Transmission of parvovirus B19 by transfusion has been linked
only to blood components or plasma products that contain high concentrations of viral DNA; only one
transmission has been documented with a product containing less than 104 IU/mL.52
Currently, there is no FDA-approved test to screen fresh blood donations for parvovirus B19 infection.
However, plasma-derivative manufacturers require screening of incoming plasma units for the presence of high-
titer parvovirus B19. This is accomplished by performing NAT on pools of samples from plasma units, with
sensitivity adjusted to detect only units with a high concentration of virus. By excluding high-titer units from
the plasma pools, the final titer in the plasma pool is kept below 104 IU/mL.
Other Agents
The AABB maintains a publicly accessible electronic resource containing expert analyses of emerging
infectious disease (EID) agents that have received attention as potential threats to the US or global blood
supply.52 This digital resource contains up-to-date fact sheets on a variety of agents. Each fact sheet includes
information about clinical manifestations and epidemiology of infection, evidence of transfusion
transmissibility, and analyses of the potential effectiveness of various mitigation strategies (eg, donor
questioning, serologic testing or NAT, or pathogen reduction). Readers are encouraged to use this rich resource.
One agent that has received recent attention is hepatitis E virus (HEV), a small, nonenveloped, single-
stranded RNA virus. HEV is thought to be primarily transmitted through food and water sources. However,
recent studies have demonstrated asymptomatic viremia
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in blood and plasma donors, and transfusion transmission has been documented.52,96 As a nonenveloped
agent, HEV is not susceptible to SD treatment. NAT screening of donors and/or heat inactivation of plasma
pools may be mitigation strategies if this agent is deemed to pose a clinically important threat.
PATHOGEN REDUCTION TECHNOLOGY
Donor screening reduces, but cannot eliminate, the infectious risks of blood transfusion. The efficacy of
blood donor testing is limited by a number of factors, including the following:
1. It is not logistically feasible to test donors for every infection that is conceivably transmissible by
transfusion.
2. For every test, there is a lag time (window period) between when a person becomes infected and when the
test detects infection.
3. Every test has limited sensitivity (concentration of the target marker that can be detected by the test).
4. Developing a donor test is a long, multiphase process that includes identification of the infectious agent,
selection of the type of test that would be effective in interdicting infectious donations (eg, serology vs NAT),
development of a test suitable for donor screening, performance of clinical trials of the test, and regulatory
approval. During this development process, infections can be transmitted.
Pathogen reduction technology (PRT) provides an attractive alternative to relying on donor testing to
interdict all infectious donations. PRT processes reduce the infectivity of residual pathogens in blood
components. This approach could reduce the transmission of infectious agents for which there are no donor
screening tests and further reduce the residual transmission risks of known agents. PRT could theoretically
enable discontinuation of some testing that is currently performed (eg, CMV
testing and bacterial testing of platelets), and some PRT methods could obviate the need for irradiation,
potentially offsetting some of the cost of PRT.
As discussed above, PRT is now an essential component of the plasma-derivative manufacturing process.
SD treatment can also be applied to pools of plasma for transfusion. An SD-treated pooled plasma product has
been approved for use in the United States.
No PRT processes are approved in the United States for treatment of individual units of plasma or for
cellular blood components, although some technologies are in use outside the United States. Processes that are
available or in development have been recently reviewed in detail and are summarized in Table
51,52,97,98
The benefit to be gained from PRT in the United States is primarily the mitigation of emerging pathogens
and platelet-associated bacterial sepsis. Currently, the quantifiable infectious risks of transfusion in the United
States are low. Therefore, it is critically important to demonstrate that PRT treatments do not introduce new
hazards to patients. Rigorous preclinical and clinical studies are required for US regulatory approval of PRT.
Toxicology studies are critical because most of these agents interact with nucleic acid, raising the theoretical
potential of carcinogenicity and mutagenicity. Treated products should be assessed for neoantigen formation
and the impact of the PRT on the final product’s clinical efficacy. The evaluation process for PRT in North
America was the subject of a recent consensus conference.97,99
Documented transmission of new infectious agents for which there are no donor screening tests could
influence the risk/benefit assessment of PRTs. It is important, though, to keep in mind that there are limits to the
viral loads that are inactivated by these processes, and not all agents are inactivated by PRTs. Pooled SD
plasma, for example, would have a reduced risk of transmitting most infectious agents but a potentially
increased risk of transmitting an agent that lacks a lipid envelope.
205
TABLE 8-7. Pathogen Reduction Technologies for Transfusable Blood Components
Component Technology Manufacturer
Plasma: commercially prepared pools Solvent/detergent treatment Octapharma
Plasma: individual units ■ Amotosalen (psoralen) + UV light Cerus
■ Riboflavin (vitamin B2) + UV light Terumo BCT
■ Methylene blue + light Macopharma
Platelets ■ Amotosalen (psoralen) + UV light Cerus
■ Riboflavin (vitamin B2) + UV light Terumo BCT
■ UV light Macopharma
Red Blood Cells ■ Frangible nucleic acid crosslinker Cerus
■ Riboflavin (vitamin B2) + UV light Terumo BCT
UV = ultraviolet.
SUMMARY
The current level of safety of blood components is based on two critical elements of donor screening: donor
questioning, which is the sole method of screening for certain agents, such as malaria and prions, and donor
testing. Testing must be performed carefully and in accordance with manufacturers’ instructions, and facilities
must have robust systems for quarantining components of donations that test positive and for retrieving prior
donations from donors whose samples have tested positive.
Current quantifiable risks of infectious disease transmission are very low; the estimated risk of HIV
transmission by transfusion in the United States is approximately 1 in 1.5 million units, the risk of HCV
transmission is approximately 1 in 1.1 million units, and the risk of HBV transmission is approximately 1 in
800,000 to 1 in 1.2 million units.46,50 However, it is critical to remain vigilant for evidence of new infectious
agents and to implement mitigation measures as quickly as feasible. PRT may, in the future, provide some
protection against emerging infectious agents for which no screening is in place.
KEY POINTS
1. Infectious disease screening of donors is accomplished by 1) questioning potential donors and excluding
those with an increased risk of infection, and 2) testing donated blood.
 2. There is a delay between the time when an individual is exposed to an infection and the time when the
donor screening test for the infection yields a positive result. Blood donated during this window period can
transmit infections.
3. The estimated window period with MP-NAT of donor samples is less than 10 days for HIV and HCV and
less than 28 days for HBV
4. The residual risk of transfusion-transmitted infection is a function of the length of the window period and
the incidence of infection in the donor population. Maintaining a donor population that has a low incidence of
infection continues to play a key role in preserving blood safety.

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5. Based on window-period and incidence calculations, the current risk of HIV transmission by transfusion
in the United States is approximately 1 in 1.5 million units, the risk of HCV transmission is approximately 1 in
1.1 million units, and the risk of HBV transmission is approximately 1 in 800,000 to 1 in 1.2 million units.
6. There are no donor screening tests approved by the FDA for malaria or vCJD. Donor questioning about
potential exposure is the sole means of protecting the US blood supply from these diseases.
7. AABB requires blood banks to have processes that limit and detect or inactivate bacteria in platelet
components. Pathogen reduction methods have replaced bacteria testing in some regions outside the United
States.
8. Infections transmitted to humans by vectors are increasingly recognized as a potential source of
transfusion-transmitted infection. These include WNY T. cruzi, Babesia species, and dengue virus.
9. PRTs may reduce the transmission of infectious agents for which there are no donor screening tests, and
PRTs may further reduce the residual transmission risks of known agents. PRTtreated plasma derivatives and
pooled plasma are available in the United States. Some PRT systems are available outside the United States for
treatment of individual platelet and plasma components, but as of early 2014, these were not approved for US
use.
10. Blood banks must have processes in place to ensure that donations with positive test results are not
released for transfusion. In some circumstances, 1) prior donations from those donors must also be retrieved and
quarantined, and 2) recipients of prior donations must be notified of their possible exposure to infection.
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83. Johnson ST, Cable RG, Tonnetti L, et al. Seroprevalence of Babesia microti in blood donors from
Babesia-endemic areas of the northeastern United States: 2000 through 2007. Transfusion 2009;49:2574-82.
84. Food and Drug Administration. Blood Products Advisory Committee, July 26,2010, Gaithersburg, MD.
Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 2010 [Available at
http://www.fda.gov/ downloads/AdvisoryCommittees/Commit teesMeetingMaterials/BloodVaccinesand
OtherBiologics/BloodProductsAdvisoryCom mittee/UCM225388.pdf (accessed December 8,2010).]
85. Moritz ED, Johnson ST, Winton C, et al. Prospective investigational blood donation screening for
Babesia microti. Transfusion 2013; 53(Suppl):13A.
86. Mali S, Steele S, Slutsker L, Arguin PM. Malaria surveillance—United States, 2007. MMWR Surveill
Summ 2009;58:1-16.
87. Eliades MJ, Shah S, Nguyen-Dinh P, et al. Malaria surveillance—United States, 2003. MMWR Surveill
Summ 2005;54:25-39.
88. Purdy E, Perry, E, Gorlin, J. et al. Transfusiontransmitted malaria: Unpreventable by current donor
guidelines? Transfusion 2003;43 (Suppl):79A.
89. Gonzalez C, Layon A, Arguin R et al. Transfusion-transmitted falciparum malaria from an
asymptomatic donor. Transfusion 2011:51 (Suppl):197A.
90. Mali S, Tan KR, Arguin PM. Malaria surveillance—United States, 2009. MMWR Morb Mortal Wkly
Rep 2011;60:1-15.
91. Spencer B, Steele W, Custer B, et al. Risk for malaria in United States donors deferred for travel to
malaria-endemic areas. Transfusion 2009;49:2335-45.
92. Food and Drug Administration. Guidance for industry: Recommendations for donor questioning,
deferral, reentry and product management to reduce the risk of transfusiontransmitted malaria. (August 2013)
Silver Spring, MD: CBER Office of Communication, Outreach and Development, 2013. [Available at http:/
/www.fda.gov/downloads/Biologics BloodVaccines/GuidanceComplianceRegula torylnformation/Guidances/
Blood /UCM 080784.pdf (accessed November 14,2013).]
93. Anez G, Rios M. Dengue in the United States of America: A worsening scenario? Biomed Res Int
2013:2013:678645.
94. Petersen LR, Busch MP. Transfusion-transmitted arboviruses. Vox Sang 2010;98:495-503.
95. Food and Drug Administration. Guidance for industry: Revised preventive measures to reduce the
possible risk of transmission of Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-Jakob disease (vCJD)
by blood and blood products. (May 2010) Silver Spring, MD: CBER Office of Communication, Outreach, and
Development, 2010. [Available at http:// www.fda.gov/downloads/biologicsbloodvac
cines/guidancecomplianceregulatoryinfor mation/guidances/UCM213415.pdf (accessed December 4,2013).]
96. Slot E, Hogema BM, Riezebos-Brilman A, et al. Silent hepatitis E virus infection in Dutch blood donors,
2011 to 2012. Euro Surveill 2013; 18.
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97. Webert KE, Cserti CM, Hannon J, et al. Proceedings of a consensus conference: Pathogen inactivation-
making decisions about new technologies. Transfus Med Rev 2008;22:1-34.
98. AuBuchon J, Prowse C, eds. Pathogen inactivation: The penultimate paradigm shift. Bethesda, MD:
AABB Press, 2010.
99. Klein HG, Anderson D, Bernardi MJ, et al. Pathogen inactivation: Making decisions about new
technologies. Report of a consensus conference. Transfusion 2007;47:2338-47.
Chapter 9
Hospital Storage, Monitoring, Pretransfusion Processing, Distribution, and Inventory Management of Blood
Components
Nancy M. Dunbar, MD
gnBLOOD COMPONENT MANUFACBIS Turing is highly regulated and must comply with Food and
Drug Administration (FDA) current good manufacturing practice (cGMP) regulations from the time of
collection to product issue.1,2 The intent of cGMP is to maintain the safety, purity, potency, quality, and identity
of blood components. Collection and transfusion facilities must develop policies, processes, and procedures and
retain records to demonstrate compliance with regulations for storage, monitoring, pretransfusion processing,
and distribution of blood components. These policies, processes, and procedures must also comply with
additional regulations (Clinical Laboratory Improvement Amendments and state laboratory statutes) and
policies and standards applicable to blood banking from voluntary laboratory accrediting organizations (eg,
College of American Pathologists, AABB,3 The Joint Commission, and American Osteopathic Association).
BLOOD AND BLOOD COMPONENT STORAGE AND MONITORING
General Considerations
Transport and storage requirements must be followed when blood components are transferred from the
collection site to the processing facility, from the supplier to the blood bank, or from the blood bank to the
patient. Storage requirements and expiration dates vary by product type and are based on factors such as in-vitro
red cell metabolism for Red Blood Cell (RBC) components in various storage solutions or coagulation protein
stabilization for plasma products (Table 9-1). Failure to adhere to these storage and expiration requirements can
result in decreased product potency and/or safety.
Temperature requirements during transport of blood components differ from those during storage.4 Shipping
from the supplier to

Nancy M. Dunbar, MD, Assistant Professor, Dartmouth-Hitchcock Medical Center, Lebanon, New
Hampshire The author has disclosed no conflicts of interest.
213

214
AABB TECHNICAL MANUAL
the hospital blood bank is considered transport, and applicable temperature requirements must be met.
When blood components are issued from the blood bank to the patient care area, maintenance of appropriate
temperature requirements allows for the possibility of returning the component to inventory if it is not
transfused.
Refrigerators, freezers, and platelet incubators for blood and blood component storage can be equipped with
continuous-temperature-monitoring devices to allow detection of temperature deviations before products are
affected. Automated electronic monitoring devices that are available include: 1) weekly pen and chart recorders,
2) sets of hard-wired or radio-frequency temperature-recording devices, and 3) centralized temperature-
monitoring systems. Thermometers or thermocouples should be strategically placed in the equipment for
optimal temperature monitoring. If an automated temperature-recording device is not used, temperatures of the
blood storage environment must be recorded manually every 4 hours. This requirement includes ambient room
temperature monitoring of platelets that are not stored in a platelet chamber or incubator.
Recorded temperatures should be checked daily to ensure proper operation of the equipment and recorder.
Deviations from acceptable temperature ranges should be documented and explained (including any actions
taken), dated, and initialed by the person noting the deviation.
Most product-storage devices are equipped with audible alarms to alert personnel that temperature ranges
are approaching unacceptable levels. Central alarm monitoring allows facilities that do not have personnel in the
vicinity of the equipment to alert designated staff at another location when an alarm is activated. Because
platelets must be gently agitated during storage, typically using horizontal flatbed or elliptical rotators, alarm
systems should also emit alerts when the platelet agitator has malfunctioned.
Transfusion services may locate blood storage refrigerators in other areas of the hos
pital to allow immediate access to blood in emergency situations. Such a practice requires that the same
blood component storage monitoring standards be met in these other areas.
If an equipment failure occurs and prevents acceptable temperature ranges from being maintained, the
facility should have policies, processes, and procedures in place to relocate blood and blood components. The
secondary storage location may be another on- or off-site refrigerator or freezer, qualified storage boxes, or
coolers used with a validated process that has been shown to maintain required storage temperatures during
storage. Because the safety, purity, potency, and quality of the blood components could be affected by delays in
relocation to a secondary storage location, it is recommended that the relocation occur before upper or lower
acceptable storage temperatures are exceeded. This can be accomplished by setting the alarm points of the
storage devices so that an alarm sounds before the unacceptable tempature limit is reached.
 Some facilities may use temperaturemonitoring indicators for each blood component container. Such
indicators monitor the liquid temperature of the immediate inner bag, not the liquid core temperature in the unit,
which may be cooler. Policies, processes, and procedures should specify how the facility will determine the
disposition of blood components when using temperature-monitoring indicators.
Specific Considerations
Holding blood components that have been dispensed from the blood bank to other hospital areas before
transfusion is considered to be “storage.” If the blood components are not kept in a monitored device, they must
be stored in containers (eg, boxes or coolers) validated to maintain the correct temperature during storage.
Red Blood Cells
RBC components are stored in plastic bags of different types with a variety of added anticoagulants and
additive solutions that modify
TABLE 9-1. Requirements for Storage, Transportation, and Expirati
CHAPTER 9 Storage, Processing, Distribution, and Inventory
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CHAPTER 9 Storage, Processing, Distribution, and Inventory
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 the cellular and protein environment. The storage temperature must be maintained at 1 to 6 C throughout the
duration of storage.3(pp51'53) Changes that may occur during storage directly affect how long RBC units may
be stored. Product approval by the FDA requires in-vivo labeling studies demonstrating that at least 75% of the
transfused red cells are present in circulation 24 hours after transfusion with less than 1% hemolysis.
During ex-vivo storage of RBC units, biochemical and morphologic changes to the red cells occur that have
been termed the “storage lesion.” These changes include cell membrane shape change and microvesiculation;
decreased pH, adenosine triphosphate, and 2,3diphosphoglycerate; and increased lysophospholipids, potassium,
and free hemoglobin.5 Although these ex-vivo observations are well described, there is currently insufficient
evidence to conclude that storage duration correlates with clinical outcomes.6,7 One situation where evidence
supports the use of fresher RBCs is large-volume transfusions (>25 mL/ kg) in neonates.8
Platelets
Platelet storage conditions and shelf life are affected by morphologic and functional changes during storage.
Metabolic changes include the glycolytic production of lactic acid and the oxidative metabolism of free fatty
acids, which results in the production of carbon dioxide. Platelet pH is maintained above 6.2 via buffering of
lactic acid by bicarbonate and promotion of oxidative metabolism facilitated by the diffusion of oxygen and
carbon dioxide across a gas-permeable storage bag during gentle agitation.9 The maximum time for platelets to
be without agitation is 24 hours.3(pp53‘55)
Platelet shelf life also is limited because of an increased risk of bacterial growth due to room-temperature
storage. Blood banks and transfusion services are required to have methods to detect or inactivate bacteria in all
platelet components.3(pll)
Granulocytes
Granulocytes are fragile, deteriorate rapidly in vitro, and should be transfused as soon as possible after
receipt from the supplier. Granulocytes are stored at 20 to 24 C, should not be agitated, and must never be
leukocyte reduced.
PRETRANSFUSION
PROCESSING
Thawing Plasma and Cryoprecipitate
Fresh Frozen Plasma (FFP), Plasma Frozen within 24 Hours After Phlebotomy (PF24), and Plasma Frozen
within 24 Hours After Phlebotomy Held at Room Temperature up to 24 Hours After Phlebotomy (PF24RT24)
must be thawed at 30 to 37 C using a waterbath or other FDA-approved device. Thawing in a waterbath
requires the frozen component to be in a plastic overwrap before insertion into the water to prevent
contamination of the container entry ports. Thawed plasma products (FFP, PF24, and PF24RT24) are stored at 1
to 6 C and expire 24 hours after thawing.
These products must be relabeled as “Thawed Plasma” if they are stored for longer than 24 hours. Although
not licensed by the FDA, Thawed Plasma is included in the AABB Standards for Blood Banks and Transfusion
Services3(p28) and the Circular of Information for the Use of Human Blood and Blood Components.10 Thawed
Plasma is stored at 1 to 6 C and expires 5 days after it was originally thawed. Facilities may label such products
as “Thawed Plasma” at the initial time of thawing. By maintaining a Thawed Plasma inventory, transfusion
services may decrease wastage of thawed plasma products.11
Levels of labile coagulation factors (Factor V and Factor VIII) and stable factors are well above 50% of
immediate post-thaw levels in Thawed Plasma that has been stored for up to 5 days.12 Thawed Plasma does,
however, contain reduced concentrations of Factor V, Factor VII, and Factor VIII. For this reason, Thawed
Plasma is not suitable for single-factor replacement when antihemophilic factor derivatives are unavailable.
222
AABB TECHNICAL MANUAL
Cryoprecipitate is thawed at 30 to 37 C, is gently resuspended, and can be pooled for ease of transfusion
using small quantities of 0.9% sodium chloride, injection (USP) to rinse the contents of the bag into the final
container (Method 6-11). Thawed cryoprecipitate is stored at 20 to 24 C and expires within 4 hours of pooling if
it is pooled in an open system or within 6 hours for single units or units pooled using an FDA-cleared sterile
connecting device.10
Thawing and Deglycerolizing RBCs
RBC units may be frozen and stored for up to 10 years following the addition of glycerol as a
cryopreservation agent (Methods 6-6 and 6-7).13,14 Frozen units can be thawed using a 37 C dry heater or 37 C
waterbath. After being thawed, the glycerol must be removed before the component is transfused. Commercial
instruments for batch or continuous-flow washing are available for deglycerolization. The manufacturer’s
instructions should be followed to ensure maximal red cell recovery and minimal hemolysis. Measurement of
free hemoglobin in the final wash can be used to confirm adequate free hemoglobin removal and as a surrogate
marker for adequate deglycerolization (Method 6-8).
Integrally attached tubing must be filled with the deglycerolized red cells and sealed appropriately so that a
segment may be detached and available for crossmatch testing.
The shelf life of Deglycerolized RBCs depends on the type of system used. Closedsystem devices allow
storage for up to 14 days, but components prepared using open systems expire within 24 hours of
deglycerolization.
Platelet Gel Production
Platelet gel is produced when thrombin and calcium are added to platelet-rich plasma to produce a glue-like
substance for surgical application.15 This product is typically prepared at the bedside immediately before use.
Facilities involved in the production of this component should refer to the current edition of the AABB
Standards for Perioperative Autologous Blood Collection and Administration for guid
ance and quality oversight of this manufacturing process.
Irradiation
Irradiation of cellular components is intended to prevent transfusion-associated graft-vshost disease (TA-
GVHD), which is caused by proliferation of donor T lymphocytes. People at increased risk of TA-GVHD
include profoundly immunocompromised patients, recipients of intrauterine transfusion, patients undergoing
marrow or peripheral blood stem cell transplantation, and recipients of cellular components from blood relatives
or donors selected for HLA compatibility.
Sources of radiation include gamma rays (cesium-137 or cobalt-60 radioisotopes) and x-rays. The required
gamma radiation dose needed to prevent proliferation of donor T lymphocytes in the recipient is a minimum of
25 Gy (2500 cGy/rad) to the central point of the blood container and 15 Gy (1500 cGy/rad) to any other part of
the container. Confirmation that the blood container has received an adequate radiation dose can be achieved
with the use of commercially available radiographic film labels.
Irradiation is associated with damage to the red cell membrane, which may result in increases in
extracellular free hemoglobin and potassium during product storage. For this reason, the expiration date of
irradiated RBCs is 28 days after irradiation or the original expiration date, whichever is earlier.
Hospital transfusion services may purchase irradiated blood components from their supplier or perform
irradiation within the blood bank using approved and monitored radiation devices. Hospitals that perform their
own irradiation may irradiate their inventory on demand or in batches. Maintenance of a dual inventory
(irradiated and nonirradiated) requires policies and procedures to ensure that transfusion recipients receive the
appropriate component for their clinical situation.
Poststorage Leukocyte Reduction
Poststorage leukocyte reduction can be performed by the blood bank before issuing a
223
component using a leukocyte reduction filter attached by a sterile connection. It can also be performed at the
bedside during transfusion using a blood-administration filter designed for this purpose. Leukocyte reduction
filters are designed to remove >99.9% of white cells (3-log reduction) and meet the AABB standard of less than
5 x 106 leukocytes in 95% of sampled units for RBCs and Apheresis Platelets.3(p24) The manufacturer’s
instructions must be followed for the filtration device used to achieve acceptable leukocyte reduction.
Prestorage leukocyte reduction is generally the preferred method for leukocyte reduction because it prevents
accumulation of cytokines during product storage. Quality control of bedside filtration is challenging, and the
process has been associated with hypotensive transfusion reactions.16
Volume Reduction
Volume reduction results when plasma and additive solutions are partially removed from RBC or platelet
components following centrifugation. This process may be used to aggressively manage volume in patients at
risk of transfusion-associated circulatory overload, reduce exposure to plasma proteins or additives, or achieve a
target hematocrit level.
Volume reduction of platelets is described in Method 6-13. The speed of centrifugation may affect the
degree of platelet loss. Higher g forces are associated with better platelet retention but raise the theoretical
concern of platelet damage and activation as platelets are forced against the container wall. When platelet
volumes are reduced, platelets should rest at room temperature for 20 to 60 minutes following centrifugation
and before resuspension in remaining plasma or added saline. The manufacturer’s instructions must be followed
regarding the minimum volume necessary to maintain proper air exchange across the gaspermeable platelet-
storage bag. The shelf life of volume-reduced platelets is 24 hours for components stored at 1 to 6 C and 4
hours for those stored at 20 to 24 C.
Washing
Cellular components are typically washed to remove plasma proteins. Washing can also be performed to
remove glycerol from frozen RBC units after thawing. Indications for washing RBC or platelet components
include a patient history of severe allergic reactions to components containing plasma, the presence of
antibodies against immune globulin A (IgA) in an IgA-deficient recipient when IgA-deficient cellular
components are not available, the presence of maternal anti-HPA-la (eg, when using maternal blood for a
neonatal transfusion), and the need for complement removal.
Washing is accomplished with the use of 1 to 2 L of sterile normal saline (preferably using automated
equipment). As with volume reduction, washed platelets should rest at room temperature without agitation
between centrifugation. Up to 20% of the red cell yield or 33% of the platelet yield may be lost during washing.
Because washing creates an “open system” and removes anticoagulant-preservative solutions, washed RBC
units expire 24 hours after washing and washed platelet units expire 4 hours after washing. It is recommended
that hospitals performing washing comply with the manufacturer’s recommendations regarding minimum
volumes needed for component storage bags to maintain optimal storage conditions.
Pooling
Certain blood components (whole-bloodderived platelets, cryoprecipitate, or RBCs and plasma to produce
reconstituted whole blood) may need to be pooled to provide clinically effective transfusion therapy without the
need to transfuse multiple single components.
Pooled whole-blood-derived platelets may contain a significant number of red cells, and, therefore, ABO
compatibility and risk for RhD alloimmunization are recipient factors that must be considered. If whole-
bloodderived platelets are pooled using an open system, the expiration time is 4 hours from the start of pooling.
A commercially available, FDAapproved, prestorage, whole-blood-derived,
224
AABB TECHNICAL MANUAL
platelet pooling system allows storage for up to 5 days and the ability to perform culturebased bacterial
testing.17 When this system is used, the pool maintains the expiration date of the earliest collected component
in the pool.
Single cryoprecipitate units are pooled after thawing in a manner similar to that used for platelets. The
expiration time of cryoprecipitate pools depends on the method used for pooling. Cryoprecipitate pooled in an
open system expires within 4 hours of pooling. Thawed single concentrates and pooled concentrates using
sterile connecting devices expire 6 hours after thawing. Thawed cryoprecipitate is stored at 20 to 24 C. As an
alternative, the blood center may pool single concentrates before freezing.
Reconstituted whole blood consists of RBCs combined with ABO-compatible FFP. This product can be
used for neonatal exchange transfusion. The conventional approach is to combine group 0 RBCs (Rh
compatible with the neonate) and group AB FFP to achieve a 50% ± 5% hematocrit of the final product. The
volumes of the two components before pooling can be adjusted to achieve a desired hematocrit level. Following
recombination, the product can be stored at 1 to 6 C for up to 24 hours.
Current FDA uniform guidelines should be followed when pooled products are labeled.18 A unique pool
number should be affixed to the final container, and all units in the pool must be documented in electronic or
manual records.
Aliquoting
Patients requiring low-volume transfusions may receive aliquots of smaller volumes derived from the
original unit via an FDA-cleared sterile connecting device or integrated transfer bags. Available products
designed for use with sterile connecting devices include transfer packs, smaller-volume bags, and tubing with
integrally attached syringes.
The expiration date of the aliquot and minimum residual volumes that must be
maintained depend on the storage container used. Hospital transfusion services must develop policies and
procedures for aliquot preparation and storage that comply with manufacturer specifications. The use of aliquots
for neonatal transfusion has been shown to result in a decreased number of donor exposures.19 The process of
preparing aliquots for small-volume transfusion in neonates and children is discussed in greater detail in
Chapter 23. Lower-volume components (split units) may also be prepared for adult patients who require slow
rates of transfusion due to concerns about fluid overload. Split units are recommended when the component
volume cannot be transfused at a rate that ensures completion of the transfusion within 4 hours.
DISTRIBUTION
Inspection
Inspection is a critical control point in blood component manufacturing and must occur before shipping,
upon receipt, and before issue for transfusion. Proper documentation of this process includes 1) date of
inspection, 2) donor identification number, 3) description of any visual abnormalities, 4) action(s) taken, and 5)
identity of the staff member performing the inspection. Visible abnormalities may include discoloration of the
segments, component, or supernatant fluid or the presence of visible clots, particulate matter, or other foreign
bodies. Detection of any such abnormalities should result in product quarantine for further investigation that
may include returning the component to the supplier.
If a component is determined to be bacterially contaminated, the component manufacturer must be notified
so that an immediate investigation can take place. Other components prepared from that collection should be
quarantined until the investigation is complete. If the component (or co-component) has been transfused, the
recipient’s attending physician should be notified, and consultation with the medical director is recommended.
225
Shipping
Blood components may be transported between blood centers, hospitals, and blood centers. All containers
used to transport blood components must be qualified before use to ensure that the proper component transport
temperature is maintained. The shipping transit time, mode of transport, and climatic conditions must also be
validated. All components should be inspected upon receipt to confirm appropriate transport conditions,
component appearance, and expiration date. Any deviation from routine shipping or component conditions
should be reported to the shipping facility and documented according to each location’s policies, processes, and
procedures.
Whole Blood, RBCs, and Thawed Plasma Products
Whole blood, RBCs, and thawed plasma products must be transported at a temperature of 1 to 10 C. A
variety of options exist for maintaining transport temperature, including bagged wet ice, commercial cooling
packs, and specially designed containers. All transport coolers must be qualified to maintain the transport
temperature when packed using a validated process.
Blood components transported at 1 to 10 C and stored at 1 to 6 C may need to be temporarily removed from
those temperatures for entry into inventory, irradiation, or other processing. The maximum number of units that
can be manipulated before the component reaches an unacceptable temperature should be determined and not
exceeded. Validation of this process may be accomplished using manual temperature monitoring indicators
affixed to the blood components or electronic devices that can measure the temperature of the blood
components without opening the bag.
Platelets, Thawed Cryoprecipitate, and Granulocytes
Platelets, thawed cryoprecipitate, and granulocytes must be transported at a temperature of 20 to 24 C. All
transport coolers must be qualified to maintain this transport tempera
ture when packed using a validated process. For platelets, the maximum time without agitation is 24 hours.
Frozen Components
Frozen components should be packaged to minimize breakage and maintain a frozen state. Dry ice in a
suitable container is routinely used for shipping these components. All transport coolers must be qualified to
maintain the transport temperature when packed using a validated process.
Receiving
The receiving facility should notify the shipping facility and document any deviation from usual shipping
container packing or appearance of the shipped blood components. Any blood component not complying with
the facility’s policies, processes, and procedures should be quarantined. Only after investigation of the deviation
and determination that the component meets acceptance criteria may the component be removed from
quarantine and released into the general inventory.
Blood components should be fully traceable from collection to final disposition. Electronic or manual
records indicating compliance with policies, processes, and procedures should be generated and maintained for
the applicable record-retention time. Any deviation must be recorded, and blood components not meeting
requirements should be quarantined. Deviations must be investigated to determine appropriate product
disposition and possible corrective action. The results of any corrective action should be reported to the blood
supplier as needed. Inventory management should consist of routine determination that all blood components
are accounted for and transfused or appropriately discarded.
Product Testing
Before transfusion, the ABO group of all units and Rh type of any units labeled “Rh negative” must be
confirmed by serologic testing for all red-cell-containing components (RBCs, whole blood, and granulocytes).
Any typing discrep
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AABB TECHNICAL MANUAL
ancies identified must be reported immediately to the supplier and resolved before the product is dispensed
for transfusion.
Issuance of Components
Ensuring that the correct blood component is transfused to the correct patient is paramount for transfusion
safety. All requests for blood components must contain two independent identifiers so that the intended
recipient can be uniquely identified. Recipient compatibilitytesting records must also be reviewed. Current
testing results must be compared with historical records, if available, and any discrepancies must be resolved
before product selection.
Personnel must visually inspect and document that the selected product is acceptable for use. This
inspection must include confirmation that the product does not have an abnormal color or appearance and that
the container is intact. Once selected for transfusion, the blood component must have an attached label or tie tag
that contains the intended recipient’s two independent identifiers, donation identification number, and
compatibility test result interpretation, if performed.
At the time of issue, there must be a final check of each unit that includes the following:
1. The intended recipient’s two independent identifiers, ABO group, and Rh type.
2. The donation identification number, donor ABO group, and, if required, Rh type.
3. The interpretation of the crossmatch test results, if performed.
4. Special transfusion requirements (eg, cytomegalovirus-reduced-risk, irradiated, or antigen-negative
components), if applicable.
5. The expiration date and, if applicable, time.
6. The date and time of issue.
The transfusion service must confirm that the recipient identifying information, transfusion request, testing
records, and blood component labeling and compatibility are accurate and in agreement. Any discrepancies
identified must be resolved before issue.
Final identification of the transfusion recipient and blood component rests with the transfusionist. This
individual identifies the patient and donor unit and certifies that the identifying information on forms, tags, and
labels is in agreement.
Documentation
The patient’s medical record must include proper documentation of all transfusions. For each transfusion,
this documentation must contain the transfusion order, consent for transfusion, component name, donation
identification number, date and time of transfusion, pre- and posttransfusion vital signs, volume transfused,
identification of the transfusionist, and, if applicable, any transfusionrelated adverse events.
Return of Blood Components and Reissue
The transfusion service may receive back into inventory units that meet acceptance specifications. These
conditions include the following:
1. The primary container has not been opened.
2. The component has been maintained at the appropriate temperature.
3. At least one sealed segment remains integrally attached to the container of RBCs.
4. Documentation indicates that the component has been inspected and is acceptable for reissue.
Individual unit temperature indicators or temperature-reading devices can be used to determine the
acceptability of products for return to inventory. Blood and blood components may also be transported or stored
in qualified containers using a validated process that has been shown to maintain acceptable temperatures for a
defined interval. If time frames are used to determine the acceptability of a product’s return to inventory, the
time frame must be validated by the individual facility. The validation should demonstrate that for the defined
period, the appropriate temperature of the product has been maintained.
 227
Components meeting the acceptance criteria may be returned to the general blood inventory and reissued.
Components not meeting the acceptance criteria must be quarantined for further investigation or discarded in a
biohazard container to prevent inadvertent return to inventory.
INVENTORY MANAGEMENT
General Considerations
A sufficient number of ABO- and Rh-compatible units should be available to meet routine hospital needs,
allow for unanticipated increases in utilization due to emergency situations, and minimize component outdating.
Factors that influence determination of blood bank component inventory levels include historic usage patterns,
outdate rates, and distance from suppliers. Inventory levels should be periodically evaluated in response to
institutional changes that may affect component usage, including expansion of inpatient beds or operating
rooms; implementation of new surgical procedures; or changes in hospital guidelines or medical practice that
may influence transfusion behavior.
The blood bank should also maintain a reserve of universally compatible RBCs for emergency use and have
a reliable emergency delivery system to ensure adequate availability of blood components in unexpected
situations when demand exceeds supply. A disaster plan should be developed and tested periodically (see
Chapter 4).
Inventory levels should be monitored daily to facilitate timely ordering from blood suppliers and maintain
adequate inventory levels. This can be particularly challenging for platelets due to their 5-day shelf life.
Inventorymanagement plans should also take into consideration desirable inventory levels of special products,
such as cytomegalovirus-seronegative, leukocyte-reduced, and irradiated components. Antigen-negative RBC
units and HLA-matched or HLA-selected platelets are typically ordered on an as-needed basis from suppliers.
Surgical Blood Ordering Practices
Component outdate rates are influenced by surgical ordering practices. For example, when RBC units are
crossmatched for surgical patients, the shelf life of the unit is shortened if the component is unused. When
crossmatchto-transfusion (C:T) ratios are monitored, a C:T ratio of >2.0 may indicate excessive ordering of
crossmatched blood.
One approach to reducing excessive C:T ratios is to identify procedures that do not typically require blood,
and use this information to develop guidelines for the use of type and screen units instead of crossmatched
units. Maximum surgical blood order schedules for common elective procedures can also be developed based
on local transfusion utilization patterns.20 This practice is particularly useful in hospital transfusion services
that lack the ability to perform electronic crossmatching. These schedules can also be applied to patients
undergoing elective surgery who are known to have clinically significant alloantibodies and require crossmatch-
compatible, antigen-negative blood. Once a surgical blood-ordering schedule has been established, the
transfusion service routinely crossmatches the predicted number of units for each patient undergoing the
designated procedures. Routine orders may need to be modified for patients with anemia, bleeding disorders, or
other conditions in which increased blood use is anticipated. As with other circumstances that require rapid
availability of blood components, the transfusion service staff should be prepared to provide additional blood
components if the need arises.
Emergent Transfusion
If transfusion is deemed medically necessary and blood is released before pretransfusion testing is complete,
the records must contain a signed statement from the requesting physician indicating that the clinical situation
was sufficiently urgent to require the release of blood components before completion of compatibility testing. If
there has been time to confirm the recipient’s ABO group, the transfusion service should issue ABO- and
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AABB TECHNICAL MANUAL
Rh-compatible blood. If the patient’s ABO group is unknown, group 0 RBCs should be issued.
A more detailed discussion of emergent transfusion that covers clinical concerns rather than inventory
management can be found in Chapter 15.
Massive Transfusion
Massive transfusion can be defined as the administration of 8 to 10 RBC units to an adult patient in less than
24 hours, acute administration of 4 to 5 RBC units in 1 hour, or exchange transfusion of an infant.
To ensure the ability to accurately interpret ABO group testing results, the patient specimen should be
obtained for testing as early as possible during massive transfusion. If the patient ABO type cannot be
determined,
continued support with group 0 RBCs is recommended. Unexpected and significant usage of group 0 RBCs
in the setting of massive transfusion should be considered when determining component inventory levels. In
massive transfusion situations where large amounts of blood may be required, policies may be developed to
provide D-positive RBCs to select patients, such as all adult males and postmenopausal females.
Many hospitals have developed massivetransfusion protocols to standardize the response to
hemorrhage.21,22 These protocols are designed to rapidly provide blood components in a balanced ratio of
plasma and platelets to RBCs, particularly when laboratory testing is not rapid enough to guide transfusion
support. Additional studies are needed to clarify whether the use of these protocols is associated with improved
patient outcomes.
KEY POINTS
1. Ensuring that the correct blood component is transfused to the correct patient is paramount for transfusion
safety. It must be confirmed that the recipient’s identifying information, transfusion request, testing records, and
blood component labeling and compatibility are accurate and in agreement. Any discrepancies identified must
be resolved before components are issued or transfused.
2. Visual inspection of the blood component is a critical control point in the manufacturing process and must
occur before shipping, upon receipt, and before issue for transfusion.
3. Refrigerators, freezers, and platelet incubators for blood component storage must be monitored to ensure
that proper storage conditions are maintained. Because the safety, purity, potency, and quality of the blood
components may be affected by improper storage, alarm settings should be configured to notify necessary
personnel before the upper or lower acceptable storage temperatures are exceeded.
4. Temperature requirements during transport of blood components differ from those during storage. Blood
components held outside the blood bank before transfusion are considered to be in storage. Validated processes
must ensure that acceptable storage temperatures are maintained.
5. Acceptable time frames for returning blood components to inventory after issue should be validated by
individual facilities. Individual unit-temperature indicators or temperaturereading devices may be used to
determine component acceptability for return to inventory.
6. Thawed FFP, PF24, and PF24RT24 expire within 24 hours of thawing. These products maybe labeled as
“Thawed Plasma” to allow for a 5-day shelf life if they were originally collected in a closed system.
 CHAPTER 9 Storage, Processing, Distribution, and Inventory
229
REFERENCES
1. Food and Drug Administration. Drugs; current good manufacturing practice in manufacture, processing,
packing, or holding. (June 19, 1963) Fed Regist 1963;133:6385-7.
2. Code of federal regulations. Title 21, CFR Parts 210 and 211. Washington, DC: US Government Printing
Office, 2014 (revised annually).
3. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
4. Nunes E. Transport versus storage: What is the difference? AABB News 2013;15(2):4-5.
5. Klein HG, Spahn DR, Carson JL. Red blood cell transfusion in clinical practice. Lancet 2007; 370:415-
26.
6. van de Watering L. Red cell storage and prognosis. Vox Sang 2011;100:36-45.
7. Zimrin AB, Hess JR. Current issues to the transfusion of RBCs. Vox Sang 2009;96:93-103.
8. Strauss RG. Data-driven blood banking practices for neonatal RBC transfusions. Transfusion
2000;40:1528-40.
9. Shrivastava M. The platelet storage lesion. Transfus Apher Sci 2009;41:105-13.
10. AABB, American Red Cross, America’s Blood Centers, Armed Services Blood Program. Circular of
information for the use of human blood and blood components. Bethesda, MD: AABB, 2013.
11. Werhli G, Taylor NE, Haines, AL, et al. Instituting a thawed plasma procedure: It just makes sense and
saves cents. Transfusion 2009;49: 2625-30.
12. Tholpady A, Monson J, Radovancevic R, et al. Analysis of prolonged storage on coagulation Factor
(F)V FVII, and FVIII in thawed plasma: Is it time to extend the expiration date beyond 5 days? Transfusion
2013;53:645-50.
13. Meryman HT, Hornblower M. A method for freezing and washing RBCs using a high glycerol
concentration. Transfusion 1972; 12: 14556.
14. Valeri CR, Ragno G, Pivacek LE, et al. A multicenter study of in vitro and in vivo values in
human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4
degrees C in AS-3: Assessment of RBC processing in the ACP 215. Transfusion 2001;41:933-9.
15. Borzini P, Mazzucco L. Platelet gels and releasates. Curr Opin Hematol 2005;12:473-9.
16. Cyr, M, Hume H, Sweeney JD, et al. Anomaly of the des-Arg9-bradykinin metabolism associated with
severe hypotensive reaticon during blood transfusions: A preliminary report. Transfusion 1999;39:1084-8.
17. Benjamin RJ, Kline L, Dy BA, et al. Bacterial contamination of whole-blood-derived platelets: The
introduction of sample diversion and prestorage pooling with culture testing in the American Red Cross.
Transfusion 2008;48: 2348-55.
18. Food and Drug Administration. Guidance: Industry consensus standard for the uniform labeling of blood
and blood components using ISBT 128 version 2.0.0, November 2005. (September 22, 2006) Silver Spring,
MD: CBER Office of Communication, Outreach, and Development, 2006.
19. Liu EA, Mannino FL, Lane TA. Prospective, randomized trial of the safety and efficacy of a limited
donor exposure transfusion program for premature neonates. J Pediatr 1994;125:92
6.
 20. Boral LI, Dannemiller FJ, Standard W, et al. A guideline for anticipated blood usage during elective
surgical procedures. Am J Clin Pathol 1979;71:680-4.
21. Young PP, Cotton BA, Goodnough LT. Massive transfusion protocols for patients with substantial
hemorrhage. Transfus Med Rev 2011; 25:293-303.
22. Hendrickson JE, Shaz BH, Pereira G, et al. Implementation of a pediatric trauma massive transfusion
protocol: One institution’s experience. Transfusion 2012;52:1228-36.

Chapter 10
Molecular Biology and Immunology in Transfusion Medicine
si
James C. Zimring, MD, PhD, and Steven L. Spitalnik, MD
gniN this chapter, the fundamental ^01 principles for analyzing deoxyribonucleic acid (DNA), ribonucleic
acid (RNA), and protein are described. In transfusion medicine, these techniques are used to 1) detect infectious
pathogens; 2) predict the phenotype of red cell, platelet, and neutrophil antigens; 3) detect and identify red cell
and platelet antibodies; 4) determine HLA type; and 5) perform relationship testing. In addition to describing
the principles behind these analyses, this chapter describes potential problems with the assay systems. This
chapter also addresses the basic mechanisms by which the immune system initiates a response to foreign
antigens.
Entire textbooks have been written to describe these processes, and it is outside the scope of this chapter to
provide detailed and comprehensive explanations. Instead, this chapter focuses on topics immediately relevant
to the practice of transfusion testing.
Moreover, this chapter predominantly focuses on humoral immunity because the role of cellular immunity
in transfusion medicine remains unclear and is rarely, if ever, of consequence in the pathophysiology of
hemolytic transfusion reactions. This chapter does not provide specific assay protocols; rather, it seeks to
provide an understanding of the scientific principles underlying the molecular biological and immunological
assays used in transfusion medicine.
NUCLEIC ACID ANALYSIS
The clinical utility of nucleic acid analysis in transfusion medicine lies in detecting infectious pathogens and
genotyping blood donors and recipients. The human genome is encoded in polymers of DNA, and, likewise,
many pathogens utilize DNA to encode their genome. Alternatively, many viral pathogens
James C. Zimring, MD, PhD, Director, Transfusion Medicine Research, Puget Sound Blood Center
Research Institute, Seattle, Washington, and Steven L. Spitalnik, MD, Professor of Pathology and Cell Biology,
and Director of Clinical Laboratories, Columbia University, New York, New York
J. Zimring has disclosed financial relationships with Immucor Inc and Haemonetics. S. Spitalnik has
disclosed no conflicts of interest.

231

232
AABB TECHNICAL MANUAL
encode their genome as RNA. In addition, because normal flora may have a substantial effect on human
biology, DNA and RNA analysis may become important to monitor normal nonpathological and commensal
microorganisms. With the possible exception of prionassociated disorders, either DNA or RNA encodes all
known genetic material relevant to transfusion medicine.
Basic Chemistry and Structure of Nucleic Acids
DNA is a nucleic acid polymer consisting of long chains of nucleotides linked together.1 Nucleotides
consist of a pentose (a carbohydrate with five carbon atoms), a phosphate group attached to the fifth carbon
atom (C5) of the pentose, and a base group attached to Cl [Fig 10-1(A)]. Variations in the chemistry of the base
group give rise to the four different nucle
otides comprising DNA: the purines (adenine and guanine) and pyrimidines (cytosine and thymine). In
addition, C3 of the pentose is modified by a hydroxyl group [Fig 10- 1(A)]. The individual nucleotides form
DNA polymers when the phosphate group on C5 of one nucleotide forms a covalent bond with the free
hydroxyl group on C3 of another nucleotide [Fig 10-1(B)]. DNA molecules vary from each other based on the
sequence of nucleotides that are incorporated into the polymer. Each DNA strand has a terminal 5' end that
contains a free phosphate (attached to C5) and a terminal 3' end that has a free hydroxyl group (attached to C3).
The human genome consists of doublestranded DNA. The bases contained within a single strand of DNA
form hydrogen bonds to complementary bases on another strand of DNA. In particular, thymidine (T) binds to
ad
A.
Phosphate
c.

base
■ sugar
Unwinding of DNA and transcription
TACTCCAGAAACGATT AT GAGGT CTTT GCT AA
 ♦
TACTCCAGAAACGATT
AUGAGGUCUUUGCUAA
AT GAGGT CTTT GCT AA
TACTCCAGAAACGATT AT GAGGT CTTT GCT AA 3,
AUGAGGUCUUUGCUAA 3,
♦
Translation into Protein

3' End
FIGURE 10-1. Chemical structure of nucleic acids and DNA.
233
enine (A), and guanine (G) binds to cytosine (C). When two strands have complementary sequences, they
can hybridize to form a double-stranded molecule [Fig 10-1(C)]. The two complementary strands hybridize
such that the 5' and 3' ends have opposite orientations and form a double helix in which the phosphodiester
backbone is on the outside of the helix and the hydrogen-bond-paired bases are on the inside.
When genes are expressed, the DNA encoding a given gene is transcribed into RNA [Fig 10-1(C)]. The
structure of RNA is similar to that of DNA with the following exceptions: 1) ribonucleotides have an additional
hydroxyl group on C2 of the pentose sugar (thus forming ribose), 2) uracil (U) replaces thymine (T), and 3)
RNA coding for gene products is typically single-stranded (although doublestranded RNA has important
regulatory roles).
Several classes of RNA exist in human cells; the type described in this paragraph, which is subsequently
translated into protein, is messenger RNA (mRNA). When a gene is expressed, the transcription machinery
unwinds the DNA double helix, synthesizes a mRNA strand of complementary sequence, and rehybridizes the
DNA in its wake [Fig 10-1(C)]. In this way, the RNA is essentially a copy of the sequence found in the DNA.
RNA is synthesized in the 5' to 31 direction, and, thus, only a single strand of the DNA is transcribed into RNA
by any given run of a polymerase. After synthesis in the nucleus, RNA is processed and exported to the
cytoplasm, where ribosomes translate it into protein.
Isolation of Nucleic Acids
The first step in most DNA and RNA analyses is the isolation of nucleic acids. All nucleated cells of an
individual contain identical genomic DNA, with some notable exceptions (eg, rearranged genes in mature T and
B cells). Genomic DNA can be isolated from readily obtainable cellular sources, such as peripheral blood
leukocytes and buccal swabs. In contrast, mRNAs are distinct in different cell populations because their
expression patterns are
important in defining the phenotype of these cells.
For mRNA analysis, then, the choice of starting cell types is critical. Multiple manufacturers offer reagents
that simplify and expedite isolation of cellular DNA and/or mRNA as well as for purifying viral nucleic acids
from plasma. Depending on the type of testing to be performed, the quantity and quality of the nucleic acids
isolated may be important, as determined by the relative purity of DNA or RNA and the absence of
contaminating protein.
Hybridization-Based Methods of Nucleic Acid Detection
Before the advent of techniques that allowed the amplification of a specific nucleic acid sequence (see
Polymerase Chain Reaction below), detection of nucleic acids depended on hybridization-based methods.
Probes with a particular sequence of nucleotides were synthesized and labeled with one of a variety of
detectable markers. The probe could then hybridize to complementary sequences of DNA or RNA, allowing the
detection and quantification of the corresponding complementary target. This could be done in the solid phase
(ie, Southern and Northern blots), the fluid phase (ie, RNAse protection assay or SI protection assay) or as a
result of enzymatic extension (ie, primer extension assay).2'5 However, compared to more recent amplification-
based techniques, hybridization-based methods have limited sensitivity and are less amenable to automation.
Although hybridization-based methods are useful in basic research and still play a minor role in some
diagnostic laboratories, most analyses of nucleic acids are performed using amplification-based methods.
The Polymerase Chain Reaction
Nucleic acid detection and analysis were revolutionized by the invention of the polymerase chain reaction
(PCR). PCR was the first amplification-based technique for generating nucleic acid fragments for direct
analysis.6 The concept of amplification-based systems has
234
AABB TECHNICAL MANUAL
grown to include many other techniques and applications.
A PCR reaction requires 1) a DNA sample to be analyzed; 2) gene-specific primers; 3) a thermostable DNA
polymerase; and 4) components that allow the DNA polymerase to enzymatically synthesize DNA, including
nucleotides (A, T, C, and G) and the proper salts and buffer. The PCR reaction involves repeat cycles of
heating/cooling (thermocycling), allowing exponential DNA amplification of the fragment of interest. This
reaction is carried out in a thermocycler that rapidly changes temperature with accuracy and precision. The
thermocycling reaction involved is 1) heat denaturation of double-stranded DNA, 2) cooling to
allow primer annealing, and 3) extension and synthesis of DNA on the primer strand. This procedure
continues for multiple cycles, typically 20 to 40, depending on the abundance of the template and the required
sensitivity of the assay.
An overview of the PCR process is presented in Fig 10-2. This example begins with a single copy of a
double-stranded DNA template. The DNA is denatured by heating to near boiling (typically at 94-95 C), which
disrupts the hydrogen bonds between complementary bases, thereby separating the two strands. The
temperature is then lowered (annealing reaction) to allow gene-specific primers to anneal to their
complementary target. Typically, an
Copies
1
Melting/annealing [=::I
Extension
Melting/annealing
I
:
Extension
FIGURE 10-2. Graphic overview of the polymerase chain reaction.
235
annealing temperature of 5 C below the melting temperature of the target DNA-primer complex increases
specificity to the correct target sequence. The exact melting temperature of a given target DNA primer complex
depends on the primer length, abundance of guanines and cytosines in the sequence (ie, GC content), and any
mismatches with the target. At this point, the temperature is raised to that at which the DNA polymerase
functions optimally (typically 72 C), and the primers are extended along the length of the DNA by the
incorporation of the correct complementary nucleotides by DNA polymerase. Thus, at the end of extension,
there are two copies of the DNA. This process repeats itself with denaturing, annealing, and extension. With
each subsequent cycle, an exponential increase in DNA copy number occurs. PCR results in a geometric
expansion of a selected DNA “amplicon,” defined as the sequence that is flanked by the two chosen primers.
PCR Considerations
Although PCR is a robust and reliable method of detecting nucleic acids, as with all methods, technical
problems can affect PCR and other amplification-based techniques.
Specimen Processing and Template Degradation
DNA is stable and can usually withstand some variations in storage temperature and handling before being
processed for genomic analysis. Exceptions include samples in which the target DNA is present in low quantity,
such as fetal typing from maternal plasma and viral testing. RNA is far less stable than DNA and is susceptible
to autocatalytic degradation by RNAse enzymes found in many biological specimens.
Inhibitors
PCR amplification depends on the enzymatic activity of DNA polymerase and can be inhibited by any
substance that negatively affects synthesis by DNA polymerase. Heparin can inhibit PCR in some situations,
and hemoglobin
or lactoferrin released from erythrocytes or leukocytes also inhibit PCR.7,8 Most analytic systems are
optimized such that the risk of interference by an inhibitor is minimized, but care should be taken not to deviate
from established protocols because such deviations may introduce unintended inhibitory substances.
Amplifying ubiquitous target sequences present in all samples (ie, conserved genomic DNA or housekeeping
genes, such as hemoglobin) and/or spiking the specimen with an internal positive control can be used to assess
whether inhibitors are present.
Primer Design
Inferior performance of primers should not be a concern in the context of commercially available tests.
Nevertheless, a thorough understanding of primer design is important in troubleshooting assays, and primer
design is a key component in developing PCR-based assays for new targets. Although ideal primers hybridize to
a target that is found in only one location in the entire genome, given the complexity of genomic DNA, this is
not always possible. Primer annealing to unintended targets can occur, resulting in ongoing consumption of
primers with each cycle and the potential to amplify unintended targets.
In addition to cross annealing to unintended genomic sites, primers can anneal to each other and form a
short amplicon. For example, if the 3' ends of two primers bind to each other, the polymerase fills in the 5'
overhangs [Fig 10-3(A)]. A primer modified in this way may still be able to anneal to its genomic target
sequence. However, the additional bases added to the 3' end no longer anneal to the genomic target and may
prohibit proper extension. This “primer-dimer formation” can take place between two different primers in a
reaction or two molecules of the same primer [Fig 10-3(B)]. Primers can also form hairpin loops and anneal to
themselves [Fig 10-3 (C)]. A self-complementary sequence in a primer may allow this to occur, particularly in
primers over 20 bp in length, and this reaction is favored because of its intramolecular nature. If self-annealing
results in a 5’ overhang, the
236
AABB TECHNICAL MANUAL
PCR Primers
Normal Amplification
5'
3'
5'
3'
A.
B.
C.
5' 3' 5' 3'
3' 5' 3' 5'
3'
5'
FIGURE 10-3. Potential problems in primer design that may inhibit polymerase chain reaction (PCR).
molecule can self-prime with the polymerase and extend the 3' end to be complementary to the 5' end,
increasing the tendency of the primer to self-anneal. The presence of the additional sequence prevents
amplification of the authentic target amplicon.
Contamination between Specimens
One of the greatest strengths of PCR is its ability to amplify very small amounts of genetic material. In
theory, single-copy sensitivity can be achieved. In practice, detection of 10 copies of DNA or fewer is not
uncommon, depending on the sensitivity of the assay readout. This level of sensitivity also makes the assay
susceptible to false-positive results due to contamination from specimens being analyzed or amplicons
generated in previous amplification reactions. Beginning with just 10 molecules of DNA, 30 rounds of
amplification in a PCR reaction yields more than 1 x 1010 amplicons. Thus, if only 0.0000001% of a previous
reaction is inadvertently introduced onto a pipette used to set up a subsequent reaction, a false-positive result
may occur.
To minimize the possibility of contamination from previously generated amplicons and a false-positive
signal, PCR laboratories routinely process samples in only one direction.
Using this approach, DNA extraction is separate from testing, PCR reactions are assembled in one room and
amplified in a second room, and downstream analysis (if required) is performed in a third room. There should
be no retrograde flow, and no materials or instruments used in the amplification or analysis (post-PCR) rooms
should make their way into the PCR setup (pre-PCR) room. Pipette filter tips that minimize carry-over
contamination and sample aerosols are routinely used.
Other ways to minimize potential contamination include the addition of deoxyuridine triphosphate (dUTP)
to PCR reactions. Polymerases incorporate dUTP in place of deoxythymidine triphosphate (dTTP), and the
added enzyme uracil-DNA glycosylase (UNG) specifically cleaves DNA containing uracil but not normal DNA
or RNA.9 Thus, adding UNG to PCR reactions destroys contaminating amplicons from previous amplifications
but not native DNA in the specimen. The UNG is heat inactivated during the initial denaturation PCR step,
allowing amplification of the new sample.
Reverse-Transcriptase PCR
When amplification and analysis of mRNA is required, a reverse-transcriptase (RT) enzyme
237
that synthesizes DNA from an RNA template is used.10,11 Like DNA polymerase, RT synthesizes in the 5'
to 3' direction and requires the annealing of a primer to initiate transcription. When RNA is transcribed, the
resulting DNA is a single-stranded complementary copy of the RNA and is referred to as “complementary
DNA” (cDNA). The cDNA is then a suitable substrate for PCR, as described above.
Transcription-Mediated Amplification and Nucleic Acid Sequence-Based Amplification
Since the advent of PCR, several additional nucleic acid amplification strategies have been devised. Among
the most useful are two related techniques, transcription-mediated amplification (TMA) and nucleic acid
sequencebased amplification (NASBA).12,13 Although TMA and NASBA have some differences (described in
this section), they are conceptually similar and, thus, are described together (see Fig 10-4).
TMA plays a large role in nucleic acid testing for human immunodeficiency virus (HIV), hepatitis C virus,
and West Nile virus (ie, using the Chiron/Gen-Probe system). Both techniques use RNA as the amplification
target. The reaction contains two primers, RT, DNA polymerase, RNAse H, and T7 polymerase. The reaction
begins when a specific downstream primer (Primer 1) hybridizes to the 3' end of the target RNA and RT
synthesizes a cDNA copy (Fig 10-4, Step 1). Primer 1 not only contains a complementary sequence at its 31 end
that hybridizes to the target RNA, but the 5' end also encodes a bacteriophage T7 promoter. The RNA template
is degraded by RT in the TMA assay or by RNAse H in an additional step with NASBA (Fig 10-4, Step 2). A
second 5'upstream primer (Primer 2) then binds to the newly synthesized cDNA (Fig 10-4, Step 3) and utilizes
DNA polymerase to synthesize a double-stranded DNA molecule (Fig 10-4, Step 4). This molecule has a T7
promoter at one end, and T7 polymerase then drives transcription of RNA (Fig 10-4, Step 5). Numerous RNA
Hybridization of Primer 1 S 5
RNA S'
Reverse transcriptase | Step!
cDNA S 5
RNA RNAse H 1 Step 2
cDNA s 5
RNA 5' Hybridization of Primer 2 1 Step 3 -3
cDNA 3' s 5
Primer2 5'
| ^ DNA polymerase I Step 4
3' 5
NewDNATemplate 5' 3
T7 polymerase | Step 5
Degraded RNA
 »- - 4 Primer 1 containing T7 promoter Primer2
RNAse H is only used in NASBA In TMA, RNA is degraded by the reverse transcriptase.
1. Direct detection
Multiple copies of *
rna antisense to 2. Additional amplification
original mRNA
FIGURE 10-4. Schematic overview of transcription-mediated amplification (TMA) and nucleic acid
sequencebased amplification.
RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid; mRNA = messenger RNA;
RNAse = ribonuclease.
238
AABB TECHNICAL MANUAL
transcripts are synthesized from a single DNA template. These new RNA molecules can reenter the
amplification cycle with Primer 2 initiating reverse transcription, followed by RNA degradation and subsequent
synthesis of DNA using Primer 1 and DNA polymerase. This leads to additional amplification with ongoing
cycles of transcription and template synthesis. One distinct advantage of NASBA compared to PCR is that
repeat nucleic acid denaturation is not required. Therefore, NASBA amplifies RNA sequences in an isothermal
reaction that does not require a thermocycler.
In addition to the amplification methods described above, several other techniques use nucleic acid
polymerizing or ligating enzymes to amplify DNA and/or RNA. These include strand displacement
amplification (SDA) and the ligase chain reaction (LCR).14'16 There are also probe/signal-amplification
methods, such as the Cleavase Invader, branched DNA, and hybrid capture assays. Although these techniques
have been adapted to detect pathogens, they are not widely used in transfusion medicine and are not described
further here.17
Detection of Nucleic Acid Amplification Products
PCR, RT-PCR, TMA, and NASBA amplify specific nucleic acid sequences. However, the amplified
sequences must still be detected. Traditionally, this has been accomplished by separating the amplification
reactions by gel electrophoresis in the presence of a fluorescent dye (eg, ethidium bromide), which makes the
nucleic acids visible in the gel under ultraviolet light. This allows visualization of the bands and, if appropriate
standards are included in the gels, provides a molecular weight by which one can distinguish amplicons of the
correct size from cross-reactive products of different sizes. To confirm that the amplified nucleic acids have the
correct sequence, the amplification products can be digested with restriction endonucleases and the sizes of the
resulting molecules determined. Because the amplicon sequence is known, one can predict the correct
restriction sites [this is known as
“restriction fragment length polymorphism” (RFLP) analysis]. Even greater specificity can be achieved by
hybridization analysis. After electrophoresis, the amplified products are transferred to nitrocellulose paper,
which is then hybridized with DNA probes specific for the amplified sequences.
Each of these approaches requires gel electrophoresis, which is time consuming, incompatible with the
throughput needs of donor-testing laboratories, and not easily automated. Moreover, it requires opening tubes
containing amplification reactions and manipulating the products, which increases the risk of contamination of
reagents and false-positive results in subsequent samples.
More advanced techniques for detecting amplification products rely on the chemistry of fluorescent
molecules. Probes that fluoresce only when the correct amplicon is present are included in the amplification
reactions. By including a fluorescence spectrophotometer in the thermocycler, the generation of amplification
products can be measured at each cycle. This approach, called “real-time PCR,” is highly sensitive; much more
quantitative than gel electrophoresis-based methods; and, by using multiple reporter dyes with distinct
fluorescence spectra, makes detection of multiple targets in a single reaction feasible (ie, multiplex nucleic acid
amplification). In addition, including fluorescent probes in the reaction allows analysis without ever opening the
tube containing the amplification products; this decreases the risk of laboratory contamination with amplicons
and subsequent false-positive results.
Two of these fluorescent methods rely on the juxtaposition of a fluorescent molecule with a quencher
molecule that prevents fluorescence. In the TaqMan system, a sequencespecific probe has the fluorescent
molecule at one end and the quencher at the other. When the probe hybridizes to its target, it essentially blocks
the DNA polymerase that is synthesizing the next round of DNA by extending from the primer. When the DNA
polymerase encounters the hybridized probe, it degrades the probe, thus separating the fluorescent and quencher
molecules [Fig 10-5(A)]. Because
239
A.
Amplicon
##
Amplicon I I I I I I I I I I I

B.
Amplicon II Mill

Amplicon IN
Amplicon
Fluorescence

FIGURE 10-5. Methods of detection by sequence-specific probes during real-time polymerase chain
reaction. Pol = polymerase enzyme.
240
AABB TECHNICAL MANUAL
this occurs only when the probe hybridizes to its target, fluorescence is generated as a function of amplicon
generation.
A second approach uses molecular beacons that also have a fluorescent molecule on one end of a DNA
probe and a quencher molecule on the other end. In this case, the sequence-specific probe is flanked by two
sequences that are complimentary to each other and form a hairpin loop, thus juxtaposing the fluorescent
molecule with its quencher. However, when the probe hybridizes to its target, the hairpin loop unfolds,
separating the quencher from the fluorescent molecule and allowing fluorescence to occur [Fig 10-5(B)].
A third approach uses two molecules that do not fluoresce unless they are in close proximity to each other.
These molecules are linked to two separate DNA probes that hybridize to adjacent sequences on the amplicon.
If the amplicon is present, the probes anneal in such a way that the two molecules are in close proximity,
providing a fluorescent signal [Fig 10-5CC)].
A fourth method uses SYBR® green dye, which fluoresces when bound to doublestranded DNA. SYBR®
green is not sequence specific and, thus, detects all amplicons. However, authentic amplicons can be
distinguished from cross-reactive products by melting curve analysis. Because the melting curve is a function of
amplicon size and GC content and the size and sequence of the correct amplicon is known, the melting curve
can be used to confirm the identity of the amplicon.
As with PCR, these fluorescent-probe techniques can be applied to other amplification technologies, such as
TMA.
Analysis of Single-Nucleotide Polymorphisms
The vast majority of blood group antigens consist of single-nucleotide polymorphisms (SNPs). The gene
product is present in most people, but the difference that determines the identity of the blood group antigen is a
small change in sequence, often a single nucleotide.
Some SNPs destroy or create recognition sites for restriction endonucleases. In these
cases, PCR-amplified material can be digested by restriction enzymes and then analyzed by agarose
electrophoresis to observe the resulting fragment sizes. RFLP analysis has low throughput and depends on the
presence of a restriction enzyme recognition site associated with the SNP in the gene of interest.
 Several different methods for detecting SNPs consist mostly of modifications of PCR or array technologies.
With these methods, primers or probes are engineered such that hybridization depends on the presence of the
correct SNP. Both multiplex PCR and DNA array systems can determine the genotype of blood group antigens
in individual specimens.17'19 These systems offer the advantage of higher throughput and automated readout
while avoiding the need for complex interpretation.
Genotyping of red cell antigens may be more efficient than traditional serologic typing. In addition, when a
patient has received multiple RBC units and it is not possible to distinguish the patient’s own red cells from
transfused red cells, genotyping the patient’s DNA may be the only reliable way to predict the patient’s red cell
phenotype. However, occasionally the genotype may not correlate with the phenotype. It is worth noting that
genotyping typically focuses on known polymorphisms but does not provide the entire sequence of the gene or
its regulatory regions. Therefore, genotyping might not predict phenotype when 1) new, hitherto unknown,
polymorphisms in the coding region alter protein structure; 2) new polymorphisms in the coding, promoter, or
other regulatory regions prevent gene expression despite the presence of the correct coding sequence; or 3)
changes alter the epitope (ie, modification by bacterial enzymes) when epitopes depend on posttranslational
modification.
PROTEIN ANALYSIS
Nucleic acid analysis, as described in the “Nucleic Acid Analysis” section above, detects the presence of
DNA or RNA that encodes genomic material and/or measures gene expression at the RNA level. However, due
to regulation of protein translation, the presence of mRNA
241
does not always correlate with the presence of the encoded protein. In addition, the detection of antibodies,
which are proteins, cannot be determined by detecting or measuring nucleic acids. Accordingly, measuring
proteinprotein and/or protein-carbohydrate interactions provides relevant biological information in transfusion
medicine that may not be obtainable by nucleic acid analysis.
Fluid-Phase Assays (AgglutinationBased Methods)
Depending on antibody isotype, immunoglobulins have 2 to 10 antigen-binding sites per molecule. Each
antibody can bind more than one target molecule, allowing antibodies to cross-link antigens present in multiple
copies on particles, such as red cells or beads. This cross-linking can aggregate particles that have the relevant
antigen on their surface, a process known as “agglutination.” Agglutination is an old, but generally reliable,
serologic method for detecting antibody-antigen interactions and is used extensively in transfusion medicine.
Agglutination can be detected by several methods. The antigen copy number and density varies depending
on the blood group. Agglutination is used for serological crossmatching (donor red cells incubated with
recipient plasma or serum), screening for unexpected antibodies (reagent red cells of known blood group
antigen composition incubated with patient plasma or serum), and blood group antigen phenotyping of the
donor or recipient (test red cells incubated with monoclonal antibodies or reagent-quality antisera of known
specificity).
Because red cells are easily visible due to their red color, several systems were devised to detect
agglutination. These include 1) tube testing in which agglutination is visually detected by the adhesion of red
cells to one another in the postcentrifuge pellet, 2) passive agglutination in microtiter plates where agglutination
is visualized by the spread pattern of red cells in individual wells, and 3) gel testing in which unagglutinated red
cells pass through a matrix but agglutinated complexes are re
tained at the top or within the matrix due to their greater size.20 In addition to blood bank serology, red cells
can be used to detect antibodies against other antigens by linking these particular antigens to the red cell
surface. Agglutination-based assays can also be engineered using particles other than red cells, such as latex
beads. Each of these techniques uses the same basic principles and has broad applicability in clinical diagnostic
testing.
Although agglutination reactions are very sensitive and easy to perform, the formation of agglutinates
depends on the proper stoichiometric ratio of antibody to antigen. When the ratio of antigen to antibody is in a
range such that agglutination readily occurs, it is referred to as the “zone of equivalence.” In this situation, each
arm of the antibody binds to a different particle, and a network (or lattice) of linked particles results in
agglutination [Fig 10-6(B)]. A false-negative result is generated if the stoichiometric ratio is outside the zone of
equivalence at either extreme.
A prozone effect can occur when such a high concentration of antibody is present that the likelihood of an
antibody being able to bind to two separate particles or red cells is diminished [Fig 10-6(A)]. This is seen most
commonly with non-red-cell-based agglutination assays, such as the rapid plasma reagin screening test for
syphilis serology. Although prozone effects are unusual in classical red cell serology, they have been observed
when titers of red cell antibodies are very high. In particular, discrepant reverse ABO typing due to prozone
effects has been reported.21,22 Diluting the serum being tested and using EDTA containing diluents decreases
the likelihood of prozone effects. One reason why prozone effects are uncommon in red cell serology is that the
use of antihuman globulin (AHG) largely overcomes these problems. However, a secondary “prozone-like”
effect occurs if there is inadequate washing before adding AHG.23 In this case, residual immune globulin G
(IgG) in solution binds the AHG and competes for AHG binding to IgG on the red cells. This may occur with
very high titers of blood group antibodies or even in the presence of highly increased levels of polyclonal
antibodies, as in hypergam
242
AABB TECHNICAL MANUAL

FIGURE 10-6. Effects of relative concentrations of antigen and antibody on the outcome of agglutination
reactions.
maglobulinemia. In this setting, additional washing steps remove the problem.23
In theory, false-negative agglutination reactions can occur due to a postzone effect in which the antigen is in
excess [Fig 10-6(C)]. In this situation, every antibody binds to multiple epitopes on the same particle, thereby
preventing the cross-linking that leads to agglutination. This could occur if a large excess of red cells were used.
This problem is easily controlled by careful attention to methods and is not common in transfusion medicine.
Solid-Phase Assays
Various solid-phase assays exist that utilize the same general principle. As opposed to fluidphase assays,
where the reaction occurs in a solution or suspension, the antigen or antibody being studied in solid-phase
assays is im
mobilized on a solid matrix. The analyte is then incubated with the coated solid phase, and adherence of the
analyte to the solid phase is measured. Several combinations of adherence and detection approaches have been
described.
Solid-Phase Red Cell Adherence Techniques
SOLID-PHASE ASSAYS FOR PHENOTYPING RED CELLS. Antibodies specific for known blood group
antigens are coated onto roundbottom microtiter plates [Fig 10-7(A)[. Cells being analyzed are added to the
microtiter plate wells and allowed to adhere. If no binding occurs (negative reaction), the red cells all cluster
together as a “button” at the bottom of the well. In contrast, specific binding results in diffusion of the red cells
over the surface of the
243

Antibodycoated well

Negative
 Positive
B.
Serum

o Antigen-containing particle

| Red cells coated with anti-IgG

Negative
Positive
FIGURE 10-7. Schematic representation of (A) phenotyping red cells and (B) detecting antibodies by
solidphase assay.
well (positive reaction). A positive reaction indicates the presence of the antigen on the red cells being
tested.
SOLID-PHASE ASSAYS FOR DETECTING ANTIBODIES TO RED CELL ANTIGENS. Antigen-coated
particles, consisting of red cells or red cell fragments, are coated onto microtiter plate wells [Fig 10-7(B)].
Patient serum is then added to each well, followed by incubation and then washing. If the patient serum has
antibodies against the red cell antigens coated on the well, then the antibodies bind to the red cells or their
fragments. Indicator red cells coated with antihuman IgG are then added. A positive reaction is demonstrated by
diffuse adherence of the indicator red cell to the well, whereas a negative reaction is demonstrated by clustering
of indicator cells in a button.
Solid-Phase Assays fo r Pla telet Tes ti ng
Solid-phase red cell adherence (SPRCA) technology has also been adapted to detect antigens on platelets,
such as HPA-la, as well as
antibodies against platelet antigens using the approaches described above.24
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay (ELISA), also referred to as “enzyme immunoassay,” can detect
antibodies and antigens. A signal is generated via an enzyme linked to a secondary antibody or antigen that
converts a substrate to a measurable product (eg, a color change or a chemiluminescent reaction). For this
reason, ELISAs have considerable signal amplification and are significantly more sensitive than fluidphase
agglutination or SPRCAs. In most cases, ELISAs use purified or recombinant antigens or antibodies, depending
on the analyte detected. However, intact red cells can be used to screen for red cell antibodies, referred to as the
"enzyme-linked antiglobulin test.”25
DETECTION OF ANTIBODIES BY ELISA (INDIRECT ELISA). To detect antibodies against a given
antigen, the antigen is coated onto microtiter plate wells [Fig 10-8(A)[. The test sample is then added,
incubated, and washed. If
244
AABB TECHNICAL MANUAL
A.
Serum
Ay
O Purified antigen
Secondary antibody

B.
 Substrate Substrate
(no color) (with color)

Antibody
C. 1
Ay
Analyte ►

O Purified antigen

Secondary antibody linked to enzyme and detection

FIGURE 10-8. Schematic representation of (A) indirect enzyme-linked immunosorbent assay (ELISA), (B)
sandwich ELISA, and (C) competitive ELISA.
antibodies against the antigen are present, they bind to the antigen-coated well and the bound antibodies are
detected by incubating with anti-Ig (eg, anti-IgG) linked to an enzyme (eg, alkaline phosphatase or horseradish
peroxidase). After further washing, enzyme substrate is added and is converted to a detectable color if enzyme
is present. Quantification is performed using a standard curve and a spectrophotometer to measure the
absorbance at the wavelength appropriate for that enzyme/ substrate product. In some cases, samples may need
to be diluted to ensure that they yield absorbance values in the linear range of the assay.
DETECTION OF ANTIGENS BY SANDWICH ELISA. In sandwich ELISA, two separate antibodies are
used that bind different epitopes on the same target antigen; the antibodies bind
the target without interfering with each other. Typically, this is accomplished by monoclonal antibodies.
Microtiter plate wells are coated with one antibody, the “capture antibody” [Fig 10-8(B)]. The specimen is then
incubated in the well; if the antigen is present, it binds to the solid-phase antibody coated in the well. The plate
is then washed and incubated with the second antibody, which is linked to a reporter enzyme (the “conjugate").
Because the second antibody is specific for the target antigen, it binds to the well only if antigen was bound to
the capture antibody. After additional washing, enzyme substrate is added and is converted to a detectable color
if enzyme is present.
DETECTION OF ANTIGENS BY COMPETITIVE ELISA. Competitive ELISA is similar to indirect
ELISA in that the target antigen is
CHAPTER 1 0 Molecular Biology and Immunology in Transfusion Medicine
245
bound to the microtiter plate well. The test sample is incubated with antibody specific for the target antigen
and this mixture is added to the well [Fig 10-8(C)]. If no antigen is in the specimen, then the reagent antibodies
bind to the solid-phase antigen. However, if antigen is present in the specimen, it combines with the reagent
antibodies and prevents them from binding to the solid-phase antigen. Therefore, as the amount of soluble
antigen in the specimen increases, the amount of reagent antibody that is free to bind to the solid-phase antigen
on the well decreases. Similarly, as the signal weakens, the amount of soluble antigen in the specimen rises.
Competitive ELISA is also used for antibody detection. In this case, the test sample along with a labeled
reagent antibody is added to an antigen-coated well. Both patient antibody and labeled reagent antibody
compete for antigen-binding sites in the well. Again, a higher signal is generated if the antibody level in the
sample is low or absent. Although more difficult to optimize, competitive ELISAs have an advantage over
sandwich ELISAs in not requiring two separate antibodies against different epitopes on the target antigen.
TECHNICAL PROBLEMS WITH ELISAS. Typically, ELISAs are straightforward and robust. Although
low signals are possible due to enzymatic inhibitors in the sample and falsepositive signals can occur due to
enzymatic activity, proper controls and washing prevent these problems. Problems also occur if the amount of
the antigen being measured exceeds the amount of antibody present. This phenomenon, called the “hook
effect,” leads to underestimates of the concentration of the antigen. Like the prozone effect (see “FluidPhase
Assays” section above), excess amounts of the analyte can cause the signal to decrease in some sandwich
ELISAs in which the analyte and detection antibody are added simultaneously. Hook effects can be overcome
by diluting the analyte. Finally, some patients have human antimouse antibodies that cross-link the capture
antibody and the detection antibody in sandwich ELISAs, resulting in very high signals for essentially all
analytes.
Protein Microarrays
Microarray technology has dramatically increased the number of substrates that can be simultaneously
assayed by solid-phase methods. By spotting numerous different protein substances on a small chip, a single
specimen can be assayed for binding activity to multiple (in some cases thousands) substances simultaneously.
For example, patient serum can be tested for antibodies to blood-group antigens by making a microarray chip
with different blood-group antigens and then incubating it with patient serum.
Although microarray approaches are very promising, like ELISA, they require that the antigens being tested
be spotted on a chip in a pure form that maintains the structural conformation required for recognition. In the
cases of carbohydrate antigens or linear protein epitopes, this is readily achieved. However, with multipass
transmembrane proteins that require membrane insertion for proper conformation (eg, the Rh antigens), it is
considerably more difficult. The application of protein microarrays to blood-bank serology is still in early stages
of development, and it remains unclear to what extent it will mature into a useful diagnostic platform.
Western Blotting
The ELISA techniques described above can be highly sensitive. However, because the antigens used may
not be pure (eg, lysates of tissue culture grown viruses), false-positive results are possible due to cross-
reactivity with other components in the antigen preparation. Western blotting is conceptually similar to ELISA,
except that instead of coating a well with the antigen, the antigen mixture (eg, viral lysate) is first separated by
high-resolution protein electrophoresis (typically using polyacrylamide gels). The separated proteins are then
transferred to nitrocellulose paper (or another suitable medium), which serves as the solid phase to be probed
with an antibody-containing patient sample. In this case, one can determine the molecular weight of the antigens
recognized by the antibodies.
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AABB TECHNICAL MANUAL
Although not often used clinically, other methods can be used to separate antigens based on physical
properties other than size. For example, isoelectric focusing separates proteins of different charges over a pH
gradient. Because the likelihood of having a crossreactive antigen with the same physical characteristics as the
authentic antigen is small, Western blotting provides more specificity than ELISA. Thus, Western blots have
been used as confirmatory tests for serologic screening assays to detect infectious agents, such as HIV. Similar
techniques were developed using recombinant, or otherwise purified, proteins for detecting other infectious
disease agents, such as hepatitis C virus.
Flow Cytometry
Flow cytometry revolutionized the analysis of cell populations. The basic principle is that antibodies against
cell-surface molecules are labeled with fluorescent tags. Cells are incubated with the antibodies, and these
“stained” cells are then passed through a flow cytometer. The individual cells are exposed to lasers that excite
the fluorochromes, causing fluorescent emissions detected by sensors in the flow cytometer. The amount of
fluorescence is determined on a cell-by-cell basis, allowing both the quantification of the number of cell-surface
molecules and the visualization of small populations of cells in a complex mixture.
Clinical flow cytometry has been applied primarily to the diagnosis of neoplasia, particularly for
hematologic malignancies. However, flow cytometry can also be applied to red cell serology. For example, red
cells can be phenotyped using labeled antibodies of known specificity. Alternatively, antibody screens or
crossmatching can be performed by mixing red cells of known phenotype with patient antisera and then staining
the cells with a secondary antihuman Ig (eg, anti-IgG) that is coupled to a fluorescent molecule. However, flow
cytometry has yet to be widely applied to human transfusion medicine and is currently utilized mostly in
experimental systems.
BASIC IMMUNOLOGY
The process by which the immune system generates antibodies against foreign antigens and yet maintains
tolerance to self-antigens is complicated and elegant, with multiple cellular players and intricate regulation. The
mechanistic details of this process fall outside the scope of this chapter but can be found elsewhere. This section
covers antibody structure, function, and role in transfusion complications.
Antibody Structure
 At its simplest, an antibody is a tetramer of two identical heavy chains and two identical light chains (Fig
10-9). Each heavy and light chain contains a variable region, the part of the molecule that varies from antibody
to antibody and binds antigen, and a constant region. Two light-chain families (kappa and lambda) are found in
humans, and any given antibody has either two identical kappa or two identical lambda light chains. Likewise,
the two heavy chains in an antibody molecule are identical and differ depending on isotype.
Immunoglobulins treated with the enzyme papain can be digested into two functional fragments. The Fab
fragment consists of the heavy- and light-chain variable regions, the light-chain constant region, and one heavy-
chain constant region domain. The Fab fragment binds antigen but does not activate effector mechanisms. In
contrast, the Fc fragment, consisting only of heavy-chain constant regions, activates effector mechanisms,
allowing destruction of the antibody target. Fc constant regions differ based on antibody isotype and subclass.
There are five different antibody isotypes— IgM, IgG, IgE, IgA, and IgD—determined by the constant
region of the heavy chain. Antibody isotypes can differ in the number of antigen-binding sites per molecule and
the potency of their effector functions [Fig 10- 10(A)]. The number of antigen-binding sites per antibody affects
binding avidity for antigen. For example, IgM is expressed early in an immune response before the onset of
affinity maturation. However, IgM has high avidity because it
Molecular Biology and Immunology in Transfusion Medicine
247
Antigen- Antigen
| Variable domains binding binding
site site

FIGURE 10-9. General structure of a monomeric immunoglobulin.

B. Y Y Y Y
IgGl lgG2 lgG3 lgG4
C' activation Strong Weak Strong No
Binds FcRs on phagocytes Yes No Yes Weakly
FIGURE 10-10. Immune globulin (Ig) isotypes (A), IgG subclasses, and their relative activation of
complement and binding to Fc-gamma receptors (FcyRs) (B).
248
AABB TECHNICAL MANUAL
consists of five Ig molecules held together by an additional protein (the J chain) and extensive disulfide
binding, resulting in 10 antigenbinding sites. The high avidity of IgM compensates for its typically low affinity
for antigen. Treatment with dithiothreitol (DTT) can destroy IgM binding because it reduces disulfide bonds,
and DTT treatment is used to distinguish IgM from IgG antibodies in the laboratory. IgM potently activates
complement by changing its three-dimensional structure after antigen binding. Although an IgM-specific Fc
receptor has long been suspected and was recendy cloned, its function is not yet fully understood. In general,
IgM (and some IgG) is known to cause hemolysis during transfusion reactions and autoimmune hemolytic
anemia.
IgG antibodies are important in mature humoral immune effector function and are divided into four
subclasses: IgGl, IgG2, IgG3, and IgG4. Each subclass has a different constant region and a different capacity to
activate complement and/or interact with Fc receptors on phagocytes [Fig 10-10(B)]. IgGl and IgG3 are
generally the most potent in these regards, IgG2 only weakly activates complement, and IgG4 largely lacks
effector activity. Consistent with these observations, patients with isolated IgG4 subclass red cell autoantibodies
typically do not exhibit hemolysis. In contrast, IgGl, IgG2, and IgG3 red cell antibodies can induce hemolysis.
IgA is the primary antibody isotype secreted at mucosal surfaces; therefore, it is largely responsible for
neutralizing pathogens encountered in the gastrointestinal, genitourinary, and respiratory tracts. Although IgA
exists in either monomeric or dimeric forms (a dimer is shown in Fig 10-10), it is often monomeric in serum.
IgA is further divided into IgAl and IgA2 subclasses (not shown). In the IgA dimeric form, IgA monomers are
connected by the J chain, similar to IgM. In rare instances, IgA red cell antibodies cause hemolysis. Most
antiglobulin (ie, Coombs) reagents do not typically detect IgA; thus, the potential presence of IgA red cell
antibodies must be considered when analyzing a patient with hemolysis and negative direct antiglobulin test-
results.
IgE antibodies bind to Fc receptors on mast cells and induce histamine release when they encounter antigen;
thus, IgE antibodies are the predominant cause of allergic and anaphylactic responses (ie, Type I
hypersensitivity). IgD primarily remains membrane bound on the B-cell surface, with only minimal levels in
serum, and its functions remain unclear.
The Role of Fc Receptors in Target Destruction
The Fc regions of antigen-bound IgGs are recognized by the gamma family of Fc receptors (FcyRs). At
least four FcyRs have been described to date, each with subdy different properties that can have opposite
functions. For example, FcyR2a and FcyR3 promote phagocytosis of targets. Due to the relatively low affinity
of these receptors, monomeric IgG does not engage FcyR2a and FcyR3, making them specific for targets bound
by multiple antibodies. In contrast, FcyR2b is an inhibitory receptor that prevents phagocytosis. FcyRl has an
unusually high affinity and binds monomeric IgG. The result is that FcyRl binds IgG whether or not it is bound
to a target; the function of this activity is currently unclear.
Thus, FcyR biology is complicated by the fact that any given IgG-bound cell or particle may simultaneously
activate multiple, potentially antagonistic, receptors. This is further complicated by the fact that each of the four
different IgG subclasses (IgGl, IgG2, IgG3, and IgG4) has a different affinity for the different FcyRs (see Fig
10-11). A mixture of IgGl-IgG4 may bind a particle or cell bearing a foreign antigen, and the net effect on
enhancing or inhibiting phagocytosis will depend on the relative binding of different IgG subclasses and
interactions with different FcyRs. Thus, direct binding of Fc domains to FcyRs can promote red cell clearance
in many cases but does not always do so.
The Role of Complement in Target Cell Destruction and Opsonization
In addition to serving as ligands for FcyRs, the Fc regions of IgG antibodies can activate com
CHAPTER 10
Molecular Biology and Immunology in Transfusion Medicine
249
A. B. C. D.

FIGURE 10-11. Mechanisms of red cell destruction by antibody binding. Upon binding (A), an immune
globulin G (IgG) represents a ligand for Fc-gamma receptors (FcyRs) on phagocytes (B). If the red cell avoids
FcyRmediated phagocytosis, opsonization may be increased by activation of complement with deposition of
C3b (C). if the combined opsonization of FcyR binding and C3b is not sufficient to mediate clearance,
completion of the complement cascade may lead to insertion of the membrane attack complex (MAC) into the
red cell surface, resulting in lysis (D). In reality, these processes likely occur simultaneously, with the ultimate
outcome being the aggregate effect of competing pathways.
CR = complement receptor.
plement. The complement system consists of a cascade of proteases that, once activated, amplifies the initial
signal, leading to the production of a large number of effector molecules. Although there are several
complementactivation pathways, this discussion focuses on the “classical pathway” initiated by Fc regions.
IgM is highly efficient in activating complement. However, to avoid indiscriminant activation, IgM
undergoes a conformational shift after binding antigen, thereby exposing complement-binding sites in the
heavy-chain constant region. This interaction is so potent that, in theory, a single antigen-bound IgM is
sufficient to lyse a target. In contrast, IgG does not require a conformational change to bind complement, but
complement activation requires clustered binding of multiple IgG molecules to the same target. This prevents
indiscriminate activation of complement by unbound circulating IgG.
Once activated, the complement system provides at least two distinct mechanisms of target destruction. The
first involves target opsonization by complement components. During early events in complement activation,
C3 covalently attaches to the antigen surface by thioester bonds, providing multiple copies of C3b, which are
recognized by the complement receptor 1 (CR1) and CRIg on phagocytes. When a phagocyte encounters a C3b-
coated molecule, it ingests and destroys it. C3b also rapidly degrades sequentially into iC3b, C3c, and C3dg.
Because C3dg is recognized by CR2, which is not on phagocytes, complement can degrade past the point of
promoting phagocytosis. In the second mechanism, downstream of C3 activation, the cascade assembles the
membrane attack complex (MAC). The MAC consists of complement proteins C5b-C9 arranged into a structure
resembling a hollow tube inserted into the membrane of the target cell. This nonselective channel between the
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AABB TECHNICAL MANUAL
inside of a target cell and its external environment results in osmotic lysis of the target (if it is sensitive to
osmotic shock).
Specific Outcomes of MAC Assembly, C3 Opsonization, and Fc Opsonization of Antibody-Coated Red
Cells
The effector mechanisms induced by antibody binding have different effects on bacteria, viruses, particles,
and various human tissues. In general, once an IgG antibody binds to a red cell, the target cell may undergo
FcyR-mediated phagocytosis by phagocytes (Fig 10-11). If the antibody initiates the complement cascade, C3b
deposition on the red cell surface contributes to opsonization, leading to phagocytosis mediated by CR1 and
CRIg. Finally, if complement activation is complete, MAC insertion causes red cell lysis. The relative
contributions of each pathway vary based on the relative amounts of antibody isotype and subclass and on the
nature of the antigen (eg, antigen density or linkage to cytoskeleton). The sections below describe what is
known about these processes with regard to red cell destruction and the clinical manifestations of hemolysis.
Extravascular Hemolysis
Consumption of antibody- and/or C3b-bound red cells by phagocytes in the reticuloendothelial system
(RES; predominantly in the spleen and liver) is referred to as “extravascular” hemolysis because the red cells
are destroyed outside of their normal compartment, the intravascular space. This process is also commonly
referred to as a “delayed hemolytic transfusion reaction” (DHTR) because it often occurs days after transfusion
in contrast to “intravascular” hemolysis (see below), which is recognized acutely during the transfusion [eg, an
acute hemolytic transfusion reaction (AHTR)]. The delayed kinetics of DHTRs are due to the milder clinical
manifestations of extravascular hemolysis and/or the need for the implicated antibodies to develop or increase
their titer before the onset of significant red cell destruction.
The term “hemolysis” in this context can cause confusion for health-care providers not accustomed to blood
bank terminology because they typically think of hemolysis as the rupture of red cells within the circulation (ie,
intravascular hemolysis). In contrast, in extravascular hemolysis, the red cells hemolyze in the digestive
compartment (ie, lysosomes) of phagocytes. This is a very important distinction because phagocytes in the RES
consume a substantial number of senescent, autologous red cells each day in the normal process of red cell
turnover. Thus, red cell consumption in this fashion uses a pathway that evolved specifically to break down and
recycle red cell contents (eg, hemoglobin and iron) in a manner that avoids tissue damage.
This does not mean, however, that extravascular removal of antibody-coated red cells is biologically
equivalent to clearance of normal, senescent red cells. On the contrary, DHTRs can cause substantial morbidity
and occasional mortality. Indeed, in murine models of incompatible transfusion, rapid clearance of antibody-
coated red cells induces systemic inflammation and cytokine storm. Nonetheless, extravascular hemolysis is
distinctly different from intravascular hemolysis in which red cell contents are direcdy released into the
circulating blood.
It is not clear why some red cell antibodies preferentially promote opsonization and phagocytosis instead of
osmotic lysis by the MAC. Substantial evidence indicates that the antibody type and/or the topographical
arrangement of the target antigen on the red cells are important. In addition, although complement may be
activated, the aggregate opsonization of red cells by C3b and antibody may result in phagocytosis before
MACinduced lysis occurs. Consistent with these explanations is the observation that extravascular hemolysis is
typically induced by IgG red cell antibodies, whereas intravascular hemolysis is typically induced by IgM red
cell antibodies. The latter is much more efficient at fixing complement and promoting MAC formation.
251
Intravascular Hemolysis
In some cases of incompatible transfusion, the MAC rapidly assembles and lyses the red cells before C3b
and/or IgG opsonization can induce phagocytosis. Because these red cells lyse while still circulating, this is
termed “intravascular hemolysis.” In addition, because antibody-mediated intravascular hemolysis occurs at a
brisker pace than extravascular hemolysis (or, at least, is more quickly noticed due to dramatic signs and
symptoms), this hemolytic transfusion reaction is called “acute.”
As discussed above, AHTRs are typically caused by IgM antibodies, which efficiently activate complement,
leading to rapid formation of the MAC. Although an IgM-specific Fc receptor has been described (ie, FctiR), it
is predominantly expressed on nonphagocytic lymphocytes; thus, it is unlikely to promote phagocytosis of IgM-
coated red cells. Nonetheless, complement activation by IgM red cell antibodies can produce opsonization by
C3b, leading to CR1- and CRIg-mediated phagocytosis. Taken together, then, it is not surprising that IgMs
predominantly induce intravascular hemolysis.
Intravascular hemolysis, unlike extravascular hemolysis, does not normally occur at any appreciable level.
The release of red cell contents directly into the circulation can be highly toxic, with free hemoglobin inducing
perhaps the greatest insult. Although much free hemoglobin is scavenged by haptoglobin, this system is easily
overwhelmed. AHTRs often result in tea-colored urine (ie, hemoglobinuria) and can induce renal dysfunction.
Moreover, the signs and symptoms of AHTRs can be very dramatic, including disseminated intravascular
coagulation, shock, and death. This type of reaction most often occurs as a result of a clerical error that leads to
an ABOincompatible transfusion, and many current practices have evolved to prevent ABO-incompatible
AHTRs.
Nonhemolytic Red Cell Antibodies
Given the redundant pathways leading to the destruction of antibody-coated red cells, it is not surprising that
transfusions of cross
match-incompatible red cells produce hemolysis. However, somewhat surprisingly, the vast majority of red
cell antibodies are not hemolytic. For some blood-group antigens, hemolysis is never, or only very rarely,
observed following incompatible transfusion (eg, JMH, Chido, and Rodgers antigens). Indeed, approximately
1% of healthy blood donors have positive direct antiglobulin test results, indicating that IgG autoantibodies are
bound to their own red cells, yet there is no evidence of hemolysis in these donors. Even for antigens known to
be involved in antibody-mediated hemolysis (eg, in the Rh, Kell, Kidd, Duffy, and Ss systems), hemolysis is
variable. Indeed, in patients mistakenly transfused with ABOincompatible Red Blood Cell (RBC) units, no
clinically significant hemolysis occurs in 50% of cases, even for this robustly hemolytic antigen/antibody
combination.
Several explanations may account for the lack of hemolysis during incompatible transfusions. For antigens
that are essentially never involved in hemolysis, the antigen density or surface topography may prevent
hemolysis. For antigens that are variably involved in hemolysis, idiosyncrasies in a given recipient’s antibodies
(ie, titer, affinity, isotype, or IgG subclass) may play a role. Based on these properties, different antibodies (of
the same antigenic specificity) may have different capacities for activating complement. This is the rationale for
including the anti-C3 component in the antiglobulin (Coombs) reagent: it provides information on whether an
antibody can fix complement. A number of different genetic polymorphisms and/or deficiencies may also
regulate hemolysis vs red cell survival on a patient-by-patient basis, including: perturbations in complement,
complement-regulatory proteins, and allelic polymorphisms in FcyRs. Thus, in some cases, regulation of
hemolysis may be independent of the nature of the antibody.
 From a practical standpoint, crossmatchincompatible RBCs may be issued for transfusion if the offending
entity is a “clinically insignificant antibody,” especially if the antigen is of very high frequency and antigen-
negative blood is difficult or impossible to obtain. The
252
AABB TECHNICAL MANUAL
blood bank must be prepared to address appropriate concerns from health-care providers managing these
patients. Also from a practical standpoint, although clinically significant antibodies may have variable
hemolysis in different patients, there are only very limited means of predicting whether hemolysis will occur in
a given recipient. Thus, transfusion of crossmatch-incompatible RBC units should not be issued for clinically
significant antibodies unless this lifesaving procedure is specifically requested by the managing physician. If
compatible RBC units are unavailable, it might be determined that hemolysis occurring over several days (eg, in
the case of a DHTR) is less dangerous than the consequences of the patient’s severe anemia. Transfusion with a
unit that is antigen matched to the patient for common, clinically significant blood-group antigens should be
considered. Close and frequent communication between managing physicians and the blood bank is required in
these cases.
Summary of Efferent Immunity
In aggregate, when an antibody binds a red cell, multiple pathways are activated that can lead to cell
destruction. Complement activation promotes phagocytosis through the opsonizing properties of C3b and direct
red cell lysis through assembly of the MAC. The presence of IgG Fc domains promotes red cell phagocytosis by
ligating FcyRs on the phagocyte surface. The relative contributions of these different pathways vary depending
on the nature of the target antigen and the properties of the cognate antibodies. Moreover, the immune-
activation events that occur and the toxicity of the released RBC units can produce a scenario in which the
negative effects of immune destruction of transfused red cells go far beyond simply losing the efficacy of the
transfused red cells. Indeed, substantial toxicity can occur, leading to morbidity and, in some cases, mortality.
Should the reader desire more fine mechanistic details on immunobiology and the immune response, additional
sources are available.26,27
KEY POINTS
1. Hybridization-based methods can be used to detect genes, gene products, and polymorphisms. However,
compared to amplification-based methods, straight hybridization methods lack sensitivity.
2. Amplification-based methods (PCR, TMA, NASBA, SDA, and LCR) are highly sensitive but are also
susceptible to problems with contamination and/or inhibitors due to the nature of geometric amplification
involved.
3. The presence of nucleic acids predicts, but does not always equate with, expression of the corresponding
protein or antigen structure.
4. Analysis of protein expression detects the actual gene product(s). Therefore, it does not suffer from the
problems of nucleic acid testing, which relies on predicting protein expression.
5. Protein analysis is less sensitive than nucleic acid testing because no amplification is involved, but
protein analysis is also less susceptible to contamination and inhibition, which can lead to false-positive and
false-negative results.
6. Methods of detecting protein suffer from nonamplification-based artifacts (ie, heterophilic antibodies,
prozone effects, and hook effects) that can lead to erroneous results.
7. There can be substantial variability in detecting antigens and antibodies based on different methods.
8. Immunoglobulins can cause destruction of red cells by several different mechanisms, based largely on the
antigen recognized and the antibody structure.
 CHAPTER 10 Molecular Biology and Immunology in Transfusion Medicine
253
9. Most IgG antibodies that cause red cell destruction induce extravascular hemolysis by promoting
consumption of red cells by phagocytes (through Fc receptors and/or complementbased opsonization). This
typically presents as a delayed hemolytic transfusion reaction.
10. IgM antibodies (and in some rare cases IgG) that cause red cell destruction typically induce
intravascular hemolysis through complement activation to the membrane attack complex. This typically
presents as an acute hemolytic transfusion reaction.
11. Despite the serious nature of hemolysis due to incompatible transfusion, many antibodies to red cell
antigens are clinically insignificant and do not result in destruction of red cells. Incompatibility is best avoided
whenever possible, but when compatible blood is not available, incompatible RBC units can be used when the
antigens are known to be clinically insignificant. Such maneuvers must be carefully considered on a-case-by-
case basis and with extensive communication with the clinicians who are requesting the blood products.
REFERENCES
1. Alberts B, Bray D, Lewis J, et al. Molecular biology of the cell. 3rd ed. New York: Garland Science,
1994:98-105.
2. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. I
Mol Biol 1975;98:503-17.
3. Alwine IC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to
diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 1977;74:5350-4.
4. Melton DA, Krieg PA, Rebagliati MR, et al. Efficient in vitro synthesis of biologically active RNA and
RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res
1984;12:7035-56.
 5. Berk Al, Sharp PA. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of SI
endonuclease-digested hybrids. Cell 1977; 12:721-32.
6. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.
Methods Enzymol 1987;155:335-50.
7. Masukawa A, Miyachi H, Ohshima T, et al. [Monitoring of inhibitors of the polymerase chain reaction for
the detection of hepatitis C virus using the positive internal control]. Rinsho Byori lap 1997;45:673-8.
8. Al-Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells.
I Clin Microbiol 2001;39: 485-93.
9. Pang I, Modlin J, Yolken R. Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the
control of contamination in the PCRbased amplification of RNA. Mol Cell Probes 1992;6:251-6.
10. Temin HM, Mizutani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature
1970;226:1211-13.
11. Baltimore D. RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature
1970;226:1209-11.
12. Compton I. Nucleic acid sequence-based amplification. Nature 1991;350:91-2.
13. Kwoh DY, Davis GR, Whitfield KM, et al. Transcription-based amplification system and detection of
amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl
Acad Sci U S A 1989;86:1173-7.
14. Walker GT, Fraiser MS, Schram JL, et al. Strand displacement amplification—an isothermal, in vitro
DNA amplification technique. Nucleic Acids Res 1992;20:1691-6.
15. Walker GT, Little MC, Nadeau JG, Shank DD. Isothermal in vitro amplification of DNA by a restriction
enzyme/DNA polymerase system. Proc Natl Acad Sci U S A 1992;89:392-6.
16. Wu DY, Wallace RB. The ligation amplification reaction (LAR)—amplification of specific DNA
sequences using sequential rounds of template-dependent ligation. Genomics 1989;4: 560-9.
17. Denomme GA, Van Oene M. High-throughput multiplex single-nucleotide polymorphism analysis for
red cell and platelet antigen genotypes. Transfusion 2005;45:660-6.
18. Bugert R McBride S, Smith G, et al. Microarraybased genotyping for blood groups: Comparison of gene
array and 5'-nuclease assay techniques with human platelet antigen as a model. Transfusion 2005;45:654-9.

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AABB TECHNICAL MANUAL
19. Hashmi G, Shariff T, Seul M, et al. A flexible array format for large-scale, rapid blood group DNA
typing. Transfusion 2005;45:680-8.
20. Langston MM, Procter JL, Cipolone KM, Stroncek DF. Evaluation of the gel system for ABO grouping
and D typing. Transfusion 1999;39: 300-5.
 21. Voak D. Observations on the rare phenomenon of anti-A prozone and the non-specific blocking of
haemagglutination due to Cl complement fixation by IgG anti-A antibodies. Vox Sang 1972;22:408-19.
22. Judd WJ, Steiner EA, O’Donnell DB, Oberman HA. Discrepancies in reverse ABO typing due to
prozone. How safe is the immediate-spin crossmatch? Transfusion 1988;28:334-8.
23. Salama A, Mueller-Eckhardt C. Elimination of the prozone effect in the antiglobulin reaction
by a simple modification. Vox Sang 1982;42:
157-9.
24. Procter JL, Vigue F, Alegre E, et al. Rapid screening of platelet donors for PIA1 (HPA-la) alloantigen
using a solid-phase microplate immunoassay. Immunohematology 1998;14:141
5.
25. Leikola J, Perkins HA. Enzyme-linked antiglobulin test: An accurate and simple method to quantify red
cell antibodies. Transfusion 1980; 20:138-44.
26. Kindt TJ, Osborne BA, Goldsby RA. Kuby immunology. 6th ed. New York: WH Freeman and
Company, 2007.
27. Murphy K, Travers R Walport M. Janeway’s immunobiology. 7th ed. New York: Garland Science, 2008.
Chapter 11
Blood Group Genetics
Christine Lomas-Francis, MSc, FIBMS
^RiTHE science of genetics is the BSI study of heredity—that is, the mechanisms by which particular
characteristics are passed from parents to offspring. This chapter describes the genetics of blood groups. The
term “blood group" can be applied to any detectable, variable characteristic of a component of the blood
including platelet and white cell groups, serum groups, red cell enzymes, and hemoglobin variants. In this
chapter, the term “blood group” applies primarily to antigens on the surface of the red cell membrane that are
defined serologically by an antibody. Platelet and white cell blood groups are discussed in Chapter 18.
That blood groups are inherited characteristics was first shown by von Dungern and Hirszfeld in 1910, 10
years after Landsteiner’s discovery of the ABO blood group. Blood groups became an ideal tool for geneticists
because they could be identified by specific antibodies in simple hemagglutination tests and, once identified,
their inheritance could easily be followed in family studies. Red cell antigens were (and still are) valuable as
markers (detectable characteristics to recognize a gene’s presence) in genetic and anthropologic studies as well
as in relationship testing.
The detection of inherited differences on the red cells from different people is the basis of safe blood
transfusion. Therefore, an understanding of the principles of human genetics (including the patterns of
inheritance and the language or terminology in use) is an important aspect of immunohematology and
transfusion medicine. This chapter outlines the fundamental principles of genetics as they apply to blood group
antigens and relates them to examples relevant to transfusion medicine. This requires the use of numerous
genetic terms; each term, when first used or when fully described, will be in bold text and usually is closely
followed by a definition.
Technical advances in genetics and molecular biology have ushered in the age of molecular genetics, when
genes are routinely sequenced and inheritance and disease are studied at the nucleic acid level.1,2 These
advances have provided an understanding of the regulatory elements and genes that control the expression of
blood groups so that the presence or absence of blood groups can be predicted through DNA-based analysis3
with the potential to revolutionize transfusion medicine and the care of patients. Knowledge of the fundamental
principles of classical genetics
Christine Lomas-Francis, MSc, FIBMS, Technical Director, Laboratory of Immunohematology and
Genomics, New York Blood Center, New York, New York The author has disclosed no conflicts of interest.
255

256
AABB TECHNICAL MANUAL
aids understanding of the molecular aspects of individual blood groups.
BASIC PRINCIPLES OF GENETICS
 Gregor Mendel established the basic techniques of genetic analysis when, in 1865, he published his classic
breeding experiments with pea plants. Mendel’s observations led him to conclude that there is a “factor” or unit
of inheritance, now known as a gene, that is passed from one generation to another according to two simple
rules: the principles of independent segregation and independent assortment (see “Inheritance of Genetic Traits”
section below).
Cytology studies, toward the end of the 19th century showed that each living cell has a characteristic set of
chromosomes in the nucleus. In the early part of the 20th century, it was realized that chromosomes carry genes.
Biochemical studies showed that the chromosomal material is primarily made up of nucleic acids and associated
proteins.1,2
Many excellent texts offer a greater insight into classical genetics.4 The fundamental principles of genetics
outlined in this chapter are intended to serve as a review of inheritance and expression of blood group antigens.
Genes (Alleles) and Chromosomes
A gene is a segment of deoxyribonucleic acid (DNA) that encodes a particular protein. A gene is the basic
unit of inheritance of any trait (defined as a genetically determined characteristic or condition), including blood
group antigens, that is passed from parents to offspring. Genes are arranged on chromosomes with each gene
occupying a specific location known as the gene locus. A locus may be occupied by one of several alternative
forms of the gene called alleles. For example, the gene that encodes the protein carrying the Jka antigen is an
alternative form (allele) of the one that encodes the Jkb antigen. The terms “gene” and “allele” can be used
interchangeably. Based on internationally accepted gene and allele terminology, the written gene or al
lele name is italicized—for example, RHD for the gene encoding the RhD protein. The name usually is not
italicized when the word “gene” or “allele” follows the name: for example, “RHD gene,” or “RHD allele.” The
International Society of Blood Transfusion (ISBT) Working Party on Red Cell Immunogenetics and Blood
Group Terminology5,6 has developed allele terminology for use in transfusion medicine. For alleles encoding
polymorphic common antigens, the name is based on the ISBT antigen name: eg, FY*01 or JK*02 refer to the
alleles encoding the Fy;i and Jkb antigens, respectively. Alternatively, where letters are commonly used,
symbols such as FY*A or JK*B are acceptable. A genotype may also be written as the italicized antigen, eg,
Fya or Jkb when the genotype has been inferred from testing by hemagglutination.
Chromosomes are the gene-carrying structures that are visible during nuclear division in the nucleus of the
cell; they contain the genetic material (DNA) necessary to maintain the life of the cell and the organism. A
human somatic cell contains 46 chromosomes that make up 23 pairs; each pair has one paternally and one
maternally derived chromosome. In males and females, 22 of the pairs are homologous chromosomes (a pair of
chromosomes in which males and females carry equivalent genes) and are referred to as the autosomes (any
chromosome that is not a sex chromosome). The remaining pair is nonhomologous and consists of the sex
chromosomes that determine a person’s sex (gender). The sex-determining chromosomes of the male are X and
Y, whereas females have two X chromosomes. The karyotype represents the chromosome complement of a
person; this is written as “46,XY” and “46,XX” for a normal male and female, respectively. The hereditary
information carried by the chromosomes is passed from a parent cell to a daughter cell during somatic cell
division and from parents to offspring (children) by the gametes during reproduction.
Chromosomes are best studied during cell division (mitosis; see “Mitosis” section below) when they
become discrete structures in the nucleus and can be visualized by various
CHAPTER 11
Blood Group Genetics
257
microscopic techniques. All chromosomes have some common morphologic features but differ in other
characteristics, including size, location of the centromere, and staining properties. Each chromosome has two
sections or arms that are joined at a central constriction called the centromere (Figs 11-1 and 11-2).
Chromosomes are distinguished by their length and the position of the centromere. These characteristics serve
as the basis for numbering the autosomes 1 through 22 such that chromosome 1 is the largest and chromosome
22 is the smallest. An internationally recognized terminology is used to describe chromosomes. The
chromosomal arms are of different length, although the difference between the arms of chromosome 1 is not
obvious (Fig 11-2). The “p” (or petite) arm is the shorter and, in diagrams, is at the top of the chromosome. The
longer arm is termed the “q” arm. Thus, the short arm of chromosome 1 is referred to as “lp,” and the long arm
of chromosome 4 is “4q.” The terminal portion of a chromosome is referred to as “ter”; “pter” and “qter”
indicate the terminus of the short (p) and long (q) arms, respectively.
 Staining techniques provide a more detailed means to distinguish individual chromosomes. Selected dyes do
not stain chromosomes uniformly and, depending on the dye used, different banding patterns are obtained so
that each human chromosome has a unique banding pattern. As shown in Fig 11-2,

FIGURE 11-1. Diagram of a metaphase chromosome. At the metaphase stage of the cell cycle, the
chromosomes have condensed and become visible by light microscopy. As shown in the diagram, metaphase
chromosomes have replicated in preparation for cell division so that each chromosome consists of two sister
chromatids connected by the centromere. The telomere is the end or terminal part of a chromosome.
Short
arm
(P)
Long
arm
(q)
ter
p36.3 \1
p36.2
p36.1 1
p35
p34.3
p34.2 —
p34.1
P iS
p31.3
p31.2
p31.1
p22.3
p22.2 ■■
p22.1
P21
pi 3.3 pi 3.2 ■■
pi 3.1
= EH
r“S
qi2
q21.1
qllli ■■■
q22
q23 ■■
q24
q25 ■■
q31
q32.1
q32.2
q33.3
■
q4i
q42.1
q42.2
 ■Ml
q42.3
q43
q44
□ RH = SC
—Centromere
3FY
_.K/V ^CROM
ter
FIGURE 11-2. Morphology and banding pattern of a Giemsa-stained human chromosome 1. The locations
of the genes controlling the expression of antigens for the Rh (RH), Scianna (SC), Duffy (FY), Knops (KN),
and Cromer (CROM) blood group systems are shown.
Giemsa staining reveals a specific pattern of dark (G) and light (reverse or R) bands. Quinacrine, used to
stain chromosomal preparations for fluorescence microscopy, results in fluorescent bands equivalent to the dark
G bands seen in light microscopy. The G bands are heterochromatin (condensed DNA) and the lighter bands are
euchromatin, which is involved in the transcription of DNA to mRNA. The bands are numbered from the
centromere outward. Using chromosome 1 as an example, the region closest to the centromere on the short or

258
AABB TECHNICAL MANUAL
long arm is numbered lpl or lql, respectively. With greater resolution, there is further distinction into
subbands (eg, lpl 1 and lpl2) that, in turn, can be subdivided (eg, lpl 1.1 and lpll.2). Genes can be individually
mapped to a specific band location (Fig 11-2), and the chromosomal location of the genes encoding the 34 red
cell systems5'9 are shown in Table 11-1.
Cell Division
As a cell divides, the chromosomes replicate and each daughter cell receives a full complement of genetic
material. In somatic cells, this occurs through mitosis; in reproductive cells a similar process called “meiosis”
takes place. A feature common to both types of cell division is that, before the start of the process, each
chromosome replicates to form two identical daughter chromatids attached to each other through the centromere
(Fig 11-1).
Mitosis
Somatic cells divide for growth and repair by mitosis (Fig 11-3). Through this process, a single cell gives
rise to two daughter cells with identical sets of chromosomes. The daughter cells, like the parent cell, are
diploid (2N); that is, they contain 46 chromosomes in 23 pairs and have all the genetic information of the parent
cell.
Meiosis
Meiosis occurs only in germ cells that are intended to become gametes (sperm and egg cells). Somatic cells
are diploid (2N), whereas gametes are haploid [having half the chromosomal complement of somatic cells
(IN)]. Meiosis is a process of cell division and replication that leads to the formation of haploid gametes.
During meiosis, diploid cells undergo DNA replication, followed by two cycles of cell division to produce four
haploid gametes (see Fig 11-4). Because sperm and egg cells fuse at fertilization, the gametes must be haploid.
If each gamete carried a diploid (2N) set of 46 chromosomes, the resulting zygote would have 92
chromosomes, which would be incompatible with life.
Meiosis ensures genetic diversity through two mechanisms: independent assortment and crossing-over.
Through independent assortment, each daughter cell randomly receives either maternally or paternally derived
homologous chromosomes. Chromosomal crossing-over involves exchange of genetic material between
homologous chromosome pairs. Such shuffling of genetic material ensures diversity and produces genetically
unique gametes that fuse to produce a unique zygote.
X Chromosome Inactivation (Lyonization)
Females have two copies of X-borne genes in their somatic cells, while males have only one copy of X-
borne genes. Because most X-borne genes do not have a homolog on the Y chromosome, there is a potential
imbalance in the dosage of X-borne genes between males and females. This difference is compensated for by X
chromosome inactivation, (also called lyonization), a process through which most of the genes on one of the
two X chromosomes in each female somatic cell are inactivated at a very early stage of embryonic
development.17 It is a matter of chance whether the maternal or paternal X chromosome is inactivated in any
one cell, but once inactivation has occurred, all descendants of that cell will have the same inactive X
chromosome. Some Xborne genes escape inactivation; the first gene found to escape inactivation was XG, the
gene encoding the antigens of the Xg blood group system. Like XG, most of the genes that escape inactivation
are located on the extreme tip of the short arm of the X chromosome, but several are clustered in regions on the
short and long arms of the chromosome. 18(pp359'370) 19
The XK gene, which encodes the Kx blood group system, is the only other X-borne gene known to encode
red cell antigens. Changes or deletions in XK result in McLeod phenotype red cells that lack Kx antigen and
have reduced expression of Kell antigens.20,21 The XK gene, unlike XG, is subject to X chromosome inacti
259
TABLE 11-1. Blood Group Systems
Gene Product and Associated Blood
ISBT Gene Name
Chromosome Component Name Group Antigens
System ISBT
Name
(HGNC)* Location [CD Number] [Null Phenotype]
(Number)
ABO ABO 9q34.2 Glycosyltransferase, A; B; A,B; A1
(001) (ABO) carbohydrate [Group 0]
MNS MNS 4q31.21 Glycophorin A (GPA) M, N,S, s, U, He, Mia,
Vw, and 38 more [En(a-);
(002) (GYPA [CD235a]
U-; MkMk]
GYPB Glycophorin B (GPB)
GYPE) [CD235b]
PI PK PI 22q13.2 Galactosyltransferase, PI, Pk, NOR
(003) (A4GALT) carbohydrate
Rh RH 1 p36.11 RhD [CD240D] D, G, Tar
(004) (RHD RhCE [CD240CE] C, E, c, e,V, Rhl 7,
RHCE) and 45 more [Rhnui|]
Lutheran Lutheran
LU 19q13.32 Lua, Lub, Lu3, Lu4, Aua,
(005) glycoprotein;
(LU) B-cell adhesion mole Aub, and 14 more
 cule [CD239] [Recessive Lu(a-b-)]
Kell KEL 7q34 Kell glycoprotein K, k, Kpa, Kpb, Ku, Jsa,
Jsb, and 28 more [K0 or
(006) (KEL) [CD238]
Knull]
Lewis LE 19p13.3 Fucosyltransferase, Lea, Leb, Leab, Lebh,
(007) (FUT3) carbohydrate ALeb, BLeb
(Adsorbed from
[Le(a-b-)]
plasma)
Duffy FY 1q23.2 Duffy glycoprotein Fya, Fyb, Fy3, Fy5, Fy6
(008) (DARC) [CD234] [Fy(a-b-)]
JK
Kidd 18q12.h3 Human urea trans Jka, Jkb, Jk3
(SLC14A1)
porter (HUT)
(009) [Jk(a-b-)]
Kidd glycoprotein
Diego Dl 17q21.31 Band 3, Anion Dia, Dib, Wra, Wrb, Wda,
(010) (SLC4A1) Exchanger 1 [CD233] Rba, and 16 more
Yt YT 7q22.1 Acetylcholinesterase Yta, Ytb
(011) (ACHE)
xg XG (XG) Xp22.33 Xga glycoprotein Xga
CD99(/W/C2
(012) (MIC2) Ypl 1.2 CD99
product)
Scianna
SC 1 p34.2 Erythroid membrane Scl, Sc2, Sc3, Rd, and
(013)
(ERMAP) associated protein 3 more
(ERMAP) [Sc:—1 ,-2,-3]
(Continued)

260
AABB TECHNICAL MANUAL
TABLE 11-1. Blood Group Systems (Continued)
Gene Product and Associated Blood
ISBT Gene Name
Chromosome Component Name Group Antigens
System ISBT
Name
(HGNC)* Location [CD Number] [Null Phenotype]
(Number)
Dombrock Do glycoprotein;
DO 12p12.3 Doa, Dob, Gya, Hy, Joa +
(014) ART 4
 (ART4) [CD297] 3 more [Gy(a—)]
Colton CO 7p14.3 Aquaporin 1 (AQP1) Coa, Cob, Co3, Co4
(015) (AQP1) [Co(a-b-)]
LW glycoprotein
Landsteiner LW 19p13.2 LWa, LWab, LWb
Intra
cellular adhesion
Wiener (ICAM4) [LW(a-b-)]
mole
cule 4 (ICAM4)
(016)
[CD242]
Chido/ CH/RG
6p21.32 Complement compo Chi, Ch2, Rgl +6
Rodgers (C4A,
(017) C4B) nent: more
C4A; C4B [Ch-Rg-]
H H 19q13.33 Fucosyltransferase, H
carbohydrate
(018) (FUT1) [Bombay (0h)]
[CD173]
Kx XK Xp21.1 XK glycoprotein Kx
(019) (XK) [McLeod phenotype]
Gerbich Glycophorin C
GE 2q 14.3 Ge2, Ge3, Ge4, and 8
(020) (GPC)
(GYPC) [CD236] more
Glycophorin D
[Leach phenotype]
(GPD)
Cromer
CROM 1q32.2 DAF Cra, Tca, Tcb, Tcc, Dra,
(021)
Esa, IFC, and 11 more
(CD55) [CD55]
[Inab phenotype]
Knops KN 1q32.2 CR1 Kna, Knb, McCa, Sla, Yka,
(022) (CR1) [CD35] and 4 more
Hermes antigen
Indian IN 11 pi 3 lna, lnb, and 2 more
[CD44]
(023) (CD44)
0k OK 19p13.3 Neurothelin, basigin 0ka, OKGV, OKGM
(024) (BSG) [CD147]
RAPH
Raph 11 pi 5.5 Tetraspanin MER2
(CD151)
(025) [CD151] [Raph-]
JMH JMH 15q24.1 Semaphorin 7A JMH and 5 more
(026) (SEMA7A) [CD108] [JMH-]
1 GCNT2 6p24.2 Glucosaminyltransfer 1
(027) (IGNT) ase, carbohydrate [l-ori adult]
Globoside
GLOB 3q26.1 Transferase, carbohy P
(028)
drate
(B3GALNT1) [P-]
(Gb4, globoside)
 CHAPTER 11 Blood Group Genetics
261
TABLE 11-1. Blood Group Systems (Continued)
Associated
Gene Product and
Blood
ISBT Gene Group
Chromosome Component Name
System Name ISBT Antigens
Name [Null
(HGNC)* Location [CD Number]
(Number) Phenotype]
Gill GIL 9p13.3 Aquaporin 3 (AQP3) GIL
(029) (AQP3) [GIL-]
Rh- Duclos, OP,
RHAG 6p21.3 Rh-associated glyco
associated DSLKk
glycoprotein protein RHAG4
[Rhnull
(030) [GD241]
(regulator type)]
Forssman10 FORS 9q34.2 Globoside 3-a-N F0RS1
acetylgalactosaminyltransferase 1
(031) (GBGT1)
Forssman glycolipid
JR11'12 JR 4q22.1 Jr glycoprotein Jra
ATP-binding cassette, sub-family G,
(032) (ABCG2) [Jr(a-)]
member 2 (ABCG2) [CD338]
Lan13 LAN 2q36 Lan glycoprotein Lan
ATP-binding cassette, sub-family B,
(033) (ABCB6) [Lan-]
member 6 (ABCB6)
Vel14'16 VEL 1p36 Small integral mem Vel
(SMI Ml) brane protein 1 (SMIM1) [Vel-]
*lf the genetic information is obtained by blood group typing, the gene name is the italicized form of the of
the blood group system ISBT name. For example, SZ.C/4/1 / would be written as JK*Aan6 JK*B orJU and J/P.
+Provisionally numbered by the ISBT because of limited genetic evidence.
 ISBT = International Society of Blood Transfusion; HGNC = Human Gene Nomenclature Committee; ATP
= adenosine triphosphate.
vation, with the result that a female who is a carrier (a person who carries one gene for a recessive trait and
one normal gene) of a gene that is responsible for the McLeod phenotype can have a dual population of Kx-
(McLeod phenotype) and Kx+ (non-McLeod) red cells. Flow cytometry, using selected Kell antibodies, shows
the weakening of Kell antigens on red cells of the McLeod phenotype and demonstrates two red cell
populations in carrier females. This mixed-cell population reflects the randomness of whether the maternal or
paternal X chromosome is inactivated in any single somatic cell lineage.
Genotype and Phenotype
The genotype of a person is the complement of genes inherited from his or her parents; the term is
frequently also used to refer to the set of alleles at a single gene locus. The phenotype is the observable
expression of the genes inherited by a person and reflects the biologic activity of the gene(s). Thus, the presence
or absence of antigens on the red cells, as determined by serologic testing, represents the phenotype. The
presence or absence of antigens on the red cells predicted by DNA-based testing represents the genotype.
Sometimes the

262
AABB TECHNICAL MANUAL
 Interphase: Individual chromosomes are not easily distinguishable because the chromatin is not contracted.
Prophase: Chromatin has condensed; individual chromosomes can be seen; each chromosome has
duplicated and consists of two sister chromatids joined at the centromere.
Metaphase: Nuclear envelope has broken down; homologous chromosomes, their centromeres still joined,
line up at the equatorial plate as cell division begins.
Anaphase: Centromeres divide; sister chromatids separate and move to opposite poles.
Telophase: The nuclear envelope reappears; the cytoplasm divides; the cell membrane pinches inward; two
identical daughter cells have been formed.
FIGURE 11-3. Diagram showing mitosis.
genotype can be predicted from the phenotype; for example, when a person’s red cells are reactive with anti-
Jka and anti-Jkb, which is a Jk(a+b+) phenotype, a JK*AIJK*B genotype can be inferred. Frequently, the
phenotype provides only a partial indication of the genotype; for example, red cells that are group B reflect the
presence of a B gene, but the genotype may be B/B or BIO. For decades, family studies were often the only way
to determine a person’s genotype, but now that most antigens and phenotypes can be defined at the DNA level,
family studies to determine genotype can be mostly replaced by DNA analysis (see “Blood Group Genomics”
section below).
Alleles
A gene at a given locus on a chromosome may exist in more than one form, that is, be allelic. Each person
has two alleles for a trait, one that is maternally derived and another that is paternally derived. At the simplest
level, the ABO gene locus can be considered to have three alleles: A, B, and 0. With three alleles, there are six
possible genotypes (A/A, A/O, MB, B/B, BIO, and O/O). Depending on the parental contribution, a person
could inherit any combination of two of the alleles and express the corresponding antigens on their red cells.
For example, inheritance of A/A and A/0 would
263
CHAPTER 11 Blood Group Genetics
2N
Diploid
4N
Parent
Cell
Nucleus
DNA Replication
4N
 Nuclear
envelope
2N
2N
Diploid

N
Haploid

Interphase: Individual chromosomes are not easily distinguishable because the chromatin is not contracted.
Prophase I:
Chromosomes have duplicated and homologous chromosomes have paired.
Crossing over occurs, resulting in an exchange of genetic material between homologous chromosomes.
Metaphase I: Homologous chromosomes line up at the equatorial plate.
Anaphase: First meiotic division occurs as homologous pairs of chromosomes separate. Process continues to
telophase and cell division.
Formation of two daughter cells.
Second meiotic division: Chromosomal duplication does not occur; previously duplicated chromosomes
separate.
Four daughter cells formed with chromosomes reduced to half.
FIGURE 11-4. Diagram showing meiosis.
result in group A red cells, A/B would result in group AB red cells, B/B and BIO would result in group B
red cells, and O/O would result in group 0 red cells.
When identical alleles for a given locus are present on both chromosomes, the person is said to be
“homozygous” for the particular allele. A person who is hemizygous for an allele has only a single copy of an
allele instead of the customary two copies; an example is the deletion of one RHD in a D-positive phenotype.
When different (ie, not identical) alleles are present at a particular locus, the person is
said to be “heterozygous." For example, a person with K-k+ red cells is homozygous at the KEL locus for
the allele (KEL*02) encoding the k antigen. A person who is heterozygous for KEL*01 and KEL*02
(KEL*01/02 genotype), would have red cells that are K+k+.
Antigens that are encoded by alleles at the same locus are said to be “antithetical” (meaning “opposite”);
thus, K and k are a pair of antithetical antigens. It is incorrect to refer to red cells that are K-k-i- or Kp(a-b+) as
being homozygous for the k or Kpb antigen; rather, it should be said that the cells have a double

264
AABB TECHNICAL MANUAL
dose of the antigen and that they are from a person who is homozygous for the allele. Genes are allelic
whereas antigens are antithetical.
The quantity of antigen expressed (antigen density) is influenced by whether a person is heterozygous or
homozygous for an allele; the antigen density is generally greater when a person is homozygous. In some blood
group systems, this difference in antigen density is manifested by antibodies giving stronger reactions with cells
that have a double dose of the antigen. Red cells with the Jk(a+b-) phenotype, encoded by a JK*AIA genotype,
have a double dose of the Jka antigen and often are more strongly reactive with anti-Jka than those that are
Jk(a+b+) and have a single dose of the antigen. Similarly, M+N- red cells tend to be more strongly reactive with
anti-M than are M+N+ red cells. Antibodies that are weakly reactive may not be detected if they are tested with
red cells expressing a single dose of the antigen. This observable difference in strength of reaction, based on
homozygosity or heterozygosity for an allele, is termed the “dosage effect."
Polymorphism
For blood group genetics, polymorphism refers to the occurrence in the population of genomes with allelic
variation (two or more alleles at one locus) producing different phenotypes, each with appreciable (greater than
1%) frequency. Some blood group systems (Rh and MNS for example) are highly polymorphic and have many
more alleles at a given locus than other systems such as Duffy, and Colton.5 An allele that is polymorphic in
one population is not necessarily polymorphic in all populations; for example, the FY allele associated with
silencing of Fyb in red cells (FY*02N.01) is polymorphic in populations of African ethnicity with a prevalence
of greater than 70%, but this allele is not found in other populations. A gene polymorphism may represent an
evolutionary advantage for a population, and a polymorphic population is likely to adapt to evolutionary change
more rapidly than if the
population had genetic uniformity. It is not yet understood what, if any, evolutionary advantages were
derived from the extensive polymorphism displayed by red cell antigens, but many publications associate
resistance to, or susceptibility for, a particular disease with a particular blood type.22
The difference between two alleles is the result of a permanent change in the DNA. An event that leads to
the production of an altered gene and a new allele or polymorphism that did not exist in the biologic parents is
referred to as a “mutation.” The mutation rate of expressed genes resulting in a new phenotype has been
estimated to be less than 10'5 (<1 in 100,000) in humans, and a mutation has to occur in the germ cells
(gametes) to be inherited.
A mutation can occur spontaneously or be brought about by agents such as radiation (eg, ultraviolet rays or
x-rays) or chemicals. A mutation may occur within a gene or in the intergenic regions. It may be silent—that is,
have no effect on the encoded protein—or it may alter the gene product and potentially cause an observable
effect in the phenotype. In the context of an allele that encodes a protein that carries red cell antigens, any
genetically induced change must be recognized by a specific antibody before an allele can be said to encode a
new antigen.
Numerous genetic events that generate red cell antigens and phenotypes have been identified. The event can
occur at the level of the chromosome (eg, deletion or translocation of part of a chromosome), the gene (eg,
deletion, conversion, or rearrangement), the exon (eg, deletion or duplication), or the nucleotide (eg, deletion,
substitution, or insertion). The mechanism that has given rise to most diversity in the human genome is single
nucleotide polymorphism (SNP)—that is, a single nucleotide change in the DNA.23'25 Accordingly, the
majority of polymorphic blood group antigens are the result of SNPs.3,26,27 DNA analysis to predict the red
cell phenotype is discussed in more detail in the section on “Blood Group Genomics.”
265
INHERITANCE OF GENETIC TRAITS
A genetic trait is the observed expression of one or more genes. The inheritance of a trait (and red cell
antigens) is determined by whether the gene responsible is located on an autosome or on the X chromosome
(sexlinked) and whether the trait is dominant or recessive.
Pedigrees
A family study follows the inheritance of a genetic characteristic—for example, an allele encoding the
expression of a red cell antigen—as it is transmitted through a kinship. A diagram that depicts the relationship
of family members and shows which family members express (are affected), or do not express, the trait under
study is termed a pedigree. A review of a pedigree should reveal the pattern or type of inheritance for the trait,
or antigen, of interest. The person who first caused the family to be investigated is considered the index case
and is often referred to as the proband or propositus/proposita (male singular form or gender unknown/female
singular form); propositi is the plural form regardless of gender. Details of the conventions and the symbols
used for the construction of pedigrees are provided in Figs 11-5 and 11-6.
Autosomal Dominant Inheritance
An antigen (or any trait) that is inherited in an autosomal dominant manner is always expressed when the
relevant allele is present, regardless of whether a person is homozygous or heterozygous for the allele. The
antigen appears in every generation and occurs with equal frequency in both males and females. A person who
carries an autosomal dominant trait, on average, transmits it to half of his or her children. The pedigree in Fig
11-7 demonstrates autosomal dominant inheritance and shows that the B allele is dominant over 0.
 FIGURE 11-5. An example of a pedigree. Males are denoted by squares and females by circles, and each
different generation in a pedigree is identified by Roman numerals. Persons in each generation are identified by
Arabic numerals; the numbering is sequential from left to right, with the eldest child for each family unit being
placed on the left of any series of siblings. Closed symbols represent family members affected by the trait,
whereas open symbols are unaffected members.
Autosomal Codominant Inheritance
Blood group antigens that appear to be autosomal dominant may be encoded by alleles that are inherited in a
codominant manner— that is, when two different alleles are present (the heterozygous condition), the products
of both alleles are expressed. Thus, when red cells have the S+s+ phenotype, the presence of one allele
encoding S and another allele encoding s [or an S/s (GYPB*S/s) genotype] can be inferred.
Autosomal Recessive Inheritance
A trait with autosomal recessive inheritance is expressed only in a person who is homozygous for the allele
and has inherited the recessive allele from both parents. When a person inherits a single copy of a recessive
allele in combination with a silent or deleted (null) allele—that is, a nonfunctioning allele or one that encodes a
product that cannot be detected—the recessive trait is expressed and the person appears to be homozygous. It is
difficult or impossible
266
AABB TECHNICAL MANUAL
□ Male O Female
Gender not specified
□—O Mating (the male is always on the left)
0=0 Consanguineous mating
Siblings (in order of birth)
I # Trait expressed
DC Heterozygote
© Carrier (heterozygote) for X-borne recessive trait
cA)
^□cfbcA]
Abortions or miscarriages Monozygotic twins Dizygotic twins
0 0 Deceased
Indicates proband
FIGURE 11-6. Symbols, and their significance, used in the construction of pedigrees.
to distinguish such a combination from homozygosity for the recessive allele through serologic testing, but
DNA-based testing can usually make this distinction.
A mating between two heterozygous carriers results in one in four of the children being homozygous for the
trait. The parents of a child who is homozygous for a recessive trait must be obligate carriers of the trait. If the
frequency of the recessive allele is low, the condition is rare and usually found only among siblings (brothers
and sisters) of the person and not in other relatives. The condition is not found in preceding or successive
generations unless consanguineous mating (ie, between blood relatives) occurs. When a recessive allele is rare,
the parents of an affected person are most likely consanguineous because a rare allele is more likely to occur in
blood relatives than in unrelated persons in a random population. When a recessive trait is one that is common,
consanguinity is not a prerequisite for homozygosity; for example, the 0 allele of the ABO system, although
recessive, is not rare, and persons who are homozygous for 0 are easily found in the random population.

idTTO
BO BOO
 []orQ = group 0 Bor® = group B

FIGURE 11-7. Autosomal dominant inheritance of the ABO alleles. Based on the ABO groups of his
children, 1-1 would be expected to have a B/0 rather than a B/B genotype (showing that the B allele is dominant
over 0) because two of his children (11-6 and 11-7) are group 0 and must have inherited an 0 allele from their
father (1-1) in addition to the 0 allele inherited from their mother (1-2). Similarly, 11-2 and 11-3 are B/0, based
on the ABO type of their children, showing the dominance of B over 0.
CHAPTER 11 Blood Group Genetics
267
In blood group genetics, a recessive trait or condition almost always means that red cells express a null
phenotype [eg, the Lu(a-b-) or Rhnull or 0 phenotypes] because of homozygosity for a “silent” or “amorphic
gene” that either results in no product or encodes a defective product. The family in Fig 11-8 demonstrates the
inheritance of a recessive silent LU gene, which in the homozygous state results in the Lu(a-b-) phenotype. The
proband, II-3, who was multitransfused, was identified because of the presence of anti-Lu3 (an antibody to a
high-prevalence Lutheran antigen) in his plasma. Because his Lu(a-b-) phenotype is the result of recessive
inheritance, any potential donors in the family can be found by testing his siblings. About 25%, or one in four of
the offspring of the mating between 1-1 and 1-2, is expected to have the Lu(a-b-) phenotype; in this case, only
the proband has Lu(a-b-) red cells.
Sex-Linked Inheritance
A sex-linked trait is one that is encoded by a gene located on the X or Y chromosome. The Y chromosome
carries few functional genes and discussion of sex-linked inheritance generally is synonymous with inheritance
of X-borne genes. In females with two X chromosomes the inheritance of X-borne genes, like the inheritance of
genes carried on the autosomes, can be dominant or recessive. Males, in contrast, have one X chromosome
(always maternally derived) and one Y chromosome (always paternally derived) and are hemizygous for genes
on the X or Y chromosome because only one chromosome (and thus one copy of a gene) is present. Most X-
borne genes do not have a homolog (a similar sequence of DNA) on the Y chromosome. As a consequence,
inheritance of an X-borne dominant trait is the same in males and females. However, an Xborne trait that is
recessive in females is

| = homozygosity for Lu Q or Q = 'acks tra't (*-°) H or C) = heterozygosity for Lu

FIGURE 11-8. Autosomal recessive inheritance. The offspring of 11-3, the Lu(a-b-) proband, and 11-4, his
Lu(a+b+) wife, demonstrate that Lu is recessive to Lif and Lub and that the presence of the silent Lu allele is
masked by the product of Luaor Lubat the phenotype level.
268
AABB TECHNICAL MANUAL
expressed by all males who carry the gene for the trait. The most striking feature of both dominant and
recessive X-linked inheritance is that there is no male-to-male transmission; that is, the trait is never transmitted
from father to son.
Sex-Linked Dominant Inheritance
 A trait encoded by an X-borne allele that has sex-linked dominant inheritance is expressed by hemizygous
males and by both heterozygous and homozygous females. A male passes his single X chromosome to all of his
daughters and all daughters will express the condition or trait. When a female is heterozygous for an allele that
encodes a dominant trait, each of her children, whether male or female, has a 50% chance of inheriting the trait.
When a female is homozygous for an X-borne allele with dominant inheritance, the encoded trait is expressed
by all her children.
The Xga antigen (Xg blood group system) is encoded by an allele on the X chromosome
and is inherited in a sex-linked dominant manner. The first indication that the Xga antigen is X-borne came
from the observation that the prevalence of the Xg(a-) and Xg(a+) phenotypes differed noticeably between
males and females; the Xga antigen has a prevalence of 89% in females and only 66% in males.5
Figure 11-9 shows the inheritance of the Xga antigen in a three-generation family. In generation I, the father
(1-1) is Xg(a+) and has transmitted Xg" to all his daughters but to none of his sons. His eldest daughter (II-2),
for example, must be heterozygous for Xg’/Xg; she received the allele encoding the Xga antigen from her
Xg(a+) father and a silent allele, Xg, from her Xg(a-) mother. 11-2 has transmitted Xg" to half her children,
regardless of whether they are sons or daughters.
Sex-Linked Recessive Inheritance
A trait encoded by an X-borne recessive allele is carried, but not expressed, by a heterozygous female. A
male inherits the trait from his
Xg(a+)
Xg°
-o2
Xg(a-)
Xg/Xg
—
Xg(a-)
Xg
Xg(a+)
XgffXg
Xg(a-)
Xg
Xg(a+)
XgffXg
fid
Xg(a-) Xg(a+) Xg(a+) Xg(a-) Xg(a+) Xg(a-)
Xg Xg3 Xgr/Xg Xg/Xg Xg/Xg3 Xg/Xg
¥
Xg(a
xg
Xg(a+) Xg(a-) Xga/Xg Xg
Xg(a+)
Xg°/Xg°
or
Xg3/Xg
Xg(a+)
Xg*
□ orO = persons have Xg(a-)red cells |orQ = persons have Xg(a+)red cells
FIGURE 11-9. Sex-linked dominant inheritance. The Xga antigen is encoded by an allele on the tip of the
short arm of the X chromosome. This family demonstrates the sex-linked dominant inheritance of the Xga
antigen.
269
CHAPTER 11
mother, who is usually a carrier (or could be homozygous for the trait if she is the offspring of a male who
expresses the trait and a carrier female). An affected male transmits the trait to all of his daughters, who in turn
transmit the trait to approximately half of their sons. Therefore, the prevalence of the expression of an Xborne
recessive trait is much higher in males than in females. A carrier female who mates with a male lacking the trait
transmits the trait to one-half of her daughters (who will also be carriers) and to one-half of her sons (who will
be affected). If mating is between an affected male and a female who lacks the trait, all of the sons will lack the
trait and all of the daughters will be carriers. If an X-borne recessive trait is rare in the population, the trait is
expressed almost exclusively in males.
The XK gene encodes the Kx protein and demonstrates X-borne recessive inheritance.
Blood Group Genetics
Mutations in XK result in red cells with the McLeod phenotype; such red cells lack Kx and have reduced
expression of Kell antigens (McLeod syndrome). McLeod syndrome is associated with late-onset clinical or
subclinical myopathy, neurodegeneration, and central nervous system manifestations as well as with
acanthocytosis and, frequently, compensated hemolytic anemia. More than 30 different XK gene mutations
associated with a McLeod phenotype have been found. Different XK mutations appear to have different clinical
effects and may account for the variability in the prognosis.28 Sequencing of XK to determine the specific type
of mutation in individuals with McLeod phenotypes has clinical prognostic value. McLeod syndrome is an X-
linked recessive condition and, as demonstrated by the family in Fig 11-10, is found only in males.

□
0
Unaffected; normal Kell antigen expression
I McLeod phenotype
0 Carrier of McLeod phenotype; mixed red cell population of McLeod phenotype and normal Kell antigen
expression
FIGURE 11-10. Sex-linked recessive inheritance. This family demonstrates that a sex-linked trait that is
recessive in females will be expressed by any male who inherits the trait. Homozygosity for such a trait is
required for it to be expressed in females. The trait skips one generation and is carried through females.
270
AABB TECHNICAL MANUAL
The Principles of Independent Segregation and Independent Assortment
The passing of a trait from one generation to the next follows certain patterns or principles. The principle of
independent segregation refers to the separation of homologous chromosomes and their random distribution to
the gametes during meiosis. Only one member of an allelic pair is passed on to the next generation, and each
gamete has an equal probability of receiving either member of a parental homologous allelic pair; these
chromosomes are randomly united at fertilization and thus segregate independently from one generation to the
next. The family in Fig 11-11 demonstrates the independent segregation of the ABO alleles on chromosome 9.
The principle of independent assortment states that alleles determining various traits are inherited
independently from each other. In other words, the inheritance of one allele (eg, a B allele, on chromosome 9,
encod
ing B antigens) does not influence the inheritance of another allele (eg, an M allele, on chromosome 4,
encoding M antigens). This is demonstrated by the family in Fig 11 -11.
Linkage and Crossing-Over
Linkage is the physical association between two genes that are located on the same chromosome and are
inherited together. Examples include RHD and RHCE encoding the antigens of the Rh system, which are both
on chromosome 1 and are linked loci that do not assort independently.
Crossing-over is the exchange of genetic material between homologous chromosome pairs (Fig 11-4). In
this process, a segment from one chromatid (and any associated genes) changes places with the corresponding
part of the other chromatid (and its associated genes); the segments are rejoined, and some genes will have
switched chromosomes. Thus, crossing-over is a means to shuffle genetic material. Because crossing-over can
result in new
II
Phenotype
Genotype
Phenotype
Genotype
B
B/0
M+N+
M/N

<y
A
NO
M+N
M/M
0
Phenotype B 0 A AB
Genotype B/0 0/0 NO NB
Phenotype M+N+ M+N+ M+N M+N
Genotype M/N M/N M/M M/M
FIGURE 11-11. Independent segregation and independent assortment are illustrated by the inheritance of
blood group alleles in one family. Parental ABO alleles were randomly transmitted (independent segregation),
and each child has inherited a different combination. The family also illustrates that the alleles encoding
antigens of the ABO and MNS blood group systems are inherited independently from each other.
271
gene combinations on the chromosomes involved, it is also referred to as recombination, and the rearranged
chromosomes can be referred to as recombinants. Crossing-over and recombination, using chromosome 1 as an
example, are explained in Fig 11-12.
Two gene loci carried by the same chromosome that are not closely linked are referred to as being syntenic.
For example, the loci for RH and FY, both located on chromosome 1, are syntenic because the distance between
them [RH on the short arm and FY on the long arm) is great enough for them to undergo crossing-over and to
assort independently.
The frequency of crossing-over involving two genes on the same chromosome is a measure of the distance
[measured in centimorgans (cM)] between them; the greater the distance between two loci, the greater the
probability that crossing-over (and recombination) will occur. In contrast, genes located very close together
(linked) tend to be transmitted together with no recombination. The degree of crossing-over between two genes
can be calculated by analyzing pedigrees of families informative for the genes of interest and observing the
extent of recombination.The traditional method of linkage analysis requires the use of lod (logarithm of the
odds) scores.29 Linkage analysis was the basis through which chromosomes were mapped and the relative
position and distance between genes established. Linkage between Lutheran [LU] and ABH secretion (SE or
FUT2) was the
first recognized example of autosomal linkage in humans and is explained in Fig 11-13.
Although crossing-over occurs readily between distant genes, rare examples of recombination have been
documented for genes that are very closely linked or adjacent on a chromosome. Such genes include those
encoding the MN (GYPA) and Ss (GYPB) antigens on chromosome 4 and are reviewed by
Daniels.18(pp96'142)
Linkage Disequilibrium
Genes at closely linked loci tend to be inherited together and constitute a haplotype (a combination of alleles
at two or more closely linked loci on the same chromosome). The alleles encoding the MNS antigens are
inherited as four haplotypes: MS, Ms, NS, or Ns. Because linked genes do not assort independently, the antigens
encoded by each of these haplotypes have a different prevalence in the population than would be expected by
random assortment. If M and S were not linked, the expected prevalence for M+ and S+ in the population would
be 17% (from frequency calculations), whereas the actual or observed prevalence (obtained from testing and
analyzing families) of the MS haplotype is 24%.18(pp96'142) This constitutes linkage disequilibrium, which is
the tendency of specific combinations of alleles at two or more linked loci to be inherited together more
frequently than would be expected by chance.
FYB
KN [Sl(a+)]

RHCE Cr FYA
KN [Sl(a-)]
Homologous pair of chromosome 1
RHCE RHD
W vf U Breakage and y y \l All
® M Recombination j^FYA Jf )\
KN [Sl(a-)] **N [Sl(a+)] 2 recombinant
chromosomes
FIGURE 11-12. Crossing-over and recombination. In the diagram, chromosome 1 is used as an example.
The very closely linked RH genes, RHD and RHCE, are located near the tip of the short arm of chromosome 1.
The loci for FY and MV are on the long arm of the chromosome and are not linked. During meiosis, crossing-
over occurs between this homologous chromosome pair, and portions of the chromosome break and become
rejoined to the partner chromosome. Crossing-over of the long arm of chromosome 1 results in recombination
between the loci for FY and KN such that the gene encoding the Fyb antigen now travels with a gene that
encodes the Sl(a-) phenotype of the Knops system.
272
AABB TECHNICAL MANUAL

Lua/Lub Lub/Lub Lub/Lub Lua/Lub Lua/Lub

se/se Se/se Se/se se/se se/se
FIGURE 11-13. Linkage between LUand SE. 1-2 is homozygous for Lub and seand must transmit these
alleles to all her offspring. 1-1 is doubly heterozygous [LuVLuband Se/se). He has transmitted Lub with Seand
Lif with se, showing linkage between Lu and Se. Several such informative families would need to be analyzed
to statistically confirm linkage.
Gene Interaction and Position Effect
Alleles that are carried on the same chromosome are referred to as being in cis position whereas those on
opposite chromosomes of a homologous pair are in trans position. Alleles that are in cis and linked are always
inherited together on the same chromosome, whereas genes in trans segregate independently.
Historically, the Rh blood group system was used to explain the meaning of cis and tram. For example, the
DCe/DcE genotype was described as having C and e alleles in cis in the DCe haplotype, with c and E alleles in
cis in the partner DcE haplotype, whereas C and E and also c and e are in opposing haplotypes and are in trans.
In this alignment, C and e, for example, are always inherited together, but C and E are not. The preceding
explanation, which implies that one gene encodes C and c antigens while another linked gene encodes E and e
antigens, was based on the Fisher-Race theory of three genes at the RH locus. In contrast, molecular analysis
indicates that only one gene IRHCE), with four alleles (RHCE*Ce, RHCE*cE, RHCE*ce, and RHCE*CE),
encodes one protein that carries the CcEe antigens. Thus, for Rh,
DCe is the haplotype, and the RHD allele is in cis to the RHCE*Ce allele.
The expression of red cell antigens may be modified or affected by gene or protein interactions that manifest
primarily as reduced antigen expression. One example in which the haplotype on one chromosome affects the
expression of the haplotype on the paired chromosome is commonly referred to as the “position effect” and can
be observed with Rh antigen expression. When a Ce haplotype (note the absence of RHD) is in tram to a D
antigen-encoding haplotype, the expression of D is dramatically reduced and a weak D phenotype can result.
When the same D-encoding haplotype is inherited with either ce or cE, D antigen is normally expressed. The
cause of this reduced antigen expression is not known, but may involve differences in gene expression levels or
altered assembly of proteins in the membrane. In the presence of the Kell system antigen Kpa, expression of
other Kell system antigens encoded by the same allele is suppressed to varying degrees (ds-modifier effect).
This is best observed in persons who have a silenced K° (a Kellnull gene) in tram. The amino acid change that
results in the expression of Kpa adversely affects trafficking of
273
the Kell glycoprotein to the red cell surface, so that the quantity of Kpa-carrying Kell glycoprotein that
reaches the red cell surface is greatly reduced.
Suppressor or modifier genes affect the expression of another gene, or genes. For example, KLF1, located
on chromosome 19pl3.3-pl3.12, encodes erythroid Kriippellike factor, which is a transcription factor essential
for terminal differentiation of red cells. Singleton et al30 first discovered that heterozygosity for nucleotide
changes in KLF1 is responsible for the dominant Lu(a-b-) phenotype,5 which is also known as the In(Lu)
phenotype. This heterozygosity is characterized by reduced expression of antigens in the Lutheran system
(Lumod) and for PI, Inb and AnWj antigens.
In kind, the transcription factor GATA-1, encoded by the X-borne GATA-1 gene, is essential for erythroid
and megakaryocyte differentiation. Changes in this gene were associated with the X-linked type of Lumod that
initially presented as an Lu(a-b-) phenotype.31 Serologic differentiation of these Lumod phenotypes from the
true Lu(a-b-) (Lunull) phenotype can be challenging, yet has clinical value. Now that the molecular basis of
these phenotypes is understood, sequencing of the relevant genes can be used to make the distinction.
In the past, independent, unidentified modifier or regulator genes were postulated to be the basis of several
null or variant phenotypes when a silent or inactive (nonfunctioning) gene was not evident. For example, the
regulator type (as opposed to the amorph type) of Rhnull was shown through family studies to result from a
gene not at the RH locus. This phenotype is now known to result from various silencing changes in RFLAG, a
gene located on chromosome 6 (and thus independent of RH). RHAG encodes the Rh-associated glycoprotein
RhAG, which is required in the red cell membrane for Rh antigen expression. Similarly, molecular analysis
indicates that the modifying gene that causes the Rhmod phenotype is a mutated RHAG.5
Several red cell antigens require the interaction of the products of two or more independent genes for their
expression. Assignment of
the high-prevalence antigen Wrb to the Diego blood group system took many years because the only red
cells that lacked Wrb were rare MNS null phenotypes [En(a-) and MkMk] and variants, yet Wra, the antigen
that is antithetical to Wrb was clearly independent of MNS. This anomaly was resolved when it was understood
that GPA (or more precisely, amino acids 75 to 99), which carries MN antigens, must be present in the red cell
membrane for Wrb expression. The Wra/Wrb polymorphism is encoded by DI and carried on band 3, whereas
GPA is the product of GYPA, a gene that is independent of DI. An absence of RhD and RhCE protein (Rhnull)
results in red cells that lack LW antigens and lack or have reduced expression of U and S or s antigens, again
demonstrating the interaction in the membrane of the products of two or more independent blood group genes.
The sequential interaction of genes at several loci is required for the expression of ABO, H, Lewis, and I
antigens on red cells and in secretions. These antigens are carbohydrate determinants carried on glycoproteins
or glycolipids, and the genetics of these antigens is more complex than that of protein-based antigens. The
carbohydrate antigens are carried on oligosaccharide chains that are assembled by the stepwise addition of
monosaccharides. ABO genes and the genes encoding the other carbohydrate-based antigens do not encode
membrane proteins but do encode an enzyme, a glycosyltransferase, that catalyzes the sequential transfer of the
appropriate immunodominant monosaccharide. Each monosaccharide structure is transferred by a separate
glycosyltransferase, such that two genes are required for a disaccharide, three for a trisaccharide, and so on. An
inactivating change at one locus can prevent or modify the expression of the other gene products. The product
encoded by the H gene is the biosynthetic precursor for A and B antigen production; if the H gene is silenced, A
or B antigens cannot be produced. A mutated A or B allele may result in a glycosyltransferase that is inactive or
that causes more or less antigen to be expressed. Details on the biosynthesis of the ABO, H, Lewis, and I
antigens maybe found in Chapter 12.
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 POPULATION GENETICS
Population genetics is the study of the distribution patterns of genes and of the factors that maintain or
change gene (or allele) frequencies. A basic understanding of population genetics, probability, and the
application of simple algebraic calculations is important for relationship (identity) testing. In transfusion
medicine, the knowledge can be applied to clinical situations such as predicting the likelihood of finding
compatible blood for a patient who has made antibody(ies) to red cell antigens. It may be helpful to define three
commonly used words so that their appropriate use is understood. “Frequency” is used to describe prevalence at
the genetic level—that is, the occurrence of an allele (gene) in a population. “Prevalence” is used to describe the
occurrence of a permanent inherited characteristic at the phenotypic level—for example, a blood group—in any
given population. “Incidence” is used when describing the rate of occurrence in a population of a condition that
changes over time, such as a disease, and is thus not suitable for use with blood groups.
Phenotype Prevalence
The prevalence of a blood group antigen or phenotype is determined by testing red cells from a large
random sample of people of the same race or ethnicity with a specific antibody and calculating the percentage
of positive and negative reactions. The larger the cohort being tested, the more statistically significant is the
result. The sum of the percentages for the prevalence of the phenotypes should equal 100%. For example, in the
Duffy blood group system, the prevalence in a random population of African ethnicity for the Fy(a+b-), Fy(a-
b+), Fy(a+b+), and Fy(a-b-) phenotypes is 9%, 22%, 1%, and 68%, respectively; together, these percentages
total 100%. If the red cells from 1000 donors of European ethnicity are tested with anti-c, and 800 of the
samples are positive and 200 are negative for the Rh antigen c, the prevalence of the c+ phenotype is 80% and
that of the c- phenotype is 20%. Thus, in this donor population, approximately 20% of ABO-compatible units of
blood, or 1 in 5,
should be compatible with serum from a patient who has made anti-c.
Calculations for Antigen-Negative Phenotypes
When blood is provided for a patient with antibodies directed at one or more red cell antigens, a simple
calculation can be used to estimate the number of units that need to be tested to find the desired antigen
combination. To calculate the prevalence of the combined antigen-negative phenotype, the prevalences of each
of the individual antigens are multiplied together because the antigens are inherited independently of each other.
An exception occurs when the antigens are encoded by alleles that are closely linked and are inherited as
haplotypes (M, N, S, s) or reside on the same carrier protein (C, c, E, e). If a patient with antibodies to K, S, and
Jka antigens requires 3 units of blood, for example, the prevalence of the antigen-negative phenotype and the
number of units that need to be tested to find them can be calculated as follows:
■ The prevalence of: K- donors = 91%; S- donors = 48%; Jk(a-) donors = 23%
■ The percentage of donors negative for each antigen is expressed as a decimal and multiplied: 0.91(K-) x
0.48(S—) x 0.23[Jk(a—)] = 0.10
■ 0.10 expressed as a % = 0.10 x 100% = 10%
■ 10% expressed as occurrence = 10%/100% = 1/10
■ Thus, approximately 1 in 10 ABO-compatible RBC units are expected to be K- S- Jk(a-)
The patient in question requires 3 units, so on average, 30 units would need to be tested to fulfill the request.
Based on these calculations, a hospital transfusion service would be able to determine the likelihood of having
the requested units in house.
The prevalence of a particular antigen (or phenotype) can vary with race,5 and the prevalence for a
combined antigen-negative phenotype calculation should be selected on the basis of the predominant race found
in the donor population.
275
Allele (Gene) Frequency
The allele frequency is the proportion of one allele relative to all alleles at a particular gene locus in a given
population at a given time. This frequency can be calculated from the prevalence of each phenotype observed in
a population. The sum of allele frequencies at any given locus must equal 100% (or 1 in an algebraic
calculation) in the population sample tested. The genotype frequency in a population is the number of
individuals with a given genotype divided by the total number of individuals in population.
The Hardy-Weinberg Equilibrium
Gene frequencies tend to remain constant from generation to generation in any relatively large population
unless they are influenced by factors such as selection, mutation, migration, or nonrandom mating, any of which
would have to be rampant to have a discernible effect. According to the principles proposed by the British
mathematician Hardy and the German physician Weinberg, gene frequencies reach equilibrium. This
equilibrium can be expressed in algebraic terms by the Hardy-Weinberg formula or equation:
p2 + 2pq + q2 = 1
If two alleles, classically referred to as A and a, have gene frequencies of p and q, the homozygotes and
heterozygotes are present in the population in the following proportions:
AA = p2 Aa = 2pq aa = q2
In such a two-allele system, if the gene frequency for one allele, say p, is known, q can be calculated by p +
q = 1.
The Hardy-Weinberg equation permits the estimation of genotype frequencies from the phenotype
prevalence in a sampled population and, reciprocally, allows the determination of genotype frequency and
phenotype prevalence from the gene frequency. The equation has a number of applications in
blood group genetics, and its use is demonstrated below. In a population of European ethnicity, the
frequencies of the two alleles encoding K or k can be determined as follows:
Frequency of the K allele = p Frequency of the k allele = q
Frequency of the genotype = p2 Frequency of the Kk genotype = 2pq
Frequency of the kk genotype = q2
The K antigen is expressed on the red cells of 9% of people of European ethnicity; therefore:
p2 + 2pq = the frequency of people who carry K and are K+
Thus, p2+ 2pq = 0.09
q2 = 1 - (p2 + 2pq) = the frequency of people who carry kk and are K
q2= 1-0.09
q2 = 0.91
q = Jom
q = 0.95 = the frequency of k
Because the sum of the frequencies of both alleles must equal 1.00:
p + q=l
p = i-q
p = 1 - 0.95
p = 0.05 = the frequency of K
Once the allele frequencies for K and k have been calculated, it is possible to calculate the percentage of k+
(both K+k+ and K-k+) and K+ (both K+k- and K+k+) people:
Prevalence of k+ = 2pq + q2
= 2(0.05 x 0.95) + (0.95)2 = 0.9975x 100
= a calculated prevalence of 99.75% (the observed prevalence of the k+ phenotype is 99.8%)
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Prevalence of K+ = 2pq + p2
= 2(0.05 x 0.95) + (0.05)2 = 0.0975
= 0.0975 x 100 = a calculated prevalence of K+ of 9.75% (the observed prevalence of the K+ phenotype is
9%)
The Hardy-Weinberg equation also can be used to calculate the frequencies of the three possible genotypes
KK, Kk, and kk from the gene frequencies K(p) = 0.05 and k (q) = 0.95:
p2 + 2pq + q2 = 1
Frequency oiKK= p2 = 0.0025 Frequency of Kk- 2pq = 0.095 Frequency of kk- q2 = 0.9025
If antibodies are available to test for the products of the alleles of interest (in this example, anti-K and anti-
k), the allele frequencies also can be obtained by direct counting as demonstrated in Table 11-2. The allele
frequencies obtained by direct testing are the observed frequencies for the population being sampled, whereas
those obtained by gene frequency calculations (above) are the expected frequencies. The various calculations
above,
when applied to a two-allele situation, are relatively simple; the calculations for three or more alleles are
much more complex and beyond the scope of this chapter.
For a given population, if the prevalence of one genetic trait, such as a red cell antigen, is known, the Hardy-
Weinberg equation can be applied to calculate allele and genotype frequencies. The Hardy-Weinberg
equilibrium principle is valid when the population is sufficiently large that chance alone cannot alter an allele
frequency and when the mating is random. A lack of selective advantage or disadvantage of a particular trait
and other influencing factors, such as mutation or migration in or out of the population, are assumed to be
absent when the Hardy-Weinberg equilibrium principle is applied. When all of these conditions are met, the
gene pool is in equilibrium and allele frequencies do not change from one generation to the next. If the
conditions are not met, changes in allele frequencies may occur over a few generations and may explain many
of the differences in allele frequencies between populations.
RELATIONSHIP TESTING
Polymorphisms are inherited characteristics or genetic markers that can distinguish between people. The
blood groups with the
TABLE 11-2. Allele Frequencies of Kand A"Calculated Using Direct Counting (assuming the absence of
null alleles)
Phenotype No. of Persons No. of Kk Alleles K k
K+k 2 4 4
K+k+ 88 176 88 88
K-k+ 910 1820 0 1820
Totals 1000 2000 92 1908
Allele frequency 0.046 0.954
A random sample of 1000 people tested for K and k antigens, have a total of 2000 alleles at the Kk locus
because each person inherits two alleles, one from each parent. Therefore, the two persons with a K+k-
phenotype (each with two alleles) contribute a total of four alleles. To this are added 88 Kalleles from the K+k+
group, for a total of 92 K alleles or an allele frequency of 0.046 (92 + 2000). The frequency of the k allele is
0.954 (1908 + 2000).

277
 greatest number of alleles (greatest polymorphism) have the highest power of discrimination and are the
most useful for determining relationships. Blood is a rich source of inherited characteristics that can be detected,
including red cell, HLA, and platelet antigens. Red cell and HLA antigens are easily identifiable, are
polymorphic, and follow Mendelian laws of inheritance. The greater the polymorphism of a system, the less
chance there is of finding two people who are identical. The extensive polymorphism of the HLA system alone
allows the exclusion of over 90% of falsely accused men in cases of disputed paternity.
Serologic methods of identity testing have been surpassed and replaced by DNA-based assays32 (referred to
as DNA fingerprinting, DNA profiling, or DNA typing) that were pioneered by Jeffreys and colleagues.33,34
Tandemly repeated sequences of DNA of varying lengths occur predominantly in the noncoding genomic DNA,
and they are classified into groups depending on the size of the repeat region. The extensive variation of these
tandemly repeated sequences between individuals makes it unlikely for the same number of repeats to be shared
by two individuals, even if these individuals are related. Minisatellite [also referred to as variable number of
tandem repeats (VNTR)] loci have tandem repeat units of nine to 80 base pairs, whereas microsatellite [also
referred to as short tandem repeat [STR)[ loci consist of two to five base pair tandem repeats.35 Microsatellites
and minisatellites are reviewed by Bennett.36
Assays for VNTR and STR sequences involve the electrophoretic separation of DNA fragments according
to size. DNA profiling involves amplification of selected, informative VNTR and STR loci using locus-specific
oligonucleotide primers with the subsequent measurement of the size of the PCR products. Hundreds of STR
loci have been mapped throughout the human genome, and many have been applied to identity testing. Analysis
of different STR loci (usually at least 12) is used to generate a person’s DNA profile that is virtually guaranteed
to be unique to that person (or to two identical twins). DNA fingerprinting is a powerful tool not only for
identity testing and
population genetics but also for monitoring chimerism after marrow transplantation.37,38 STR analysis also
has been used to monitor patients for graft-vs-host disease after organ transplantation, particularly after a liver
transplant.39
In a case of disputed paternity, if an alleged father cannot be excluded from paternity, the probability of his
paternity can be calculated. The calculation compares the probability that the alleged father transmitted the
paternal obligatory genes with the probability that any other randomly selected man from the same racial or
ethnic group transmitted the genes. The result is expressed as a likelihood ratio (paternity index) or as a
percentage. The AABB has developed standards and guidance documents for laboratories that perform
relationship testing.40
BLOOD GROUP GENE MAPPING
Gene mapping is the process through which a gene locus is assigned to a location on a chromosome. The
initial mapping of blood group genes was accomplished by testing many families for selected red cell antigens.
Pedigrees were analyzed for evidence of recombination between the genes of interest to rule out or establish
linkage of a blood group with another marker, with a known chromosomal location.
The gene encoding the antigens of the Duffy blood group system was the first to be assigned to a
chromosome, by showing that the gene is linked to an inherited deformity of chromosome 1. More recently,
recombinant DNA methods were used to establish the physical locations of genes, and today, with sequencing
of the human genome, determining the location of a gene involves a computer database sequence search. The
Human Genome Project (http://www.ornl.gov/sci/tech resourc es/Human_Genome/home.shtml) has resulted in
construction of a physical gene map indicating the position of gene loci and the distance between loci is
expressed by the number of base pairs of DNA.
Currently, 34 blood group systems are recognized by the ISBT.6 The genes for all of them have been cloned
and assigned to their respec
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AABB TECHNICAL MANUAL
tive chromosomes (see Table 11-1). Traditionally, genes were mapped to chromosomes according to the
metaphase banding patterns produced through staining with Giemsa (G banding) or quinacrine (Q banding).
Other methods used in chromosome mapping have included deletion mapping (partial or total loss of a
chromosome related to the presence or absence of a gene), somatic cell hybridization (based on the fact that
human-rodent hybrid cell lines randomly shed human chromosomes, permitting the association of a trait with
the presence or absence of a chromosome), in-situ hybridization (use of fluorescent DNA probes with a
preparation of intact chromosomes), and chromosome walking (a technique to clone a gene from its known
closest markers). Details on gene mapping procedures are beyond the scope of this chapter, but reviews are
available.7
Advances in genetic technology and information generated through the Human Genome Project make it
feasible to construct a physical gene map of the absolute positions of gene loci in which the distance between
loci is expressed by the number of base pairs of DNA.
CHIMERISM
The observation that a sample gives mixedfield agglutination is not unusual in a laboratory. Often this is the
result of artificially induced chimerism through the transfusion of donor red cells or a stem cell transplant. On
rare occasions, the observation of mixed-field agglutination identifies a true chimera, that is, a person with a
dual population of cells derived from more than one zygote. Indeed, the first example of a human chimera was a
female blood donor discovered through mixed-field agglutination during antigen typing. Most human chimeras
can be classified as either twin chimeras or tetragametic (dispermic) chimeras. Chimerism is not a hereditary
condition.41
Twin chimerism occurs through the formation of placental blood vessel anastomoses, which results in the
mixing of blood between two fetuses. This vascular bridge allows hematopoietic stem cells to migrate to the
marrow of the opposite twin. Each twin may
have two distinct populations of cells (red cells and leukocytes), that of their true genetic type and that of
their twin. The percentage of the two cell lines in each twin tends to vary; the major cell line is not necessarily
the autologous cell line, and the proportions of the two cell lines may change throughout life. Chimeric twins
have immune tolerance; they do not make antibody against the A or B antigens that are absent from their own
red cells but are present on the cells of the engrafted twin. This tolerance extends beyond red cells to negative
mixed lymphocyte cultures and the mutual acceptance of skin grafts.
In twin chimeras, the dual cell population is strictly confined to blood cells. Tetragametic or dispermic
chimeras present chimerism in all tissues and are more frequently identified because of infertility than because
of mixed populations of red cells. The mechanism (s) leading to the development of tetragametic chimeras are
unknown, but they arise through the fertilization of two maternal nuclei by two sperm followed by fusion of the
two zygotes and development into one person containing two cell lineages.
More commonly, chimeras occur through medical intervention and arise from the transfer of actively
dividing cells, such as through hematopoietic transplantation.41 However, chimerism may be more prevalent
than once thought. DNA analysis to confirm the Rh-negative status of Northern European blood donors
identified a donor with a dual red cell population of which 95% was Rh negative and 5% was Rh positive. The
donor was confirmed to be a chimera. Chimerism was found to be the cause of a discrepancy observed when
ABO was determined, and news headlines were made by a case of disputed maternity when a woman was
falsely excluded as the mother of her children because of chimerism.42'43
BLOOD GROUP TERMINOLOGY
Antigens were originally named using an alphabetical (eg, A/B, C/c) notation or they were named after the
proband whose red cells carried the antigen or who made the first known
279
antibody (eg, Duclos). A symbol with a superscript letter (eg, Lua, Lub; Jka, Jkb) was used, and a numerical
(eg, Fy3, Jk3, Rh32) terminology was introduced. In blood group systems, antigens are named using more than
one scheme (eg, the Kell blood group system: K, k, Jsa, Jsb, Kll, K17, TOU).
In 1980, the ISBT established its Working Party on Terminology for Red Cell Surface Antigens. The
working party was charged to develop a uniform nomenclature that would be “both eye and machine readable”
and “in keeping with the genetic basis of blood groups.” A blood group system consists of one or more antigens
under the control of a single gene locus or of two or more homologous genes that are so closely linked that
virtually no recombination occurs between them. Thus, each blood group system is genetically independent
from every other blood group system. The failure of an antibody to be reactive with red cells of a particular null
phenotype is not sufficient for assignment of the corresponding antigen to a system. Some null phenotypes are
the result of inhibitor or modifying genes that may suppress the expression of antigens from more than one
system [eg, the Rhnull phenotype lacks not only Rh antigens but also LW system antigens, Fy5 antigen (Duffy
system), and sometimes U (MNS system) antigen]. Similarly, a blood group antigen must be shown to be
inherited through family studies, or the expression of the antigen must be demonstrated to be associated with a
variation in the nucleotide sequence of the gene controlling the system, to be assigned antigen status by the
ISBT terminology working party. A blood group antigen must be defined serologically by an antibody; a
polymorphism that is detectable only by DNA analysis and for which there is no corresponding antibody cannot
be called a blood group antigen.
The working party established a terminology consisting of uppercase letters and Arabic numerals to
represent blood group systems and antigens.6,44 Each system can also be identified by a set of numbers (eg,
ABO system = 001; Rh system = 004). Similarly, each antigen in the system is assigned a number (eg, A
antigen = 001; B antigen = 002; D antigen = 001).
Thus, 001001 identifies the A antigen and 004001 identifies the D antigen. Alternatively, the sinistral zeros
may be omitted so that the A antigen becomes 1.1 and the D antigen becomes 4.1. Each system also has an
alphabetical abbreviation (Table 11-1); thus KEL is the ISBT symbol for the Kell system, the Rh ISBT system
symbol is RH, and an alternative name for the D antigen is RH1. This alphanumeric terminology, which was
designed primarily for computer use, is not ideal for everyday communication. To achieve uniformity, a
recommended list of user-friendly alternative names was compiled.45
The ISBT working party meets periodically to assign names and numbers to newly discovered antigens. For
terminology criteria; tables listing the systems, antigens, and phenotypes; and other information see ISBT Red
Cell Immunogenetics and Blood Group Terminology web resources.6 A comprehensive review of the
terminology and its usage is found in Garratty et al.45
The working party is charged to develop, maintain, and monitor a terminology for blood group genes and
their alleles.6 The terminology takes into account the guidelines for human gene nomenclature published by the
Human Genome Organization (HUGO), which is responsible for naming genes based on the International
System for Human Gene Nomenclature.46 For information regarding the current status of gene and allele
terminology see ISBT Red Cell Immunogenetics and Blood Group Terminology web resources.6 An example
of the traditional and current ISBT terminology as it applies to alleles, genotypes, phenotypes, and antigens is
shown in Table 11-3.
BLOOD GROUP GENOMICS
As discussed in earlier sections of this chapter, the antigens expressed on red cells are the products of genes
and can be detected directly by hemagglutination techniques (as long as relevant antisera are available). Their
detection is an important aspect of the practice of transfusion medicine because an antigen can, if it is
introduced into the circulation of an
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AABB TECHNICAL MANUAL
TABLE 11-3. Example of Allele, Genotype, Phenotype and Antigen Terminology
Duffy System Traditional ISBT
Allele Fya, Fyb, Fy FY*01 or FY*A, FY*02or FY*B, FY*N or FY*01N or FY*02N
Genotype/haplotype FyVFyb FY*A/FY*B or FY*01/FY*02
Phenotype Fy(a+b+) FY:1,2
Antigen Fya, Fyb FY1, FY2
ISBT = International Society of Blood Transfusion; N denotes “null”; FY*01N or FY*02I\I indicates that
the null allele is on a FY*A or FY*B background, respectively.
individual who lacks that antigen, elicit an immune response.
It is the antibody from such an immune response that causes problems in clinical practice, such as
patient/donor blood transfusion incompatibility, maternal-fetal incompatibility, and the reason why antigen-
negative blood is required for safe transfusion in these patients. Hemagglutination is simple, quick, and
relatively inexpensive. When carried out correctly, it has a specificity and sensitivity that is appropriate for most
testing. However, hemagglutination has limitations; for example, it is difficult and often impossible to obtain an
accurate phenotype for a recently transfused patient or to type red cells that are coated with IgG, and some
typing reagents are in short supply or not available. Because the genes encoding the 34 known blood group
systems have been cloned and sequenced and the molecular bases of most blood group antigens and phenotypes
are known, DNA-based methods (genotyping) are increasingly being used as an indirect method to predict a
blood group phenotype. This approach has introduced blood group genomics, often referred to as “molecular
immunohematology,” into the practice of transfusion medicine. Prediction of a blood group antigen by testing
DNA is simple and reliable for the majority of antigens because most result from SNPs that are inherited in a
straightforward Mendelian manner. For example, the antithetical antigens S and s arise from GYPB alleles that
differ by one nucleotide—143T for S and 143C for s—and the re
 sulting proteins differ by one amino acid, methionine at residue 48 for S and threonine for s (designated
c,143T>C p.Met48Thr). As a result, assay design and interpretation are fairly straightforward for the prediction
of most phenotypes.
However, detailed serologic and molecular studies have shown that there are far more alleles than
phenotypes for some systems, especially ABO and Rh. More than 100 different alleles encoding the
glycosyltransferases responsible for the four ABO types have been identified, and a single nucleotide change in
an A or B allele can result in an inactive transferase and a group 0 phenotype (see Chapter 12). Testing for the
common Rh antigens D, C/c, and E/e is uncomplicated for most populations, but antigen expression is more
complex in some ethnic groups. There are more than 200 RHD alleles encoding weak D or partial D
phenotypes, and more than 100 RHCE alleles encoding altered, or novel, hybrid Rh proteins, some of which
result in weakened antigen expression (see Chapter 13). RH genotyping, particularly in minority populations,
requires sampling of multiple regions of the gene(s) and algorithms for interpretation.
The basis of DNA assays is the amplification of a target gene sequence through the polymerase chain
reaction (PCR), followed by manual, semi-automated, or automated downstream analysis. Commonly used
methods are sequence-specific PCR (SSP-PCR) and allele-specific PCR (AS-PCR). For manual methods, gel
electrophoresis is used to sepa

281
rate the PCR products for fragment size determination. As an alternative, the assay may include digestion of
the PCR products with a restriction fragment length polymorphism (RFLP), followed by electrophoresis and
visualization of the fragments. Semi-automated approaches include real-time PCR using fluorescent probes with
quantitative and qualitative automated read-out. Manual methods are labor-intensive, and each assay is
performed separately on each sample. Automated DNA arrays allow for higher throughput with a larger number
of target alleles in the PCR reaction, which makes possible the determination of numerous antigens in a single
assay. Most available platforms are based on fluorescent bead technology or mass spectrometry. Routine ABO
and RhD testing is not currently available on most automated platforms because the expression of these
antigens is complex and further development is required.
To resolve discrepancies and identify new alleles, specialty referral laboratories use methods that are similar
to those used for high-resolution HLA typing, ie, gene-specific amplification of coding exons followed by
sequencing, or gene-specific cDNA amplification and sequencing. These methods are used to investigate new
alleles and resolve serologic and molecular discrepancies. The application of these methods has been reviewed
by several groups.47’49
Clinical Application of the Prediction of Blood Groups by DNA Analysis
A major use of DNA-based assays is to predict the red cell phenotype of a fetus, or of a patient who has
been transfused, or when red cells are coated with IgG. Additional applications include the resolution of
discrepancies in the ABO and Rh systems and identification of the molecular basis of unusual serologic results.
DNA analysis also affords the capability of distinguishing alloantibodies from autoantibodies. This section
gives an overview of some of the major applications of DNA-based analysis that are currently employed in
patient and donor testing. These, and additional clinical applications, are summarized in Table 11-4.
DNA-Based Assays to Predict the Red Cell Phenotype: Recently Transfused Patients
In patients receiving chronic or massive transfusions, the presence of donor red cells often makes typing by
hemagglutination inaccurate. Time-consuming and cumbersome cell separation methods that are often
unsuccessful in isolating and typing the patient’s reticulocytes can be avoided when DNA typing is used.
PCRbased assays mostly use DNA extracted from WBCs isolated from a sample of peripheral blood.
Interference from donor-derived DNA is avoided by targeting and amplifying a region of the gene that is
common to all alleles so that the minute quantity of donor DNA is not detected. This approach makes possible
reliable blood group determination with DNA prepared from a blood sample collected after transfusion. DNA
isolated from a buccal smear or urine sediment is also suitable for testing. In transfusion-dependent patients
who produce alloantibodies, an extended antigen profile is important to determine additional blood group
antigens to which the patient can become sensitized.
In the past, when a patient with autoimmune hemolytic anemia was transfused before the patient’s red cell
phenotype for minor antigens was established, time- and resourceconsuming differential allogeneic absorptions
were required to determine the presence or absence of alloantibodies underlying the autoantibody. Establishing
the patient’s most probable phenotype through DNA-based assays makes it possible to match the antigen profile
of the absorbing red cells to that of the patient, thereby reducing the number of cell types required for
absorption. This approach also allows matching of the antigen profile of the donor to that of the patient for the
most clinically significant, common antigens (eg, Jka, Jkb; S, s) when transfusion is required. This matching
avoids the use of “least incompatible” blood for transfusion, and allows transfusion of units that are “antigen-
matched for clinically significant blood group antigens” to prevent delayed transfusion reactions and
circumvent additional alloimmunization.
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TABLE 11-4. Applications of DNA-Based Assays for Patient and Donor Testing
To predict a patient’s red cell phenotype:
■ After a recent transfusion.
- Aid in antibody identification and RBC unit selection.
- Select RBCs for adsorption.
■ When antibody is not available (eg, anti-Doa, -Dob, -Jsa, -V, -VS).
■ Distinguish an alloantibody from an autoantibody (eg, anti-e, anti-Kpb).
■ Help identify alloantibody when a patient’s type is antigen-positive and a variant phenotype is possible
(eg, anti-D in a D-positive patient, anti-e in an e-positive patient).
■ When the patient’s red cells are coated with immunoglobulin (DAT+).
- When direct agglutinating antibodies are not available.
- When the antigen is sensitive to the IgG removal treatment (eg, antigens in the Kell system are denatured
by EDTA-glycine-acid elution).
- When testing requires the indirect antiglobulin test and IgG removal techniques are not effective at
removing cell-bound immunoglobulin.
- When antisera are weakly reactive and reaction is difficult to interpret (eg, anti-Doa, anti-Dob, anti-Fyb).
■ After allogeneic stem cell transplantation.
- If an antibody problem arises, test stored DNA samples from the patient and the donor(s).
■ To detect weakly expressed antigens (eg, Fyb with the Fyx phenotype); where the patient is unlikely to
make antibodies to transfused antigen-positive RBCs.
■ Identify molecular basis of unusual serological results, especially Rh variants.
■ Resolve discrepancies, eg, A, B, and Rh.
■ Aid in the resolution of complex serologic investigations, especially those involving high-prevalence
antigens when reagents are not available.
■ Identify if a fetus is or is not at risk for hemolytic disease of the fetus and newborn.
- Predict if the partner of a prospective mother with anti-D is homozygous or heterozygous for RHD.
To predict the donor’s red cell phenotype:
■ Screen for antigen-negative donors.
■ When antibody is weak or not available (eg, anti-Doa, -Dob; -Jsa, -Jsb; -VA/S).
■ Mass screening to increase antigen-negative inventory.
■ Find donors whose red cells lack a high-prevalence antigen.
■ Resolve blood group A, B, and Rh discrepancies.
■ Detect genes that encode weak antigens.
■ Type donors for reagent red cells for antibody screening cells and antibody identification panels (eg, Doa,
Dob, Jsa, V, VS).
■ Determine zygosity of donors on antibody detection/identification reagent panels, especially D, S, Fya,
and Fyb.
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DNA-Based Assays to Predict the Red Cell Phenotype: When Red Cells Are Coated with IgG
In patients with or without autoimmune hemolytic anemia, the presence of immunoglobulin bound to the
red cells [positive result on direct antiglobulin testing (DAT)] often makes antigen typing results by serologic
methods invalid. Certain methods, such as treatment of the red cells with chloroquine diphosphate or EDTA-
glycine acid (EGA) may be employed to remove the red-cell-bound IgG. These methods are not always
successful, the antigen of interest may be denatured by the treatment (eg, EGA destroys antigens of the Kell
blood group system), and direct agglutinating antibodies for the antigen of interest may not be available. DNA
testing allows determination of an extended antigen profile to select antigennegative red cell units for
transfusion.
DNA-Based Assays to Distinguish Alloantibody from Autoantibody
When an antibody specificity is found in a patient whose red cells express the corresponding antigen, it is
essential to know whether the antibody is an alio- or autoantibody and a DNA-based investigation is helpful for
transfusion management. If DNA typing predicts the red cells to be antigen positive, further investigation by
high-resolution gene sequencing should be considered because the sample may have a novel amino acid change
in the protein carrying the blood group antigen. These novel amino acid changes result in new epitopes and
altered (weakened or partial) expression of the conventional antigen.
This situation is especially relevant in patients with sickle cell disease (SCD) or thalassemia who require
long-term transfusion support and are at risk of alloimmunization that is often complicated by the presence of
autoantibodies. In patients of African ancestry who have SCD, partial expression of common Rh antigens (D, C,
c, and e) is prevalent. Such patients frequently present with a combination of anti-D, -C, and -e and yet their red
cells type serologically as D+, C+, and e+. Although such
patients may make alloantibodies to these antigens, autoantibody production with Rhrelated specificity is
prevalent, and distinguishing between the two is critical for safe transfusion practice to avoid hemolytic
transfusion reactions.50,51 Delayed hemolytic transfusion reaction in particular, places patients with SCD at
risk for life-threatening anemia, pain crisis, acute chest syndrome, and/or acute renal failure. Patients may also
experience hyperhemolysis, in which hemoglobin levels drop below pretransfusion levels due to bystander
hemolysis of the patients’ own antigen-negative red cells. RH genotyping has revealed that many of these
patients have variant RHD and/or RHCE alleles that encode amino acid changes in Rh proteins that result in
altered or partial antigens. For details on RHD and RHCE alleles that encode partial antigen, refer to Chapter
13.
Reports of autoantibodies to Jka and Jkb are not uncommon. With the discovery of variant JK alleles that
encode partial Jka and Jkb antigens, it is probable that some previously identified autoantibodies were
alloantibodies (see Chapter 14). DNA analysis for JK variants is helpful to clarify the situation. As in other
blood group systems, Kidd system genetic diversity is higher in populations of African ancestry.
DNA-Based Testing in Prenatal Practice
DNA-based testing has affected prenatal practice in the areas that are discussed below. Hemagglutination,
including antibody titers, gives only an indirect indication of the risk and severity of hemolytic disease of the
fetus and newborn (HDFN). Antigen prediction by DNAbased assays can be used to identify the fetus who is
not at risk of HDFN (ie, who is predicted to be antigen-negative) so that the mother need not be aggressively
monitored. Testing of fetal DNA should be considered when a mother's serum contains an IgG alloantibody that
has been associated with HDFN and the father's antigen status for the corresponding antigen is heterozygous or
indeterminable, or he is not available for testing.
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To Identify a Fetus at Risk for Anemia of the Neonate
The first application of DNA-based assays for the prediction of blood group phenotype occurred in the
prenatal setting and was reported by Bennett et al52 who tested fetal DNA for the presence of RHD. Because of
the clinical significance of anti-D, RHD is probably the most frequent target gene, but DNA-based assays can
be used to predict the antigen type of the fetus for any antigen if the molecular basis is known. When the
implicated IgG antibody in the maternal circulation is not anti-D, it is prudent, when possible, to also test the
fetal DNA for RHD to preempt unnecessary requests for D- blood for intrauterine transfusion; this is
particularly relevant to avoid the use of rare r'r' or r"r" blood when anti-c or anti-e is the implicated antibody.
PCR analyses for the prediction of fetal D phenotype are based on detecting the presence or absence of
specific portions of RHD. In populations of European ancestry, the molecular basis of the D-negative phenotype
is usually associated with deletion of the entire RHD, but several other molecular bases have been described. In
populations of Asian ancestry 15% to 30% of D-negative people have an intact but inactive RHD, while others
with red cells that are nonreactive with anti-D have the Del phenotype. Approximately a quarter of Dnegative
people of African ethnicity have an RHD pseudogene (RHD\|/), which does not encode the D antigen, and many
others have a hybridRHD-CE-Dgene (eg, the r,s phenotype). Predicting the D type by DNA analysis requires
probing for multiple nucleotide changes. The choice of assays depends upon the patient’s ethnicity and the
degree of discrimination desired. Establishing the fetal KEL genotype is also of great clinical value in
determining whether a fetus is at risk for severe anemia, because the strength of the mother's anti-K antibody
often does not correlate with the severity of the infant’s anemia. The same is true for anti-Ge3.53
Amniocytes, harvested from amniotic fluid, are the most common source of fetal DNA. Chorionic villus
sampling and cordocentesis
are not favored because of their more invasive nature and associated risk to the fetus. A noninvasive sample
source is the cell-free fetal DNA that is present in maternal plasma as early as 5 weeks of gestation; the amount
of DNA increases with gestational age and reliable results in DNA-based assays are obtained starting at about
15 weeks of gestation (sometimes earlier, depending on the gene of interest).54,55 These assays are particularly
successful for D typing because the D-negative phenotype in the majority of samples is due to the absence of
the RHD gene.
Testing for the presence or absence of a gene is less demanding than testing for a single gene polymorphism
or SNP to predict, for example, the K/k antigen status. Cell-free fetal DNA from the maternal plasma is
routinely tested in Europe for the presence of a fetal RHD gene to eliminate the unnecessary administration of
antepartum RhIG to the approximately 40% of D-negative women who are carrying a D-negative fetus. In the
United States, due to intellectual property ownership, testing of cell-free fetal DNA is limited to women with
active anti-D and is available only commercially.
DNA-Based Testing for D in Pregnant Women
 Serologic typing for D cannot easily distinguish women whose red cells lack some epitopes of D (partial D)
and are at risk for D immunization, from those with a weak D phenotype who are not at risk for D
immunization. Red cells with partial D type as D+, some in direct tests and others by indirect tests. These
women might benefit from receiving RhIG prophylaxis if they deliver a D+ fetus. RHD genotyping can
distinguish weak D from partial D to guide RhIG prophylaxis and blood transfusion recommendations.
DNA-Based Testing of Paternal Samples
The father’s red cells should be tested for the antigen corresponding to the antibody in the maternal plasma.
If the red cells are negative, the fetus is not at risk. If the father is positive
285
for the antigen, zygosity testing can determine whether the father is homozygous or heterozygous for the
gene encoding the antigen, particularly when there is no allelic antigen or no antisera to detect the allelic
product.
Zygosity testing of paternal samples is most often performed when testing for possible HDFN due to anti-D
or anti-K. If the paternal red cells are K+ and the mother has anti-K, they can be tested serologically for
expression of the allelic k antigen. However, many centers do not have a licensed reagent available and genetic
counselors often request DNA testing. If the paternal red cells are K-, the maternal anti-K is mostly likely the
result of immunization through transfusion.
For maternal anti-D, DNA testing of RHD zygosity is the only method to determine the paternal gene copy
number. Several different genetic events cause a D-negative phenotype, and multiple assays must be conducted
to accurately determine RHD zygosity, especially in minority ethnic groups. If the father is RHD homozygous,
all of his children will be D+ and any pregnancy in his partner needs to be monitored. If the father is
heterozygous, the fetus has a 50% chance of being at risk. The D type of the fetus should be determined to
prevent invasive and unnecessary testing so the mother need not be aggressively monitored or receive immune-
modulating agents.
DNA-Based Testing for AntigenNegative Blood Donors
DNA-based typing to predict donor antigen profiles in the search for antigen-negative units is now a
standard procedure for blood centers, especially when suitable antibodies are not available. Because red cell
typing for Dombrock antigens is notoriously difficult, one of the most frequent approaches is to type for Doa
and Dob. Many other antibody specificities are unavailable for mass donor screening. These specificities
include anti-Hy, -Joa, -Jsa, -Jsb, -Cw, -V, and -VS. Even specificities considered to be common, such as anti-S
and antiFyb, are not always readily available.
DNA arrays can be used to screen for multiple minor antigens in a single assay format
and have the potential to be used for mass screening of donors. This practice not only increases the antigen-
negative inventory by expanding combinations of the minor antigens and some high-prevalence antigens, but
also allows consideration of the possibility of providing donor components that are DNA matched to the
patient’s type. Although DNA methods are not yet licensed by the Food and Drug Administration for the
labeling of donor units in the United States, DNA testing is a valuable screening tool and only negative test
results need to be confirmed using a licensed reagent when one is available.
DNA-Based Testing to Confirm D Type of Donors
Donor centers must test donors for weak D to avoid labeling a product as D-negative that might result in
anti-D in response to transfused RBCs. Some donor red cells with very weak D expression (weak D type 2, and
especially those with the Del phenotype) are not typed as D+ using current methods and are labeled as D-. The
prevalence of weak D red cells not detected by serologic reagents is approximately 0.1% (but may vary
depending on the test method and population). Although the clinical significance has not been established,
donor red cells with weak D expression have been associated with alloimmunization. RHD genotyping would
improve donor testing by confirming D- phenotypes,56 but a highthroughput and cost-effective platform is not
yet available.
Discrepancies between Serologic (Phenotype) and DNA (Genotype) Testing
Differences between serologic and DNA testing results do occur and must be investigated. Often, these
discrepancies lead to interesting discoveries such as the presence of a novel allele or genetic variant, particularly
when people of diverse ethnicities are tested. Causes of discrepancies include recent transfusions, stem cell
transplantation, and natural chimerism. Stem cell transplantation and natural
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 chimerism also may cause results of testing DNA from somatic cells to differ from results of testing DNA
from extracted from peripheral WBCs. Thus, when using DNA testing, it is important to obtain an accurate
medical history. Many genetic events can cause apparent discrepant results between hemagglutination and DNA
test results and the genotype does not always predict the phenotype (see Table 11-5 for examples).3,5,27
Silenced or Nonexpressed Genes
DNA testing interrogates a single SNP or a few SNPs associated with antigen expression and cannot sample
every nucleotide in the gene. Although a gene may be detected by DNA testing, there are times when the gene
product is
not expressed on the red cells because of a mutation that silences the gene. Such changes result in
discrepancies in the typing of patients and donors. Homozygosity (or compound heterozygosity) for a silenced
gene results in a null phenotype and most null phenotypes have more than one molecular basis.5
With donor typing, the presence of a grossly normal gene whose product is not expressed on the red cell
surface results in the donor being falsely typed as antigen-positive. Although this situation means loss of an
antigen-negative donor, it does not jeopardize the safety of blood transfusion. However, if a grossly normal
gene is detected in a patient but the gene is not expressed, the patient could produce an antibody if he or she
receives a transfusion of antigen-positive blood.
TABLE 11-5. Examples of Some Molecular Events Where Analysis of Gene and Phenotype Do Not Agree
Molecular Event Mechanism Observed Blood Group Phenotype
Transcription Nt change in GATA box Fy(b-)
Alternative Nt change in splice site: Partial/
S—s—; Gy(a-)
splicing complete skipping of exon
Deletion of nt(s) Dr(a-)
Premature stop Fy(a-b-); D-; c-E-; Rhnull; Gy(a-);
Deletion of nt(s) -» frameshift
codon GE:-2-3,-4; K0; McLeod
Insertion of nt(s) -> frameshift D-; Co(a-b-)
Nt change Fy(a-b-); r'; Gy(a-); K0; McLeod
Amino acid
Missense nucleotide change D-; Rhnuli; K0; McLeod
change
Reduced amount
Missense nucleotide change Fyx; Co(a-b-)
of protein
Hybrid genes Cross-over GP.Vw; GP.Hil; GP.TSEN
Gene conversion GP.Mur; GP.Hop; D--; R0Har
Interacting
Absence of RhAG Rhnull
protein
Absence of Kx Weak expression of Kell antigens
Absence of aas 75 to 99 of GPA Wr(b-)
Absence of protein 4.1 Weak expression of Ge antigens
Modifying gene In(Jk) Jk(a-b-)
Nt = nucleotide; aas = amino acids.
 CHAPTER 11 Blood Group Genetics
287
To avoid misinterpretation, routine assays must include appropriate tests to detect a change that silences
gene expression if prevalent in the population tested. Silenced alleles can be specific to a particular ethnic
group. For example, in the Duffy blood group system, a single nucleotide change (-67T>C) within the promoter
region (GATA box) of FYprevents transcription of FY*A and/or FY*B in red cells but not in other tissues.
Although silencing of FY*A is rare, silencing of FY*B is frequent in persons of African ethnicity where
homozygosity for the -67T>C change in FY*B results in the Fy(a-b-) phenotype, which has a prevalence of
60% or higher. To ensure accuracy, testing for the GATA box mutation must be included in typing for Duffy in
persons of African ethnicity.
When the assay is used to predict the presence or absence of D antigen, particularly in populations of
African ancestry, it is essential to include a test for the complete but inactive RHD pseudogene (RHDy), which
has a 37-bp sequence duplication. If the assay tests for GYPB*S (S antigen), additional testing should be
performed to detect a C>T change at nucleotide 230 in GYP*B exon 5 or a change in intron 5 (+5g>t); both
changes prevent expression of S antigen when testing persons of African ancestry. Homozygosity, or compound
heterozygosity, for the 230T, or the +5g>t change, results in the S-s-U+w phenotype.
Other common causes of discrepancies include the presence in the sample of an altered FY*B allele that
encodes an amino acid change causing an Fyx phenotype with greatly reduced expression of Fyb antigen. The
red cells type as Fy(b-) with most serologic reagents. The prevalence of the allele encoding
the Fyx phenotype in people of European ancestry is as high as 2%, and the allele has been found in persons
of African ancestry also. Silencing mutations associated with the loss of Kidd antigen expression occur more
often in people of Asian ancestry, whereas nucleotide changes encoding amino acid changes that weaken Kidd
expression occur in people of African ethnicity.
 The routine detection of some blood group polymorphisms by DNA analysis is complex and not practical at
this time. This includes situations where 1) a large number of alleles encode one phenotype (eg, ABO, Rh, and
null phenotypes in many blood group systems), 2) a phenotype results from alleles with a large deletion (eg,
GE:-2,3 and GE:-2,-3,-4), or 3) a phenotype results from hybrid alleles (eg, in the Rh and MNS systems). In
addition, there is a high probability that not all alleles in all ethnic populations are known.
Blood group genomics has become an essential component of the practice of transfusion medicine.57
Genomics has provided a greater understanding of genetic blood group variants, including the complexity of the
molecular basis of Rh variants, such as those that encode the Hr-hrs- and hrB-HrBphenotypes58,59 and the
associated partial Rh antigens that are a daily challenge for the management of patients with SCD.50,51 RH
genotyping expands and extends matching for Rh in this patient population. High-throughput platforms provide
a means to test relatively large numbers of donors, thereby opening up the possibility of changing the way
antigen-negative blood is provided to patients to prevent immunization or to eliminate transfusion reactions for
those who are already immunized.
KEY POINTS
1. Genetics is the study of heredity, that is, the mechanisms by which a particular characteristic, such as a
blood group, is passed from parents to offspring.
2. A gene is a segment of DNA and is the basic unit of inheritance; it occupies a specific location on a
chromosome (the gene locus). Alleles are alternative forms of a gene at the same gene locus (eg, alleles JK*A
and JK*B encode the Jka and Jkb antigens, respectively).

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3. A human somatic (body) cell is diploid, containing 46 chromosomes in 23 pairs: 22 pairs are alike
(homologous) in males and females and are termed autosomes. The remaining pair is the sex chromosomes: X
and Y for males, or two X chromosomes for females.
4. Somatic cells divide for growth and repair by mitosis. Mitosis replicates the chromosomes and produces
two identical nuclei in preparation for cell division. The new cells are diploid, like the parent cell, and have all
the genetic information of the parent cell.
5. Meiosis is the process by which germ cells divide to become gametes (sperm and egg cells); diploid cells
undergo DNA replication and two divisions to form four gametes, each of which is haploid and has half the
chromosomal complement of the parent somatic cell.
6. The term “genotype” traditionally refers to the complement of genes inherited by each person from his or
her parents; the term is also used to refer to the set of alleles at a single gene locus. Whereas the genotype of a
person is his or her genetic constitution, the phenotype is the observable expression of the genes and reflects the
biologic activity of the gene(s). Thus, the presence or absence of antigens on the red cells, as determined by
serologic testing, represents the phenotype.
7. When identical alleles for a given locus are present on both chromosomes, a person is homozygous for
the particular allele, whereas when nonidentical alleles are present at a particular locus, the person is
heterozygous. Antigens encoded by alleles at the same locus are said to be antithetical. Thus, genes are allelic
but not antithetical, whereas antigens are antithetical but not allelic.
8. The expression of blood group antigens on the red cell may be modified or affected by gene interaction.
Homozygosity (or compound heterozygosity) for a silenced gene results in a null phenotype and most null
phenotypes have more than one molecular basis.
9. A blood group system consists of one or more antigens under the control of a single gene locus (eg, KEL
encodes the Kell blood group antigens) or of two or more homologous genes (eg, RHD and RHCE encode the
Rh blood group antigens) so closely linked that virtually no recombination occurs between them; thus, each
blood group system is genetically independent. Currently, 34 blood group systems are recognized.
10. The genes encoding the 34 blood group systems have been sequenced and the molecular bases of most
antigens and phenotypes are known so that DNA-based methods (genotyping) can be used to predict a blood
group phenotype.
11. DNA-based assays (blood group genotyping) have major applications in patient and donor testing. They
can be used to predict the red cell phenotype of a fetus, or of a patient who was transfused, or when red cells are
coated with IgG; they can be used to resolve ABO and Rh discrepancies and to identify the molecular basis of
unusual serologic results. DNA analysis aids in distinguishing alloantibodies from autoantibodies and is being
applied to highthroughput screening of donors.
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39. Domiati-Saad R, Klintmalm GB, Netto G, et al. Acute graft versus host disease after liver
transplantation: Patterns of lymphocyte chimerism. Am J Transplant 2005;5:2968-73.
40. Mount M, ed. Standards for relationship testing laboratories. 11th ed. Bethesda, MD: AABB, 2014.
41. Bluth MH, Reid ME, Manny N. Chimerism in the immunohematology laboratory in the molecular
biology era. Transfus Med Rev 2007; 21:134-46.
42. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genet
2001;2:10.
43. Cho D, Lee JS, Yazer MH, et al. Chimerism and mosaicism are important causes of ABO phenotype and
genotype discrepancies. Immunohematology 2006;22:183-7.
44. Daniels GL, Anstee DJ, Cartron J-P, et al. Blood group terminology 1995. ISBT Working Party on
Terminology for Red Cell Surface Antigens. Vox Sang 1995;69:265-79.
45. Garratty G, Dzik WH, Issitt PD, et al. Terminology for blood group antigens and genes: Historical
origins and guidelines in the new millennium. Transfusion 2000;40:477-89.
46. HGNC searches. Cambridge, UK: HUGO Gene Nomenclature Committee, 2013 [Available at
http://www.genenames.org/ (accessed August 12,2013).]
47. Avent ND. Large scale blood group genotyping. Transfus Clin Biol 2007;14:10-15.
48. Monteiro F, Tavares G, Ferreira M, et al. Technologies involved in molecular blood group genotyping.
ISBT Science Series 2011;6:1-6.
49. Gassner C, Meyer S, Frey BM, et al. Matrix-assisted laser desorption/ionization, time of flight mass
spectrometry-based blood group genotyping - the alternative approach. Transfus Med Rev 2013;27:2-9.
50. Chou ST, Westhoff CM. The role of molecular immunohematology in sickle cell disease. Transfus
Apher Sci 2011;44:73-9.
51. Noizatt-Pirenne F, Tournamille C. Relevance of RH variants in transfusion of sickle cell patients.
Transfus Clin Biol 2011;18:527-35.
52. Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal determination of fetal RhD type by DNA
amplification. N Engl J Med 1993;329:607-10.
53. Pate LL, Myers J, Palma J, et al. Anti-Ge3 causes late-onset hemolytic disease of the newborn: The
fourth case in three Hispanic families. Transfusion 2013;53:2152-7.
54. Daniels G, Finning K, Martin P Soothill P. Fetal blood group genotyping from DNA from maternal
plasma; an important advance in the management and prevention of haemolytic disease of the fetus and
newborn. Vox Sang 2004;87:225-32.
55. Clausen FB, Christiansen M, Steffensen R, et al. Report of the first nationally implemented clinical
routine screening for fetal RHD in Dpregnant women to ascertain the requirement for antenatal RhD
prophylaxis. Transfusion 2012;52:752-8.
56. Wagner FF. RHD PCR of D-negative blood donors. Transfus Med Hemother 2013;40:172-81.
57. Hillyer C, Shaz B, Winkler A, Reid ME. Integrating molecular technologies for red blood cell typing
and compatibility testing into blood centers and transfusion services. Transfus Med Rev 2008;22:117-32.
58. Pham B-N, Peyrard T, Tourret S, et al. Anti-HrB and anti-hrB revisited. Transfusion 2009;49: 2400-05.
59. Reid ME, Hipsky CH, Velliquette RW, et al. Molecular background of RH in Bastiaan, the RH:-31,-34
index case, and two novel RHD alleles. Immunohematology 2012;28:97-103.
Chapter 12
ABO, H, and Lewis Blood Groups and Structurally Related Antigens

Laura Cooling, MD, MS

|MK| THE ANTIGENS OF the ABO, H, LewM is, I, and P blood group systems are defined by small
carbohydrate epitopes on glycoproteins and glycolipids. Because the epitopes represent posttranslational
modifications, the synthesis of those antigens requires the action of several enzymes known as
“glycosyltransferases.” Glycosyltransferases reside in the Golgi apparatus and are responsible for adding
specific sugars, in a specific sequence and steric or anomeric linkage (a-linked or [3linked), to growing
oligosaccharide chains on glycolipids and glycoproteins.
 Transcriptional regulation coupled with the enzymatic specificity of these glycosyltransferases for both
sugar donors [nucleotide sugars; eg, uridine diphosphate (UDP)-galactose] and acceptor substrates (eg, Type 1
chain vs Type 2 chain) is responsible for the tissuespecific distribution of many blood group antigens.1,2
Because they are widely distributed on many tissues, including embryonic stem cells, such antigens are
considered to be histoblood-group antigens.2’5 Several studies have shown that these antigens have a role in
devel
opment, cell adhesion, malignancy, and infectious disease.1,6,7
ABO SYSTEM
The ABO system, initially described by Karl Landsteiner in 1900, remains the most important blood group
system in transfusion and organ transplantation medicine.7 In blood, ABO antigens are found on red cells,
platelets, and many circulating proteins. As histo-blood-group antigens, ABO antigens are also present on many
tissues, including those of the endothelium, kidney, heart, bowel, pancreas, and lung.2
Transfusion of ABO-incompatible blood can be associated with acute intravascular hemolysis, renal failure,
and death. Likewise, transplantation of ABO-incompatible organs is associated with acute humoral rejection.7
Because of the dire clinical consequences associated with ABO incompatibilities, ABO typing and ABO
compatibility testing remain the foundation of pretransfusion testing and an important component of typing
before transplantation.
Laura Cooling, MD, MS, Associate Professor, Department of Pathology, and Associate Medical Director,
Transfusion Medicine, University of Michigan, Ann Arbor, Michigan The author has disclosed no conflicts of
interest.
291

292
AABB TECHNICAL MANUAL
The ABO system contains four major ABO phenotypes: A, B, 0, and AB. The four phenotypes are
determined by the presence or absence of two antigens (A and B) on red cells (see Table 12-1). The ABO
system is also characterized by the presence or absence of naturally occurring antibodies, termed
“isohemagglutinins,” directed against missing A and B antigens. As shown in Table 12-1, an inverse reciprocal
relationship exists between the presence of A and/or B antigens on red cells and the presence of anti-A, anti-B,
or both, in sera. For example, group 0 individuals, who lack A and B antigens on red cells, possess both anti-A
and anti-B. It is believed that the immunizing sources for such naturally occurring antibodies are gut and
environmental bacteria, such as the Enterobacteriaceae, which have been shown to possess ABO-like structures
on their lipo-polysaccharide coats.8,9
Biochemistry
The A and B antigens are defined by three sugar terminal epitopes on glycolipids and glycoproteins.7 As
shown in Fig 12-1, H antigen is characterized by a terminal al->2 fucose; it is the immediate biosynthetic
precursor for both A and B antigens and is required for their expression. In group A individuals, an Af-
acetylgalactosamine is added, in an al->3 linkage,
to the subterminal galactose of the H antigen to form A antigen. In group B individuals, an al->3 galactose
is added to the same subterminal galactose to form B antigen. In group AB individuals, both A and B structures
are synthesized. In group 0 individuals, neither A nor B antigens are synthesized as a result of a mutation in the
ABO gene.7,10 As a consequence, group 0 individuals express only H antigen. A and B antigens are also absent
in the very rare Bombay phenotype because of the absence of the H-antigen precursor (see section below on the
H system).
As terminal motifs, the A and B antigens can be displayed on a number of oligosaccharide scaffolds that
differ in their size, composition, linkages, and tissue distribution. On red cells, ABH sites are present on
AMinked (65%75%) and O-linked (5%-15%) glycoproteins, polyglycosylceramides (10%-15%), and simpler
glycosphingolipids (Fig 12-2). ABO antigens are subclassified by the carbohydrate sequence immediately
upstream of the ABO motif. In humans, ABH is expressed predominantly on four different oligosaccharide
backbones (see Table 12-2). On human red cells, the majority of endogenous ABH antigen synthesized is
present on Type 2 chain structures.
 The ability to synthesize and use different carbohydrate chains is genetically determined and can contribute
to antigenic differences in
TABLE 12-1. Routine ABO Grouping
Reaction of Reaction of
Prevalence
Red Cells with Serum with Interpre
(%) in US
Antisera (Red Reagent Red Cells tation
Population
Cell Grouping) (Serum Grouping)
European African
A1 0 ABO
Anti-A Anti-B B Cells Ethnicity Ethnicity
Cells Cells Group
0 0 + + 0 0 45 49
+ 0 0 + 0 A 40 27
0 + + 0 0 B 11 20
+ + 0 0 0 AB 4 4
0 0 + + + Bombay* Rare Rare
*H null phenotype (see section on H antigen). + = agglutination; 0 = no agglutination.

293
 Gal = Galactose GIcNAc = N-acetylglucosamme Fuc = Fucose
GolNAc = N-acetylgalactosamine
FIGURE 12-1. GalNAc added to the subterminal Gal confers A activity to the sugar; Gal added to the
subterminal Gal confers B activity. Unless the fucose moiety that determines H activity is attached to the
number 2 carbon, galactose does not accept either sugar on the number 3 carbon.
weak ABO subgroups.10'12 For instance, Type 3 (repetitive A) and Type 4 (globo-A) A antigens are present
on A,, but not A2, red cells. Differences in cis carbohydrate sequences upstream of the terminal ABO motif can
also influence antibody reactivity.12 As an example, ABO antigens on Type 1 chain substrates can be
recognized by antibodies directed against both ABO and Leb antigen (see “Lewis System” section below).10,13
ABO in Development and Aging
ABO antigens can be detected on red cells of embryos as early as 5 to 6 weeks of gestation.13 The quantity
of ABO antigens on umbilical cord red cells is less than that of adults as the
result of the immaturity of Type 2 chain precursors on cord red cells (see “I and i Antigens” section
below).14 With increasing age, precursor chains become increasingly branched, thereby allowing more A and B
antigen to be expressed. Adult levels of ABO expression are generally present by age 2 to 4 years.13,14
Anti-A and anti-B are not present at birth or, if present, are of maternal origin. Endogenous synthesis of
anti-A and anti-B can develop as early as age 3 to 6 months, with nearly all children displaying the appropriate
isohemagglutinins in their sera at 1 year of age.13,15 Titers of anti-A and anti-B continue to increase during
early childhood and achieve adult levels within 5 to 10 years.
Among healthy adults, ABO titers can naturally vary from 4 to 2048 or higher.13,15,16 Hightiter ABO
antibodies can be present in group 0 multiparous women and in patients taking certain bacteria-based nutritional
supplements.7,9,13 Although older reports indicated a fall in isoagglutinin titers in the elderly, more recent
studies have disputed these findings.15 In industrialized countries, isoagglutinin titers have generally decreased
with increasing consumption of processed foods.16
Genetics
The ABO gene is located on chromosome 9q34 and is fairly large, consisting of seven exons spread over 18
kb.7 The open reading frame of the protein is located primarily in exons 6 and 7. A study of the promoter region
indicates that ABO gene expression is transcriptionally regulated by several mechanisms, including
methylation, tissue-specific transcriptionfactor-binding proteins, antisense RNA, and, possibly, a minisatellite
enhancer region 4 kb upstream of exon l.7 ABO expression is also regulated by the H gene, which is
responsible for the synthesis of H antigen substrate, the precursor of A and B antigens. The H gene is tightly
regulated in a tissue-specific manner through tissue-specific transcription factors and promoters.17 In the
absence of H, no A or B antigen is expressed regardless of ABO genotype (Bombay or Oh phenotype).7,10
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AABB TECHNICAL MANUAL

Single-pass protein
 GPI-linked
Multipass protein protein
FIGURE 12-2. Schematic representation of the red cell membrane showing antigen-bearing glycosylation of
proteins and lipids.
GPI = glycosylphosphatidylinositol. (Courtesy of ME Reid)
A series of elegant studies over the past decade has identified the molecular basis for A, B, 0, ris-AB, and
weak ABO subgroups.7,10,18 Fundamentally, three common ABO alleles are responsible for the A, B, and 0
phenotypes. The A and B consensus alleles are autosomal codominant and differ by only seven nucleotides and
four amino acids.7,18 Three amino ac
ids (A—>B; Gly235Ser, Leu266Met, and Gly268Ala) are critical in determining whether the
glycosyltransferase uses UDP-Af-acetyl-Dgalactosamine or UDP-D-galactose donor sugars to synthesize A or
B antigens, respectively.7,10 The rare ds-AB phenotype is a chimeric enzyme with a mix of A-specific and B-
specific amino acids at those or other amino acid posi
TABLE 12-2. Chain Variants of A Antigen in Humans
Antigen Oligosaccharide Sequence*
A epitope GalNAcal -3(Fuca1 -2)Gaipi -R
Type 1 A GalNAcal -3(Fuca1 -2)Gal|31-3Glcl\IAc(31 -3-R
Type 2 A GalNAcal -3(Fuca1-2)Gaipi-4GlcNAcP1 -3-R
GalNAcal -3(Fuca1 -2)Gaipi-3GalNAca1 -3(Fuca1 -2)Gaipi -4GlcNAcpi -3-R
Type 3 A (repetitive A) v- . . y v _ j
VY
Type 4 A (globo-A) GalNAcal -3(Fuca1 -2)Gal|31-3GalNAca1 -4Gala1 -4Gaipi -4Glc-Cer
‘Underlined sequences denote the critical differences between Type 1,2, and 4 chains. Linkages and
anomery (a or p linked) of the A antigen galactose are in bold. Bracketed sequences denote repetitive A antigen
characteristic of Type 3 chain A antigen.
Cer = ceramide; Fuc = fucose; Gal = galactose; GallMAc = /V-acetyl-galactosamine; Glc = glucose;
GIcNAc = /V-acetyl-glucosamine; R = upstream oligosaccharide.

295
tions.18 A plethora of mutations associated with weak A and B subgroups has been described. As an
example, group A2 (a weak A subgroup) is commonly the result of a nucleotide deletion and frameshift,
resulting in an enzyme with an additional 21 amino acids at the C-terminus of the molecule.10,18
The 0 allele is an amorph, encoding a nonfunctional enzyme. The group 0 phenotype, therefore, is an
autosomal recessive trait, representing inheritance of two nonfunctional ABO genes. Overall, more than 50 0
alleles have been identified.7,18 The two most common 0 alleles (OOl and 002) contain a nucleotide deletion
and frameshift, leading to a truncated, 117-amino-acid protein. Another common 0 allele is 003 (or O2), a group
of nondeletional 0 alleles that contains a mutation at amino acid 268 (Gly268Arg), which is a critical residue for
donor binding (UDP-galactose or UDP-lV-acetylgalactosamine). One German study found that 003 and a
related allele (Aw08) were responsible for 25% of all ABO typing discrepancies caused by reverse-grouping
problems in healthy donors.19 It was speculated that weak anti-A and anti-B could reflect weak residual
glycosyltransferase activity; however, a later study was unable to demonstrate A antigen or enzyme activity in
individuals with the 003 allele.20
ABO Subgroups
ABO subgroups are phenotypes that differ in the amount of A and B antigen carried on red cells and present
in secretions. In general, A subgroups are more common than B subgroups. Clinically, the two most common
subgroups encountered are A, and A2. A, represents the majority of group A donors (80%) and is characterized
by approximately 1 million A antigen epitopes per red cell. A2 is the second most common subgroup (20%) and
possesses only one fifth (2.2 x 105) the number of A antigen sites as Ar Both A, and A2 are strongly
agglutinated by reagent anti-A in routine direct testing. A! can be distinguished from A2 by the lectin Dolichos
biflorus, which agglutinates A1 red cells but not A2 red cells. In addition, 1% to 8% of individuals with A2 and
 22% to 35% of individuals with A2B possess an alloanti-A, in their sera. Because the A2 phenotype reflects
the inefficient conversion of H->A antigen, A2 red cells have increased reactivity with the anti-H lectin Ulex
europaeus. Enzyme studies comparing A1 and A2 glycosyltransferase activity show that the A, enzyme is 5 to
10 times more active than the A2 enzyme, resulting in quantitative and qualitative differences in A antigen
expression.7,10 The latter includes the synthesis of unusual Type 3 and Type 4 chain A antigen on A, red cells
that is not present on A, or weaker A subgroups.10,11
In addition to A2, several weaker A subgroups have been described (eg, A3, Ax, Am, and Ael). The
extremely weak A (and B) subgroups are infrequently encountered and are usually recognized by apparent
discrepancies between red cell (forward) and serum (reverse) grouping results. Most weak A and B subgroups
were originally described before the advent of monoclonal typing reagents and were based on reactivity with
human polyclonal anti-A, anti-B, and anti-A,B reagents. Weak A subgroups are frequently nonreactive with
human polyclonal anti-A (see Table 12-3) and can show variable reactivity with human polyclonal anti-Al, anti-
A,B, and murine monoclonal antibodies (not shown).10,13,18 The degree of reactivity with commercial murine
monoclonal reagents is clone dependent; however, most commercially available anti-A agglutinates A3 red
cells. Because of the reciprocal relationship between H and synthesis of A and B antigens, nearly all weak A
and B subgroups have higher H expression.7 In clinical practice, it is seldom necessary to identify a patient’s
specific A or B subgroup.
When performed, classification of weak A subgroups is typically based on the following:
1. Degree of red cell agglutination by anti-A and anti-Al.
2. Degree of red cell agglutination by human and some monoclonal anti-A,B.
3. Degree of H antigen (anti-H lectin and Ulex europaeus) expression.
4. Presence or absence of anti-Aj (Method 29).
5. Presence of A and H in saliva.
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AABB TECHNICAL MANUAL
TABLE 12-3. Serologic Reactions Observed in A and B Subgroups
Red Red Cell Serum
Cell Reactions with Reactions with Saliva
Phenotype Antisera or Lectins Reagent Red Cells
Anti- Anti- B 0
Anti-A Anti-A,B A1 Cells (Secretors)
B H Cells Cells
A
A, 4+ 0 4+ 0 0 4+ 0
&H
A
a2 4+ 0 4+ 2+ 0/2+* 4+ 0
&H
A
a3 2+mf 0 2+nf 3+ 0/2+* 4+ 0
&H
Ax 0/± 0 1-2+ 4+ 0/2+ 4+ 0 H
Aei 0 0 0 4+ 0/2+ 4+ 0 H
B
B 0 4+ 4+ 0 4+ 0 0
&H
B
b3 0 1+mf 2+nf 4+ 4+ 0 0
&H
Bx 0 0/± 0/2+ 4+ 4+ 0 0 H
B
B(A) ±/2+t 4+ 4+ 0 4+ 0 0
&H
‘The occurrence of anti-AI is variable in these phenotypes. tMost often detected with anti-A clones
containing the MH04 clone.
1 + to 4+ = agglutination of increasing strength; + = weak agglutination; mf = mixed-field agglutination; 0 =
no agglutination.
6. Adsorption and elution studies.
7. Family (pedigree) studies.
 B(A) andA(B) Phenotypes
The B(A) phenotype is an autosomal dominant
phenotype characterized by weak A expression
on group B red cells.13,21 Serologically, red cells
from B (A)-phenotype individuals are strongly
reactive with anti-B and weakly reactive with
monoclonal anti-A (<2+), and they possess a
strong anti-A that is reactive with both A, and A2 red cells in their sera. B(A) red cells can
show varying reactivity with monoclonal antiA reagents; however, most cases are detectable with
monoclonal typing reagents containing
the MH04 clone. In general, the agglutination
is weak with fragile, easily dispersed aggluti
nates. Testing the sample with polyclonal antiA or a different monoclonal anti-A should resolve the
discrepancy. Amino acid polymorphisms have been identified in some B(A) in
dividuals near (Pro234Ala) or at (Ser235Gly)
critical amino acids.10,18 The B glycosyltransfer
ase in these individuals has an increased capacity to use UDP-Al-acetylgalactosamine in
addition to UDP-galactose, resulting in detectable A antigen synthesis.
An A(B) phenotype has also been described with monoclonal anti-B. The A(B) phenotype was associated
with elevated H antigen and plasma H-transferase activity.13 It is hypothesized that the increased H precursor
on these cells may permit the synthesis of some B antigen by the A glycosyltransferase.
Acquired B Phenotype
The acquired B phenotype phenomenon is a transient serologic discrepancy in group A individuals that is a
cause of red cell grouping discrepancies.6 7 * * * * * * * * * * * * * * 22 Acquired B should be suspected when
a patient or donor who has historically typed as group A now presents with weak B expression on forward or
red cell grouping. Serologically, the acquired B phenotype shows strong agglutination with anti-A, shows weak
agglutination (2+ or less) with monoclonal anti-B, and contains a strong anti-B in serum. Despite reactivity of
the patient’s red cells with reagent anti-B, the patient’s serum is not reactive with autologous red cells.

297
Chemically, acquired B is the result of deacetylation of the A antigen’s N-acetylgalactosamine, yielding a B-
like galactosamine sugar.23,24 In patients’ samples, acquired B is often present in the setting of infection by
gastrointestinal bacteria. Many enteric bacteria possess a deacetylase enzyme capable of converting A antigen
to a B-like analog.24 Identification of the acquired B phenotype can also be influenced by reagent pH and
specific monoclonal anti-B typing reagents.22 In the past, anti-B reagents containing the ES-4 clone were
associated with an increased incidence of acquired B.
To resolve a patient’s true red cell type and confirm the presence of acquired B, red cells should be retyped
using a different monoclonal anti-B or acidified (pH 6.0) human anti-B. Acidified human anti-B does not react
with acquired B antigen. The ability of monoclonal anti-B to recognize acquired antiB should be noted in the
manufacturer’s insert.
ABO Antibodies
Anti-A and Anti-B
Immune globulin M (IgM) is the predominant isotype found in group A and group B individuals, although
small quantities of IgG antibody can be detected. In group 0 serum, IgG is the major isotype for anti-A and anti-
B. As a consequence, hemolytic disease of the fetus and newborn (HDFN) is more common among the
offspring of group 0 mothers than of mothers with other blood types because IgG can cross the placenta but
IgM cannot.
Both IgM and IgG anti-A and anti-B preferentially agglutinate red cells at room temperature (20 to 24 C) or
cooler, and both can efficiently activate complement at 37 C. The complement-mediated lytic capability of these
antibodies becomes apparent if serum testing includes an incubation phase at 37 C. Hemolysis caused by ABO
antibodies should be suspected when either the supernatant serum is pink to red or the cell button is smaller or
absent. Hemolysis must be interpreted as a positive result. The use of plasma for testing or of reagent red cells
suspended in solutions
 that contain EDTA prevents complement activation and hemolysis.
Anti-A, B
Sera from group 0 individuals contain an antibody known as “anti-A,B” because it is reactive with both A
and B red cells. Such anti-A and anti-B reactivity cannot be separated by differential adsorption, suggesting that
the antibody recognizes a common epitope shared by the A and B antigens.7 Saliva containing secreted A or B
substance can inhibit the activity of anti-A,B against both A and B red cells.
Anti-Al
Anti-Al is present as an alloantibody in the serum of 1% to 8% of A2 individuals and 22% to 35% of A,B
individuals. Anti-Al is sometimes present in the sera of individuals with other weak A subgroups. Group 0
serum contains a mixture of anti-A and anti-Al.24 Anti-Al can cause ABO discrepancies during routine testing
and lead to incompatible crossmatches with Aj and A, B red cells. Anti-Al is usually of IgM isotype, reacting
best at room temperature or below, and is usually considered clinically insignificant. Anti-Al is considered
clinically significant if reactivity is observed at 37 C.24 Group A2 patients with an anti-Al that is reactive at 37
C testing should be transfused with group 0 or A2 red cells only; group A2B patients should receive group 0,
A2, A,B, or B red cells.
Routine Testing for ABO
Donor blood samples are routinely typed for ABO at the time of donation and on receipt of Red Blood Cell
(RBC) units in the hospital transfusion service (confirmatory typing). Recipient samples are typed before
transfusion. ABO grouping requires both antigen typing of red cells for A and B antigen (red cell grouping or
forward type) and screening of serum or plasma for the presence of anti-A and anti-B isoagglutinins (serum
grouping or reverse type). Both red cell and serum or plasma grouping are required for donors and patients
because each grouping serves as a check on
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AABB TECHNICAL MANUAL
the other. Reverse or serum grouping is not required in two circumstances: 1) for confirmation testing of
labeled, previously typed donor red cells and 2) in infants younger than 4 months of age. As previously
discussed, isoagglutinins are not present at birth and develop only after 3 to 6 months of age.
Commercially available anti-A and anti-B for red cell typing are extremely potent and agglutinate most
antigen-positive red cells directly, even without centrifugation. Most monoclonal typing reagents have been
formulated to detect many weak ABO subgroups (see manufacturers’ inserts for specific reagent
characteristics). Additional reagents (anti-Al and anti-A,B) and special techniques to detect weak ABO
subgroups are not necessary for routine testing but are helpful for resolving ABO-typing discrepancies.
In contrast to commercial ABO typing reagents, human anti-A and anti-B in the sera of patients and donors
can be relatively weak, requiring incubation and centrifugation. Tests for serum grouping, therefore, should be
performed using a method that adequately detects human anti-A and anti-B. Several methods are available for
determining ABO group, including slide, tube, microplate, and column agglutination techniques.
ABO Discrepancies
Table 12-1 shows the results and interpretations of routine red cell and serum tests for ABO. A discrepancy
exists when the results of red cell tests do not agree with those of serum tests, usually due to unexpected
negative or positive results in either the forward or reverse typing (see Table 12-3). ABO discrepancies may
arise from intrinsic problems with either red cells or serum or from technical errors in performing the test (see
Table 12-4 and section on Resolving ABO Discrepancies).
When a discrepancy is encountered, the discrepant results must be recorded, but interpretation of the ABO
group must be delayed until the discrepancy has been resolved. If the specimen is from a donor unit, the unit
must be quarantined and cannot be released for transfusion. When an ABO discrepancy is
identified in a patient, it may be necessary to transfuse group 0 red cells pending an investigation. It is
important to obtain a sufficient pretransfusion blood sample from the patient to complete any additional studies
that maybe required.
Red Cell Testing Problems
ABO testing of red cells may give unexpected results for many reasons, including the following:
1. Weak ABO expression that results from inheritance of a weak ABO subgroup. Some patients with
leukemia and other malignancies can also show weakened ABO expression.25
2. Mixed-field agglutination with circulating red cells of more than one ABO group following out-of-group
red cell transfusion or hematopoietic progenitor cell (HPC) transplantation (eg, group 0 to group A). Mixed-
field agglutination is also present in some ABO subgroups (A3), blood group chimerism in fraternal twins, and
very rare cases of mosaicism arising from dispermy.
3. Neutralization of anti-A and anti-B typing reagents by high concentrations of A or B blood group
substance in serum, resulting in unexpected negative reactions with serum- or plasma-suspended red cells.
4. Spontaneous agglutination or autoagglutination of serum- or plasma-suspended red cells caused by heavy
coating of red cells by potent autoagglutinins.
5. Nonspecific aggregation of serum- or plasma-suspended red cells caused by abnormal concentrations of
serum proteins or infused macromolecular solutions.
6. False-positive reactions caused by a pHdependent autoantibody, a reagentdependent antibody (eg, EDTA
or paraben), or rouleaux.
7. Anomalous red cell grouping resulting from acquired B, B(A), or A(B) phenotypes.
299
TABLE 12-4. Possible Causes of ABO Typing Discrepancies Category Causes
Weak/missing red cell reactivity ABO subgroup
Leukemia/malignancy
Transfusion
Intrauterine fetal transfusion Transplantation
Excessive soluble blood group substance
Extra red cell reactivity Autoagglutinins/excess protein coating red cells
Unwashed red cells: plasma proteins
Unwashed red cells: antibody in patient’s serum to reagent constituent
Transplantation
Acquired B antigen
B(A) phenomenon
Out-of-group transfusion
Mixed-field red cell reactivity Recent transfusion
Transplantation Fetomaternal hemorrhage Twin or dispermic (tetragametic) chimerism Weak/missing serum
reactivity Age related (<4-6 months old, elderly)
ABO subgroup
Hypogammaglobulinemia
Transplantation
Extra serum reactivity Cold autoantibody
Cold alloantibody
Serum antibody to reagent constituent Excess serum protein Transfusion of plasma components
Transplantation
Infusion of intravenous immune globulin
 300
AABB TECHNICAL MANUAL
8. Polyagglutination (eg, T activation) resulting from inherited or acquired abnormalities of the red cell
membrane, with exposure of “cryptic autoantigens.”24 Because all human sera contain naturally occurring
antibodies to such cryptic antigens, those abnormal red cells are agglutinated by sera from group A, B, and AB
individuals. Monoclonal anti-A and antiB reagents do not detect polyagglutination.
Problems with Serum or Plasma Testing
Problems may arise during ABO testing of serum or plasma, including the following:
1. Small fibrin clots in plasma or incompletely clotted serum that can be mistaken for red cell agglutinates.
2. Lack of detectable isoagglutinins in infants younger than 4 to 6 months. Children do not develop
isoagglutinins until 3 to 6 months of age. Isoagglutinins present at birth are passively acquired from the mother.
3. Unexpected absence of ABO agglutinins caused by a weak A or B subgroup (see Table 12-3).
4. Unexpected absence in children of anti-B agglutinins that are sterile, free of bacteria, and result from
long-term parenteral and enteral nutrition.26
5. Unexpected absence of anti-A agglutinins in patients receiving equine-derived immunoglobulins.27
6. ABO-incompatible HPC transplantation with induction of tolerance. For example, a group A patient
receiving a group 0 marrow transplant will have circulating group 0 red cells but will produce only anti-B in
serum. (Refer to Chapter 25 for more information on ABO-mismatched transplants.)
7. Severe hypogammaglobulinemia secondary to inherited immunodeficiency or disease therapy.
Hypogammaglobulinemia with dilution of isoagglutinins can also
occur after several courses of plasma exchange with albumin replacement.
8. Cold alloantibodies (eg, anti-M) or autoantibodies (eg, anti-I) reactive with reverse-grouping cells,
leading to unexpected positive reactions.
9. Antibodies directed against constituents in the diluents used to preserve reagent A: and B red cells.24
10. Nonspecific aggregation or agglutination caused by high-molecular-weight plasma expanders, rouleaux,
high serum-protein concentrations, or altered serum-protein ratios.
11. Recent transfusion of out-of-group plasma-containing components (eg, a group A patient transfused with
platelets from a group 0 donor, causing unexpected anti-A in the patient’s plasma).
 12. Recent infusion of intravenous immunoglobulin, which can contain ABO isoagglutinins.
Technical Errors
Technical problems with a sample or during
testing can also lead to problems in ABO
grouping, including:
1. Specimen mix up.
2. Too heavy or too light red cell suspensions.
3. Failure to add reagents.
4. Missed observation of hemolysis.
5. Failure to follow the manufacturer’s instructions.
6. Under- or overcentrifugation of tests.
7. Incorrect interpretation or recording of test results.
Resolving ABO Discrepancies
The first step in resolving an apparent serologic testing discrepancy should be to repeat the test with the
same sample to exclude the possibility of a technical error during testing. Additional studies may include testing
washed red cells; testing a new sample; testing for unexpected red cell alloantibodies; and reviewing
301
the patient’s medical record for conditions, medications, or recent transfusions that may have contributed to
the conflicting test results (Method 2-4). Samples with apparent weak or missing ABO antigens and/or
antibodies may require tests using methods that enhance antigen-antibody binding, including incubating red
cells at 4 C (Method 2-5), using enzyme-treated red cells (Method 2-6), and conducting adsorption and elution
studies (Method 2-7). In some instances, it may be necessary to test for the secretion of ABO antigens in saliva
(Method 2-8). Patients with suspected B(A), acquired B, or A(B) phenotypes should be retested using different
monoclonal and human polyclonal reagents.
ABO discrepancies caused by unexpected serum reactions are not uncommon. Commonly encountered
causes of serum-grouping discrepancies include cold autoantibodies, rouleaux, cold-reacting alloantibodies (eg,
anti-M), and weak A subgroups with an antiAl. To resolve an ABO discrepancy caused by an anti-Al in a group
A individual, red cells should be tested with Dolichos biflorus lectin, which agglutinates group A, but not A2
and weaker A subgroups. The presence of an antiAl should be confirmed by testing serum against Ap A,, and 0
red cells (Method 2-9). Reverse-grouping problems resulting from either a cold alloantibody (Method 2-10) or
autoantibody can be identified with a roomtemperature antibody detection test and a direct antiglobulin test.
Techniques to identify ABO antibodies in the presence of cold autoantibodies include testing at 37 C without
centrifugation (Method 2-11) and cold autoadsorption (Method 4-5). Serum or plasma properties can induce
rouleaux formation that resembles agglutination with A1 and B red cells. Saline replacement or saline dilution
(Method 3-7) can be used to distinguish rouleaux from agglutination and identify ABO antibodies.
Cold autoantibodies can cause autoagglutination of red cells and unexpected reactions during red cell
typing. Red cells heavily coated with autoantibodies can spontaneously agglutinate and cause false-positive
reactions in tests with anti-A and anti-B. Usually,
false-positive reactions caused by autoantibodies can be eliminated by washing red cells with warm saline
(Method 2-17). Autoagglutination caused by IgM can also be inhibited or dispersed by incubating red cells in
the presence of either dithiothreitol or 2-aminoethylisothiouronium bromide (Method 3-16). These reagents
reduce the disulfide bonds on IgM molecules, decreasing their polyvalency and ability to directly agglutinate
red cells.
THE H SYSTEM
H antigen is ubiquitously expressed on all red cells except the rare Bombay phenotype. Because H antigen
serves as the precursor to both A and B antigens, the amount of H antigen on red cells depends on an
individual’s ABO type. H antigen is highly expressed on group 0 red cells because group 0 individuals lack a
functional ABO gene. In group A and B individuals, the amount of H antigen is considerably less because H is
converted to the A and B antigens, respectively. The amount of H antigen on red cells, based on agglutination
with the anti-H lectin Ulex europaeus, is represented thus: 0>A2>B>Aj>AjB. H antigen is present on HPCs, red
cells, megakaryocytes, and other tissues.2,3,28 H antigen has been implicated in cell adhesion, normal
hematopoietic differentiation, and several malignan
Biochemistry and Genetics
 H antigen is defined by the terminal disaccharide fucose al->2 galactose. Two different fucosyltransferase
(FUT) enzymes are capable of synthesizing H antigen: FUT1 (H gene) and FUT2 (secretor gene). FUT1
specifically fucosylates Type 2 chain oligosaccharides on red cell glycoproteins and glycolipids to form Type 2
chain H. In contrast, FUT2 or secretor recognizes Type 1 chain precursors to form Type 1 chain H and Leb
antigens in secretions (Fig 123).10 Secretion of Type 1 chain ABH antigens in saliva and other fluids requires a
functional FUT2 gene. FUT2 is not expressed in red cells but is expressed in salivary glands, gastrointestinal
tissues, and genitourinary tissues.1,7,10
TYPE 2 CHAIN ABH TYPE 1 CHAIN ABH & LEWIS
302
AABB TECHNICAL MANUAL
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Fuc = fucose; Gal = galactose; GalNAc = /V-acetylgalactosamine; GIcNAc = /V-acetylglucosamine; R =
other upstream carbohydrate sequence. Reproduced with permission from Cooling.30
303
Type 1 chain ABH antigen present on red cells is passively adsorbed from circulating glycolipid antigen
present in plasma (see “Lewis System” section).24
Null Phenotypes
Bombay (Oh) Phenotype
Originally described in Bombay, India, the 0h or Bombay phenotype is a rare, autosomal recessive
phenotype characterized by the absence of H, A, and B antigens on red cells and in secretions. Genetically, Oh
individuals are homozygous for nonfunctional H (hh) and secretor (sese) genes, resulting in a complete absence
of Type 1 and Type 2 chain H, A, and B. Oh red cells type as H negative with the anti-H lectin Ulex europaeus
and monoclonal anti-H. Because these individuals lack a functional secretor gene necessary for Leb synthesis,
Oh individuals also type as Le(b-) (see “Lewis System” section). Genotyping studies have described a wide
range of inactivating mutations in both the H and secretor genes in Oh individuals.10,18 The Oh phenotype is
also present in leukocyte adhesion deficiency 2 (LAD2) due to a mutation in the GDP-fucose transporter
gene.25
Because they lack all ABH antigens, Oh individuals possess natural isohemagglutinins to A, B, and H (see
Table 12-1). In routine ABO typing, these individuals initially type as group 0. The Oh phenotype becomes
apparent during antibody-detection tests with group 0 red cells, which are rich in H antigen. The anti-H present
in Oh individuals strongly agglutinates all group 0 red cells and sometimes demonstrates in-vivo hemolysis.
The Oh phenotype can be confirmed by demonstrating an absence of H antigen on red cells and the presence of
an anti-H in serum that is reactive with group 0 red cells but not with Oh red cells from other individuals.
Para-Bombay Phenotype
Individuals with the para-Bombay phenotype are H-deficient secretors.7,10 Genetically, these individuals
are homozygous for a nonfunctional H gene (hh), but they have inherited at
least one functional secretor gene (Se). The red cells from H-deficient secretors lack serologically detectable
H antigen but can carry small amounts of A and/or B antigen because, unlike persons with classic Bombay
phenotype, paraBombay persons express Type 1 chain ABH antigens in their secretions and plasma (Method 2-
8).24 Type 1 chain A or B antigen in plasma is then passively adsorbed onto red cells, resulting in weak A or B
antigen expression. ParaBombay can also occur in group 0 individuals, as evidenced by trace Type 1 chain H on
their red cells and in their secretions. Red cells from para-Bombay individuals are designated “Ah,” “Bh,” and
“ABh.”
In laboratory testing, red cells from paraBombay individuals may (or may not) have weak reactions with
anti-A and anti-B reagents. In some cases, A and B antigens may be detected only after adsorption and elution.
Para-Bombay red cells are nonreactive with anti-H lectin, monoclonal anti-H, and human anti-H from Oh
individuals. The sera of paraBombay individuals contain anti-H, anti-HI, or both and, depending on their ABO
type, anti-A and anti-B.13,24
Anti-H
Alloanti-H (Bombay and Para-Bombay)
The anti-H in Bombay and para-Bombay phenotypes is clinically significant and associated with acute
hemolytic transfusion reactions. The antibody is predominantly of IgM isotype and exhibits a broad thermal
range (4 to 37 C) with all red cells except Oh red cells. As with anti-A and anti-B, alloanti-H is capable of
activating complement and causing red cell hemolysis.
Autoanti-H and Autoanti-HI
Autoantibodies to H and HI antigens can be encountered in healthy individuals. When present, these
autoantibodies are most common in Aj individuals, who have very little H antigen on their red cells. Autoanti-H
and autoanti-HI are usually of IgM isotype and are reactive at room temperature.
304
AABB TECHNICAL MANUAL
Transfusion Practice
Alloanti-H is highly clinically significant and is capable of fixing complement and causing hemolytic
transfusion reactions. As a result, patients with alloanti-H caused by a Bombay or para-Bombay phenotype must
be transfused with H-negative (Oh) RBCs.
In contrast, autoantibodies against H and HI are generally clinically insignificant. In most patients,
transfused group-specific or group 0 RBCs should have normal in-vivo survival. Occasionally, autoanti-HI can
result in decreased red cell survival and hemolytic transfusion reactions after transfusion of group 0 RBCs.13,24
In most instances, hemolysis follows transfusion of group 0 RBCs to a group Aj or B patient with an unusually
potent high-titer anti-HI that is reactive at 37 C.24 In such patients, it may be advisable to transfuse group-
specific (A,, B, or AB) RBCs.
THE LEWIS SYSTEM
The Lewis blood group system consists of two main antigens, Lea (LEI) and Leb (LE2), and three common
phenotypes, Le(a+b-), Le(ab+), and Le(a-b-). Four additional Lewis antigens represent composite reactivity
between Lea, Leb, and ABO antigens: Leab (LE3), LebH (LE4), ALeb (LE5), and BLeb (LE6).18,25 In
addition to being present on red cells, Lewis antigens are widely expressed on platelets, endothelium, and the
kidney as well as on genitourinary and gastrointestinal epithelium.
Lewis antigens are not synthesized by red cells but are passively adsorbed onto red cell membranes from a
pool of soluble Lewis glycolipid present in plasma.25 The gastrointestinal tract, which is rich in Lewis-active
glycolipid and glycoprotein, is thought to be the primary source of Lewis glycolipid in plasma. Because Lewis
antigens are passively adsorbed onto red cell membranes, they can be eluted from red cells after transfusion or
by increases in plasma volume and increased circulating lipoproteins, which also adsorb Lewis glycolipid. For
example, Lewis antigen is often decreased on red cells during pregnancy, with some women transiently typing
as Le(a-b-).
The latter is attributed to an increase in circulating plasma volume and a fourfold increase in lipoprotein.24
Leb expression and immunoreactivity are also influenced by ABH type as a result of the synthesis of hybrid
structures with both Lewis and ABH activity (Fig 12-3).2,10,28
Biochemistry and Synthesis
Lewis antigen synthesis depends on the interaction of two distinct fucosyltransferases (Fig 12-3): Lewis
(FUT3) and secretor (FUT2).25,30 Unlike H or FUT1, which is relatively specific for Type 2 chain substrates,
Lewis and secretor preferentially fucosylate Type 1 chain substrates. Secretor (FUT2) can add a terminal al->2
fucose to a Type 1 chain precursor to form Type 1 chain H antigen. The Lewis gene encodes an al->3/4
fucosyltransferase that transfers a fucose, in an a 1—>4 linkage, to the penultimate IV-acetyl-glucosamine of
Type 1 chain precursor (“Lewis c”) to form Lea antigen. Lewis (FUT3) can also add a second fucose to Type 1
H antigen to form Leb antigen. Note that Leb cannot be formed from Lea because the presence of a subterminal
fucose on Lea sterically inhibits binding by the secretor enzyme.1
In individuals with both Lewis and secretor enzymes, Type 1 chain H is favored over Lea synthesis. As a
result, most of the Lewis antigen synthesized is Leb [Le(a—b+) phenotype]. In group Aj and B individuals, Leb
and Type 1 chain H can be further modified by ABO glycosyltransferases to form LE5, LE6, Type 1 A, and
Type 1 B antigens.2,30 In group A1 individuals, the majority of Lewis antigen in plasma is actually ALeb.31
Genetics and Lewis Phenotypes
The three Lewis phenotypes commonly encountered represent the presence or absence of Lewis and secretor
enzymes (see Table 125). Le(a+b-) individuals have inherited at least one functional Lewis gene (Le) but are
homozygous for nonfunctional secretor alleles (sese). As a result, such individuals synthesize and secrete Lea
antigen but lack Leb and Type 1 chain ABH antigens.
305
The Le(a-b+) phenotype reflects inheritance of both Le and Se alleles, leading to the synthesis of Lea, Leb,
and Type 1 chain ABH. Because most Type 1 chain precursor is converted to Leb in those individuals, they
appear to type as Le(a-). An Le(a+b+) phenotype is transiently observed in infants because secretor activity
increases with developmental age. An Le(a+b+) phenotype is also present in 16% of Japanese individuals as a
result of inheritance of a weak secretor gene (Sew) ,18 In the absence of a functional Lewis gene (/e/e), neither
Lea nor Leb is synthesized, leading to an Le(ab-) or null phenotype. Type 1 chain ABH antigens may still be
synthesized and secreted in individuals who have inherited at least one functional Se allele (Method 2-8). The
Le(ab-) phenotype is significantly more common in persons of African ethnicity. Although rare, an Le(a-b-)
phenotype is also present in individuals with LAD2 due to defects in fucose transport.25
Several inactivating mutations have been identified in both Lewis and secretor genes.18 Many of the
mutations are geographically and racially distributed, with many populations displaying a few predominant
alleles. Of the nonfunctional Le alleles described, most have more than one mutation.10,18
 Lewis Expression in Children
Table 12-5 shows the distribution of Lewis types in adults. In contrast, most newborns type as Le(a-b-) with
human anti-Lewis typing reagents. Approximately 50% of newborns subsequently type as Le(a+) after ficin
treatment. The prevalence of Leb antigen, however, is low in newborns compared to in adults because of
developmental delays in secretor CFUT2) activity. An Le(a+b+) phenotype can be transiently present in
children as the level of secretor activity approaches adult levels. A valid Lewis phenotype is not developed until
age 5 or 6.13
Lewis Antibodies
Antibodies against Lewis antigens are generally of IgM isotype and occur naturally. Clinically, Lewis
antibodies are most often encountered in the sera of Le(a-b-) individuals and may contain a mixture of anti-Lea,
anti-Leb, and anti-Leab, an antibody capable of recognizing both Le(a+) and Le(b+) red cells. Because small
amounts of Lea are synthesized in the Le(a-b+) phenotype, Le(a-b+) individuals do not make anti-Lea. Anti-
Leb is present infrequently in the Le(a+b-) phenotype. Lewis antibodies, accompanied by a transient Le(a-b-)
TABLE 12-5. Adult Phenotypes and Prevalence in the Lewis System
Red Cell Prevalence
Genotype*
Reactions (%)
Anti-
Anti-Lea Phenotype Whites Blacks Lewis Secretor Saliva1
Leb
+ 0 Le(a+b—) 22 23 Le sese Lea
Lea,
0 + Le(a-b+) 72 55 Le Se
Leb, ABH
Type 1
0 0 Le(a-b-) 6 22 lele sese
precursor
Type 1
lele Se
ABH
Lea,
+ + Le(a+b+)* Rare Rare Le Sew
Leb
'Probable genotype at the Lewis (FUT3) and Secretor (FUT2) loci.
Hype 1 chain antigens present in saliva and other secretions.
*Le(a+b+) is present in 16% ot Japanese individuals and is also transiently observed in infants.
Le = gene encoding functional Lewis enzyme; lele = homozygous for gene encoding an inactive enzyme; Se
= gene encoding sactive secretor enzyme; sese = homozygous for gene encoding an inactive enzyme; Sew =
gene encoding weak secretor enzyme.
 306
AABB TECHNICAL MANUAL
phenotype, are present during pregnancy. Finally, anti-Leb can demonstrate ABO specificity (anti-LebH,
anti-ALeb, and anti-BLeb), and is preferentially reactive with Le(b+) red cells of a specific ABO group.24,28
Anti-LebH, the most common reactivity, is more strongly reactive with Le(b+) group 0 and A2 red cells than
with group Aj and B red cells, which have low H antigen levels. Anti-LebL is strongly reactive with all Le(b+)
red cells, regardless of ABO group.
Most examples of Lewis antibodies are saline agglutinins that are reactive at room temperature. Unlike
ABO, the agglutination is relatively fragile and easily dispersed, requiring gentle resuspension after
centrifugation. Agglutination is sometimes observed after 37 C incubation, but the reaction is typically weaker
than that at room temperature. On occasion, Lewis antibodies can be detected in the antihuman globulin (AHG)
phase. Such detection may reflect either IgG or bound complement (if polyspecific AHG reagent is used). Very
rarely, Lewis antibodies can cause in-vitro hemolysis. Hemolysis occurs more often when fresh serum
containing anti-Lea or anti-Leab is tested, particularly against enzyme-treated red cells.
Transfusion Practice
In general, Lewis antibodies are not considered clinically significant. Red cells that are compatible in tests at
37 C, regardless of Lewis phenotype, are expected to have normal invivo survival. It is not necessary to
transfuse antigen-negative RBCs in most patients. Unlike ABO antigens, Lewis antigens are extrinsic glycolipid
antigens that are readily eluted and shed from transfused red cells within a few days of transfusion.25
Furthermore, Lewis antigens in transfused plasma can neutralize Lewis antibodies in the recipient. For these
reasons, hemolysis is rare following transfusion of either Le(a+) or Le(b+) red cells.
Lewis antibodies are not a cause of HDFN.13 Lewis antibodies are predominantly of IgM isotype and do
not cross the placenta. In addition, Lewis antigens are poorly expressed on neonatal red cells, with many
newborns
typing as Le(a-b-) with human Lewis antibodies (see above).
Disease Associations
 The Leb and H antigens are receptors for Helicobacter pylori, a gram-negative bacterium implicated in
gastritis, peptic ulcer disease, gastric carcinoma, mucosa-associated lymphoma, and idiopathic
thrombocytopenia. Leb and Type 1 H antigens are also receptors for noroviruses, common causes of acute
gastroenteritis. An Le(a-b-) phenotype is associated with increased susceptibility to infections by Candida and
uropathogenic Escherichia coli, cardiovascular disease, and possibly decreased graft survival after renal
transplantation.6,25
I AND i ANTIGENS
The I and i antigens are ubiquitous, structurally related antigens present on all cell membranes. The
minimum epitope common to both i and I is a terminal repeating lactosamine (Gaipi->4GlcNAc) or Type 2
chain precursor. The minimum i antigen is a linear, nonbranched structure containing at least two successive
lactosamine structures.14 The I antigen is a polyvalent, branched glycan derived from the i antigen (Fig 12-4).
Both i and I serve as substrate and scaffold for the synthesis of ABO, Lewis X [Gal(Sl-4(Fucal-3) GlcNAc], and
other Type 2 chain antigens.1,2,14 On red cells, i and I antigens are present on AM inked glycoproteins and
glycolipids.
Phenotypes
Two phenotypes are recognized according to the presence or absence of I antigen: I and i (I-). The i
phenotype is characteristic of neonatal red cells, whereas 1+ is the common phenotype in adults. With
increasing age, there is a gradual increase in I antigen accompanied by a reciprocal decrease in i antigen; most
children develop an adult 1+ phenotype by age 2.14 An increase in i antigen can occur in people with chronic
hemolytic disorders and is a sign of stressed erythropoiesis.32
307
GCNT2 Gene (IGnT, I gene)

i Antigen Gal(31^4GlcNAcp1^3Gal(31^4GlcNAcpi-R
GCNT2
1f
GIcNAcpi—>6
I Antigen Gaipi^4GicNAcpi-R
Gaipi—»4GlcNAcpi—>3
pi, 4GalT
Galp1->4GlcNAcp1^6
I Antigen Gaipi->4GicNAcpi-R
Gaipi^4GlcNAcpi->3
FUT1
ir
Galpl ^4GlcNAcp1 -»6
HI Antigen Gaipi->4GicNAcpi-R
Fuca1-»2Gaipi-4GlcNAcpi->3
FIGURE 12-4. Structure of I gene (GCNT2) (above) and synthesis of I antigen from i antigen (below). Gal
= galactose; GIcNAc = /V-acetylglucosamine; R = other upstream carbohydrate sequence.
Two genetic disorders are associated with an increase in i antigen.14 The iadult phenotype (I—i+) is an
autosomal recessive phenotype caused by mutations in the 1 gene (GCNT2 or IGnT). In populations of Asian
ancestry, the hduit phenotype can be associated with congenital cataracts. Increased i antigen levels are also
present in people with congenital dyserythropoietic anemia Type 2 (also known as hereditary erythroblastic
multinuclearity with positive acidified serum lysis test). The latter is a genetic defect in Golgi transport associat
ed with abnormal glycosylation, chronic hemolysis, splenomegaly, and erythroid multinuclearity.
Genetics
The I gene (GCNT2) encodes a pi—>6 IV-acetylglucosaminyltransferase that converts the linear i antigen
into the branched I antigen.14,18 The gene resides on chromosome 6p24 and contains five exons, including
three tissuespecific exons (exons 1A, IB, and 1C). As a result, three slightly different mRNA transcripts
308
AABB TECHNICAL MANUAL
can be synthesized, depending on which exon 1 is used.
 In the iadult phenotype without cataracts, there is a mutation in exon 1C that is specific for I antigen
synthesis in red cells. As a consequence, I antigen is missing on red cells but is still synthesized in other tissues
that use either exon 1A or exon IB. In the iadult phenotype with cataracts, there is a loss of I antigen synthesis
in all tissues caused by either gene deletion or mutations in exons 2 and 3.
Antibodies
Anti-I
Anti-I is common in the serum of healthy individuals. Anti-I is usually of IgM isotype and is strongly
reactive at 4 C with titers of <64. Samples with higher titers may also be detectable at room temperature. Anti-I
is identified by strong reactions with adult red cells but weak or no agglutination with cord red cells (see Table
12-6). Anti-I can be enhanced by 4 C incubation, the presence of albumin, or use of enzyme-treated red cells.
An alloanti-I can be seen in the iadult phenotype.
Some examples of anti-I can demonstrate complex reactivity and are more strongly reactive with red cells of
specific ABO, Pj, or Lewis phenotypes. Many of those antibodies appear to recognize branched
oligosaccharides that have been further modified to express additional blood group antigens. Anti-IH is
commonly present in the serum of A1 individuals. Anti-IH is more strongly reactive with
TABLE 12-6. Comparative Serologic Behavior of the
Suspensions
group 0 and group A, red cells, which are rich in H antigen, than with group A, red cells. AntiIH is
suspected when serum from a group A individual directly agglutinates all group 0 red cells but is compatible
with most group A donor blood tested. Other examples of complex reactivity include anti-IA, -IP1( -IBH, and -
ILebH.24
Anti-i
Autoanti-i is a relatively uncommon cold agglutinin in sera from healthy individuals. Like anti-I, anti-i is
primarily of IgM isotype but is weakly reactive at 4 to 10 C. Anti-i is most strongly reactive with cord and
iadult red cells and more weakly reactive with 1+ adult red cells (Table 12-6). Patients with infectious
mononucleosis often have transient but potent anti-i. As with anti-I, complex reactivity can sometimes occur
(eg, anti-iH).
Cold Agglutinin Syndrome
Autoanti-I and autoanti-i are pathologically significant in cold agglutinin syndrome (CAS) and mixed-type
autoimmune hemolytic anemia. In those disorders, autoanti-I (or anti-i) behaves as a complement-binding
antibody with a high titer and wide thermal range. Primary CAS occurs with lymphoproliferative disorders (eg,
Waldenstrom macroglobulinemia, lymphoma, and chronic lymphocytic leukemia). A potent autoanti-I can also
occur in the setting of infection. Mycoplasma pneumoniae infections are a common cause of autoanti-I and can
be accompanied by a transient hemo
l/i Blood Group Antibodies with Saline Red Cell
Temperature Cell Type Anti-I Anti-i
4C 1 adult 4+ 0-1 +
i cord 0-2+ 3+
i adult 0-1 + 4+
22 C 1 adult 2+ 0
i cord 0 2-3+
i adult 0 3+
 309
lysis. (See Chapter 17 for additional information on CAS.)
The specificity of the autoantibody in CAS may not be apparent when undiluted samples are tested.
Titration and thermal amplitude studies maybe required to discern the specificity of the autoantibodies and their
potential clinical significance. Table 12-6 illustrates the serologic behavior of anti-I and anti-i at 4 C and 22 C.
(See Chapter 17 and Method 4-7 for additional information regarding titration and thermal amplitude studies.)
Transfusion Practice
Autoanti-I can interfere with ABO typing, antibody screening, and compatibility testing. In laboratory
testing, these antibodies can be reactive in the antiglobulin phase of testing, particularly when polyspecific
AHG is used. Such reactions rarely indicate antibody activity at 37 C but are the consequence of antibody
binding, followed by complement binding, at low temperatures. Usually, avoiding room-temperature testing and
using anti-IgG-specific AHG prevents detection of nuisance cold autoantibodies. For stronger antibody
samples, autoantibody can be removed from serum by cold autoadsorption techniques (see Method 4-5). Cold
autoadsorbed serum can also be used for ABO testing.
P BLOOD GROUPS/GLOB COLLECTION
The first antigen of the P blood group system was discovered by Landsteiner and Levine in 1927 in a series
of experiments that also led to the discovery of M and N antigens. Several related glycosphingolipid antigens
belong to P1PK (P,, Pk, NOR), GLOB (P, PX2), and 209 collection (LKE).18 Pk, P, and LKE are high-
incidence antigens expressed on nearly all red cells except rare null phenotypes, which lack P (Pk phenotype) or
both P and Pk antigens (p phenotype) (see Table 12-7). Red cells are particularly rich in P antigen, which makes
up nearly 6% of total red cell lipid. Pk and P antigens are also widely expressed on nonerythroid cells, including
lymphocytes; platelets; and plasma, kidney, lung, heart, endothelium, placenta, and synovium cells.33 In
contrast, P2 antigen is uniquely expressed on red cells.33
Phenotypes
 More than 99% of donors have the Pj (P,+) or P2 (P,-) phenotype (see Table 12-7). Both phenotypes
synthesize Pk and P antigens and differ only in the expression of the P, antigen. Three rare, autosomal recessive
phenotypes have been identified (p, Ptk, P2k) as well as weak
TABLE 12-7. Phenotypes and Prevalence in the PI PK and GLOB Group
Red Cell
Antibodies Prevalence
Reactions with Phenotype
in Serum (%)
Antisera
Anti-PPI European African
Anti-Pi Anti-P Anti-Pk
Pk Ethnicity Ethnicity
+ + 0 + None Pi 79 94
0 + 0 + PI* P2 21 6
PP1 Pk
0 0+ 0 0 P Rare Rare
0T)
+ 0 + + P p; Rare Rare
0 0 + + P p2k Rare Rare
*An anti-PI is
detected in
approximately 25%
of P2 individuals.
Msually negative. Some examples may be weakly positive as a result of crossreactivity of anti-P with X2
and sialosyl-X2 glycolipid on p red cells.

310
AABB TECHNICAL MANUAL
variants.34 Analogous to the ABO system, the rare p and Pk phenotypes are associated with the presence of
naturally occurring antibodies against missing antigens (anti-P,, anti-P, and anti-PPjP^.
Biochemistry
 The synthesis of the Pk, R and P, antigens proceeds through the stepwise addition of sugars to
lactosylceramide, a ceramide dihexose (CDH). (See Fig 12-5 and Table 12-8.) The first step in this process is
the synthesis of the
Pk antigen, the ultimate precursor of all globotype glycosphingolipids. To make the Pk antigen, al,4
galactosyltransferase 1 (A4GALT1) adds a terminal galactose, in an a l-»4 linkage, to CDH. The Pk antigen can
then serve as a substrate for pi,3 Af-acetylgalactosaminyltransferase I (B3GALNACT1), which adds a pi—>3
N-acely 1 galaclosamine to the terminal galactose of Pk (Gb3) to form P antigen. In some cells, including red
cells, the P antigen is further elongated to form additional, globofamily antigens, such as Luke (LKE), Type 4
GlcCer
y
Neolacto Family LacCer Globo Family
(CDH)
A4GALT1

Paragloboside
(PG, nLc4)
A4GalT 1

X2 SPG P2
(G b3)
B3GalNAcT 1 ^
P
A4GALT1
NOR
(G b4)
V B3GalT5
Gb5
ST3GAL2 FUT1/2
V
Sialosyl-X2

LKE Globo-H
FIGURE 12-5. Synthesis of PI, Pk, P, and related glycosphingolipid antigens. Carbohydrate structures are
shown in Table 12-8.
311
TABLE 12-8. Structures of PI, GLOB, and Related Glycosphingolipids
Family* Name Oligosaccharide Structure
CDH Gaipi -4Glcpi-1 Cer
Globo (Gb) Gb3, Pk Galocl -4Gaipi -4Glcpi-1 Cer
Gb4, P GalNAcpi-3Gala1-4Gaipi-4Glcpi-1Cer
Gb5 Gaipi -3GalNAcpi -3Gala1 -4Gaipi -4Glcpi -1 Cer
N0R1 Galal -4GalNAcpi -3Gala1 -4Gaipi -4Glcpi -1 Cer
Globo-H Fucal -2Gaipi -3GalNAcpi -3Galcc1 -4Gaipi -4Glcpi -1 Cer
LKE NeuAca2-3Gaipi-3GalNAcpi-3Gala1-4Gaipi-4Glcpi-1Cer
N0R2 Galal -4GalNAcpi -3Gala1 -4GalNAcpi -3Gala1 -4Gaipi -4Glcpi -1 Cer
Neolacto
Lc3 GIcNAcpi -3Gaipi -4Glcpi -1 Cer
(nLc)
nLc4, PG Gaipi-4GlcNAcpi-3Gaipi-4Glcpi-1 Cer
PI Galal -4Gaipi -4GlcNAcpi -3Gaipi -4Glcpi -1 Cer
SPG NeuAca2-3Gaipi-4GlcNAcpi-3Gaipi-4Glcpi-1Cer
 X2 GalNAcpi-3Gaipi-4GlcNAcpi-3Gaipi-4Glcpi-1Cer
Sialosyl-X2 NeuAca2-3GalNAcpi-3Gaipi-4GlcNAcpi-3Gaipi-4Glcpi-1 Cer
*Glycosphingolipid family. Note: Neolacto are type 2 chain glycosphingolipids.
CDH = ceramide dihexose or lactosylceramide; Cer = ceramide; Gal = galactose; GalNAc = /(/-
acetylgalactosamine; Glc = glucose, GIcNAc = /V-acetylglucosamine; NeuAc = /V-acetylneuramanic acid
(sialic acid); PG = paragloboside; SPG = sialosylparagloboside.
chain ABH antigens (globo-ABH), and NOR. NOR, a rare polyagglutinable red cell phenotype, is the result
of unusual globo-family antigens characterized by the addition of an a l->4 galactose to the terminus of P and
related longchain globo-glycolipids (see Table 12-8).35
Unlike Pk and P antigens, the P, antigen is not a globo-glycosphingolipid but is a member of the neolacto
family (Type 2 chain glycosphingolipids). In Pj individuals, A4GALT1 adds an al->4 galactose to the terminus
of paragloboside. The P, antigen is not expressed on red cell glycoproteins.36 The weak P-like activity on p red
cells is believed to be X2 (PX2), a related Type 2 chain glycolipid. Recent studies have shown that
B3GALNACT1 is capable of synthesizing PX2-active structures.37
Molecular Biology
Several inactivating mutations have been identified in both a4GALTl and P3GALNACT1.18,38 The p
phenotype is the consequence of mutations in the protein-coding sequence of A4GATT1. In the absence of
A4GALT1 activity, there is a loss of all globo-family and PI antigens. These patients have a compensatory
increase in Type 2 chain glycolipid synthesis, as evidenced by increased paragloboside, sialoparagloboside, and
PX2.37 Mutations in B3GalNACTl give rise to the Pk phenotype, which is characterized by a loss of P and
LKE antigens and by increased Pk expression. The P2 phenotype arises from a point mutation that introduces
an alternate start codon. It is hypothesized that this alternate A4GALT1

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AABB TECHNICAL MANUAL
transcript or peptide may downregulate A4GAT1 transcription with low enzyme levels and decreased
affinity for paragloboside. Interestingly, individuals with weak Pj expression are heterozygous for PJP2
alleles.34
P Blood Group Antibodies
Anti-PI
Anti-Pj is a present in the sera of one-quarter to two-thirds P2 donors.24 Anti-Pj is a naturally occurring
antibody of IgM isotype and is often detected as a weak, room-temperature agglutinin. In rare cases, anli-P, is
reactive at 37 C or shows in-vitro hemolysis. Because anti-P, is nearly always IgM, anti-P, does not cross the
placenta and has not been reported to cause HDFN. Anti-P, has only rarely been reported to cause in-vivo
hemolysis. Anti-P, titers are often elevated in patients with hydatid cyst disease or fascioliasis (liver fluke) and
in bird handlers. It is believed that Prlike substance in bird excrement can stimulate anti-P, levels.25 Some
people with anti-Pj also have I blood group specificity (anti-IPl).24
Pj expression varies in strength among individuals and has been reported to decrease during in-vitro
storage.24 As a consequence, anti-Pj may not be reactive with all P,+ red cells tested. Anti-P, can be enhanced
by incubation at low temperatures (eg, 4 C) or by testing serum against enzyme-treated red cells. Anti-P,
reactivity can be inhibited in the presence of hydatid cyst fluid or P, substance derived from pigeon eggs.
Inhibiting Pj activity may be helpful when testing sera containing multiple antibodies.
Alloanti-PPand Alloanti-P
Anti-PP,Pk (historically known as anti-Tf) is a separable mixture of anti-P, anti-P,, and antiPk in the sera of
p individuals. Alloanti-P is present in the sera of P,k and P2k individuals, occurs naturally, and is predominantly
of IgM isotype or a mixture of IgM and IgG (see Table 12-7). The antibodies are potent hemolysins and are
associated with hemolytic transfusion reactions and, occasionally, HDFN. There is an association between anti-
PP,Pk and early
spontaneous abortion. The placenta, which is of fetal origin, is rich in Pk and P antigen and is a target for
maternal cytotoxic IgG antibodies.
Autoanti-P (Donath-Landsteiner)
An autoantibody with P specificity is present in patients with paroxysmal cold hemoglobinuria (PCH), a
clinical syndrome that most commonly occurs in children following viral infection. In PCH, autoanti-P is an
IgG biphasic hemolysin capable of binding red cells at colder temperatures, followed by intravascular
hemolysis at body temperature. This characteristic can be demonstrated in vitro in the Donath-Landsteiner test
(see Chapter 17 and Method 4-11 for further details).
Transfusion Practice
Alloanti-PPjPk and alloanti-P are clinically significant antibodies associated with acute hemolytic
transfusion reactions and spontaneous abortion. Rare individuals of p and Pk phenotypes should be provided
with antigennegative, crossmatch-compatible RBCs for transfusion.
In general, anti-P, is a clinically insignificant, room-temperature agglutinin. Patients with anti-Pj, which is
reactive only at room temperature or below, can be safely transfused with P,+ RBCs, which results in normal red
cell survival. It is not necessary to provide antigen-negative units to these patients. Very rarely, anti-P, can cause
decreased red cell survival and hemolytic transfusion reactions.
Anti-Pj that is capable of fixing complement at 37 C and is strongly reactive in the AHG phase of testing is
considered potentially clinically significant. In such rare instances, units selected for transfusion should be
nonreactive at 37 C and in an indirect antiglobulin test with either polyspecific AHG or anti-C3.24
Disease Associations
The Pk antigen is a receptor for Shiga toxins, the causative agent of shigella dysentery, and Escherichia co/i-
associated hemolytic uremic syndrome.33 The Pk antigen is also a receptor for Streptococcus suis, a species of
bacteria that
313
triggers zoonotic illness capable of causing bacterial meningitis.33 Pk expression may also modulate host
resistance to human immunodeficiency virus.39 The P antigen is a receptor for parvovirus B19, the etiologic
agent of erythema infectiosum (fifth disease). In some patients, B19 can cause a transient anemia or
aplastic crisis. P,, Pk, P and LKE antigens can all serve as receptors for P-fimbriated uropathogenic E. coli, a
cause of chronic urinary tract infections.33 LKE is a common oncofetal marker present on tumors and
pluripotent embryonic, mesenchymal, and very small embryonic-like stem cells.4,34
KEY POINTS
1. The antigens of the ABO, H, Lewis, I, and P blood group systems are defined by small carbohydrate
epitopes on glycoproteins and glycolipids. They are widely distributed on many tissues, including embryonic
stem cells, and are considered histo-blood-group antigens.
2. The ABO system contains four major ABO phenotypes: A, B, 0, and AB. The four phenotypes are
determined by the presence or absence of two antigens (A and B) on red cells. An inverse reciprocal
relationship exists between the presence of A and B antigens on red cells and the presence of anti-A, anti-B, or
both, in sera.
3. ABO grouping requires both antigen typing of red cells for A and B antigen (red cell grouping or forward
type) and screening of serum or plasma for the presence of anti-A and anti-B isoagglutinins (serum grouping or
reverse type).
 4. H antigen is ubiquitously expressed on all red cells, except the rare Bombay phenotype.
5. H antigen is the precursor to both A and B antigens; thus, the amount of H antigen on red cells depends
on the person’s ABO group. H antigen is highly expressed on group 0 red cells because group 0 persons lack a
functional ABO gene. In group A and B persons, the amount of H antigen is considerably less because H is
converted to the A and B antigens, respectively.
6. Lewis antigens are not synthesized by red cells but are passively adsorbed onto red cell membranes from
a pool of soluble Lewis glycolipid present in plasma.
7. The three common Lewis phenotypes indicate the presence or absence of Lewis and secretor enzymes.
8. With increasing age, there is a gradual increase in I antigen accompanied by a reciprocal decrease in i
antigen. Most children develop an adult 1+ phenotype by age 2.
9. Autoanti-I and autoanti-i are pathologically significant in cold agglutinin syndrome and mixed-type
autoimmune hemolytic anemia.
10. More than 99% of donors have the (Pj+) or P2 (Pr) phenotype. Both phenotypes synthesize Pk and P
antigens and differ only in the expression of the P, antigen.
REFERENCES
1. Lowe IB, Marth JD. A genetic approach to mammalian glycan function. Annu Rev Biochem
2003;72:643-91.
2. Marionneau S, Cailleau-Thomas A, Rocher I, et al. ABH and Lewis histo-blood group antigens: A model
for the meaning of oligosaccharide diversity in the face of a changing world. Biochimie 2001;83:565-73.
3. Molne I, Bjorquist R Andersson K, et al. Blood group ABO antigen expression in human embryonic stem
cells and in differentiated hepa
tocyte- and cardiomyocyte-like cells. Transplantation 2008;86:1407-13.
4. Gang El, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone
marrow. Blood 2007; 109:1743-51.
5. Hirvonen T, Suila H, Kotovuori A, et al. The i blood group antigen as a marker for umbilical cord blood-
derived mesenchymal stem cells. Stem Cells Develop 2012;21:455-64.
6. Anstee JD. The relationship between blood groups and disease. Blood 2010;115:4635-43.

314
AABB TECHNICAL MANUAL
 7. Storry JR, Olsson ML. The ABO blood group system revisited: A review and update.
Immunohematology 2009;25:48-59.
8. Springer GF. Blood-group and Forssman antigenic determinants shared between microbes and
mammalian cells. Prog Allergy 1971;15:977.
9. Daniel-Johnson J, Leitman S, Klein H, et al. Probiotic-associated high-titer anti-B in a group A platelet
donor as a cause of severe hemolytic transfusion reactions. Transfusion 2009; 49:1845-9.
10. Daniels G. Human blood groups. 3rd ed. Oxford: Wiley-Blackwell, 2013.
11. Clausen H, Levery SB, Nudelman E, et al. Repetitive A epitope (Type 3 chain A) defined by group Aj-
specific monoclonal antibody TH-1: Chemical basis of qualitative Aj andA2 distinction. Proc Natl Acad Sci U
S A 1985;82:1199203.
12. Svensson L, Rydberg L, Hellberg A, et al. Novel glycolipid variations revealed by monoclonal antibody
immunochemical analysis of weak ABO subgroups of A. Vox Sang 2005;89:27-38.
13. Klein HG, Anstee DJ. ABO, H, LE, P1PK, GLOB, I, and FORS blood group systems. In: Mollison’s
blood transfusion in clinical medicine. 12th ed. Oxford: Wiley-Blackwell, 2014:118-66.
14. Cooling L. Polylactosamines, there more than meets the “Ii”: A review of the I system.
Immunohematology 2010;26:133-55.
15. Auf Der Maur C, Hodel M, Nydegger UE, Rieben R. Age dependency of ABO histo-blood group
antibodies: Reexamination of an old dogma. Transfusion 1993;33:915-18.
16. Mazda T, Yabe R, NaThalang O, et al. Differences in ABO antibody levels among blood donors: A
comparison between past and present Japanese, Laotian, and Thai populations. Immunohematology
2007;23:38-41.
17. Koda Y, Soejima M, Kimura H. Changing transcription start sites in H-type a(l,2)fucosyltransferase
gene (FUT1) during differentiation of the human erythroid lineage. Eur J Biochem 1998;256:379-87.
18. Reid ME, Lomas-Francis C. The blood group antigen factsbook. 2nd ed. San Diego, CA: Academic
Press, 2004.
19. Wagner FF, Blasczyk R, Seltsam A. Nondeletional ABO*0 alleles frequently cause blood group typing
problems. Transfusion 2005;45: 1331-4.
20. Yazer MH, Hult AK, Hellberg A, et al. Investigation into A antigen expression on 02 heterozy
gous group O-labeled red blood cell units. Transfusion 2008;48:1650-7.
21. Beck ML, Yates AD, Hardman J, Kowalski MA. Identification of a subset of group B donors reactive
with monoclonal anti-A reagent. Am J Clin Pathol 1989;92:625-9.
22. Garratty G, Arndt P, Co A, et al. Fatal hemolytic transfusion reaction resulting from ABO mistyping of a
patient with acquired B antigen detectable only by some monoclonal anti-B reagents. Transfusion 1996;36:351-
7.
23. Okubo Y, Seno T, Tanaka M, et al. Conversion of group A red cells by deacetylation to ones that react
with monoclonal antibodies specific for the acquired B phenotype (letter). Transfusion 1994;34:456.
24. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific
Publications, 1998.
25. Combs MR. Lewis blood group system review. Immunohematology 2009;25:112-18.
26. Cooling LW, Sitwala K, Dake LR, et al. ABO typing discrepancies in children requiring longterm
nutritional support (abstract). Transfusion 2007;47(3S): 10A.
27. Shastry S, Bhat SS, Singh K. A rare case of missing antibody due to anti-snake venom. Transfusion
2009;49:2777-8.
28. Larson G, Svensson L, Hynsjo L, et al. Typing for the human Lewis blood group system by quantitative
fluorescence-activated flow cytometry: Large differences in antigen presentation on erythrocytes between A1(
A2, B, O phenotypes. Vox Sang 1999;77:227-36.
29. Hosoi E, Hirose M, Hamano S. Expression levels of H-type a(l,2)-fucosyltransferase gene and histo-
blood group ABO gene corresponding to hematopoietic cell differentiation. Transfusion 2003;43:65-71.
30. Cooling L. Carbohydrate blood group antigens and collections. In: Petrides M, Stack G, Cooling L,
Maes L, eds. Practical guide to transfusion medicine. 2nd ed. Bethesda, MD: AABB Press, 2007:59-91.
31. Lindstrom K, Breimer ME, Jovall P-A, et al. Non-acid glycosphingolipid expression in plasma of an A2
Le(a-b+) secretor human individual: Identification of an ALeb heptaglycosylceramide as major blood group
component. J Biochem 1992;111:337-45.
 32. Navenot JM, Muller JY, Blanchard D. Expression of blood group i antigen and fetal hemoglobin in
paroxysmal nocturnal hemoglobinuria. Transfusion 1997;37:291-7.
CHAPTER 12 ABO, H, and Lewis Blood Groups
315
33. Cooling L, Downs T. Immunohematology. In: McPherson RA, Pincus MR, eds. Henry’s clinical
diagnosis and management by laboratory methods. 22nd ed. Philadelphia: Saunders, 2007:618-68.
34. Thuresson B, Westman JS, Olsson ML. Identification of a novel A4GALT1 exon reveals the genetic
basis of the P1/P2 histo-blood groups. Blood 2011;117:678-87.
35. Duk M, Singh S, Reinhold VN, et al. Structures of unique globoside elongation products present in
erythrocytes with a rare NOR phenotype. Glycobiology 2007;17:304-12.
36. Yang Z, Bergstrom J, Karlsson K-A. Glycoproteins with Gala4Gal are absent from human
erythrocyte membranes, indicating that glycolipids are the sole carriers of blood group P activities. J Biol
Chem 1994;269:14620-4.
37. Westman JS, Storry JR, Hult AD, et al. Identification of the genetic basis of PX2, a recently reported
glycolipid blood group antigen (abstract). Transfusion 2013; 53 (S): 15A.
38. Hellberg A, Ringressi A, Yahalom V et al. Genetic heterogeneity at the glycosyltransferase loci
underlying the GLOB blood group system and collection. Br J Haematol 2004;125:528-36.
39. Lund N, Olsson ML, Ramkumar S, et al. The human P(k) histo-blood group antigen provides protection
against HIV-1 infection. Blood 2009;133:4980-91.
 Chapter 13
The Rh System
Gregory A. Denomme, PhD, FCSMLS(D), and Connie M. Westhoff, PhD, SBB

is the most complex of the 34 huKSl man blood group systems. The clinical importance of Rh is
underscored by the D antigen, which is considered to be the most immunogenic of all antigens. Anti-D is
present in more than 50% of Rh-negative recipients transfused with Rh-positive blood, and the prevalence of
alloimmunization in Rh-negative females with an Rh-positive fetus was a significant risk prior to the
availability of Rh Immune Globulin (RhIG) prophylaxis. A historical account of the Rh system and its
confusion with the LW system has been described by Rosenfeld.1 It was in 1939, when Levine and Stetson
made key observations that the serum of a pregnant woman agglutinated 80% of samples within her ABO blood
group, that a new blood group antigen was suspected to exist. These authors also proposed that “products of the
disintegrating fetus” and an adverse transfusion reaction in the mother to a blood transfusion from her husband
were related.2
Today, the terms “Rh positive” and “Rh negative” refer to the presence or absence, respectively, of the D
antigen. Four additional Rh
antigens—antithetical C and c, and E and e— are the system’s principal antigens. The antigens were named
by Fisher using the next available letters of the alphabet. Thus, the five principal Rh antigens—D, C, c, E, and e
—are responsible for the majority of clinically significant Rh antibodies among the 61 Rh antigens that have
been characterized (Table 13-1).
A true success story in transfusion medicine therapy, the development of RhIG prophylaxis arose from the
observation that ABO incompatibility between a mother and fetus had a partial protective effect against
immunization to D.4 The administration of IgG anti-D obtained from human plasma was effective in the
prevention of hemolytic disease of the fetus and newborn (HDFN).5 Beginning in the mid-1960s,
alloimmunization to D in pregnancy was reduced to about 1 in 2000 live births.
CHARACTERIZATION OF RH
Rh proteins, unlike most membrane proteins, are neither glycosylated nor phosphorylated.6,7 The use of
immunoprecipitation followed by
Gregory A. Denomme, PhD, FCSMLS(D), Director of Immunohematology and Transfusion Services,
Diagnostic Laboratories, BloodCenter of Wisconsin, Milwaukee, Wisconsin, and Connie M. Westhoff, PhD,
SBB, Director of Immunohematology, Genomics, and Rare Blood, New York Blood Center, New York, New
York G. Denomme disclosed no conflicts of interest. C. Westhoff has disclosed financial relationships with
Novartis, BioArray, and Grifols.
317

318
AABB TECHNICAL MANUAL
TABLE 13-1. Terminology for Rh Antigens
ISBT Antigen or Numerical Antigen or
Prevalence Prevalence
Designation Symbol(s) Designation Symbol(s)
85% Whites 1% Blacks RN with
RH1 D RH32#
92% Blacks DBT
68% Whites R0Har,
RH2 C RH33 0.01% Germans
27% Blacks DHAR
29% Whites
RH3 E RH34** HrB High
22% Blacks
80% Whites
RH4 c RH35 Low
96% Blacks
RH5 e 98% RH36 Bea Low
65% Whites Low (several D/CE
RH6 ce orf RH37 Evans
92% Blacks or CE/D hybrids)
RH7 Ce or rhj 68% Whites RH39 C-like High
 27% Blacks
Low, 2%
RH8 gw RH40 Tar Low (DVII)
Whites
Low, 1.8%
RH9 Cx RH41 Ce-like 70% Whites
Finns
RH10 V 30% Blacks RH42 Ces 2% Blacks
RH11 Low RH43 Crawford 0.1% Blacks
84% Whites
RH12* G RH44 Nou High
92% Blacks
RH17+ Hr0 High RH45 Riv Low
RH18* Hr High RH46 Sec High
RH19§ hrs 98% RH47 Dav High
RH20 VS 32% Blacks RH48 JAL Low
RH21 CG 68% Whites RH49n STEM 6% Blacks
<1% (DCE,
RH22 CE RH50 FPTT Low (DFR, R0Har)
CE)
RH23" Dw Low (DVa) RH51 MAR High
RH26 c-like High (most c+) RH52" BARC Low (DVI)
28% Whites
RH27 cE RH53 JAHK Low
22% Blacks
Low (Dili3, DOL,
RH28 hrH Low RH5411 DAK
RN)
RH29'1 total Rh 100% RH55 LOCR Low
RH3011 Goa Low RH56 CENR Low
High (antithetical to
RH31§ hrB High RH57 CEST
JAL)
 319
TABLE 13-1. Terminology for Rh Antigens (Continued)
ISBT Antigen or Numerical Antigen or
Designation Symbol(s) Prevalence Designation Symbol(s) Prevalence
RH58 CEL0 High (antithetical to
RH60 PARG Low
Crawford)
RH59 CEAG High RH61 CEVF
Note: RH13 through RH16, RH24, RH25, and RH38 are obsolete.
‘Present on red cells expressing C or D antigen.
Antibody made by individuals with D-deletion phenotypes D-- Dc-, and DC*-.
Antibody made by individuals with altered e and/or D phenotypes prevalent in groups of African ethnicity.
Absent from red cells with DcE/DcE (R2R2) phenotype or variant e found in groups of African ethnicity, 'low-
prevalence antigen associated with the partial D indicated.
Antibody made by individuals with Rhnull red cells.
'Low-prevalence antigen expressed by red cells with RN or the partial DBT antigen.3
“Antibody made by individuals with altered C, E, and/or D phenotypes prevalent in groups of African
ethnicity.
^Associated with 65% of hrs— Hr— and 30% of hrB- HrB- red cells.
sodium dodecylsulfate polyacrylamide gel electrophoresis led to the discovery that Rh proteins have a
molecular weight of 30,000 to 32,000 kDa.8'10 N-terminal amino acid sequencing of Rh was accomplished in
the late 1980s.11 The findings led to the cloning of the RHCE gene by Cartron12 and colleagues in 1990 and of
RHD in 1992.13,14 The different RHCE alleles responsible for the Core and E or e antigens were determined in
1994.15
TERMINOLOGY
Early Rh nomenclature reflects the differences in opinion concerning the number of genes that encode DCE
antigens. The Fisher-Race terminology was based on the premise that three closely linked genes, C/c, E/e, and
D, were responsible. In contrast, the Wiener nomenclature (Rh-Hr) was based on the belief that a single gene
encoded several blood group factors. There are actually two genes, RHD and RHCE, as proposed by Tippett.16
The Fisher-Race CDE terminology is often preferred for written communication, but a modified version of
Wiener’s nomenclature makes it possible to identify the Rh antigens present on one chromosome, that is, a
haplo
type, using a single term (Table 13-2). In the modified Wiener’s nomenclature, “R” indicates that D is
present, and a subscripted number or letter indicates the C/c and E/e antigens: Rj for Ce, R2 for cE, R0 for ce,
and Rz for CE. In addition, “r” indicates haplotypes that lack D, and the C/c and E/e antigens are indicated
using superscripted symbols: r' for Ce, r" for cE, and ry for CE (Table 13-2).
The International Society of Blood Transfusion (ISBT) Working Party on Terminology for Red Cell Surface
Antigens adopted six-digit numbers to indicate red cell antigens. The first three numbers represent the system,
and the remaining three digits refer to the antigenic specificity; the Rh system was assigned Number 004. In
2008, the ISBT committee recognized the antigens of the Rh-associated glycoprotein (RHAG) as a new blood
group system (Number 30).17
RH GENES AND Rh PROTEINS
Two genes (RHD and RHCE) are closely linked on chromosome 1 and encode 416 amino acids. One gene
encodes the D antigen, and the other encodes CE antigens in four combinations (ce, cE, Ce, or CE) [Fig 13-
1(A)]. Each

320
AABB TECHNICAL MANUAL
TABLE 13-2. Prevalence of the Principal Rh Haplotypes
Fisher-Race Modified Prevalence (%)
 Haplotype Wiener
Haplotype
White Black Asian
Rh positive
DCe R, 42 17 70
DcE r2 14 11 21
Dee Ro 4 44 3
DCE Rz <0.01 <0.01 1
Rh negative
ce r 37 26 3
Ce r' 2 2 2
cE r" 1 <0.01 <0.01
CE rv <0.01 <0.01 <0.01
A. RH genes
Rh positive
RHD RHCE

D antigen C/c and E/e antigens

Rh negative
X Deleted X

B. Rh proteins
A
RhD
T7
A
I
Kj
A
V.
C/c
E/e
RhCE

FIGURE 13-1. (A) RHD and RHCE genes. The inverted gene orientation, the antigens they encode, and the
deletion of RHD that results in the D-negative red cell phenotype are shown. (B) Predicted 12-transmembrane
domain model of the RhD and RhCE proteins. The amino acid differences between RhD and RhCE are shown
as grey circles. The zigzag lines represent the location of possible palmitoylation sites. Positions 103 and 226 in
RhCE critical for C or c and E or e expression are indicated as open circles.18

321
gene has 10 exons, and the two genes share 97% identity. The two proteins created by these genes differ by
32 to 35 amino acids [Fig 13-1 (B), shown as circles on RhD], depending on whether D is compared to C or c.
The last decade has witnessed the development of an abundance of information on the genetic diversity of
the RH locus, and the antigens identified by molecular testing have far exceeded the number of antigens
identified by serology. More than 275 RHD and 50 RHCE alleles have been documented. A directory of RHD
alleles is maintained on a Rhesus-database,19 and RHCE and RHD alleles are listed on the National Center for
Biotechnology Information human blood group mutation website.19,20 The ISBT Working Party on Red Cell
Immunogenetics and Blood Group Terminology maintains, names, and catalogs new alleles.21
 Most D-negative (Rh-negative) phenotypes are the result of complete deletion of the RHD gene [Fig 13-
1(A)]. These phenotypes provide the immunological rationale for why the transfusion of Rh-positive blood to a
Dnegative individual often results in production of anti-D. The immunogenicity of a protein correlates with the
degree of foreignness to the host, and the large number of amino acid differences in D explains why exposure
can result in a potent immune response.
RHCE [found in all but rare D- - individuals, where the dashes represent missing antigens] encodes both C/c
and E/e antigens on a single protein. C and c antigens differ by four amino acids, but only the serine to proline
at position 103 is predicted to be extracellular [Fig 13-1(B)], The E and e antigens differ by one amino acid, a
proline to alanine at amino acid position 226, located on the fourth extracellular loop of the protein. The RH
genes and proteins shown in Fig 13-1 (B) are typical of the majority of individuals. Commercial antibody
reagents are available to detect the expression of the principal Rh antigens—D, C, c, E, and e (Table 13-3).
The five principal antigens are responsible for the majority of Rh incompatibilities, although the Rh system
is more complex, with 61 antigens identified to date (Table 13-1).
New antigens may result from single nucleotide polymorphisms (SNPs) to major gene rearrangements. For
example, the genetic exchanges between RHD and RHCE can create hybrid proteins that express an RhD
protein with a portion of RhCE, or vice versa. Rearrangements are common and thought to be facilitated by the
inverted orientation of the RH genes [Fig 13-1(A)].18 The structure promotes hairpin loop formation and
subsequent genetic exchange via template conversion; one member acts as a donor template during replication
but remains unchanged in the process. The donated region can span several base pairs, single exons, or multiple
exons.
ANTIGENS
Routine donor and patient typing tests only for D. Testing for other common Rh antigens is performed
primarily to resolve or confirm antibody identification. Exceptions include patients receiving chronic
transfusion, such as in some sickle cell disease (SCD) programs, to provide antigen-matched donor units to
minimize alloimmunization.
Rh Phenotypes
Testing of red cells with the five principal Rh antisera has been used to predict the RH genotype (Table 13-
3). Some haplotypes are more common in certain ethnic groups. The frequencies of the predicted RH genotypes
are uncertain (eg, the frequencies of R0R0 vs R0r in persons of African ancestry), and frequencies are more
uncertain in people of mixed ethnicity. Serologic testing cannot determine whether red cells are from a
homozygous (D/D) or heterozygous (D/—) person because anti-D seldom shows any difference in reactivity
between red cells with a single or a double dose of D antigen. RHD zygosity can be determined by DNA
molecular testing for the presence of a RHD deletion or an inactive RHD.18
The Rh haplotype influences the level of D antigen expression. Less D is expressed in the presence of C, a
phenomenon called the “Ceppellini effect.”22 Red cells from a DCe/DCe (RjRj) individual express significantly
fewer D
322
AABB TECHNICAL MANUAL
TABLE Results of Tests with Five
13-3. Principal Rh Antisera with Phenotype
Genotype and Predicted RH
Antisera Predicted Alternative
Anti-D Anti-e
Anti-c Genotype Genotype
Anti-C Anti-E Phenotype
Rh
positive*
+ D, C, c,
++ 0 + Rir R1R0
e
DCe/ce DCe/Dce
Ro r'
Dce/Ce
+ D, C,
++ 0 0 RiRt Ri r'
G
 DCe/DCe DCe/Ce
+ D, C, c,
++ + + R1R2 R,r"
E, e
DCe/DcE DCe/cE
Rz r'
DcE/Ce
Rzr
DCE/ce
Ro Rz
Dce/DCE
+0 0 + + D, c, e Ro r Ro Ro
Dce/ce Dce/Dce
+ D, c, E,
+0 + + R2 r R2 Ro
e
DcE/ce DcE/Dce
Ror"
Dce/cE
+0 + + 0 D,c, E Rz R2 Rz r"
DcE/DcE DcE/cE
+ D,C, E,
++ + 0 RiRz Rzr'
e
DCe/DCE DCE/Ce
0 D, C, c,
++ + + R2 Rz Rzr"
E
DcE/DCE DCE/cE
++ + 0 0 D,C, E RzRz Rzry
DCE/DCE DCE/CE

323
TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH Genotype
(Continued)
Antisera Predicted Alternative
Anti-D Anti-C Anti-c Anti-e Phenotype
Anti-E Genotype Genotype
Rh negative1
rr
00 0 + + c, e
ce/ce
 0+ 0 + + 0, c, e r' r Ce/ce
00 + + + c, E, e r" r cE/ce
0+ + + + C, c, E, e r' r Ce/cE
*Rare genotypes (R°rv, flVy, and R2ry) not shown (prevalence of <0.01%). fRare genotypes (rry, r'ry, r"ry,
and ryry) not shown (prevalence of <0.01%).
antigen sites than red cells from a DcE/DcE (R,R2) individual. Therefore, it is important to choose a
consistent haplotype when titrating anti-D in the antenatal setting because significantly different titers can be
obtained.
D Antigen
The D antigen is composed of numerous epitopes (designated by “epD”) that were originally defined by
antibodies from D-positive people who made anti-D. Subsequent studies with monoclonal antibodies defined 30
or more epitopes designated as “epDl” to “epD9.”23 Each epitope has additional subdivisions (eg, epD6.1). D
epitopes are highly conformational and consist of more than simple linear amino acid residues. Amino acid
changes in intracellular regions of the protein may alter D epitopes.
D-Positive (Rh-Positive) Phenotypes
Most D-positive red cell phenotypes have a conventional RhD protein [Fig 13- 1(B)]. However, more than
275 different RHD alleles that
encode proteins with amino acid changes have been reported. These alleles can cause numerous variations
in the expression of D, and red cells with some form of altered D expression are encountered in routine
transfusion practice. An estimated 1% to 2% of individuals of European ethnicity carry RHD alleles that encode
altered D antigens, and the incidence in individuals of African ethnicity is higher. Altered D is organized into
four groups: weak D, partial D (including category D), Del, and nonfunctional RHD.24'27
WEAK D. Traditionally, weak D red cells were defined as having a reduced amount of D antigen (formerly
called “Du”) that required an indirect antiglobulin test (IAT) for detection. However, the number of samples
identified as being weak D depended on the typing reagent and method used, which have changed over the
years. In 1999, Wagner and Flegel proposed a system to classify altered D red cells on the basis of their
nucleotide substitutions (reviewed in Flegel and Denomme28). Weak D types are the result of an SNP that
encodes a single amino acid change predicted to be

324
AABB TECHNICAL MANUAL
located in the intracellular or transmembrane region of the protein, rather than on the outer surface of the red
cell (Fig 13-2). The amino acid changes may affect the insertion of the protein in the membrane and, thus,
reflect the reduced number of D antigen sites on the red cells.
Uniquely different SNPs cause weak D Types 1-84.19 The most common is weak D Type 1, which has a
valine-to-glycine amino acid substitution at position 270. Types 1, 2, and 3 represent approximately 90% of the
weak D types in persons of European ethnicity.25 Weak D types can be further weakened when C is present in
trans to a weak D type (eg, r' in trans with weak D Type 2).
PARTIAL D. Red cells with “category D” have historically been classified by epitope studies. Category D
individuals type as D positive, but can make anti-D when they are exposed to conventional D antigen. Category
D phenotypes are now classified as partial D. The majority of partial D phenotypes are due to hybrid genes in
which portions of RHD are replaced by corresponding portions of RHCE (Fig 13-3). The novel sequences of
the hybrid
protein resulting from regions of RhD joined to RhCE can result in the loss of D epitopes and also generate
new antigens. For example, DVI red cells carry the BARC antigen. A few partial D phenotypes are the result of
multiple nucleotide changes. Some partial D types are detected only by the 1AT. in contrast to weak D types,
partial changes are predicted to be located on the exterior membrane surface (Fig 13-2).29
del. Red cells that express extremely low levels of D antigen that cannot be detected by routine serologic
methods are designated as “D-elution” or Del. Red cells have enough D antigens to adsorb and elute anti-D. Del
cells are found in 10% to 30% of D-negative people of Asian ancestry and result from several different RHD
mutations that severely reduce RhD expression in the membrane. Del cells are much less common in
individuals of European ancestry (0.027%) and carry different nucleotide substitutions than those of Asian
ancestry.26,27 Del red cells can usually be characterized by RHD genotyping and careful adsorption-elution
studies.
Weak D
Partial D
Exterior
Plasma
membrane
Interior

FIGURE 13-2. Structural models of weak D and partial D. The locations of amino acid changes are shown
as solid circles in the plasma membrane or on the interior. Weak D Types 1 and 2 (shown by the arrows) are
found in approximately 80% of people with weak D phenotypes. Partial D types are encoded by single amino
acid changes that are generally present on the exterior of the cell.
325
Conventional
RHD
RHCE
ce
African Ethnicity
RHD negative
D/CE/DQdKJlUh^^hHhOQO
No D antigen, altered C antigen
Partial RHD
diiu ommooooo
L62FA137V N152TT201R F223V
DIVa IZKEHIIhCIHZI^IHrhCIhEhn
Go(a+) L62F A137V N152T D350H
DAR DCOKK}{|}000
T201R F223V I342T
DOL Doomooom
DAK+ M170T F223V
Altered RHCE*ce (hr8-)
[DilHlihl^^ ces
W16C L245V G336C V-VS+
lE-nHZHDiEHDHDHZHZhn ces
W16C L245V V+VS+
(hrs-)
DDDODQDC'D □ ceMO
W16C V223F
ITHDiZHZKniHZHZl-QiZHD ceEK
W16C M238V R263G M267K
[HOOOiDIKQOOOO ceAR
W16C M238V L245V R263G M267K 1306V
 W16C M238V A273V L378V
ceBI
pyi European Ethnicity
Type i QdKH ■ ■ QdhQOQ
Type 2 Q-Q-D ■ ■ ■ IZZUZZHZZHZZIBARC+
Type 4 III Q-EHZhQQ barc+
FIGURE 13-3. RHD and RHCE genes. The 10 exons of RHD and RHCE are depicted as white and grey
boxes, respectively. Also shown are some examples of RHD encoding partial D and of RHCE with mutations
often found in people of African ethnicity. These mutations complicate transfusions in patients with sickle cell
disease.
NONFUNCTIONAL RHD. RHD genes that cannot produce a full-length polypeptide are deemed
nonfunctional and have been given allele designations.21
D EPITOPES ON RHCE (RHCE). Expression of D epitopes by the protein product of the RHCE gene, in
the absence of RHD, further complicates serologic determination of D status. Several Rhce (RHCE) proteins
have D-specific amino acids result in epitopes that are reactive with some monoclonal anti-D. They are more
often found in a specific population. Examples include DHAR, which is found in individuals of German
ancestry, and Crawford (ceCF), found in individuals of African ancestry. These two examples are notable
because the red cells show strong reactivity with some monoclonal reagents but are nonreactive with others,
including polyclonal anti-D reagents
(Table 13-4). D^and Crawford phenotypes can cause D typing discrepancies. Less dramatic are changes in
Rhce [RHCE) proteins that mimic a D-epitope structure encoded by alleles designated as “ceRT" and
“ceSL.”30,31 The red cells are weakly reactive with some, but not all, monoclonal anti-D. Most importantly,
individuals with D"Ali and Crawford lack RhD and can be sensitized to D.32,33
ELEVATED D. Several rare deletion phenotypes, designated as “D- “Dc-,” and “DC"-,”
have an enhanced expression of D antigen and no, weak, or altered C/c and E/e antigens.34 These variants
are the converse of partial D (discussed above) and result from the replacement of portions of RHCE by RHD.
The additional RHD sequences in RHCE result in the additional expression of (hybrid) D along with
TABLE 13-4. Reactivity of FDA-Licensed Anti-D Reagents with Some D Variant Red Cells
326
AABB TECHNICAL MANUAL
 TEnzyme-treated cells.
FDA = Food and Drug Administration; IgM = immune globulin M; IS = immediate spin; AHG = antihuman
globulin; pos = positive; neg = negative.
 327
normal D, which explains the enhanced D expression and reduced or missing C/c and E/e antigens.
D-Negative (Rh-Negative) Phenotype
The D-negative phenotype is most common in people of European ancestry (15-17%), is less common in
people of African ethnicity (3-5%), and is rare in people of Asian ancestry (<0.1%).35 The D-negative
phenotype has arisen multiple times, as evidenced by the different nonfunctional alleles responsible for the lack
of D expression in various ethnic groups.
In most people of European ancestry, the D-negative phenotype results from a deletion of the entire RHD
gene.36 There are exceptions, however, and red cell samples with uncommon haplotypes [r' (Ce) or r" (cE)] are
more likely to carry a nonfunctional RHD. In other ethnic groups, D-negative phenotypes are primarily caused
by inactivating changes in RHD. In individuals of African ancestry, 66% have a 37-bp insertion in RHD,37
which results in a premature stop codon. D-negative phenotypes in people of Asian ancestry result from
mutations in RHD that are most often associated with a Ce (r') haplotype, although 10% to 30% of people of
Asian ancestry who type as D negative are actually Del.26,27
Testing for D
Monoclonal antibody technology introduced in the 1980s freed manufacturers from reliance on human
source material to manufacture anti-D reagents. However, these antibodies are specific for a single D epitope
and do not detect all D-positive red cells. Consequendy, most Food and Drug Administration (FDA)approved
anti-D reagents combine a monoclonal IgM, which causes direct agglutination at room temperature, with a
monoclonal or polyclonal IgG that is reactive by IAT for the determination of weak D. Anti-D for column
agglutination testing is an exception and contains only a monoclonal IgM. Three of the five FDA-licensed
reagents contain unique IgM clones, and these may exhibit different reactivity with red cells that have weak D,
partial D, or D-like epitopes (Table 13-4).
Typing Donors for D
The goal of D typing of Red Blood Cell (RBC) donors, including the identification of units with weak D or
partial D types, is to prevent anti-D immunization of recipients. Therefore, the AABB Standards for Blood
Banks and Transfusion Services requires donor blood to be tested using a method that is designed to detect
weak expression of D. There is no requirement that the typing be done using an IAT. If the test results are
positive, the unit must be labeled as “Rh positive.”38(p31) Most weak D or partial D antigen units are detected
but infrequently, some very weak D red cells are not detected, and Delred cells appear nonreactive with anti-D.
Red cells with weak D antigen are less immunogenic than normal Dpositive red cells, and Del donor units can
stimulate anti-D in some D-negative recipients.39'43 A unit labeled Rh negative must be confirmed by testing
an integrally attached segment before transfusion. Weak D testing is not required. The RhD type of the units
labeled Rh positive do not require confirmatory test
ing 38(p35)
Typing Patients for D
When the D type of a patient is determined, a weak D test is not necessary except to assess the red cells of
an infant whose mother is at risk of D immunization. Today, monoclonal IgM reagents type many samples as
being D positive in direct testing that would have previously been detected only by IAT. As a result, some of the
concerns regarding the unnecessary use of Rh-negative blood and RhIG have been addressed.
DVI is the most common partial D found in people of European ancestry, and anti-D produced by women
with partial DVI has resulted in fatal hemolytic disease.44 Current FDA-licensed monoclonal IgM reagents are
selected to be nonreactive with RBCs from partial DVI in direct tests (Table 13-4). Therefore, performing only
the direct test on red cells from female children and women of childbearing potential avoids the risk of
sensitization by classifying DVI as D negative for transfusion and RhIG prophylaxis. However,
328
AABB TECHNICAL MANUAL
because no IAT is performed, the results of positive rosetting tests (to detect fetomaternal hemorrhage) must
be carefully evaluated; maternal weak D types that are reactive only in the IAT phase have a false-positive
rosette test result.
D Typing Discrepancies
D typing discrepancies should always be investigated and resolved (see “Resolving D Typing
Discrepancies” below). An Rh-negative blood transfusion is an appropriate option for patients needing
immediate transfusion, but a thorough clerical and serologic investigation should be performed. RHD
genotyping is also useful to resolve D typing discrepancies.45
Because donor centers test for weak D, a donor who is correctly classified as Rh positive may be classified
as Rh negative as a recipient. This discrepancy should not be considered problematic but, rather, should be
communicated to the patient and health-care staff and be noted in the patient’s medical record.
Clinical Considerations
The long history of transfusing patients who have weak D red cells with D-positive RBCs suggests that
weak D Types 1, 2, and 3, which are present in the majority of people of European ancestry with weak D, are
unlikely to make anti-D. People with these weak D types can therefore safely receive D-positive blood. The less
common weak D Types 11 and 15 have been reported to make anti-D, suggesting that they have altered D
epitopes.3 Empirical data are needed to determine the risk of production of anti-D in people with other weak D
types.
Unfortunately, serologic reagents cannot typically be used to distinguish people with partial D that is
reactive only with enhanced methods and techniques from D-positive individuals. Many partial D red cells (eg,
Dllla, the most common partial D type in people of African ancestry) type as strongly D positive in direct tests
and are recognized only after the patients produce anti-D.
Policies regarding D typing procedures and selection of blood components for trans
fusion should be based on the patient population, risk of immunization to D, and limited supply of D-
negative blood components. These policies should address what should be done when a partial D type is found
before the patient makes anti-D. Anti-D is a clinically significant antibody, and preventing immunization in
females of childbearing potential is important to avoid the complications of HDFN. For other patients, the
complications of anti-D are less serious, and the decision to transfuse Rh-positive or Rh-negative blood should
take into consideration dependence on D-negative blood transfusions for future bleeding episodes and a
possible increased risk of multiple blood group antibodies in addition to anti-D.46
Not all D-negative patients make anti-D when they are exposed to D-positive RBCs. The incidence in D-
negative hospitalized patients switched to D-positive blood components is much lower, than anticipated.47
AABB Standards requires that transfusion services must have policies that address the administration of D-
positive red cells to D-negative patients and the use of RhIG, which is a human blood product that is not
entirely without risk.38(p37)
G Antigen
The G antigen is found on red cells possessing C or D and maps to the 103Ser residue on RhD, RhCe, and
RhCE (Fig 13-1). Antibodies to G appear as anti-D plus anti-C and cannot be separated. However, the antibody
can be adsorbed by either D-C+ or D+C- red cells. The presence of anti-G can explain why a D-negative person
who was transfused with D- (C+) blood or a Dnegative woman who delivered a D- (C+) child and subsequently
appeared to have made anti-D. Anti-D, -C, and -G can be distinguished by adsorption and elution studies.48
These analyses are not usually necessary in the pretransfusion setting. However, it is important to provide RhIG
prophylaxis to pregnant women who have anti-G only.
C/c and E/e Antigens
The common RHCE alleles encode the principal C or c and E or e antigens. However, more
329
than 50 different RHCE alleles are known, and many are associated with altered or weak expression of the
principal antigens and, in some cases, loss of high-prevalence antigens.37 Partial C and many partial e antigens
are well recognized, and the majority are reported in individuals of African ethnicity.
Compound Antigens (ce, Ce, cE, and CE)
Compound antigens define epitopes that depend on conformational changes that result from amino acids
associated with both C/c and E/e. These antigens were previously referred to as “cis products” to indicate that
the antigens were expressed from the same haplotype. However, it is now known that these antigens are
expressed on a single protein. These antigens are shown in Table 13-5 and include ce or f, Ce or rhi( cE or
Rh27, and the uncommon CEorRh22.
Altered or Variant C and e Antigens
Nucleotide changes in RHCE result in quantitative and qualitative changes in C/c or E/e antigen expression;
altered C and altered e are encountered most frequently. In persons of European ancestry, altered C is associated
with amino acid changes on the first extracellular loop of RhCe and the expression of Cw (Gln41Arg) or Cx
(Ala36Thr) antigens. Altered C is also associated with changes that result in the expression of the novel
antigens JAHK (Serl22Leu) and JAL (Argll4Trp). These red
TABLE 13-5. Compound Rh Antigens on Rh Proteins
Compound
Antigen Rh Protein Present on Red Cells with These Haplotypes
Designation
ce orf Rhce Dee (R0) or ce (r)
Ce or rhj, Rh7 RhCe DCe (R,) orCe (r)
cE or Rh27 RhcE DcE (R2) orcE (r")
CE or Rh22 RhCE DCE (Rz) or CE (ry)
cells type as C positive, but these patients can make apparent anti-C or anti-Ce (rhj) when they are
stimulated.
In individuals of African ancestry, altered C expression most often results from the inheritance of a
RHDIIIa-CE(4-7)-D hybrid and less often of a RHD-CE(4-7)-D hybrid.37 These hybrids do not encode D
antigen, but they do encode C antigen on a hybrid background that differs from the normal background (Fig 13-
3). The gene has an incidence of approximately 20% in people of African ancestry. It is inherited with an RHCE
allele designated as “RHCE*ces” that encodes altered e antigen and a V-VS+ phenotype.50 The expressed
product of the hybrid RHDIIIa-CE(4-7)-D linked to RHCE*ces is referred to as the “(C) ces” or “r's” haplotype.
Red cells with the r's haplotype often type as strongly C-positive with monoclonal reagents, and the presence of
the altered C is undetected.
Patients with these types of red cells frequently make alloantibodies with C-like, and occasionally e-like,
specificities that may appear to be autoantibodies. The red cells may also lack the high-prevalence hrB antigen
(hrB-).51,52 Altered or partial e expression is associated with other RHCE*ce alleles.37,53 These alleles are
found primarily in people of African ethnicity; some examples are shown in Fig 133. Some people lack the
high-prevalence hrs antigen (hrs—). Anti-hrs is a clinically significant antibody and has caused transfusion
fatalities.53 Not all red cells designated as hrB- or hrs- by serologic testing are compatible with antibodies
produced by patients with altered or partial e expression.
 An additional complication is that altered RHCE*ce is often inherited with a partial RHD (eg, Dill, DAU, or
DAR).54 As discussed above, patients with partial D red cells are at risk of producing anti-D.
Clinical Considerations
It has long been recognized that alloimmunization represents a significant problem in patients with SCD
because 25% to 30% or more of those who are chronically transfused develop red cell antibodies. To address
the problem,

330
AABB TECHNICAL MANUAL
many programs determine the pretransfusion red cell phenotype in patients with SCD and transfuse RBCs
that are antigen matched for D, C, E, and K because these antigens are considered to be the most immunogenic.
In addition, some programs attempt to supply RBCs from donors of African ancestry whenever possible.
Although there is no consensus on the need to perform red cell phenotype matching for all patients with SCD,55
transfusion programs that transfuse RBCs that are antigen matched for D, C, E, and K have reduced the
incidence of alloantibody production.56 Unfortunately, despite matching for D, C/c, and E/e, some patients
become sensitized because they express Rh variants.57
RH GENOTYPING
RH genotyping is a powerful adjunct to serologic testing for the typing of transfused patients, RHD zygosity
determination, noninvasive fetal D typing, determination of D status, and identification of antigen-matched
blood for patients with SCD.
Typing Multitransfused Patients
In patients receiving chronic or massive transfusions, the presence of donor red cells in the peripheral blood
often makes red cell phenotyping by agglutination difficult or inaccurate. Genotyping overcomes this limitation
because blood grouping can be determined with DNA prepared from a blood sample, even if the sample was
collected after transfusion.58
RHD Zygosity Testing
RHD zygosity can be determined by assaying RHD dosage or confirming the presence of a hybrid rhesus
box.18,59 In prenatal practice, paternal RHD zygosity testing is important to predict fetal D status when the
mother has anti-D. Care must be taken in the interpretation of testing results using either approach. Several
different genetic events cause an apparent D-negative haplotype, and multiple targets must be tested to
accurately determine zygosity.59,60
Fetal RHD Typing
To determine the D antigen status of a fetus, fetal DNA can be isolated from cells obtained by amniocentesis
or chorionic villus sampling. An alternative, noninvasive approach is to test the maternal plasma, which
contains cell-free, fetal-derived DNA beyond 5 weeks’ gestation.61,62 In the future, determination of fetal
RHD status using this noninvasive procedure could become routine in clinical practice to eliminate the
unnecessary administration of antepartum RhIG to women who are carrying a D-negative fetus.62
Confirming D Status
RHD genotyping is useful to distinguish partial D from weak D or to resolve serologic D typing
discrepancies. Although patients with an uncertain D status can be treated as D negative for transfusion and
RhIG administration, this approach may be unsatisfactory for females of childbearing potential who face
unnecessary RhIG injections and puts a strain on the limited Rh-negative blood supply. RHD genotyping allows
informed decisions to be made in this setting (see “Weak D Types” above).
For donors, D typing discrepancies must be resolved because errors in determining their D status may be
reportable to the FDA and result in the recall of blood components. D-negative, first-time donors are screened
for RHD to detect red cells with very weak D in some European centers.3,63,64
RH Genotyping for Patients with SCD
Currently, extensive RH genotyping is time consuming and is used to find compatible donors in the
American Rare Donor Program for patients with antibodies to high-prevalence Rh antigens.52 The future
availability of highthroughput RH genotyping platforms enables donors to be identified by genotyping.
However, RH genotype matching patients with SCD who have rare Rh variant types may not be possible for
chronic prophylactic transfusions.65 These patients may be candidates for stem cell transplantation.66
331
 rhnull syndrome and rhag
BLOOD GROUP SYSTEM
Erythrocytes express a third Rh-protein, RhAG. RhAG shares 38% of its identity with RhD/ RhCE, has the
same topology in the membrane, and is encoded by a single gene on chromosome 6. RhAG associates with the
Rh blood group proteins in the membrane to form an Rh-core complex. Three red cell antigens resulting from
single amino acid substitutions form the RHAG blood group system: Duclos is RHAG1,01(a) is RHAG2, and
DSLK is RHAG3 and RHAG4.87
Red cells lacking all Rh antigens are designated as “Rhnull.” Although uncommon, the phenotype most
often results from nucleotide changes in RHAG, known as a “regulator” type Rhnull, indicating that the RhAG
protein plays a critical role in trafficking RhCE and RhD to the membrane. Less often, Rhnull individuals have
RHCE nucleotide changes along with the common deletion of RHD, and these individuals are called
“amorph.”49
Rhnull red cells are stomatocytic and associated with mild anemia, suggesting that the Rh proteins have an
important structural role in the erythrocyte membrane. The Rh complex is linked to the membrane skeleton
through CD47-protein 4.2 interactions and Rh/RhAGankryin interactions.68,69
RH ANTIBODIES
Most Rh antibodies are IgG, although some sera may have an IgM component. Typically, Rh antibodies do
not activate complement, although rare exceptions have been reported. As a result, in transfusion reactions
involving Rh antibodies, hemolysis is primarily extravascular rather than intravascular.
Most Rh antibodies have the potential to cause clinically significant HDFN and transfusion reactions. Anti-
c, which is clinically the most important Rh antibody after anti-D, may cause severe HDFN. Anti-C, -E, and -e
do not often cause HDFN, and when they do, it is usually mild. The reactivity of Rh antibodies is enhanced by
enzyme treatment of red cells, and
most Rh antibodies are optimally reactive at 37 C.
Concomitant Rh Antibodies
Some Rh antibodies are often found together. For example, a DCe/DCe (R,R,) patient with anti-E most
certainly has been exposed to the c antigen as well. Anti-c may be present in addition to anti-E but the anti-c
may be weak and undetectable at the time of testing. When seemingly compatible E-negative blood is
transfused, it is most likely to be c positive and may elicit an immediate or delayed transfusion reaction.
Therefore, some experts advocate for avoiding the transfusion of c-positive blood in this situation. In contrast,
testing for anti-E in serum containing anti-c is not warranted because the patient has probably been exposed to c
without being exposed to E. In addition, the vast majority of c-negative donor blood is E negative (see Table 13-
3).
Antibodies to High-Prevalence Rh Antigens
Alloantibodies to high-prevalence Rh antigens include anti-Rh29, made by some Rhnull individuals who
lack Rh antigens, and others (antihrs, -hrB, -Hr®, and -Hr) that are most often encountered in transfused
patients with SCD.
TECHNICAL CONSIDERATIONS FOR RH TYPING
High-Protein Reagents and Controls
Some Rh reagents for use in slide, rapid tube, or microplate tests contain high concentrations of protein
(20% to 24%) and other macromolecular additives. These reagents are prepared from pools of human sera and
give reliable results; however, high-protein levels and macromolecular additives may cause false-positive
reactions (see “Causes of FalsePositive and False-Negative Rh Typing Results” below). These reagents must be
used according to the manufacturers’ instructions and with the appropriate controls. False-positive results could
cause a D-negative patient to receive Dpositive blood and become immunized. If red
332
AABB TECHNICAL MANUAL
cells exhibit aggregation in the control test, the results of the test are not valid.
Low-Protein Reagents and Controls
Most Rh reagents in routine use are low-protein reagents formulated predominantly with IgM monoclonal
antibodies. Spontaneous agglutination causing a false-positive result can occur, although this happens much less
frequently than with high-protein reagents. A negative result from a test that was performed concurrently with a
similar reagent serves as a control. For example, for ABO and Rh typing, the absence of agglutination by anti-A
or anti-B serves as a negative control for spontaneous aggregation. For red cells that show agglutination with all
reagents (eg, type AB or D+), a control performed as described by the reagent manufacturer is required (with
the exception of donor retyping). In most cases, a suitable control is a suspension of the patient’s red cells with
autologous serum or 6% to 10% albumin. Indirect antiglobulin testing is not valid for red cells with a positive
direct antiglobulin test (DAT) result unless a method is used to remove the IgG antibody. Positive and negative
controls should be tested, and the positive control cells should have a single dose of the antigen or be known to
show weak reactivity.
Rh Testing Considerations in HDFN
Red cells from an infant with HDFN are coated with immunoglobulin, and a low-protein reagent is usually
necessary to test these cells. Occasionally, red cells with a strongly positive DAT result may be so heavily
coated that they are not agglutinated by a reagent with the same specificity as the bound antibody. This
“blocking” phenomenon probably results from steric hindrance, and occupied antigen sites show false-negative
results. Heat elution of the antibody performed at 45 C permits red cell typing, but elution must be performed
with caution to avoid denaturing the antigen. Although detection of the antibody in an eluate confirms the
presence of the antigen on the eluted red cells, RHD genotyping can be used for confirmation of D typing.
Causes of False-Positive and False
Negative Rh Typing Results
False-positive typing results can be caused by:
1. Immunoglobulin coating of the cells as a result of warm or cold autoagglutinins. Wash the red cells
several times and retest them with low-protein reagents by direct methods. If an IAT is required, IgG coating on
the red cells can be removed by treating the cells with glycine/EDTA (Method 2-21) or chloroquine (Method 2-
20) and retesting.
2. Induction of rouleaux by serum factors that can be eliminated by thoroughly washing the red cells and
retesting.
3. Use of the wrong reagent.
4. Contamination with reagent from another vial.
5. Nonspecific aggregation of the red cells due to some component of the reagent other than the antibody
(ie, a preservative, antibiotic, or dye).
6. Testing of polyagglutinable red cells agglutinated with reagents that contain human serum.
False-negative typing results can be caused by:
1. Failure to add the reagent. It is good practice to add typing reagent to all test tubes or wells before adding
the red cells.
2. Use of the wrong reagent.
3. A red cell suspension that is too heavy for a tube test or too weak for a slide test.
4. Failure to detect a weak D reaction with direct testing (immediate centrifugation).
5. Nonreactivity of a reagent with a weak or partial form of the antigen.
6. Aggressive resuspension of the red cell button dispensing the agglutination.
7. Contamination, improper storage, or outdating of the reagent.
8. Red cells with a strongly positive DAT result and antigen sites blocked because of a large amount of
bound antibody (most common in severe HDFN caused by anti-D).
333
Resolving D Typing Discrepancies
To investigate D typing discrepancies, errors in sample identification or of a clerical nature should be
eliminated by obtaining and testing a new sample. Beyond clerical errors, multiple variables contribute to D
typing discrepancies. These variables include the use of different methods (ie, slide, tube, microplate, gel, and
automated analyzers using enzyme-treated red cells), phase of testing (direct or IAT), different IgM clones in
manufacturers’ reagents, and large number of RHD gene variations that affect the level of expression and
epitopes of the D antigen.
It is important to know the characteristics of the D typing reagent used and to always consult and follow the
manufacturer’s instructions during D typing. The FDA has drafted recommendations that require manufacturers
to specify the reactivity of their reagents with partial DIV, DV, and DVI red cells.70
The IgM anti-D in all of the tube reagents currently licensed by the FDA is reactive by direct testing (initial
spin) with DIV and DV red cells but has been selected to be nonreactive with partial DVI red cells in direct
testing. Limited studies have been performed to characterize the reactivity of anti-D reagents with other partial
D and weak D red cells. These studies have shown that the anti-D reagents cannot reliably predict whether a D
variant is a weak or partial D antigen.71,72 Table 13-5 shows the reactivity of D variant red cells that have
predictable patterns among the different antiD reagents. In general, an individual with partial D should be
considered to be a D-positive blood donor but a D-negative transfusion recipient.
KEY POINTS
1. The Rh system is highly immunogenic, complex, and polymorphic. More than 50 Rh antigens have been
characterized, although the five principal antigens—D, C, c, E, and e—are responsible for the majority of
clinically significant antibodies.
2. “Rh positive” and “Rh negative” refer to the presence or absence, respectively, of the D antigen.
3. Contemporary Rh terminology distinguishes between antigens (such as D and C), genes (such as RHD
and RHCE), alleles (such as RHCE*ce and RHCE*Ce), and proteins (such as RhD and Rhce).
4. Most D-negative (Rh-negative) phenotypes result from complete deletion of the RHD gene. Exposure of
D-negative individuals to RhD often results in the development of anti-D.
5. RHCE encodes both C/c and E/e antigens on a single protein. C and c differ by four amino acids, whereas
E and e differ by one amino acid.
6. Routine donor and patient Rh typing procedures test only for D. Testing for other common Rh antigens is
used to resolve or confirm antibody identification and, for many SCD transfusion programs, or for other
patients receiving chronic transfusions to match patients and donors for D, C, and E.
7. Weak D phenotypes are defined as having a reduced amount of D antigen and are detected by IAT. Weak
D usually results from amino acid changes that impair the insertion of the protein in the membrane. Many
different mutations cause weak expression of D.
8. RHD genotyping can identify those pregnant females and blood transfusion recipients with a serologic
weak D phenotype who can be managed safely as Rh positive.
9. Most anti-D reagents approved by the FDA combine a monoclonal IgM (that is reactive at room
temperature for routine testing) and a monoclonal or polyclonal IgG (that is reactive by IAT for the
determination of weak D). The exception is anti-D for column agglutination testing, which contains only IgM.
These reagents may show different reactivity with red cells that have weak D, partial D, or D-like epitopes.
 334
AABB TECHNICAL MANUAL
10. When determining the D type of a patient, an IAT for weak expression of D is not necessary except
when testing the red cells of an infant born to a mother at risk of D immunization. Rh-negative donors must be
tested by a method that detects weak D.
11. Most Rh antibodies are IgG, although some may have an IgM component. With rare exceptions, Rh
antibodies do not activate complement and, thus, cause primarily extravascular rather than intravascular
hemolysis. Antibodies almost always result from red cell immunization through pregnancy or transfusion.
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the human blood group RhD polypeptide. Proc Natl Acad Sci U S A 1992;89:10925-9.
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in RhD-positive, but not RhDnegative individuals. Blood 1993;82:651-5.
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Terminology for Red Blood Cell Surface Antigens: Macao report. Vox Sang 2009;96:153-6.
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ISBT, 2014. [Available at: http:// www.isbtweb.org/fileadmin/user_upload/
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22. Ceppellini R, Dunn LC, Turri M. An interaction between alleles at the RH locus in man which weakens
the reactivity of the Rh(0) Factor (D). Proc Natl Acad Sci USA 1955;41:283-8.
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CHAPTER 13 The Rh System

335
24. Ye L, Wang P, Gao H, et al. Partial D phenotypes and genotypes in the Chinese population. Transfusion
2012;52:241-6.
25. Wagner FF, Gassner C, Muller TH, et al. Molecular basis of weak D phenotypes. Blood 1999; 93:385-
93.
26. Shao CP Maas JH, Su YQ, et al. Molecular background of Rh D-positive, D-negative, D(el) and weak D
phenotypes in Chinese. Vox Sang 2002;83:156-61.
27. Sun CF, Chou CS, Lai NC, Wang WT. RHD gene polymorphisms among RhD-negative Chinese in
Taiwan. Vox Sang 1998;75:52-7.
28. Flegel WA, Denomme GA. Alio- and autoantiD in weak D types and in partial D. Transfusion
2012;52:2067-9.
29. Wagner FF, Frohmajer A, Ladewig B, et al. Weak D alleles express distinct phenotypes. Blood
2000;95:2699-708.
30. Wagner FF, Ladewig B, Flegel WA. The RHCE allele ceRT: D epitope 6 expression does not require D-
specific amino acids. Transfusion 2003;43:1248-54.
31. Chen Q, Hustinx H, Flegel WA. The RHCE allele ceSL: The second example for D antigen expression
without D-specific amino acids. Transfusion 2006;46:766-72.
32. Beckers EA, Porcelijn L, Ligthart P et al. The Ro1™* antigenic complex is associated with a limited
number of D epitopes and alloanti-D production: A study of three unrelated persons and their families.
Transfusion 1996;36:104-8.
33. Westhoff CM. Review: The Rh blood group D antigen: Dominant, diverse, and difficult.
Immunohematol 2005;21:155-63.
34. Daniels G. Human blood groups. 2nd ed. Cambridge, MA: Blackwell Science, 2002.
35. Race RR, Sanger R. Blood groups in man. 6th ed. Oxford: Blackwell, 1975.
36. Colin Y, Cherif-Zahar B, Le Van Kim C, et al. Genetic basis of the RhD-positive and RhDnegative
blood group polymorphism as determined by Southern analysis. Blood 1991;78: 2747-52.
37. Reid ME, Lomas-Francis C, Olsson ML. The blood group antigen factsbook. 3rd ed. San Diego, CA:
Academic Press, 2012.
 38. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
39. Schmidt PJ, Morrison EC, Shohl J. The antigenicity of the Rh0 (Du) blood factor. Blood 1962;20:196-
202.
40. Wagner T, Kormoczi GF, Buchta C, et al. Anti-D immunization by DEL red blood cells. Transfusion
2005;45:520-6.
41. Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Secondary anti-D immunization by Del red blood cells.
Transfusion 2005;45:1581-4.
42. Flegel WA, Khull SR, Wagner FF. Primary antiD immunization by weak D type 2 RBCs. Transfusion
2000;40:428-34.
43. Mota M, Fonseca NL, Rodrigues A, et al. Anti-D alloimmunization by weak D type 1 red blood cells
with a very low antigen density. Vox Sang 2005;88:130-5.
44. Lacey PA, Caskey CR, Werner DJ, Moulds JJ. Fatal hemolytic disease of a newborn due to anti-D in an
Rh-positive Du variant mother. Transfusion 1983;23:91-4.
45. Flegel WA, Denomme GA, Yazer MH. On the complexity of D antigen typing: A handy decision tree in
the age of molecular blood group diagnostics. J Obstet Gynaecol Can 2007;29: 746-52.
46. Schonewille H, van de Watering LM, Brand A. Additional red blood cell alloantibodies after blood
transfusions in a nonhematologic alloimmunized patient cohort: Is it time to take precautionary measures?
Transfusion 2006; 46:630-5.
47. Frohn C, Dumbgen L, Brand J-M, et al. Probability of anti-D development in D-patients receiving D+
RBCs. Transfusion 2003;43:893-8.
48. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific
Publications, 1998.
49. Singleton BK, Green CA, Avent ND, et al. The presence of an RHD pseudogene containing a 37 base
pair duplication and a nonsense mutation in Africans with the Rh D-negative blood group phenotype. Blood
2000;95:12-18.
50. Daniels GL, Faas BH, Green CA, et al. The VS and V blood group polymorphisms in Africans: A
serologic and molecular analysis. Transfusion 1998;38:951-8.
51. Reid ME, Storry JR, Issitt PD, et al. Rh haplotypes that make e but not hrB usually make VS. Vox Sang
1997;72:41-4.
52. Vege S, Westhoff CM. Molecular characterization of GYPB and RH in donors in the American Rare
Donor Program. Immunohematol 2006;22:143-7.
53. Noizat-Pirenne F, Lee K, Pennec PY, et al. Rare RHCE phenotypes in black individuals of
AfroCaribbean origin: Identification and transfusion safety. Blood 2002;100:4223-31.
336
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54. Westhoff CM, Vege S, Halter-Hipsky C, et al. DUIa and Dill Type 5 are encoded by the same allele and
are associated with altered RHCE*ce alleles: Clinical implications. Transfusion 2010;50:1303-11.
55. Ness PM. To match or not to match: The question for chronically transfused patients with sickle cell
anemia. Transfusion 1994;34:558-60.
56. Vichinsky ER Luban NL, Wright E, et al. Prospective RBC phenotype matching in a stroke prevention
trial in sickle cell anemia: A multicenter transfusion trial. Transfusion 2001;41: 1086-92.
57. Chou ST, Jackson T, Vege S, et al. High prevalence of red blood cell alloimmunization in sickle cell
disease despite transfusion from Rhmatched minority donors. Blood 2013;122: 1062-71.
58. Reid ME, Rios M, Powell VI, et al. DNA from blood samples can be used to genotype patients who
have recently received a transfusion. Transfusion 2000;40:48-53.
59. Pirelli KJ, Pietz BC, Johnson ST, et al. Molecular determination of RHD zygosity: Predicting risk of
hemolytic disease of the fetus and newborn related to anti-D. Prenat Diagn 2010; 12-13: 1207-12.
60. Matheson KA, Denomme GA. Novel 3' rhesus box sequences confound RHD zygosity assignment.
Transfusion 2002;42:645-50.
61. Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum.
Lancet 1997;350:485-7.
62. Van der Schoot CE, Soussan AA, Koelewijn J, et al. Non-invasive antenatal RHD typing. Transfus Clin
Biol 2006;13:53-7.
 63. Gassner C, Doescher A, Drnovsek TD, et al. Presence of RHD in serologically D-, C/E+ individuals: A
European multicenter study. Transfusion 2005;45:527-38.
64. Polin H, Danzer M, Hofer K, et al. Effective molecular RHD typing strategy for blood donations.
Transfusion 2007;47:1350-5.
65. Chou St, Westhoff CM. The role of molecular immunohematology in sickle cell disease. Transfus Apher
Sci 2011;44:73-9.
66. Fasano RM, Monaco A, Meier ER, et al. RH genotyping in a sickle cell disease patient contributing to
hematopoietic stem cell transplantation donor selection and management. Blood 2010;116:2836-8.
67. Tilley L, Green C, Poole J, et al. A new blood group system, RHAG: Three antigens resulting from
amino acid substitutions in the Rh-associated glycoprotein. Vox Sang 2010;98:151-9.
68. Dahl KN, Parthasarathy R, Westhoff CM, et al. Protein 4.2 is critical to CD47-membrane skeleton
attachment in human red cells. Blood 2004;103:1131-6.
69. Nicolas V Le Van Kim C, Gane P, et al. RhRhAG/ankyrin-R, a new interaction site between the
membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation. J Biol Chem
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71. Judd WJ, Moulds M, Schlanser G. Reactivity of FDA-approved anti-D reagents with partial D red blood
cells. Immunohematol 2005;21:1468.
72. Denomme GA, Dake LR, Vilensky D, et al. Rh discrepancies caused by variable reactivity of partial and
weak D types with different serologic techniques. Transfusion 2008;48:473-8.
Chapter 14
Other Blood Group Systems and Antigens
Jill R. Storry, PhD, FIBMS
THE INTERNATIONAL SOCIETY of
12191 Blood Transfusion (ISBT) currently recognizes 339 antigen specificities, of which 297 belong to 1 of
34 blood group systems.1'3 Each system represents either a single gene or two or three closely linked
homologous genes. The ABO and Rh systems are the best known and clinically most important systems and are
described in detail in Chapters 12 and 13. The antigens of the H, Lewis, I, P1PK, and Globoside systems are
carbohydrate structures that are biochemically closely related to ABO antigens and are discussed in Chapter 12.
The remaining systems are described in this chapter; some, generally the most important in transfusion
medicine, are described in some detail, others in a few lines. They are listed in ISBT order, as in Table 14-1.
In addition to the 34 systems, some groups of antigens that are serologically, biochemically, or genetically
related but not eligible to join a system are classified together as collections. Other antigens that are not eligible
to join a system or collection are of either low or high prevalence in most major populations
and make up the 700 and 901 series, respectively.1 They are all discussed at the end of this chapter.
The full ISBT classification can be found on the ISBT website and in Appendix 6.4 Many more references
to blood group systems and antigens than can be provided here are available in various textbooks and
reviews.5'7
The most important aspect of blood group antigens in transfusion medicine is whether their corresponding
antibodies are hemolytic and therefore have the potential to cause hemolytic transfusion reactions (HTRs) and
hemolytic disease of the fetus and newborn (HDFN).5 A guide to the potential clinical significance of blood
group antibodies is provided in Table 14-1.
THE MNS SYSTEM
MNS is a highly complex blood group system consisting of 46 antigens. As with the Rh system, much of its
complexity arises from recombination between closely linked homologous genes.

fill R. Storry, PhD, FIBMS, Associate Professor, Region Skane Office of Medical Services, Department of
Clinical Immunology and Transfusion Medicine, Lund, Sweden.
The author has disclosed no conflicts of interest.
337
TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens
338
AABB TECHNICAL MANUAL
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CHAPTER 14 Other Blood Groups
339
(Continued)
340
AABB TECHNICAL MANUAL
 341
The MNS Glycoproteins and the Genes that Encode Them
The antigens of the MNS system are located on one or both of two glycoproteins: glycophorin A (GPA,
CD235A) and glycophorin B (GPB, CD235B). Each crosses the membrane once and has an external N-terminal
domain and a C-terminal cytosolic domain. The extracellular domains of both molecules have many sialic acid-
rich O-glycans; GPA is N-glycosylated at asparagine-45 (26 in the mature protein), whereas GPB is not N-
glycosylated. The long cytosolic tail of GPA interacts with the cytoskeleton. GPA is abundant, with about 106
copies per red cell, whereas GPB has only about 200,000 copies per cell. GPA forms an association in the
membrane with Band 3 (Diego blood group system), and both GPA and GPB appear to be part of the Band 3/Rh
ankyrin complex (Fig 14-1).8,9
GYPA and GYPB, the genes encoding GPA and GPB, comprise seven and five exons, respectively. A
region of intron 3 of GYPB is homologous to exon 3 of GYPA but is not expressed because of a defective splice
site (Fig 14-2).10 Exon 1 of each gene encodes a 19-amino-acid signal peptide that is not present in the mature
protein. A third gene, GYPE, probably produces a third glycoprotein, glycophorin E, but this plays little or no
part in MNS antigen expression.
GPA is restricted to blood cells of erythroid origin and is often used as an erythroid marker. Both GPA and
GPB are exploited by the malarial parasite Plasmodium falciparum as receptors for binding to red cells and may
be critical to the invasion process.11,12 A GPAlike molecule has been detected on renal endothelium.
M (MNS1), N (MNS2), S (MNS3), AND s (MNS4)
M and N (as detected by most anti-N reagents) are antithetical antigens and polymorphic in all populations
tested (Table 14-2). M and N are located at the N-terminus of GPA. M-active GPA has serine and glycine at the
first and fifth
TABLE 14-2. Frequencies of Some Phenotypes of the MNS System
Frequency (%)
Phenotype Whites Blacks
M+ N 30 25
M+N+ 49 49
M-N+ 21 26
S+ s 10 6
S+ s+ 42 24
S- S+ 48 68
S- s 0 2
positions of the mature protein (positions 20 and 25); N-active GPA has leucine and glutamic acid at those
positions.
S and s are another pair of polymorphic antithetical antigens of the MNS system (Table 14-2). Family
studies show tight linkage between M/N and S/s. The S and s antigens represent a Met48Thr (29 in mature
protein) polymorphism in GPB. The amino-terminal 26 amino acids of GPB mature protein are usually identical
to those of the N form of GPA. Consequently, in almost all people of European ancestry and most people of
other ethnicities, GPB expresses ‘N.’ Elowever, because GPB is much less abundant than GPA, most anti-N
reagents do not detect the ‘N’ antigen on GPB. The N-terminal region of GPA is cleaved from intact red cells by
trypsin, whereas that of GPB is not. Consequently, M and N antigens on GPA are trypsin sensitive, and S, s, and
‘N’ on GPB are trypsin resistant. In contrast, with achymotrypsin treatment of red cells, M and N activity is
only partially reduced, whereas S, s, and ‘N’ expression is completely destroyed. M, N, S, s, and ‘N’ are all
destroyed by treatment of the red cells with papain, ficin, bromelin, or pronase, although this effect with S and s
is variable.
342
AABB TECHNICAL MANUAL
 FIGURE 14-1. Model of two proposed membrane complexes containing Band 3 and Rh proteins: 1)
containing tetramers of Band 3 and heterotrimers of RhD, RhCE, and RhAG, and linked to the spectrin matrix
of the cytoskeleton through Band 3, protein 4.2, and ankyrin; and 2) containing Band 3, RhD, and RhCE, and
linked to the spectrin/actin junction through glycophorin C (GPC), p55, and protein 4.1 and through Band 3 and
adducin.
343
GYPA
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M/N
GPA
A2A3A4 A5A6A7 S/s
GPB
B2 B4 B5 B6 Mur
^ils
.GP(B-A-B)
Mur
B2/ \B4B5B6 B3 A3
Membrane
Extracellular Cytosol
FIGURE 14-2. GYPA, GYPB, and the hybrid GYPB-A-B gene responsible for GP.Mur, and a
representation of the proteins they encode, showing the regions of proteins encoded by the various exons.
Y = pseudoexon not represented in the mRNA or the encoded protein.
S-s-U- Phenotype
 The red cells of about 2% of Americans of African ancestry and a higher proportion of Africans are S-s- and
lack the high-prevalence antigen U (MNS5). The S-s-U- phenotype often results from homozygosity for a
deletion of the coding region of GYPB, but other, more complex molecular phenomena involving hybrid genes
may also give rise to an S-s- phenotype with expression of a variant U antigen. U is generally resistant to
denaturation by proteases—papain, ficin, trypsin, a-chymotrypsin—although anti-U is not reactive with papain-
treated red cells in rare cases.
M, N, S, s, and U Antibodies and Their Clinical Significance
Anti-M is a relatively common antibody, whereas anti-N is quite rare. Most anti-M and -N are not active at
37 C and are not clinically significant.13 They can generally be ignored in transfusion practice. If room-
temperature incubation is eliminated from compatibility
testing and screening for antibodies, these antibodies are not detected. When M or N antibodies active at 37
C are encountered, antigennegative or red cells that are compatible by an indirect antiglobulin test (IAT) should
be provided.5 Very occasionally, anti-M and -N have been implicated as the cause of acute and delayed HTRs,
and anti-M has very rarely been responsible for severe HDFN. A few cases of warm autoimmune hemolytic
anemia (AIHA) caused by autoanti-N have been described, one of which had a fatal outcome. Autoanti-M
responsible for warm AIHA has not been reported.
Anti-S and -s are usually immune globulin G (IgG) antibodies that are active at 37 C. They have been
implicated in HTRs and have caused severe and fatal HDFN. Autoanti-S has caused AIFIA. If immunized,
individuals with S-s-U- red cells may produce anti-U. Anti-U has been responsible for severe and fatal HTRs
and HDFN. Autoanti-U has been implicated in AIHA.
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AABB TECHNICAL MANUAL
Other MNS Antigens and Antibodies
The other MNS antigens are either of high or low prevalence in most populations. The similarity of
sequence between certain regions of GYPA and GYPB may occasionally lead to GYPA pairing with GYPB
during meiosis. If recombination then occurs, either by crossing over or by a less well-defined mechanism
called “gene conversion,” a hybrid gene can be formed consisting partly of GYPA and partly of GYPB. A large
variety of these rare hybrid genes exists, and they give rise to low-prevalence antigens and, in the homozygous
state, to phenotypes that lack high-prevalence antigens.10 Red cells of some of the phenotypes resulting from
hybrid genes react with an antibody called “anti-Mia.” The phenotypes were grouped together as the
Miltenberger series, but this classification is obsolete because the hybrids are now well defined.14
One example that is relatively common in Southeast Asia is the hybrid gene that is responsible for the
GP.Mur (previously Mi.III) phenotype. The hybrid gene is mostly GYPB, but a small region of GYPB
encompassing the 3' end of the pseudoexon and the 5' end of the adjacent intron has been replaced by the
equivalent region from GYPA. This means that the defective splice site in GYPB is now replaced by the
functional splice site from GYPA, and the new, composite exon is expressed in the mRNA and represented in
the protein.10 This provides an unusual amino acid sequence that is immunogenic and represents the antigen
Mur. The amino acid sequence that results from the junction of exons B3 and A3 gives rise to Hil and MINY
(Fig 14-2).
Mur antigen is rare in people of European and African ethnicity but has a prevalence of about 7% in people
of Chinese and 10% in people of Thai ancestry. Anti-Mur has the potential to cause severe HTRs and HDFN. In
Hong Kong and Taiwan, anti-Mur is the most common blood group antibody after anti-A and -B. In Southeast
Asia, it is important that red cells for antibody detection include a Mur+ sample.15
Antibodies to regions of CPA with the generic name Enathat may be made by very rare
individuals who lack all or part of CPA have caused severe HTRs and HDFN.
THE LUTHERAN SYSTEM
Lutheran is a complex system consisting of 20 antigens, including four antithetical pairs: Lua/ Lub
(His77Arg); Lu6/Lu9 (Ser275Phe); Lu8/ Lul4 (Met204Lys); and Aua/Aub (Thr529Ala).16 The other Lutheran
antigens are highly prevalent in all populations tested. Lua (LU1) has a prevalence of about 8% in people of
European or African ethnicity but is rare elsewhere; its antithetical antigen, Lub (LU2), is common everywhere.
In the other Lutheran polymorphism, Aua and Aub have a prevalence of around 80% and 50%, respectively, in
people of European ancestry.
Lutheran antigens are destroyed by treatment of the red cells with trypsin or a-chymotrypsin, whereas
papain and ficin have little effect. Most Lutheran antibodies are not reactive with red cells treated with the
sulfhydryl reagents 2-aminoethylisothiouronium bromide (AET) or dithiothreitol (DTT), which reduce the
disulfide bonds of the immunoglobulin superfamily (IgSF) domains (Method 3-18).
The Lutheran antigens are located on a pair of glycoproteins that span the membrane once, have five
extracellular IgSF domains, and differ by the length of their cytoplasmic domains as a result of alternative RNA
splicing. The isoform with the longer cytoplasmic domain interacts with spectrin of the membrane skeleton.17
The location of the Lutheran antigens on the IgSF domains is shown in Fig 14-3. The Lutheran glycoproteins
are adhesion molecules that bind isoforms of laminin that contain a-5 chains. Laminin is a glycoprotein of the
extracellular matrix, and Lutheran-laminin interactions may play a role in the migration of mature erythroid
cells from the marrow to the peripheral blood at the latest stages of erythropoiesis. Upregulation of Lutheran
glycoproteins on red cells of patients with sickle cell disease could play a part in adhesion of these cells to the
vascular endothelium and the resultant crises of vascular occlusion.17
345
NH,

Lua/Lub LURC Lu21 Lu5 Lu17 Lu12

Lu4
Lu8/Lu14 Lu16
Lu6/Lu9 Lu20
Lu13
Aua/Aub
COOH
FIGURE 14-3. Diagram of the two isoforms of the Lutheran glycoprotein, showing the five extracellular
immunoglobulin superfamily domains and the location of the Lutheran antigens on these domains, the single
membrane-spanning domain, and the cytoplasmic domains.
The extremely rare Lunull phenotype arises from homozygosity for inactive LU gene.18 The red cells lack
any expression of Lutheran antigens, and individuals with this phenotype
may produce anti-Lu3, which is reactive with all red cells except those from other Lu(a-b-) individuals.
Heterozygosity for inactivating mutations in the erythroid transcription factor KLF1 is responsible for In(Lu), a
Lumod phenotype with extremely weak expression of Lutheran antigens that are detectable only by
adsorption/elution techniques. Mutations in KLF1 also affect other blood group genes and cause weakened
expression of several other antigens, including PI, Inb, and AnWj.19 The In(Lu) phenotype has a prevalence of
around 0.03%. In one family, hemizygosity for a mutation in the X-linked gene for the major erythroid
transcription factor GATA-1 resulted in an Lumotl phenotype with an X-linked mode of inheritance.20
Lutheran antibodies are most often IgG and demonstrate reactivity best by IAT, but have generally been
implicated only in mild delayed HTRs and have not caused severe HDFN. Anti-Lua may be “naturally
occurring” or immune, and it is often IgM but may also be IgG and IgA. These antibodies are usually reactive
by direct agglutination of Lu(a+) red cells but often also reactive by an IAT and may show a mixed-field-like
agglutination that is characteristic of this and other antibodies to Lutheran antigens.
THE KELL AND KX SYSTEMS
The antigen often referred to as “Kell,” but correctly named “K” or “KEL1,” is the original antigen of the
Kell system and the first blood group antigen to be identified following the discovery of the antiglobulin test in
1946. Its antithetical antigen, k or KEL2, was identified 3 years later. The Kell system now consists of 35
antigens numbered from KEL1 to KEL38, of which three are obsolete.21,22 The Kell system includes six pairs
(K/k, Jsa/Jsb, K11/K17, K14/ K24, VLAN/VONG, and KYO/KYOR) and one triplet (Kpa/Kpb/Kpc) of Kell
antithetical antigens. Initially, most antigens joined the Kell system through genetic associations observed in
family studies. These associations have now been confirmed by DNA sequencing of the KEL gene.

346
AABB TECHNICAL MANUAL
The Kell Glycoprotein and the KEL Gene
The Kell antigens are located on a red cell membrane glycoprotein (CD238) with four or five N-glycans but
no O-glycosylation. Kell is unique as a blood group antigen because it is a Type II membrane glycoprotein; it
spans the membrane once and has a short N-terminal domain in the cytosol and a large C-terminal domain
outside the membrane (Fig 14-4).23 The extracellular domain has 15 cysteine residues and is extensively folded
by disulfide bonding, although crystallographic studies are required to determine the molecule’s
threedimensional structure. Kell system antigens depend on the conformation of the glycoprotein and are
sensitive to disulfide-bond-reducing agents, such as DTT and AET.
The Kell glycoprotein is linked through a single disulfide bond to the Xk protein (Fig 14
TABLE 14-3. Frequencies of Some Kell Phenotypes
Frequency (%)
Phenotype Whites Blacks
K-k+ 91.0 98
K+ k+ 8.8 2
K+k 0.2 Rare
Kp(a-b+) 97.7 100
Kp(a+b+) 2.3 Rare
Kp(a+b—) Rare 0
Js(a—b+) 100.0 80
Js(a+b+) Rare 19
Js(a+b—) 0 1
K, Kpa, and Jsa are extremely rare in populations of
Asian ancestry.
Xk Kell

FIGURE 14-4. Diagram of the Kell and Xk proteins linked by a single disulfide bond. The Xk protein has
cytoplasmic N- and C-terminal domains and 10 membrane-spanning domains. The Kell glycoprotein has a
large, folded, extracellular, C-terminal domain and an intracellular N-terminal domain.
4), an integral membrane protein that expresses the Kx blood group antigen (XK1). Absence of Xk protein
from the red cell results in reduced expression of the Kell glycoprotein and weakened Kell antigens (McLeod
phenotype, see below).
The KEL gene is located on chromosome 7q34. It is organized into 19 exons: exon 1 encodes the probable
translation-initiating methionine; exon 2, the cytosolic domain; exon 3, the membrane-spanning domain; and
exons 4 through 19, the large extracellular domain.
Kell Antigens
K has a prevalence of about 9% in people of European ancestry and about 1.5% in people of African
ancestry. It is rare in East Asia (Table 14-3). The k antigen is highly prevalent in all populations. K and k result
from a single nucleotide polymorphism (SNP) in exon 6, which encodes Metl93 in K and Thrl93 in k. Asnl91
347
 is N-glycosylated in the product of the k but not the JSCallele.
Kpa (KEL3) is found in about 2% of people of European ancestry and is not present in people of African or
Japanese ancestry (Table 14-3); Kpb (KEL4) has high prevalence in all populations. Whereas 2.3% of people of
European ancestry are Kp(a+) only 1.2% of K+ persons of that same ancestry are Kp(a+). Nine percent of
people of European ancestry are K+, but only 2.7% of Kp(a+) people from the same population are K+. This
strong allelic association has been confirmed by family studies. Only one example of the KKpa haplotype has
been found.24 Kpc (KEL21), an antigen with very low prevalence, is the product of another allele of Kpa and
Kpb. KEL alleles encoding the three Kp antigens differ by single base substitutions within codon 281 (exon 8):
Kpa, TGG, Trp281; Kpb, CGG, Arg281; and Kpc, CAG, Gln281. The mutation associated with Kpa expression
appears to reduce the quantity of Kell glycoprotein in the red cell membranes, giving rise to a slight reduction in
expression of Kell antigens in Kpa/Kpa homozygotes but a more obvious weakening of Kell antigens in
individuals who are heterozygous for Kpa and the null allele K°.
Jsa (KEL6) is almost completely confined to people of African ethnicity. The prevalence of Jsa in African
Americans is about 20% (Table 14-3). Jsb (KEL7) is highly prevalent in all populations, and Js(a+b—) has not
been found in persons of non-African ethnicity. Jsa represents Pro597; Jsb, Leu597.
K17 (Wka) (Ala302) has a prevalence of 0.3% in English donors; Kll (Val302), its antithetical antigen, has
very high prevalence. K14 (Argl80) and K24 (Prol80) are very high- and very low-prevalence antigens,
respectively. VLAN and VONG are low-prevalence antigens associated with Arg248Gln and Arg248Trp,
respectively.
The presence of the low-prevalence antigens Ula, KYO, and K23 and the absence of the high-prevalence
antigens K12, K13, K18, K19, K22, TOU, RAZ, KALT, KTIM, KUCI, KANT, KASH, KELP KETI, and
KHUL result from single amino acid substitutions in the Kell glycoprotein.
Kell antigens are resistant to papain, ficin, trypsin, and a-chymotrypsin but are destroyed by a mixture of
trypsin and a-chymotrypsin. They are also destroyed by DTT and AET (see above) and by EDTA glycine.
Kell Antibodies and Their Clinical Significance
Kell antibodies are usually IgG, predominantly IgGl.13 They should be considered potentially clinically
significant from the perspective of causing severe HDFN and HTRs. Patients with Kell antibodies should be
transfused with antigen-negative blood whenever possible.
Anti-K is the most common immune red cell antibody outside the ABO and Rh systems; one-third of all
non-Rh red cell immune antibodies are anti-K. An antiglobulin test is usually the method of choice for detecting
anti-K, although occasional samples may agglutinate red cells directly. Most anti-K appears to be induced by
blood transfusion. Because anti-K can cause severe HDFN, it is usual practice in some countries for girls and
women of childbearing potential to receive only K- red cells. Anti-K, -k, -Kpa, -Kpb, -Jsa, -Jsb, -Ku, -Ula, -Kll,
-K19, and -K22 are all reported to have caused severe HDFN. Anti-K, -k, -Kpb, -Jsa, -Jsb, -Ku, and -K19 have
all been implicated in acute or delayed HTRs.
The pathogenesis of HDFN caused by anti-K differs from that resulting from anti-D. Anti-K HDFN is
associated with lower concentrations of amniotic fluid bilirubin than anti-D HDFN of comparable severity.
Postnatal hyperbilirubinemia is not prominent in infants with anemia caused by anti-K. There is also reduced
reticulocytosis and erythroblastosis in the anti-K disease compared with anti-D HDFN. These symptoms
suggest that anti-K HDFN is associated with a lower degree of hemolysis and that fetal anemia in anti-K HDFN
results predominantly from a suppression of erythropoiesis. The Kell glycoprotein appears on erythroid
progenitors at a much earlier stage of erythropoiesis than do Rh antigens. Consequently, anti-K probably
facilitates phagocytosis of K+ erythroid progenitors at an early stage of development by macrophages in
348
AABB TECHNICAL MANUAL
the fetal liver, before the erythroid cells produce hemoglobin.25
Antibodies mimicking Kell specificities have been responsible for severe AIHA. Presence of the
autoantibody is often associated with apparent depression of all Kell antigens. Although most examples of anti-
K are stimulated by pregnancy or transfusion, a few cases of apparently non-red-cell immune anti-K have been
described. In some cases, the antibodies were found in untransfused, healthy, male blood donors; in others,
microbial infection was implicated as an immunizing agent.
Null (K0) and Mod Phenotypes
Like most blood group systems, Kell has a null phenotype (K0) in which none of the Kell antigens is
expressed and the Kell glycoprotein cannot be detected in the membrane. Immunized K0 individuals may
produce anti-Ku (anti-KEL5), an antibody that is reactive with all cells except those of the K0 phenotype.
Homozygosity for a variety of nonsense, missense, and splice-site mutations have been associated with K0
phenotype.26
Kmod red cells have only very weak expression of Kell antigens, and individuals with this phenotype are
homozygous (or doubly heterozygous) for missense mutations, resulting in single-amino-acid substitutions
within the Kell glycoprotein.27 Some Kmod individuals make an antibody that resembles anti-Ku but differs in
being nonreactive with Kmod red cells. Other phenotypes in which Kell antigens have substantially depressed
expression result from KpaIK0 heterozygosity (see above), absence of Xk protein (see below), and absence of
the Gerbich antigens Ge2 and Ge3, which are located on the glycophorins C and D. The reason for this
phenotypic association between Kell and Gerbich is not known, although Kell glycoproteins, Xk, and
glycophorins C and D could all be located within the same membrane protein complex (Fig 14-1).
Functional Aspects
The Kell protein has structural and sequence homology with a family of zinc-dependent en
dopeptidases that process a variety of peptide hormones. Although the function of the Kell glycoprotein is
not known, it is enzymatically active and can cleave the biologically inactive peptide big-endothelin-3 to create
the biologically active vasoconstrictor endothelin-3. Consequently, Kell might play a role in regulating vascular
tone, but there is no direct evidence for this.28 No obvious pathogenesis is associated with the K0 phenotype.
In addition to erythroid cells, Kell antigens may be present on myeloid progenitor cells, and Kell
glycoprotein has been detected in testis, lymphoid tissues, and with Xk protein in skeletal muscle.
Kx Antigen (XK1), McLeod Syndrome, and McLeod Phenotype
Kx is the only antigen of the Kx blood group system. It is located on a polytopic protein that spans the red
cell membrane 10 times and is linked to the Kell glycoprotein by a single disulfide bond (Fig 14-4). Xk protein
is encoded by the XK gene on chromosome Xp21.1.
McLeod syndrome is a very rare X-linked condition that develops almost exclusively in males and is
associated with acanthocytosis and a variety of late-onset muscular, neurologic, and psychiatric symptoms. It
results from hemizygosity for inactivating mutations and deletions of the XK gene.29 McLeod syndrome is
associated with McLeod phenotype, in which Kell antigens are expressed weakly and Km (KEL20) as well as
Kx are absent. When transfused, people with McLeod phenotype without chronic granulomatous disease (CGD)
produce anti-Km only, which is compatible with both McLeod and K0 phenotype red cells. The function of the
Xk-Kell complex is not known, but Xk has structural resemblance to a family of neurotransmitter transporters.
Deletion of part of the X chromosome that includes XK may also include CYBB, absence of which is
responsible for X-linked CGD. When transfused, CGD patients with McLeod syndrome usually produce anti-
Kx plus anti-Km, making it almost impossible to find compatible donors. It is recommended,
CHAPTER 14 Other Blood Groups
349
therefore, that transfusion of males with CGD and McLeod syndrome be avoided.
THE DUFFY SYSTEM
The Duffy system officially includes five antigens that reside on a glycoprotein known as the Duffy antigen
receptor for chemokines (DARC). The DARC gene consists of two exons, with exon 1 encoding only the first
seven amino acids of the Duffy glycoprotein.21,22 DARC is on chromosome lq23.2.
Fya (FY1) and Fyb (FY2)
In people of European and Asian ancestry, the Duffy polymorphism consists of two antigens, Fya and Fyb,
giving rise to three phenotypes: FyCa+b—), Fy(a+b+), andFy(a-b+) (Table 14-4). The Fy1 and Fy1’ alleles
represent an SNP in exon 2 of DARC that encodes Gly42 and Asp42, respectively, in the N-terminal
extracellular domain of the glycoprotein (Fig 14-5). Fy3 and Fyb are very sensitive to most proteolytic
enzymes, including bromelin, a-chymotrypsin, ficin, papain, and pronase, but are not destroyed by trypsin.
People of African ethnicity have a third allele, Fy, that is more abundant than Fya and Fy6. Fy produces no
Duffy glycoprotein on red cells and, hence, neither Fy3 nor Fyb. Individuals who are homozygous for Fy have
the red cell phenotype Fy(a-b-), whose prevalence varies from about 70% in African Americans to 100% in
residents of Gambia (Table 14-4).
The coding region for the Fy allele in people of African ancestry is identical to that of the Fyb allele, which
encodes Asp42. Fy produces no Duffy glycoprotein and no Fyb antigen in red cells because of an SNP in the
promoter region of DARC. This mutation disrupts the binding site for the erythroid-specific GATA-1
transcription factor and prevents expression of the gene in erythroid tissue.30 Duffy glycoprotein is present on
many cells throughout the body; thus, Fy(a-b-) people of African ethnicity lack Duffy glycoprotein on their red
cells but not on cells from other tissues. This explains why they do not make anti-Fyb and only very rarely
make anti-Fy3 or anti-Fy5 (see below). Although the GATA-1 binding site mutation in people of African
ancestry has been found only in Duffy genes with the Fyb sequence, the mutation has been detected in silent
Fy1 alleles in Papua New Guinea and Brazil.
Fy31 is a weak form of Fyb that results from an Fyb allele with a missense mutation encoding an Arg89Cys
substitution in a cytosolic domain of the glycoprotein (Fig 14-5). Fyb antigen may be undetected in some
samples of anti-Fyb, although the antigen can be detected by adsorption/elution.
Fy3, Fy4, Fy5, and Fy6
Very rare people of non-African ancestry with Fy(a-b-) red cells are homozygous for inactivating mutations
in their DARC genes. These individuals have no Duffy glycoprotein on
TABLE 14-4. Duffy Phenotypes and Genotypes in Selected Populations
Frequency
Genotype
(%)
European or
Phenotype Asian Ethnicity African Whites Blacks Japanese
Ethnicity
Fy(a+b—) Fy/Fy Fy/Fy3 or Fy/Fy 20 10 81
Fy(a+b+) Fy3/Fyb Fy/Fyb 48 3 15
Fyb/Fyb or
Fy(a-b+) Fyb/Fyb 32 20 4
Fyb/Fy
Fy(a-b-) Fy/Fy Fy/Fy 0 67 0
 350
AABB TECHNICAL MANUAL

FIGURE 14-5. Diagram of the Duffy glycoprotein (DARC), with a glycosylated external N-terminal
domain, seven membrane-spanning domains, and cytoplasmic C-terminus. The positions of the Fya/Fyb
polymorphism and of the amino acid substitution responsible for the Fyx phenotype are shown.
their red cells and would not be expected to have it in any other tissues. All were found through the presence
in their sera of anti-Fy3, an antibody that is reactive with all red cells except those of the Fy(a-b-) phenotype.
FY6, like Fy3 and Fyb, is sensitive to protease treatment, whereas FY3 and FY5 are resistant. Fy5 is also absent
from cells of the Fy(a-b-) phenotype, but unlike Fy3, Fy5 is also absent from cells of the Rhnull phenotype. The
Duffy glycoprotein may belong to the junctional membrane protein complex, which also contains Rh proteins
(Fig 14-1).9 Anti-Fy5 has been found only in multitransfused individuals of African ethnicity. Anti-Fy6 was the
designation given to a monoclonal antibody that produced serologic reactions very similar to those of anti-Fy3.
Anti-Fy4 appeared to be reactive with red cells of individuals with the silent Fy allele. However, the work could
not be repeated, the antibody is no longer available, and Fy4 is now obsolete.
Duffy Antibodies and Their Clinical Significance
Anti-Fy3 is a relatively common antibody, and anti-Fyb is about 20 times less common.31 IgGl
usually predominates, and these antibodies are generally detected by an antiglobulin test. Naturally
occurring examples are very rare.
Anti-Fy3 and anti-Fyb may cause acute or delayed HTRs. Although generally mild, some have proved fatal.
These antibodies have also been responsible for from mild to severe HDFN. Anti-Fy3 has been responsible for
acute and delayed HTRs and anti-Fy5 for delayed HTRs.
The Duffy Glycoprotein, a Receptor for Chemokines
The Duffy glycoprotein is a red cell receptor for a variety of chemokines, including interleukin8, monocyte
chemotactic protein-1, and melanoma growth stimulatory activity.32 It traverses the membrane seven times, and
a 63-aminoacid extracellular N-terminal domain contains two potential N-glycosylation sites and a cytoplasmic
C-terminal domain (Fig 14-5). This arrangement is characteristic of the G proteincoupled superfamily of
receptors that includes chemokine receptors.
The function of the DARC on red cells is not known. It has been suggested that it might act as a clearance
receptor for inflammatory mediators and that Duffy-positive red cells function as a “sink,” or as scavengers, for
the removal of unwanted chemokines.33 If so, this function must be of limited importance because Duffy is not
present on the red cells of most individuals of African ancestry. It has been suggested that DARC on red cells
reduces angiogenesis and, consequently, the progression of prostate cancer by clearing angiogenic chemokines
from the tumor microenvironment. This potential effect of erythroid DARC could provide an explanation for
the substantially higher levels of prostate cancer in men of African ancestry compared with those of European
ancestry.34
DARC is present in many organs, where it is expressed on endothelial cells lining postcapillary venules.35
Duffy glycoprotein on vascular endothelium may be involved in the inhibition of cancer-cell metastasis and
induction of cellular senescence.34 DARC may also
CHAPTER 14
Other Blood Groups
351
facilitate movement of chemokines across the endothelium.
The Duffy Glycoprotein and Malaria
The Duffy glycoprotein is a receptor for merozoites of Plasmodium vivax, the parasite responsible for a
form of malaria that is widely distributed in Africa but is less severe than malaria resulting from P. falciparum
infection. Red cells with the Fy(a-b-) phenotype are resistant to invasion by P vivax merozoites. Consequently,
the Fy allele confers a selective advantage in geographic areas where P vivax is endemic; this advantage
probably balances out any potential disadvantage resulting from the absence of the chemokine receptor on red
cells.33
THE KIDD SYSTEM
The Kidd system consists of three antigens located on a glycoprotein with 10 membranespanning domains,
cytoplasmic N- and C-termini, and one extracellular N-glycosylation site (Fig 14 - 4).22,36 The Kidd gene
(SLC14A1) contains 11 exons, 4 through 11 encoding the mature protein, and is located on chromosome
18ql2.3.
Jka (JK1) and Jkb (JK2)
Jka and Jkb are the products of antithetical alleles and represent Asp280 and Asn280 in the fourth external
loop of the Kidd glycoprotein (Fig 14-6). They have similar prevalence in populations of European and Asian
ancestry, but Jka is more common than Jkb in people of African ancestry (Table 14-5). Kidd antigens are
resistant to proteolytic enzymes, such as papain and ficin.
Jk(a-b-) and Jk3
The null phenotype, Jk(a-b-) Jk:-3, usually results from homozygosity for a silent gene at the JKlocus.
Although very rare in most populations, the null phenotype is relatively common in people of Polynesian
ethnicity, with a prevalence of around 1 in 400, but as high as 1.4% in those of Niuean ancestry. The Polyne

nh2 cooh
FIGURE 14-6. Diagram of the Kidd glycoprotein, a urea transporter, with cytoplasmic N- and C-terminal
domains, 10 membrane-spanning domains, and an N-glycan on the third extracellular loop. The position of the
Jka/Jkb polymorphism is shown on the fourth external loop.
sian null allele contains a splice site mutation in intron 5 that results in the loss of exon 6 from the mRNA.
In people of Finnish ancestry, where Jk(a-b-) is less rare than in other populations of European ancestry, the
mutation responsible encodes a Ser291Pro substitution. Immunized individuals with the Jk(a-b-) phenotype may
produce anti-Jk3. An extremely rare form of Jk(a-b-) phenotype found in
TABLE 14-5. Kidd Phenotypes in Three Populations
Frequency (%)
Phenotype Whites Blacks Asians
Jk(a+b—) 26 52 23
Jk(a+b+) 50 40 50
Jk(a-b+) 24 8 27
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AABB TECHNICAL MANUAL
people of Japanese ethnicity results from heterozygosity for a dominant inhibitor gene, named In(Jk) in
analogy with the In(Lu) dominant inhibitor of Lutheran and other antigens. Very weak expression of Jka and/or
Jkb can be detected on In(Jk) red cells by adsorption/elution tests.
Kidd Antibodies and Their Clinical Significance
Anti-Jka and -Jkb are not common antibodies and are generally found in antibody mixtures. They are
usually IgGl and IgG3, but some are partly IgG2, IgG4, or IgM. About 50% of anti-Jka and -Jkb bind
complement.13
Kidd antibodies are often difficult to detect. Some agglutinate antigen-positive cells directly, but the
reactions are usually weak. Generally, an antiglobulin test is required, and use of enzyme-treated cells may be
necessary to detect weaker antibodies.
Kidd antibodies may cause severe acute HTRs. They are also a very common cause of delayed HTRs,
probably because they are often not detected in pretransfusion testing due to their tendency to drop to low or
undetectable levels in plasma. Anti-Jk3 can also cause acute or delayed HTRs. Despite their hemolytic
potential, Kidd antibodies only very rarely cause severe HDFN.
Kidd antibodies have been implicated in acute renal transplant rejection, suggesting that Kidd antigens can
behave as histocompatibility antigens.37
The Kidd Glycoprotein in Urea Transportation
The Kidd antigens are located on a red cell urea transporter, also known as “human urea transporter 11”
(HUT11 orUT-Bl}.38 When red cells approach the renal medulla, which contains a high concentration of urea,
the urea transporter permits rapid uptake of urea and prevents the cells from shrinking in the hypertonic
environment. As the red cells leave the renal medulla, urea is transported rapidly out of the cells, preventing the
cells from swelling and carrying urea away from the kidney.
HUT11 has been detected on endothelial cells of the vasa recta, the vascular supply of the renal medulla, but
it is not present in renal tubules.
Normal red cells are rapidly lysed by 2M urea because urea transported into the cells makes them
hypertonic and they burst as a result of the osmotic influx of water. Because of the absence of the urea
transporter, Jk(a-b-) cells are not hemolyzed by 2M urea, and this can be used as a method for screening for
Jk(ab-) donors.39
The Jk(a-b-) phenotype is not associated with any clinical defect, although two unrelated Jk(a-b-)
individuals had a mild urineconcentrating defect.40
THE DIEGO SYSTEM
Band 3, the Red Cell Anion Exchanger
The 22 antigens of the Diego system are located on Band 3, the common name for the red cell anion
exchanger or solute carrier family 4A1 (SLC4A1).41 Band 3 is a major red cell membrane glycoprotein with
approximately 106 copies per red cell. Band 3 has a transmembrane domain that traverses the membrane about
14 times, with an N-glycan on the fourth extracellular loop. Band 3 also has a long cytoplasmic N-terminal
domain that interacts with the membrane skeleton proteins ankyrin, 4.1R, and protein 4.2 and functions as a
binding site for hemoglobin (Figs 14-1 and 14-7). The short cytoplasmic C-terminal domain binds carbonic
anhydrase II.
Band 3 in red cells has at least two major functions: the rapid exchange of HC03” and Cl” ions, which are
important in C02 transport, and attachment of the red cell membrane to the cytoskeleton.9 Tetramers of Band 3
form the core of the Band 3/Rh ankyrin macrocomplex of red cell membrane proteins, which could function as
a gas channel for 02 and C02.8 Band 3 is also a component of the junctional complex that links the red cell
membrane to the membrane skeleton via glycophorin C (Fig 14-1).9 The Band 3 gene (SLC4A1)
353

FIGURE 14-7. Diagram of Band 3, the Diego glycoprotein and anion exchanger, with cytoplasmic N- and
C-terminal domains, 14 membrane-spanning domains, and an N-glycan on the fourth extracellular loop
(although the precise conformation is still controversial). The locations of 22 antigens of Diego system on the
extracellular loops are shown.
consists of 20 exons of coding sequence and is on chromosome 17.
Dia (Dll) and Dib (DI2); Anti-Dia and -Dib
Dia, the original Diego antigen, is very rare in people of European or African ancestry but has a prevalence
of 5% in people of Chinese or Japanese ancestry and an even higher prevalence in the indigenous peoples of
North and South America, reaching 54% in the Kainganges Indians of Brazil. Dib is a high-prevalence antigen
in almost all populations. Dia and Dib represent an amino acid substitution in the seventh extracellular loop of
Band 3—Leu854 in Dia and Pro854 in Dib.
 Anti-Dia and -Dib are usually IgGl plus IgG3 and typically require an antiglobulin test for detection,
although a few directly agglutinating samples have been found. Anti-Dia occasionally binds complement and
lyses un
treated red cells. Anti-Dia, which is present in 3.6% of multitransfused patients in Brazil, can cause severe
HDFN. Anti-Dib has, very rarely, been responsible for serious HDFN. Apart from one example of anti-Dia
causing a delayed reaction, neither anti-Dia nor anti-Dib has been reported to be responsible for an HTR.13,21
Antigens of the Diego system are not destroyed by proteolytic enzymes, such as papain, ficin, or trypsin;
however, the antigens carried on the third extracellular loop (Rba, Tra, WARR, Vga, Wda, BOW, NFLD, Wu,
DISK, Jna, KREP, and Bpa) are sensitive to a-chymotrypsin.
Wra (DI3) and Wrb (DI4); Anti-Wra and -Wrb
The low-prevalence antigen Wra and its antithetical antigen of extremely high prevalence, Wrb, represent an
amino acid substitution in the fourth loop of Band 3—Lys658 in Wra and Glu658 in Wrb. Wrb expression,
however, also depends on the presence of GPA. Despite its homozygosity for the Glu658 codon in the Band 3
gene, Wrb is not expressed in the rare phenotypes associated with a complete absence of the MN glycoprotein
GPA or of the part of GPA that is close to insertion into the red cell membrane. This provides strong evidence
for an interaction between Band 3 and GPA within the red cell membrane.
Anti-Wrais a relatively common antibody, usually detected by an antiglobulin test but sometimes by direct
agglutination of red cells. Wra antibodies are mostly IgGl but sometimes IgM or IgM plus IgG. Anti-Wra has
been responsible for severe HDFN and HTRs. Alloanti-Wrb is rare and little is known about its clinical
significance, but autoanti-Wrb is a relatively common autoantibody and may be implicated inAIHA.
Other Diego Antigens
Since 1996,17 antigens of very low prevalence have been shown to represent amino acid substitutions in
Band 3 and have joined the Diego system: Wda, Rba, WARR, ELO, Wu, Bpa, Moa, Hga, Vga, Swa, BOW,
NFLD, Jna, Krep, Tra, Fra,
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AABB TECHNICAL MANUAL
and SW1. Anti-DISK detects a high-prevalence antigen that is antithetical to Wu. Anti-ELO and anti-BOW
have caused severe HDFN.
THE YT SYSTEM
Yta (YT1; His353) and Ytb (YT2; Asn353) are antithetical antigens on acetylcholinesterase, an enzyme that
is important in neurotransmission but has unknown function on red cells. Ytb has a prevalence of about 8% in
people of European or African ancestry but has not been found in people of Japanese ancestry; Yta has
relatively high prevalence in all populations. Yta is not affected by trypsin but is destroyed by a-chymotrypsin
treatment of the red cells; papain and ficin may also destroy the antigen, but this ability appears to depend on
the antiYta used. Yta and Ytb are sensitive to the disulfide-bond-reducing agents AET and DTT.
Yt antibodies are usually IgG and require an IAT for detection. They are not generally considered clinically
significant, although anti-Yta may cause accelerated destruction of Yt(a+)-transfused red cells and has been
implicated in acute and delayed HTRs.21,41
THE XG SYSTEM
Xga (XG1) is the only polymorphic blood group antigen encoded by an X-linked gene.41 Xgahas a
prevalence of about 66% in males and 89% in females. Part of the XG gene is within the X chromosome
pseudoautosomal region, a section at the tip of the short arm that pairs with the Y chromosome. CD99 (XG2) is
the second antigen of the Xg system. The CD99 gene is homologous to XG and is located on both X and Y
chromosomes, with pairing occurring at meiosis. Both CD99 and Xga expression appear to be controlled by a
common regulator gene, XGR. Although Xga antibodies occasionally agglutinate red cells directly, they are
generally IgG and are reactive by an IAT. They are not reactive with red cells treated with proteolytic enzymes.
Anti-Xga is not clinically significant. CD99 antibodies in common use are mostly monoclonal and of mouse
origin; a few human alloanti-CD99 occur, although little is known about their characteristics.
THE SCIANNA SYSTEM
The Scianna system consists of seven antigens on erythrocyte membrane-associated protein, a member of
the IgSF that has one IgSF domain.41,42 Scl (Gly57) and Sc2 (Arg57) are antithetical antigens of high and low
prevalence, respectively. Rd (SC4) has very low prevalence; Sc3, STAR, SCER, and SCAN all have very high
prevalence. Anti-Sc3 is produced by individuals with the very rare Scianna-null phenotype.
 No Scianna antibody has been implicated in an HTR or severe HDFN, although evidence is limited because
of the scarcity of the antibodies. Although directly agglutinating SCI antibodies are known, Scianna antibodies
are generally reactive by an IAT. Treatment of red cells by proteolytic enzymes has little effect on their
reactivity with Scianna antibodies, but disulfide-bond-reducing agents (AET and DTT) substantially reduce
reactivity.
THE DOMBROCK SYSTEM
The Dombrock system consists of eight antigens: the polymorphic antithetical antigens Doa (DOl; Asn265)
and Dob (D02; Asp265) and the high-prevalence antigens Gy3, Hy, Joa, DOYA, DOMR, and DOLG.3,43 Doa
and Dobhave a prevalence of 66% and 82%, respectively, in populations of European ancestry (Table 146). The
prevalence of Doais somewhat lower in populations of African ancestry and substantially lower in people of
East Asian ancestry. Anti-Gy3 is the antibody that is characteristically produced by immunized individuals with
the Dombrock-null [Gy(a—)] phenotype that results from various inactivating mutations. Two uncommon
phenotypes are present in individuals of African ethnicity: Hy- Jo (a-) (Glyl08Val) andHy+wJo(a-) (Thrl 17Ile),
which are usually associated with weak expression of Dob and Doa, respectively (Table 14-6). The Dombrock
glycoprotein (CD297) has a structure that is characteristic of an adenosine diphosphate ribosyltransferase, and
the Dombrock gene has the designation “ART4.”
Dombrock antigens are resistant to papain and ficin treatment of the red cells but are sensitive to trypsin, a-
chymotrypsin, and pro
355
TABLE 14-6. Phenotypes of the Dombrock System and Their Approximate Frequencies
Frequency (%)
Phenotype Doa Dob Gya Hy Joa Whites Blacks
Do(a+b—) + + + + 18 11
Do(a+b+) + + + + + 49 44
Do(a-b+) + + + + 33 45
Gy(a-) Rare 0
Hy +w +w 0 Rare
Jo(a-) +w -/+w + +w 0 Rare
DOYA +w +w +w Rare Rare
DOMR + +w +w Rare Rare
DOLG + +w + + Rare Rare
+w = weakened expression of antigen.
nase. They are also sensitive to the disulfidebond-reducing agents AET and DTT.
Dombrock antibodies are usually IgG and reactive by an IAT. They are in short supply and are often of poor
quality, with very weak reactivity. Screening for Dombrock-compatible donors, therefore, is best performed by
molecular genetics (SNP testing).
Anti-Doaand -Dob have been responsible for acute and delayed HTRs. There is little information regarding
the clinical significance of other Dombrock antibodies. No Dombrock antibody has caused HDFN.
THE COLTON SYSTEM
Coa (COl; Ala45) is a high-prevalence antigen; Cob (C02; Val45), its antithetical antigen, has a prevalence
of about 8% in people of European ancestry but is less common in other ethnic groups.41 Anti-Co3 is reactive
with all red cells except those of the extremely rare Co(a-b-) phenotype that results from various inactivating
mutations. Co4 (Gln47) is a high-prevalence antigen whose presence is required for the expression of Coa due
to the proximity of the polymorphism.44 The Colton antigens are located on aquaporin-1, a water channel (Fig
14-8).45 Colton antibodies are usually IgG and reactive by an IAT, although agglutinating IgM anti-Coa has
been found. Colton antibodies have been implicated in severe HDFN and HTRs. Colton antigens are resistant to
proteolytic enzymes.
THE LAND STEINER-WIENER SYSTEM
LW3 (LW5) and LWb (LW7; GlnlOOArg) are antithetical antigens of high and low prevalence,
respectively.41 Anti-LWab is reactive with all red cells except those of the extremely rare LWnull phenotype
and Rhnull cells, which are also LW(a-b-). LW antigens are expressed more strongly on D+ than D- red cells
and more strongly on umbilical cord red cells than on those of adults. LW antigens are unaffected by treatment
of the red cells with papain, ficin, trypsin, or a-chymotrypsin but are destroyed by pronase. Disulfide-bond-
reducing agents (AET and DTT) either destroy or greatly reduce LW3 or LWab (LW6) on red cells.
The LW glycoprotein is intercellular adhesion molecule-4 (ICAM-4), an IgSF adhesion molecule. ICAM-4
binds integrins on macrophages and erythroblasts, and it is probably

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AABB TECHNICAL MANUAL

FIGURE 14-8. A three-dimensional model of aquaporin-1, showing the six membrane-spanning domains as
cylinders. The first extracellular loop is glycosylated and contains the Coa/Cob polymorphism. The third
extracellular loop and first intracellular loop contain alanine (A)-proline (P)-asparagine (N) motifs and form a
channel in the membrane through which water molecules pass.
involved in the stabilization of erythroblastic islands in the marrow during the later stages of
erythropoiesis.45 ICAM-4 is also part of the Band 3/Rh ankyrin macrocomplex (Fig 14-1) of red cell surface
antigens and might maintain close contact between the red cell surface and the vascular endothelium.8
Upregulation of ICAM-4 on red cells of patients with sickle cell disease could play a part in the adhesion of
these cells to the vascular endothelium and the resultant crises of vascular occlusion.17,46
Most LW antibodies are reactive by an IAT. They are not generally considered to be clinically significant
and have not been implicated in HTRs or HDFN. Acquired and often tempo
rary LW-negative phenotypes sometimes occur with production of anti-LW' or anti-LW1*, a phenomenon
that is usually associated with pregnancy or hematologic malignancy. The transient antibodies behave like
alloantibodies but, strictly speaking, should be considered autoantibodies.
THE CHIDO/RODGERS SYSTEM
The nine antigens of the Chido/Rodgers system are not true blood group antigens because they are not
produced by erythroid cells. They are located on a fragment of the fourth component of complement (C4d) that
attaches to
357
the red cells from the plasma. Chi to Ch6, Rgl, and Rg2 each have a prevalence greater than 90%; WH has a
prevalence of about 15%. A complex relationship exists between the nine determinants and SNPs in C4A and
C4B, the genes encoding the C4a chains. The expression of Chido/Rodgers on red cells is destroyed by
treatment of the cells with proteolytic enzymes, such as papain or ficin.
No Chido/Rodgers antibodies are known to have caused HTRs or HDFN, and antigennegative blood is not
required for transfusion. Chido/Rodgers antibodies are generally IgG. Detection of these antibodies with native
red cells usually requires an IAT, but they often directly agglutinate red cells coated artificially with C4d.
Binding of Chido/Rodgers antibodies to red cells is readily inhibited by plasma from Ch/Rg+ individuals; this is
a useful aid to identification of these antibodies (Method 317).
THE GERBICH SYSTEM
The Gerbich system consists of six highly prevalent antigens—Ge2, Ge3, Ge4, GEPL, GEAT, and GETI—
and five antigens with very low prevalence—Wb, Lsa, Ana, Dha, and GEIS. These antigens are located on the
sialoglycoproteins glycophorin C (GPC), glycophorin D (GPD), or both. These two glycoproteins are produced
by the same gene, GYPC, by initiation of translation at two different sites on the mRNA. GPD lacks the N-
terminal 21 amino acids of GPC. GPC and GPD are part of the junctional complex of membrane proteins. Their
C-terminal cytoplasmic domains interact with the membrane skeleton through 4.1R, p55, and adducin and serve
as an important link between the membrane and its skeleton.9,45
GPC appears to be exploited as a receptor by some strains of the malarial parasite P. falciparum. There are
three types of “Gerbich-negative” phenotypes (Table 14-7). The first of these phenotypes, Ge:-2,-3,-4, is the
true null in which both GPC and GPD are absent from the red cells, and the cells are elliptocytic. In the other
phenotypes, Ge:-2,3,4 and Ge:-2, -3,4, GPD is absent and an abnormal form of GPC is present. Ge2, Ge3, and
Ge4 are de
stroyed by trypsin treatment of red cells. Whereas Ge2 and Ge4 are also sensitive to papain, Ge3 is resistant
to papain treatment. Consequently, papain-treated red cells can be used for distinguishing anti-Ge2 from
antiGe3 in the absence of the very rare Ge:-2,3,4 phenotype red cells.
Gerbich antibodies may be IgM and directly agglutinating, but most are IgG and require an IAT for
detection. They are not generally considered to be clinically significant and have not caused HTRs. However,
anti-Ge3 has caused HDFN that tends to manifest 2 to 4 weeks after birth. Some autoantibodies with
specificities resembling anti-Ge2 or -Ge3 have been responsible for AIHA.
THE CROMER SYSTEM
The 18 Cromer antigens are located on the complement-regulatory glycoprotein, decay accelerating factor
(DAF or CD55).3,47 They include the antithetical antigens Tca (Arg52), Tcb (Leu52), Tcc (Pro52), WESa
(Arg82), and WESb (Leu82). Tca and WESb have high prevalence, and Tcb, Tcc, and WESa, have low
prevalence, although both Tcb and WESa are present in approximately 0.5% of people of African ancestry and
WESa is present in 0.6% of people of Finnish ancestry. Other antigens—Cra, Dra, Es, 1FC, UMC, GUTI,
SERF, ZENA, CROV, CRAM, and CROZ, CRUE, and CRAG—have high prevalence.
Anti-IFC is the antibody made by individuals with the very rare Cromer-null phenotype (Inab phenotype),
and it is reactive with all red cells, apart from those of individuals with the
TABLE 14-7. Phenotypes Lacking HighPrevalence Gerbich Antigens and the Antibodies that May Be
Produced
Phenotype Antibodies
Ge:-2,3,4 (Yus type) Anti-Ge2
Ge:-2 —3,4 (Ge type) Anti-Ge2 or -Ge3
Ge:-2-3,-4 (Leach Anti-Ge2, -Ge3, or -Ge4
type)

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AABB TECHNICAL MANUAL
Inab phenotype. Cromer antigens are readily destroyed by a-chymotrypsin treatment of red cells but not by
papain, ficin, or trypsin treatment. The disulfide-bond-reducing agents AET and DTT reduce antigen expression
only slightly.
CD55 helps protect the red cells from lysis resulting from autologous complement by inhibiting the action
of C3-convertases. Inabphenotype red cells do not undergo undue hemolysis, however, because of the activity
of another complement regulatory glycoprotein, CD59. CD55 and CD59 are both linked to the red cell
membrane by a glycosylphosphatidylinositol (GPI) anchor. Pathological levels of hemolysis occur in
paroxysmal nocturnal hemoglobinuria, which is associated with a clonal defect in GPI biosynthesis and the
absence of both CD55 and CD59 in affected red cells.
Cromer antibodies are not usually considered to be clinically significant because there is no firm evidence
that any of them has caused an HTR, and the evidence from functional cellular assays is equivocal. No Cromer
antibodies have been implicated in HDFN, and they are probably sequestered by high levels of CD55 in the
placenta. Cromer antibodies are usually IgG and require an IAT for detection. They are inhibited by serum or
concentrated urine from antigen-positive individuals and are removed from serum by platelet concentrates.
THE KNOPS SYSTEM
The nine antigens of the Knops system are located on the complement-regulatory glycoprotein complement
receptor 1 (CR1 or CD35).48 All are polymorphic, although Kit', McCa, Sll, and Yka have relatively high
prevalence (Table 14-8).
 Kna/Knb represent Val 1561Met, McCa/ McCb, Lysl590Glu, S11/S12, and Argl601Gly. S13 requires the
presence of Serl610 and Argl601 (S12) for expression. Absence of KCAM results from an Ilel615Val
substitution. An apparent null phenotype, the Helgeson phenotype, indicates very low levels of red cell CR1 and
very weak expression of Knops antigens. Knops an
TABLE14-8. Approximate Frequencies of Knops Antigens in Two Populations
Frequency (%)
Antigen Whites Blacks
Kna KN1 99 100
Knb KN2 6 0
McCa KN3 98 94
Sll (Sla) KN4 98 60
Yka KN5 92 98
McCb KN6 0 45
SI2 KN7 0 80
SI3 KN8 100 100
KCAM KN9 98 20
tigens are generally resistant to papain and ficin, although this may depend on the antibodies used, and are
destroyed by trypsin or achymotrypsin treatment. They are also destroyed, or at least weakened, by AET and
DTT.
CR1 appears to be involved in the resetting of red cells that is associated with severe P. falciparum malaria.
The McCb and S12 alleles, present almost exclusively in individuals of African ancestry, may confer a degree
of protection from the parasite. This might explain the very strong difference in the prevalence of some
antigens, especially Sll, McCb, S12, and KCAM, among populations of European and African ethnicity (Table
14-8).
Knops antibodies are not clinically significant and can be ignored when selecting blood for transfusion.5
They are usually difficult to work with, often making it difficult to distinguish antigen-negative cells from those
with weak expression. They are generally IgG and reactive only by an IAT.
THE INDIAN SYSTEM
The low-prevalence antigen Ina (Arg46) and its antithetical antigen Inb (Pro46) plus two other high-
prevalence antigens, INFI and INJA, are located on CD44, the predominant cell surface

CHAPTER 14 Other Blood Groups

359
receptor for the glycosaminoglycan hyaluronan, a component of the extracellular matrix.41 AnWj (901009),
an antigen with very high prevalence, may also be located on or associated with CD44, but the evidence is
incomplete. Indian antigens have reduced expression on red cells with the In(Lu) phenotype, and AnWj is
virtually undetectable on In(Lu) cells. In' and Inb are sensitive to treatment of red cells with proteolytic
enzymes—papain, ficin, trypsin, achymotrypsin—and are also destroyed by the disulfide-bond-reducing agents
AET and DTT. AnWj, however, is resistant to all these enzymes but shows variable outcomes with reducing
agents.
Anti-Ina and -Inb often agglutinate red cells directly, but the reaction is usually enhanced by an IAT. Indian
antibodies are not generally considered to be clinically significant, although there is one report of anti-Inb
causing an HTR. AnWj, however, has caused severe HTRs, and In(Lu) red cells should be selected for
transfusion.5
THE OK SYSTEM
Oka, OKGV, and OKVM have very high prevalence and are located on the IgSF molecule CD147 or
basigin, which has two IgSF domains. Oka is resistant to proteolytic enzymes and disulfide-bond-reducing
agents. Only two alloanti-Oka antigens and a single example each of anti-OKGV and -OKVM are known; all
are reactive by an IAT.49 In-vivo survival tests and cellular functional assays with one antiOka have suggested
that it could be clinically significant, but no clinical information exists.
THE RAPH SYSTEM
 MER2 (RAPH1), which is located on the tetraspanin CD151, was initially defined by mouse monoclonal
antibodies that recognized a quantitative polymorphism, and about 8% of the population has undetectable levels
of MER2 on their mature red cells. Alloanti-MER2 was found in three Israeli Jews originating from India who
had a RAPH-null phenotype resulting from a single nucleotide deletion that led to a premature stop codon.
These three in
dividuals were CD 151 deficient and had endstage renal failure, sensorineural deafness, and pretibial
epidermolysis bullosa, suggesting that CD151 is essential for the proper assembly of basement membranes in
kidney, inner ear, and skin.50 MER2-negative individuals with anti-MER2 but only single amino acid
substitutions in CD151 do not have these symptoms.
MER2 antigen is resistant to treatment of red cells with papain but is destroyed by trypsin, a-chymotrypsin,
and pronase and by AET and DTT. MER2 antibodies react in an IAT. There is no evidence that anti-MER2 is
clinically significant.
THE JOHN MILTON HAGEN SYSTEM
This system consists of six antigens with very high prevalence—JMH, JMHK, JMHL, JMHG, JMHM, and
JMHQ—on the semaphorin glycoprotein CD108 (Sema7A). Anti-JMH is typically produced by individuals
with an acquired loss of CD108. This most often occurs in elderly patients and is associated with a weakly
positive direct antiglobulin test result. The absence of the other JMH antigens results from different missense
mutations in SEMA7A.51 JMH antigens are destroyed by proteolytic enzymes and disulfide-bond-reducing
agents. They are not detected on cord red cells.
JMH antibodies are usually reactive in an IAT. They are not generally considered to be clinically significant,
although one example was implicated in an acute HTR.
THE GILL SYSTEM
GIL antibodies detect a very high-prevalence antigen, GIL, located on aquaporin 3 (AQP3), a member of
the aquaporin superfamily of water and glycerol channels (like the Colton antigen).52 AQP3 enhances the
permeability of the red cell membrane by glycerol and water.
GIL antigen is resistant to proteolytic enzymes and disulfide-bond-reducing agents. GIL antibodies are
reactive by an IAT. Anti-GIL has not been implicated in HTRs or HDFN,
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AABB TECHNICAL MANUAL
although monocyte monolayer assays have suggested a potential to cause accelerated destruction of GIL+
red cells.
THE RHAG SYSTEM
The four antigens of the RHAG system are located on the Rh-associated glycoprotein (RhAG), which is also
described in Chapter 13.53 RhAG is closely associated with the Rh protein in the membrane as part of the Band
3/Rh/ankyrin macrocomplex (Fig 14-1). 01a is very rare, and homozygosity for the allele encoding 01a is
associated with an Rhmod phenotype. Duclos and DSLK have high prevalence, and absence of these antigens is
associated with an aberrant U (MNS5) antigen. RHAG4 is a low-prevalence antigen whose antibody was
associated with a single case of severe HDFN.3
THE FORS SYSTEM
FORS is a new blood group system consisting of a single antigen, Forssman glycosphingolipid antigen
(FORS1). The presence of FORS1 on human erythrocytes is unusual and was shown to be the result of an
enzyme-activating amino acid substitution arising from a missense mutation in the human Forssman synthase
gene GBGT1. FORS1 was demonstrated biochemically on the red cells of two blood donors from different
families with the Apae phenotype, which was first described in 1987.54,55Apae had been previously thought to
constitute a subgroup of A in the ABO system but has now been shown to be based on the presence of FORS1
antigen on red cells. Forssman synthase adds a terminal 3-a-Ai-acetylgalactosamine to its globoside acceptor.
F0RS1 is not usually present on the red cells of primates but is highly expressed on the red cells and
uroepithelia of lower mammals, such as dogs and sheep. As with other carbohydrate blood group antigens,
naturally occurring antibodies to FORS1 are present in all human sera, with the exception of rare FORS1+
individuals, and
have the potential to cause a positive crossmatch.
THE JR SYSTEM
The high-prevalence antigen Jra has been promoted to a new blood group system, JR, following the
independent findings of two groups demonstrating that the Jr(a-) phenotype was due to inactivating nucleotide
changes in ABCG2,56,57 The gene encodes ABCG2, a multipass membrane protein family member of the
adenosine triphosphate (ATP)-binding cassette transporters that is broadly distributed throughout the body. Jra
has long been associated with drug resistance in cancer and resistance to xenobiotics, and it might be important
for porphyrin homeostasis.58
The Jr(a-) phenotype is present predominantly in people of Japanese ancestry. Jra antigen is resistant to
proteolytic enzymes and disulfide-bond-reducing agents. Anti-Jra is reactive by an IAT and has caused HTR. It
is not usually implicated in HDFN, although one case has been reported.
THE LAN SYSTEM
Lan, another high-prevalence antigen, was also elevated to a new blood group system following the
discovery that it was carried on ABCB6, another ATP-binding cassette transporter molecule on the erythrocyte
membrane.59 Unlike Jra, Lan is not associated with any single geographic or ethnic group, and this is mirrored
by the diversity of mutant alleles in the Lan- individuals studied. ABCB6 is associated with porphyrin transport
and was thought to have an important role in heme synthesis.60 However, the existence of ABCB6deleted
individuals indicates that there maybe compensation by other transporters in the absence ofABCB6.
Lan antigen is expressed variably on red cells in different individuals but is resistant to proteolytic enzymes
and disulfide-bondreducing agents. Anti-Lan is reactive by an IAT
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CHAPTER
and has been implicated in HTR but not generally in HDFN.
THE VEL SYSTEM
Vel is a high prevalence blood group antigen that has been shown to depend on the presence of small
integral protein 1 (SMIM1), a protein of unknown function newly discovered on the erythrocyte surface.60'62
Absence of Vel antigen in the vast majority of individuals regardless of ethnic background, is due to a 17base
pair deletion in SMIM1, which results in the absence of the protein at the cell membrane.
Vel antigen expression is generally weak on cord RBCs and differs substantially from one individual to
another. Patterns of expression are a consequence both of zygosity for the 17-bp deletion and also for a SNP in a
GATA-1 transcription factor site in intron 2.62 Serological expression is not affected by protease treatment
although sensitivity to reducing agents such as 0.2 M DTT is variable. Anti-Vel are often a mixture of IgG and
IgM, readily activate complement and have been implicated in mild to severe HTRs, although HDFN is rare.
ANTIGENS THAT DO NOT BELONG TO A BLOOD GROUP SYSTEM
CD59—A Potential New Blood Group System
An antibody to a high-prevalence antigen detected in the plasma of a transfused CD59deficient child was
shown to be specific for CD59.63 The antibody was readily inhibited with soluble protein. Sequence analysis of
samples from the family revealed that the parents (first-degree cousins) were heterozygous and the child
homozygous for a silencing mutation in CD59. The antibody was an IgG antibody, and although the child’s
RBCs had been weakly DAT-positive following transfusion, incompatible blood was well-tolerated. Thus,
CD59 has been proposed as a blood group system but is as yet, unconfirmed.
14 Other Blood Groups
Blood Group Collections
Although many antigens belong to blood group systems, others have not been shown to belong to a system.
These are mostly antigens with either very high or very low prevalence. Some of them are included in blood
group collections that contain two or more antigens that are related serologically, biochemically, or genetically
but do not fit the criteria for system status.1
The high-prevalence antigen ABTI is serologically related to Vel. However, it has been excluded from
SMIM1 by sequencing analysis and thus remains in a collection. Like Vel, ABTI expression differs
substantially and it is generally expressed only weakly on cord red cells. ABTI is resistant to treatment of red
cells with proteolytic enzymes or disulfide-bond-reducing agents. Anti-ABTI has not caused HDFN and clinical
data are limited.
The Cost collection contains Csa and Csb, antithetical antigens with relatively high and low prevalence,
respectively. These antigens are serologically related to those of the Knops system but do not appear to be
located on CR1. Cost antibodies are not clinically significant.
Era and Erb are antithetical antigens with very high and low prevalence, respectively. Anti-Er3 is produced
by individuals with Er(ab-) red cells. There is no evidence that Er antibodies are clinically significant.
Carbohydrate antigens of the Ii and GLOB collections are described in Chapter 12.
 Carbohydrate antigens associated with MNS antigens that are not encoded by GYPA or GYPB are included
in a seventh collection, MNS CHO. These antigens have been shown to be due to altered glycosylation of the
O-linked sugars on CPA and GPB.2
High-Prevalence Antigens (901 Series)
The 901 series of the ISBT classification contains six antigens (Table 14-9): five have a prevalence well in
excess of 99%, whereas Sda has a prevalence of about 91%. All are inherited, and none is eligible to join a
system.1 All six antigens are resistant to papain, trypsin, achymotrypsin, and AET treatment of the red
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TABLE 14-9. Antigens of the 901 Series of High-Frequency Antigens and Their Clinical Significance
Antigen Number Clinical Significance
Ata 901003 Reported to have caused an HTR and mild HDFN
Emm 901008 No evidence of clinical significance
AnWj 901009 Severe AHTRs
Sda 901011 No evidence of clinical significance
PEL 901014 No evidence of clinical significance
MAM 901016 Severe HDFN
HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; AHTR = acute
HTR.
cells, and all except AnWj and Sda are expressed strongly on cord cells.
Sda represents carbohydrate structures on red cells, and the product of the Sda gene is probably a |:U 1,4)A?
-aceLylgalactosairiinyltransferase. The strength of Sda on red cells is highly variable, and Sda is not detected on
cord red cells. Agglutination of Sd(a+) red cells has a characteristic “mixed-field” appearance of agglutinates
and free red cells; when viewed microscopically, the agglutinates are refractile. Anti-Sda is inhibited by urine
from Sd(a+) individuals (Method 3-19), and by guinea pig urine.
Low-Prevalence Antigens (700 Series)
Eighteen antigens of very low prevalence in all of the populations tested constitute the 700 series of the
ISBT classification: By, Chra, Bi, Bxa, Toa, Pta, Rea, Jea, Lia, Milne, RASM, JFV, Kg, JONES, HJK, HOFM,
SARA, and REIT. All are inherited and do not fit any criteria for joining or forming a system.1
Antibodies to low-prevalence antigens do not present transfusion problems because compatible blood is
readily available. These antibodies remain undetected if a serologic crossmatch is not employed. Anti-JFV, -Kg,
-JONES, -JJJK, and -REIT have all caused HDFN.
HLA Antigens on Red Cells
“Bg” is the name given to HLA Class I antigens expressed on mature red cells. Bga represents
HLA-B7; Bgb, HLA-B17 (B57 or B58); and Bgc, HLA-A28 (A68 or A69, which cross-reacts with HLA-
A2). Many individuals, however, do not express Bg antigens on their red cells, despite having the corresponding
HLA antigens on their lymphocytes.
There are a few reports of Bg antibodies causing HTRs.64 These antibodies are sometimes present as
contaminants in reagents. HLA antigens on red cells are not destroyed by papain, ficin, pronase, trypsin, a-
chymotrypsin, AET, or DTT. They can be stripped from red cells with chloroquine (Method 2-20) or
EDTA/glycine-HCl (Method 2-21).
ERYTHROIDPHENOTYPES CAUSED BY MUTATIONS IN TRANSCRIPTION FACTOR GENES
Mutations in genes encoding erythroid transcription factors are emerging as important modifiers of blood-
group-antigen expression. As described in the “Lutheran System” section above, heterozygosity for different
mutations in KLF1 has been identified in individuals with the In(Lu) phenotype. In these individuals,
expression of antigens carried on CD44 (Ina/Inb) and the AnWj and PI antigens is weak.19 However, KLF1
mutations have also been shown to affect other genes, notably the /3-globin gene, resulting in the hereditary
persistence of fetal hemoglobin syndrome.65 Affected individuals have elevated HbF levels, some >30%, and
demonstrate an In(Lu) phenotype. Further
 363
more, discrete mutations in KLF1 appear to give rise to different phenotypes; for example, the change of
Glu325Lys does not result in the In(Lu) phenotype but is associated with severe congenital dyserythropoietic
anemia. These red cells demonstrate weakened expression of antigens in the Colton (AQP1), Cromer (DAF),
and LW (ICAM-4) blood group systems.66
Although mentioned above in the “Lutheran System” section, it is worth repeating that a mutation in GATA-
1 resulted in the Xlinked Lu(a-b-) phenotype in one family.20 It is likely that additional mutations in these and
other erythroid-specific transcription factors will be identified as the causes of altered blood group antigen
expression.
KEY POINTS
1. Of 339 recognized antigen specificities, 297 belong to 1 of 34 blood group systems representing either a
single gene or two or three closely linked homologous genes. Some groups of antigens that are not eligible to
join a system are classified together as collections. Antigens not classified in a system or collection have either
low or high prevalence and make up the 700 and 901 series, respectively.
2. M and N are antithetical, polymorphic antigens. M, N, S, s, and ‘N’ are all destroyed by treatment of the
red cells with papain, ficin, bromelin, or pronase, although this effect with S and s is variable. M and N—but
not S, s, or ‘N’—are destroyed by trypsin treatment.
3. Anti-M is relatively common, and anti-N is quite rare. Most anti-M and -N are not clinically significant.
When M or N antibodies active at 37 C are encountered, antigen-negative or compatible red cells should be
provided. Anti-S, -s, and -U are generally IgG antibodies that are active at 37C. They have been implicated in
HTRs and severe and fatal HDFN.
4. The antigen often referred to as “Kell” is correctly named “K” or “KEL1”; its antithetical antigen is k or
KEL2.
5. Because Kell antibodies can cause severe HDFN and HTRs, patients with Kell antibodies should be
transfused with antigen-negative blood whenever possible. Anti-K is the most common immune red cell
antibody not in the ABO and Rh systems.
6. In people of European or Asian ancestry, the Duffy polymorphism consists of two antigens, Fy1 * 3 and
Fyb, and three phenotypes, Fy(a+b—), Fy(a+b+), and Fy(a-b+). Fy3 and Fybare very sensitive to most
proteolytic enzymes. In people of African ancestry, a third allele, Fy, may result in neither Fy3 nor Fyb.
Individuals who are homozygous for Fy have the red cell phenotype Fy(a-b-).
7. Anti-Fy3 (common) and anti-Fyb (rare) are generally detected by an IAT and may cause acute or delayed
HTRs that are usually mild, although some have been fatal.
8. Kidd antigens are resistant to proteolytic enzymes, such as papain and ficin.
9. Kidd antibodies anti-Jk3 and -Jkb are not common, are generally present in antibody mixtures, and are
often difficult to detect. An IAT is usually required, and use of enzyme-treated cells may be necessary to detect
weaker antibodies. Kidd antibodies may cause severe acute HTRs and are a common cause of delayed HTRs.
10. The 22 antigens of the Diego system are located on Band 3, the red cell anion exchanger. Anti-Di3 and -
Wr3 can cause severe HDFN. Anti-Wr3 can also cause HTRs.
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1. Daniels GL, Fletcher A, Garratty G, et al. Blood
group terminology 2004: From the Interna
tional Society of Blood Transfusion committee
on terminology for red cell surface antigens. Vox Sang 2004;87:304-16.
 364
AABB TECHNICAL MANUAL
2. Storry JR, Castilho L, Daniels G, et al. International Society of Blood Transfusion Working Party on Red
Cell Immunogenetics and Blood Group Terminology: Berlin report. Vox Sang 2011;101:77-82.
3. Storry JR, Castilho L, Daniels G, et al. International Society of Blood Transfusion Working Party on Red
Cell Immunogenetics and Terminology: Cancun report. Vox Sang 2014 (in press).
4. International Society of Blood Transfusion. Blood group terminology. Amsterdam: ISBT, 2013.
[Available at http://www.isbtweb.org/ working-parties/red-cell-immunogeneticsand-blood-group-terminology
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5. Poole J, Daniels G. Blood group antibodies and their significance in transfusion medicine. Transfus Med
Rev 2007;21:58-71.
6. Reid ME, Lomas-Francis C, Olsson ML. The blood group antigen factsbook. London: Academic Press,
2012.
7. Daniels G. Human blood groups. 3rd ed. Oxford: Wiley Blackwell, 2013.
8. Bruce LJ, Beckmann R, Ribeiro ML, et al. A Band 3-based macrocomplex of integral and peripheral
proteins in the RBC membrane. Blood 2003;101:4180-8.
9. Mohandas N, Gallagher PG. Red cell membrane: Past, present, and future. Blood 2008; 112:3939-48.
10. Blumenfeld OO, Huang CH. Molecular genetics of the glycophorin gene family, the antigens for MNSs
blood groups: Multiple gene rearrangements and modulation of splice site usage result in extensive
diversification. Hum Mutat 1995;6:199-209.
11. Sim BK, Chitnis CE, Wasniowska K, et al. Receptor and ligand domains for invasion of erythrocytes by
Plasmodium falciparum. Science 1994;264:1941-4.
12. Mayer DC, Cofie J, Jiang L, et al. Glycophorin B is the erythrocyte receptor of Plasmodium falciparum
erythrocyte-binding ligand, EBL-1. Proc Natl Acad Sci U S A 2009; 106:5348-52.
 13. Klein HG, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 12th ed. Oxford: Wiley-
Blackwell, 2014.
14. Tippett P, Reid ME, Poole J, et al. The Miltenberger subsystem: Is it obsolescent? Transfus Med Rev
1992;6:170-82.
15. Broadberry RE, Lin M. The incidence and significance of anti- “Mia” in Taiwan. Transfusion
1994;34:349-52.
16. Crew VK, Green C, Daniels G. Molecular bases of the antigens of the Lutheran blood group system.
Transfusion 2003;43:1729-37.
17. Eyler CE, Telen MJ. The Lutheran glycoprotein: A multifunctional adhesion receptor. Transfusion
2006;46:668-77.
18. Karamatic Crew V Mallinson G, Green C, et al. Different inactivating mutations in the LU genes of
three individuals with the Lutherannull phenotype. Transfusion 2007;47:492-8.
19. Singleton BK, Burton NM, Green C, et al. Mutations in EKLF/KLF1 form the molecular basis of the
rare blood group In(Lu) phenotype. Blood 2008;112:2081-8.
20. Singleton BK, Roxby DJ, Stirling JW, et al. A novel GATA1 mutation (Stop414Arg) in a family with the
rare X-linked blood group Lu(a-b-) phenotype and mild macrothrombocytic thrombocytopenia. Br J Haematol
2012;161: 139-42.
21. Westhoff CM, Reid ME. Review: The Kell, Duffy, and Kidd blood group systems. Immunohematol.
2004;20:37-49.
22. Lee S, Zambas ED, Marsh WL, Redman CM. Molecular cloning and primary structure of Kell blood
group protein. Proc Natl Acad Sci U S A 1991;88:6353-7.
23. Kormoczi GF, Scharberg EA, Gassner C. A novel KEL*1,3 allele with weak Kell antigen expression
confirming the cis-modifier effect of KEL3. Transfusion 2009;49:733-9.
24. Daniels G, Hadley A, Green CA. Causes of fetal anemia in hemolytic disease due to anti-K. Transfusion
2003;43:115-16.
25. Lee S, Russo DC, Reiner AR et al. Molecular defects underlying the Kell null phenotype. J Biol Chem
2001;276:27281-9.
26. Lee S, Russo DC, Reid ME, Redman CM. Mutations that diminish expression of Kell surface protein
and lead to the Kmod RBC phenotype. Transfusion 2003;43:1121-5.
27. Lee S, Debnath AK, Redman CM. Active amino acids of the Kell blood group protein and model of the
ectodomain based on the structure of neutral endopeptidase 24.11. Blood 2003;102: 3028-34.
28. Danek A, Rubio JR Rampoldi L, et al. McLeod neuroacanthocytosis: Genotype and phenotype. Ann
Neurol 2001;50:755-64.
29. Tournamille C, Colin Y, Cartron JP, Le Van KC. Disruption of a GATA motif in the Duffy gene promoter
abolishes erythroid gene expression in Duffy-negative individuals. Nat Genet 1995; 10:224-8.
CHAPTER 14 Other Blood Groups
365
30. Inoue H, Kozlowski SD, Klein JD, et al. Regulated expression of renal and intestinal UT-B urea
transporter in response to varying urea load. Am J Physiol Renal Physiol 2005;289:F451-F8.
31. Hadley TJ, Peiper SC. From malaria to chemokine receptor: The emerging physiologic role of the Duffy
blood group antigen. Blood 1997; 89:3077-91.
32. Horuk R, Chitnis CE, Darbonne WC, et al. A receptor for the malarial parasite Plasmodium vivax: The
erythrocyte chemokine receptor. Science 1993;261:1182-4.
33. Daniels G. The molecular genetics of blood group polymorphism. Hum Genet 2009; 126: 729-42.
34. Hadley TJ, Lu ZH, Wasniowska K, et al. Postcapillary venule endothelial cells in kidney express a
multispecific chemokine receptor that is structurally and functionally identical to the erythroid isoform, which is
the Duffy blood group antigen. J Clin Invest 1994;94:985-91.
35. Daniels G, Bromilow IM. Essential guide to blood groups. 2nd ed. Oxford: Wiley Blackwell, 2010.
36. Holt S, Donaldson H, Hazlehurst G, et al. Acute transplant rejection induced by blood transfusion
reaction to the Kidd blood group system. Nephrol Dial Transplant 2004;19:2403-6.
37. Sands JM. Molecular mechanisms of urea transport. J Membr Biol 2003; 191:149-63.
38. Heaton DC, McLoughlin K. Jk(a-b-) red blood cells resist urea lysis. Transfusion 1982;22:70-1.
39. Sands JM, Gargus JJ, Frohlich O, et al. Urinary concentrating ability in patients with Jk(a-b-) blood type
who lack carrier-mediated urea transport. J Am Soc Nephrol 1992;2:1689-96.
 40. Byrne KM, Byrne PC. Review: Other blood group systems—Diego, Yt, Xg, Scianna, Dombrock,
Colton, Landsteiner-Wiener, and Indian. Immunohematology 2004;20:50-8.
41. Wagner FF, Poole J, Flegel WA. Scianna antigens including Rd are expressed by ERMAP. Blood
2003;101:752-7.
42. Reid ME. Complexities of the Dombrock blood group system revealed. Transfusion 2005;45: 92S-9S.
43. Arnaud L, Helias V, Menanteau C, et al. A functional AQP1 allele producing a Co(a-b-) phenotype
revises and extends the Colton blood group system. Transfusion 2010;50:2106-16.
44. Daniels G. Functions of red cell surface proteins. Vox Sang 2007;93:331-40.
45. Zennadi R, Moeller BJ, Whalen EJ, et al. Epinephrine-induced activation of LW-mediated
sickle cell adhesion and vaso-occlusion in vivo. Blood 2007;110:2708-17.
46. Storry JR, Reid ME, Yazer MH. The Cromer blood group system: A review. Immunohematology
2010;26:109-18.
47. Moulds JM. The Knops blood-group system: A review. Immunohematology 2010;26:2-7.
48. Smart EA, Storry JR. The OK blood group system: A review. Immunohematology 2010;26:
124-6.
49. Karamatic Crew V Burton N, Kagan A, et al. CD151, the first member of the tetraspanin (TM4)
superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in
kidney and skin. Blood 2004;104:2217-23.
50. Seltsam A, Strigens S, Levene C, et al. The molecular diversity of Sema7A, the semaphorin that carries
the JMH blood group antigens. Transfusion 2007;47:133-46.
51. Roudier N, Ripoche P, Gane P, et al. AQP3 deficiency in humans and the molecular basis of a novel
blood group system, GIL. J Biol Chem 2002;277:45854-9.
52. Tilley L, Green C, Poole J, et al. A new blood group system, RHAG: Three antigens resulting from
amino acid substitutions in the Rh-associated glycoprotein. Vox Sang 2010;98:151-9.
53. Svensson L, Hult AK, Stamps R, et al. Forssman expression on human erythrocytes: Biochemical and
genetic evidence of a new histo-blood group system. Blood 2013;121:1459-68.
54. Stamps R, Sokol RJ, Leach M, et al. A new variant of blood group A. Apae. Transfusion 1987; 27:315-
18.
55. Saison C, Helias V Ballif BA, et al. Null alleles of ABCG2 encoding the breast cancer resistance protein
define the new blood group system Junior. Nat Genet 2012;44:174-7.
56. Zelinski T, Coghlan G, Liu XQ, Reid ME. ABCG2 null alleles define the Jr(a-) blood group phenotype.
Nat Genet 2012;44:131-2.
57. Robey RW, To KK, Polgar O, et al. ABCG2: A perspective. Adv Drug Deliv Rev 2009;61:3-13.
58. Helias V Saison C, Ballif BA, et al. ABCB6 is dispensable for erythropoiesis and specifies the new
blood group system Langereis. Nat Genet 2012;44:170-3.
59. Krishnamurthy P, Schuetz JD. The role of ABCG2 and ABCB6 in porphyrin metabolism and cell
survival. Curr Pharm Biotechnol 2011; 12:647-55.
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60. Ballif BA, Helias V Peyrard T, et al. Disruption of SMIM1 causes the Vel- blood type. EMBO Mol Med
2013;5:751-61.
61. Storry JR, Joud M, Christophersen MK, et al. Homozygosity for a null allele of SMIM1 defines the Vel-
negative blood group phenotype. Nat Genet 2013;45:537-41.
62. Cvejic A, Haer-Wigman L, Stephens JC, et al. SMIM1 underlies the Vel blood group and influences red
blood cell traits. Nat Genet 2013; 45:542-5.
63. Anliker M, von Zabern I, Hochsmann B, et al. A new blood group antigen is defined by anti
CD59, detected in a CD59-deficient patient. Transfusion 2014 (inpress).
64. Nance ST. Do HLA antibodies cause hemolytic transfusion reactions or decreased RBC survival?
Transfusion 2003;43:687-90.
65. Borg J, Papadopoulos G, Georgitsi M, et al. Haploinsufficiency for the erythroid transcription factor
KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 2010;42:801-5.
66. Arnaud L, Saison C, Helias V et al. A dominant mutation in the gene encoding the erythroid
transcription factor KLF1 causes a congenital dyserythropoietic anemia. Am J Hum Genet 2010;87:721-7.
Chapter 16
 Identification of Antibodies to Red Cell Antigens
Phyllis S. Walker; MS, MT(ASCP)SBB, and Janis R. Hamilton, MS, MT(ASCP)SBB
NATURALLY OCCURRING ANTI-A Off and anti-B are the only red cell antibodies that are commonly
found in human serum or plasma. All other antibodies are called “unexpected red cell antibodies.” This chapter
discusses methods for detecting and identifying unexpected red cell antibodies.
There are two types of unexpected red cell antibodies: alloantibodies and autoantibodies. When someone
produces an antibody to an antigen that he or she lacks, the antibody is called an “alloantibody.” When someone
produces an antibody to an antigen that he or she possesses, the antibody is called an “autoantibody.” Therefore,
by definition, alloantibodies react only with allogeneic red cells that express the corresponding antigens—not
with the antibody producer’s red cells. Conversely, autoantibodies are reactive with the red cells of the antibody
producer. In fact, autoantibodies usually are reactive with most reagent red cells as well as with autologous red
cells.
Immunization to red cell antigens may result from pregnancy, transfusion, transplantation, needle sharing, or
injections of immunogenic material. The frequency of alloimmunization is extremely variable depending on the
patients or donors being studied. Healthy blood donors have very low immunization rates. In chronically
transfused patient populations, such as those with sickle cell anemia or thalassemia, as many as 14% to 50% of
individuals are reported to be alloimmunized.1'3
In some instances, no specific immunizing event can be identified. In such cases, naturally occurring
antibodies have presumably resulted from exposure to environmental, bacterial, or viral antigens that are similar
to blood group antigens. Also, antibodies detected in serologic tests may be passively acquired from injected
immunoglobulin, donor plasma, passenger lymphocytes in transplanted organs, or hematopoietic progenitor
cells (HPCs).

Phyllis S. Walker, MS, MT(ASCP)SBB, retired from Reference Laboratory, Blood Centers of the Pacific-
Irwin Center, San Francisco, California, and Janis R. Hamilton, MS, MT(ASCP)SBB, Manager, Reference
Laboratory, American Red Cross Blood Services, Detroit, Michigan
P. Walker has disclosed no conflict of interest. J. Hamilton has disclosed a financial relationship with Bio-
Rad Laboratories, Inc.
391

392
AABB TECHNICAL MANUAL
SIGNIFICANCE OF ALLOANTIBODIES
Alloantibodies to red cell antigens may be detected initially with any test that uses serum or plasma (eg, an
ABO test, antibody detection test, or compatibility test) or in an eluate prepared from red cells coated with
alloantibody. Serum and plasma are interchangeable for antibody testing unless complement is required for
antibody detection. In such rare cases, only serum provides complement. Throughout this chapter, serum can be
considered to be interchangeable with plasma unless the text indicates otherwise.
After an antibody has been detected, its specificity should be determined and its clinical significance
assessed. A clinically significant red cell antibody is defined as an antibody that is frequently associated with
hemolytic disease of the fetus and newborn (HDFN), hemolytic transfusion reactions, or a notable decrease in
transfused red cell survival. The degree of clinical significance varies among antibodies with the same
specificity; some cause destruction of incompatible red cells within hours or even minutes, whereas others
decrease red cell survival by only a few days, and still others do not shorten red cell survival discernibly. Some
antibodies are known to cause HDFN, whereas others may cause a positive direct antiglobulin test (DAT) result
in the fetus without clinical evidence of HDFN.
PREANALYTICAL
CONSIDERATIONS
Before antibody identification testing is started, the patient’s medical history should be considered if such
information can be obtained. Exposure to foreign red cells through blood transfusion or pregnancy is the usual
cause of red cell immunization. It is uncommon for patients who have never been transfused or pregnant to
produce clinically significant alloantibodies, although “naturally occurring” antibodies may be present. If the
patient has been transfused, it is critical to know when the most recent transfusion was
 given. If the patient was transfused during the past 3 months, primary immunization to red cell antigens may
be a risk. In addition, the presence of circulating donor red cells may cause mixed-field results in antigen-typing
tests, and autologous adsorption techniques should not be used because alloantibodies could be adsorbed onto
transfused donor red cells. In general, women who may have been sensitized by pregnancy are more likely to
have alloantibodies than men. Infants who are younger than 6 months usually do not produce alloantibodies, but
newborns may have passive antibody of maternal origin.
Certain diseases have been associated with red cell antibodies; depending on the methods used, such
antibodies may be detectable in antibody detection and identification tests. Cold agglutinin syndrome, Raynaud
phenomenon, and infections with Mycoplasma pneumoniae are often associated with anti-I. Infectious
mononucleosis is sometimes associated with anti-i. Patients with paroxysmal cold hemoglobinuria, which is
associated with syphilis in adults and viral infections in children, may demonstrate autoantibodies with anti-P
specificity. Warm autoantibodies often accompany diagnoses such as warm autoimmune hemolytic anemia,
systemic lupus erythematosus, multiple myeloma, chronic lymphocytic leukemia, or lymphoma. Patients who
have received solid-organ or HPC transplants may demonstrate passive antibodies that originate from donor
passenger lymphocytes.
Drugs are known to cause antibody identification problems. (See Chapter 17 for a discussion of drug-related
mechanisms and drugs that are associated with serologic problems.) Administration of intravenous immune
globulin (IVIG) and Rh Immune Globulin (RhIG) can interfere with antibody-screening tests. Some lots of
IVIG have been reported to contain unexpected antibodies, including anti-A and anti-B. Intravenous RhIG,
which is sometimes used to treat thrombocytopenia, could explain the presence of anti-D in an Rhpositive
patient.
Finally, when a patient is suspected of having an antibody to a high-prevalence anti
393
gen, the patient’s ethnic origin may provide clues to the specificity of the antibody. Table 16-1 lists some
rare blood types that are more commonly associated with certain ethnic groups.4
ANALYTICAL PHASE OF ANTIBODY IDENTIFICATION
Specimen Requirements
Either serum or plasma may be used for antibody detection and identification; however, plasma may not be
suitable for detecting complement-activating antibodies. Depending on the test methods used, a 5-mL to 10-mL
aliquot of whole blood usually contains enough serum or plasma for identifying simple antibody specificities;
more whole blood may be required for complex studies. When autologous red cells are studied, the use of
samples anticoagulated with EDTA avoids problems associated with the in-vitro uptake of complement
components by red cells, which may occur when clotted samples are used.
Reagents and Test Methods
Antibody Detection Red Cells
Group 0 red cells suitable for pretransfusion antibody screening are commercially available and offered as
sets of either two or three reagent single-donor red cells. Pooled red cells for antibody detection are usually
obtained from two different donors and may be used only when donor serum is tested. Each laboratory should
decide whether to use two or three reagent donor red cells for antibody-detection testing. All reagent red cells
licensed by the Food and Drug Administration (FDA) for this purpose must express the following antigens: D,
C, E, c, e, M, N, S, s, PI, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb. Three-cell antibody detection sets usually offer
red cells from presumed homozygous donors with double-dose expression for the following common antigens:
D, C, E, c, e, M, N, S, s, Fya, Fyb, Jka, and Jkb. Some weakly reactive antibodies are reactive only with red
cells from donors who are homozygous for the genes encoding the antigens; this serologic
phenomenon is known as “dosage.” Antibodies in the Rh, MNS, Duffy, and Kidd systems most commonly
demonstrate dosage. Reagent red cells should be refrigerated when not in use, and they should not be used for
antibody detection after their expiration date.
Antibody Identification Panels
Identification of an antibody to red cell antigen (s) requires testing the serum against a panel of selected red
cell samples (typically 814 samples) with known antigenic composition for the major blood groups. Usually, the
red cell samples are obtained from commercial suppliers, but institutions may assemble their own panels using
red cells from local sources. Except in special circumstances, panel cells are group 0, thereby allowing the
serum of any ABO group to be tested.
 Each reagent red cell of the panel is from a different donor. The reagent red cells are selected so that if one
takes all of the examples of red cells into account, a distinctive pattern of positive and negative reactions exists
for each of many antigens. To be functional, a reagent red cell panel must make it possible to identify with
confidence those clinically significant alloantibodies that are most commonly encountered, such as anti-D, -E, -
K, and -Fy3. The phenotypes of the reagent red cells should be distributed so that single specificities of the
common alloantibodies can be clearly identified and most others can be excluded. Ideally, patterns of reactivity
for most examples of single alloantibodies should not overlap with any other (eg, all of the K+ samples should
not be the only ones that are also E+). Also, it is important to include reagent red cells with double-dose antigen
expression to detect common antibodies that frequently show dosage. Commercial panels are accompanied by a
sheet that lists the phenotypes of the reagent red cells. The combination of reagent red-cell samples varies from
lot to lot, so it is essential to use the correct phenotype sheet when interpreting panel results. Commercial
reagent red cells for tube testing are diluted to a 2% to 5% suspension in a preservative solution that can be used
directly from the bottle. Washing the
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AABB TECHNICAL MANUAL
TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations
Phenotype Population
AnWj Transient>lsraeli Arabs (inherited)
At(a-) Blacks
Cr(a-) Blacks
Di(b-) South Americans>Native AmericansxJapanese
DISK Dutch>Europeans>any
Dr(a-) Jews from Bukhara>Japanese
En(a-) Finns>Canadians>English>Japanese
Es(a-) Mexicans, South Americans, Blacks
Fy(a-b-) Blacks>Arabs/Jews>others of Mediterranean ancestry>Whites
Ge:-2,-3 (Gerbich phenotype) Papua New Guineans>Melanesians>Whites>any
Ge:-2,3 (Yus phenotype) Mexicans>lsraelis>Mediterraneans>any
Ge:-2,-3,-4 (Leach phenotype) Any
GUTI Chileans
Gy(a-) Eastern Europeans (Romany)>Japanese
hrB Blacks
hrs Blacks
Hy Blacks
IFC (Crnull, Inab) Japanese>any
In(b-) lndians>lranians>Arabs
Jk(a-b-) Polynesians>Finns>Japanese>any
Jo(a-) Blacks
Jr(a-) Japanese>Asians>Europeans>Bedouin Arabs>any
Js(b-) Blacks
k Whites>any
K„(Knull) Reunion lslanders>Finns>Japanese>any
K12 Whites
K14 French-Cajuns
K22 Israelis
KCAM Blacks>any
Kn(a-) Whites>Blacks>any
 395
TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations (Continued)
Phenotype Population
Kp(b-) WhitesxJapanese
KUCI Native Americans
Lan Whites>Japanese>Blacks>any
Lu(a-b-) Any
Lu20 Israelis
Lu21— Israelis
LW(a-b-) Transient in any>inherited in Canadians
LW(a-) Those of Baltic Sea region
MAM Arabs>any
MAR Finns>any
McC(a-) Blacks>Whites>any
MER2 Indian Jews, Turks, Portuguese
MkMk SwissxJapanese
0h (Bombay) lndians>Japanese>any
Ok(a-) Japanese
P Japanese>Finns>lsraelis>any
Para-Bombay Reunion lslanders>lndians>any
PEL French-Canadians
PP1 Pk Swedes>Amish>lsraelis>Japanese>any
Sl(a-) Blacks>Whites>any
Tc(a-b+c-) Blacks
Tc(a-b-c+) Whites
SERF Thais
U- and S-s-U+var Blacks
UMC Japanese
Vel Swedes>any
WES(b-) Finns>Blacks>any
Yk(a-) Whites>Blacks>any
Yt(a-) Arabs>Jews>any
Used with permission from Reid, Lomas-Francis, and Olsson.4 Any = may be found in any population; > =
more prevalent than.
 396
AABB TECHNICAL MANUAL
red cells before use is usually unnecessary unless the preservative solution is suspected of interfering with
antibody identification.
Panel cells should not be used after the expiration date; however, this restriction is not always practical.
Most serologists use in-date reagent cells for initial antibody identification panels and, if necessary, use expired
reagent red cells to exclude or confirm specificities. Each laboratory should establish a policy for using expired
reagent red cells and validate any procedures associated with this practice.5[p21)
Antiglobulin Reagents
Most antibody detection and identification studies include an indirect antiglobulin test (IAT) phase.
Antihuman globulin (AHG) can be specific only for human immunoglobulin G (IgG), or a polyspecific reagent
that contains anti-IgG and anti-complement maybe used. A polyspecific reagent may detect—or may detect
more readily—antibodies that bind complement. To detect complement binding, serum rather than plasma must
be used because the anticoagulant binds calcium, making it unavailable for complement activation. Although
complement binding may be advantageous in some instances, such as the detection of certain JK system
antibodies, many serologists prefer the routine use of IgG-specific AHG reagents to avoid unwanted reactivity
resulting from in-vitro complement binding by cold-reactive antibodies.6
Enhancement Media
Although the test system may consist solely of serum and red cells (either reagent red cells as provided by
the manufacturer or saline-suspended red cells), most serologists use some type of enhancement medium.
Several different enhancement media are available, including low-ionic strength saline (LISS), polyethylene
glycol (PEG), and 22% bovine albumin. Additional enhancement techniques may be used for complex studies.
Enhancement techniques are discussed in more detail later in this chapter.
Test Methods
Hemagglutination testing performed in tubes has been the gold standard of immunohematology testing for
decades, but testing by gelcolumn agglutination and solid-phase technology are commonplace. These methods
offer stable and possibly less subjective endpoints, work-flow standardization, and the ability to be incorporated
into semiautomated or automated systems. They provide a sensitive detection system for most blood-group
antibodies. Both gel and solid-phase methods have also been shown to enhance serologic reactivity that may not
be clinically significant in the selection of units for transfusion, including the reactivity of warm autoantibodies.
Reactivity that is dependent on the diluent in commercially prepared reagent cells can also be seen in gel-
column tests. Published studies have compared the various methods for detection of wanted and unwanted red
cell alloantibodies.7'9
Recent reports have illustrated the potential effect of using red cell membranes vs intact red cells to coat the
microwells in solid-phase tests.10,11 In selected samples, reactivity was found with the disrupted red cell
membranes, but the samples were not reactive when the same intact red cells were used. Laboratories using
these techniques for routine testing and initial problem solving should be familiar with the unique reactivity
characteristics of their chosen method. Testing algorithms that involve both nontube and tube techniques are
frequently developed to address anomalous reactivity in either gel-column or solid-phase testing if specificity
cannot be assigned. When careful review of initial and extended testing using these methods does not allow
identification of alloantibody specificity(ies), a tube test performed with LISS or PEG enhancement media has
been used to further evaluate the sample for clinically significant alloantibodies.
Basic Antibody Identification
Identification Panel
For initial antibody identification panels, it is common to use the same methods and test
397
phases as in the antibody-detection test or crossmatch. The gel and solid-phase methods involve a single
reading of the test at the indirect antiglobulin phase. Tube-testing protocols have greater flexibility for readings
at different test phases but many serologists also utilize this single test reading because the IAT detects the
overwhelming majority of clinically significant alloantibodies.
Some serologists using tube methods may choose to include an immediate centrifugation reading, a room-
temperature incubation that is read before an enhancement medium is added, or both. Such an approach may
enhance the detection of certain antibodies (eg, anti-M, -N, -PI, -I, -Lea, or-Leb) and may help explain reactions
detected in other phases. These steps are frequently omitted in initial antibody-identification studies because
most antibodies that are reactive only at lower temperatures have little or no clinical significance.
Test observation after 37 C incubation in tube testing is influenced by the enhancement media used. Tests
employing PEG enhancement cannot be centrifuged and read because the reagent causes nonspecific
aggregation of all red cells. LISS, albumin, and saline (no enhancement) tests do not have this restriction. A 37
C reading can detect some antibodies (eg, potent anti-D, -E, or -K) that may cause direct agglutination of red
cells. Other antibodies (eg, anti-Lea or -Jka) may occasionally be detected by the lysis of antigen-positive red
cells during the 37 C incubation if serum is tested. Omitting centrifugation and the reading at 37 C should
lessen the detection of unwanted positive reactions caused by clinically insignificant cold-reactive
autoantibodies and alloantibodies. However, in some instances, potentially clinically significant antibodies are
detected only by their 37 C reactivity. Examples of 103 such antibodies (63-E, 27-K, 5-Jk\ 4-D, 3-cE, and 1-C)
were identified in 87,480 samples.12 If the 37 C reading is desired in a specific antibody study, an alternative
strategy to avoid the uptake of cold antibodies is to set up duplicate tests. One test is read after the 37 C
incubation, and the other test is read only at the AHG phase.
Selected Cell Panel
If a patient has antibodies that were identified previously, the known antibodies should be considered when
selecting panel cells to test. For example, if the patient has known anti-e, it will not be helpful to test the
patient’s serum with a routine panel, in which 9 of 10 cells are e-positive. Testing a selected panel of e-negative
red cells is a better approach to find any newly formed antibodies. It is not necessary to test e-positive cells to
reconfirm the previously identified anti-e.
If the patient’s red cell phenotype is known, reagent red cells selected to detect only those alloantibodies that
are potentially formed by the patient may be tested. For example, if the patient’s Rh phenotype is CE+c+e-,
cells selected to exclude anti-E and anti-c should not be necessary or can be limited to a single selected cell
because the patient is not expected to form alloantibodies to these antigens. Exceptions include patients with
weak or altered (partial) Rh antigens, which are usually found in minority ethnic groups, and patients whose Rh
phenotype was determined by deoxyribonucleic acid (DNA) testing rather than serology and who could be
carrying a silenced allele. This approach can minimize the amount of testing required.
INTERPRETING RESULTS. Antibody-detection results are interpreted as positive or negative according to
the presence or absence of reactivity (ie, agglutination or hemolysis), respectively. Interpretation of panel results
can be a more complex process combining technique, knowledge, and intuitive skills. Panel results generally
include both positive and negative results, sometimes at different phases of testing; each positive result should
be explained by the final conclusion. The patient’s phenotype and the probability of the antibody specificity are
also taken into account in the final interpretation.
POSITIVE AND NEGATIVE REACTIONS. Both positive and negative reactions are important in antibody
identification. The phase and strength of positive reactions can suggest certain specificities. (See Method 1-9 for
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AABB TECHNICAL MANUAL
grading agglutination.) Positive reactions are compared to the antigen patterns of the panel cells to help
assign specificity. A single alloantibody usually produces a clear pattern with antigen-positive and antigen-
negative reagent red cells. For example, if a serum sample is reactive only with red cell samples 3 and 5 of the
reagent red cell panel shown in Table 16-2, anti-E is very likely present. Both reactive red cells express the
antigen, and all nonreactive cells lack it. When there is no discernible pattern to explain the reactivity, possible
explanations include multiple antibodies, dosage, and variations in antigen expression. These factors are
discussed in more detail later in this chapter. Negative reactions are important in antibody identification because
they allow tentative exclusion of antibodies to antigens expressed on the nonreactive red cells. Exclusion of
antibodies is an important step in the interpretation process and must be performed to ensure proper
identification of all of the antibodies present.
EXCLUSION, “RULE OUT,” OR “CROSS OUT.” A widely used first approach to the interpretation of
panel results is to exclude specificities on the basis of nonreactivity of the patient’s serum with red cells that
express the antigen. Such a system is sometimes referred to as a “cross-out” or “rule-out” method. Once results
have been recorded on the work sheet, the antigen profile of the first nonreactive red cell is examined. If an
antigen is present on the red cell sample and the serum was not reactive with it, the presence of the
corresponding antibody may tentatively be excluded. Many technologists actually cross out such antigens from
the list at the top of the panel sheet to facilitate the process. After all of the antigens on the list for that red cell
have been crossed out, the same process is performed with the other nonreactive red cells; additional
specificities are then excluded. In most cases, this process leaves a group of antibodies that have not been
excluded.
Next, the red cells that are reactive with the serum are evaluated. The pattern of reactivity for each
specificity that was not excluded is compared to the pattern of reactivity ob
tained with the test serum. If there is an antigen pattern that matches the test serum pattern exactly, this
pattern most likely identifies the specificity of the antibody in the serum. If there are remaining specificities that
were not excluded, additional testing is needed to eliminate the possibilities and confirm the suspected
specificity. This process requires testing of the serum with additional selected red cells.
Although the exclusion (rule-out) approach often identifies simple antibodies, it should be considered only
as a provisional step, particularly if the rule out was based on the lack of reactivity with red cells that have
weaker expression of the antigen (eg, cells from heterozygous donors).
SELECTED CELLS. Selected cells are red cells that have been chosen because they express some specific
antigens and lack others. Selected red cells with different antigen combinations can be used to confirm or rule
out the presence of antibodies. For example, if a pattern of reactive red cells fits anti-Jka exactly, but anti-K and
anti-S were not excluded, the serum should be tested with selected red cells. Ideally, red cells with the following
phenotypes should be chosen: Jk(a-), K+, S-; Jk(a-), K-, S+; and Jk(a+), K-, S-. The reaction pattern with these
red cells should both confirm the presence of anti-Jka and include or exclude anti-K and anti-S. Whenever
possible, selected red cells should have a strong expression of the antigen being tested (ie, from homozygous
donors or red cells with double-dose expression). Such red cells help ensure that nonreactivity with the selected
red cell indicates the absence of the antibody and not that the antibody was too weak to be reactive with a
selected red cell that had a weak expression of the antigen.
PROBABILITY. To ensure that an observed pattern of reactions is not the result of chance alone, conclusive
antibody identification requires the serum to be tested against a sufficient number of reagent red cell samples
that lack—and express—the antigen that corresponds with the antibody’s apparent specificity. A standard
approach (which is based on Fisher’s exact method) has been to require that
399
 400
AABB TECHNICAL MANUAL
three antigen-positive red cell samples are reactive and that three antigen-negative red cell samples are not
reactive for each specificity identified.13 In some cases, the use of two reactive and two nonreactive red cell
samples is also an acceptable approach for antibody confirmation.5,14 When that approach is not possible, a
more liberal approach (which is derived from calculations by Harris and Hochman15) allows the minimum
requirement for a probability (p) value of <0.05 to be met with two reactive and three nonreactive red cell
samples or with one reactive and seven nonreactive red cell samples (or the reciprocal of either combination).
Comparative p-value calculations are shown in Table 16-3. Additional details on calculating probability may be
found in the suggested readings list at the end of this chapter. The possibility of false-negative results with
antigen-positive red cells must be considered as well as that of unexpected positive results (ie, caused by the
presence of an additional antibody or an error in the presumptive antibody identification).
Autologous Control
The autologous control (autocontrol), in which serum and autologous red cells are test
ed under the same conditions as serum and reagent red cells, is an important part of antibody identification.
The autocontrol is not the same as or equivalent to a DAT (Method 3-14). Incubation and the presence of
enhancement reagents may cause reactivity in the autocontrol that is only an in-vitro phenomenon. If the
autocontrol is positive in the antiglobulin phase, a DAT should be performed. If the DAT result is negative,
antibodies to an enhancement medium constituent or autoantibodies that are reactive only in the enhancement
medium should be considered. Warm autoantibodies and cold autoantibodies, such as anti-I, -IH, or -Pr, may be
reactive in an IAT when certain enhancement media are used; therefore, testing should be repeated in another
medium. If the DAT result is positive, it must be interpreted with careful attention to the transfusion history.
Autoantibodies or drugs could explain a positive DAT result; however, if the patient has an alloantibody and
was recently transfused with blood that expressed the corresponding antigen, the circulating donor red cells
could be coated with alloantibody, resulting in a positive DAT result associated with a clinically significant
delayed transfusion reaction.
TABLE 16-3. Probability Values for Selected Antibody Identification Approaches
No. No. No.
Fisher13 Harris-Hochman15
Tested Positive Negative
5 3 2 0.100 0.035
6 4 2 0.067 0.022
6 3 3 0.050 0.016
7 5 2 0.048 0.015
7 4 3 0.029 0.008
8 7 1 0.125 0.049
8 6 2 0.036 0.011
8 5 3 0.018 0.005
8 4 4 0.014 0.004

CHAPTER 16 Identification of Antibodies to Red Cell Antigens

 401
PhenotypingAutologous Red Cells
Determining the phenotype of the autologous red cells is an important part of antibody identification. When
an antibody is tentatively identified in the serum, the corresponding antigen is expected to be absent from the
autologous red cells. For example, if serum from an untransfused individual appears to contain anti-Fy" but the
autologous red cells have a negative DAT result and type as Fy(a+), the results are clearly in conflict and further
testing is indicated. Thus, knowing the patient’s phenotype can help guide exclusion testing.
It may be difficult to determine the patient’s phenotype if he or she was transfused in the past 3 months. A
pretransfusion specimen, if available, should be used to determine the phenotype. If a pretransfusion sample is
not available, the patient’s newly formed autologous red cells can be separated from the transfused red cells and
then typed (Method 222). Separation of young red cells by centrifugation is based on the difference in the
densities of new and mature red cells. Centrifugation is most successful when 3 or more days have elapsed since
the last transfusion, which will provide time for new autologous red cell production. New autologous red cells
must be isolated from the sample while it is fresh. The technique is ineffective if the sample is too old (>24
hours), the patient is not producing new red cells, or the patient has sickle cell anemia.
Sickle cells are quite dense, and centrifugation is not an effective technique for separating the autologous
red cells from the transfused donor red cells in a patient with sickle cell disease. However, autologous sickle
cells may be separated from donor red cells using washes with hypotonic saline (Method 2-23). Sickle cells
containing hemoglobin SS are resistant to lysis by hypotonic saline, whereas donor red cells containing
hemoglobin AA are lysed.
The use of potent blood-typing reagents, appropriate controls, and observation for mixed-field reactions can
allow a specimen contaminated with donor red cells to be phenotyped. Phenotyping results on posttransfusion
samples can be misleading, however, and
should be interpreted with caution.16 If the specificity of the antibody(ies) in the patient’s plasma is clear,
extensive efforts to separate and type the patient’s own red cells are not necessary. A compatible AHG
crossmatch using antigen-negative donor units provides additional confirmation of the antibody’s
specificity.17(p37) Definitive typing can be performed on the patient’s red cells 3 months after the last
transfusion if the patient is not transfusion dependent or experiencing marrow aplasia or failure.
Cold and warm autoantibodies may also complicate red cell typing. When red cells are coated with cold
autoantibodies, it may be possible to remove the autoantibodies with warm (37 C) saline washes (Method 2-17).
If the cold autoantibodies are very potent, it may be necessary to treat the red cells with dithiothreitol (DTT) to
dissociate IgM molecules that cause spontaneous agglutination (Method 2-18). When red cells are coated with
IgG autoantibodies, it is not possible to perform typing that requires an IAT without first removing the bound
IgG. However, it is often possible to type antibody-coated red cells with direct-agglutinating antisera, such as
IgM monoclonal reagents. With rare exceptions, most direct-agglutinating monoclonal reagents give valid
phenotyping results despite a positive DAT result.18 For antisera that require an IAT (eg, anti-Fya and anti-
Fyb), it is necessary to remove the IgG from the test red cells before typing. Common techniques for removing
IgG antibodies include gentle heat elution (Method 2-19), treatment with chloroquine diphosphate (Method 2-
20), and treatment with acid glycine/EDTA (Method 2-21).
Molecular DNA genotyping offers an alternative to serologic typing and is especially useful when the
patient has been recently transfused or the patient’s red cells are heavily coated with IgG. Molecular testing
relies on the extraction of DNA from white cells. Because of the short life-span of white cells in vivo and the
assay design, the presence of transfused white cells from donors is not a limiting factor in determining the
patient’s genotype.
402
AABB TECHNICAL MANUAL
There are situations, however, where the genotype of a person may not predict the red cell phenotype.
Mutations that inactivate gene expression or rare new alleles may not be identified by the specific assay
performed. The genotype obtained from DNA isolated from leukocytes and other hematopoietic cells may differ
from that of other tissues in people with a history of transplantation.19
When DNA testing is used as a tool in antibody identification, the predicted red cell phenotype should be
used judiciously. If a sample appears to contain an antibody specificity when the predicted phenotype of the
patient is antigen positive, the apparent antibody should be further investigated. This discrepancy may indicate
that the patient’s red cells do not actually express the antigen due to a gene mutation not detected by the assay.
Alternatively, the patient might have an altered or partial antigen due to an additional gene polymorphism. It is
important to remember that the antibody in the sample could be, in fact, an alloantibody.
Complex Antibody Problems
Not all antibody identifications are simple. The exclusion procedure does not always lead directly to an
answer, and additional testing may be required. When an antibody screen or incompatible crossmatch detects an
unexpected antibody, the next step maybe to determine whether the antibody is an autoantibody or alloantibody.
An autocontrol, which may not have been performed in the initial testing, can start the identification process.
Figure 161 shows some approaches to identifying antibodies in a variety of situations when the auto control is
negative, and Fig 16-2 shows some approaches to identifying antibodies when the autocontrol is positive.
Selected Serologic Procedures
Many techniques and methods are used in complex antibody identification. Some of the methods described
in this chapter are used routinely by many laboratories; others are used selectively and may apply only in
special circumstances. It is important to remember
that no single method is optimal for detecting all antibodies. Any laboratory that performs antibody
detection or identification should use routine methods and have access to some alternative approaches.
When a pattern of weak reactions fails to indicate specificity or the presence of an antibody is suspected but
cannot be confirmed, the use of enhancement techniques or testing of panel cells treated with enzymes or
chemicals may be helpful. An autocontrol should always be included with each technique.
LISS and PEG
LISS and PEG techniques (Methods 3-4 and 35) are used to enhance reactivity and reduce incubation time.
LISS may be used to suspend test red cells for use in tube or column agglutination tests or as an additive
medium for tube or solid-phase tests. Care should be taken to follow the instructions in the manufacturer’s
product insert closely to ensure that the appropriate proportion of serum to LISS is achieved. Commercially
prepared LISS additives or PEG additives may contain additional enhancing agents. Because LISS and PEG
enhance autoantibodies, their use may complicate alloantibody identification in samples that also contain
autoantibodies.20,21
Enzymes
Ficin and papain are the most frequently used enzymes for complex antibody identification. They destroy or
weaken antigens, such as M, N, Fy2, Fyb, Xg2, JMH, Ch, and Rg (Table 16-4). Antibodies to these antigens are
nonreactive with treated red cells. Conversely, ficin-treated and papain-treated red cells show enhanced
reactivity with other antibodies (eg, Rh, P I, Kidd, and Lewis). Additional enzymes that are commonly used in
immunohematology laboratories include trypsin, a-chymotrypsin, and pronase. Depending on the enzyme and
method used, other antigens may be altered or destroyed. Antigens that are inactivated by one proteolytic
enzyme may not be inactivated by other enzymes. The clinical significance of antibodies that are reactive only
with enzyme-treated cells is questionable; such
CHAPTER 16
Identification of Antibodies to Red Cell Antigens
403
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404
AABB TECHNICAL MANUAL
 FIGURE 16-2. Antibody identification with positive autocontrol.
HPCs = hematopoietic progenitor cells; IVIG = intravenous immune globulin.
405
TABLE 16-4. Alterations of Antigens
by Various Agents
Agent Antigens Usually Denatured or Altered*
M, N, S, Fya, Fyb, Yta, Ch, Rg, Pr, Tn, Mg, MiYVw, Cla, Jea,
Proteolytic enzymes1
l\lya, JMH, some Ge, and lnb
Dithiothreitol (DTT) or 2- Yta, JMH, Kna, McCa, Yka, LWa, LWb, all Kell, Lutheran,
aminoethylisothiouronium bromide (AET) Dombrock, and Cromer blood group antigens
‘Some antigens listed may be weakened rather than completely denatured. Appropriate controls should be
used with modified red cells.
+Different proteolytic enzymes may have different effects on certain antigens.
“enzyme-only” antibodies may not have clinical significance.22
In addition to enhancing the reactivity of certain antibodies, enzyme techniques may be used to separate
mixtures of antibodies. For example, if a serum sample contains anti-Fya and anti-Jka, many of the red cell
samples on the initial panel would be reactive. Then, if a panel of enzyme-treated red cells were tested, the anti-
Jka reactivity would be enhanced, whereas the anti-Fya reactivity would be destroyed. Procedures for the
preparation and use of proteolytic enzymes are given in Methods 3-8 to 3-13.
Temperature Reduction
Some antibodies (eg, anti-M, -N, -PI, -Lea, -Leb, and -Al) react better at room temperature or below, and
their specificity may be apparent only at a temperature below 22 C. An autocontrol is especially important for
tests at low temperatures because many sera also contain anti-I or other cold-reactive autoantibodies.
Increased Serum-to-Cell Ratio
Increasing the volume of serum incubated with a standard volume of red cells may enhance the reactivity of
antibodies that are present in low concentrations. One acceptable procedure involves mixing four volumes
(drops) of serum with one volume of a 2% to
5% saline suspension of red cells and incubating the mixture for 60 minutes at 37 C. Periodic mixing during
the incubation promotes contact between the red cells and the antibodies. It is helpful to remove the serum
before washing the cells for an IAT because the standard three to four washes may be insufficient to remove all
of the unbound immunoglobulin if increased amounts of serum are used. More than four washes are not
recommended because bound antibody molecules may dissociate. Increasing the serum-to-red-cell ratio is not
appropriate for tests using LISS or commercial PEG, which may contain LISS. Tests performed in a low-ionic-
strength medium require specific proportions of serum and additive.
Increased Incubation Time
For some antibodies, a 15-minute incubation period may not be sufficient to achieve equilibrium; therefore,
the reactions may be negative or weak, particularly in saline or albumin media. Extending the incubation time to
between 30 and 60 minutes for albumin or saline tests often improves the reactivity and helps clarify the pattern
of reactions. Extended incubation may be contraindicated when LISS or PEG is used. If the incubation period
exceeds the recommended times for these methods, the reactivity may be diminished or lost. Care must be taken
to use all reagents according to the manufacturer’s directions.

406
AABB TECHNICAL MANUAL
Alteration in pH
Altering the pH of the test system can change the reactivity of certain antibodies, enhancing the reactivity of
some and decreasing that of others.
Some examples of anti-M are enhanced when the pH of the test system is lowered to 6.5.23 If anti-M is
suspected because the only reactive red cells are M+N-, a definitive pattern (ie, reactivity with M+N+ red cells
also) maybe seen if the serum is acidified. The addition of one volume of 0.1 N HC1 to nine volumes of serum
lowers the pH to approximately 6.5. The acidified serum should be tested with known M-negative cells to
control for nonspecific agglutination.
Lowering the pH, however, significantly decreases the reactivity of other antibodies.24 If unbuffered saline
with a pH <6.0 is used to prepare red cell suspensions or for washing in an IAT, antibodies in the Rh, Duffy,
Kidd, and MNS blood groups may lose reactivity. Phosphate-buffered saline (Method 1-8) can be used to
control the pH and enhance the detection of antibodies that are poorly reactive at a lower pH.25
Inhibition Techniques
Soluble forms of some blood group antigens exist in body fluids, such as saliva, urine, and plasma. These
substances are also present in other natural sources, and they can be prepared synthetically. Soluble substance
can be used to inhibit the reactivity of the corresponding antibody that could mask the presence of underlying
nonneutralizable antibodies. Also, when an antibody is suspected, inhibition of the reactivity by a soluble
substance can help with the identification of the antibody. For example, if a suspected anti-Pl does not produce a
definitive pattern of agglutination, the loss of reactivity after the addition of soluble PI substance strongly
suggests that the specificity is anti-Pl if a parallel dilution control with saline. Inhibition results can be
interpreted only when the test is nonreactive and the dilution control that substitutes an equal volume of saline
for the soluble substance is reactive.
The most commonly used substances for inhibition include the following:
1. Lewis substances. Lea substances, Leb substances, or both are present in the saliva of individuals who
possess the Lewis gene CFUT3). Lea substance is present in the saliva of Le (a+b—) individuals, and both Lea
and Leb substances are present in the saliva of Le(a-b+) individuals (Method 2-8). Commercially prepared
Lewis substance is available.
2. PI substance. Soluble PI substance is present in hydatid cyst fluid and the ovalbumin of pigeon eggs.
Commercially prepared PI substance is available.
3. Sda substance. Soluble Sda blood group substance is present in various body fluids, but urine has the
highest concentration of Sda.26 To confirm the presence of anti-Sda in a serum sample, urine from a known
Sd(a+) individual (or a pool of urine specimens) can be used to inhibit the antibody reactivity (Method 3-19).
 4. Chido and Rodgers substances. Ch and Rg antigens are epitopes on the fourth component of human
complement (C4).27,28 Most normal red cells have a trace amount of C4 on their surface. Anti-Ch and anti-Rg
are reactive with this C4 in an IAT. If red cells are coated in vitro with excess C4, these antibodies may cause
direct agglutination.29 A useful test to identify anti-Ch and anti-Rg is inhibition of the antibodies with plasma
from Ch+, Rg+ individuals (Method 3-17).
Inactivation of Blood Group Antigens
Certain blood group antigens can be destroyed or weakened by chemical treatment of the cells (Table 16-4).
Modified red cells can be useful for both confirming the presence of suspected antibodies and detecting
additional antibodies. The use of modified red cells can be especially helpful if a sample contains an antibody to
a high-prevalence antigen because antigen-negative red cells are rare. Proteolytic enzymes, described above, are
commonly used to alter red cell antigens. Sulfhydryl re
407
agents, such as 2-aminoethylisothiouronium bromide (AET), 2-mercaptoethanol (2-ME), or DTT, can be
used to weaken or destroy antigens in the Kell system and some other antigens (Method 3-18).30,31 ZZAP
reagent, which contains proteolytic enzyme and DTT, denatures antigens that are sensitive to DTT (eg, all Kell
system antigens) as well as antigens that are sensitive to enzymes (Method 4-8).32Glycine-HCl/EDTA
treatment of red cells destroys Bg and Kell system antigens as well as the Era antigen (Methods 2-21 and 4-
2).33Chloroquine diphosphate can be used to weaken the expression of Class I HLA antigens (Bg antigens) on
red cells.34 Chloroquine treatment also weakens some other antigens, including Rh antigens (Method2-20).
Sulfhydryl Reagents
Sulfhydryl reagents, such as DTT and 2-ME, can be used to cleave the disulfide bonds that join the
monomeric subunits of the IgM pentamer. Intact 19S IgM molecules are cleaved into 7S Ig subunits, which
have altered serologic reactivity.35 The interchain bonds of 7S Ig monomers are relatively resistant to such
cleavage. Sulfhydryl reagents (DTT and 2-ME as well as AET) can also be used to cleave disulfide bonds that
are responsible for the conformation of certain blood group antigens and, therefore, are used to destroy certain
red cell antigens.
Uses of sulfhydryl reagents include the following:
1. Determining the Ig class of an antibody (Method 3-16).
2. Identifying antibodies in a mixture of IgM and IgG antibodies, particularly when an agglutinating IgM
antibody masks the presence of IgG antibodies.
3. Determining the relative amounts of IgG and IgM components of a given specificity (eg, anti-A or-B).
4. Dissociating red cell agglutinates caused by IgM autoantibodies (Method 2-18).
5. Dissociating IgG antibodies from red cells using a mixture of DTT and proteolytic enzyme (ZZAP
reagent) (Method 4-8).
6. Destroying selected red cell antigens for use in antibody investigations (eg, antigens in the Kell,
Dombrock, Cartwright, LW, and Knops systems) (Method3-18).
Adsorption
Antibody can be removed from a serum sample by adsorption onto red cells that express the corresponding
antigen. After the antibody attaches to the membrane-bound antigens and the serum and cells are separated, the
specific antibody remains attached to the red cells. It may be possible to harvest the bound antibody by elution
or examine the adsorbed serum for antibody(ies) remaining after the adsorption process.
Adsorption techniques are useful for the following purposes:
1. Separating multiple antibodies present in a single serum.
2. Removing autoantibody to permit the detection or identification of underlying alloantibodies.
3. Removing unwanted antibody (often antiA, anti-B, or both) from serum that contains an antibody that is
suitable for reagent use.
4. Confirming the presence of specific antigens on red cells by their ability to remove antibody of
corresponding specificity from previously characterized serum.
5. Confirming the specificity of an antibody by showing that it can be adsorbed onto red cells of only a
particular blood group phenotype.
Adsorption serves different purposes in different situations; no single procedure is satisfactory for all
purposes (Methods 4-5, 4-8, 4-9, and 4-10). A basic procedure for antibody adsorption can be found in Method
3-20. The usual serum-to-cell ratio is one volume of serum to an equal volume of washed, packed red cells. To
enhance antibody removal, a larger volume of red cells increases the proportion of antigen. The incubation
temperature should be that at which the antibody is optimally reactive. Pretreating red cells with a proteolytic
enzyme may enhance antibody uptake and
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AABB TECHNICAL MANUAL
reduce the number of adsorptions required to remove an antibody completely. Because enzymes destroy
some antigens, antibodies directed against those antigens are not removed by enzyme-treated red cells. To
ensure that an adsorption process is complete (ie, that no unadsorbed antibody remains), it is essential to
confirm that the adsorbed serum is nonreactive with a reserved sample of the adsorbing red cells that was not
used for adsorption. Adsorption requires a substantial volume of red cells, and vials of reagent red cells are
usually not sufficient. Blood samples from donor units or staff members are the most convenient sources.
When separating mixtures of antibodies, the selection of red cells of the appropriate phenotype is extremely
important. If one or more antibodies have been previously identified, red cells that express the corresponding
antigens can be used to remove the known antibodies and leave the unknown antibody(ies) in the adsorbed
serum. For example, if a person who types K+k-, Fy(a-b+) has produced anti-k, it may be necessary to adsorb
the anti-k onto K-k+, Fy(a-b+) reagent red cells to remove the anti-k. Then, the adsorbed serum can be tested
with common K-k+, Fy(a+b-) red cells to detect possible anti-Fya.
Elution
Elution dissociates antibodies from sensitized red cells. Bound antibody may be released by changing the
thermodynamics of antigenantibody reactions, neutralizing or reversing forces of attraction that hold antigen-
antibody complexes together, or disturbing the structure of the antigen-antibody binding site. The usual
objective is to recover bound antibody in a usable form.
Various elution methods have been described in the literature. Selected procedures are given in Methods 4-1
through 4-4. No single method is best for all situations. Heat or freeze-thaw elution techniques are usually
restricted to the investigation of HDFN caused by ABO incompatibility because these elution procedures rarely
work well for other antibodies. Acid or organic solvent methods are used
for eluting warm-reactive auto- and alloantibodies. Commercial kits are also available for performing
elution. (See Table 17-2 for a list of elution methods and their uses, advantages, and disadvantages.)
Elution techniques are useful for the following:
1. Investigation of a positive DAT result (Chapter 17).
2. Concentration and purification of antibodies, detection of weakly expressed antigens, and identification
of multiple antibody specificities. Such studies are used in conjunction with an appropriate adsorption
technique, as described below and in Method 2-7.
3. Preparation of antibody-free red cells for phenotyping or autologous adsorption studies. Procedures used
to remove coldand warm-reactive autoantibodies from red cells are discussed in Methods 4-5 and 4-8.
Technical factors that influence the outcome of elution procedures include the following:
1. Incomplete washing. Sensitized red cells should be thoroughly washed before an elution to prevent
contamination of the eluate with unbound residual antibody. Six washes with saline are usually adequate, but
more washes may be needed if the serum contains a high-titer antibody (the considerations in Item 3 below
should be kept in mind). To confirm the efficacy of the washing process, supernatant fluid from the final wash
should be tested for antibody activity and found to be nonreactive.
2. Binding of protein to glass surfaces. If an eluate is prepared in the test tube that was used during the
sensitization or washing phases, antibody that nonspecifically binds to the test tube surface may dissociate
during the elution. Similar binding can also occur from a whole blood sample when a patient has a positive
DAT result and has free antibody in the serum. To avoid such contamination, red cells used to prepare an
409
eluate should be transferred to a clean test tube before washing and then to another clean tube before the
elution procedure is initiated.
3. Dissociation of antibody before elution. IgM antibodies, such as anti-A or anti-M, or low-affinity IgG
may spontaneously dissociate from the red cells during the wash phase. To minimize the loss of bound antibody,
cold (4 C) saline or wash solution provided by the manufacturer should be used for washing.
4. Incorrect technique. Such factors as incomplete removal of organic solvents or failure to correct the
tonicity or pH of an eluate may cause the reagent red cells used to test the eluate to hemolyze or appear “sticky.”
The presence of stromal debris may interfere with the reading of test results. Careful technique and strict
adherence to procedures should eliminate such problems.
 5. Instability of eluates. Dilute protein solutions, such as those obtained by elution into saline, are unstable.
Eluates should be tested as soon after preparation as possible. Alternatively, bovine albumin may be added to a
final concentration of 6% w/v, and the preparation may be frozen during storage. Eluates can also be prepared in
antibody-free plasma, 6% albumin, or a similar protein medium. When commercial elution kits are used, the
manufacturer’s instructions for preparation and storage should be followed.
Combined Adsorption-Elution
Combined adsorption-elution tests can be used to separate a mixture of antibodies in a single serum sample,
detect weakly expressed antigens on red cells, or help identify weakly reactive antibodies. The process consists
of first incubating serum with selected red cells and then eluting antibody from the adsorbing red cells.
Care must be taken when selecting the adsorbing cells to separate a mixture of antibodies. The cells should
express only one of the antigens corresponding to an antibody in the mixture so that the eluate from the cells
will contain only that antibody. Both the eluate and adsorbed serum can be used for further testing.
Unmodified red cells are generally used for adsorptions when subsequent elutions are being prepared.
Titration
The titer of an antibody is usually determined by testing serial twofold dilutions of the serum with selected
red cells. Results are expressed as the reciprocal of the highest serum dilution that shows macroscopic
agglutination. Titration values can provide information about the relative amount of antibody present in a serum
sample or the relative strength of antigen expression on red cells.
Titration studies are useful for the following purposes:
1. Prenatal studies. When the antibody has a specificity that is known to cause HDFN or the antibody’s
clinical significance is unknown, the results of titration studies may contribute to the decision about performing
additional procedures (eg, Doppler sonography or amniocentesis). (See Chapter 22 and Method 5-3.)
2. Antibody identification. Some antibodies that agglutinate virtually all reagent red cells may give an
indication of specificity by demonstrating reactivities of different strengths with different red cell samples in
titration studies. For example, potent undiluted autoanti-I may be reactive with both adult and umbilical cord
blood red cells, but titration studies may reveal reactivity with adult 1+ cells at a higher dilution than with cord
blood I- red cells. The reactivity of most antibodies weakens progressively with serial dilutions (ie, a 2+
reaction becomes 1+ in the next dilution), and weak antibodies (<1+) may lose their reactivity when diluted.
However, some antibodies that have weak reactions when undiluted continue to react at dilutions as high as 1 in
2048. Such antibodies include anti-Ch, -Rg, -Csa, -Yka, -Kna, -McCa, and -JMH. When weak reactions are
observed in an IAT, titration studies may be performed to determine whether the
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AABB TECHNICAL MANUAL
reactivity is consistent with the antibodies in this group; however, not all examples of these antibodies
demonstrate such high titer, low-avidity characteristics. Thus, the serologic characteristics may suggest certain
specificities, but failure to do so does not eliminate these possibilities. The antibodies listed above are not
expected to cause shortened red cell survival, although there are examples of other antibodies (eg, antiLub, -Hy,
and -Yta) that may mimic these serologic characteristics and cause shortened red cell survival. Details about
titration are given in Method 3-15.
3. Separating multiple antibodies. Titration results may suggest that one antibody is reactive at higher
dilutions than another antibody. That information can allow the serum to be diluted before it is tested with a red
cell panel, which effectively removes one antibody and allows the other to be identified. For example, if a
serum contains anti-c that is reactive to a titer of 2 and antiJka that is reactive to a titer of 16, it may be possible
to eliminate the anti-c reactivity by diluting the serum to a titer of 8.
Other Methods
Methods other than traditional tube, gel, or solid-phase techniques may be used for antibody identification.
Some methods are especially useful for testing small volumes of serum or reagents. Such methods include
testing in capillary tubes, microplates, or enzyme-linked immunosorbent assays. Other methods that are useful
in laboratories with specialized equipment include radioimmunoassay, immunofluorescence, flow cytometry,
and immunoblotting.
Factors Affecting Antibody Identification
Variation in Antigen Expression
For a variety of reasons, antibodies are not always reactive with all red cells that express the corresponding
antigen. Basic interpretation by exclusion, as described previously, may result in an antibody being excluded
because the se
rum is nonreactive with an antigen-positive red cell sample, despite the presence of the antibody. Technical
error, weak antibody reactivity, and variant or weak antigenic expression are all possible causes. Therefore,
whenever possible, antibody exclusions should be based only on red cells that strongly express the antigen.
Enhancement techniques often help resolve problems associated with variations in antigen expression (Methods
3-3 through 313).
ZYGOSITY. Reaction strength of some antibodies may vary because of dosage, in which antibodies are
more strongly reactive (or only) with red cells that possess a “double-dose” expression of the antigen. Double-
dose antigen expression occurs when an individual is homozygous for the gene that encodes the antigen. Red
cells from individuals who are heterozygous for the gene may express fewer antigens and, therefore, may be
weakly reactive or nonreactive with a weak example of the corresponding antibody. Alloantibodies vary in their
tendency to demonstrate dosage. Many antibodies to antigens in the Rh, Duffy, MNS, and Kidd blood groups
demonstrate dosage.
VARIATION IN ADULTS AND INFANTS.
Some antigens (eg, I, PI, Lea, and Sda) show variable expression on red cells from different adults. The
antigenic differences can be demonstrated serologically; however, the variability is unrelated to zygosity.
Certain antibodies (eg, anti-I or -Lub) demonstrate weaker reactivity with cord red cells than with red cells from
adults (Table 16-5).
CHANGES WITH STORAGE. Blood group antibodies may be more weakly reactive with stored red cells
than with fresh red cells. Some antigens (eg, Fya, Fyb, M, PI, Kna/McCa, and Bg) deteriorate more rapidly than
others during storage, and the rate varies among red cells from different individuals.36 Because red cells from
donors are often fresher than commercial reagent red cells, some antibodies have stronger reactions with donor
red cells than with reagent red cells. Similarly, storage of red cells in a freezer may cause antigens to deteri
411
TABLE 16-5. Antigen Expression on Cord Red Blood Cells*
Expression Antigens
Negative Lea, Leb, Sda, Ch, Rg, and AnWj
Weak 1, H, PI, Lua, Lub, Yta, Vel, Bg, KN and DO antigens, Yka, Csa, and Fy3
Strong i, LWa, and LWb
‘Modified with permission from Reid, Lomas-Francis and Olsson.4
orate, thus producing misleading antibody identification results.
The pH or other characteristics of the storage medium can affect the rate of antigen deterioration.36,37 For
example, Fya and Fyb antigens may weaken when the red cells are stored in a medium with low pH and low
ionic strength. Thus, certain antibodies may demonstrate differences in reactivity with red cells from different
manufacturers if the suspending media are different.
The age and nature of the specimen must be considered when red cells are typed. Antigens on red cells from
clotted samples tend to deteriorate more quickly than antigens on red cells from donor units that are collected in
citrate anticoagulants, such as acid-citrate-dextrose or citrate-phosphate-dextrose. Red cells in donor units
collected in approved anticoagulants usually retain their antigens throughout the standard shelf life of the blood
component. EDTA samples up to 14 days old are suitable for antigen typing.38 However, the manufacturer’s
instructions should be consulted when commercial typing reagents are used.
No Discernible Specificity
Factors other than variation in antigen expression may contribute to difficulty in interpreting results of
antibody identification tests. If the reactivity with the serum is very weak, the pattern of reactivity and cross-out
process have excluded all likely specificities, or both, alternative approaches to interpretation should be used.
PRESENCE OF ANTIGENS IN COMMON. Instead of excluding antibodies to antigens on nonreactive red
cells, it might be helpful to ob
serve which antigens the reactive red cells have in common. For example, if all of the red cells reactive at
room temperature are P1+ but the anti-Pl pattern is not complete, the antibody could be anti-Pl that is not
reactive with red cells with a weaker expression of the antigen. (Sometimes, such red cells are designated on the
panel sheet as “+w.”) In this case, it might be helpful to use a method that enhances anti-Pl, such as testing at a
colder temperature.
If all of the reactive red cells are Jk(b+) but not all Jk(b+) red cells are reactive, the reactive red cells might
be Jk(a-b+) with a double-dose expression of the antigen. In this case, enhancement techniques, such as
enzymes, LISS, or PEG, might help demonstrate reactivity with all of the Jk(b+) red cells. Typing the patient’s
red cells to confirm that they lack the corresponding antigen is also very helpful.
Finally, the presence of some antigens in common may suppress the expression of certain antigens. This
suppression can cause weak antibodies to be missed or certain cells to be unexpectedly nonreactive when a
suspected antibody fails to show reactivity with all antigen-positive cells. For example, In(Lu) is known to
suppress the expression of Lutheran antigens, PI, Inb, andAnWj. Similarly, Kpais known to weaken the
expression of Kell antigens. (See Chapter 14 for a more detailed discussion.)
INHERENT VARIABILITY. Nebulous reaction patterns that do not appear to fit any particular specificity
are characteristic of certain antibodies, such as anti-Bga, -Kna, -McCa, -Sla, -Yka, -Csa, and -JMH. Antigens
corresponding to these antibodies vary markedly in their

412
AABB TECHNICAL MANUAL
expression on red cells from different individuals. For example, the expression of Knops blood group
antigens shows marked differences between individuals of either African or European ethnicity, and this
difference in expression is caused by variations in the CR1 copy numbers on the red cells.39 Rarely, a pattern of
reactive and nonreactive red cells cannot be interpreted because the typing result for a reagent red cell is
incorrect or the reagent red cell has a positive DAT result. If the red cell sample is from a commercial source,
the manufacturer should be notified immediately of the discrepancy.
UNLISTED ANTIGENS. Sometimes, a serum reacts with an antigen that is not routinely listed on the
antigen profile supplied by the reagent manufacturer—Ytb is an example. Even though serum studies yield
clearly reactive and nonreactive test results, anti-Ytb may not be suspected. In such circumstances, it is useful to
ask the manufacturer for additional phenotype information. If only one cell is unexpectedly reactive, this
reaction is most likely caused by an antibody to a low-prevalence antigen. These antibodies are discussed in
more detail later in this chapter.
ABO TYPE OF RED CELLS TESTED. A serum sample may be reactive with many or all of the group 0
reagent red cells but not with red cells of the same ABO group as the autologous red cells. Such a reaction
occurs most frequently with anti-H, anti-IH, or anti-LebH. Group 0 and A2 red cells have more H antigen than
A: and AtB red cells, which express very little H. (See Chapter 12 for more information.) Thus, sera containing
anti-H or anti-IH are strongly reactive with group 0 reagent red cells, whereas autologous Aj or AjB red cells or
donor red cells used for crossmatching may be weakly reactive or nonreactive. Anti-LebH is strongly reactive
with group 0, Le(b+) red cells but weakly reactive or nonreactive with Le(b+) red cells from Aj or A,B
individuals.
Multiple Antibodies
When a serum sample contains two or more alloantibodies, it may be difficult to interpret
the results of testing performed with a single panel of reagent red cells. Perhaps the easiest way to identify
multiple antibodies is to determine the phenotype of the patient’s pretransfusion autologous red cells and then
use a selected cell panel to identify or exclude all common antibodies to the red cell antigens that the patient
lacks. (See the discussion on selected cells earlier in this chapter.) The presence of multiple antibodies may be
suggested by a variety of test results, such as the following:
1. The observed pattern of reactive and nonreactive red cells does not fit a single antibody. When the
exclusion approach fails to indicate a specific pattern, it is helpful to determine whether the pattern matches two
combined specificities. For example, if the reactive red cells on the panel in Table 16-2 are numbers 3, 5, 6, 9,
and 10, none of the specificities remaining after crossing out fits a pattern exactly. However, if both E and K are
considered together, a pattern is discerned, with cells 3 and 5 showing reactivity because of anti-E, and cells 6,
9, and 10 because of anti-K. If the reaction pattern does not fit two combined specificities, the possibility that
more than two antibodies are present must be considered. The more antibodies a serum sample contains, the
more complex identification and exclusion become, but the basic process remains the same.
2. Reactivity occurs at different test phases. When reactivity occurs at several phases, each phase should be
analyzed separately. The pattern at room temperature may indicate a different specificity from the pattern at the
AHG phase. It is also helpful to look for variations in the strength of the reactions at each phase of testing.
Table 16-6 provides information about the characteristic reactivity of several antibodies.
3. Unexpected reactions occur when attempts are made to confirm the specificity of a suspected single
antibody. If a serum suspected to contain anti-e is reactive with some enegative cells, another antibody may be
present or the suspected antibody may not
TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies
Immuno- Reactivity Associated with
DTT
globulin Papain/
Antibody 4C 22 C 37 C AHG (200 HDFN HTR
Class Ficin
mM)
IgG >
Anti-M Most Most Rare Sensitive Resistant Rare Rare
IgM
IgM >
Anti-N Most Most Rare Sensitive Resistant No Rare
IgG
IgG >
Anti-S Most Most Variable Resistant Yes Yes
IgM
IgG >
Anti-s Most Variable Resistant Yes Yes
IgM
Anti-U IgG Most Resistant Resistant Yes Yes
IgM
Anti-Pi (IgG Most Most Resistant Resistant No Rare
rare)
IgG >
Anti-D IgM (IgA Some Some Most Resistant Resistant Yes Yes
rare)
IgG >
Anti-C Some Some Most Resistant Resistant Yes Yes
IgM
IgG >
Anti-E Some Some Most Resistant Resistant Yes Yes
IgM
IgG >
Anti-c Some Some Most Resistant Resistant Yes Yes
IgM
IgG >
Anti-e Some Some Most Resistant Resistant Yes Yes
IgM
IgM > Resistant
Anti-Lua Most Most Variable Rare No
IgG or weakened
IgG > Resistant
Anti-Lub Some Most Variable Mild Yes
IgM or weakened
IgG >
Anti-K Some Most Resistant Sensitive Yes Yes
IgM
IgG >
Anti-k Most Resistant Sensitive Yes Yes
IgM
Anti-Kpa IgG Most Resistant Sensitive Yes Yes
Anti- IgG >
Most Resistant Sensitive Yes Yes
Kpb IgM
IgG >
Anti-Jsa Most Resistant Sensitive Yes Yes
IgM
Anti-Jsb IgG Most Resistant Sensitive Yes Yes
 Anti-Lea IgM > Most Most Most Most Resistant Resistant No Rare
IgG
IgM >
Anti-Leb Most Most Most Most Resistant Resistant No No
IgG
IgG >
Anti-Fya Most Sensitive Resistant Yes Yes
IgM
IgG >
Anti-Fyb Most Sensitive Resistant Yes Yes
IgM
IgG >
Anti-Jka Most Resistant Resistant Yes Yes
IgM
IgG >
Anti-Jkb Most Resistant Resistant Yes Yes
IgM
Anti-Dia IgG Most Resistant Resistant Yes Yes
Anti-Dib IgG Most Resistant Resistant Yes Yes

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AABB TECHNICAL MANUAL
TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies (Continued)
DTT Associated
Immuno Reactivity
(200 mM) with
4 C 22 C
Antibody Class AHG Ficin HDFN HTR
37 C
Sensitive or
Anti-Yta igG Most Variable No Yes
weakened
Sensitive or
Anti-Ytb igG Most Variable No No
weakened
IgG >
Anti-Xga Some Most Sensitive Resistant No No
IgM
Anti-Scl IgG Most Resistant Variable No No
Anti-Sc2 IgG Most Resistant Variable No No
Anti-Doa IgG Most Resistant Variable No Yes
Anti-
IgG Most Resistant Variable No Yes
Dob
IgG >
Anti-Coa Most Resistant Resistant Yes Yes
IgM
Anti-Cob IgG Most Resistant Resistant Yes Yes
AHG = antiglobulin reagent; DTT = dithiothreitol; HDFN = hemolytic disease of the fetus and newborn;
HTR = hemolytic transfusion reaction; Ig = immunoglobulin.
be anti-e. Testing a panel of selected e-negative red cells may help identify an additional specificity.
4. No discernible pattern emerges. When variable reaction strengths are observed and dosage or other
variations in antigen strength do not provide an explanation, additional approaches and methods of testing are
needed. Some helpful steps include the following:
a. If strongly positive results are obtained, use the exclusion method with nonreactive cells to eliminate
some specificities from initial consideration.
 b. If weak or questionable positive results are obtained, test the serum against cells with a strong expression
of the antigen that corresponds with any suspected specificity, and combine this approach with methods that
enhance the reactivity.
c. Type the patient’s red cells for common red cell antigens, and eliminate from consideration specificities
that correspond to antigens on the patient’s autologous red cells. This step may not be possible if the patient has
been transfused recently or has had a positive DAT result.
d. Use a method to inactivate certain antigens on the reagent cells. Enzyme treatment renders cells negative
for such antigens as Fya and Fyb (see Table 16-4).
e. Use adsorption or elution methods to separate antibodies (Methods 3-20,4-1, and 4-2).
f. Enhance antibody reactivity by using a more sensitive method (eg, PEG, enzymes, increased incubation
time, or increased serum-to-cell ratio; see Methods 3-5 and 3-8 through 3-13).

415
Antibodies to High-Prevalence Antigens
If all reagent red cells are reactive but the autocontrol is nonreactive, an alloantibody to a high-prevalence
antigen should be considered, especially if the strength and test phase of reactions are uniform for all of the red
cells tested. Antibodies to high-prevalence antigens can be identified by testing selected red cells of rare
phenotypes and by typing the patient’s autologous red cells with antisera to highprevalence antigens. Knowing
the race or ethnic origin of the antibody producer can be helpful when selecting additional tests to perform
(Table 16-1). Red cells that lack all of the antigens in a blood group system (eg, Rhnull or Ko) or chemically
modified red cells (eg, DTTtreated red cells) can help limit possible specificities (Table 16-4).
If red cells negative for a particular highprevalence antigen are not available, red cells that are positive for
the lower-prevalence antithetical antigen can sometimes be helpful. For example, if a serum contains anti-Coa,
which reacts with a high-prevalence antigen, weaker reactions may be observed with Co(a+b+) red cells than
with Co(a+b-) red cells because of a dosage effect.
Antibodies to high-prevalence antigens may be accompanied by antibodies to common antigens, which can
make identification much more difficult. In such cases, it may be necessary to determine the patient’s phenotype
for common antigens, choose a phenotypically similar red cell sample (ie, one that lacks the patient’s common
antigens) that is incompatible with the patient’s plasma, and adsorb the antibody to the high-prevalence antigen
onto that red cell sample. This approach leaves antibodies to common red cell antigens in the adsorbed plasma,
where they can be identified with a routine selected red cell panel. Because the identification of antibodies to
high-prevalence antigens is complicated, it may be necessary to refer such specimens to a reference laboratory.
SEROLOGIC CLUES. Knowing the serologic characteristics of particular antibodies to high-prevalence
antigens and/or the preva
lence of such antigens in different populations may help with identification.
 1. Reactivity in tests at room temperature suggests anti-H, -I, -IH, -P, -PPlPk (-Tja), -Ena, -LW (some), -Ge
(some), -Sda, or -Vel.
2. Lysis of reagent red cells during testing with fresh serum is characteristic of anti-Vel, -P, -PPlPk (-Tja), -
Jk3, and some examples of anti-H and -I.
3. Reduced or absent reactivity with enzymetreated red cells occurs with anti-Ch, -Rg, -Ena, -Inb, -JMH, -
Ge2, and some examples of anti-Yta. Weak nebulous reactions in the AHG phase are often associated with anti-
Kna, -McCa, -Yka, and -Csa.
4. Complement-binding autoantibodies, such as anti-I and -IH, or alloantibodies, such as anti-Lub, -Vel, and
-Yta, may give similar results when polyspecific AHG reagent is used.
ETHNIC CLUES. Antibodies, such as anti-U, -McCa, -Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and-Ata, should be
considered if the serum is from an individual of African ethnicity because the antigen-negative phenotypes
occur almost exclusively in persons of African ethnicity. Individuals with anti-Kpb are almost always of
European ethnicity. Anti-Dib is usually found among populations of Asian, South American Indian, and Native
American ethnicity (Table 16-1).
INTERPRETING A POSITIVE DAT RESULT. When a patient produces an antibody to a high-prevalence
antigen after transfusion, the patient’s posttransfusion red cells may have a positive DAT result, and both serum
and eluate may be reactive with all red cells tested. Because this pattern of reactivity is identical to that of many
warm-reacting autoantibodies that appear after transfusion, the two scenarios can be very difficult to
differentiate. A posttransfusion alloantibody to a high-prevalence antigen would be expected to produce a DAT
result of mixed-field appearance (ie, some red cells agglutinated among many unagglutinated red cells) because
only the transfused red cells would be coated with antibody. In practice,
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AABB TECHNICAL MANUAL
however, weak sensitization and mixed-field agglutination can be difficult to differentiate. If a
pretransfusion specimen is not available, it may be helpful to use red cell separation procedures to isolate
autologous red cells for testing or perform a DNA-based genotype, as described earlier in this chapter.
Performing a DAT on autologous red cells, testing the posttransfusion serum with DAT-negative autologous red
cells, or both may help distinguish an autoantibody from an alloantibody. If a DAT result from autologous red
cells is negative, the reactivity is consistent with an alloantibody. If the posttransfusion serum is reactive with
DAT-negative autologous red cells, the reactivity is consistent with an autoantibody (Chapter 17 and Fig 16-2).
Antibodies to Low-Prevalence Antigens
If a serum sample is reactive only with a single donor or reagent red cell sample, an antibody to a low-
prevalence antigen should be suspected. To identify such an antibody, one can test the serum with a panel of
reagent red cells that express low-prevalence antigens. Alternatively, the one reactive red cell sample can be
tested with known antibodies to low-prevalence antigens. Unfortunately, sera that contain antibodies to low-
prevalence antigens often contain multiple antibodies to low-prevalence antigens. Although low-prevalence
antigens are rare by definition, antibodies that recognize some of them are much less rare. Many antibodies to
low-prevalence antigens are reactive only at temperatures below 37 C and therefore have doubtful clinical
significance. To confirm the suspected specificities, one may need the expertise and resources of a reference
laboratory.
If an antibody to a low-prevalence antigen is suspected, transfusion should not be delayed while
identification studies are performed. Because antisera to type donor units for low-prevalence antigens are rarely
available, it is usually necessary to rely on the crossmatch to avoid transfusion of antigen-positive units. When
the serum is reactive with only one donor unit or reagent red cell, the most likely cause is an antibody to a low-
prevalence
antigen; however, other possible explanations include that the red cells may be ABO incompatible, have a
positive DAT result, or be polyagglutinable.
If an antibody in the serum of a pregnant woman is suspected of reacting with a lowprevalence antigen,
testing the father’s red cells with maternal plasma (if the plasma is ABO compatible) can predict the possibility
that the fetal red cells also carry the paternal antigen and are incompatible with the maternal antibody, even if
the antibody specificity is unknown. If a newborn has a positive DAT result, testing the mother’s serum or an
eluate from the infant’s red cells against the father’s red cells can implicate an antibody to a lowprevalence
antigen as the probable cause. That test can be performed only if the mother’s plasma is ABO compatible with
the father’s red cells or if the eluate from the infant’s red cells does not contain anti-A or -B that would be
reactive with the father’s red cells. Some reference laboratories do not attempt to identify antibodies to low-
prevalence antigens because the antibodies are not clinically meaningful. Antibodies may be identified when
time permits and suitable reagents are available.
Antibodies to Reagent Components and Other Anomalous Serologic Reactions
Antibodies to a variety of drugs and additives can cause positive results in antibody detection and
identification tests. The mechanisms are probably similar to those discussed in Chapter 17. Most of these
anomalous reactions are in-vitro phenomena and have no clinical significance in transfusion therapy, other than
causing laboratory problems that delay transfusions. The reactions rarely cause erroneous interpretations of
ABO typing that could endanger the patient. For a more detailed discussion, see the suggested reading by
Garratty.
ROULEAUX. Rouleaux are aggregates of red cells that often look like a stack of coins when viewed
microscopically. Rouleaux formation is an in-vitro phenomenon produced by abnor
417
mal serum protein concentrations. It maybe difficult to detect antibodies by direct agglutination in a test
serum that contains rouleauxproducing proteins; however, it is not a problem in an IAT, where the serum is
washed away. The saline replacement technique can be used to detect direct-agglutinating antibodies in the
presence of rouleaux (Method 3-7).
PRESERVATIVE SOLUTIONS. Antibodies that are reactive with an ingredient in the solution used to
preserve reagent red cells (eg, chloramphenicol, neomycin, tetracycline, hydrocortisone, EDTA, sodium
caprylate, or various sugars) may agglutinate red cells suspended in that solution. The autologous control is
often nonreactive unless a suspension of autologous red cells is prepared with the manufacturer’s red cell
diluent or a similar preservative. Such reactions can often be avoided by washing the reagent red cells with
saline before testing. Adding the medium to the autologous control and converting a nonreactive test to a
reactive test can often confirm the role of the preservative. In some cases, however, washing the reagent red
cells does not circumvent the reactivity, and the resolution may be more complex.
ENHANCEMENT MEDIA. Antibodies that are reactive with ingredients in other reagents, such as
commercially prepared LISS additives or albumin, can cause agglutination in tests that use reagent red cells,
donor red cells, autologous red cells, or all three. Ingredients that have been implicated include parabens (in
some LISS additives), sodium caprylate (in some albumins), and thimerosal (in some LISS/saline preparations).
Antibody to ingredients in enhancement media may be suspected if the autologous control is positive but the
DAT result is negative. Omitting the enhancement medium usually circumvents the reactivity.
In some cases, antibodies to reagent ingredients show blood group specificity (eg, paraben-dependent anti-
Jka, paraben-dependent antibody to Rh protein, or caprylate-dependent autoanti-e).40-42The autocontrol may
be reactive if the patient’s own red cells express
the antigen, but the DAT result should be negative.
RED-CELL-RELATED ANOMALIES. The age of red cells can cause anomalous serologic reactions.
Antibodies exist that are reactive only with stored red cells. Such antibodies can cause agglutination of reagent
red cells by all techniques, and the reactivity may be enhanced with enzyme-treated red cells. Such reactivity is
not affected by washing the red cells, and the autocontrol is usually nonreactive. No reactivity is seen when
freshly collected red cells (ie, from freshly drawn donor or autologous blood samples) are tested.
Patients with a Positive Autocontrol
Reactivity of a patient’s serum with the autologous cells may indicate the presence of warm or cold
autoantibodies, antibodies to certain drugs, alloantibodies to transfused red cells if the patient was recendy
transfused, or antibodies to a constituent of the test medium. When the autocontrol is positive, a DAT should be
performed (Chapter 17 and Fig 16-2).
NO recent TRANSFUSIONS. If the reactivity in the serum occurs at room temperature or below, the cause
is often anti-I or another cold autoagglutinin. If the reactivity occurs in the AHG phase, the reactivity is usually
associated with a positive DAT result and possible autoantibodies. If, in addition, the serum is reactive with all
cells tested, autoadsorption or other special procedures may be necessary to determine whether there are
underlying alloantibodies that are masked by the autoantibodies. If the serum is nonreactive or shows only weak
reactivity, an eluate may demonstrate more potent autoantibody reactivity. (See “Cold Autoantibodies” and
“Warm Autoantibodies” sections below and Chapter 17 for a more detailed discussion.)
recent TRANSFUSIONS. If the autocontrol is positive in the AHG phase, there may be antibody-coated
donor red cells in the patient’s circulation, resulting in a positive DAT result that may show mixed-field
reactivity. An elution should be performed, especially when
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AABB TECHNICAL MANUAL
tests on serum are inconclusive. For example, a recently transfused patient may have a positive autocontrol
and serum that is weakly reactive with most but not all Fy(a+) red cells. It may be possible to confirm anti-Fya
specificity in an eluate because more antibody is often bound to donor red cells than is free in the plasma.
Furthermore the preparation of an eluate concentrates the antibody. It is rare for transfused red cells to make the
autocontrol positive at other test phases, but this can occur, especially with a newly developing or cold-reacting
alloantibody.
Some patients may form warm autoantibodies following red cell transfusion; therefore, if the positive DAT
test does not have a mixed-field appearance—and especially if the serum is reactive with all red cells—then the
possibility of an autoantibody should be considered. Detection of alloantibodies in samples with autoantibody
may require allogeneic adsorptions (Method 3-20). If the patient’s phenotype is known for the common red cell
antigens, an allogeneic adsorption may require only one adsorption using a reagent cell that is matched for the
antigens that the patient lacks. If the patient’s phenotype is unknown, it is necessary to perform multiple
allogeneic adsorptions using a combination of reagent cells, each of which lacks some of the common red cell
antigens. Cells that are homozygous for the common red cell antigens, such as Rj (DCe), R2 (DcE), and r (ce),
are frequently used. The adsorbed serum is tested with reagent cells to rule out the common antibodies that
correspond to the antigens that are missing from the adsorbing cell. For example, if the adsorbing cell types R1
(DCe), K-, Fy(a+b-), ]k(a-b+), and S-s+, the adsorbed plasma could be tested for anti-c, anti-E, antiK, anti-Fyb,
anti-Jka, and anti-S. It is never possible, however, to rule out antibodies to high-prevalence antigens when
allogeneic adsorption techniques are used.
If the DAT result does have a mixed-field appearance and the serum is reactive with all cells tested, a
transfusion reaction caused by an alloantibody to a high-prevalence antigen should be considered (Fig 16-2).
COLD AUTOANTIBODIES. Potent cold autoantibodies that are reactive with all red cells, including the
patient’s own, can create special problems—especially when the reactivity persists at temperatures above room
temperature. Cold autoantibodies may be benign or pathologic. (See Chapter 17 for a more detailed discussion.)
There are different approaches to testing sera with potent cold agglutinins. To detect underlying and
potentially clinically significant antibodies, methods that circumvent the cold autoantibody can be used.
Procedures for the detection of alloantibodies in the presence of cold-reactive autoantibodies include the
following:
1. Prewarming techniques in which red cells and serum are prewarmed to 37 C separately before they are
combined (Method 36).
2. The use of anti-IgG rather than polyspecific AHG reagent.
3. Cold autoadsorption of the patient’s serum with autologous red cells to remove autoantibodies but not
alloantibodies.
4. Adsorption with rabbit erythrocytes or rabbit erythrocyte stroma.
WARM AUTOANTIBODIES. Patients with warm-reactive autoantibodies in their sera create a special
challenge because the antibody is reactive with virtually all red cells tested. The majority of warm
autoantibodies are IgG, although some warm autoantibodies are IgM. IgM warm autoantibodies are unusual, but
they have caused severe (often fatal) autoimmune hemolytic anemia.43 If a patient with warm autoantibodies
requires a transfusion, it is important to detect any underlying clinically significant alloantibodies that the
autoantibodies may mask. (For more information, see Chapter 17 and Methods 4-8 through 4-10.) The
reactivity of most warm-reactive autoantibodies is greatly enhanced by certain methods such as PEG and
enzymes and, to a lesser extent, by LISS. It is often helpful to omit the enhancement media when testing sera
that contain warm autoantibodies. If such tests are nonreactive, the same procedure can be used
419
for compatibility testing without the need for adsorptions.
Frequency of Antibody Testing
After a clinically significant antibody has been identified, antigen-negative Red Blood Cell (RBC) units
must be selected for all future transfusions, even if the antibodies are no longer detectable.17(p37) In addition,
an AHG crossmatch must be performed. It is rarely necessary to repeat the identification of known antibodies.
AABB Standards for Blood Banks and Transfusion Services states that in patients with previously identified
antibodies, testing methods should be used that identify additional clinically significant antibodies.17(p36) Each
laboratory should define and validate methods for the detection of additional antibodies in these patients.
Immunohematology Reference Laboratories
When antibody problems cannot be resolved or rare blood is needed, a reference laboratory can provide
consultation and assistance through its access to the American Rare Donor Program (ARDP). (See Method3-
21.)
POSTANALYTICAL CONSIDERATIONS: SELECTING BLOOD FOR TRANSFUSION
After an antibody has been identified, it is important to determine its clinical significance. Antibodies that
are reactive at 37 C, in an IAT, or both are potentially clinically significant. Antibodies that are reactive at room
temperature and below are usually not clinically significant; however, there are many exceptions. For example,
anti-Vel, -P, and -PP1Pk (-Tja) may be reactive only at cold temperatures yet may cause red cell destruction in
vivo. Anti-Ch, anti-Rg, and many of the Knops and Cost antibodies have little or no clinical significance despite
their reactivity in an IAT. Reported experience with examples of antibodies with the same specificity can be
used in assessing the
clinical significance. Table 16-6 summarizes the expected reactivity and clinical significance of commonly
encountered alloantibodies. For some antibodies, little or no data exist, and the decision about clinical
significance must be based on the premise that clinically significant antibodies are those that are active at 37 C,
in an IAT, or both.
Certain laboratory tests have been used to predict the clinical significance of antibodies. The monocyte
monolayer assay, which quantifies phagocytosis, adherence of antibody-coated red cells, or both, can be used to
predict the in-vivo clinical significance of some antibodies. The test for antibody-dependent cellular
cytotoxicity, which measures lysis of antibodycoated red cells, and the chemiluminescence assay, which
measures the respiratory release of oxygen radicals after phagocytosis of antibody-coated red cells, have been
helpful in predicting in-vivo antibody reactivity—particularly for predicting the severity of HDFN. For cold-
reactive antibodies, in-vitro thermal amplitude studies may predict the likelihood of in-vivo hemolysis.44
In-vivo tests may also be used to evaluate the significance of an antibody. The most common technique is a
red cell survival study in which radiolabeled, antigen-positive red cells (usually labeled with 51Cr) are infused
into the patient. After a specified period has elapsed, a sample of blood from the patient is tested for
radioactivity. With this technique, it is possible to measure the survival of 1 mL or less of infused cells. Another
in-vivo technique, flow cytometry, can also be used to measure the survival of infused red cells, but a larger
aliquot of red cells (about 10 mL) is usually required. Interpretation of in-vivo survival test results is
complicated by the fact that small aliquots of incompatible red cells may have a faster rate of destruction than
an entire transfused RBC unit. Comparison with documented cases in the literature and consultation with a
reference laboratory should provide guidance about previous examples of similar specificities.
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AABB TECHNICAL MANUAL
Phenotyping Donor Units
Whenever possible, RBC units selected for transfusion to a patient with potentially clinically significant
antibodies should be tested and found negative for the corresponding antigen(s). Even if the antibodies are no
longer detectable, all subsequent RBC transfusions to that patient should lack the antigen to prevent a secondary
immune response. The transfusion service must maintain records of all patients in whom clinically significant
antibodies have been previously identified, and an AHG crossmatch procedure is required if the serum contains
—or has previously contained—a clinically significant antibody.17(pp37'38,75)
A potent example of the antibody should be used to identify antigen-negative blood. Often, the antibody is a
commercial antiserum, but to save expensive or rare reagents, units can be tested first for compatibility with the
patient’s serum. Then, the absence of the antigen in compatible units can be confirmed with commercial
reagents. If the antibody is unusual and commercial antiserum is not available, a stored sample from the
sensitized patient can be used to select units for transfusion at a later time, especially if the patient’s later
samples lose reactivity. If a patient’s serum is used as a typing reagent, the antibody should be well
characterized and retain its reactivity after storage. Appropriate negative and weak-positive controls (eg, from
heterozygous donors) should be used at the time of the testing. The following criteria, established by the FDA
for licensing some reagents, should be used as guidelines for human-source reagents used in lieu of commercial
reagents45,46:
1. Anti-K, -k, -Jka, -Fya, and -Cw: dilution of 1:8 must produce at least a 1+ reaction.
 2. Anti-S, -s, -PI, -M, -I, -c (saline), -e (saline), and -Al: dilution of 1:4 must produce at least a 1+ reaction.
3. Most other specificities: undiluted must produce at least a 2+ reaction.
When selecting units for patients with clinically significant antibodies, some serologists recommend typing
the units with anti
bodies from two different sources, but others consider this step unnecessary—especially when potent
reagents are available and an AHG crossmatch will be performed. Different lots of antibody from the same
manufacturer and even different reagents from different manufacturers may have been prepared from the same
source material because manufacturers often share these resources.
If a donor unit is tested for selected antigens and labeled by the blood center, the use of licensed
(commercial) reagents, if available, is required. If no licensed reagent is available, the unit may be labeled with
appropriate wording (eg, “Tested and found negative for XX antigen using unlicensed typing reagents”).47
Except for results of ABO and D typing, there is no requirement that the hospital repeat testing of donor minor
antigen typing if the results are attached to the units on the label or by a tag. Minor antigen typing results on
packing slips or not physically attached to the donor unit should be confirmed by the hospital if the unit is used
for clinical care.
Antigen-Negative Blood vs Crossmatch for Compatibility
For certain antibodies, typing the donor units may not be necessary, and the patient’s serum can be used to
select serologically compatible RBC units. This is especially true for antibodies that characteristically are
reactive below 37 C (eg, anti-M, -N, -PI, -Lea, -Leb, and -Al) and that do not ordinarily produce a secondary
immune response following the transfusion of antigen-positive RBC units.
It can sometimes be a best practice to provide phenotypically matched, antigen-negative RBC units as a
prophylactic measure when the patient has no detectable antibody. When a patient of the RjRj phenotype
produces anti-E, some serologists suggest that RBC units should be negative for both the E and c antigens. This
recommendation is based on the assumption that the stimulus to produce anti-E may also have stimulated anti-c
or anticE that remains undetected by routine tests.48 Similarly, for an R,R2 patient with demonstra
421
ble anti-C, the use of e-negative donor blood may be considered.
When a patient has potent warm autoantibody and compatibility cannot be demonstrated by routine testing
or when an antibody has not been specifically demonstrated but cannot be conclusively excluded, it may be
prudent to select RBC units that are phenotypically matched for clinically significant antigens.
When Rare Blood Is Needed
Rare blood includes units that are negative for high-prevalence antigens or are negative for a combination of
common antigens. When a patient has multiple antibodies, it can be helpful to determine the prevalence of
compatible donors. To calculate this prevalence, one must multiply the prevalence of donors who are negative
for one antigen by the prevalence of donors who are negative for each of the other antigens. For example, if a
serum contains anti-c, anti-Fy1, and anti-S and if the prevalence of antigen-negatives are c-negative = 18%,
Fy(a-) = 34%, and S-negative = 45%, then the prevalence of compatible units is 0.18 x
0.34 x 0.45 = 0.028, or 2.8%. If the patient is group O, the prevalence of group O donors (45%) is factored
into the calculation as follows: 0.028 x 0.45 = 0.013, or 1.3%.
If any of these antibodies is present alone, finding compatible blood is not very difficult; but the
combination requires a large number of units to provide one compatible unit. The calculation above uses the
prevalence in populations of European ethnicity, and prevalence may be different in populations of
nonEuropean ethnicity. In calculating the probability of compatible donors, one should use the prevalence that
corresponds with the eth
nic composition of the donor population, if available.
When units of rare (<1 in 5000) or uncommon (<1 in 1000) phenotypes are needed, the ARDP can be very
helpful. This program, which is accessible only to personnel from an accredited reference laboratory, can
identify blood suppliers that are known to have potentially compatible units (usually frozen RBC units) and
donors who maybe eligible to donate (Method 3-21).
Family members are another potential source of rare blood donors. The absence of high-prevalence antigens
is usually associated with the inheritance of the same rare recessive blood group gene from each heterozygous
parent. Children from the same parents have one chance in four of inheriting the same two rare genetic
mutations, making siblings much more likely than the general population to have the rare blood type. In most
cases, blood from the patient’s parents, children, and half of the patient’s siblings express only one rare gene. If
transfusion is essential and there is no alternative to transfusing incompatible blood, these heterozygous (single-
dose) donors are preferable to random donors. For infants with HDFN resulting from multiple antibodies or an
antibody to a high-prevalence antigen, the mother (if she is ABO compatible) is often the logical donor.
If the clinical situation allows, autologous RBC transfusions should be considered for patients with rare
phenotypes who are expected to need rare blood in the future. For some patients with multiple antibodies who
are not able to donate autologous units, it may be necessary to determine whether any of the antibodies is less
likely to cause red cell destruction and, in a critical situation, to give blood that is incompatible for that
particular antigen.
KEY POINTS
1. It is important to consider the patient’s medical history (transfusions, pregnancies, transplantations,
diagnoses, drugs, and ethnic origin) before starting antibody identification testing.
2. An antibody may be tentatively excluded or ruled out if an antigen is present on a reagent cell and the
patient’s serum does is not reactive with it.

422
AABB TECHNICAL MANUAL
3. Common clinically significant alloantibodies that should be excluded during antibody identification
testing are anti-D, -C, -E, -c, -e, -K, -Fy'\ -Fyb, -Jka, -Jkb, -S, and -s.
4. Based on probability, the use of two reactive and two nonreactive red cell samples is an acceptable
approach for antibody confirmation.
5. The autologous control, in which serum and autologous red cells are tested under the same conditions as
the serum and reagent red cells, is an important part of antibody identification. The autologous control is not the
same as a DAT.
6. The phenotype of the autologous red cells is an important part of antibody identification. When an
antibody has been tentatively identified in the serum, the corresponding antigen is expected to be absent from
the autologous red cells, although exceptions can occur.
 7. An antibody can be removed from a serum sample by adsorption onto red cells that express the
corresponding antigen.
8. Elution dissociates antibodies from sensitized red cells. Bound antibody may be released by changing the
thermodynamics of an antigen-antibody reaction, neutralizing or reversing forces of attraction that hold antigen-
antibody complexes together, or disturbing the structures of the antigen-antibody binding site.
9. A clinically significant red cell antibody is an antibody that is frequently associated with HDFN,
hemolytic transfusion reactions, or a notable decrease in the survival of transfused red cells.
10. Whenever possible, RBC units selected for transfusion to a patient with a potentially clinically
significant antibody should be tested and found negative for the corresponding antigents). Even if the antibody
is no longer detectable, all subsequent RBC transfusions to the patient should lack the antigen to prevent a
secondary immune response.
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RBCs with monoclonal Rh reagents (abstract). Transfusion 1995;35(Suppl):67.
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Their application, interpretation, and correlation of results. Immunohematology 2008;24:180-90.
20. Reisner R, Butler G, Bundy K, Moore SB. Comparison of the polyethylene glycol antiglobulin test and
the use of enzymes in antibody detection and identification. Transfusion 1996; 36:487-9.
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red cell autoantibodies. Transfusion 1996;36:481-6.
22. Issitt PD, Combs MR, Bredehoeft SJ, et al. Lack of clinical significance of “enzyme-only” red cell
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 23. Beattie KM, Zuelzer WW. The frequency and properties of pH-dependent anti-M. Transfusion
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24. Bruce M, Watt AH, Hare W, et al. A serious source of error in antiglobulin testing. Transfusion
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30. Advani H, Zamor J, Judd WJ, et al. Inactivation of Kell blood group antigens by 2-
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33. Liew YW, Uchikawa M. Loss of Era antigen in very low pH buffers. Transfusion 1987;27:4423.
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35. Freedman J, Masters CA, Newlands M, Mollison PL. Optimal conditions for use of sulphydryl
compounds in dissociating red cell antibodies. Vox Sang 1976;30:231-9.
36. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific
Publications, 1998.
37. Malyska H, Kleeman JE, Masouredis SP, Victoria EJ. Effects on blood group antigens from storage at
low ionic strength in the presence of neomycin. Vox Sang 1983;44:375-84.
38. Westhoff CM, Sipherd BD, Toalson LD. Red cell antigen stability in K3EDTA. Immunohematol
1993;9:109-11.
39. Moulds JM, Zimmerman PA, Doumbo OK, et al. Molecular identification of Knops blood group
polymorphisms found in long homologous region D of complement receptor 1. Blood 2001;97:2879-85.
40. Judd WJ, Steiner EA, Cochran RK. Paraben-associated autoanti-Jka antibodies: Three examples
detected using commercially prepared low-ionic strength saline containing parabens. Transfusion 1982;22:31-5.
41. Judd WJ, Storry JR, Amnesley TD, et al. The first example of a paraben-dependent anti
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body to an Rh protein. Transfusion 2001;41: 371-4.
42. Dube VE, Zoes C, Adesman R Caprylate-dependentauto-anti-e. Vox Sang 1977;33:359-63.
43. Arndt PA, Leger RM, Garratty G. Serologic findings in autoimmune hemolytic anemia associated with
immunoglobulin M warm autoantibodies. Transfusion 2009;49:235-42.
44. Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia: Churchill Livingstone, 2004.
45. Food and Drug Administration. 7342.001: Inspection of licensed and unlicensed blood banks, brokers,
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SUGGESTED READINGS
Dake L, ed. Standards for immunohematology reference laboratories. 8th ed. Bethesda, MD: AABB, 2013.
 Daniels G. Human blood groups. 3rd ed. Hoboken, NJ: Wiley-Blackwell, 2013.
Daniels G, Poole J, deSilva M, et al. The clinical significance of blood group antibodies. Transfus Med
2002;12:287-95.
Engelfriet CP Overbeeke MA, Dooren MC, et al. Bioassays to determine the clinical significance of red cell
antibodies based on Fc receptorinduced destruction of red cells sensitized with IgG. Transfusion 1994;34: 617-
26.
Garratty G. In-vitro reactions with red blood cells that are not due to blood group antibodies: A review.
Immunohematology 1998; 14:1-11.
Harmening DM. Modem blood banking and transfusion practices. 6th ed. Philadelphia: FA Davis, 2012.
Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific
Publications, 1998.
Judd WJ, Johnson S, Storry J. Judd’s methods in immunohematology. 3rd ed. Bethesda, MD: AABB Press,
2008.
Kanter MH. Statistical analysis. In: Busch MP, Brecher ME, eds. Research design and analysis. Bethesda,
MD: AABB, 1998:63-104.
Klein HG, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 12th ed. Oxford: WileyBlackwell,
2014.
Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
Lomas-Frances C, DePalma H. 2007 Rock 0yen Symposium. DNA-based assays for patient testing: Their
application, interpretation, and correlation of results. Immunohematology 2008;24:180-90.
Menitove JE. The Hardy-Weinberger principle: Selection of compatible blood based on mathematic
principles. In: Fridey JL, Kasprisin CA, Chambers IA, Rudmann SV eds. Numbers for blood bankers. Bethesda,
MD: AABB, 1995: 111.
Reid ME, Lomas-Francis C, Olsson M. The blood group antigen factsbook. 3rd ed. London: Elsevier
Academic Press, 2012.
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Rudmann SV ed. Serologic problem-solving: A systematic approach for improved practice. Bethesda, MD:
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Weisbach V Kohnhauser T, Zimmermann R, et al. Comparison of the performance of microtube column
systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in
the detection of red cell alloantibodies. Transfus Med 2006; 16: 276-84.
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practice. Immunohematology 2008; 24:190-5.
Chapter 17
The Positive Direct Antiglobulin Test and Immune-Mediated Hemolysis
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
OHITHE DIRECT ANTIGLOBULIN test (DAT) is a simple test used to determine if red cells have been
coated in vivo with immunoglobulin (Ig), complement, or both. The DAT is used primarily for the investigation
of hemolytic transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic
anemia (AIHA), and drug-induced immune hemolysis. A positive DAT result may or may not be associated
with immune-mediated hemolysis. As shown in Table 17-1, there are many causes of a positive DAT result.
THE DAT
The DAT should be performed on every patient in whom the presence of hemolysis has been established to
distinguish immune from nonimmune hemolytic anemia. The DAT should also be performed when a positive
autocontrol is found in antibody identification studies (see Chapter 16), but there is no bene
fit to performing a DAT (or autocontrol) as part of routine pretransfusion testing. The predictive value of a
positive DAT result is 83% in a patient with hemolytic anemia, but only 1.4% in a patient without, hemolytic
anemia.1
Small amounts of IgG and complement that are lower than the detection limit of routine testing techniques
appear to be present on all red cells. Using sensitive testing techniques, 5 to 90 IgG molecules/red cell2 and 5 to
40 C3d molecules/red cell3 have been detected in healthy individuals. Depending on the technique and reagents
used, the DAT can detect 100 to 500 molecules of IgG/red cell and 400 to 1100 molecules of C3d/red cell.
Positive DAT results are reported in 1:1000 to 1:14,000 blood donors and 1% to 15% of hospital patients.4
These large differences in incidence are probably related to the different DAT techniques used.
 Most blood donors with a positive DAT result appear to be perfectly healthy, and most patients with positive
DAT results have no

Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ), Research Associate, American Red Cross
Blood Services, Southern California Region, Pomona, California The author has disclosed no conflicts of
interest.
425

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AABB TECHNICAL MANUAL
TABLE 17-1. Some Causes of a Positive DAT Result Autoantibodies to intrinsic red cell antigens
Hemolytic transfusion reactions Hemolytic disease of the fetus and newborn Drug-induced antibodies
Passively acquired alloantibodies (eg, from donor plasma, derivatives, or immunoglobulin)
Nonspecifically adsorbed proteins (eg, hypergammaglobulinemia, high-dose intravenous immune globulin,
or modification of red cell membrane by some drugs)
Complement activation due to bacterial infection, autoantibodies, or alloantibodies
Antibodies produced by passenger lymphocytes (eg, in transplanted organs or hematopoietic components)
DAT = direct antiglobulin test
obvious signs of hemolytic anemia. However, a careful evaluation may show evidence of increased red cell
destruction. A recent study suggests that a positive DAT result in a healthy blood donor may be a marker of risk
of future development of malignancy.5,6
A positive DAT result in a patient with hemolytic anemia indicates that the most likely diagnosis is one of
the immune hemolytic anemias. However, the DAT result can be positive, coincidentally, in patients with
hemolytic anemia that is not immune mediated. Conversely, some patients with immune hemolytic anemia have
a negative DAT result (see “DAT-Negative AIHA” section below).
The DAT can also be positive for IgG or complement without a clear correlation with anemia in patients
with sickle cell disease, beta-thalassemia, renal disease, multiple myeloma, autoimmune disorders, AIDS, or
other diseases associated with elevated serum globulin or blood urea nitrogen levels.7'9 The interpretation of a
positive DAT result should take into consideration the patient’s history, clinical data, and results of other
laboratory tests.
Initial transfusion reaction investigations include a DAT on a posttransfusion specimen. In the presence of
immune-mediated hemolysis, the DAT result may be positive if sensitized red cells have not been destroyed or
negative if hemolysis and rapid clearance have occurred. Preparation and testing of an eluate from DAT
positive posttransfusion-reaction red cells is indicated. Even if the DAT result is only weakly positive or
negative, testing of an eluate may be informative. If the DAT result is positive on the postreaction specimen, a
DAT should also be performed on the pretransfusion specimen for comparison and appropriate interpretation.
The Principles of the DAT
The DAT is based on the test developed by Coombs, Mourant, and Race10 for the detection of antibodies
attached to red cells that do not produce direct agglutination. This test, an indirect antiglobulin test, was initially
used to demonstrate antibody in serum, but it was later applied to demonstrate the in-vivo coating of red cells
with antibody or complement components (the DAT).
Most of the antiglobulin reactivity is directed at the heavy chains (eg, Fc portion of the sensitizing antibody)
or the complement component, thus bridging the gap between adjacent red cells to produce visible
agglutination. The strength of the observed agglutination is usually proportional to the amount of bound protein.
The DAT is performed by testing freshly washed red cells directly with antiglobulin reagents containing
anti-IgG and anti-C3d. In the United States, only polyspecific anti-IgG, -C3d and monospecific anti-IgG, anti-
C3d,
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CHAPTER 17 DAT/Immune Hemolysis
and anti-C3b,-C3d reagents are currendy licensed. The red cells need to be washed to remove free plasma
globulins and complement; otherwise, the antiglobulin reagent can be neutralized, leading to a false-negative
result. The saline used for washing the red cells should be at room temperature; washing red cells with warm
(eg, 37 C) saline can result in the loss of red-cell-bound, low-affinity IgG. The red cells should be tested
immediately after washing to prevent false-negative results due to the elution of IgG.
Although any red cells may be tested, EDTA-anticoagulated blood samples are preferred. The EDTA
prevents in-vitro fixation of complement by chelating the calcium that is needed for Cl activation. If red cells
from a clotted blood sample have a positive DAT result due to complement, the results should be confirmed on
red cells from freshly collected blood kept at 37 C or an EDTA-anticoagulated specimen if these results are to
be used for diagnostic purposes.
The DAT can be initially performed with a polyspecific antihuman globulin (AHG) reagent that is capable
of detecting both IgG and C3d (see Method 3-14). If the results are positive, tests with monospecific reagents
(anti-IgG and anticomplement) need to be performed to appropriately characterize the immune process involved
and determine the diagnosis. Because polyspecific reagents are usually blended and testing conditions for
optimally detecting IgG and C3d on red cells may differ, some laboratories perform the DAT initially with anti-
IgG and anti-C3d reagents separately. If the polyspecific reagent is polyclonal, proteins other than IgG or C3d
(eg, IgM, IgA, or other complement components) can occasionally be detected; however, specific reagents to
distinguish these other proteins by serologic techniques are not readily available. If umbilical cord blood
samples are to be tested, it is appropriate to use anti-IgG only because HDFN results from fetal red cell
sensitization with maternally derived IgG antibody and complement activation rarely occurs.4
It is important to follow the reagent manufacturer’s instructions and recognize any product limitations.
False-negative or weaker
results can be obtained if the washed red cells are allowed to sit before they are tested with anti-IgG or if the
reading of the results is delayed. Some anticomplement reagents, in contrast, demonstrate stronger reactivity if
centrifugation is delayed for a short time after the reagent has been added. When the DAT result is positive with
both anti-IgG and anti-C3, the red cells should be tested with an inert control reagent (eg, 6% albumin or
saline). Lack of agglutination of the red cells in the control reagent provides some assurance that the test results
are accurately interpreted. If the control is reactive, the DAT result is invalid [see sections below on warm
AIHA (WAIHA) and cold agglutinin disease (CAD)]. Reactivity with this control reagent can indicate
spontaneous agglutination caused by heavy coating of IgG or rare warm-reactive IgM, or it can indicate IgM
cold autoagglutinins that were not dissociated during routine washing.
Evaluation of a Positive DAT Result
A positive DAT result alone is not diagnostic of hemolytic anemia. Understanding the significance of this
positive result requires knowledge of the patient’s diagnosis; recent drug, pregnancy, and transfusion history;
and the presence of acquired or unexplained hemolytic anemia. Dialogue with the attending physician is
important. Clinical considerations together with laboratory data should dictate the extent to which a positive
DAT result is evaluated.
Patient History
The following situations may warrant further investigation of a positive DAT result.
1. Evidence of in-vivo hemolysis (ie, red cell destruction). If a patient with anemia who has a positive DAT
result shows evidence of hemolysis, testing to evaluate a possible immune etiology is appropriate.
Reticulocytosis; spherocytes observed on the peripheral blood film; hemoglobinemia; hemoglobinuria;
decreased serum haptoglobin; and elevated levels of serum unconjugated (indirect) bilirubin or lactate
dehydroge
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AABB TECHNICAL MANUAL
 nase (LDH), especially LDH1, may be associated with increased red cell destruction. These factors are
indicative of hemolytic anemia but not specifically immune hemolytic anemia. If there is no evidence of
hemolytic anemia, no further studies are necessary unless the patient requires a red cell transfusion and the
serum contains incompletely identified antibodies to red cell antigens. Testing an eluate may be helpful for
antibody identification (see “Elution” section below and Chapter 16).
2. Recent transfusion. When a patient has recently been transfused, a positive DAT result may be the first
indication of a developing immune response. The developing antibody sensitizes the transfused red cells that
have the corresponding antigen, and the DAT result becomes positive. The antibody may not be present in
sufficient quantity to be detected in the serum. Antibody may appear as early as 7 to 10 days after transfusion in
a primary immunization or as early as 1 to 2 days in a secondary response.4,11 These alloantibodies could
shorten the survival of red cells that have already been transfused or administered in subsequent transfusions. A
mixed-field appearance in the posttransfusion DAT result (ie, agglutination of donor red cells and no
agglutination of the patient’s red cells) may or may not be observed.
3. Administration of drugs associated with immune-mediated hemolysis. Many drugs have been reported to
cause a positive DAT result and/or immune-mediated hemolysis, but this occurrence is not common.12 (See
“Drug-Induced Immune Hemolytic Anemia” section below.)
4. History of hematopoietic progenitor cell or organ transplantation. Passenger lymphocytes of donor origin
produce antibodies directed against ABO or other blood group antigens on the recipient’s red cells, causing a
positive DAT result.4
5. Administration of intravenous Ig (IVIG) or IV anti-D. MG may contain ABO antibodies, anti-D, or,
sometimes, other antibodies.13 Intravenous anti-D used to treat
immune thrombocytopenia (previously known as “immune thrombocytopenic purpura”) causes Rh-positive
patients to develop a positive DAT result. IV anti-D may also contain other antibodies, including ABO
antibodies.14
Serologic Investigation
Three investigative approaches are helpful in the evaluation of a positive DAT result.
1. Test the DAT-positive red cells with anti-IgG and anti-C3d reagents to characterize the type of protein(s)
coating the red cells. This will help classify an immune-mediated hemolytic anemia.
2. Test the serum/plasma to detect and identify clinically significant antibodies to red cell antigens.
Additional tests that are useful in classifying the immune hemolytic anemias and procedures for detecting
alloantibodies in the presence of autoantibodies are described later in this chapter.
3. Test an eluate prepared from the DAT-positive red cells with reagent red cells to determine whether the
coating protein has red cell antibody specificity. When the only coating protein is complement, the eluate is
likely to be nonreactive. However, an eluate from the patient’s red cells coated only with complement should be
tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion. The eluate
preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient’s
plasma.
Results of these tests combined with the patient’s history and clinical data should assist in classification of
the problem involved.
Elution
Elution can be informative in the following situations:
■ Clinical signs and symptoms of immune hemolysis are present.
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CHAPTER 17 DAT/Immune Hemolysis
■ Serum test results are negative or inconclusive for a patient who has been recently transfused.
■ HDFN is suspected but no alloantibodies were detected in the maternal plasma.
Performing an elution routinely on the red cells of all patients who have a positive DAT result is not
recommended. The majority of pretransfusion patients with a positive DAT result have a nonreactive eluate that
is often associated with an elevated serum globulin level.7'9
Elution frees antibody from sensitized red cells and recovers antibody in a usable form. Multiple elution
methods have been described and reviewed.15 Many laboratories use commercial acid elution kits, primarily for
ease of use and decreased exposure to potentially harmful chemicals; these kits are suitable to recover antibody
in most cases. False-positive eluate results associated with high-titer antibodies have been reported when the
low ionic wash solution supplied with the commercial acid eluates was used.16 Because no single elution
method is ideal in all situations, an alternative elution method (eg, an organic solvent) may be used in some
high-complexity reference laboratories when a nonreactive acid eluate result is not in agreement with clinical
data.17
Table 17-2 lists the uses of some common elution methods. Typically, eluates are tested
only at the antiglobulin phase. If an IgM antibody is being investigated or suspected, however,
centrifugation and reading after the 37 C incubation should be performed. Technical considerations for elution
are discussed in Chapter 16.
In cases of hemolytic transfusion reactions or HDFN, specific antibody (or antibodies) is usually detected in
the eluate that may or may not be detectable in the serum. For transfusion reactions, newly developed antibodies
that are initially detectable only in the eluate are usually detectable in the serum after about 14 to 21 days.18 If
the eluate is nonreactive and a non-group-0 patient has received plasma containing anti-A or anti-B (as a result
of the transfusion of group 0 platelets, for example) and the recipient appears to have immune hemolysis, the
eluate should be tested against Aj and/or B cells. It may be appropriate to test the eluate against red cells from
recently transfused donor units, which could have caused immunization to a rare antigen. For cases of HDFN
when no maternal antibody has been detected and paternal red cells are ABO incompatible with maternal
plasma, testing an eluate prepared from the infant’s red cells with the paternal red cells may detect a maternally
derived antibody to a low-prevalence antigen,
When the eluate reacts with all cells tested, autoantibody is the most likely explana
TABLE17-2. Antibody Elution Methods
Method Use Comments
Quick, small volume of red cells needed, poor
Lui freeze-thaw ABO HDFN
recovery of other antibodies
ABO HDFN, IgM
Heat (56 C) Easy, poor recovery of IgG alloand autoantibodies
agglutinating antibodies
Acid elution kits Warm auto- and Easy, possible false-positive eluate results when
(commercial) alloantibodies high-titer antibody is present16
Chemical/organic Warm auto- and Chemical hazards, eg, flammability, toxicity, or
solvent alloantibodies carcinogenicity
HDFN = hemolytic disease of the fetus and newborn; IgM = immunoglobulin M.
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AABB TECHNICAL MANUAL
tion, especially if the patient has not been transfused recently. However, if the patient has been recently
transfused, an antibody to a high-prevalence antigen should be considered. When no unexpected antibodies are
present in the serum and the patient has not been transfused recently, no further serologic testing of an
autoantibody detected only in the eluate is necessary.
The patient’s complete history, including the presence of potential passive antibodies, needs to be reviewed
when the serologic test results are evaluated. If both the serum and eluate are nonreactive, there is evidence of
immune hemolysis, and the patient has received a drug reported to have caused immune-mediated hemolysis,
testing to demonstrate drugrelated antibodies should be considered (see “Laboratory Investigation of Drug-
Induced Immune Hemolysis” section below).
AUTOIMMUNE HEMOLYTIC ANEMIA
Immune-mediated hemolysis is the shortening of red cell survival as a result of an immune response. If the
marrow is able to adequately compensate, the reduced red cell survival may not result in anemia. Immune-
mediated hemolysis is only one cause of hemolytic anemia, and many causes of hemolysis are unrelated to
immune reactions.
The serologic investigations carried out in the blood bank do not determine whether a patient has a
“hemolytic” anemia. The diagnosis of hemolytic anemia rests on clinical findings and laboratory data, such as
hemoglobin or hematocrit values; reticulocyte count; red cell morphology; bilirubin, haptoglobin, and LDH
levels; and, sometimes, red cell survival studies. The serologic findings help determine whether the hemolysis
has an immune basis and, if so, what type of immune hemolytic anemia is present. This is important because the
treatment for each type is different.
In some cases, the destruction of red cells takes place in the intravascular space with the release of free
hemoglobin into the plasma. The red cells are ruptured following activation of the classical complement
cascade. The
 characteristic features of this rare type of hemolysis are hemoglobinemia, and, when the plasma hemoglobin
level exceeds the renal threshold, hemoglobinuria. Conversely and more commonly, extravascular hemolysis
results when macrophages in the spleen and liver phagocytose red cells completely or partially (producing
spherocytes) or destroy red cells by cytotoxic events, resulting in an increase in serum bilirubin. This distinction
is a simplification, however, because hemoglobin can also be released into the plasma following extravascular
destruction if hemolysis is brisk.
Immune hemolytic anemias can be classified in various ways. One classification system is shown in Table
17-3. The AIHAs are subdivided into the major types: WAIHA, CAD, mixed- or combined-type AIHA, and
paroxysmal cold hemoglobinuria (PCH). Not all cases fit neatly into these categories. Table 17-4 shows the
typical serologic characteristics of the AIHAs. Drugs (discussed in the “Drug-Induced Immune Hemolytic
Anemia” section below) may also induce immune hemolysis; the effects of drug-induced uu/oantibodies are
serologically indistinguishable from WAIHA.
Warm Autoimmune Hemolytic Anemia
The majority of AIHA cases are caused by warm-reactive autoantibodies that are opti
TABLE17-3. Classification of Immune Hemolytic Anemias
Autoimmune hemolytic anemia (AIHA)
Warm AIHA Cold agglutinin disease Mixed-type AIHA Paroxysmal cold hemoglobinuria Alloimmune
hemolytic anemia Hemolytic transfusion reaction Hemolytic disease of the fetus and newborn Drug-induced
immune hemolytic anemia
CHAPTER 17 DAT/Immune Hemolysis
431
TABLE 17-4. Typical Serologic Findings in AIHA
WAIHA CAD Mixed-Type AIHA PCH
DAT IgG
03 only IgG + 03 03 03 only
(routine) IgG + C3 C3
lg type IgG IgM IgG, IgM IgG
IgG
Eluate Nonreactive IgG antibody Nonreactive
antibody
IAT, 35% IgM agglutinating IgG lAT-reactive Negative routine IAT
agglutinate antibody, titer >1000 antibody plus IgM result, IgG biphasic
Serum
untreated red (60%) at 4 G, reactive agglutinating antibody hemolysin in Donath-
cells at 20 C at 30 0 reactive at 30 C Landsteinertest
Broadly
reactive,
Specificity multiple Usually anti-l Usually unclear Anti-P
specificities
reported
AIHA = autoimmune hemolytic anemia; WAIHA = warm AIHA; CAD = cold agglutinin disease; PCH =
paroxysmal cold hemoglobinuria; DAT = direct antiglobulin test; IgG = immunoglobulin G; IAT = indirect
antiglobulin test.
mally reactive with red cells at 37 C. The autoantibody is usually IgG, but it can be IgM or IgA.
Serologic Characteristics
The DAT result may be positive due to IgG plus complement (67% of cases), IgG without complement
(20%), or complement without IgG (13%).4 Performing an elution at initial diagnosis and/or during
pretransfusion testing is useful to demonstrate that the IgG coating the patient’s red cells is autoantibody.
Typically in WAIHA, the eluate is reactive with virtually all red cells tested, and reactivity is enhanced in
tests against enzyme-treated red cells, with polyethylene glycol (PEG) enhancement, or in column agglutination
and solid-phase tests. The eluate usually has no serologic activity if the only protein coating the red cells is
complement.
If the autoantibody has been adsorbed by the patient’s red cells in vivo, the serum may not contain
detectable free antibody. The serum contains free antibody when the amount
of autoantibody exceeds the available binding sites on the patient’s red cells. The DAT result in such cases is
usually strongly positive.
 Autoantibody in the serum typically is reactive against all cells by an IAT. Approximately 60% of patients
with WAIHA have serum antibodies that react with untreated salinesuspended red cells. When tested with PEG,
enzyme-treated red cells, column agglutination, or solid-phase methods, more than 90% of these sera can be
shown to contain autoantibody. Agglutination at room temperature is present in about one-third of patients with
WAIHA, but these cold agglutinins have normal titers at 4 C and are nonreactive at 30 C and 37 C. Thus, these
cold agglutinins are nonpathogenic and the patient does not have CAD in addition to WAIHA.4
An unusual subcategory of WAIHA is associated with IgM agglutinins in the plasma that are reactive at 37
C.4,19 This type of WAIHA is characterized by severe hemolysis, and the prognosis for these patients can be
poor. The red cells are typically spontaneously agglutinated in the DAT; that is, the washed red cells

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AABB TECHNICAL MANUAL
are reactive with all reagents tested, including a control, such as 6% albumin (see “Serologic Problems”
section below). Complement is usually detected on the red cells; IgG or IgM may or may not be detected. IgM
agglutinins are often detected in an eluate (eg, acid) when it is inspected for agglutination after the 37 C
incubation and before the antiglobulin test is conducted. Some serum IgM warm autoagglutinins may be
difficult to detect; some are enhanced in the presence of albumin or at low pH. Optimal reactivity of the
agglutinin sometimes occurs between 20 C and 30 C rather than at 37 C. These antibodies have low titers at 4
C, usually <64, which easily differentiates this IgM warm antibody from those in CAD. To prevent
misinterpretation of titration results, titrations at different temperatures (eg, 37 C, 30 C, room temperature, and
4 C) need to be carried out with separate sets of tubes to avoid carry-over agglutination.419
Serologic Problems
Warm autoantibodies can cause technical difficulties during red cell testing. Spontaneous agglutination can
occur if the red cells are heavily coated with IgG and the reagent contains a potentiator, such as albumin. This
has been observed when high-protein Rh typing sera are used. If the control reagent provided by the
manufacturer for these antisera is reactive, the typing is invalid. IgG can less commonly cause spontaneous
agglutination in lower protein reagents (eg, monoclonal typing sera); this reactivity is often weaker or more
fragile than true agglutination and may not be detected by a 6% albumin control.20
Warm-reactive IgM agglutinins can also cause spontaneous agglutination, resulting in ABO and Rh typing
problems and/or reactivity with the negative control reagent for the DAT.19 In these cases, treatment with
dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) (Method 2-18) to disrupt the IgM agglutinin is required to
accurately interpret typing and DAT results. When the spontaneous agglutination is disrupted, the control
reagent is nonreactive.
When the DAT result is positive due to IgG, antiglobulin-reactive typing reagents cannot be used unless the
red-cell-bound IgG is first removed (see Methods 2-20 and 2-21). An alternative is to use low-protein antisera
(eg, monoclonal reagents) that do not require an antiglobulin test (refer to the manufacturer’s instructions for
the detection of spontaneous agglutination). It is helpful to know which of the common red cell antigens are
lacking on the patient’s red cells to predict which clinically significant alloantibodies the patient may have
produced or may produce in the future. Antigens absent from autologous cells could well be the target of
present or future alloantibodies.
The presence of autoantibody in the serum increases the complexity of the serologic evaluation and the time
needed to complete pretransfusion testing. If a patient who has warm-reactive autoantibodies in the serum needs
a transfusion, it is important to determine whether alloantibodies are also present. Some alloantibodies may
make their presence known by reacting more strongly or at different phases than the autoantibody, but quite
often, routine testing may not suggest the existence of masked alloantibodies.21,22
Methods to detect alloantibodies in the presence of warm-reactive autoantibodies are used to attempt to
remove, reduce, or circumvent the autoantibody. Antibody detection methods that use PEG, enzymes, column
agglutination, or solid-phase red cell adherence usually enhance autoantibodies. Antibody detection tests using
low-ionic-strength saline (LISS) or saline tube methods may not detect autoantibodies but they do detect most
clinically significant alloantibodies. Other procedures involve adsorption; two widely used adsorption
approaches are discussed below.
Adsorption with Autologous Red Cells
In a patient who has not been transfused recendy, adsorption with autologous red cells (autologous
adsorption; see Method 4-8) is the best way to detect alloantibodies in the presence of warm-reactive
autoantibodies. Only
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CHAPTER 17 DAT/Immune Hemolysis
autoantibodies are removed, and alloantibodies, if present, remain in the serum.
Autologous adsorption typically requires some initial preparation of the patient’s red cells. At 37 C, in-vivo
adsorption has occurred, and all antigen sites on the patient’s own red cells may be blocked. A gentle heat
elution at 56 C for 3 to 5 minutes can dissociate some of the bound IgG. This can be followed by treatment of
the autologous red cells with proteolytic enzymes to increase their capacity to adsorb autoantibody. Treatment
of the red cells with ZZAP, a mixture of papain or ficin and DTT, accomplishes both of these actions in one
step. It is proposed that the sulfhydryl component makes the IgG molecules more susceptible to the protease
and dissociates the antibody molecules from the cell.23 Multiple sequential autologous adsorptions with new
aliquots of red cells may be necessary if the serum contains high levels of autoantibody. Once autoantibody has
been removed, the adsorbed serum is tested for alloantibody reactivity.
Autologous adsorption is not recommended for patients who have been transfused within the last 3 months
because a blood sample may contain some transfused red cells that might adsorb alloantibody. Red cells
normally survive for about 110 to 120 days. In patients with AIHA, autologous and transfused red cells can be
expected to have shortened survival. However, determining how long transfused red cells remain in circulation
in patients who need repeated transfusions is not feasible. It has been demonstrated that very small amounts
(<10%) of antigen-positive red cells are capable of removing alloantibody reactivity in in-vitro studies.24
Therefore, it is recommended to wait for 3 months after transfusion before performing autologous adsorptions.
Adsorption with Allogeneic Red Cells
The use of allogeneic red cells for adsorption (allogeneic adsorption) may be helpful when the patient has
been recently transfused or insufficient autologous red cells are available. The goal is to remove autoantibody
and leave
the alloantibody in the adsorbed serum. The adsorbing red cells must not have the antigens against which
the alloantibodies are reactive. Because alloantibody specificity is unknown, red cells of different phenotypes
are usually used to adsorb several aliquots of the patient’s serum.
Given the number of potential alloantibodies, the task of selecting the red cells may appear formidable.
However, red cell selection is based only on those few antigens for which alloantibodies of clinical significance
are likely to be present. These include the common Rh antigens (D, C, E, c, and e), K, Fya and Fyb, Jka and
Jkb, and S and s. Red cell selection is made easier by the fact that some of these antigens can be destroyed by
appropriate pretreatment (eg, with enzymes or ZZAP) before use in adsorption procedures (see Table 16-4).
Antibodies to high-prevalence antigens cannot be excluded by allogeneic adsorptions because the adsorbing red
cells are expected to express the antigen and adsorb the alloantibody along with autoantibody.
When the patient’s phenotype is not known, group 0 red cell samples of three different Rh phenotypes
(RjR1( R2R2, and rr) should be selected (see Method 4-9). One sample should lack Jka and another, Jkb. As
shown in Table 17-5, ZZAP or enzyme pretreatment of the adsorbing red cells reduces the phenotype
requirements. Untreated red cells may be used, but the adsorbing red cells must include at least one sample that
is negative for the S, s, Fy'\ Fyb, and K antigens in addition to the Rh and Kidd requirements stated above.
If the patient’s phenotype is known or can be determined, adsorption with a single sample of red cells may
be possible. Red cells can be selected that match the patient’s phenotype or at least match the Rh and Kidd
phenotypes if ZZAP treatment is used. For example, if a patient’s phenotype is E- K- S- Fy(a-) Jk(a-), untreated
adsorbing red cells need to lack all five antigens, but enzyme-treated red cells only need to be E- K- Jk(a-) and
ZZAP-treated red cells only need to be E- Jk(a-). Adsorption using untreated red cells in the presence of PEG
(Method 4-10) or LISS25,26 are modifications that have been used to decrease the
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AABB TECHNICAL MANUAL
TABLE 17-5. Selection of Red Cells for Allogeneic Adsorption
Step 1. Select red cells for each Rh phenotype.
R2R2
rr
Step 2. On the basis of the red cell treatment, or lack of treatment (below), at least one of the Rh-phenotyped
cells should be negative for the antigens listed below.
ZZAP-Treated Red Cells Enzyme-Treated Red Cells Untreated Red Cells
 Jk(a-) Jk(a-) Jk(a-)
Jk(b-) Jk(b-) Jk(b-)
K K
Fy(a-)
Fy(b-)
S
s
incubation time for adsorptions and increase efficiency.
Testing of Adsorbed Serum
In some cases, each aliquot of serum may need to be adsorbed two or three times to remove the
autoantibody. The fully adsorbed aliquots are then tested against reagent red cells known to either lack or carry
common antigens of the Rh, MNS, Kell, Duffy, and Kidd blood group systems (eg, antibody detection cells). If
an adsorbed aliquot is reactive, the aliquot should be tested to identify the antibody. Adsorbing several aliquots
with different red cell samples provides a battery of potentially informative specimens. For example, if the
aliquot adsorbed with Jk(a-) red cells subsequently is reactive only with Jk(a+) red cells, the presence of
alloanti-Jka can be inferred confidently.
Occasionally, autoantibody is not removed by three sequential adsorptions. Additional adsorptions can be
performed, but the performance of multiple adsorptions has the potential to dilute the serum. If the adsorbing
cells do not appear to remove the antibody, the
autoantibody may have an unusual specificity that is not reactive with the red cells used for adsorption. For
example, autoantibodies with Kell, LW, or EnaFS specificity are not removed by ZZAP-treated red cells (see
Table 16-4 for a list of antigens altered by various agents). The possibility that the sample contains an autoor
alloantibody to a high-prevalence antigen should always be considered when adsorption fails to remove the
reactivity.
Autoantibodies sometimes have patterns of reactivity that suggest the presence of alloantibody. For
example, the serum of a D- patient may have apparent anti-C reactivity. The anti-C reactivity may reflect warm-
reactive autoantibody even if the patient’s red cells lack C. The apparent alloanti-C would, in this case, be
adsorbed by C- red cells, both autologous and allogeneic. This is unlike the behavior of a true alloanti-C, which
would be adsorbed only by C+ red cells. In one study, the serum adsorbed with autologous red cells often
retained autoantibodies that mimicked alloantibodies in addition to the true alloantibody(ies) present, whereas
serum adsorbed with allogeneic red cells most often contained only alloantibodies.27 This reflects an
inefficiency of autologous

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CHAPTER 17 DAT/Immune Hemolysis
adsorption that is primarily caused by limited volumes of autologous red cells available for removing all of
the autoantibody reactivity from the serum.
Specificity of Autoantibody
 In many cases of WAIHA, no autoantibody specificity is apparent. The patient’s serum reacts with all of the
red cell samples tested. If testing is performed with cells of rare Rh phenotypes, such as D- - or Rhnull, some
autoantibodies are weakly reactive or are nonreactive, and the autoantibody appears to have broad specificity in
the Rh system. Apparent specificity for simple Rh antigens (D, C, E, c, and e) is occasionally seen, especially in
saline or LISS indirect antiglobulin tests. A “relative” specificity based on stronger reactivity with cells of
certain phenotypes may also be seen; relative specificity may also be apparent after adsorption. Autoantibody
specificities are clearer in the serum than in the eluate.
Apart from Rh specificity, warm autoantibodies with many other specificities have been reported (eg,
specificities in the LW, Kell, Kidd, Duffy, and Diego systems).28,29 Patients with autoantibodies of Kell, Rh,
LW, Ge, Sc, Lu, and Lan specificities may have transiently depressed expression of the respective antigen, and
the DAT result may be negative or very weakly positive.29 In these cases, the autoantibody may initially appear
to be alloantibody. Proof that it is truly autoantibody is demonstration that after the antibody and hemolytic
anemia subside, the antigen strength returns to normal and stored serum containing antibody is reactive with the
patient’s red cells.
Tests against red cells of a rare phenotype and by special techniques to determine autoantibody specificity
have limited clinical or practical application. If the autoantibody reacts with all red cells except those of a rare
Rh phenotype (eg, Rhnull), compatible donor blood is unlikely to be available. Such blood, if available, should
be reserved for alloimmunized patients of that uncommon phenotype.
Selection of Blood for Transfusion
The most important consideration is to exclude the presence of potentially clinically significant
alloantibodies before selecting Red Blood Cell (RBC) units for transfusion. There are multiple reports in the
literature demonstrating that patients who have warm autoantibodies in their sera have a higher rate of
alloimmunization (eg, 12% to 40%, with a mean of 32%) ,21,30'33 Although these patients present a serologic
challenge, they deserve the same protection from hemolytic transfusion reactions as any other patient.
Autoantibodies that react with all reagent red cells, even weakly, are capable of masking alloantibody reactivity
(ie, reactivity of red cells with both alloantibody and autoantibody may not be any stronger than with
autoantibody alone) .21,22
It is the exclusion of newly formed alloantibodies that is of concern. Because of the presence of
autoantibodies, all crossmatches are incompatible. This is unlike the case of clinically significant alloantibodies
without autoantibodies, where a compatible crossmatch with antigen-negative red cells is possible. Monitoring
for evidence of red cell destruction caused by alloantibodies is difficult in patients who already have AIHA;
these patients’ own red cells and transfused red cells have shortened survival.
If no alloantibodies are detected in adsorbed serum, random units of the appropriate ABO group and Rh
type may be selected for transfusion. If clinically significant alloantibodies are present, the transfused cells
should lack the corresponding antigen(s). For patients facing long-term transfusion support, it is prudent to
obtain an extended phenotype or genotype. Consideration can then be given to transfusion with donor units that
are antigen matched for clinically significant blood group antigens to avoid additional alloimmunization and
potentially decrease the number of adsorptions required and the complexity of pretransfusion workup.
If the autoantibody has clear-cut specificity for a single antigen (eg, anti-e) and active hemolysis is ongoing,
blood lacking that antigen should be selected. There is evidence that
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AABB TECHNICAL MANUAL
such red cells survive longer than the patient’s own red cells.4 In the absence of hemolysis or evidence of
compromised survival of transfused cells, autoantibody specificity is not important. However, donor units that
are negative for the antigen may be chosen because this is a simple way to circumvent the autoantibody and
detect potential alloantibodies.
If the autoantibody shows broader reactivity—reacting with all cells but showing some relative specificity
(eg, preferentially reacting with e+ red cells)—whether to transfuse blood lacking the corresponding antigen is
debatable. It may be undesirable to expose the patient to Rh antigens absent from autologous cells, especially D
and especially in females of childbearing potential, merely to improve serologic compatibility testing results
with the autoantibody. (For example, when a D- patient has autoanti-e, available e- units are likely to be D+; D-
e- units are extremely rare.) Referral of the sample for molecular investigation to determine the risk for alio- or
autoantibody production will aid in decision making in these complex cases and potentially improve patient
care.
 Some laboratories use the adsorbed serum to screen and select nonreactive units (units that are antigen-
negative for clinically significant alloantibodies, if detected) for transfusion. Other laboratories do not perform a
crossmatch with the adsorbed serum because all units will be incompatible in vivo due to the autoantibody.
Issuing a unit that is serologically compatible with adsorbed serum may provide some assurance that the correct
unit has been selected and avoid incompatibility because of additional antibodies (eg, anti-Wra), but this
practice can also provide a false sense of security about the safety of the transfusion for these patients.
A transfusion management protocol using prophylactic antigen-matched units for patients with warm
autoantibodies, where feasible, in combination with streamlined adsorption procedures has been described.34
The same antigens for the commonly occurring, clinically significant antibodies (D, C, E, c, e, K, Fy", Fyb, Jka,
Jkb, and S and s) are taken into account, as discussed in the previous section on
adsorptions. The ability to implement such a protocol depends on the ability of the transfusion service, and
more often the blood supplier, to maintain an adequate inventory of phenotyped units to meet the antigen-
matching needs.
In recent years, molecular technologies have been applied to red cell genotyping for patients with warm
autoantibodies to determine which alloantibodies the patient can make. DNA tests are attractive for determining
the predicted phenotype of patients with a positive DAT (IgG) result because IgG is not always successfully
removed and some red cell antigens are sensitive to IgG removal treatment.35'37 Recent transfusions do not
interfere with molecular testing. It must be remembered that genotyping may not accurately predict the
phenotype if uncommon or rare silencing mutations are present or the patient has received a stem cell
transplant.
Some experts propose that an electronic crossmatch can be safely used for patients with autoantibodies
when the presence of common, clinically significant alloantibodies has been excluded.38,39 This approach
circumvents the need to issue units that are labeled “incompatible”; however, as discussed above, this practice
can also lead to a false sense of security.
Although resolving serologic problems for these patients is important, delaying transfusion in the hope of
finding serologically compatible blood may, in some cases, cause greater danger to the patient. Only clinical
judgment can resolve this dilemma; therefore, dialogue with the patient’s physician is important.
Transfusion of Patients with WarmReactive Autoantibodies
Patients with warm-reactive autoantibodies may have no apparent hemolysis or may have life-threatening
anemia. Patients with little or no evidence of significant hemolysis tolerate transfusion quite well. The risk of
transfusion is somewhat increased in these patients because of the difficulties with pretransfusion testing. The
duration of survival of the trans
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CHAPTER 17 DAT/Immune Hemolysis
fused red cells is about the same as that of the patient’s own red cells.
In patients with active hemolysis, transfusion may increase hemolysis, and the transfused red cells may be
destroyed more rapidly than the patient’s own red cells. This is related to the increased red cell mass available
from the transfusion and the kinetics of red cell destruction.4 Destruction of transfused cells may increase
hemoglobinemia and hemoglobinuria. Disseminated intravascular coagulation can develop in patients with
severe posttransfusion hemolysis.
The transfusion of patients with AIHA is a clinical decision that should be based on the balance between the
risks and clinical need. Transfusion should not be withheld solely because of serologic incompatibility. The
volume transfused should usually be the smallest amount required to maintain adequate oxygen delivery and not
necessarily the amount required to reach an arbitrary hemoglobin level.4 The patient should be carefully
monitored throughout the transfusion.
DAT-Nega ti ve AIHA
Clinical and hematologic evidence of WAIHA is present in some patients whose DAT result is negative. The
most common causes of AIHA associated with a negative DAT result are redcell bound IgG below the detection
threshold of the antiglobulin test, red-cell-bound IgM and IgA that are not detectable by routine AHG reagents,
and low-affinity IgG that is washed off the red cells during the washing phase for the DAT.4'40
Nonroutine tests can be applied in these situations. Unfortunately, these assays require standardization and
many have a low predictive value. One of the easier tests is for lowaffinity antibodies. Washing with ice-cold
(eg, 4 C) saline or LISS may help retain antibody on the cells; a control (eg, 6% albumin) is necessary to
confirm that cold autoagglutinins are not causing the positive results.4,40 Complement fixation antibody
consumption assay, enzyme-linked antiglobulin test, radiolabeled anti-IgG, flow cytometry, solid-phase, direct
PEG test, direct Polybrene test, column agglu
tination, and concentrated eluates are all methods that have been used to detect lower levels of red cell-
bound IgG.40
Anti-IgG, anti-C3d, and the combined anti-C3b,-C3d reagents are the only licensed products available in the
United States for use with human red cells. AHG reagents that react with IgA or IgM are available
commercially but probably have not been standardized for use with red cells in agglutination tests. They must
be used cautiously, and their hemagglutination reactivity must be carefully standardized by the user.4 In
countries outside the United States, AHG reagents for the detection of IgM and IgA in tube tests or column
agglutination tests may be available.
Cold Agglutinin Disease
CAD, which is less common than WAIHA, is the hemolytic anemia that is most commonly associated with
autoantibodies that react preferentially in the cold. CAD occurs as an acute or chronic condition. The acute form
is often secondary to Mycoplasma pneumoniae infection. The chronic form is often seen in elderly patients and
is sometimes associated with lymphoma, chronic lymphocytic leukemia, or Waldenstrom macroglobulinemia.
Acrocyanosis and hemoglobinuria may occur in cold weather. CAD is often characterized by agglutination, at
room temperature, of red cells in an EDTA specimen, sometimes to the degree that the red cells appear to be
clotted.
Serologic Characteristics
Complement is the only protein detected on red cells in almost all cases of CAD. If the red cells have been
collected properly and washed at 37 C, there will be no immunoglobulin on the cells and no reactivity in the
eluate. If other proteins are detected, a negative control for the DAT (eg, 6% albumin or saline) should be tested
to ensure that the cold autoagglutinin is not causing a false-positive result. The cold-reactive autoagglutinin is
usually IgM, which binds to red cells in the lower temperature of the peripheral circulation and causes
complement components to attach to the red cells. As
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AABB TECHNICAL MANUAL
the red cells circulate to warmer areas, the IgM dissociates but the complement remains.
IgM cold-reactive autoagglutinins associated with immune hemolysis usually react at 30 C, and 60% have a
titer of >1000 when tested at 4 C.4 If 22% to 30% bovine albumin is included in the test system, pathologic cold
agglutinins will react at 30 C or 37 C.4 On occasion, pathologic cold agglutinins have a lower titer (ie, <1000),
but they have a high thermal amplitude (ie, reactive at 30 C with or without the addition of albumin). The
thermal amplitude of the antibody has greater significance than the titer. Hemolytic activity against untreated
red cells can sometimes be demonstrated at 20 C to 25 C. Except in rare cases with Pr specificity, enzyme-
treated red cells are hemolyzed in the presence of adequate complement.
To determine the true thermal amplitude or titer of the cold autoagglutinin, the specimen is collected and
maintained strictly at 37 C until the serum and red cells are separated to avoid in-vitro autoadsorption.
Alternatively, plasma can be used from an EDTA-anticoagulated specimen that has been warmed for 10 to 15
minutes at 37 C (with repeated mixing) and then separated from the cells, ideally at 37 C. This process should
release autoadsorbed antibody back into the plasma.
In chronic CAD, the IgM autoagglutinin is usually a monoclonal protein with kappa light chains. In the
acute form induced by Mycoplasma or viral infections, the antibody is polyclonal IgM with normal kappa and
lambda light-chain distribution. Rare examples of IgA and IgG cold-reactive autoagglutinins have also been
described.4
Serologic Problems
Problems with ABO and Rh typing and other tests are not uncommon. Often, it is only necessary to
maintain the blood sample at 37 C immediately after collection and to wash the red cells with warm (37 C)
saline before testing. Alternatively, an EDTA sample can be warmed to 37 C for about 10 minutes, after which
the red cells are washed with warm saline. It is helpful to perform a parallel control
test with 6% bovine albumin to determine whether autoagglutination persists. If the control test result is
nonreactive, the results obtained with anti-A and anti-B are usually valid. If autoagglutination still occurs, it
may be necessary to treat the red cells with sulfhydryl reagents. Because cold-reactive autoagglutinins are
almost always IgM and sulfhydryl reagents denature IgM molecules, reagents (such as 2ME or DTT) can be
used to abolish autoagglutination (see Method 2-18). Treating the red cells with ZZAP reagent, as in the
preparation for adsorptions, can also be used (see Method 4-8).
When the serum agglutinates group O reagent red cells, ABO serum tests are invalid. Repeating the tests
using prewarmed serum and group A,, B, and O red cells and allowing the red cells to “settle” after incubation
at 37 C for 1 hour (instead of centrifuging the sample) often resolves any discrepancy (see Method 211). By
eliminating the centrifugation step, interference by cold-reactive autoantibodies might be avoided. Weak anti-A
and/or -B in some patients’ sera may not react at 37 C. Alternatively, adsorbed serum (either autoadsorbed or
adsorbed with allogeneic group O red cells) can be used. Rabbit erythrocyte stroma should not be used for ABO
serum tests because anti-B and anti-Al maybe removed.41,42
Detection ofAlloantibodies in the Presence of Cold-Reactive Autoantibodies
Cold-reactive autoagglutinins rarely mask clinically significant alloantibodies if serum tests are conducted
at 37 C and IgG-specific reagents are used for the antiglobulin phase. The use of potentiators (eg, albumin or
PEG) is not recommended because they may increase the reactivity of the autoantibodies. In rare instances, it
may be necessary to perform autologous adsorption at 4 C (see Method 4-5). Achieving the complete removal
of potent cold-reactive autoagglutinins is very time consuming and usually unnecessary. Removal of sufficient
cold autoagglutinins may be facilitated by treating the patient’s cells with enzymes or ZZAP before adsorption.
One or two cold autologous adsorptions should remove
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CHAPTER 17 DAT/Immune Hemolysis
enough autoantibody to make it possible to detect alloantibodies at 37 C that were otherwise masked by the
cold-reactive autoantibody. As an alternative, the allogeneic adsorption process used for WAIHA can be
performed at 4 C. Rabbit erythrocyte stroma, which removes autoanti-I and -1H from sera, should be used with
caution because this method can remove clinically significant alloantibodies—notably anti-D, -E, -Vel, and IgM
antibodies regardless of blood group specificity.43'44
Specificity of Autoantibody
The autoantibody specificity in CAD is most often anti-I but is usually of academic interest only. Anti-i is
found less commonly, and it is usually associated with infectious mononucleosis. On rare occasions, other
specificities are seen.
Autoantibody specificity is not diagnostic for CAD. Autoanti-I may be seen in healthy individuals as well as
in patients with CAD. The nonpathologic forms of autoanti-I, however, rarely react at titers above 64 at 4 C and
are usually nonreactive with I- (cord i and adult i) red cells at room temperature. In contrast, the autoanti-I of
CAD may react quite strongly with I- red cells in tests at room temperature, and equal or even stronger reactions
occur with 1+ red cells. Autoanti-i reacts in the opposite manner, demonstrating stronger reactions with I- red
cells than with red cells that are I+. Anti-Ir, originally thought to recognize a transition state of i to 1 (explaining
the designation “IT”), reacts strongly with cord red cells, weakly with normal adult I red cells, and most weakly
with the rare adult i red cells. In rare cases, the cold agglutinin specificity may be anti-Pr, which reacts equally
well with untreated red cells of I or i phenotypes but does not react with enzyme-treated red cells. Procedures to
determine the titer and specificity of cold-reactive autoantibodies are given in Methods 4-6 and 4-7. Typical
reactivity patterns of cold autoantibodies are shown in the table in Method 4-6.
Mixed-Type AIHA
Although about one-third of patients with WAIHA have nonpathologic IgM antibodies that agglutinate at
room temperature, another group of patients with WAIHA have cold agglutinins that react at or above 30 C.
This latter group is referred to as having “mixed” or “combined warm and cold” AIHA and can be subdivided
into patients with high-titer, high-thermal-amplitude IgM cold antibodies (the rare WAIHA plus classic CAD)
and patients with normal-titer (<64 at 4 C), high-thermal-amplitude cold antibodies.45’47 Patients with
mixedtype AIHA often present with hemolysis and complex serum reactivity in all phases of testing.
Serologic Characteristics
In mixed-type AIHA, both IgG and C3 are usually detectable on patients’ red cells; however, C3, IgG, or
IgA alone may be detectable on the red cells.4 An eluate contains a warm-reactive IgG autoantibody. Both
warm-reactive IgG autoantibodies and cold-reactive, agglutinating IgM autoantibodies are present in the serum.
These autoantibodies usually result in reactivity at all phases of testing and with virtually all cells tested. The
IgM agglutinating autoantibody reacts at 30 C or above. If adsorptions are performed to detect alloantibodies, it
may be necessary to perform them at both 37 C and 4 C.
Specificity of Autoantibodies
 The unusual cold-reactive IgM agglutinating autoantibody can have specificities that are typical of CAD (ie,
anti-I or -i) but often has no apparent specificity.45,46 The warm-reactive IgG autoantibody often appears to be
serologically indistinguishable from autoantibodies encountered in typical WAIHA.
Transfusion of Patients with Mixed-Type AIHA
If blood transfusions are necessary, the considerations for the exclusion of alloantibodies and the selection
of blood for transfusion are identical to those described for patients with
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AABB TECHNICAL MANUAL
acute hemolysis caused by WAIHA and CAD (see above).
Paroxysmal Cold Hemoglobinuria
PCH is the rarest form of DAT-positive AIHA. Historically, PCH was associated with syphilis, but this
association is now unusual.48 More commonly, PCH presents as an acute transient condition that is secondary
to a viral infection, particularly in young children. In such cases, the biphasic hemolysin may be only transiently
detectable. PCH can also occur as an idiopathic chronic disease in older people.
Serologic Characteristics
PCH is caused by a cold-reactive IgG complement-binding antibody. As with IgM cold-reactive
autoagglutinins, reactivity occurs with red cells in colder areas of the body (usually the extremities) and causes
C3 to bind irreversibly to red cells. The antibody then dissociates from the red cells as the blood circulates to
warmer parts of the body. Red cells washed in a routine manner for the DAT are usually coated only with
complement, but IgG may be detectable on cells that have been washed with cold saline and tested with cold
anti-IgG reagent.4 Keeping the test system close to its optimal binding temperature allows the coldreactive IgG
autoantibody to remain attached to its antigen. Because complement components are usually the only globulins
present on circulating red cells, eluates prepared from the red cells of patients with PCH are almost always
nonreactive.
The IgG autoantibody in PCH is classically described as a biphasic hemolysin because binding to red cells
occurs at low temperatures but hemolysis does not occur until the complement-coated red cells are warmed to
37 C. This is the basis of the diagnostic test for the disease, the Donath-Landsteiner test (see Method 4-11). The
autoantibody may agglutinate normal red cells at 4 C but rarely to titers greater than 64. Because the antibody
rarely reacts above 4 C, pretransfusion antibody detection tests are usually nonreactive and the serum is usually
compatible with random donor cells by routine crossmatch procedures.
Specificity of Autoantibody
The autoantibody of PCH has most frequently been shown to have P specificity. The autoantibody reacts
with all red cells by the DonathLandsteiner test (including the patient’s own red cells), except those of the very
rare p or Pk phenotypes.
Transfusion of Patients with PCH
Transfusion is rarely necessary for adult patients with PCH, unless their hemolysis is severe. In young
children, the thermal amplitude of the antibody tends to be much wider than in adults and hemolysis is often
more brisk, so transfusion may be required as a lifesaving measure. Although there is some evidence that p red
cells survive longer than P+ (P1+ or P1-) red cells, the prevalence of p blood is approximately 1 in 200,000, and
the urgent need for transfusion usually precludes attempts to obtain this rare blood. Transfusion of donor blood
should not be withheld from patients with PCH whose need is urgent. Transfusion of red cells that are negative
for the P antigen should be considered only for those patients who do not respond adequately to randomly
selected units of donor blood.4
DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA
Drugs rarely cause immune hemolytic anemia; the estimated incidence is 1 in 1 million people.49 Many
drugs have been implicated in hemolytic anemia over the years, as can be seen in the list provided in Appendix
17-1 and reviewed elsewhere.12
Drugs sometimes induce the formation of antibodies—against the drug, red cell membrane components, or
an antigen formed by the drug and the red cell membrane. These antibodies may cause a positive DAT result,
immune red cell destruction, or both.12,40 In some instances, a positive DAT result can be caused by
nonimmunologic protein adsorption (NIPA) onto the red cell, which is caused by the drug.12
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CHAPTER 17 DAT/Immune Hemolysis
Theoretical Mechanisms of DrugInduced Antibodies
 Numerous theories have been suggested to explain how drugs induce immune responses and what relation
such responses may have to the positive DAT result and immune-mediated cell destruction observed in some
patients.4 For many years, drug-associated positive DAT results were classified by four mechanisms: drug
adsorption (penicillin-type), immune complex formation, autoantibody production, and NIPA. This
classification has been useful serologically, but many aspects lack definitive proof. In addition, some drugs
demonstrate serologic reactivity that appears to involve more than one mechanism. A more comprehensive
approach, termed a “unifying hypothesis,” is shown in Fig 17-1. One or more populations of antibodies may be
present. In addition, NIPA, which is independent of antibody production, appears to play a role in drug-induced
immune hemolytic anemia.12
Serologic Classification
Drug-induced antibodies can be classified into two groups: drug dependent (those that require the presence
of the drug in the test system to be detected) and drug independent (those that do not require the in-vitro
addition of the drug for detection).4 Drug-dependent antibodies are subdivided into those that react with drug-
treated red cells (eg, antibodies to penicillin and some cephalosporins) and those that react with untreated red
cells in the presence of a solution of the drug (eg, antibodies to quinine and ceftriaxone). Drug-independent
antibodies (eg, autoantibodies induced by methyldopa and fludarabine) have serologic reactivity that is
independent of the drug despite the fact that the drug originally induced the immune response. Because the drug
does not need to be added to the test system, drugindependent antibodies behave like autoantibodies that are
serologically indistinguishable from idiopathic warm autoantibodies.
ANTIBODY TO DRUG
ANTIBODY TO (MAINLY) MEMBRANE COMPONENTS

ANTIBODY TO DRUG AND MEMBRANE COMPONENTS

RED CELL MEMBRANE
FIGURE 17-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by Habibi
as cited by Garratty28). The thicker lines represent antigen-binding sites for the Fab region of the drug-induced
antibody. Drugs (haptens) bind loosely or firmly to cell membranes, and antibodies may be made to 1) the drug
[producing in-vitro reactions typical of a drug adsorption (penicillin-type) reaction]; 2) membrane components
or mainly membrane components (producing in-vitro reactions typical of autoantibody); or 3) part-drug,
partmembrane components (producing an in-vitro reaction typical of the so-called immune complex
mechanism).28(p55)
442
AABB TECHNICAL MANUAL
If a patient is suspected of having druginduced immune hemolytic anemia (DIIHA), then the suspected drug
should be stopped. Laboratory testing to detect drug-dependent antibodies can be performed, but DIIHA caused
by drug-independent antibodies or NIPA can only be suggested by showing a temporal association of the drug
administration and hemolysis.
The historical details of penicillin- and methyldopa-induced antibodies are not described in this chapter but
have been extensively reviewed elsewhere.4,49 DIIHA caused by high-dose intravenous penicillin therapy is no
longer seen, and methyldopa, the prototype for drug-independent antibodies, is not used as frequently as in the
past. Currently, the drugs most commonly associated with DIIHA are piperacillin, ceftriaxone, and cefotetan.50
Drug-Dependen t An tibodies tha t Are Reactive with Drug-Treated Red Cells
Some drugs (eg, penicillin, ampicillin, and many cephalosporins) covalently bind to red cells, thus making it
possible to coat red cells in the laboratory with the drug. Antibodies directed to these drugs will react with the
drugtreated red cells but not with untreated red cells (unless the patient also has alloantibodies to red cell
antigens present on these cells).
Penicillin and the cephalosporins are beta-lactam antibiotics. It was thought for some time that antibodies to
any drug in the penicillin and cephalosporin families could be detected by testing red cells with drug-treated
cells using methods previously described for penicillin and cephalothin. It is now known that this is not the
case. Synthetic penicillins and newer cephalosporins cannot be assumed to have the same red cell binding
characteristics as penicillin and cephalothin (a first-generation cephalosporin). Cefotetan (a second-generation
cephalosporin) binds very well to red cells, and antibodies caused by cefotetan typically react to very high titers
with cefotetan-treated red cells. However, ceftriaxone (a third-generation cephalosporin) does not bind well to
red cells; therefore, antibodies to ceftriaxone cannot be tested by this meth
od.50 Piperacillin, a semisynthetic penicillin, binds to red cells at high pH. However, a large percentage of
plasma from healthy blood donors and patients react with piperacillin-treated red cells, so this method is not
recommended for testing for piperacillin antibodies.51 For drug antibodies detected using drug-treated red cells,
the following are expected:
■ The DAT result is usually positive for IgG, but complement may also be present.
■ The serum contains an antibody that reacts with the drug-treated red cells but not the untreated red cells.
■ Antibody eluted from the patient’s red cells reacts with drug-treated red cells but not untreated red cells.
Hemolysis develops gradually but may be life threatening if the etiology is unrecognized and drug
administration is continued. The patient may or may not have been previously exposed to the drug and, in the
case of cefotetan, only a single dose given prophylactically can result in severe hemolysis. Normal plasma has
been shown to react with some drug-treated red cells (eg, red cells treated with cefotetan,4 piperacillin,51 or
oxaliplatin52), suggesting prior exposure to these drugs through environmental routes.
Drug-Dependen t An tibodies that Are Reactive with Untreated Red Cells in the Presence of Drug
Antibodies to many drugs that have been reported to cause immune hemolytic anemia are detected by
testing untreated red cells in the presence of the drug. Piperacillin and some of the second- and third-generation
cephalosporins react by this method; anti-ceftriaxone has been detected only by testing red cells in the presence
of drug.49 The following observations are characteristic:
■ Complement may be the only protein easily detected on the red cells, but IgG may be present.
■ The serum antibody can be IgM, IgG, or IgM with IgG.
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CHAPTER 17 DAT/Immune Hemolysis
■ A drug (or metabolite) must be present in vitro for the antibody in the patient’s serum to be detected.
Antibodies may cause hemolysis, agglutination, and/or sensitization of red cells in the presence of the drug.
■ The patient need only take a small amount of the drug (eg, a single dose).
■ Acute intravascular hemolysis with hemoglobinemia and hemoglobinuria is the usual presentation. Renal
failure is quite common.
■ Once antibody has been formed, severe hemolytic episodes may recur after exposure to very small
quantities of the drug.
On occasion, it appears that a patient’s serum contains an “autoantibody” in addition to a drug antibody
reacting in the presence of the drug. Rather than a true autoantibody, it is believed that this reactivity is due to
the presence of circulating drug or drug plus antibody complexes.50,53 In these cases, an eluate is usually
nonreactive when the drug is not present in the system. However, in some cases involving piperacillin, the
eluate reacts while the patient is still taking the drug. A sample collected several days after the drug has been
discontinued will be nonreactive. A true warm autoantibody is expected to be reactive in an eluate prepared
from the patient’s red cells, and autoantibody in the serum persists. Consequently, DIIHA caused by piperacillin
can be misdiagnosed as WAIHA, especially if the eluate reacts. Differentiation of warm-reactive autoantibody
from drug-induced immune hemolytic anemia is important for clinical management.53
Drug-Independen t An tibodies: Autoantibody Production
Some drugs induce autoantibodies that appear serologically indistinguishable from those of WAIHA. Red
cells are coated with IgG, and the eluate as well as the serum react with virtually all cells tested in the absence
of the drug. The antibody has no direct or indirect invitro interaction with the drug. The prototype drug for such
cases is methyldopa, which is now used much less frequently than in the past. Currently, fludarabine, used to
treat
chronic lymphocytic leukemia, is the most commonly used drug to produce drug-independent antibodies
and AIHA.49
Non-Immunologic Protein Adsorption
The positive DAT result associated with some drugs is caused by modification of the red cell membrane by
the drug and is independent of antibody production. Hemolytic anemia associated with this mechanism is rare.
 Cephalosporins (primarily cephalothin) are the drugs with which positive DAT results and NIPA were
originally associated. In vitro, red cells coated with cephalothin in pH 9.8 buffer and incubated with normal
plasma adsorb albumin, IgA, IgG, IgM, C3, and other proteins in a nonimmunologic manner. For this reason,
the indirect antiglobulin test result with virtually all plasma will be positive. Other drugs that cause NIPA and a
positive DAT result include diglycoaldehyde, cisplatin, oxaliplatin, and beta-lactamase inhibitors (clavulanic
acid, sulbactam, and tazobactam).12
NIPA should be suspected when a patient’s plasma/serum and most normal plasma/sera are reactive in an
indirect antiglobulin test with drug-treated red cells but the eluate from the patient’s red cells is nonreactive with
the drug-treated cells.
Laboratory Investigation of DrugInduced Immune Hemolysis
The drug-related problems that are most commonly encountered in the blood bank are those associated with
a positive DAT result and a nonreactive eluate. Recent red cell transfusions and/or dramatic hemolysis may
result in a weak DAT result by the time hemolysis is suspected. When other, more common causes of immune-
mediated hemolysis have been excluded and a temporal relationship exists between the administration of a drug
and the hemolytic anemia, a drug antibody investigation should be pursued.
The patient’s serum should be tested for unexpected antibodies by routine procedures. If the serum does not
react with untreated red cells, the tests should be repeated with the
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AABB TECHNICAL MANUAL
drug(s) suspected of causing the problem. Some drug formulations contain inert ingredients (eg, pill or
capsule forms), and other drugs are combinations of two drugs (eg, piperacillin plus tazobactam). Although it
would seem logical to test the patient’s serum with the actual drug that the patient received, inert ingredients or
drug combinations can make preparation of drug-treated red cells difficult or make the results confusing. It is
preferable to test serum using pure drug formulations as well as separate components of combination drugs.
If the drug has already been reported to cause hemolytic anemia, testing methods may be described in the
case reports. Far more drug antibodies are detected by testing serum in the presence of drug; therefore, when a
previous report of antibodies to a drug is not available, an initial screening test can be performed with a solution
of the drug at a concentration of approximately 1 mg/mL in phosphate-buffered saline (see Method 4-13).
Serum, rather than plasma, is the preferred specimen for testing for hemolysis to be observed; this also allows
for the addition of fresh normal serum (as a source of complement) to the test system. The addition of the fresh
complement increases the sensitivity of the test for the detection of in-vitro hemolysis resulting from
complement activation.
If these tests are not informative, attempts can be made to coat normal red cells with the drug. The patient’s
serum and an eluate from the patient’s red cells can be tested against the drug-treated red cells (see Method 4-
12). This is the method of choice when cephalosporins (except for ceftriaxone) are thought to be implicated.
Results that are definitive for a drug-induced positive DAT result are reactivity of the eluate with drug-treated
red cells and absence of reactivity with untreated red cells.
Drug-treated red cells should always be tested with saline and normal serum (or plasma) as negative
controls. This approach ensures that the observed reactivity with the patient’s serum/plasma is appropriately
interpreted. Antibodies reactive with red cells treated with some drugs (eg, beta-lactams and platinums) have
been detected in the plasma from blood donors and patients without hemolytic anemia thought to be due to
environmental exposure. Therefore, misinterpretation of reactivity in a patient’s serum is possible.51-52
Whenever possible, a positive control should be tested with drug-treated red cells. Negative results of a
patient’s serum and eluate without a positive control can only be interpreted as showing that antibodies to that
drug were not detected. The drug may or may not be bound to the test red cells.
If the drug in question is known to cause NIPA, the patient’s serum and the controls (negative and positive)
should also be tested at a dilution of 1 in 20. Normal sera at this dilution do not usually contain enough protein
for NIPA to be detected.
An immune response may be caused by a metabolite of a drug rather than the drug itself. If the clinical
picture is consistent with immune-mediated hemolysis and the above tests are noninformative, it may be helpful
to test metabolites of the parent drug that are present in the serum or urine of an individual who is taking that
drug.54 Antibodies to some nonsteroidal antiinflammatory drugs have required testing in the presence of
metabolite.55 The metabolism and half-life of the drug determines when the drug metabolite should be
collected. Pharmacology information for the metabolite (s) detectable in serum or urine and to previous reports
for the drug under investigation should be consulted.
KEY POINTS
1. The DAT is used to determine whether red cells have been coated in vivo with immunoglobulin,
complement, or both. The DAT is used primarily for the investigation of hemolytic transfusion reactions,
HDFN, AIHA, and drug-induced immune hemolysis.
2. The DAT should be used to determine whether a hemolytic anemia has an immune etiology.

445
3. A positive DAT result may or may not be associated with hemolysis.
4. Performance of the DAT on postreaction specimens is part of the initial investigation of a transfusion
reaction. The DAT result maybe positive if sensitized red cells have not been destroyed, or may be negative if
hemolysis and rapid clearance have occurred.
5. The DAT is performed bytesting freshly washed red cells directly with antiglobulin reagents containing
anti-IgG and anti-C3d. False-negative or weaker results can be obtained if the washed red cells are allowed to
sit before testing with anti-IgG or if the reading is delayed.
6. When the DAT result is positive with both anti-IgG and anti-C3, the red cells should be tested with an
inert control reagent (eg, 6% albumin or saline). If the control is reactive, the DAT result is invalid, possibly
indicating spontaneous agglutination from heavy coating of IgG or rare warm-reactive IgM. The invalid DAT
result could also be due to IgM cold autoagglutinins that were not dissociated during routine washing.
7. A positive DAT result alone is not diagnostic of hemolytic anemia. The interpretation of the significance
of this positive result requires additional patient-specific information. Dialogue with the attending physician is
important. Clinical considerations together with laboratory data should dictate the extent to which a positive
DAT result is evaluated.
8. The following situations may warrant further investigation of a positive DAT:
a. Evidence of in-vivo red cell destruction.
b. Recent transfusion.
c. Administration of drugs that have previously been associated with immune-mediated hemolysis.
 d. History of hematopoietic progenitor cell or organ transplantation.
e. Administration of MG or intravenous anti-D.
9. Elution frees antibody from sensitized red cells and recovers antibody in a usable form. Elution is useful
in certain situations for implicating an autoantibody, detecting specific antibodies that may not be detectable in
the serum, and deciding to test patient’s serum for drug-related antibodies.
10. AIHAs are subdivided into the major types: WAIHA, CAD, mixed- or combined-type AIHA, and PCH.
Drugs may also induce immune hemolysis.
REFERENCES
1. Kaplan HS, Garratty G. Predictive value of direct antiglobulin test results. Diagnostic Med 1985;8:29-32.
2. Garratty G. The significance of IgG on the red cell surface. Transfus Med Rev 1987;1:47-57.
3. Freedman J. The significance of complement on the red cell surface. Transfus Med Rev 1987; 1:58-70.
4. Petz LD, Garratty G. Immune hemolytic anemias. 2nd ed. Philadelphia: Churchill-Livingstone, 2004.
5. Rottenberg Y, Yahalom Y Shinar E, et al. Blood donors with positive direct antiglobulin tests are at
increased risk for cancer. Transfusion 2009;49:838-42.
6. Hannon JL. Management of blood donors and blood donations from individuals found to
have a positive direct antiglobulin test. Transfus Med Rev 2012;26:142-52.
7. Toy PT, Chin CA, Reid ME, Burns MA. Factors associated with positive direct antiglobulin tests in
pretransfusion patients: A case control study. Vox Sang 1985;49:215-20.
8. Heddle NM, Kelton JG, Turchyn KL, Ali MAM. Hypergammaglobulinemia can be associated with a
positive direct antiglobulin test, a nonreactive eluate, and no evidence of hemolysis. Transfusion 1988;28:29-33.
9. Clark JA, Tanley PC, Wallas CH. Evaluation of patients with positive direct antiglobulin tests and
nonreactive eluates discovered during pretransfusion testing. Immunohematology 1992;8:9-12.
 446
AABB TECHNICAL MANUAL
10. Coombs RRA, Mourant AE, Race RR. A new test for the detection of weak and “incomplete” Rh
agglutinins. Br J Exp Pathol 1945;26:255-66.
11. Heddle NM, Soutar RL, O’Hoski PL, et al. A prospective study to determine the frequency and clinical
significance of alloimmunization post-transfusion. Br J Haematol 1995;91:10005.
12. Garratty G, Arndt PA. An update on drug-induced immune hemolytic anemia. Immunohematology
2007;23:105-19.
13. Morgan S, Sorensen P, Vercellotti G, Zantek ND. Haemolysis after treatment with intravenous
immunoglobulin due to anti-A. Transfus Med 2011;21:267-70.
14. Rushin J, Rumsey DH, Ewing CA, Sandler SG. Detection of multiple passively acquired alloantibodies
following infusions of IV Rh immune globulin. Transfusion 2000;40:551-4.
15. Judd WJ. Elution—dissociation of antibody from red blood cells: Theoretical and practical
considerations. Transfus Med Rev 1999; 13: 297-310.
16. Leger RM, Arndt PA, Ciesielski DJ, Garratty G. False-positive eluate reactivity due to the lowionic
wash solution used with commercial acid-elution kits. Transfusion 1998;38:565-72.
17. Judd WJ, Johnson ST, Storry JR. Judd's methods in immunohematology. 3rd ed. Bethesda, MD: AABB
Press, 2008.
18. Judd WJ, Barnes BA, Steiner EA, et al. The evaluation of a positive direct antiglobulin test (autocontrol)
in pretransfusion testing revisited. Transfusion 1986;26:220-4.
19. Arndt PA, Leger RM, Garratty G. Serologic findings in autoimmune hemolytic anemia associated with
immunoglobulin M warm autoantibodies. Transfusion 2009;49:235-42.
20. Rodberg K, Tsuneta R, Garratty G. Discrepant Rh phenotyping results when testing IgG-sensitized
RBCs with monoclonal Rh reagents (abstract). Transfusion 1995;35(Suppl):67S.
21. Leger RM, Garratty G. Evaluation of methods for detecting alloantibodies underlying warm
autoantibodies. Transfusion 1999;39:11-16.
22. Church AT, Nance SJ, Kavitsky DM. Predicting the presence of a new alloantibody underlying a warm
autoantibody (abstract). Transfusion 2000;40(Suppl):121S.
23. Branch DR, Petz LD. A new reagent (ZZAP) having multiple applications in immunohematology. Am J
Clin Pathol 1982;78:161-7.
24. Laine ER Leger RM, Arndt PA, et al. In vitro studies of the impact of transfusion on the de
tection of alloantibodies after autoadsorption. Transfusion 2000;40:1384-7.
25. Chiaroni J, Touinssi M, Mazet M et al. Adsorption of autoantibodies in the presence of LISS to detect
alloantibodies underlying warm autoantibodies. Transfusion 2003;43:651-5.
26. Magtoto-Jocom J, Hodam J, Leger RM, Garratty G. Adsorption to remove autoantibodies using
allogeneic red cells in the presence of low ionic strength saline for detection of alloantibodies (abstract).
Transfusion 2011;51 (Suppi) :174A.
 27. Issitt PD, Combs MR, Bumgarner DJ, et al. Studies of antibodies in the sera of patients who have made
red cell autoantibodies. Transfusion 1996;36:481-6.
28. Garratty G. Target antigens for red-cell-bound autoantibodies. In Nance SJ, ed. Clinical and basic
science aspects of immunohematology. Arlington, VA: AABB, 1991:33-72.
29. Garratty G. Specificity of autoantibodies reacting optimally at 37° C. Immunohematology 1999;15:24-
40.
30. Branch DR, Petz LD. Detecting alloantibodies in patients with autoantibodies (editorial). Transfusion
1999;39:6-10.
31. Young PR Uzieblo A, Trulock E, et al. Autoantibody formation after alloimmunization: Are blood
transfusions a risk factor for autoimmune hemolytic anemia? Transfusion 2004; 44:67-72.
32. Maley M, Bruce DG, Babb RG, et al. The incidence of red cell alloantibodies underlying panreactive
warm autoantibodies. Immunohematology 2005;21:122-5.
33. Ahrens N, Pruss A, Kahne A, et al. Coexistence of autoantibodies and alloantibodies to red blood cells
due to blood transfusion. Transfusion 2007;47:813-16.
34. Shirey RS, Boyd JS, Parwani AV) et al. Prophylactic antigen-matched donor blood for patients with
warm autoantibodies: An algorithm for transfusion management. Transfusion 2002;42:1435-41.
35. Hillyer CD, Shaz BH, Winkler AM, Reid M. Integrating molecular technologies for red blood cell
typing and compatibility testing into blood centers and transfusion services. Transfus Med Rev 2008;22:117-32.
36. Anstee DJ. Red cell genotyping and the future of pretransfusion testing. Blood 2009; 114:24856.
447
37. Reid ME, Denomme GA. DNA-based methods in the immunohematology reference laboratory. Transfus
Apher Sci 2011 ;44:65-72.
38. Lee E, Redman M, Burgess G, Win N. Do patients with autoantibodies or clinically insignificant
alloantibodies require an indirect antiglobulin test crossmatch? Transfusion 2007;47:1290-5.
39. Richa EM, Stowers RE, Tauscher CD, et al. The safety of electronic crossmatch in patients with warm
autoantibodies (letter). Vox Sang 2007:93:92.
40. Leger RM, Co A, Hunt P, Garratty G. Attempts to support an immune etiology in 800 patients with
direct antiglobulin test-negative hemolytic anemia. Immunohematology 2010;26: 156-60.
41. Waligora SK, Edwards JM. Use of rabbit red cells for adsorption of cold autoagglutinins. Transfusion
1983;23:328-30.
42. Dzik WH, Yang R, Blank J. Rabbit erythrocyte stroma treatment of serum interferes with recognition of
delayed hemolytic transfusion reaction (letter). Transfusion 1986;26:303-4.
43. Mechanic SA, Maurer JL, Igoe MJ, et al. AntiVel reactivity diminished by adsorption with rabbit RBC
stroma. Transfusion 2002;42:11803.
44. Storry JR, Olsson ML, Moulds JJ. Rabbit red blood cell stroma bind immunoglobulin M antibodies
regardless of blood group specificity (letter). Transfusion 2006;46:1260-1.
45. Sokol RJ, Hewitt S, Stamps BK. Autoimmune haemolysis: An 18-year study of 865 cases referred to a
regional transfusion centre. Br Med J 1981;282:2023-7.
46. Shulman LA, Branch DR, Nelson JM, et al. Autoimmune hemolytic anemia with both cold
and warm autoantibodies. JAMA 1985;253: 1746-8.
47. Garratty G, Arndt PA, Leger RM. Serological findings in autoimmune hemolytic anemia (AIHA)
associated with both warm and cold autoantibodies (abstract). Blood 2003:102 (Suppl) :563a.
48. Eder AF. Review: Acute Donath-Landsteiner hemolytic anemia. Immunohematology 2005; 21:56-62.
49. Garratty G. Immune hemolytic anemia associated with drug therapy. Blood Rev 2010;24: 143-50.
50. Arndt PA, Leger RM, Garratty G. Serologic characteristics of ceftriaxone antibodies in 25 patients with
drug-induced immune hemolytic anemia. Transfusion 2012;52:602-12.
51. Leger RM, Arndt PA, Garratty G. Serological studies of piperacillin antibodies. Transfusion
2008;48:2429-34.
52. Leger RM, Garratty G. Antibodies to oxaliplatin, a chemotherapeutic, are found in plasma of healthy
blood donors. Transfusion 2011;51: 1740-4.
53. Bandara M, Seder DB, Garratty G, et al. Piperacillin-induced immune hemolytic anemia in an adult with
cystic fibrosis (article 10 161454). Case Report Med 2010.
54. Salama A, Mueller-Eckhardt C, Kissel K, et al. Ex vivo antigen preparation for the serological detection
of drug-dependent antibodies in immune haemolytic anaemias. Br J Haemat 1984;58:525-31.
 55. Johnson ST, Fueger JT, Gottschall JL. One center’s experience: The serology and drugs associated with
drug-induced immune hemolytic anemia—a new paradigm. Transfusion 2007; 47:697-702.
448
AABB TECHNICAL MANUAL
■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia
Drug Method of Detection
Aceclofenac +Drug
Acetaminophen + Drug
Acyclovir DT
Aminopyrine DT
Amoxicillin DT
Amphotericin B + Drug
Ampicillin DT + Drug
Antazoline + Drug
Azapropazone AA DT
Butizide + Drug
Carbimazole AA DT + Drug
Carboplatin AA DT + Drug NIPA
Carbromal DT
Cefamandole DT
Cefazolin DT
Cefixime DT + Drug
Cefotaxime DT + Drug
Cefotetan AA DT + Drug NIPA
Cefoxitin AA DT + Drug
Ceftazidime AA DT + Drug
Ceftizoxime DT + Drug
Ceftriaxone + Drug
Cefuroxime DT
Cephalexin DT
Cephalothin DT + Drug NIPA
Chloramphenicol AA DT
Chlorinated hydrocarbons AA DT + Drug
Chlorpromazine AA + Drug
Chlorpropamide + Drug
Cimetidine DT +Drug
449
■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Ciprofloxacin +Drug
Cisplatin DT +Drug NIPA
Cladribine AA
Clavulanate NIPA
Cyanidanol AA DT + Drug
Cyclofenil AA + Drug
Cyclosporin DT
Diclofenac AA DT + Drug
Diethylstilbestrol + Drug
Diglycoaldehyde NIPA
Dipyrone DT + Drug
Erythromycin DT
Etodolac + Drug
Fenoprofen AA + Drug
Fluconazole DT + Drug
Fludarabine AA
Fluorescein DT + Drug
Fluorouracil + Drug
Furosemide + Drug
Hydralizine DT
Hydrochlorothiazide DT + Drug
Hydrocortisone DT + Drug
9-Hydroxy-methyl-ellipticinium + Drug
Ibuprofen +Drug
Imatinib mesylate DT
Insulin DT
Isoniazid DT + Drug
Levodopa AA
Levofloxacin DT +Drug
Mefenamic acid AA
(Continued)

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AABB TECHNICAL MANUAL
■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Mefloquine DT + Drug
Melphalan + Drug
6-Mercaptopurine DT
Methadone DT
Methotrexate AA DT + Drug
Methyldopa AA
Nabumetone +Drug
Nafcillin DT
Naproxen + Drug
Oxaliplatin DT + Drug NIPA
p-Aminosalicylic acid + Drug
Penicillin G DT
Phenacetin + Drug
Phenytoin DT
Piperacillin DT + Drug
Probenicid + Drug
Procainamide AA
Propyphenazone + Drug
Pyrazinamide DT + Drug
Pyrimethamine DT
Quinidine DT + Drug
Quinine + Drug
Ranitidine DT + Drug
Rifabutin + Drug
Rifampicin DT + Drug
Sodium pentothal/thiopental + Drug
Stibophen + Drug
Streptomycin AA DT + Drug
Sulbactam NIPA
Sulfamethoxazole + Drug

CHAPTER 17 DAT/Immune Hemolysis

451
■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
+
Sulfasalazine
Drug
Sulfisoxazole +Drug
+
Sulindac AA DT
Drug
+
Suprofen AA
Drug
Tazobactam NIPA
 Teicoplanin AA +
Drug
+
Teniposide AA
Drug
Tetracycline DT
Ticarcillin AA DT
Tolbutamide DT
+
Tolmetin AA
Drug
+
Triamterene DT
Drug
+
Trimethoprim
Drug
+
Vancomycin
Drug
+
Zomepirac AA
Drug
AA = drug-independent = testing with drug-treated red = testing in the presence
autoantibody; DT cells; + Drug of drug;
NIPA = nonimmunologic protein adsorption.
 Chapter 18
Platelet and Granulocyte Antigens and Antibodies
Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB
jgKjTHIS CHAPTER DISCUSSES antiM gens expressed on platelets and granulocytes together with
antibodies that arise when individuals are sensitized to these markers. These antigens and the immune responses
to them are of importance in alloimmune, autoimmune, and drug-induced immune syndromes involving
platelets and granulocytes.
PLATELET ANTIGENS AND ANTIBODIES
Platelets express a variety of antigenic markers on their surface. Some of these antigens are shared with
other cells, as with ABO and HLA, whereas others are essentially platelet specific, like human platelet
alloantigens (HPAs).
HPAs
Platelets reportedly play roles in inflammation; innate, adaptive, and autoimmunity; cardiovascular disease;
and even cancer.1,2 However, they are most well known for their function in forming clots to stem bleeding.
Platelets perform these functions through
multiple ligand-receptor interactions involving glycoproteins (GPs) expressed on their cell-surface
membranes.
Platelet membrane GPs are expressed in different forms due to single nucleotide polymorphisms (SNPs) in
the genes that encode them. The amino acid changes resulting from these SNPs induce changes in the
glycoprotein structure to form antigens that can elicit antibodies through exposure from pregnancy or platelet
transfusions. Currently, 33 different HPAs expressed on six different platelet membrane glycoproteins—GPIIb,
GPIIIa, GPIba, GPIbp, GPIa, and CD109—have been identified (Table 18-1).3 These antigens are often
referred to as “platelet specific,” but some are found on cells other than platelets (especially leukocytes and
endothelial cells), although their chief clinical importance appears to be linked to their presence on platelets.
Twelve antigens are clustered into six biallelic groups (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-
15). The nomenclature for HPAs consists of numbering the antigens in their order of discovery, with the higher

Brian R. Curtis PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet and Neutrophil Immunology Lab,
BloodCenter of Wisconsin, Milwaukee, Wisconsin
B. Curtis has disclosed a financial relationship with Gen-Probe Incorporated.
453

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AABB TECHNICAL MANUAL
TABLE 18-1. Human Platelet Alloantigens
Phenotypic Glycoprotein Amino Acid Encoding Nucleotide
Antigen
Frequency* (GP) Change Gene Change
HPA-la 72% a/a GPIIIa Leu33Pro ITGB3 176T>C
HPA-lb 26% a/b
2% b/b
HPA-2a 85% a/a GPIba Thrl 45Met GPIBA 482C>T
HPA-2b 14% a/b
1% b/b
HPA-3a 37% a/a GPIIb Me847Ser ITGA2B 2621T>G
HPA-3b 48% a/b
15% b/b
HPA-4a >99.9% a/a GPIIIa Arg143Gln ITGB3 506G>A
HPA-4b <0.1% a/b
<0.1% b/b
HPA-5a 88% a/a GPIa Glu505Lys ITGA2 1600G>A
HPA-5b 20% a/b
1% b/b
HPA-6bw <1% b/b GPIIIa Arg489Gln ITGB3 1544G>A
HPA-7bw <1% b/b GPIIIa Pro407Ala ITGB3 1297C>G
HPA-8bw <1% b/b GPIIIa Arg636Cys ITGB3 1984C>T
HPA-9bw <1% b/b GPIIb Val837Met ITGA2B 2602G>A
HPA-
<1% b/b GPIIIa Arg62Gln ITGB3 263G>A
IObw
HPA-llbw <1% b/b GPIIIa Arg633His ITGB3 1976G>A
HPA-
<1% b/b GPIbp Glyl 5Glu GPIBB 119G>A
12bw
HPA-
<1% b/b GPIa Met799Thr ITGA2 2483C>T
13bw
HPA- 1909_1911
<1% b/b GPIIIa Lys611 del ITGB3
14bw delAAG
HPA-
35% a/a CD109 Ser682Tyr CD109 2108C>A
15bw
 42% a/b
23% b/b
HPA-
<1% b/b GPIIIa Thrl 40lle ITGB3 497C>T
16bw
HPA-
<1% b/b GPIIIa Thrl 95Met ITGB3 662C>T
17bw
HPA-
< 1 % b/b GPIa Gln716His ITGA2 2235G>T
18bw
HPA-
< 1 % b/b GPIIIa Lys137Gln ITGB3 487A>C
19bw
HPA-
<1% b/b GPIIb Thr619Met ITGA2B 1949C>T
20bw

455
TABLE 18-1. Human Platelet Alloantigens (Continued)
Phenotypic Nucleotide
Antigen Glycoprotein (GP) Amino Acid Change
Frequency* Change
HPA-21bw < 1 % b/b GPIIIa Glu628Lys ITGB3 1960G>A
HPA-22bw < 1 % b/b GPIIb Lys164Thr ITGA2B 584A>C
HPA-23bw <1% b/b GPIIIa Arg622Trp ITGB3 1942C>T
HPA-24bw < 1 % b/b GPIIb Ser472Asn ITGA2B 1508G>A
HPA-25bw <1% b/b GPIa Thrl 087Met ITGA2 3347C>T
HPA-26bw < 1 % b/b GPIIIa Lys580Asn ITGB3 1818G>T
HPA-27bw < 1 % b/b GPIIb Leu841Met ITGA2B 2614C>A
'Phenotypic frequencies are for people of European ancestry who live in North America. Human platelet
alloantigen (HPA) frequencies in other races and ethnic groups can be found in the IPD-lmmuno Polymorphism
Database.3
frequency antigens designated “a” and the lower-frequency antigens designated “b.”4 HPAs for which
antibodies against only one of the two antithetical antigens have been detected are labeled with a “w” for
“workshop,” such as HPA-6bw.
Platelet Alloantigens on GPIIb/IIIa
HPA-la is the platelet alloantigen that was discovered first and is most familiar.5 Originally named “Zw3
and more commonly referred to as “P1A1,” it is expressed on GPIIIa, the p-subunit of the integrin GPIIb/IIIa
(a2b/p3) complex.
Integrins are a broadly distributed family of adhesion molecules consisting of an a and a P chain held
together by divalent cations in a heterodimeric complex.6 Integrins are essential for platelet adhesion and
aggregation because they serve as receptors for ligands, such as fibrinogen, collagen, fibronectin, von
Willebrand factor (vWF), and other extracellular matrix proteins.
Binding of fibrinogen by GPIIb/IIIa results in platelet aggregation, which leads to the formation of the
“platelet plug” to stop bleeding. The importance of GPIIb/IIIa in hemostasis is demonstrated by the serious
bleeding that occurs in patients with the rare disorder
Glanzmann thrombasthenia, in which platelet GPIIb/IIIa is absent or dysfunctional due to inherited
mutations in ITGA2B and/or ITGB3.7 Patients with Glanzmann thrombasthenia who are exposed to normal
platelets by transfusion or pregnancy can make isoantibodies against GPIIb/IIIa.
GPIIb/IIIa is the most abundantly expressed (approximately 80,000 molecules/ platelet) glycoprotein
complex on the platelet membrane, making it highly immunogenic. Antibodies against HPA-la account for the
vast majority (>80%) of the HPA-specific platelet antibodies detected in the sera of alloimmunized people of
European ancestry. HPA-la antibodies are produced by the 2% of individuals with the platelet type HPA-lb/lb.
Antibodies specific for HPA-lb are commonly detected in patients with posttransfusion purpura (PTP).
Twenty of the 33 HPAs are carried by GP1Ib (6) and GPIIIa (14). Like HPA-la/lb, the HPA-4a/4b antigens
are also expressed on GPIIIa and have been implicated in fetal and neonatal alloimmune thrombocytopenia
(FNAIT), PTP, and platelet transfusion refractoriness. The low-frequency HPA-4b antigen is more common in
populations of Japanese and Chinese ancestry.

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AABB TECHNICAL MANUAL
The HPA-3a/3b antigens are expressed on GPIIb, but despite the high rate of incompatibility for both
antigens in the general population, detection of HPA-3 antibodies is uncommon. Some HPA-3 antibodies are
difficult to detect in monoclonal antigen capture assays, such as the modified antigen capture enzymelinked
immunosorbent assay (MACE) and monoclonal antibody immobilization of platelet antigens (MAIPA), in
which GPIIb is extracted from platelets with detergents that can denature the antigenic epitopes recognized by
HPA-3 antibodies.8,9 X-ray crystallography studies could not determine the portion of the domain of GPIIb
where the HPA-3 antigens are located, which might explain the difficulties with detection of HPA-3
antibodies.10
An additional 17 different low-frequency platelet antigens are expressed on either GPIIb or GPIIIa (Table
18-1). These antigens were all discovered in cases of FNAIT when specific antibodies in maternal sera were
detected that were reactive only with the fathers’ GPIIb/IIIa. The vast majority of these antigens are private
antigens restricted to the single families in which they were discovered. HPA-6bw and HPA-21bw are
exceptions, having antigen frequencies of 1% and 2%, respectively, in people of Japanese ancestry, and HPA-
9bw has been implicated in several cases of FNAIT.11'14
Platelet Alloantigens on GPIb/V/IX
The GPIb/V/IX complex forms the vWF receptor on platelets, and platelets express approximately 25,000
copies of the complex. Following vascular injury, binding of the GPIb/V/IX complex to vWF facilitates platelet
adhesion to vascular subendothelium and initiates signaling events within adherent platelets that lead to platelet
activation, aggregation, and hemostasis. GPIb is composed of a (GPIba) and p (GPIbp) subunits that form
noncovalent associations with GPIX and GPV. GPIba carries HPA-2a/2b, and GPIbp carries HPA-12bw.
Antibodies against HPA-2a, -2b, and -12bw have all been implicated in causing FNAIT.
Deficiency of the entire GPIb/V/IX complex can occur due to mutations in the encoding genes GPIBA,
GPIBB, or GP9, and is the
 cause of Bernard Soulier syndrome (BSS). BSS is a disorder characterized by prolonged bleeding time,
thrombocytopenia, and the presence of “giant platelets” that affects approximately 1 in 1 million people.7,15
BSS patients whose platelets are devoid of GPIb/V/IX can produce antibodies when they are exposed to the
protein complex on normal platelets through transfusions or pregnancy.
Platelet Alloantigens on GPIa/IIa
The integrin GPIa/IIa, also known as integrin a2PP is a major collagen receptor on platelets. The GPIa
protein carries the HPA-5a/5b antigens. Antibodies against HPA-5 antigens are the second most frequently
detected, after anti-HPA-la, in patients with FNAIT and are also frequently detected in patients with PTP. About
3000 to 5000 molecules of the GPIa/IIa heterodimeric complex are expressed on platelets, and higher
expression correlates with the presence of both HPA-5b and threonine at amino acid 807 in GPIa.16 HPA-13bw,
-18bw, and -25bw are low-frequency antigens that are also expressed on GPIa and have all been implicated in
FNAIT. Interestingly, the HPA-13bw polymorphism has been reported to cause functional defects that reduce
platelet responses to collagen-induced aggregation and spreading on collagen-coated surfaces.4
Platelet Alloantigens on CD109
CD109 is a glycosylphosphatidylinositol (GPI)linked protein and a member of the a2-
macroglobulin/complement superfamily. Its function is still not completely understood, but CD109 has been
reported to bind to and negatively regulate the signaling of transforming growth factor beta. CD109 is also
expressed on activated T lymphocytes, CD34+ hematopoietic cells, and endothelial cells, and it carries the
HPA-15 antigens.
The clinical significance of HPA-15 antibodies is uncertain because platelets express only about 1000
molecules of CD109. Studies show the presence of HPA-15 antibodies in 0.22% to 4% of maternal sera in
patients with suspected FNAIT, and one report suggests that HPA-15 antibodies are more frequently detect
457
ed in sera from patients with immune platelet refractoriness.17'19
Other Antigens on Platelets
ABO and Other Blood Groups
Most of the ABO antigen on platelets is carried on saccharides attached to the major platelet membrane
glycoproteins (Table 18-2). GPIIb and platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31) carry the
largest amounts of A and B antigens.20 Platelet A and B antigen levels are quite variable from individual to
individual, with 5% to 10% of non-group0 individuals expressing extremely high levels of Aj or B on their
platelets.20,21 These “high expresses” have highly active glycosyltransferases, which are much more efficient
at attaching A or B antigens.20
Interestingly, although individuals with the subgroup A2 red cell phenotype express lower levels of A on
their red cells than A1 individuals, they do not express detectable A antigens on their platelets. As a result, A2
platelets have been successfully transfused to group 0 patients, even those with high-titer anti-A.22
Although platelets are often transfused without regard to ABO compatibility, the use of mismatched
platelets frequently results in lower posttransfusion recovery rates.23,24 In some cases, high-titer
immunoglobulin G (Ig G) A,B antibodies in blood group 0 recipients are reactive with transfused platelets
carrying large amounts of A or B antigens, resulting in platelet transfusion refractoriness.21 Recovery of
transfused platelets can also be influenced by the transfusion of group 0 platelets to group A recipients. Anti-A
and/or anti-B in the donor plasma might be reactive with soluble A or B in the recipient plasma to form immune
complexes that bind to transfused (and autologous) platelets via FcyRIIa, thereby influencing the survival of the
transfused platelets.25 Clinical trials comparing ABO-identical to -unmatched platelets in patients with cancer
who require multiple platelet transfusions have suggested that rates of refractoriness are significantly higher
when unmatched components are used.22,25 Although other red cell antigens (eg, Lea, Leb, I, i, R Pk, and
Cromer) are also present on platelets, there is no evidence that these antigens significantly reduce platelet
survival in vivo.26,27
TABLE 18-2. Other Platelet Antigens
Phenotypic Glycoprotein Amino Encoding Nucleotide
Antigen
Frequency (GP)* Acid Change4 Gene Change*
GPIIb, Ilia, IV, laJ
ABO Same as for red cells Multiple ABO Multiple
lla, GPIb/V/IX, CD31
HLA-
Same as for leukocytes Class 1 HLA Multiple MHC Multiple
A, B, and C
 GPIV 90-97% (Africans) 90- CD36 Tyr325Thr* CD36 1264T>G*
97% (Asians) 99.9% Pro90Ser* 478C>T*
(Caucasians) Exons 1-3 del
GPVI N/A GPVI N/A GP6/N/A N/A
*AB0 saccharides are attached to platelet GPs during their glycosylationj. +0nly the most common changes
are shown.
♦Only the most common mutations are shown.
N/A = not applicable.

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AABB TECHNICAL MANUAL
GPIV/CD36
Platelets, monocytes/macrophages, and nucleated erythrocytes are the only blood cells that express
GPIV/CD36 (Table 18-2). GPIVbelongs to the Class B scavenger receptor family and binds a number of
different ligands, including low-density lipoprotein cholesterol, thrombospondin, Types I and IV collagen, and
malaria-infected red cells. A number of mutations in CD36 have been described that result in a complete lack of
protein expression on both platelets and monocytes in populations of Asian and African ancestry.28'30 CD36-
deficient individuals exposed to normal platelets can produce antibodies to CD36 that have been reported to
cause FNAIT, PTP, and platelet refractoriness.29,31,32
GPVI
GPVI is a major collagen receptor on platelets and a member of the Ig superfamily. GPVI interactions with
collagen exposed on the extracellular matrix result in platelet activation and aggregation. To date, no HPAs have
been identified on GPVI, but platelet autoantibodies formed against GPVI have been reported to cause a mild
form of autoimmune thrombocytopenia.33,34 Interestingly, GPVI autoantibodies induce shedding of GPVI
from platelets, resulting in reduced collagen binding and clinically significant bleeding.
HLA
HLA is present on all nucleated cells of the body (see Chapter 19). HLA associated with platelets is the
main source of Class I HLA in whole blood.35 Most Class I HLA on platelets is expressed as integral
membrane proteins, whereas smaller amounts may be adsorbed from surrounding plasma. HLA-A and -B locus
antigens are significantly represented, but there appears to be only minimal platelet expression of HLA-C.36
With rare exceptions, Class II HLA is not present on the platelet membrane.
Several factors influence the likelihood that HLA antibodies will develop after transfusions, and
sensitization may be clinically im
portant in patients receiving multiple platelet transfusions. Transfusion-associated HLA alloimmunization
appears to be influenced by the underlying disease, immunosuppressive effects of treatment regimens, and
whether the blood components contain a significant amount of leukocytes. With widespread use of leukocyte-
reduced (LR) blood components for transfusion, associated HLA alloimmunization has been reduced
considerably. HLA antibodies also commonly develop following pregnancy and are present in the sera of more
than 32% of women who have had four or more pregnancies.37 HLA antibodies have also been identified in
1.7% of never-pregnant or -transfused women and men with no previous transfusions.37 Sensitization to HLA
antigens becomes important in managing patients receiving platelet transfusions when HLA antibodies cause
destruction of transfused platelets and, especially, when patients develop platelet transfusion refractoriness.
Immune Platelet Disorders
Platelet Transfusion Refractoriness
A less-than-expected increase in platelet count occurs in about 20% to 70% of multitransfused patients with
thrombocytopenia.38 Those treated for malignant hematopoietic disorders are particularly likely to become
refractory to platelet transfusions. Responses to platelet transfusions are often determined 10 to 60 minutes after
transfusion by calculating either a corrected platelet count increment (CCI) or a posttransfusion platelet
recovery (PPR), both of which normalize transfusion responses for patient blood volume and platelet dose (see
Chapter 20, Table 5). Most experts would agree that a 1-hour posttransfusion CCI of less than 5000 to 7500
after two consecutive transfusions adequately defines the refractory state.
The CCI and PPR measurements are commonly used to assess the success of platelet transfusions. However,
these calculations may be misleading, particularly when smaller doses of platelets are administered, as can
occur during LR transfusions. Therefore, absolute posttransfusion platelet count increments are more accurate
than CCI or PPR.23
459
HLA sensitization is the most common immune cause of refractoriness and can be diagnosed by
demonstration of significant levels of antibodies to Class I HLA in the refractory patient’s serum (see Chapter
19 for more information on detecting HLA antibodies). Other immune causes to be considered include
antibodies to HPA, ABO incompatibility, and antibodies in the sera of patients with congenital platelet
glycoprotein deficiencies (eg, Glanzmann’s thrombasthenia).
Although platelet alloimmunization is one cause of refractoriness, there are multiple nonimmune-related
reasons why transfused platelets may not yield the expected increase in platelet count, such as sepsis,
disseminated intravascular coagulation, and the administration of certain drugs. It cannot be stressed enough
that these nonimmune factors are more often implicated in transfusion refractoriness than alloimmunization.39
Some of the most commonly cited nonimmune factors associated with refractoriness are listed in Table 18-3.
Even when possible immune causes of refractoriness are identified, nonimmune factors are often
simultaneously present.
TABLE 18-3. Some Nonimmune Causes of Platelet Refractoriness
■ Massive bleeding
■ Fever
■ Sepsis
■ Splenomegaly (splenic sequestration)
■ Disseminated intravascular coagulation
■ Allogeneic transplantation
■ Poor storage of platelets before transfusion
 ■ Effects of drugs (may include immune mecha
nisms)
■ Intravenous amphotericin B
■ Thrombotic thrombocytopenic purpura
■ Treatment regimen (ie, total body irradiation or
chemotherapy)
■ Liver dysfunction
Selection of Platelets for Transfusion in Patients with Alloimmune Refractoriness
Several strategies may be considered when selecting platelets for transfusion to patients with alloimmune
refractoriness. When HLA antibodies are present, a widely used approach is to supply apheresis platelets from
donors whose Class 1 HLA closely matches those of the patient. This approach is primarily performed using
molecular methods. A disadvantage of relying on HLA-matched platelets is that a pool of 1000 to 3000 or more
random, HLA-typed, potential apheresis donors is generally necessary to find sufficient HLA-compatible
donors to support a typical patient.40 Moreover, donor selection on the basis of HLA type can lead to the
exclusion of donors whose HLA types, although different from that of the recipient, may still be suitable for
transfusion to the patient.
An additional concern when using HLAselected platelets is that requesting “HLAmatched” platelets does
not necessarily lead to receiving HLA-identical or even wellmatched platelets. It is important to understand the
degree of matching that may be provided (Table 18-4). Platelets received following an HLA-matched request
are typically the closest match obtainable within the constraints of time and donor availability. In one study,
43% of platelets provided as HLA matched were relatively poor Grade B or C matches.41 In alloimmune
refractory patients, the best increases in CCI occur with the subset of Grade A and B1U or B2U HLA-matched
platelets, but platelets mismatched for some antigens (eg, B44,45) that are poorly expressed on platelets can
also be successfully transfused.
According to AABB Standards for Blood Banks and Transfusion Services, HLA-matched platelets should
be irradiated to prevent transfusion-associated graft-vs-host disease (GVHD).42(p40) HLA-matched platelets
are selected to minimize incompatible antigens. Therefore, these components are more likely to cause GVHD
than random platelets because the recipient’s immune system may fail to recognize
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AABB TECHNICAL MANUAL
TABLE 18-4. Degree of Matching for HLA-Matched Platelets
Match Examples of Donor Phenotypes for a Recipient Who
Description
Grade Is A1,3; B8, 27
B8,
A 4-antigen match A1, 3
27
B8,
B1U 1 antigen unknown or blank A1,
27
CO
BIX 1 cross-reactive group A1,3
oo
1 antigen blank and 1 cross- B8,
B2UX At,
reactive 7
B8,
C 1 mismatched antigen present A1,3
35
2 or more mismatched
D A1.32; B8, 35
antigens present
R Random A2, 28; B7, 35
the donor T lymphocytes as foreign. Treating the component with gamma irradiation effectively eliminates
this risk by inactivating the donor lymphocytes so that they cannot proliferate.
An alternative approach for supplying HLA-compatible transfusions is to determine the specificity of the
patient’s HLA antibodies and select donors whose platelets lack the corresponding antigens. This antibody
specificity prediction (ASP) method is as effective as selection by HLA matching or platelet crossmatching and
is superior to random selection of platelets.43 Furthermore, many more potential HLA-typed donors are
identified by the ASP method than are available using traditional HLA-matching criteria. A variation of the
ASP method involves analysis of antibody specificities detected in sensitive flow cytometry or Luminex-based
assays, where single HLAs are represented on discrete and identifiable populations of beads.44 Reactivity of the
patient’s serum with the specific bead populations yields both the specificity and the relative strength of the
antibodies. Bead populations that lack reactivity with patient serum are used to identify antigens that could be
used in donor selection, even though they may not be matched to the patient’s HLA type using classic criteria
for HLA matching.
In the absence of information concerning which specific platelet donor HLA to avoid, permissive HLA-
mismatched antigens can also be identified using an HLA Matchmaker
algorithm that is based on knowledge of shared HLA epitopes.45 The identification of so-called permissive
HLA epitopes is modeled on a strategy used for identifying potentially compatible deceased donor kidney grafts
for HLA-sensitized recipients.46 This is yet another way to expand the donor pool to provide platelets that are
compatible with the patient’s HLA antibodies.
Pretransfusion crossmatching of the patient’s serum against platelets from potential donors is an additional
approach to provide effective platelet transfusions to patients who are alloimmune refractory. Each potential
platelet product is tested in the crossmatch assay with a current sample of the patient’s serum. The solid-phase
red cell adherence (SPRCA) test is the most widely used method. Good correlation between test results and
posttransfusion platelet counts have been achieved.47 Compared with HLA matching, crossmatching can be
more convenient and cost effective. It avoids exclusion of HLA-mismatched but compatible donors and has the
added advantage of facilitating the selection of platelets when platelet-specific antibodies are present.
Platelet crossmatching, however, will not always be successful, particularly when patients are highly
alloimmunized, which can make finding sufficient amounts of compatible platelets problematic. Although the
incidence of platelet-specific antibodies causing patients to be refractory to most or all platelet

461
transfusions is very small, this possibility should be investigated when most of the crossmatches are positive
or HLA-matched transfusions fail. If platelet-specific antibodies are present, donors of known platelet antigen
phenotype or family members, who may be more likely to share the patient’s phenotype, should be tested.
Platelet crossmatching or HPA genotyping should be considered for patients who do not respond to ABO- and
HLAcompatible platelets.
Fetal and Neonatal Alloimmune Thrombocytopenia
FNAIT (also known as neonatal alloimmune thrombocytopenia and abbreviated as “NATP,” “NAT,”
“NAIT,” or “NIT”) is a syndrome involving immune destruction of fetal platelets by maternal antibody that is
analogous to red cell destruction in hemolytic disease of the fetus and newborn. During pregnancy, a mother
may become sensitized to an incompatible fetal platelet antigen inherited from the father of the fetus. IgG
specific for the platelet antigen crosses the placenta, causing thrombocytopenia.
FNAIT is the most common cause of severe fetal/neonatal thrombocytopenia, and affected infants are at risk
of major bleeding complications, especially intracranial hemorrhage. The most commonly implicated platelet
antigen incompatibility in FNAIT is for HPA-la, but all HPAs identified to date have been implicated.48 A
serologic diagnosis of FNAIT maybe made by 1) testing maternal serum for platelet antibodies using assays that
can differentiate platelet-specific from nonplatelet-specific reactivity and 2) performing platelet genotyping on
parental deoxyribonucleic acid (DNA).49 Demonstration of both a platelet-specific (HPA) antibody in the
maternal serum and the corresponding incompatibility for the antigen in the parental platelet types confirms the
diagnosis.
Treatment of acutely thrombocytopenic newborns includes the administration of intravenous immune
globulin (MG) with or without antigen-compatible platelet transfusions that are sometimes provided by the
mother as washed platelet products.50 Once the diagnosis of FNAIT has been made in a family, subsequent
fetuses are at risk. Antenatal treatment with MG with or without steroids has proven to be an effective means of
reducing fetal thrombocytopenia and preventing intracranial hemorrhage.51 (For a more indepth discussion of
FNAIT, see Chapter 22.)
Posttransfusion Purpura
PTP is a rare syndrome characterized by the development of dramatic, sudden, and selflimiting
thrombocytopenia 5 to 10 days after a blood transfusion in patients with a previous history of sensitization by
pregnancy or transfusion.52 Coincident with the thrombocytopenia is the development of a potent
plateletspecific alloantibody, usually anti-HPA-la, in the patient’s serum. Other specificities have been
implicated; these are almost always associated with antigens on GPIIb/IIIa. PTP differs from transfusion
reactions caused by red cell antibodies in that the patient’s own antigennegative platelets as well as any
transfused antigen-positive platelets are destroyed. The pathogenesis of autologous platelet destruction in PTP
is not fully understood; however, mounting evidence suggests that distinct platelet autoantibodies transiently
arise, along with the alloantibodies, and cause both autologous and transfused antigen-negative platelet
destruction.52 These panreactive autoantibodies often target the same glycoprotein that expresses the HPA
against which alloantibodies were made.
Platelet antibody assays usually reveal an antibody in the serum with HPA-la specificity. Genotyping
documents the absence of HPA-la or other platelet-specific antigens. Plasma exchange—once the treatment of
choice for PTP—has now largely been supplanted by MG as first-line therapy. Transfusion of antigen-negative
platelets may be of value during the acute phase of PTP; however, such platelets have reduced in-vivo survival
due to destruction by the aforementioned platelet autoantibodies.53
Following recovery, future transfusions should be from HPA-la-negative donors if
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AABB TECHNICAL MANUAL
possible. Washed red cells may offer some protection against recurrence; however, this is controversial and
there is at least one report of PTP being precipitated by a frozen deglycerolized red cell transfusion.54
Moreover, according to recent data reported from the Serious Hazards of Transfusion surveillance program, the
frequency of PTP has rather dramatically decreased coincidently with the introduction of leukocyte-reduced
blood products.55 Although no data have been reported to explain this trend, use of leukocyte-reduced products
may also be of benefit in reducing the risk of PTP recurrence.
Drug-Induced Thrombocytopenia
Thrombocytopenia caused by drug-induced platelet antibodies is a recognized complication of drug therapy.
Drugs commonly implicated include quinine, sulfa drugs, vancomycin, GPIIb/IIIa antagonists, and
heparin.56,57 Both drug-dependent and non-drug-dependent antibodies may be produced. Non-drugdependent
antibodies, although stimulated by drugs, do not require the continued presence of the drug to be reactive with
platelets and are serologically indistinguishable from other platelet autoantibodies.
Although several mechanisms for druginduced antibody formation have been described, most clinically
relevant drug-dependent platelet antibodies are thought to result when a drug interacts with platelet membrane
glycoproteins, inducing conformational changes recognized by drug-dependent antibodies.58,59 These
antibodies can cause a thrombocytopenia of sudden and rapid onset that usually resolves within 3 to 4 days after
the drug is discontinued.
Among the drug-induced immune responses to platelets, those triggered by exposure to heparin have
particular clinical importance because of both the widespread use of this anticoagulant and the devastating
thrombotic complications associated with the heparin-induced thrombocytopenia (HIT) syndrome.60 The
incidence of HIT is unknown, but
it may develop in up to 5% of patients treated with unfractionated heparin. Low-molecularweight heparin is
less likely to be associated with HIT than unfractionated heparin.
A reduction in baseline platelet count by 30% to 50% occurs generally within 5 to 14 days after primary
exposure to heparin and sooner if the patient has been exposed to heparin within the last 3 months. The platelet
count is often less than 100,000/pL but usually recovers within 5 to 7 days upon discontinuation of heparin.
More than 50% of patients with HIT develop thrombosis, which can occur in the arterial, venous, or both
systems.61 Patients may develop stroke, myocardial infarction, limb ischemia, deep venous thrombosis, or
ischemia of other organs. The thrombotic complications may lead to limb amputation or prove fatal. Because
the rate of HIT-associated thrombosis is so substantial, it is of critical importance to discontinue heparin therapy
when a diagnosis of HIT is suspected. Moreover, strong consideration should be given to using an alternative
(nonheparin) anticoagulant (eg, a direct thrombin inhibitor) to prevent thrombosis.61
The mechanism of HIT includes formation of a complex between heparin and platelet factor 4 (PF4), a
tetrameric protein released from platelet a granules. Antibodies (IgG, IgA, and some IgM) are produced to the
complex, and IgG in the complex attaches secondarily to platelet receptor FcyRIIa via its Fc, resulting in
platelet activation with subsequent thrombin generation. The antibody may also bind to complexes formed at
other sites, notably on endothelial cells and monocytes. Thus, HIT might involve activation and damage not
only of platelets but also of endothelium and monocytes/macrophages, causing increased susceptibility to
thrombosis. This understanding of the mechanism of HIT antibodies is exploited by enzyme-linked
immunosorbent assay (ELISA) tests in which microtiter wells are coated with the complexes of PF4 and
heparin (or heparin-like molecules) rather than with the platelets themselves.62
463
Autoimmune or Immune Thrombocytopenic Purpura
Immune thrombocytopenic purpura (ITP) is an immune platelet disorder in which autoantibodies are
directed against platelet antigens, resulting in platelet destruction. Chronic ITP, which is most common in
adults, is characterized by an insidious onset and moderate thrombocytopenia that may exist for months to years
before diagnosis. Females are twice as likely to be affected as males.
Spontaneous remissions are rare, and treatment is usually required to raise the platelet count. First-line
therapy consists of steroids or MG followed by more potent immunosuppressive agents or splenectomy in
nonresponders. Many other therapies have been used in patients who do not respond to splenectomy, with
variable results.
Chronic autoimmune thrombocytopenia may be idiopathic or associated with other conditions, such as
human immunodeficiency virus infection, malignancy, or other autoimmune diseases. Acute ITP is mainly a
childhood disease characterized by the abrupt onset of severe thrombocytopenia and bleeding symptoms, often
after a viral infection. The majority of cases resolve spontaneously over a 2- to 6-month period. If treatment is
required, MG or anti-D immunoglobulin infusions given to D-positive patients are usually effective in raising
platelet counts. Steroids are used less often because of their serious side effects in children. Splenectomy, if
used, is reserved for children whose disease is severe and lasts longer than 6 months; this condition is similar to
chronic ITP in adults. Rituximab and various thrombopoietin receptor agonists have been used as second-line
therapies for acute ITP63
Studies of both sera and washed platelets from patients with ITP have identified autoantibodies of IgG, IgM,
and IgA that are reactive with a number of platelet surface-membrane structures, most often including GP
complexes Ilb/IIIa, Ia/IIa, and Ib/IX but that can also include GPIV GP\j and GPVI.64 In the majority of cases,
platelet-associated autoantibodies are reactive with two or more platelet glycopro
teins.85 There is no compelling evidence to date suggesting that a patient’s profile of autoantibody
specificities correlates with the severity of the disease or predicts that patient’s response to therapy.
Testing for Platelet Antigens and Antibodies
Detection of platelet antibodies in the laboratory provides important results to aid in making a clinical
diagnosis of an immune platelet disorder. The leaders of the International Society of Blood Transfusion (ISBT)
platelet immunology workshops, designed to establish best practices in platelet antibody and antigen testing,
have determined that a comprehensive work-up for platelet antibodies requires the use of multiple test methods,
including a glycoprotein-specific assay, a test employing intact/whole platelets, and HPA genotyping.66
Glycoprotein-specific assays are the most sensitive and specific for identifying the HPA specificity of serum
antibodies (Fig 18-1). The inclusion of assays that use intact platelet targets is critical for detection of antibodies
that can be missed by glycoprotein-specific tests because the process of platelet lysis with detergent and capture
of GP with a specific monoclonal antibody can disrupt HPA epitopes recognized by some antibodies. HPA
genotyping by DNA methods is helpful to confirm the HPA specificity of the antibodies and for prenatal typing
of a fetus in suspected cases of FNAIT. The test methods that follow are examples of the current state-of-the-art
methods used by reference laboratories. For indepth descriptions of platelet antibody and antigen testing,
readers should consult recent reviews.49,67,68
 Assays Using Intact Platelets
An assay that is widely used for the detection of platelet-specific antibodies for platelet crossmatching is the
SPRCA.47 Intact platelets are immobilized in round-bottomed wells of a microtiter plate and are then incubated
with the patient’s serum. After washing, detector red cells coated with antihuman IgG are
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AABB TECHNICAL MANUAL

FIGURE 18-1. Antigen capture enzyme-linked immunosorbent assay (ELISA) testing. Modified antigen
capture ELISA (MACE) involves incubation of a patient's serum with target platelets, washing and
solubilization of the platelets in nonionic detergent, and addition of lysate to micrototer plate well for capture of
platelet glycoprotein (GP) by a specific mouse immunoglobulin G (IgG) monoclonal antibody (MoAb).
Platelet-specific antibodies [human platelet alloantigen (HPA)-Aby] in the patient’s serum bound to the GP are
detected by the addition of an enzymelabeled goat antihuman IgG (Enz-AHG). The monoclonal antibody
immobilization of platelet antigens (MAIPA) assay is very similar to the MACE, but the patient’s serum and
MoAb are incubated with platelets before washing and platelet solubilization, and GP/HPA-Aby complex is
captured by goat antimouse IgG (G-AMIgG) adhered to the bottom of the well.
added, centrifuged, and examined visually. The method’s main limitations are its subjective endpoint and
failure to distinguish platelet-specific from non-platelet-specific antibodies.
Flow cytometry is commonly used for immunofluorescent detection of platelet antibodies utilizing intact
platelets.49 Following incubation of platelets with the patient’s serum, platelet-bound antibodies are detected
with a fluorescent-labeled antiglobulin reagent that is specific for human IgG or IgM. The results can be
expressed as a ratio of the mean or median channel fluorescence of platelets sensitized with patient serum to
that of platelets incubated with negative control serum. Platelet autoantibodies coating the patient’s platelets can
also be detected in a direct flow-cytometry
69
assay.
Flow cytometry has proven to be a very sensitive method for detection of antibodies to platelets.
Alloantibodies that are specific for labile epitopes and unreliably detected by antigen capture assays (ACAs)
can be detected with intact platelets using flow cytometry.8 Flow cytometry does not differentiate between
platelet-specific (ie, platelet-glycoprotein-directed/HPA) and non-platelet-specific antibodies (ie, HLAs or
autoantibodies). This is a drawback when suspected FNAIT or PTP is investigated because the more relevant
plateletspecific antibodies that are characteristic of these syndromes can be obscured by nonplatelet-specific
reactivity.
Antigen Capture Assays
Platelet glycoprotein ACAs are used to determine the HPA that is recognized by platelet antibodies in a
patient’s serum. The assays developed for this purpose include MACE and MAIPA (Fig 18-1).49,70 The assays
require the use of monoclonal antibodies that recognize the target antigens of interest but do not compete with
the patient’s antibody. These assays capture specific platelet glycoproteins on plastic wells of a microplate after
sensitization with patient’s serum. The patient’s bound antibody is detected with an enzyme-labeled antihuman
Ig. Because only the glycoproteins of interest are immobilized, interference by reactions from non-platelet-
specific antibodies, especially anti-HLA, is eliminated.
465
Platelet Genotyping
 Genotyping for the SNPs in the genes encoding HPA can be performed by any of the myriad molecular
methods available. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism
analysis and allele-specific PCR are two methods that have been used successfully.67 These techniques are
reliable, but they are also laborious and time consuming. Higher-throughput methods have been developed,
such as real-time PCR and allele-specific fluorescent probes.67
Testing for Platelet Autoantibodies
Numerous assays have been developed to detect platelet autoantibodies in patients with ITR Although many
tests are quite sensitive, particularly in detecting cell-surface, plateletassociated Igs, none has been sufficiently
specific to be particularly useful in either the diagnosis or management of ITP. The American Society of
Hematology practice guidelines for ITP state that serologic testing is unnecessary, assuming that the clinical
findings are compatible with the diagnosis.71 However, platelet antibody tests may be helpful in the evaluation
of patients suspected of having ITP when nonimmune causes may be present. The goal of serologic testing in
ITP is to detect autoantibody bound to the patient’s own platelets with or without demonstration of similar
reactivity in the patient’s plasma.
Newer assays are designed to detect immunoglobulin binding to platelet-specific epitopes found on platelet
GPIIb/IIIa, GPIa/GPIIa, and/or GPIb/IX complexes. These solid-phase, GP-specific assays appear to have
improved specificity in distinguishing ITP from nonimmune thrombocytopenia, but this benefit is often
balanced by a decrease in sensitivity.65,72 One commercially available test uses eluates prepared from the
patient’s washed platelets.65 The eluates are tested against a panel of monoclonal-antibody-immobilized
platelet glycoprotein complexes, and platelet antibodies are detected using an enzyme-linked antihuman Ig. In
the indirect phase of the assay, patient plasma is tested against the same glycoprotein panel. Although
autoantibodies are
most often detected in the eluates, they are infrequently detected (in approximately 17% of cases) in the
plasma. Patients with ITP may have antibodies that are reactive with one or several GP targets.61
Testing for Drug-Dependent Platelet Antibodies
Any platelet serology test that is used to detect platelet-bound Ig can be modified to detect platelet drug-
dependent antibodies. Each patient serum or plasma sample must be tested against normal platelets in the
presence and absence of the drug. Moreover, at least one normal control serum sample should be tested with
and without the drug to control for the nonspecific antibody binding that might be induced by the drug’s
presence. A positive control serum sample that is known to be reactive with the drug being assayed should be
tested with and without the drug to complete the evaluation. A positive result shows that the serum is positive
(or more positive) against normal platelets in the presence of the drug vs without the drug and that the drug did
not nonspecifically cause a positive result with normal serum controls. Flow cytometry is the most sensitive and
most commonly used method to detect both IgG and IgM drugdependent antibodies.49,73 Limitations to
detection of drug-dependent platelet antibodies include that 1) for many drugs, the optimal concentration for
antibody detection has not been determined and hydrophobic drugs are difficult to solubilize; 2) the presence of
nondrug antibodies can mask the antibodies; and 3) a patient may be sensitized to a drug metabolite and not the
native drug.
Assays for heparin-dependent antibodies include the PF4 ELISA, which involves the addition of the
patient’s serum diluted at a 1:50 ratio alone and in the presence of high-dose (100 U/mL) heparin to plastic
microplate wells to which bound complexes of PF4 and heparin or heparin-like molecules (eg, polyvinyl
sulfonate) are affixed.62 Heparin-dependent antibodies bind to the complexes and are detected via enzyme-
conjugated antihuman Ig. An optical density value, generally above 0.4, in the
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AABB TECHNICAL MANUAL
PF4-heparin well that can be inhibited by high-dose heparin confirms the presence of heparin-dependent
antibodies. Although IgG antibodies are the most clinically relevant antibodies, a few patients with HIT appear
to have only IgM or IgA antibodies. PF4 ELISAs are available in two forms: those that detect but do not
differentiate IgG, IgM, and IgA heparin-dependent antibodies, and those that detect only IgG.
The 14C-serotonin release assay (SRA) is a functional assay that uses washed platelets for detection of
heparin-dependent antibodies.74 Normal, fresh platelets are incubated with 14C-serotonin, which is taken up
into their dense granules. The platelets are then exposed to the patient’s serum in the presence of low (0.1
U/mL) and high (100 U/mL) concentrations of heparin. Release of at least 20% of the radioactivity at the low
dose of heparin and inhibition of this release below 20% at the high dose confirms the presence of heparin-
dependent antibodies.
Other functional tests used to detect heparin-dependent antibodies include the heparin-induced platelet-
aggregation test and the heparin-induced platelet-activation test. The PF4 ELISA and the SRA are both more
sensitive and specific than the platelet-aggregation test for the detection of heparin-dependent platelet
antibodies in patients for whom there is a clinical suspicion that these antibodies are present. However, in
asymptomatic patients receiving heparin or who have not yet received the drug, neither test is sufficiendy
predictive of HIT to warrant its use in screening.75
GRANULOCYTE ANTIGENS AND ANTIBODIES
Antibodies against granulocyte (neutrophil) antigens are implicated in the following clinical syndromes:
neonatal alloimmune neutropenia (NAN), transfusion-related acute lung injury (TRALI), immune neutropenia
after HPC transplantation, refractoriness to granulocyte transfusion, and chronic benign autoimmune
neutropenia of infancy (AIN). To date, nine neutrophil antigens carried on five different glycoproteins have
been character
ized and given human neutrophil alloantigen (HNA) designations by the Granulocyte Antigen Working
Party of the ISBT (Table 18-5).76 This nomenclature system follows a similar convention to that used for HPA
nomenclature. Several of the antigens on granulocytes are shared with other cells and are not granulocyte
specific.
HNA
Antigens on FcyRIIIb
The first granulocyte-specific antigen detected was NA1, later named “HNA-la.” Three alleles of HNA-1
have now been identified—HNA-la, HNA-lb, and HNA-lc—and are located on the protein FcyRIIIb
(CD16b).77 FcyRIIIb is a GPIlinked protein receptor for the Fc of IgG and is present only on the surfaces of
neutrophils. Neutrophils express 100,000 to 200,000 molecules of FcyRIIIb, but there are rare individuals
(approximately 0.1%) whose neutrophils express no FcyRIIIb (CD16 null) and who can produce antibodies that
are reactive with FcyRIIIb when they are exposed to it through transfusion or pregnancy.78,79 Antibodies to
HNA-la and -lb have been implicated in TRALI, NAN, and AIN.77
Antigens on CD177
HNA-2 (previously known as “NB1”) is not an alloantigen because HNA-2 antibodies are isoantibodies that
recognize common epitopes on CD177 protein, which is missing from the neutrophils of immunized
individuals. Neutrophils from approximately 3% to 5% of people lack expression of CD 177 on neutrophils.77
Lack of CD 177 is thought to be caused by a messenger ribonucleic acid splicing defect that results in a
truncated protein that cannot be expressed.80 Interestingly, CD177 is expressed only on a subpopulation of
neutrophils in CD177-positive individuals.81 The proportion of the CD177-positive neutrophil population
ranges from 0% to 100%.82 CD177 is expressed only on neutrophils and belongs to the Ly-6/uPAR/snake-toxin
family of proteins.81
467
TABLE 18-5. Human Neutrophil Antigens
Phenotypic Amino Acid Nucleotide
Encoding
Antigen Frequency* Glycoprotein Change Change
Gene
HNA-la 12% a/a CD16 Multiple1 FCGR3B Multiple1
HNA-
54% a/b
lb
46% b/b
HNA-lc 5%
HNA-2 97% CD177+ CD177 N/A CD 177 N/A
3% CD177
HNA-
56%-59% a/a CTL2 Arg152Gln SLC44A2 455G>A
3a
HNA-
34%-40% a/b
3b
3%-6% b/b
 HNA- 78.6% a/a CDIIb Arg61 His ITGAM 230G>A
4a
HNA-
19.3% a/b
4b
2.1% b/b
HNA-
54.3% a/a CDIIa Arg766Thr ITGAL 2466G>C
5a
HNA-
38.6% a/b
5b
7.1% b/b
Nucleotide Amino Acid
Changes Changes
141 147 227 266 277 : 349 36 38 65 78 82 106
Leu Asn
HNA-la GCA CG G Arg Asp Val
Ala
HNA-
CTG CA A Ser Leu Ser Ala Asn lie
lb
HNA-lc CTG AA A Ser Leu Ser Asp Asn lie
‘Phenotypic frequencies are for people of European ancestry who live in North America. fHNA-1 amino
acid and nucleotide changes are shown in a separate section of the table. HNA = human neutrophil antigen; N/A
= not applicable.
A recent report documented cation-dependent heterophilic interactions between CD 177 and the endothelial
cell membrane protein PECAM-1(CD31), suggesting a role for CD177 in neutrophil transendothelial migration
to sites of infection.83 Antibodies against HNA-2 have been implicated in NAN, TRALI, and neutropenia in
marrow transplant recipients.84-86
Antigens on CTL2
HNA-3a and HNA-3b are carried on the choline transporter-like protein 2 (CTL2), and a SNP in the gene
(SLC44A2) accounts for the polymorphism (Table 18 - 5).87,88 CTL2 is also expressed on both T and B
lymphocytes, and small amounts are present on platelets. HNA3a antibodies are usually agglutinins. They
 468
AABB TECHNICAL MANUAL
occasionally develop in women after pregnancy, and HNA-3a antibodies are the most frequent cause of fatal
TRALI.89 HNA-3b antibodies are rarely detected, but several have been found during screening of the serum of
multiparous blood donors.
Antigens on CDlla and CD11 b
HNA-4a and HNA-5a, both high-prevalence antigens, are present on monocytes and lymphocytes as well as
granulocytes. HNA-4a is carried on the CDllb/18 (Mac-1, CR3, am(32) glycoprotein.77 CDllb/18 plays a role
in neutrophil adhesion to endothelial cells and phagocytosis of C3bi opsonized microbes. There is some
evidence showing that alloantibodies against HNA-4a interfere with CDllb/ 18-dependent neutrophil adhesion
and enhance neutrophil respiratory burst.90 Antibodies against HNA-4a have been implicated in NAN, and
autoantibodies against CDllb/18 have also been described.91,92
HNA-5a is carried on CDlla/18 (LFA-1, aLp2) glycoprotein.93 CDlla/18, like CDllb/ 18, plays a role in
neutrophil adhesion to endothelial cells. Antibodies that are reactive with HNA-5a have been found in a
chronically transfused patient with aplastic anemia and have also been reported to be associated with NAN.94
The patient who made the original HNA-5a antibody experienced prolonged survival of an HLA nonidentical
skin graft that was attributed to the HNA-5a antibody.93
Other Neutrophil Antigens
Neutrophils do not express ABH or other red cell group antigens, but they do express modest amounts of
Class I and II HLA only upon activation.
Immune Neutrophil Disorders
Neonatal Alloimmune Neutropenia
NAN is caused by maternal antibodies against antigens on fetal neutrophils; the most frequent specificities
are against HNA-la, HNAlb, and HNA-2 antigens. NAN may also occur
in the children of women who lack the FcyRIIIb protein. Neutropenia in NAN can occasionally be life-
threatening because of increased susceptibility to infection.95 Management with antibiotics, MG, granulocyte
colony-stimulating growth factor, and/or plasma exchange may be helpful.
TRALI
TRALI is an acute, often life-threatening reaction characterized by respiratory distress, hypo- or
hypertension, and noncardiogenic pulmonary edema that occurs within 6 hours of a blood component
transfusion.96 TRALI has been the most common cause of transfusionrelated death for more than 10 years.97
In TRALI, the causative antibodies are most often found in the plasma of the blood donor. When these
antibodies are transfused, they cause activation of primed neutrophils that are sequestered in the lungs of certain
patients. The activated neutrophils undergo oxidative burst, releasing toxic substances that damage pulmonary
endothelium and resulting in capillary leak and pulmonary edema. Class I and II HLA and HNA antibodies
have all been implicated in TRALI. However, in a recent large clinical study of TRALI, HNA and Class II HLA
antibodies, but not Class I HLA antibodies, were significantly associated with TRALI.98 (For a more in-depth
discussion of TRALI, see Chapter 27.)
Autoimmune Neutropenia
Autoimmune neutropenia may occur in adults or in infants. When present in adults, it may be idiopathic or
be secondary to such diseases as rheumatoid arthritis or systemic lupus erythematosus or to bacterial
infections.99 In autoimmune neutropenia of infancy, usually occurring in children between the ages of 6 months
and 2 years, the autoantibody has neutrophil antigen specificity (usually HNA-la or -lb) in about 30% of the
patients. The condition is generally self-limiting, with recovery usually occurring in 7 to 24 months, and is
relatively benign and manageable with antibiotics.87
469
Testing for Granulocyte Antibodies and Antigens
Granulocyte antibody testing is technically complex and labor intensive. The inability to maintain the
integrity of granulocytes for testing that are stored at room temperature, in refrigerated conditions, or by
cryopreservation requires that cells be isolated from fresh blood on each day of testing. This demands that
readily available blood donors typed for the various granulocyte antigens be available. Class I HLA antibodies
that are often present in patient sera complicate detection and identification of granulocyte antibodies. For these
reasons, it is critical that granulocyte antibody and antigen testing be performed by an experienced laboratory
using appropriate controls.
GRANULOCYTE AGGLUTINATION TEST. This was one of the first tests developed for the detection of
granulocyte antibodies. It is typically performed by overnight incubation of small volumes of isolated fresh
neutrophils with the patient’s serum in a microplate. The wells are viewed under an inverted phase microscope
for neutrophil agglutination or aggregation.
GRANULOCYTE IMMUNOFLUORESCENCE TEST. This test also requires fresh target cells that are
incubated, usually at room tempera
ture for 30 minutes, and washed in EDTA and phosphate-buffered saline. Neutrophil-bound antibodies are
then detected with fluorescein isothiocyanate-labeled antihuman IgG or IgM with either a fluorescence
microscope or a flow cytometer.89 100 A combination of agglutination and immunofluorescence tests is
beneficial.79,88 Other methods include chemiluminescence, SPRCA, and the monoclonal antibody
immobilization of granulocyte antigens (MAIGA) assay, which is similar to the MAIPA assay, but uses
monoclonal antibodies to capture the various glycoproteins that express HNA. The MAIGA assay is used to
differentiate between HLA- and HNA-specific antibodies.
HNA TYPING. As with HPA, typing for HNA is largely performed using molecular methods to detect the
allelic variants that determine the antigens. Any methods used in HPA typing can be applied to HNA typing
with simple modifications to the primer and probe sequences. Readers are referred to several publications on
this subject.101,102 Because the splicing defect that results in CD 177 deficiency is not known, typing for
HPA-2/CD177 requires testing for CD177 on freshly isolated neutrophils using specific monoclonal antibodies
and the granulocyte immuno fluorescence test.
KEY POINTS
1. Platelets express a variety of antigenic markers on their surfaces. Some of these antigens, such as ABH
and HLA, are shared with other cells, whereas HPAs are essentially platelet specific. There are currently 33
HPAs carried on six platelet glycoproteins.
2. HLA sensitization is the most common immune cause of platelet refractoriness and can be diagnosed by
the demonstration of significant levels of antibodies to HLA Class I in a patient’s serum. When antibodies to
HLA antigens are demonstrated, a widely used treatment approach is to supply apheresis platelets from donors
whose Class I HLAs are similar to those of the patient. HLA-matched platelets should be irradiated to prevent
transfusion-associated GVHD.
3. Compared with HLA matching for platelet-refractory patients, crossmatching can be both more
convenient and cost effective. It avoids exclusion of HLA-mismatched but compatible donors and has the added
advantage of selecting platelets when the antibodies involved are directed at platelet-specific rather than HLA
antigens.
4. Although blood group antibodies (anti-A and -B) and platelet-specific antibodies can be responsible for
poor responses to platelet transfusion, they are less commonly implicated in the refractory state than HLA
antibodies.
 470
AABB TECHNICAL MANUAL
5. Sensitization to HPA is the most common cause of FNAIT, a syndrome involving immune destruction of
fetal platelets by maternal antibody. Platelet-specific antibodies are also involved in PTP, a rare syndrome
characterized by severe thrombocytopenia that occurs 5 to 10 days after a blood transfusion. The most
commonly implicated antibody in both conditions is anti-HPA-la. Serologic testing using intact platelets and
antigen capture assays together with HPA genotyping is used to confirm both of these diagnoses.
6. Autoantibodies directed against platelet antigens may result in ITP. Chronic ITP which is most common
in adults, is characterized by an insidious onset and moderate thrombocytopenia that may be present for months
to years before diagnosis. Females are twice as likely to be affected as males. The goal of serologic testing in
ITP is to detect autoantibody bound to the patient’s own platelets.
7. Granulocyte (neutrophil) antigens are implicated in the clinical syndromes NAN, TRALI, immune
neutropenia after hematopoietic progenitor cell transplantation, refractoriness to granulocyte transfusion, and
chronic benign autoimmune neutropenia of infancy.
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neutrophil-specific antigen NB1 following marrow transplantation. Transfusion 1993;33:158-63.
87. Curtis BR, Cox NJ, Sullivan MJ, et al. The neutrophil alloantigen HNA-3a (5b) is located on choline
transporter-like protein 2 and appears to be encoded by an R>Q154 amino acid substitution. Blood
2010;115:2073-6.
88. Greinacher A, Wesche J, Hammer E, et al. Characterization of the human neutrophil alloantigen-3a. Nat
Med 2010;16:45-8.
89. Reil A, Keller-Stanislawski B, Gunay S, Bux J. Specificities of leucocyte alloantibodies in transfusion-
related acute lung injury and results of leucocyte antibody screening of blood donors. Vox Sang 2008;95:313-
17.
90. Sachs UJ, Chavakis T, Fung L, et al. Human alloantibody anti-Mart interferes with Mac-1 dependent
leukocyte adhesion. Blood 2004; 104:727-34.
 91. Fung YL, Pitcher LA, Willett JE, et al. Alloimmune neonatal neutropenia linked to antiHNA-4a.
Transfus Med 2003;13:49-52.
92. Hartman KR, Wright DG. Identification of autoantibodies specific for the neutrophil adhesion
glycoproteins CDllb/CD18 in patients with autoimmune neutropenia. Blood 1991; 78:1096-104.
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93. Simsek S, van der Schoot CE, Daams M, et al. Molecular characterization of antigenic polymorphisms
(Ond(a) and Mart (a)) of the beta 2 family recognized by human leukocyte alloantisera. Blood 1996;88:1350-8.
94. Porcelijn L, Abbink F, Terraneo L, et al. Neonatal alloimmune neutropenia due to immunoglobulin G
antibodies against human neutrophil antigen-5a. Transfusion 2011;51:574-7.
95. Davoren A, Saving K, McFarland JG, et al. Neonatal neutropenia and bacterial sepsis associated with
placental transfer of maternal neutrophil-specific autoantibodies. Transfusion 2004;44:1041-6.
96. Kleinman S, Caulfield T, Chan B et al. Toward an understanding of transfusion-related acute lung
injury: Statement of a consensus panel. Transfusion 2004;44:1774-89.
97. Fatalities reported to FDA following blood collection and transfusion. Annual summary for fiscal year
2012. Silver Spring, MD: CBER Office of Communication, Outreach, and Development, 2013. [Available at
http://www.fda.
gov/BiologicsBloodVaccines/SafetyAvailabili ty/ReportaProblem/TransfusionDonationFa
talities/ucm346639.htm (accessed September 2,2013).]
98. Toy P, Gajic O, Bacchetti P, et al. Transfusionrelated acute lung injury: Incidence and risk factors. Blood
2012;119:1757-67.
99. Akhtari M, Curtis B, Waller EK. Autoimmune neutropenia in adults. Autoimmun Rev 2009;9: 62-6.
100. Curtis BR, Reno C, Aster RH. Neonatal alloimmune neutropenia attributed to maternal
immunoglobulin G antibodies against the neutrophil alloantigen HNA-lc (SH): A report of five cases.
Transfusion 2005;45:1308-13.
101. Stroncek DF, Fadeyi E, Adams S. Leukocyte antigen and antibody detection assays: Tools for assessing
and preventing pulmonary transfusion reactions. Transfus Med Rev2007;21:27386.
102. Bux J. Molecular genetics of granulocyte polymorphisms. Vox Sang 2000;78(Suppl 2):125-30.
Chapter 19
The HLA System
Robert A. Bray; PhD; Marilyn S. Pollack, PhD; and Howard M. Gebel, PhD
imr|The hla system is composed of a |Qj3 complex array of genes located within the human major
histocompatibility complex (MHC) on the short arm of chromosome 6. Their protein products, the HLA
antigens, contribute to the recognition of self and nonself, the immune responses to antigenic stimuli, and the
coordination of cellular and humoral immunity.
HLA antigens are cell-surface glycoproteins that are divided into two groups (Class I and Class II)
according to their coding gene locus, function, tissue distribution, and biochemistry. HLA Class I molecules
contain one copy each of two polypeptides: a heavy chain that is an integral cell-membrane protein and a
noncovalently associated light chain called “P2-microglobulin” (the P2-microglobulin gene resides on
chromosome 15). HLA Class II molecules are composed of one copy each of an achain and a p-chain, both of
which are integral membrane proteins. Class I molecules are
found on the surface of platelets and, with a few exceptions, on all nucleated cells of the body. Mature red
cells usually lack HLA antigens that are identifiable by conventional methods, whereas nucleated immature
erythroid cells do express HLA antigens. MHC Class II antigen expression is typically restricted to a few cell
types, including B lymphocytes, monocytes, macrophages, dendritic cells, and activated T lymphocytes.
HLA molecules play a key role in antigen presentation and the initiation of the immune response. The HLA
system is generally viewed as second in importance only to the ABO antigens in influencing the survival of
transplanted solid organs. In hematopoietic progenitor cell (HPC) transplantation, the HLA system is paramount
with regard to graft rejection and graft-vs-host disease (GVHD). HLA antigens and antibodies are also
important in complications of transfusion therapy, such as platelet refractoriness, febrile nonhemolytic transfu
Robert A. Bray, PhD, Professor of Pathology, and Co-director, Histocompatibility and Molecular
Immunogenetics Laboratory, Department of Pathology, Emory University, Atlanta, Georgia; Marilyn S. Pollack,
PhD, Professor of Pathology, University of Texas Health Science Center, and Director, University Health
System Histocompatibility and Immunogenetics Laboratory, San Antonio, Texas; and Howard M. Gebel, PhD,
Professor of Pathology, and Co-director, Histocompatibility and Molecular Immunogenetics Laboratory,
Department of Pathology, Emory University, Atlanta, Georgia The authors have disclosed no conflicts of
interest.

475

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AABB TECHNICAL MANUAL
sion reactions (FNHTRs), transfusion-related acute lung injury (TRALI), and transfusionassociated GVHD
(TA-GVHD).
Interest in HLA polymorphisms has extended beyond their role as transplantation antigens. Studies
correlating them with disease susceptibility and disease resistance began soon after serologic techniques for
HLA Class I typing were developed. Historically, HLA antigen typing had been of value in relationship
assessments and forensic investigations. However, with the development of deoxyribonucleic acid (DNA)
typing, molecular methods replaced the mixed leukocyte culture (MLC) as the preferred method to select
matched donor-recipient pairs for HPC transplantation. Molecular HLA testing also fostered a resurgence in the
investigation of disease associations that permitted the analysis of peptide-binding restriction parameters needed
for effective vaccine development and provided a more accurate tool for anthropologic population studies.
Because of the polymorphic nature of the HLA genes, a complex nomenclature was developed (and is
continually evolving) to define unique allele sequences based on the relationship of each allele’s protein
sequence to the serologic specificity of the corresponding antigen.1,2
GENETICS OF THE MHC
Class I and II HLA antigens are cell-surface glycoproteins that are products of closely linked genes mapped
to the p21.3 band on the short arm of chromosome 6 (Fig 19-1). That genomic region is called the “MHC” and
is usually inherited en bloc as a haplotype. Each of the many loci has multiple alleles with codominant
expression of the products from each chromosome. With the exception of immunoglobulin (Ig) idiotypes, the
HLA system is the most polymorphic genetic system described in humans.
The genes HLA-A, HLA-B, and HLA-C encode the corresponding Class I A, B, and C antigens. The genes
HLA-DRB1, -DRB3, -DRB4, -DRB5; HLA-DQA1, -DQB1; and HLADPA1, -DPB1 encode the corresponding
Class II antigens. Located between the Class I and
Class II genes is a group of non-HLA genes that code for molecules that include the complement proteins
C2, Bf, C4A, and C4B; a steroid enzyme (21-hydroxylase); and a cytokine (tumor necrosis factor). This non-
HLA region is often referred to as “MHC Class III” even though it does not contain any HLA genes.
Organization of HLA Genetic Regions
The HLA Class I region contains (in addition to the classical genes HLA-A, HLA-B, and HLA-C) other
gene loci designated HLA-E, HLA-F, HLA-G, HLA-H, HFE, HLA-J, HLA-K, HLA-L, MICA, and MICB. The
latter genes encode nonclassical, or Class lb, HLA proteins, which have limited polymorphism and low levels of
expression.4 Some Class lb genes express nonfunctional proteins or no proteins whatsoever. Genes that are
unable to express a functional protein product are termed “pseudogenes” and presumably represent an
evolutionary dead end. In contrast, other nonclassical HLA proteins that are expressed have been associated
with a variety of functions. For example, HLA-E is associated with the surveillance system of one subset of
natural killer cells. HLA-G is expressed by the trophoblast and may be involved in the development of maternal
immune tolerance of the fetus. Hereditary hemochromatosis (HH), an iron overload disorder with a 10% carrier
frequency in people of Northern European ancestry, is associated with two missense mutations in a Class I-like
gene.5 The gene that causes HH was initially named “HLA-H"-, however, the HLA-H designation had already
been assigned to an HLA Class I pseudogene by the World Health Organization (WHO) Nomenclature
Committee.6 The gene that is responsible for HH is now called “HFE.” Additional Class I-like genes that code
for molecules, such as CD1, are also located outside the MHC. These molecules present nonprotein antigens
(such as lipids) to T cells.
The genomic organization of the MHC Class II (HLA-D) region is more complex. An MHC Class II
molecule consists of a noncovalent complex of two structurally similar chains: the a-chain and the p-chain. Both
of
LMP2
 FIGURE 19-1. The HLA complex is located on the short arm of chromosome 6. The centromere is to the
top left of the figure, the telomere to the bottom right. The organization of the Class I, II, and III regions is
shown.3

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these chains are encoded within the MHC. The polymorphism of HLA Class II molecules results from
differences in both the a-chain and the p-chain; this polymorphism depends on the Class II isoform. For
example, with HLADR, the a-chain is essentially monomorphic, but the p-chain is very polymorphic. Multiple
loci code for the a- and p-chains of the Class II MHC proteins.
Different haplotypes have different numbers of Class II genes and pseudogenes. The proteins coded by
DRA1 and DRB1 result in HLA-DR1 through HLA-DR18 antigens. The products of DR AI and DRB3 (if
present) express HLA-DR52; those of DRA1 and DRB4 (if present) express HLA-DR53; and those of DRA1
and DRB5 (if present) express HLADR51. The HLA-DQ1 through DQ9 antigens are expressed on the
glycoproteins coded by DQA1 and DQB1 in the DQ cluster. Many of the other genes of the DQ cluster are
probably pseudogenes. A similar organization is found in the HLA-DP gene cluster.
 Although not generally considered part of the HLA system, the MHC Class III region contains four
complement genes with alleles that are typically inherited together as a unit, termed a “complotype.” More than
10 different complotypes are inherited in humans. Two of the Class III genes, C4A and C4B, encode for
variants of the C4 molecule and antigens of the Chido/Rodgers blood group system. These variants have distinct
protein structures and functions; the C4A molecule (if present) carries the Rg antigen, and the C4B molecule (if
present) carries the Ch antigen. Both of these antigens are adsorbed onto the red cells of individuals who
possess the gene(s).
Patterns of Inheritance
Although MHC organization is complicated, its inheritance follows the established principles of Mendelian
genetics. Every person has two different copies of chromosome 6 and possesses two HLA haplotypes, one from
each parent. The expressed gene products constitute the phenotype, which can be determined by typing for HLA
antigens or alleles. Because HLA genes are autosomal and codominant,
the phenotype represents the combined expression of both haplotypes. However, to define haplotypes,
parents (and possibly other family members) must also be phenotyped to determine which alleles are inherited
together. Figure 19-2 illustrates inheritance of haplotypes.
Finding HLA-Identical Siblings
A child inherits one copy of chromosome 6 from each parent; hence, one MHC haplotype is inherited from
each parent. Because each parent has two different copies of chromosome 6, four different combinations of
haplotypes are possible in the offspring (assuming that no recombination occurs). The inheritance pattern is
important in predicting whether family members will be compatible donors for transplantation. The chance that
two siblings will be genotypically HLA identical is 25%. The chance that any one patient with “n” siblings will
have at least one HLAidentical sibling is 1 - (3/4)n. Having two siblings provides a 44% chance, and having
three siblings provides a 58% chance, that one sibling will be HLA identical. Moreover, each time a new sibling
is tested, that new sibling has (only) a 25% chance of being a match, no matter how many siblings have
previously been tested.
Absence of Antigens
Before the advent of molecular-based HLA typing, the absence of an antigen in serologic phenotyping
results was attributed to homozygosity at a locus (eg, inheritance of A1 from both parents, which in reality
represented only an apparent absence of the antigen as a result of limitations of phenotyping methods) or to a
null (nonexpressed) allele. With DNA sequencing and other molecular HLA typing methods, homozygosity can
now be presumed with a higher degree of confidence. However, homozygosity still can be proven only through
family studies or methods permitting hemizygous typing (ie, typing of an individual haplotype). A null allele is
characterized by one or more substitutions that are within or outside the gene’s coding region and that prevent
expres
479
Chromosome 6
en bloc inheritance of the HLA complex

••••
IT It It It
bb
c
d
cd
cd
FIGURE 19-2. The designations a/b and c/d represent paternal and maternal HLA haplotypes, respectively.
Except for crossovers, the HLA complex is transmitted en bloc from parent to offspring.
sion of a functional protein at the cell surface. Such inactivation of a gene may be caused by nucleotide
substitutions, deletions, or insertions that lead to a premature cessation in the antigen’s synthesis. In the absence
of a family study, a phenotyping study revealing a single allele at any locus offers only presumptive evidence
for homozygosity. In this situation, the allele should be listed only once because it is unknown whether that
allele is present twice (a true homozygote) or there is another allele not detected by the available method.
Crossovers
The genes of the HLA region occasionally demonstrate chromosome crossover, in which segments
containing linked genetic material are exchanged between the two chromosomes during meiosis or
gametogenesis. The recombinant chromosomes are then transmitted as new haplotypes to the offspring.
Crossover frequency is related partly to the physical distance between the genes and partly to the resistance or
susceptibility of specific A, B, and
DR antigens to recombination (see below). The HLA-A, HLA-B, and HLA-DR loci are close together, with
0.8% crossover between the A and B loci and 0.5% between the B and DR loci. Crossovers between the HLA-B
and HLA-C loci or between the HLA-DR and the HLA-DQ loci are extremely rare, whereas crossovers
between the DQ and DP loci are relatively common.7,8 In family studies and relationship evaluations, the
possibility of recombination should always be considered.
Linkage Disequilibrium
The MHC system is so polymorphic that the number of possible unique HLA phenotypes is theoretically
greater than that of the global human population. Moreover, new HLA alleles are constantly being discovered
and characterized. As of March 2014, 2579 HLA-A alleles, 3285 HLA-B alleles, 1512 DRB1 alleles, and 509
DQB1 alleles had been identified.9 However, because HLA genes are inherited as an entire chromosome, in
reality, many HLA haplotypes are overrepresented compared with what
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would be expected if the distribution of HLA genes were random. The phenomenon of linkage
disequilibrium accounts for the discrepancy between expected and observed HLA haplotype frequencies.
Expected frequencies for HLA haplotypes are derived by multiplication of the frequencies of each allele.
For example, in individuals of European ancestry, the overall frequency of the gene coding for HLA-A1 is 0.15
and that for HLA-B8 is 0.10; therefore, 1.5% (0.15 x 0.10) of all HLA haplotypes in people of European
ethnicity would be expected to contain genes coding for both HLA-A1 and HLA-B8 if the haplotypes were
randomly distributed. The actual haplotype frequency of the A1 and B8 combination, however, is 7% to 8% in
that population.
Certain allelic combinations occur with increased frequency in different racial groups and constitute
common haplotypes in those populations. These common haplotypes are called “ancestral haplotypes” because
they appear to be inherited from a single common ancestor or to be conserved within the population because of
resistance to recombination or survival advantage. The most common ancestral haplotype in people of Northern
European ancestry—Al, B8, DR17 (DRB 1*03:01), DQ2— includes both Class I and Class II regions.
Some haplotypes in apparent linkage disequilibrium may represent relatively young haplotypes that have
not had sufficient time to undergo recombination, whereas some old haplotypes are resistant to recombination
because of selection or physical limitations. For example, the Al, B8, DRB1*03:01 haplotype appears to be
resistant to recombination because that sequence of DNA has a different length than most other comparable
sequences due to the deletion of the complement gene C4A. Linkage disequilibrium in the HLA system is
important in relationship studies because haplotype frequencies in the relevant population make the
transmission of certain gene combinations more likely than others. Linkage disequilibrium also affects the
likelihood of finding suitable unrelated donors for HLA-matched platelet transfusions and HPC transplantation.
BIOCHEMISTRY, TISSUE DISTRIBUTION, AND STRUCTURE
Characteristics of Class I and Class II Antigens
Class I antigens (HLA-A, -B, and -C) have a molecular weight of approximately 57,000 Daltons and consist
of two protein chains: a glycoprotein heavy chain (45,000 Daltons) encoded on the short arm of chromosome 6
and, as a light chain, the p,-microglobulin molecule (12,000 Daltons) encoded by a gene on chromosome 15.
The heavy chain penetrates the cell membrane, whereas (Syuicroglobulin does not. Rather, P2microglobulin
associates (noncovalently) with the heavy chain through the latter’s nonvariable (a3) domain (see Fig 19-3). The
external portion of the heavy chain consists of three amino acid domains (al, a2, and a3), of which the
outermost al and a2 domains contain the majority of polymorphic regions conferring serologic HLA antigen
specificity.
Class I molecules are present on platelets and most nucleated cells in the body, with some exceptions that
include neurons, corneal epithelial cells, trophoblasts, and germinal cells. Only vestigial amounts remain on
mature red cells, with certain allotypes better expressed than others. These Class I types were independently
recognized as red cell antigens by serologists and designated as “BennettGoodspeed" (Bg) antigens. The
specificities called “Bga," “Bgb,” and “Bgc” are actually HLAB7, HLA-B17 (B57 or B58), and HLA-A28 (A68
or A69), respectively. Platelets express primarily HLA-A and HLA-B antigens. HLA-C antigens are present at
very low levels, and Class II antigens are generally not present.
Class II antigens (HLA-DR, -DQ, and -DP) have a molecular weight of approximately 63,000 Daltons and
consist of two structurally similar glycoprotein chains (a and p), both of which traverse the membrane (see Fig
19-3). The extramembranous portion of each chain has two amino acid domains, of which the outermost one
contains the variable regions of the Class II alleles. The expression of Class II

FIGURE 19-3. Stylized diagram of Class I and Class II major histocompatibility complex molecules
showing a and p polypeptide chains, their structural domains, and attached carbohydrate units.
antigens is more restricted than that of Class I antigens. Class II antigens are expressed constitutively on B
lymphocytes, monocytes, macrophages, dendritic cells, intestinal epithelium, and early hematopoietic cells.
There is also constitutive expression of Class II antigens on some endothelial cells, especially those lining the
microvasculature. However, in general, endothelium, particularly that of larger blood vessels, is negative for
Class II antigen expression, although Class II antigen expression can be readily induced (for instance, by
interferon-y during immune activation). Resting T lymphocytes are negative for Class II antigen expression but
can become positive when activated.
Soluble HLA Class I and Class II antigens shed from cells are present in blood and body fluids and may
play a role in modulating immune reactivity.10 Levels of soluble HLA increase with infection [including with
human immunodeficiency virus (HIV)], inflammatory disease, and transplant rejection, but HLA levels decline
with progression of malignancy. Levels of soluble HLA in blood components are proportional to the number of
residual do
nor leukocytes and the duration of storage.11 Soluble HLA in blood components may be involved in the
immunomodulatory effect of blood transfusion.
Configuration
A representative three-dimensional structure of Class I and Class II molecules can be obtained by x-ray
crystallographic analysis of purified HLA antigens (see Fig 19-4). The outer domains, which contain the regions
of amino acid variability and the antigenic epitopes of the molecules, form a structure known as the "peptide-
binding groove.” Alleles that are defined by polymorphisms in the HLA gene sequences encode unique amino
acid sequences and therefore form unique binding grooves, each of which is able to bind peptides of different
sequences. The peptide-binding groove is critical for the functional aspects of HLA molecules (see “Biologic
Function” section below).
Nomenclature for HLA Antigens
An international committee sponsored by the WHO establishes the nomenclature of the HLA
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Class I Class II
 FIGURE 19-4. Ribbon diagram of HLA Class I and Class II molecule. Note the peptide in the groove of
each molecule.
system. This nomenclature is updated regularly to incorporate new HLA alleles.2 HLA antigens are
designated by a number following the letter that denotes the HLA series (eg, HLA-A1 or HLA-B8). Previously,
antigenic specificities that were not fully confirmed carried the prefix “w” (eg, HLA-Aw33) for “workshop.”
When the antigen’s identification became definitive, the WHO Nomenclature Committee dropped the “w” from
the designation. (The Committee meets regularly to update nomenclature by recognizing new specificities or
genetic loci.) The “w” prefix is no longer applied in this manner and is now used only for the following: 1) Bw4
and Bw6, to distinguish such “public" antigens (see “‘Public’ Antigens” section below) from other B locus
alleles; 2) all serologically defined C locus specificities, to avoid confusion with components of the complement
system; and 3) Dw specificities that were defined by mixed leukocyte reactions but are now known to be caused
by HLA-DR, HLA-DQ, and HLA-DP polymorphisms. The numeric designations for the HLA-A and HLA-B
specificities were assigned according to the order of their discovery.
Splits and Cross-Reactive Groups
Refinement of serologic methods permitted antigens that were previously believed to rep
resent a single specificity to be “split” into specificities that were serologically (and, later, genetically)
distinct. The designation for an individual antigen that was split from an earlier recognized antigen often
includes the number of the parent antigen in parentheses [eg, HLAB44 (12)].
In addition to “splits,” certain apparently distinct HLA antigens may have other epitopes in common.
Antibodies that are reactive with these shared determinants often cause crossreactions in serologic testing. The
collective term for a group of HLA antigens that exhibit such cross-reactivity is “cross-reactive group” (CREG).
“Public” Antigens
In addition to splits and CREGs, HLA proteins have reactivity that is common to many different HLA
specificities. Called “public” antigens, these common amino acid sequences appear to represent the less variable
portion of the HLA molecule. Two well-characterized public antigens, HLA-Bw4 and HLA-Bw6, are present in
almost all HLA-B molecules.12 The HLA-A locus molecules A23, A24, A25, and A32 also have a Bw4-like
epitope.
Public antigens are clinically important because patients exposed to them through pregnancy, transfusion, or
transplantation can
483
make antibodies to these antigens if the patients do not express the epitopes themselves. A single antibody,
when directed against a public antigen, can resemble multiple discrete alloantibodies, and this has significant
consequences for identifying compatible donors for transplantation and platelet transfusion.
Nomenclature for HLA Alleles
Because nucleotide sequencing is used to investigate the HLA system, increasing numbers of HLA alleles
are being identified, many of which share a common serologic phenotype. The minimum requirement for
designation of a new allele is the sequence of exons 2 and 3 for HLA Class I and exon 2 for HLA Class II
(.DRB1). These exons encode the variable amino acids that confer HLA antigen specificity.
A uniform nomenclature has been adopted that takes into account the locus, major serologic specificity, and
allele group determined by molecular typing techniques. For example, although many alleles have been
sequenced only for exon 2, nucleotide sequencing has identified at least 165 unique amino acid sequence
variants (alleles) of HLA-DR4 as of March 2014. The first HLA-DR4 variant is designated “DRB1*04:01,”
indicating the locus (DR), protein ((31 chain), major serologic specificity (04 for HLA-DR4), and sequence 2
variation allele number (variant 01). The asterisk indicates that an allele name follows (and that
the typing was determined by molecular techniques).
A similar system is used for naming Class
1 alleles. The name of the locus—for example, “HLA-B"—is followed by an asterisk and then by several
digits separated by colons. The first two digits correspond to the antigen’s serologic specificity in most cases.
The next group of digits makes up the code for a unique amino acid sequence in exons 2 and 3, with numbers
being assigned in the order in which the DNA sequences were determined. Therefore, B*27:04 represents the
HLA-B locus, has a serologic specificity of B27, and was the fourth unique sequence 2 and 3 allele described in
this family (see Table 19-1). A third “place” in the allele name is added for alleles that differ only by
synonymous (“silent”) nucleotide substitutions in exons 2 and 3 for Class I or in exon
 2 for Class II. For example, A*01:01:02 differs from A*01:01:01 only in that the codon for isoleucine in
position 142 is ATT instead of ATC. A fourth “place” in the allele name can be added for alleles that differ only
in sequences within introns or in 3' or 5' untranslated regions. Finally, the nomenclature accommodates alleles
with null or low expression or other characteristics by the addition of an “N” or “L," respectively, or another
letter as appropriate to the end of the allele name. The other official expression modifiers are as follows: S
(secreted, not on cell surface), Q (expression level
TABLE 19-1. HLA Nomenclature 2013
Antigen Silent Outside Expression
Species Locus Equivalent Allele Mutation Exon Modifier
HLA DRB1 * 04 01 : 01 01:02 N,L,S,Q
Examples:
DR4 - Serology
DRB1*04:xx - Serologic Equivalent
DRB1 *04:02 - Allele
DRB1 *04:01:01; DRB1 *04:01:02 - Silent Mutations
A*02:15N; DRB4*01:03:01:02N - Null Alleles (exon, intron)
A*24:02:01:02L
B*44:02:01:02 - Expression Modifiers
B*32:11Q

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AABB TECHNICAL MANUAL
 questionable), A (unknown but aberrant expression, perhaps null), and C (cytoplasmic expression only). The
last two have not been used to date.
Biologic Function
The essential function of the HLA system is self/nonself discrimination, which is accomplished by the
interaction of T lymphocytes with peptide antigens presented by HLA proteins. T lymphocytes interact with
peptide antigens only when the T-cell receptor (TCR) for antigen engages both an HLA molecule and the
antigenic peptide contained within the TCR’s peptide-binding groove. This limitation is referred to as “MHC
restriction.”13
In the thymus, T lymphocytes with TCRs that bind to a self HLA molecule are selected (positive selection),
with the exception of those with TCRs that also bind to a peptide derived from a self-antigen, in which case the
T lymphocytes are deleted (negative selection). Some self-reactive T cells escape negative selection. If not
functionally inactivated (for instance, by the mechanism of anergy), such self-reactive T cells may become
involved in an autoimmune process.
Role of Class I Molecules
Class I molecules are synthesized, and peptide antigens are inserted into the peptide-binding groove, in the
endoplasmic reticulum. Peptide antigens that fit into the Class I peptide-binding groove are typically eight or
nine amino acids in length and are derived from proteins made by the cell (endogenous proteins). Such
endogenous proteins—which may be normal self-proteins; altered self-proteins, such as those in cancer cells; or
viral proteins, such as those in virus-infected cells—are degraded in the cytosol by a large multifunctional
protease (LMP) and are transported to the endoplasmic reticulum by a transporter associated with antigen
processing (TAP). The LMP and TAP genes are both localized to the MHC.
Class I molecules are transported to the cell surface, where the molecules are available to interact with CD8-
positive T lymphocytes. If the TCR of a CD8 cell can bind the antigenic
peptide in the context of the specific Class I molecule displaying it, then TCR binding activates the
cytotoxic properties of the T cell, which attacks the cell, characteristically eliciting an inflammatory response.
The presentation of antigen by Class I molecules is especially important in host defense against viral pathogens
and malignant transformation. Tumor cells that do not express Class I antigens escape this immune surveillance.
Role of Class II Molecules
Like Class I molecules, Class II molecules are synthesized in the endoplasmic reticulum, but peptide
antigens are not inserted into the peptide-binding groove there. Instead, an invariant chain (Ii) is inserted as a
place holder. The Class II-invariant chain complex is transported to an endosome, where the invariant chain is
removed by a specialized Class II molecule called “DM.” The DM locus is also localized to the MHC. A Class
II antigenic peptide is then inserted into the peptide-binding groove.
Peptide antigens that fit into the Class II peptide-binding groove are typically 12 to 25 amino acids in length
and are derived from proteins that are taken up by the cell through endocytosis (of exogenous proteins).
Exogenous proteins, which may be normal self-proteins or proteins derived from pathogens, such as bacteria,
are degraded to peptides by enzymes in the endosomal pathway. Class II molecules are then transported to the
cell surface, where the molecules are available to interact with CD4-positive T lymphocytes, which secrete
immunostimulatory cytokines in response. That mechanism is especially important for the production of
antibodies.
IDENTIFICATION OF HLA ANTIGENS AND ALLELES
Methods for the identification of HLA antigens and alleles fall into two categories: 1) molecular (DNA
based) and 2) serologic (antibody based). Historically, cell-based assays were also used.
Detailed procedures for commonly used assays are provided in the ASHI Laboratory
485
Manual from the American Society for Histocompatibility and Immunogenetics.14 Depending on the
clinical situation, a particular HLA antigen/allele detection or typing method maybe preferable (see Table 19-2).
DNA-Based Assays
DNA-based typing has several advantages over serologic assays: 1) high sensitivity and specificity, 2) use of
small sample volumes, and 3) no need for cell-surface-antigen expression or cell viability. Although serologic
methods can distinguish among a limited number of HLA specificities, high-resolution DNA-based methods
have the capability to identify all known alleles.
Polymerase Chain Reaction Testing
 Polymerase chain reaction (PCR) technology allows amplification of large quantities of a particular target
segment of genomic DNA. Low- to intermediate-resolution typing detects the HLA serologic equivalents with
great accuracy (eg, it distinguishes DR15 from
DR16), whereas high-resolution typing distinguishes individual alleles (eg, DRB1*01:01:01 from
DRB1*01:02:01). Several PCR-based methods have been developed; three general approaches are described
below.
OLIGONUCLEOTIDE PROBES. This method for establishing HLA genotypes features arrays of
oligonucleotide probes that have been individually attached to a solid-phase matrix— for example, each probe
is attached to a different microbead or well of an enzyme-linked immunosorbent assay (ELISA) plate. DNA for
an entire locus is then amplified and the binding to different probes is evaluated. The microbead array assay is a
sequence-specific oligonucleotide probe method for HLA Class I and Class II low-to-high-resolution, DNA-
based, tissue typing. This method allows high throughput with a reduction in sample processing time.15
SEQUENCE-SPECIFIC PRIMERS. A second major technique uses sequence-specific primer (SSP) pairs
that target and amplify a particu
TABLE19-2. HLA Typing Methods and Appropriate Applications
Method Clinical Application Resolution
Solid-organ transplantation, evaluation of
Microlymphocytotoxicity platelet refractoriness, HLA typing (Glass 1 only) Serologic specificity
of platelet recipients and platelet donors
Serologic to allele level,
Solid-organ, related and unrelated HPC
SSP (PCR) higher resolution with large
transplantation
number of primers
Forward SSOP Solid-organ and HPC transplantation (can
Serologic to allele level
Hybridization accommodate high-volume testing)
Serologic, higher
Reverse SSOP Solid-organ, related and unrelated HPC
resolution with larger
Hybridization transplantation
number of probes
Unrelated HPC transplantation, resolution of
DNA Sequencing typing problems with other methods, Allele level
characterization of new alleles
SSP = sequence-specific primer; PCR = polymerase chain reaction; HPC = hematopoietic progenitor cell;
SSOP = sequencespecific oligonucleotide probe.
 486
AABB TECHNICAL MANUAL
lar DNA sequence.16 The sequence-specific method requires the performance of multiple PCR assays in
which each reaction is specific for a particular allele or group of alleles. The amplified alleles are directly
visualized after agarose gel electrophoresis. Because SSPs have such specific targets, the amplified material
indicates the presence of the allele or alleles that have that sequence. The pattern of positive and negative PCR
amplifications is examined to determine the actual HLA allele(s) present. Primer pair sets are commercially
available that can determine a complete HLA-A, -B, -C, -DR, -DQA1, -DQB1, and -DPB1 allele-level
phenotype.
SEQUENCE-BASED TYPING. High-resolution nucleic acid sequencing of HLA alleles generates allele-
level sequences. Sequence-based typing (SBT) can be used to characterize new allele(s). Although SBT is
considered the “gold standard” for HLA typing, ambiguities often occur when two different base pairs at the
same position can result in two different possible combinations of alleles. These ambiguities occur because SBT
evaluates both maternal and paternal HLA genes (haplotypes) simultaneously, and which haplotype to assign
each base pair to is not always certain. New techniques for high-resolution SBT allow separation of HLA
haplotypes before sequencing, thereby avoiding such ambiguities.
Serologic (Lymphocytotoxicity) Assays
The microlymphocytotoxicity test can be used to detect HLA-A, -B, -C, -DR, and -DQ antigens.
Lymphocytes are used for testing because they are readily obtained from anticoagulated peripheral blood and
are a more reliable target than granulocytes. Lymphocytes obtained from lymph nodes or spleen may also be
used. HLA-typing sera are obtained primarily from multiparous women. Some mouse antihuman HLA
monoclonal antisera are also available.
HLA sera of known specificities are placed in different wells of a microdroplet test plate. A suspension of
lymphocytes is added to each well. Rabbit complement is then added; if suf
ficient antibody has bound to the lymphocyte membranes, the complement cascade is activated through the
membrane attack complex, leading to lymphocytotoxicity. Damage to the cell membrane can be detected by the
addition of dye. Cells that have no attached antibody, activated complement, or damage to the membrane keep
the vital dyes from penetrating; however, cells with damaged membranes allow the dye to enter. The cells are
examined for dye exclusion or uptake under phase contrast microscopy. If a fluorescent microscope is available,
fluorescent vital dyes can also be used.
Because HLA-Class II antigens (DR, DQ, and DP) are expressed on B cells and not on resting T cells,
typing for these antigens usually requires manipulation of the initial lymphocyte preparation to yield an
enriched B-cell population before testing.
The interpretation of serologic reactions requires skill and experience. The extreme polymorphic nature of
the HLA system; variation in antigen frequencies among different racial groups; reliance on biologic antisera
and living target cells; and complexities introduced by splits, CREGs, and “public” antigens all contribute to
difficulties in accurate serologic HLA typing. Given these problems, most US laboratories now rely primarily
on DNA-based methods for HLA typing. However, although the more common “null alleles,” such as
DRB4*01: 03:01:02N and C*04:09N, can now be identified by routine molecular typing methods, rare null
alleles may not be as easily identified. It is important to stay current on the ever-changing array of expressed
and nonexpressed HLA polymorphisms. A very useful resource is the International Immunogenetics Project’s
IMGT/ HLA Database.2,9
Cellular Assays
 Historically, the MLC [also called “mixed leukocyte reaction” (MLR)] and primed lymphocyte typing (PLT)
assays were used to detect genetic differences in the Class II region. In the MLC, mononuclear cells from
different individuals are cultured together; the ability of one population (the responder) to recognize the
487
HLA-D (combined DR, DQ, and DP) antigens/ alleles of the other (the target) as foreign can be detected by
measuring the proliferation of the responders. In PLT, reagent cells previously stimulated by specific Class II
mismatched types allow the identification of those types by their accelerated proliferative responses to
stimulator cells sharing the original mismatches.
These classical cellular assays have been largely replaced by molecular typing methods for HLA antigens.
However, these assays are still used in some laboratories to monitor immune function, assess relative functional
compatibility, or select a partially mismatched unrelated donor for HPC transplantation.
CROSSMATCHING AND DETECTION OF HLA ANTIBODIES
Serologic Assays
The same microlymphocytotoxicity testing used for serologic HLA typing can be used to test serum
specimens for antibodies against selected target cells. This type of testing is referred to as “lymphocyte
crossmatching.” Crossmatching consists of incubating serum from a potential recipient with lymphocytes
(unfractioned or separated into T and B lymphocytes) from prospective donors. A variation of the
microlymphocytotoxicity test uses an antiglobulin reagent, which increases assay sensitivity. Flow cytometry is
yet another crossmatch method with even greater sensitivity than the antiglobulin-enhanced crossmatch.
Testing the patient’s serum against a panel of 30 to 60 (or more) different target cells was historically used
to assess the extent of HLA alloimmunization. A positive result was target cell death. The percentage of panel
cells that died after reacting with the cytotoxic antibodies in patient serum was referred to as the “panel-reactive
antibody” (PRA) level in that patient. Determination of cytotoxic PRA was (and may still be) useful for
following patients awaiting deceased-donor solid-organ transplantation, conducting the work-up of pa
tients for platelet refractoriness, and investigating FNHTR or TRALI cases. The PRA “HLA antibody
screen” not only detects the presence of cytotoxic HLA antibodies but can often also identify the specificity of
those antibodies.
Although they have long been the gold standard for antibody identification, complement-dependent
cytotoxic assays are not optimal tests. Their sensitivity and specificity are marginal, but more importantly, most
cytotoxic assays cannot identify all of the possible HLA antibody specificities in highly sensitized patients
whose cells are reactive with 80% or more of test panel cells or in patients with Class II antibodies.
Solid-Phase Assays
The current approach to identify HLA antibodies (especially for highly sensitized solid-organ transplant
candidates) relies on the use of beads or microparticles (ie, solid-phase methodology) coated either with clusters
of HLA Class I or Class II antigens (ie, an HLA phenotype) or with recombinantly expressed, individually
purified HLA antigens (single antigen beads).17 Antibody binding is detected by staining with a fluorescently
labeled antihuman globulin (AHG). Flow cytometry and microarray methods are more sensitive than
lymphocytotoxicity or ELISA testing and focus on the detection of IgG antibodies. The use of single-antigen
bead assays are of particular importance for highly sensitized patients where multiple HLA antibody
specificities cannot be reliably distinguished and identified with either cell-based cytotoxic assays or solid-
phase assays using clusters of HLA molecules.
The clinical significance of the low-level antibodies that are detectable by the more sensitive solid-phase
assays and that may not cause a positive cytotoxicity or flow-cytometric crossmatch is controversial. Newer
adaptations of the solid-phase technology can determine whether these antibodies do or do not fix complement.
However, use of this method is also controversial because the significance of noncomplement-fixing antibodies
is also unknown.
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AABB TECHNICAL MANUAL
THE HLA SYSTEM AND TRANSFUSION
HLA system antigens and antibodies play important roles in a number of transfusion-related events,
including platelet refractoriness, FNHTRs, TRALI, and TA-GVHD. HLA antigens are highly immunogenic. In
response to pregnancy, transfusion, or transplantation, immunologically competent individuals are more likely
to form antibodies to HLA antigens than to any other antigens.
Platelet Refractoriness
 The incidence of HLA alloimmunization and platelet refractoriness among patients receiving repeated
transfusions of cellular blood components ranges from 20% to 71%.18 The refractory state exists when a
transfusion of suitably preserved platelets fails to increase the recipient’s platelet count. Platelet refractoriness
may be caused by clinical factors, such as sepsis, high fever, disseminated intravascular coagulopathy,
medications, hypersplenism, complement-mediated destruction, or a combination of these factors; alternatively,
it may have an immune basis.
Antibody Development
Antibodies against HLA Class I antigens are a common cause of immune-mediated platelet refractoriness,
but antibodies to platelet-specific or ABH antigens may also be involved. HLA alloimmunization can follow
pregnancy, transfusion, or organ transplantation because the foreign antigens are the donor MHC antigens
themselves. A common example is the development of HLA antibodies directed against Class I and Class II
antigens that occurs with platelet transfusion, even though platelets express only Class I antigens. It is likely
that the presence of donor leukocytes (bearing Class I and II antigens) elicits alloimmunization. The likelihood
of HLA immunization can be lessened (but not eliminated) by transfusion of leukocyte-reduced blood
components.
The threshold level of leukocytes required to provoke a primary HLA alloimmune re
sponse is unclear and probably varies among different recipients. Some studies have suggested that 5 x 106
leukocytes per transfusion may represent an immunizing dose. In patients who have been sensitized by prior
transplantation, pregnancy, or transfusion, exposure to even lower numbers of allogeneic cells is likely to
provoke an anamnestic antibody response.
Identifying Compatible Donors
The HLA antibody response of transfused individuals may be directed against individual specificities or
public alloantigens. Precise characterization may be difficult and is best accomplished using a single-antigen,
solidphase assay as described in the “Solid-Phase Assays” section above. Platelet-refractory patients with
broadly reactive antibodies may be difficult to support with platelet transfusions. HLA-matched platelets,
obtained by plateletpheresis, benefit some, but not all, of those refractory patients. Donors who are
phenotypically matched with the immunized recipient for four Class I antigens (two alleles each at the HLA-A
and HLA-B loci) are difficult to find, and strategies to obtain HLA-compatible platelets vary. Although
selection of partially mismatched donors (based on serologic CREGs) has been emphasized, such donations
may fail to provide an adequate transfusion response in vivo.
An alternative approach to the selection of donors is based on matching for immunogenic epitopes as
described by the “HLA Matchmaker” program developed at the University of Pittsburgh. HLA Matchmaker is
used to consider epitopes on all the patient’s HLA Class I (and if indicated, Class II) molecules regardless of
which allele encodes them. HLA Matchmaker is an excellent tool to predict compatibility between a specific
donor-recipient pair, but, unfortunately, the tool does not generate accurate predictions 100% of the time. Use of
single-antigen beads or microarrays to identify HLA antibody specificities can also help improve the selection
of donors with acceptable mismatched antigens.19
489
Approaches such as HLA Matchmaker can be referred to as “pull” approaches, wherein donors are selected
(or pulled into) the process. In contrast, antibody specificity identification strategies can be considered “push”
approaches that exclude (push away) inappropriate donors. Either approach has a similar net effect:
identification of donors who have the greatest likelihood of optimizing posttransfusion platelet counts.
As an alternative approach, HLA-alloimmunized patients often respond to crossmatch-compatible platelets
selected by using patient serum and samples of apheresis platelets in a platelet antibody assay. Crossmatching
techniques may assess compatibility for both HLA and platelet-specific antibodies.20 Histocompatible platelet
components are discussed further in Chapter 18.
Febrile Nonhemolytic Transfusion Reactions
HLA, granulocyte, and platelet-specific antibodies have been implicated in the pathogenesis of FNHTRs.
The recipient’s antibodies, reacting with transfused antigens, elicit the release of cytokines (eg, interleukin-1)
that are capable of causing fever. Serologic investigation, if undertaken, may require multiple techniques and
target cells from a number of different donors (see Chapter 27).
TRALI
In TRALI (a potentially fatal transfusion reaction that is recognized with increasing frequency), acute
noncardiogenic pulmonary edema develops in response to transfusion (see Chapter 27). The pathogenesis of
TRALI appears to reflect the presence of HLA or neutrophil antibodies in donor blood. Studies have shown that
up to one-third of blood components can contain detectable amounts of HLA antibodies.21 If present, such
antibodies can be reactive with and fix complement to the recipient’s granulocytes, leading to severe capillary
leakage and pulmonary edema. Rarely, the recipient’s HLA antibodies are reactive with transfused leukocytes
from the donor.
Cases of TRALI have been reported that appear to be caused by donor antibodies against Class I or Class II
antigens in recipients. Because Class II antigens are not expressed on resting neutrophils, an alternate
explanation for activation of neutrophils is required in these instances. One hypothesis is that Class II antigens
on the recipient’s pulmonary macrophages are targeted by the complement-activating antibodies. The
subsequent release of cytokines and chemokines results in the recruitment and activation of neutrophils in the
lungs.22 Yet another explanation is that activated neutrophils, which can express HLA Class II antigens in
response to a patient’s inflammatory state, could be further activated with the binding of donor anti HLA Class
II antibodies, resulting in TRALI.
Chimerism and TA-GVHD
“Chimerism” refers to the presence of two cell populations, such as transfused or transplanted donor cells in
a recipient, in an individual. Persistent chimerism after blood transfusion may lead to the development of TA-
GVHD in the recipient. The development of TA-GVHD depends on the following factors: 1) the degree to
which the recipient is immunocompromised, 2) the number and viability of lymphocytes in the transfused
component, and 3) the degree of HLA similarity between the donor and recipient. The development of TA-
GVHD with the use of fresh blood components from blood relatives has highlighted the pathogenic role of the
HLA system.
Figure 19-5 illustrates the conditions for increased risk of TA-GVHD. The parents have one HLA haplotype
in common. Each child, therefore, has one chance in four of inheriting the same haplotype from each parent,
and child 1 is homozygous for the shared parental HLA haplotype. Transfusion of blood from child 1 to an
unrelated recipient with different haplotypes would have no untoward consequences. If, however, child 1 were a
directed donor for a relative who was heterozygous for that haplotype (eg, one of the parents or child 3), the
recipient’s body would fail to recognize the antigens on the transfused lymphocytes as
490
AABB TECHNICAL MANUAL
Father
A1 A3
B8 B7
DR17 DR11
Child 1
A1 A1
B8 B8
DR17 DR17
Mother

FIGURE 19-5. HLA haplotypes in a family at risk for transfusion-associated graft-vs-host disease (GVHD).
In contrast to the family shown in Fig 19-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17.
Child 1 is homozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child 1 are
capable of producing posttransfusion GVHD if they are transfused to either parent or to child 3.
foreign and would not eliminate them. The donor cells would recognize the recipient’s other haplotype as
foreign and would become activated, proliferate, and attack the host.
To avoid this situation, it is recommended that all cellular components from blood relatives be irradiated
before transfusion. Other specially chosen donor units, including HLAmatched platelets, may also present an
increased risk of TA-GVHD. Rarely, TA-GVHD has occurred after the transfusion of blood from an unrelated
donor, usually within populations with relatively limited genetic diversity, such as in Japan.
Chimerism is proposed to be responsible for the maintenance of tolerance in some organ transplant
recipients as well as for the maintenance of HLA sensitization.23,24 It has
been postulated that scleroderma is a form of GVHD resulting from chimeric cells derived from fetal cells
transferred across the placenta during pregnancy.25 Furthermore, the persistence of donor lymphocytes
originally present in and transplanted with a solid-organ allograft has been documented to cause fatal GVHD in
recipients of these organs.26
Hemolytic Transfusion Reactions
HLA incompatibility has rarely been implicated in shortened red cell survival in patients with antibodies to
HLA antigens, such as Bga (B7), Bgb (B17-B57 or B58), and Bgc (A28-A68 or A69). These antigens are
expressed, although weakly, on red cells. Such incompatibility may not be detected by conventional
pretransfusion testing.
491
HLA TESTING AND TRANSPLANTATION
HLA testing is an integral part of solid-organ and HPC transplantation. The extent of testing differs
depending on the type of transplantation (see Chapters 29 and 30).
Hematopoietic Progenitor Cell Transplants
It has long been recognized that disparity within the HLA system is an important barrier to successful HPC
transplantation.27 HLA similarity and compatibility between the donor and the recipient are required for
engraftment and to help prevent GVHD. However, some degree of rejection or GVHD is a common problem for
recipients of allogeneic HPCs, despite immunosuppressive conditioning.
Candidate donors and recipients are typed for their HLA-A, -B, -C, -DR, and -DQ alleles and, in some
cases, for their HLA-DP alleles. The goal of HLA typing is to match the alleles of the prospective donor and
recipient at the HLA-A, -B, -C, and -DRB1 loci.28 Some transplant programs also attempt to match donors and
recipients for HLA-DQ alleles, HLA-DP alleles, or both.
Although HLA-identical sibling donors remain the best choice for HPC transplantation, there is increasing
use of unrelated donors identified by searching the files of more than 10 million HPC donors listed in the
National Marrow Donor Program’s registry of volunteer donors or other international registries. The use of
umbilical cord HPCs and HPC grafts from mismatched donors that have undergone T-cell depletion may allow
an increased number of donor-recipient mismatches.29,30
Kidney Transplants
ABO compatibility is the most important factor in determining the immediate outcomes of kidney
transplants. Because ABH antigens are expressed in varying amounts on all of the body’s cells, transplanted
ABO-incompatible tissue comes into continuous contact with the recipient’s ABO antibodies. Of particular im
portance is the expression of ABH antigens on vascular endothelial cells because the vascular supply in the
transplant is a common site for rejection. Currently, the use of non-A] A blood group organs for group B and
group 0 recipients with low anti-A blood group titers has become acceptable.31,32 In fact, several
transplantation programs have used ABO-incompatible donors with higher anti-A titers following protocols that
include various combinations of rituximab, splenectomy, plasmapheresis with intravenous Ig, and other
treatments to remove preexisting antibodies and promote accommodation of the transplanted organ.
Both recipients and donors are routinely typed for ABO and HLA-A, -B, and -DR antigens. HLA-C and -
DQ typing is usually also performed. Before surgery, a crossmatch between recipient serum and donor
lymphocytes is required. The ASHI Standards for Accredited Laboratories requires that the crossmatch be
performed using a method that is more sensitive than routine microlymphocytotoxicity testing, such as
prolonged incubation, washing, augmentation with AHG reagents, or flow cytometry.33 Flow cytometry is the
most sensitive method and has been credited with predicting early acute rejection and delayed graft function,
both of which are strong predictors of chronic rejection (if results are positive) and long-term allograft survival
(if results are negative).34
In a study of patients undergoing deceased-donor kidney retransplantation, the 7year graft survival rate
using a negative T-cell flow crossmatch to select the donor kidney was comparable to that of patients
undergoing primary deceased-donor transplantation (68% vs 72%) and was significantly better than that of
regraft patients for whom only the antiglobulin lymphocytotoxicity crossmatch was used (45%).35
Because HLA antibody responses are dynamic, the serum used for the crossmatch is often obtained within
48 hours of surgery for sensitized potential recipients and is retained in the frozen state for any required
subsequent testing. An incompatible crossmatch with unfractionated or T lymphocytes is typically a
contraindication to kidney transplantation. A
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AABB TECHNICAL MANUAL
positive B-cell crossmatch is significant when caused by donor-specific HLA Class I or Class II antibodies.
Serum from a patient awaiting deceaseddonor kidney transplant surgery is tested at regular intervals for the
degree of alloimmunization by determining the percent PRA as well as the specificities of the detected
antibodies. If an antibody with a defined HLA specificity is identified in a recipient, a common practice is to
avoid donors who express the corresponding HLA antigen (s). Such antigens are deemed “unacceptable.” A
more recent approach is the use of solid-phase assays to identify HLA antibodies and unacceptable antigens and
then calculate the patient’s PRA (cPRA) using a database of more than 12,000 HLA-typed donors.36 Frozen
serum samples used for periodic PRA testing are often stored so that “historic” samples with the highest PRA
can be used in addition to the preoperative sample for pretransplantation crossmatching.
Prospective crossmatching is often not performed for recipients who are conclusively devoid of HLA
antibodies (ie, cPRA = 0%). Prompt transplantation with reduced coldischemia time for the renal allograft may
provide greater benefit to the patient than prospective crossmatching, provided that 1) a very sensitive method
for antibody detection, such as flow cytometry or microarrays, has been used, and 2) it is certain that the patient
has had no additional sensitizing event (ie, immunizations or transfusions in the 2 weeks before or at any time
after that serum was screened).37
The approach to kidney transplants with living donors is different. In the past, when several prospective
living donors were being considered, MLC testing of recipients and donors was sometimes performed, but this
is rarely (if ever) the case today. HLA matching of recipients with kidney donors (both living and deceased)
contributes to long-term allograft survival by decreasing the likelihood of chronic rejection. According to the
Scientific Registry for Transplant Recipients, recent 1-year graft survival rates from living and deceased renal
donors were 96.5% and 91.7%, respectively, and the half-lives of living-donor and
deceased-donor renal allografts were 21.6 years and 13.8 years, respectively.38
The significantly better graft survival rate for recipients of living- vs deceased-donor renal allografts, even
when donors and recipients are completely unrelated, coupled with inadequate numbers of deceased-organ
donors has led to a relatively new practice, kidney paired donation (KPD).39 Recently, KPDs have been
facilitated through local and national registries, which permit patients with ABO- or HLA-incompatible
potential living donors to exchange these donors for the donors of other patients in the same situation. As a
simple example, a blood group A transplant candidate with an incompatible blood group B potential living
kidney donor could exchange that donor for the incompatible blood group A living kidney donor of a blood
group B transplant candidate. Patients with HLA-incompatible potential donors have similar possibilities for
donor exchange, and multiple “pairs” can be involved in one continuous exchange (chain) process.
The introduction of altruistic donors (ie, individuals who choose to donate a kidney without having a
specific intended recipient) can significantly expand KPD options. Briefly, the altruistic donor donates a kidney
to a patient with an incompatible potential living donor who then donates to a different recipient with an
incompatible donor, starting a “chain” with the possibility of a large number of livingdonor transplants. One
chain of 10 transplants was recently reported.40 Currently, there is a movement to establish a national KPD
program.
Other Solid-Organ Transplants
For liver, heart, lung, and heart/lung transplants, ABO compatibility remains the primary immunologic
concern for donor selection, and determining pretransplant ABO compatibility between the donor and recipient
is required. However, it has been shown that young pediatric heart or liver transplant recipients, who have low
levels of ABO isoagglutinins, have successful outcomes with ABO-incompatible hearts or livers.41,42
Although it is not a
493
requirement, HLA typing of potential recipients of the above organs is recommended. Furthermore, a
crossmatch should be available before transplantation when the recipient has demonstrated presensitization,
except for emergency situations. Although a degree of HLA compatibility correlates with graft survival after
heart, lung, small-intestine, and liver transplantations, prospective HLA matching is generally not performed for
these procedures.43 Pancreas transplantation generally follows the same guidelines as kidney transplantation.
Relationship and Other Forensic Testing
HLA typing (particularly DNA-based HLA typing) has been useful in forensic testing. Although rarely used
now for relationship testing, DNA-based HLA typing alone can exclude more than 90% of falsely accused
males. Haplotype frequencies, rather than gene frequencies, are used in such calculations because linkage
disequilibrium is very common in the HLA system. It is important, however, to consider the racial differences
that exist in HLA haplotype frequencies in the calculations used; the possibility of recombination events should
also be considered.
More commonly used DNA-based assays for relationship testing and forensic detection of polymorphisms
between individuals are employed to measure variations in the number of short tandem repeats, which are used
to assess other polymorphic, non-HLA genetic regions, and single nucleotide polymorphisms (SNPs). DNA-
based assays, including HLA typing, allow identification of individuals on the basis of extremely small samples
of fluid or tissue, such as hair, epithelial cells, or semen.
OTHER CLINICALLY SIGNIFICANT ASPECTS OF HLA
For some conditions, especially those believed to have an autoimmune etiology, an association exists
between HLA phenotype and the occurrence of, or resistance to, clinical disease (see Table 19-3).43'46 HLA-
associated disease
TABLE 19-3. HLA-Associated Diseases
Disease HLA RR43 46
Celiac disease DQ2 >250
Ankylosing spondylitis B27 >150
Narcolepsy DQ6 >38
Subacute thyroiditis B35 14
Type 1 diabetes DQ8 14
Multiple sclerosis DR15, DQ6 12
Rheumatoid arthritis DR4 9
Juvenile rheumatoid DR8 8
arthritis
Grave disease DR17 4
RR = Relative Risk
susceptibilities have several features in common. The susceptibilities are known or suspected to be
inherited, display a clinical course with acute exacerbations and remissions, and usually have characteristics of
autoimmune disorders. Furthermore, their exact cause is often unknown.
Evidence has been accumulating that implicates the HLA molecules themselves, rather than linked genes, in
most cases of disease susceptibility. One mechanism that could lead to the association of HLA phenotype and
disease is the presence of a Class I or Class II heterodimer encoded by a specific allele that preferentially
presents autoantigens to the TCR. The ancestral haplotype HLA-A1, B8, DR17 (DRB1 *03:01), DQ2,
discussed in the “Linkage Disequilibrium” section above, is associated with susceptibility to Type I diabetes,
systemic lupus erythematosus, celiac disease, common variable immunodeficiency, IgA deficiency, and
myasthenia gravis.47 This haplotype is also associated with an accelerated course of HIV infection that is likely
caused by the presence of multiple genes.
However, HLA typing has only limited value in assessing the risk of most diseases because the association
is incomplete. The association of HLA-B27 and ankylosing spondylitis

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 AABB TECHNICAL MANUAL
in patients of European ancestry is instructive. The test is highly sensitive; more than 90% of patients with
ankylosing spondylitis possess the HLA-B27 antigen. Conversely, the test’s specificity is low; only 20% of
individuals with the B27 antigen develop ankylosing spondylitis. A second condition, narcolepsy, is strongly
associated with the HLA allele DQB1 *06:02.48 As with HLA-B27 and ankylosing spondylitis, more than 90%
of individuals with narcolepsy are positive for HLA-DQB1*06:02, but only a minority of individuals with that
HLA allele develop the disease. For some autoimmune diseases, the specific peptide that might trigger the
autoimmune response has been at least tentatively identified: a gluten peptide, gliadin, for celiac disease; cyclic
citrullinated peptides for rheumatoid arthritis; and a peptide from glutamic acid decarboxylase for type I
diabetes.49'51 Resistance to cerebral malaria seems to result from a strong cytotoxic T-cell response to
particular malarial peptides that are restricted by (ie, fit into the peptide-binding grooves of) two specific HLA
molecules.52
A similar peptide-binding specificity is important to consider in the development of vaccines. For example,
a vaccine to enhance immune responses to melanoma using a melanoma-specific peptide that binds only to the
cells of individuals with the HLA type HLAA*0201 was selected for development because A*02:01 is the most
common allele in virtually all populations.53
HLA typing is also important in pharmacogenomic applications. For example, the presence of certain HLA
alleles confers increased risk for several hypersensitivity reac
tions to certain drugs: HLA-B*57:01 confers sensitivity to abacavir, HLA-B* 15:02 to carbamazepine, and
HLA-B*58:01 to allopurinol.5455
The degree of association between a given HLA type and a disease is often described in terms of relative
risk (RR), which is a measure of how much more frequently a disease occurs in individuals with a specific HLA
type than in individuals not having that HLA type. Calculation of RR is usually based on the cross-product ratio
of a 2 x 2 contingency table. However, because the HLA system is so polymorphic, there is an increased
possibility of finding an association between an HLA antigen and a disease by chance alone. Therefore,
calculating RRs for HLA disease associations is more complex and is typically accomplished by use of
Haldane’s modification of Woolf’s formula.56,57 The RR values for some diseases associated with HLA types
are shown in Table 19-3.
SUMMARY
In conclusion, the HLA system is a complex and highly polymorphic set of genes that are collectively
involved in all aspects of the immune response. The recent development of molecular tools to explore this
genetic oasis is providing additional information, such as the elucidation of unrecognized polymorphisms
within the HLA complex (ie, SNPs). In the future, the translation of this basic information will undoubtedly
lead to new clinical applications in transplantation, autoimmune diseases, vaccine development,
pharmacogenetics, and infectious diseases.
KEY POINTS
1. Genes encoded by the major histocompatibility complex (or HLA complex in humans) are critical
components of the immune system and play a major role in distinguishing self from nonself.
2. HLA genes are located within multiple loci on chromosome 6. Each locus is extremely polymorphic.
3. HLA genes encode multiple Class I (eg, HLA-A, -B, and -C) and Class II (eg, HLA-DR, -DQ, and -DP)
cell-surface proteins.
4. Class I proteins are expressed ubiquitously, but Class II proteins have restricted tissue distribution.
 495
5. Everyone inherits a set of HLA genes from his or her mother and father, referred to as a “maternal
haplotype” and “paternal haplotype,” respectively.
6. Together, the maternal and paternal haplotypes are referred to as a “genotype." The cell-surface
expression of proteins encoded by these HLA genes is referred to as a “phenotype.”
7. Class I and Class II HLA proteins are strongly immunogenic and can induce an immune response, eg,
formation of HLA antibodies.
8. Donor-directed HLA antibodies are associated with graft dysfunction and/or loss.
9. Solid-phase assays (eg, flow cytometry and Luminex) have become the gold standard for detecting and
identifying HLA antibodies.
10. Identification of donor-directed HLA antibodies can be used to perform a virtual (in-silico) crossmatch.
REFERENCES
1. Holdsworth R, Hurley CK, Marsh SG, et al. The HLA dictionary 2008: A summary of HLA-A, -B, -C, -
DRB1/3/4/5, and -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR, and -DQ
antigens. Tissue Antigens 2009;73:95-170.
2. HLA-DRB protein sequence alignments. Hampstead, United Kingdom: Anthony Nolan Research
Institute, 2013. [Available at http:// hla.alleles.org/data/txt/drb_prot.txt (accessed November 22,2013).]
3. Janeway CA, Travers R Walport M, et al. The immune system in health and disease. 5th ed. New York:
Garland Science, 2001.
4. Braud VM, Allan DSJ, McMichael AJ. Functions of nonclassical MHC and non-MHC-encoded class I
molecules. Curr Opin Immunol 1999; 11:100-8.
5. Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with
hereditary haemochromatosis. Nat Genet 1996;13:399-408.
6. Bodmer JG, Parham P Albert ED, Marsh SG. Putting a hold on “HLA-H.” Nat Genet 1997; 15:234-5.
 7. Termijtelen A, Meera Khan P Shaw S, van Rood JJ. Mapping SB in relation to HLA and GLOl using
cells from first-cousin marriage offspring. Immunogenetics 1983;18:503-12.
8. Buchler T, Gallardo D, Rodriguez-Luaces M, etal. Frequency of HLA-DPB1 disparities detected by
reference strand-mediated conformation analysis in HLA-A, -B, and -DRB1 matched siblings. Hum Immunol
2002;63: 13942.
9. IMGT/HLA database. [Available at http:// www.ebi.ac.uk/ipd/imgt/hla/stats.html (accessed March
31,2014).]
10. McDonald JC, Adamashvili I. Soluble HLA: A review of the literature. Hum Immunol 1998; 59:387-
403.
11. Ghio M, Contini B Mazzei C, et al. Soluble HLA class 1, HLA class II, and Fas ligand in blood
components: A possible key to explain the immunomodulatory effects of allogeneic blood transfusion. Blood
1999;93:1770-7.
12. Voorter CE, van der Vlies S, Kik M, van den Berg-Loonen EM. Unexpected Bw4 and Bw6 reactivity
patterns in new alleles. Tissue Antigens 2000;56:363-70.
13. Zinkernagel RM, Doherty PC. The discovery of MHC restriction. Immunol Today 1997; 18: 1417.
14. Phelan DL, Mickelson EM, Noreen HS, et al, eds. ASHI laboratory manual. 4th ed. Mt. Laurel, NJ:
American Society for Histocompatibility and Immunogenetics, 2001.
15. Petrik J. Microarray technology: The future of blood testing? Vox Sang 2001;80:1-11.
16. Welsh K, Bunce M. Molecular typing for the MHC with PCR-SSP. Rev Immunogenet 1999; 1:157-76.
17. Bray RA, Gebel HM. Strategies for human leukocyte antigen antibody detection. Curr Opin Organ
Transplant 2009; 14:392-7.
18. Triulzi DJ, Dzik WH. Leukocyte-reduced blood components: Laboratory and clinical aspects. In: Simon
TL, Snyder EL, Solheim BG, et al, eds. Rossi’s principles of transfusion medicine. 4th ed. Bethesda, MD:
AABB Press, 2009:22846.
19. Duquesnoy R. Structural epitope matching for HLA-alloimmunized thrombocytopenic patients: A new
strategy to provide more effective platelet transfusion support. Transfusion 2008; 48:221-7.

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20. Friedberg RC. Independent roles for platelet crossmatching and HLA in the selection of platelets for
alloimmunized patients. Transfusion 1994;34:215-20.
21. Bray RA, Harris, SB, Josephson CD, et al. Unappreciated risk factors for transplant patients: HLA
antibodies in blood components. Hum Immunol 2004;65:240-4.
22. Kopko PM, Popovsky MA, MacKenzie MR, et al. HLA class II antibodies in transfusion-related acute
lung injury. Transfusion 2001 ;41: 1244-8.
23. Starzl TE, Demetris AJ, Murase N, et al. Chimerism after organ transplantation. Curr Opin Nephrol
Hypertens 1997;6:292-8.
 24. Sivasai KSR, Jendrisak M, Duffy BF, et al. Chimerism in peripheral blood of sensitized patients waiting
for renal transplantation. Transplantation 2000;69:538-44.
25. Artlett CM, Smith JB, Jimenez SA. Identification of fetal DNA and cells in skin lesions from women
with system sclerosis. N Engl J Med 1998;338:1186-91.
26. Pollack MS, Speeg KM Callander NS, et al. Severe, late-onset graft vs. host disease in a liver transplant
recipient documented by chimerism analysis. Hum Immunol 2005;66:28-31.
27. Thomas ED. Bone marrow transplantation: A review. Semin Hematol 1999;36:95-103.
28. Mickelson EM, Petersdorf E, Anasetti PM, et al. HLA matching in hematopoietic cell transplantation.
Hum Immunol 2000;61:92-100.
29. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for
transplantation into unrelated recipients. N Engl J Med 1996;335:157-66.
30. Aversa F, Tabilio A, Velardi A. Treatment of high-risk acute leukemia with T cell depleted stem cells
from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998;339:1186-93.
31. Bryan CF, Winklhofer FT, Murillo D, et al. Improving access to kidney transplantation without
decreasing graft survival: Long-term outcomes of blood group A2/A2B deceased donor kidneys in B recipients.
Transplantation 2005; 80:75-80.
32. Tyden G, Donauer J, Wadstrom J, et al. Implementation of a protocol for ABO-incompatible kidney
transplantation—a three-center experience with 60 consecutive transplantations. Transplantation 2007;83:1153-
5.
33. Standards for accredited laboratories. Mt Laurel, NJ: American Society for Histocompatibility and
Immunogenetics, 2009.
34. Bryan CF, Baier KA, Nelson PW, et al. Longterm graft survival is improved in cadaveric renal
retransplantation by flow cytometric crossmatching. Transplantation 2000;66:1827-32.
35. Taylor Cl, Smith SI, Morgan CH, et al. Selective omission of the donor crossmatch before renal
transplantation: Efficacy, safety, and effects of cold storage time. Transplantation 2000;69: 719-23.
36. Cecka JM. Calculated PRA (CPRA): The new measure of sensitization for transplant candidates. Am J
Transplant 2010;10:26-9.
37. Gebel HM, Bray RA. Sensitization and sensitivity: Defining the unsensitized patient. Transplantation
2000;69:1370-4.
38. Scientific Registry of Transplant Recipients. Minneapolis, MN: SRTR, 2013. [Available at
www.ustransplant.org (accessed November 22,2013).]
39. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival rates of kidney transplants from
spousal and living unrelated donors. N Engl J Med 1995;333:333-6.
40. Rees MA, Kopke JE, Pelletier RP, et al. A nonsimultaneous, extended, altruistic-donor chain. N Engl J
Med 2009;360:1096-101.
41. Daebritz SH, Schmoeckel M, Mair H, et al. Blood type incompatible cardiac transplantation in young
infants. Eur J Cardiothorac Surg 2007;31:339-43.
42. Heffron T, Welch D, Pillen T, et al. Successful ABO-incompatible pediatric liver transplantation
utilizing standard immunosuppression with selective postoperative plasmapheresis. Liver Transpl 2006;12:972-
8.
43. Ketheesan N, Tay GK, Witt CS, et al. The significance of HLA matching in cardiac transplantation. J
Heart Lung Transplant 1999; 18:226,30.
44. Thorsby E. Invited anniversary review: HLA associated diseases. Hum Immunol 1997;53:111.
45. Pile KS. HLA and disease associations. Pathology 1999;31:202-12.
46. Howell WM, Jones DB. The role of human leukocyte antigen genes in the development of malignant
disease. J Clin Pathol Mol Pathol 1995;48:302-6.
47. Price P, Witt C, Allcock R, et al. The genetic basis for the association of the 8.1 ancestral haplotype (Al,
B8, DR3) with multiple immuno
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497
pathological diseases. Immunol Rev 1999;167: 257-74.
48. Pelin Z, Guilleminault C, Risch N, et al. HLADQB1*0602 homozygosity increases relative risk for
narcolepsy but not for disease severity in two ethnic groups. US Modafinil in Narcolepsy Multicenter Study
Group. Tissue Antigens 1998;51:96-100.
 49. Cinova J, Palova-Jelinkova L, Smythies LE, et al. Gliadin peptides activate blood monocytes from
patients with celiac disease. J Clin Immunol 2007;27:201-9.
50. Van Gaalen FA, van Aken J, Huizinga TW, et al. Association between HLA class II genes and
autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis
Rheum 2004;50:2113-21.
51. Mayr A, Schlosser M, Grober N, et al. GAD autoantibody affinity and epitope specificity identify
distinct immunization profiles in children at risk for type 1 diabetes. Diabetes 2007; 56:1527-33.
52. Hill AV The immunogenetics of resistance to malaria. Proc Assoc Am Physicians 1999;111: 272-7.
53. Slingluff CL Jr, Yamshchikov G, Neese P et al. Phase I trial of a melanoma vaccine with gpl00(280-288)
peptide and tetanus helper peptide in adjuvant: Immunologic and clinical outcomes. Clin Cancer Res
2001;7:3012-24.
54. Hughes DA, Vilar FJ, Ward CC, et al. Cost-effectiveness analysis of HLA B*5701 genotyping in
preventing abacavir hypersensitivity. Pharmacogenetics 2004;14:335-42.
55. Chung W-H, Hung S-I, Chen YT. Human leukocyte antigens and drug hypersensitivity. Curr Opin
Allergy Clin Immunol 2007;7:31723.
56. Haldane JBS. The estimation and significance of the logarithm of a ratio of frequencies. Ann Hum
Genet 1955;20:309-11.
57. Woolf B. On estimating the relation between blood groups and disease. Ann Hum Genet 1955;19:251-3.

Chapter 20
 Hemotherapy Decisions and Their Outcomes
Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD
gn AS with other medical intervenBfiuions, transfusion offers both benefits and risks that must be balanced
for each patient. Although the myriad situations in which transfusion might be considered prevent the
promulgation of “one-size-fits-all” transfusion guidelines, the accumulating data in the medical literature can
certainly help clinicians make hemotherapy decisions based on increasingly sound evidence.
This chapter provides a review of the current literature on the indications for, and outcomes of, transfusion
of blood components. When health-care providers use this chapter in combination with information presented
elsewhere in the Technical Manual on the content of blood components and the risks they pose, they should
have sufficient data to form evidence-based decisions about hemotherapy.
RED CELL TRANSFUSION
At first glance, the decision to transfuse a Red Blood Cell (RBC) unit appears to be easy; for
example, a patient with acute anemia requires an RBC transfusion to restore the body to its baseline state.
For the trauma patient with massive injuries, death will ensue without a transfusion. For the critically ill patient
with anemia stemming from a variety of causes, it would seem reasonable to maintain adequate oxygen stores
in the tissues through transfusion.
Yet the decision to transfuse a stored RBC unit has become more complex in the last decade, with
substantial evidence suggesting that RBC transfusion may be detrimental to some patients. For example, a
sentinel 1999 study randomly assigned critically ill patients (who are among the most frequent recipients of
RBC products) to a restrictive or liberal transfusion strategy (thresholds of hemoglobin = 7 vs 10 g/dL).1 The
lower threshold reduced morbidity and mortality significantly and was not associated with increased length of
stay or morbidity in patients with heart disease or with prolonged ventilation for those requiring a respirator.
The authors concluded
Theresa Nester, MD, Associate Medical Director, Puget Sound Blood Center, and Associate Professor of
Laboratory Medicine, University of Washington, Seattle, Washington; Shweta fain, MD, Transfusion Medicine
Fellow, Puget Sound Blood Center, Seattle, Washington; and fessica Poisson, MD, Director of Transfusion
Services, USC Medical Center, Los Angeles, California The authors have disclosed no conflicts of interest.

499

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AABB TECHNICAL MANUAL
that a hemoglobin threshold of 7 g/dL was appropriate for all critically ill patients, except perhaps those
with unstable cardiac conditions. Similarly, a 2008 systematic review of the critical care literature determined
that in most cohort studies in 272,586 patients, RBC transfusions were an independent predictor of death,
infection, multiorgan dysfunction, and acute respiratory distress syndrome.2
Most randomized, controlled trials (RCTs) conducted to date have demonstrated that a restrictive RBC
transfusion strategy is not inferior to a liberal transfusion strategy. A recent RCT that evaluated transfusion
strategies in patients with severe upper gastrointestinal bleeding showed a superior outcome with a restrictive
strategy for patients with cirrhosis or Child-Pugh Class A or B disease.3 Because of such evidence, many
studies now compare the risk of anemia to that of transfusion and the degree to which each risk affects clinical
outcomes.
The appropriate RBC transfusion threshold has become a central point of discussion. The hemoglobin level
in any individual patient only tells a portion of the story and, by itself, does not reflect the compensatory
mechanisms used to respond to the anemia. Stable tissue oxygenation may be maintained by increased cardiac
output (depending on the coronary arteries’ ability to dilate) and increased oxygen extraction, which result in
lower venous oxygen content.4 Clinicians are appropriately looking for ways to monitor a patient’s response to
anemia before deciding to administer a transfusion. At the same time, hospital-utilization and blood-
management committees look for guidance on the levels of hemoglobin, below which to consider transfusion in
a given patient population.
Parallel to the RCTs performed thus far is the observation that patients without evidence of cardiovascular
disease who refuse transfusion on religious grounds and undergo elective surgery tolerate hemoglobin
reductions of 6 to 7 g/dL with only a minimal rise in perioperative mortality risk.5
 Although many otherwise-healthy adults can tolerate significant anemia equivalent to a halving of their red
cell mass, comorbidities in
many patients may limit their reserve. So what should the indication be for RBC transfusion in the setting of
acute anemia? A recent metaanalysis of 19 RCTs evaluated the effects of different RBC transfusion thresholds
on clinical outcomes between 1956 to 2011, a period during which the definitions of liberal vs restrictive RBC
transfusion strategies varied.6,7 Of the 6264 patients in these studies, most were in surgical or intensive care
units. The lower transfusion threshold (hemoglobin = 7.0-10.0 g/dL, and most commonly 7.0-8.0 g/dL) was not
inferior to the higher hemoglobin level (9.0-13.3 g/dL, and most commonly 9.5-10.0 g/dL). In the lower
threshold groups, fewer RBC units were transfused without an adverse impact on mortality, morbidity, or time
to functional recovery. There were also no significant differences in the incidence of major complications, such
as stroke, pulmonary edema, or infection. Caution must be used in extending these findings to all patient
populations because some high-risk groups (eg, patients with acute brain injury or renal failure) were not
adequately represented.
Although the findings of this meta-analysis suggest that a restrictive transfusion strategy is appropriate for
many hospitalized, stable patients, many clinicians still pause at the thought of withholding transfusion from a
patient with coronary artery disease. In the past, large observational studies gave conflicting results regarding
whether a higher or lower red cell transfusion threshold was beneficial.8'10 More recently, an RCT known as
“FOCUS” included 2016 patients (average age = 82 years) with risk factors for, or a history of, cardiovascular
disease who required hip surgery.11 The results showed that the restrictive strategy (hemoglobin <8 g/dL) was
not inferior to a liberal transfusion strategy (hemoglobin <10 g/dL) with respect to mortality, morbidity, or
function. The primary outcome of death or inability to walk independently across the room at 60 days was
similar in the two groups (34.7% in the restrictive vs 35.2% in the liberal groups). Rates of in-hospital cardiac
events or death as well as other complications were also similar.
The 1999 multicenter Transfusion Requirements in Critical Care (TRICC) trial in 838
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CHAPTER 20 HemotherapyDecisions
critically ill patients, including 326 with atherosclerotic disease but not acute, unstable myocardial
conditions showed that mortality at 30 days was no different in the subgroup with a primary or secondary
diagnosis of cardiac disease between the two strategies (hemoglobin <7 g/dL vs <10 g/dL).1 Cardiac events
(pulmonary edema and myocardial infarction) were more frequent in the liberal strategy group, although there
were no significant differences in rates of these events during the 48 hours prior to death in the patients who
died. The subgroup analysis showed a trend toward increased mortality in patients with ischemic heart disease
in the restrictive arm, leading the investigators to suggest that patients with active coronary ischemic syndromes
may need a higher transfusion threshold than 7 g/dL.
The FOCUS and TRICC trials account for 60% of the available data on the risk of myocardial infarction
following restrictive vs liberal RBC transfusion strategies in patients who have coronary artery disease. An
analysis of combined data from these trials and six smaller trials did not find an elevated risk [risk ratio (RR) =
0.88; 95% confidence interval (Cl) = 0.38, 2.04] for myocardial infarction using a restrictive strategy.12 The
statistical power of the combined data was sufficient to detect moderate to large differences in risk of
myocardial infarction, but the analysis could have missed a twofold higher risk of myocardial infarction with a
restrictive transfusion strategy. In light of the available data, the Clinical Transfusion Medicine Committee of
the AABB published guidelines suggesting that transfusion should be considered for hospitalized patients with
preexisting cardiovascular disease who have symptoms or a hemoglobin level of 8 g/dL or less.12
For patients with acute coronary syndrome, no clinical trials have compared restrictive and liberal
transfusion strategies. For this reason, the development of a guideline based on hemoglobin levels is difficult for
patients with this diagnosis.
The most appropriate hemoglobin threshold for transfusion is a patient-specific, and even situation-specific,
parameter. A he
moglobin level <6 g/dL almost always requires a transfusion, whereas a level >10 g/dL rarely does so. Of
course, most patients have a hemoglobin level between these limits, and many have one or more comorbidities
that affect their tolerance of anemia. The assemblage of experience and knowledge must be blended into the art
of medicine to address this need. In addition to symptoms, research continues to identify consistent signs, such
as venous oxygen saturation, that reflect how an individual patient is tolerating anemia.13
Transfusion in Patients with Hemorrhagic Shock
 A brief history of the changes in transfusion support over the last 40 years for patients experiencing
hemorrhagic shock can be reviewed elsewhere.14 Currently, in the civilian setting, such patients represent 2%
to 3% of all trauma admissions. Signs of acute blood loss in such patients include tachycardia, hypotension,
increased respiratory rate, and mental status changes (Table 20-1). When these symptoms are not addressed
quickly, the mortality rate in patients experiencing hemorrhagic shock ranges from 40% to 70%.
Before 1995, aggressive resuscitation using crystalloid and RBCs was the standard of
TABLE 20-1. Signs and Symptoms of Acute Blood Loss
Tachycardia Palpitations Cooling of extremities Pallor
Hypotension
Reduced arterial pressure
Reduced central venous (jugular) pressure
Acidosis
Increased respiratory rate Decline in urinary output Mental status changes
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AABB TECHNICAL MANUAL
care in civilian hospitals. In the late 1990s, trauma surgeons started to recognize the potential deleterious
effects of too much crystalloid, including increased risk of acute respiratory distress syndrome, multiple-organ
failure, and abdominal compartment syndrome. Gradually, and after new data became available from the
military experience supporting combat casualties in the Iraq and Afghanistan wars, a new approach to
hemorrhagic shock, damage control resuscitation, was adopted. This approach included the early transfusion of
plasma and platelets in addition to RBCs while minimizing crystalloid use. More emphasis was also placed on
addressing the lethal triad of acidosis, hypothermia, and coagulopathy early in patient resuscitation.
Since publication in 2007 of a seminal study involving 252 military personnel with combat casualties and
using almost a 1:1 ratio of plasma to RBCs, many publications from civilian hospitals have echoed the idea that
using an increased ratio of plasma or platelets to RBCs can improve outcomes.15 In 2012, the Prospective,
Observational, Multi-Center Massive Transfusion Study evaluated 905 patients in 10 Level I US civilian trauma
centers who survived for 30 minutes after admission and received at least 1 RBC unit within the first 6 hours
and at least three other blood products within 24 hours of admission.16 An increased ratio of plasma to RBCs or
platelets to RBCs was independently associated with decreased 6-hour mortality. Furthermore, patients with
plasma to red cell ratios less than 1:2 during the first 6 hours were three to four times more likely to die than
patients with ratios of 1:1 or higher.
The Army Surgeon General has established a clinical policy requiring transfusions of Fresh Frozen Plasma
(FFP), platelets, and RBCs in a 1:1:1 ratio for patients injured in combat who require a massive transfusion of 6
whole blood platelet units or 1 apheresis platelet unit following transfusion of 6 RBC units. This emphasis on
addressing coagulopathy early in a massive resuscitation pushes laboratorians to develop faster ways of
evaluating coagulation results to provide optimal transfusion support.17 The benefit of cryoprecipitate
infusion for low fibrinogen levels, although not widely emphasized in these protocols, has also been
established.18
Transfusion in Patients with Chronic Anemia
Transfusion is much less commonly indicated when anemia has persisted for weeks or months because
compensatory mechanisms have had time to work than in patients with anemia of more recent onset. These
longerterm anemias are usually best treated by addressing their etiologies, such as providing supplementation to
treat a nutritional deficiency (eg, of iron) or reducing the rate of autoimmune hemolysis. Congenital
hemoglobinopathies, such as sickle cell disease, are treated according to disease-related protocols for purposes
that are not necessarily related to oxygen delivery. Hypoproliferative anemias secondary to chemotherapy or
end-stage renal disease are often treated with marrow stimulants, such as recombinant erythropoietin. Any of
these diseases could conceivably require a transfusion if patient symptomatology requires more rapid reversal
than would be possible by treating the underlying mechanisms, but transfusion is usually considered only as a
last resort in these patients.
Some patients become transfusion dependent because of their inability to create and maintain an adequate
red cell mass. These patients often “declare” the hemoglobin at which their symptoms are best controlled. The
symptomatology reported by patients with chronic anemia does not generally correlate well with laboratory
values in different patients but often corresponds well with these values in an individual over time.
Selected Clinical Issues
 Use of Whole Blood
Whole blood provides oxygen-carrying capacity, stable coagulation factors (concentrations of Factors V and
VIII decrease during storage), and blood volume expansion. Thus, it is potentially useful for patients with
concomitant red cell and volume deficits, such as actively
503
CHAPTER 20 HemotherapyDecisions
bleeding patients, and helps support coagulation if coagulation factor consumption is the primary cause of
coagulopathy.19 However, whole blood is rarely available for allogeneic transfusion. Component therapy has
evolved as an improved way to expand the blood component supply to meet the demand. Whole blood is mostly
commonly used in the United States today for autologous transfusion. The ABO type of transfused whole blood
must be identical to that of the recipient.
Duration of Storage
With the rapid disappearance of 2,3-diphosphoglycerate (DPG) from stored red cells and the concomitant
increase in hemoglobin’s affinity for 02, a concern is that RBC units stored for more than 1 to 2 weeks may not
provide the intended increased availability of 0, at the tissue level, at least for the first 12 to 24 hours after
transfusion.20,21 Beyond 7 to 10 days of storage, the P50 of hemoglobin decreases from 27 to 16 mmHg,
markedly shifting the dissociation curve to the left.
There are other known changes to red cells that occur with storage over time, sometimes referred to
collectively as the “storage lesion.” These changes include shape changes, such as decreased deformability of
the red cell, and decreased adenosine triphosphate. In addition, a loss of nitric oxide occurs within a few hours
after collection.22 Whether these changes are clinically important is unclear.
The corollary question regarding whether the age of blood matters for clinical outcomes is an important and
controversial one. A recent meta-analysis that evaluated both observational studies and RCTs in which death
was the primary outcome showed that transfusions of older RBC units (>21 days old) were associated with a
significantly increased risk of death [odds ratio (OR) = 1.16, 95% Cl = 1.07, 1.24]. The majority of studies in
this analysis were observational, and the three RCTs analyzed had only small numbers of patients. Therefore,
the conclusion that transfusions of older blood components caused the increased mortality is weakened by the
confounding and unintentional bias that arises with observa
tional studies.23,24 At least one of the studies was difficult to interpret because of clinical differences in the
two groups of patients.25 The authors of an earlier meta-analysis that focused on homogeneous subgroups
concluded that the available data were inadequate to prove causality between older stored blood components
and increased morbidity and mortality.26
Three larger RCTs have been undertaken to clarify the issue. The Age of Red Blood Cells in Premature
Infants trial included 377 very low-birthweight premature infants requiring transfusion. Transfusions of fresh
RBC units (average age = 5.1 days) vs standard-issue RBC units (average age = 14.6 days) were compared,
with rates of major neonatal morbidities as the primary outcome measure and the rate of nosocomial infection
as a secondary outcome. In this study, there were no clinically meaningful or statistically significant differences
in either outcome.27 The Red Cell Storage Duration Study randomly assigned patients aged at least 12 years
who required complex cardiac surgery to receive RBCs less than 11 days old or greater than 20 days old
throughout their perioperative course.28 The primary outcome measure is an assessment of clinical outcomes
using the Multiple Organ Dysfunction Score. One ongoing RCT, the Age of Blood Evaluation study, involves
critically ill patients who require transfusion; this prospective randomized trial will compare all-cause mortality
at 90 days in patients receiving RBCs less than 8 days old vs standard-issue RBC units.29
Certainly, if the storage lesion is detrimental to a patient’s clinical course, research must determine whether
and, if so, why certain patient populations are more affected than others. With the tenuous balance that already
exists between the safety and supply of this human-derived resource, having fewer blood components available
to support patients may prove more detrimental than having stored blood components available. Determining a
better way to store RBCs—one that prevents the storage lesion—seems to be a prudent approach. Opportunities
to improve RBC storage, prevent cell damage, and
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AABB TECHNICAL MANUAL
improve the preservation of 2,3-DPG continue to be pursued.30
ABO Matching
 Although matching patient and donor ABO types ensures compatibility in that blood group system,
inventory considerations may suggest that an ABO-compatible rather than an ABO-identical RBC unit should
be transfused (Table 20-2). Although a compatible but nonidentical RBC unit introduces some isoagglutinins
directed against the recipient’s A and/or B antigens, the small amount of plasma in an additive system or packed
RBC unit (or even multiple units) is insufficient to cause hemolysis.
Components containing larger amounts of plasma, such as platelets (see below) or whole blood units, raise
the potential for isoagglutinins to cause hemolysis. Whole blood, when used, would be transfused to an
ABOidentical recipient because of this issue. In the setting of ABO-incompatible heart transplantation in an
infant, the transfusion service may be asked to plasma-reduce or wash an RBC unit, largely because the original
protocol called for this modification rather than because solid evidence indicates that incompatible passive ABO
isoagglutinins can impair a new graft.31
Leukocyte Reduction
The most appropriate use of leukocyte reduction remains controversial. Ardent proponents on both sides
argue about whether all cellular
TABLE 20-2. Selection of ABO-Compatible Red Blood Cell Units
Recipient Blood Compatible Red Blood
Group Cell Units*
A A, 0
B B, 0
AB AB, A, B, 0
0 0
*Red Blood Cells prepared as additive system or “packed” units.
components should have their white cells reduced.32 Although initially introduced in several European
countries to decrease the transmission of prions, universal leukocyte reduction is most often used to reduce the
risk of postoperative infection and improve posttransfusion survival by reducing transfusionrelated
immunomodulation (TRIM).
The benefit of removing leukocytes for patients who will be multitransfused and/or transplanted is clearly
evident in terms of reduced rates of alloimmunization, episodes of refractoriness to platelet transfusion, and
febrile reactions.33-36 However, universal implementation of leukocyte reduction may not cause a measureable
drop in febrile reaction rates, given the proportion of patients who are susceptible to these reactions.37 A
positive clinical impact of universal leukocyte reduction has been difficult to identify and was not seen in the
only large-scale, prospective RCT of its implementation.38 Even in more defined situations, such as cardiac
surgery, prospective trials have reached contradictory conclusions, including when they were performed by the
same research team.39-42 The many retrospective analyses ascribing benefits to leukocyte reduction have often
been limited by confounders.43,44 However, some support strong arguments that removing leukocytes does
lead to reduced lengths of stay, reduced infection rates, and improved outcomes.45,46 Other potential concerns
associated with the presence of leukocytes in blood components, such as increased red cell adhesive properties,
raise new questions about their impact.47 For a thorough examination of this complex subject, the reader is
referred to several excellent metaanalyses and other publications.48-51
Many studies have addressed the possibility that leukocyte reduction can reduce the incidence of clinical
outcomes due to TRIM, but the results have been contradictory.50 One hypothesis was that the savings resulting
from reduced immunomodulation could offset the costs of leukocyte reduction, but this was not demonstrable in
a large prospective RCT.42 Nonetheless, several countries maintain a leukocyte-reduced blood supply, and the
need for leukocyte reduction remains controversial.43

CHAPTER 20
Hemotherapy Decisions
505
The value of leukocyte reduction as a means of preventing cytomegalovirus (CMV) transmission has been
well documented.52,53 Studies using polymerase chain reaction (PCR) indicate that the highest risk of CMV
transmission through blood transfusion may be from recently seroconverted donors rather than donors who have
been seropositive for more than a year.54,55 This information is interesting but difficult to translate into an
operational policy. Because of a 1% to 2% risk of “breakthrough” transmission with either seronegative or
leukoreduced components, patients at high risk of disseminated CMV infection should be actively monitored
for evidence of CMV using antigenemia assays or reversetranscription PCR techniques.56
The leukocyte content of units varies by component type (Table 20-3).57,58 Current federal guidelines and
AABB Standards for Blood Banks and Transfusion Services define a leukocyte-reduced component as one with
<5 x 106 residual donor leukocytes per final product (including RBCs; apheresis platelets, and pooled
platelets).59,60(p26) The AABB Standards calls for <8.3 x 105 residual leukocytes in platelets prepared from a
single unit of whole blood, and 5 x 106 in Pooled Platelets Leukocytes Reduced.60(p29) The Food and Drug
Ad
ministration (FDA) recommends quality-control steps to indicate with 95% confidence that at least 95% of
units meet these criteria.59 By comparison, European guidelines define leukocyte-reduced components as those
with <1 x 106 residual leukocytes per unit and require no more than a 10% failure rate in the leukoreduction
process.61
Matching Units to Patient Hemoglobin Targets
For adult patients, the discussion of when or whether to transfuse RBCs has not usually proceeded to a
consideration of how much to transfuse. Although pediatricians commonly prescribe blood components based
on the patient’s size, the typical order for adult RBC transfusion until recently had been 2 units, regardless of
patient size and usually without regard to a desired target hemoglobin level. In addition, the widely variable
hemoglobin content of RBC units usually is not considered.62
A more physiologic approach would be to identify the target hemoglobin desired after transfusion and then
base the dose (ie, number of units or g of hemoglobin to be transfused) on the patient’s current hemoglobin, any
ongoing blood loss, and the calculated blood volume. A pilot study, however, showed
TABLE 20-3. Approximate Leukocyte Content of Blood Components (Per Unit)
Whole blood 109
Red Blood Cells 108
Red Blood Cells, washed 107
Red Blood Cells, deglycerolized 106-107
O
X
Red Blood Cells, leukocyte reduced (by filtration)*
LO
V
Apheresis Platelets 106-108
Apheresis Platelets, leukocyte reduced <5 x 106
Platelets1 107
Platelets, leukocyte reduced <8.3 xIO5
Platelets, pooled, leukocyte reduced <5 x 106
Fresh Frozen Plasma, thawed 0.6 x 106 -1.5 x 107
‘Leukocyte reduction with third-generation leukocyte adsorption filter, derived from 1 unit of whole blood
via platelet-rich plasma process.
 506
AABB TECHNICAL MANUAL
that the number of units transfused using this approach to a group of patients could be reduced, thus reaping
benefits for inventory management as well as donor exposure rates.63 Issues that remain to be resolved for this
approach include the following:
■ Whether the approach could be implemented successfully for all patients in an institution.
■ Whether blood collection establishments would be able to provide the needed hemoglobin content for all
RBC units.
■ Whether the approach could be sustained in an era when most units are produced by apheresis, with a
standardized hemoglobin content.
Emergency Transfusion
The unexpected need to transfuse possibly a large amount of RBCs may require application of alternative
procedures. Release of group 0 RBCs or antigen-negative, uncrossmatched units may be necessary if waiting to
complete standard pretransfusion testing routines could induce anemic morbidity (see Chapter 15).
An emergency-release protocol might be integrated into one that allows rapid provision of a large number of
RBC units to accommodate the needs of patients who are bleeding rapidly. Elements of such a block-release
system might include completing the paper work for multiple units in advance and prepackaging these units to
facilitate immediate delivery to the patient. The need for and components of such a system vary according to the
types of patients served by the institution and the logistics of blood delivery. The expectations of clinicians
(particularly surgeons, anesthesiologists, and emergency physicians) for rapid delivery of large volumes of
RBCs should be discussed with them so that appropriate requirements can be met reliably. In evaluating these
clinical needs, attention should be given not just to the time required to issue RBCs from inventory to the
laboratory but also for the units to reach the patient.
Incompatible RBC Transfusion
 Occasionally, transfusion of RBCs may be necessary when no serologically compatible units are available
for a patient. This most often occurs in patients with autoantibodies that typically are reactive with red cells
from all donors; however, as long as the presence of alloantibodies can be ruled out, the transfused cells should
survive as long as autologous cells.
The key point in ensuring a safe and successful transfusion in such a situation is the exclusion of the
presence of alloantibodies. Because this may be difficult and the beneficial effects of the transfusion may be
temporally limited, a conservative transfusion strategy is usually recommended for patients with autoimmune
hemolytic anemia. Determination of the patient’s phenotype before transfusion simplifies subsequent
investigations of the presence of alloantibodies. (Chapters 1517 contain a more complete discussion of this
issue.)
Other situations in which all units appear to be incompatible include the presence of alloantibodies to high-
frequency antigens and/ or multiple antibody specificities. If serologic testing fails to resolve the problem or the
problem is identified but sufficient time is not available to acquire compatible units, consultation between the
transfusion service physician and the patient’s clinician is advised to weigh the risks and benefits of transfusion
and to consider which alternative therapies are suitable. If the need is sufficiently urgent, ABO-compatible but
crossmatch-incompatible RBCs may need to be used.
Depending on the alloantibody’s specificity or possible specificities that have not been ruled out,
incompatible RBC transfusion does not always result in immediate hemolysis, and the incompatible cells may
remain in the circulation long enough to provide therapeutic benefit.64
If time permits and equipment is available, the survival of a radiolabeled aliquot of the incompatible RBCs
can be determined. However, this is beyond the capability of most laboratories and is rarely needed. An in-vivo
crossmatch can be performed by cautiously
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CHAPTER 20 HemotherapyDecisions
transfusing 25 to 50 mL of the incompatible cells, watching the patient’s clinical response, and checking a
30-minute posttransfusion specimen for hemoglobin-tinged serum. Such an assessment does not guarantee
normal red cell survival, but it can indicate whether an acute reaction might occur. If no adverse symptoms or
hemolysis are observed, the remainder of the unit can be transfused slowly and with careful clinical monitoring.
If the condition requiring a transfusion intervention is life threatening, RBC units may sometimes be given
without special testing, but clinical staff should be prepared to treat any reaction that may result.
RBC Substitutes
A variety of means have been explored to provide the 02-carrying capacity of hemoglobin without utilizing
red cells. Development has proceeded furthest using Hb solutions derived from red cell lysates in which the
hemoglobin molecules have undergone one or more chemical modifications to optimize their 02 dissociation
and prevent renal damage. Two of the greatest challenges with cell-free hemoglobin are its vasoconstrictive
properties and its short half-life within the intravascular circulation. The goal of creating a hemoglobin
substitute that the FDA considers to be as safe as donor red cells continues to be an elusive one.65,66
PLATELET TRANSFUSION
Prophylactic vs Therapeutic Transfusion
Before the advent of platelet therapy, bleeding exceeded infection as the most common cause of death in
patients with acute leukemia. The availability of platelet concentrates led to the natural consideration that
prophylactic transfusion to prevent or limit the most severe thrombocytopenia would prevent the onset of severe
hemorrhage. Today, approximately 80% of all platelet transfusions are administered to patients with
hypoproliferative thrombocytopenia.
However, the value of this approach has never been definitively documented. Early studies yielded
equivocal results or utilized statistical approaches that were less rigorous than would be expected today.67,68 In
fact, an RCT in children with leukemia prior to the era of leukocyte reduction indicated that the use of
prophylactic platelet transfusion from the outset of chemotherapy increased the likelihood of bleeding due to the
development of platelet refractoriness.67
The alternative strategy of directing platelet resources to those patients who are bleeding may be a wiser and
more effective use of a limited resource.69 In fact, a recently published Cochrane review showed that overall, a
therapeutic platelet transfusion strategy might not be inferior to a prophylactic strategy, but the evidence
supporting this approach is not robust.67 The majority of studies reviewed were conducted in the 1970s, and the
conclusions did not take into account practice changes since that time.
 Overall, the role of prophylactic platelet transfusions for the prevention and control of thrombocytopenic
bleeding is not yet clear. When bleeding risk is identified in patients with thrombocytopenia, several factors in
addition to platelet count should be considered. These include altered platelet function due to intrinsic or
acquired defects and hemostatic defects involving the coagulation system. The disease process itself must also
be considered. For example, in acute leukemia, intracranial hemorrhage is more often associated with high
circulating blast counts than with low platelet counts.70 The leukemic blasts may cause sludging of cells in
capillaries and possible hemorrhagic infarction, particularly in the presence of a concomitant coagulopathy.
Transfusion Thresholds
If prophylactic platelet transfusions are to be given to patients with hypoproliferative thrombocytopenia,
what is the most appropriate threshold for transfusion? The first study to address this question sought to define a
clinically useful threshold by observing and categorizing bleeding and associating the risks of
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AABB TECHNICAL MANUAL
hemorrhage with patients’ platelet counts.71 The researchers were unable to identify a platelet count
threshold below which the risk of hemorrhage increased rapidly. However, this paper was often cited as the
source of the dictum that patients’ platelet counts should be kept above 20,000/pL. In the 1960s, when this
study was conducted, aspirin was commonly used to treat fever, pain, and transfusion reactions among patients
with neutropenia. But with current knowledge about the platelet dysfunction induced by aspirin and associated
changes in hematology practice, the study results are less applicable today (Table 20-4).73
Over the last decade, several prospective studies using either historical or randomized control groups have
documented the successful application of 10,000/pL as a prophylactic transfusion threshold in
inpatients.67,74'76 These results parallel the finding that stool blood loss does not accelerate until a platelet
count of approximately 5000/pL is reached.77 Many institutions have now adopted 10,000/ pL as their standard
prophylactic platelet transfusion threshold, but others have opted to combine laboratory data with the patient’s
clinical status to determine the most appropriate transfusion point72 (Table 20-4). Furthermore, because
platelets adsorb circulating thrombopoietin (TPO) and higher platelet counts are associated with lower levels of
free TPO,78 maintaining patients at a lower platelet count has been suggested to lead to shorter intervals of
thrombocytopenia, which is a potential additional benefit.
For patients who are already bleeding or are about to undergo a hemostatic challenge, such as a surgical
procedure, attempts are made to keep the platelet count higher, usually in the vicinity of 50,000/pL.79 Although
there are no trials to document that this is the most appropriate level, it has become generally accepted as
adequate.80 An even higher target, up to 100,000/pL, is often used for intracerebral, pulmonary, and ophthalmic
hemorrhage. This is to provide a greater “cushion” to ensure adequate hemostasis in these vital and susceptible
organs even if the platelet count should drop. Higher cut points might also be used in massive transfusion or
disseminated intravascular coagulation (DIC), where the platelet count may drop rapidly.
The role of anemia should also be considered in the prevention or treatment of hemorrhage. In a 2001 study
in healthy volunteers, a reduction in hematocrit from 41% to 35% resulted in almost a doubling of the bleeding
time, whereas a decrease in the platelet count by one-third had no effect.81 Traditionally, this result has been
attributed to the rheologic properties of flowing blood. Given their larger size and higher density, red cells tend
to occupy the central (axial) portion of the blood flow, pushing platelets to the periphery in proximity with the
vessel wall. This makes teleologic sense because it is only along the vessel wall that a rent in the endothelium
can occur where the platelets would then perform their hemostatic functions.
Recent research has focused on elucidating the mechanisms of red cell and platelet
TABLE 20-4. Current Prophylactic Platelet Transfusion Thresholds
All patients 10,000/pL
- or
Stable patients 5,000/pL
Patients with fever or recent hemorrhage 10,000/pL
Patients with coagulopathy, on heparin, or with anatomic lesion likely to bleed72 20,000/pL
Note: These thresholds are most commonly applied to inpatients. Adjustment of the
transfusion threshold may be necessitated by unusual clinical situations.
 509
CHAPTER 20 HemotherapyDecisions
communication, which might also explain the hemostatic effect of a higher hematocrit. These newer studies
have shown, for example, that the red cell membrane augments thrombin generation,82 adenosine diphosphate
released from red cells may be a chemical messenger for platelet activation,83 and red cell phosphatidyl serine
expression might be another pathway for thrombin generation. Regardless of the postulated mechanism,
correction of anemia may be an additional tool to use for bleeding prevention, particularly in patients with
thrombocytopenia. In patients with uremia, RBC transfusion or the administration of erythropoietin to increase
the hematocrit improves hemostasis similarly.84 Thus, although the patient’s cardiovascular system might be
tolerating the anemia associated with chemotherapy (or hemorrhage), the hemostatic system may not.85
Transfusion to Correct Thrombocytopathy
Patients whose platelets are not able to complete all of the complex metabolic steps necessary for activation,
granular release, and aggregation may have an increased likelihood of bleeding and/or an inability to respond to
hemorrhage appropriately. These abnormalities may be congenital (eg, Glanzmann thrombasthenia) or acquired
as the result of disease (eg, myelodysplasia) or drug treatment (eg, with aspirin or glycoprotein Ilb/IIIa
antagonists). In addition, patients who have recently undergone extracorporeal circulation (eg, during cardiac
bypass surgery) may have platelet counts that appear to be adequate for hemostasis but, in fact, are
dysfunctional due to prolonged exposure to roller pumps and foreign surfaces, leading to partial activation and
degranulation.86
In all of these circumstances, the decision whether to transfuse platelets probably needs to be based on the
patient’s clinical status rather than his or her platelet count. Patients undergoing surgery while still under the
effect of previously ingested aspirin do not necessarily bleed more than other patients, although clinician
knowledge that the patient has been
taking aspirin has been associated with increased blood component usage.87'92 The prophylactic use of
platelets (and plasma) after cardiac surgery has been shown to be neither necessary nor beneficial, but a patient
experiencing excessive blood loss postoperatively whose heparin level has been reversed may benefit from a
dose of platelets even before his or her postoperative platelet count is known. Patients treated with irreversible
platelet antagonists (eg, clopidogrel) during cardiac catheterization and who proceed directly to cardiac surgery
may need one or more doses of platelets (as well as increased RBC transfusions) because their own platelets are
no longer functional.93,94 Antiplatelet therapies continue to be used in catheterization because they appear to
improve outcomes even if the patient requires immediate surgery.95 The effect of these drugs can be
antagonized through pharmacologic intervention, including the use of desmopressin acetate.96
Given the high proportion of patients treated with aspirin for a variety of reasons, patients who experience
bleeding while taking aspirin may be encountered frequently. Although the daily consumption of 81 mg aspirin
for cardiac prophylaxis is unlikely to contribute to hemostatic difficulties, some patients are hyperresponders to
aspirin and their bleeding times may be dramatically extended. Platelet transfusions are occasionally requested
to correct the effect of aspirin. In these cases, only a small proportion—approximately 20%—of circulating
platelets need to be functional to correct the deficiency.97 Therefore, neither achievement of donor platelet
levels of 50,000/pL nor a full therapeutic dose of platelets is required in most patients.
Patients with significant renal disease (ie, a creatinine level exceeding 3 mg/dL) may also have
dysfunctional platelets due to the uremic environment. Transfusion of platelets to these patients is of little value,
however, because they, too, rapidly succumb to the same metabolic derangement. Desmopressin acetate
(1deamino-8-D-arginine vasopressin, or DDAVP) treatment is usually recommended to augment the
responsiveness of these platelets.98 Alternatively, cryoprecipitate may provide the
510
AABB TECHNICAL MANUAL
increased amount of von Willebrand factor (vWF) that is believed to be helpful in activating these platelets
if tachyphylaxis to multiple doses of desmopressin acetate precludes further treatment." Dialysis to decrease the
uremia is often indicated in clinical scenarios where optimal platelet function is desired.
Dosage
The amount of platelets considered a therapeutic dose remains undecided, but recent research is elucidating
this issue.100 Much early research was performed using platelet units with significantly lower platelet counts
than units currently being produced. Initial platelet separation efficiencies were significantly lower than those
achieved through evolutionary improvements in both component production processes and larger whole-blood
collections. Accordingly, many blood centers now report mean contents that are 20% to 40% above the required
minimum of 5.5 x 1010 platelets per unit derived from whole blood. As a result, fewer units need to be pooled
to obtain the same platelet dose. At the same time, acceptance of lower platelet counts in patients has facilitated
the progressive reduction in the standard dose from 10 to 8, 6, or even 4 units per pool (or transfusion).
The amount of platelets that can be collected by apheresis also merits consideration. The standard of 3.0 x
1011 apheresis platelets per unit is the amount that could be practically collected with early instruments and
may also have accounted for the maximum collection time that donors would tolerate. These apheresis units
yielded an increment similar to the transfusion of a pool of 6 to 8 units of wholeblood-derived platelet
components. Today, the increased efficiency of apheresis instruments allows the collection of two or even three
times the standard quantity. Although blood centers derive a significant economic boost from splitting platelet
apheresis units, whether patients are better served by receiving larger platelet units remains to be determined.
The question of what the optimal platelet dose is has been approached in several ways. A mathematical
model was used to calculate the
smallest number of platelets that would need to be transfused if patients received just enough platelets to
achieve the desired target level when they reached the transfusion threshold.101 This analysis argued for the use
of small therapeutic doses administered more frequently. The use of larger units in a clinical study resulted in
longer intertransfusion intervals, although this temporal increase was smaller than the increase in the number of
platelets transfused.102 In a paired study of marrow transplant patients, larger units provided the expected
lengthening of intertransfusion intervals and, unexpectedly, resulted in both count increments and corrected
count increments (CCIs) that were higher than those achieved with smaller units.103
The Strategies for Transfusion of Platelets study104 was a multicenter, prospective RCT designed to show
that a lower platelet dose for prophylactic transfusions was not inferior to the standard dose; the primary
outcome was incidence of WHO Grade 2 (or higher) bleeding. The trial enrolled patients undergoing
hematopoietic stem cell transplantation and nontransplant patients with chemotherapyinduced
thrombocytopenia. The experimental arm received low-dose prophylactic platelet transfusions (1.5-2.9 x 1011
platelets per transfusion, defined as half the standard dose) and the control arm received a standard dose (3.0-6.0
x 1011 platelets per transfusion). Although sample size calculations indicated that approximately 270 patients
would be necessary in each treatment arm, the study was stopped after enrolling 130 patients based on a
preestablished safety threshold because the cumulative incidence of Grade 4 bleeding exceeded 5% between the
two study arms. The main trends of this study did not support the hypotheses that patients in the low-dose arm
would require fewer products and have a shorter duration of thrombocytopenia.
Another recent multicenter, prospective, RCT, the platelet dose (PLADO) study,105 did proceed to
completion. Patients with hypoproliferative thrombocytopenia secondary to chemotherapy for hematologic
malignancies or undergoing either autologous or allogeneic stem cell transplantation were randomly
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CHAPTER 20 HemotherapyDecisions
assigned to a prophylactic platelet transfusion dose of 1.1 (low dose), 2.2 (medium dose), or 4.4 x 10n/m2
platelets (high dose). The patients were transfused with their assigned dose prophylactically for morning platelet
counts of <10,000/pL. Additional platelet transfusions could be given for active bleeding or planned invasive
procedures. The primary endpoint was the percentage of patients in each group with at least one episode of
WHO Grade 2 or higher bleeding. Of the 1272 patients who received at least one platelet transfusion, the
primary endpoint was achieved in 71%, 69%, and 70% in the low-, medium-, and high-dose groups,
respectively. These differences were not statistically significant. Those in the lowdose arm did receive an
average of one more platelet transfusion, however.
The lifespan of transfused platelets appears to be abnormally short in patients with thrombocytopenia.
Although autologous platelets stored for 5 or 7 days and then reinfused to healthy people usually survive for
more than 5 days, the lifespan of platelets in patients with severe thrombocytopenia may be only 2 days. This
shortened lifespan may be attributable to the fixed loss of platelets of approximately 7100/pL per day from
circulation for maintenance of normal hemostasis.106 When the patient’s platelet count is low (and, of course,
still subject to daily reductions due to senescence), this obligatory loss represents a large proportion of
circulating platelets and leads to the need for transfusions every 2 to 3 days even in patients achieving good
increments from transfusions.107 More RCTs similar to the PLADO study are needed to clarify which dosage
provides optimal hemostasis.
As with adult RBC transfusions, the size of the patients (and their spleens) are usually not considered in
selecting the platelet unit dose to be transfused. One of the dose studies mentioned above attempted to set the
dose based on patient weight, and the outcomes of the two studies may be helpful in determining the clinical
importance of such a strategy. The patient’s size and the content of the unit are routinely assessed through the
CCI (Table 20-5). This measure, or a similar approach of calculating the recovery rate of transfused platelets
based on content and blood volume,108 can provide a yardstick to determine whether special efforts, such
as selection of antigennegative units to combat alloimmunization, maybe helpful.
Unit Type and Age
The types of platelet units used for routine transfusion varies by institution. Currently, about 90% of platelet
transfusions in the United States are derived from apheresis collections, and the usage of these platelets has
increased in a steady, linear manner for the past two decades.109 Local pricing policies and the preference of
transfusion services to avoid the extra processing steps associated with wholeblood-derived units are probably
significant factors in this choice (Table 20-6). Some invitro measures have identified a larger platelet storage
lesion in platelets produced via the platelet-rich plasma (PRP) method than by apheresis,110 and one paired
reinfusion study has confirmed these findings.111 However, the radiolabeled autologous recovery and survival
rates of the two types of platelets do not appear dissimilar even after 7 days of storage.112,113 Platelets derived
from whole-blood huffy coats may have the advantages of reduced activation during centrifugal preparation
steps, but these are currently not available in the United States, although they are probably the most widely used
platelet component around the world. From a clinical efficacy standpoint, all three platelet preparations yield
good clinical results.
All platelet unit types accumulate a storage lesion over time that leaves them less responsive to physiologic
agonists. Typically, these platelets also bear markers of platelet activation, although none of these markers has
proven to be a useful predictor of recovery or survival after transfusion.114'117 Studies in which radiolabeled
platelets at the limit of the storage period are reinfused are commonly required for FDA licensure of new
techniques of collecting or storing platelets. Both recovery and survival appear to become shorter with increased
storage time in such radiolabeling studies. However, not all series of clinical
512
AABB TECHNICAL MANUAL
 TABLE 20-5. Determination of Platelet Response
CCI
CCI =
01 (per |iL) x BSA unit content
Platelet Recovery
Platelet recovery (%) =
Cl x patient mass (kg) x blood volume (estimated at 75 mL/kg) x 100
unit content
Sample Calculations
Patient mass: 80 kg blood volume = 80 kg x 75 mL/kg = 6000 mL
Patient BSA: 2.0 m2 (determined from a table or nomogram, available in many textbooks)
Pretransfusion platelet count: 5000/pL _
Posttransfusion platelet count: 25,000/pL " ’ ^
Platelet count in unit: 1.5 x 106/pL
Volume of unit: 267 mL * unit content = 4.0x10” platelets
CCI = (20,000/pL x 2.0 m2)/4.0 = 10,000 Successful transfusion: >7500
Refractory patient: Two or more transfusions with CCI <7500
Recovery = (20,000/pL x 1000 pL/mL x 6000 mL x 100%)/(4.0 x 1011) = 30%
Maximum achievable if the patient has a spleen: 65% to 70%
CCI = corrected count increment; Cl = count increment; BSA = body surface area.
TABLE 20-6 Characteristics of Platelet Unit Types Available in the United States
Whole-Blood Derived
Prestorage
Platelet Unit Characteristic Individual Unit Apheresis
Pooled*
Cost of preparation Lower Higher
Ease of bacterial testing Lower Higher Higher
Ease of leukoreduction Lower Higher Higher
Hospital preparation required More Less Less
Donor exposures Greater Fewer
HLA selection possible No Yes
Platelet content known No Yes
‘Storage of pools of platelets for longer than 4 hours requires bacteria detection by a culture technique
approved by the Food and Drug Administration.
 513
CHAPTER 20 HemotherapyDecisions
observations have shown a decrement in CCI with increased storage time, perhaps due to limited study sizes
and the inherent variability between patients and outcomes of transfusions at different points during the course
of illness.118,119 Therefore, platelet-transfusion policies in most institutions call for the most efficient use of
the short-lived and scarce resource by transfusing the most appropriate units that are closest to their outdate.
Some patients may appear to be more sensitive to the storage lesion than others and may benefit from receipt of
platelets that have been stored for less time; this can only be determined empirically.
Out-of-Group Transfusions
The importance of providing platelet transfusions using units of the same ABO group as that of the recipient
is an unresolved issue. There are several points to consider: 1) the presence of ABH antigens on platelets that
could be targeted by the recipient’s isoagglutinins,120 2) the presence of plasma in the unit that could lead to
hemolysis of the recipient’s red cells, and 3) the potential that the incompatibility could have immunologic
effects that could affect patient outcomes. Typically, the presence of ABO-incompatible donor red cells in the
platelet product is not a significant concern because of their very low levels.
A recently published systematic review121 showed that ABO-identical and ABO-compatible platelet
transfusions (eg, group A platelets in an AB recipient) result in a higher CCI than ABO-incompatible platelet
transfusions (eg, group 0 platelets in a non-group 0 recipient).
Whether the benefits of transfusing ABOidentical or -compatible platelets also translates into improved
clinical outcomes in terms of decreased bleeding severity or need for transfusions is not clear from the existing
data. These data do indicate that a subset of recipients with a higher isoagglutinin titer, particularly anti-A, have
poorer recovery after transfusion with a high-ABH-antigen-expression unit, especially if the platelets comes
from an Aj donor.122'126 Any diminution in response appears to be more pronounced with repeated
out-of-group transfusions, possibly due to the stimulation of higher isoagglutinin titers in the recipients.127
Failure to achieve expected platelet increments with an out-of-group transfusion should prompt a trial of ABO-
identical transfusions, particularly if the patient is not alloimmunized to platelet-specific or HLA antigens (Fig
20-1).128
An out-of-group platelet transfusion, such as of a group 0 unit to a group A recipient, results in the
transfusion of about 300 mL of plasma containing isoagglutinins that are directed against antigens present on
the recipient’s red cells. Although it is not standard practice to transfuse a group 0 plasma unit to a group A
recipient, this practice is common for platelet transfusions.80 Many patients show no signs or symptoms of the
mismatch, although the majority may have a transiently positive direct antiglobulin test result that, at least,
causes some immunohematologic confusion regarding its cause.129
A high titer of isoagglutinins in a platelet unit can cause hemolysis of recipient red cells that may be
clinically significant and even fatal. This outcome may be more likely in situations where the recipient is
smaller (and, thus, the volume transfused represents a greater proportion of the recipient’s plasma volume),
when apheresis units are used (and all the plasma comes from the same donor and is not “diluted” by plasma of
lower isoagglutinin titers from other units in the pool or neutralized by the presence of soluble ABO antigens in
the other units), and in patients undergoing multiple transfusions.130 The risk of hemolysis with apheresis
platelet units is in the range of 1:3000 to 1:10,OOO.131132 Whether ABO-compatible but nonidentical plasma
poses a risk through circulating immune complexes has been considered.133
Different approaches may be used to prevent acute hemolysis when incompatible plasma must be transfused
as part of a platelet transfusion. One approach is to limit the amount of plasma being transfused by centrifuging
the unit and expressing off the majority of the plasma shortly before transfusion (see Method 6-13). This
reduces the potential severity of any hemolysis but may not prevent it
514
AABB TECHNICAL MANUAL

FIGURE 20-1. One approach to management of patients with thrombocytopenia.

and, in any case, does result in the loss of some proportion of the unit’s platelet content. An approach that
achieves a similar result is to use platelet additive solutions, which allow platelets to be stored in electrolyte
solutions that replace up to 65% of plasma, reducing the amount of incompatible isoagglutinins. Another
approach is to avoid out-of-group transfusions of units having a dangerously high titer of isoagglutinins. This
approach usually involves identifying units with amounts of anti-A above a particular threshold, often a titer of
200. This approach lacks a solid evidence base, its outcomes vary by method,134 and it does not identify all
potentially harmful units.135 Avoidance of out-of-group plasma or amelioration of the situation through volume
reduction is especially important for pediatric recipients, in whom the volume transfused is relatively greater,
and in neonates, where hyperbilirubinemia may have particularly adverse consequences.
The possibility exists that the transfusion of incompatible plasma may also have other, less-immediate but
still untoward effects on recipients’ immune systems. It is postulated
that these effects might be mediated by the formation of circulating immune complexes.133 For example,
minimization of exposure to incompatible plasma was associated with improved survival probability in marrow
transplant recipients in one retrospective analysis,135 and another retrospective study suggested that such a
strategy reduced in-hospital mortality after cardiac surgery by twothirds.136 This finding was not replicated in a
subsequent, larger study, however.137 There is also some suggestion that transfusion with ABO-incompatible
platelets may hasten the development of alloimmunization and platelet refractoriness138 and may reduce
survival after marrow transplantation.139
Any delayed immunologic impact of the transfusion of mismatched plasma with platelets remains to be
fully elucidated, although avoidance of out-of-group transfusion when possible would obviate this concern.
Weighed against the practical issue of a human-derived therapeutic product that expires in 4 to 5 days issuing a
platelet unit with incompatible plasma generally carries a lower risk than withholding transfusion.
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CHAPTER 20 HemotherapyDecisions
Rh Matching
Platelets themselves do not express or carry Rh antigens. Despite improvements in apheresiscollection and
whole-blood-processing techniques, a small but immunogenic dose of red cells can be contained in a platelet
unit. This is more likely in whole-blood-derived than apheresis platelet units. In one study, an internal quality
control check of platelet units using flow cytometry found that mean red cell content of platelet concentrates
from whole-blood donations and from apheresis were 0.036 mL and 0.00043 mL, respectively.138
The risk of developing alloimmunization to the D antigen from a platelet transfusion is also dependent on a
multitude of other platelet unit factors (eg, ABO compatibility and whether the unit was leukoreduced) and
recipient factors (eg, gender, immunologic status, whether the patient needs a massive transfusion, and whether
a concurrent febrile transfusion reaction occurs). The development of anti-D has been studied in many
observational and retrospective studies.140'142
 The clinical magnitude of this issue is far less than one might expect. Most patients receiving platelet
transfusions are severely immunosuppressed, and a primary response to the D or other red cell antigens is very
uncommon.140,142 In addition, for most patients, the formation of anti-D has minimal impact on their
subsequent hemotherapy support. However, if the patient is a female of childbearing potential, the formation of
anti-D could have a significant impact on her future pregnancies. Therefore, attempts are usually made to
provide Rh-negative recipients with Rh-negative platelet units even though platelets do not express or carry Rh
antigens. When an Rh-negative patient must receive an Rh-positive unit of platelets, a dose of Rh Immune
Globulin (RhIG) may be administered to prevent Rh(D) alloimmunization.
Rather than provide RhIG to all Rh-negative recipients of Rh-positive platelets, many centers supply RhIG
only to premenopausal females. Given the 3-week half-life of IgG, a single dose should provide prophylaxis for
multiple transfusions over a 2- to 4-week peri
od and certainly for the period during which anti-D is detectable serologically. Because the recipient had,
and probably still has, thrombocytopenia, an intravenous form of RhIG may be administered to avoid a
hematoma, particularly if the platelet count remains below 50,000/pL.143
Alloimmunization: Prevention and Response
Although patients who are receiving multiple platelet transfusions are usually immunosuppressed by virtue
of their underlying disease and/or therapy, they are still able to mount an immune response against antigens on
platelets. This response is usually in the form of antibodies to HLA Class I antigens, but some patients may
make antibodies to platelet-specific antigens. The former require presentation of the antigens via donor
lymphocytes. Leukocyte reduction of both platelet and RBC units has proven to be highly effective in reducing
the risk of primary HLA alloimmunization.33 However, if a patient has previously been sensitized, such as
through pregnancy or a prior, nonleukoreduced transfusion, transfusion of the Class I antigens on the platelets
may be sufficient to provoke an anamnestic response and the appearance of platelet refractoriness.
For patients who can be expected to require multiple platelet transfusions, such as those who will undergo
hematopoietic stem cell transplantation, advance knowledge of their (current) alloimmunization status may help
blood services prepare to provide specially selected units. Usually, however, the hunt for alloantibodies begins
when alloimmunization is suspected from poor responses to platelet transfusions that cannot be explained by
other, nonimmunologic factors, such as splenomegaly or sepsis. The availability of kits to screen for HLA and
other platelet-directed antibodies by enzyme immunoassay or other simple approaches has expanded the
number of laboratories able to perform this testing and provide clinically useful information about the results
with a short turnaround time. Identification of the specificities of multiple HLA anti
516
AABB TECHNICAL MANUAL
bodies, however, may still require lymphocytotoxicity testing in some circumstances.
Before the ready availability of such testing, the detection or supposition of immunologic platelet
refractoriness led to a call for HLA-matched platelets.144,145 Even a large donor registry may not be able to
provide a completely matched unit for a given patient; many “HLA-matched” units actually carry antigens
against which the patient may have an alloantibody. (The presence of serologic cross-reactive groups prevents
recognition of some antigens as foreign but, at the same time, reduces the range of donors whose platelets are
truly compatible.) Platelet crossmatching is another approach that can be utilized, with the lack of in-vitro
reactivity used as a predictor of good in-vivo compatibility. This technique is often still used when
alloimmunization is directed at a platelet antigen because few blood-collection agencies have phenotyped their
donors for these antigens. However, neither approach can guarantee a good result more than 50% to 80% of the
time, although a “good” HLA match (A or BU grade) provides the best prediction of transfusion success.146
An alternative, simpler approach that parallels the practice with patients who are alloimmunized against red cell
antigens is the selection of platelet units that lack the antigen(s) against which the recipient is immunized and
those from cross-reacting groups (Fig 20-1).147
Antibodies provoked by or directed at a wide variety of drugs can lead to thrombocytopenia.148 These
drug-dependent platelet antibodies (DDPAs) can lead to mild or profound thrombocytopenia and to
refractoriness to transfused platelets.149 Although often thought of as rare, DDPAs can be caused by many
drugs and are present in many patients. For example, in one study, approximately 10% of patients receiving
gentamicin had a drop in their platelet counts, and almost half of these patients (5.9% overall) had an antibody
that interacted with platelets in the presence of gentamicin.150 When refractoriness to platelet transfusion
cannot be explained by the patient’s condition or alloimmunization to HLA or platelet antigens, consideration
should be given to the presence of DDPAs.
Dealing with Platelet Refractoriness
Responses to platelet transfusion are most often quantitated using the CCI 1 hour after transfusion (Table
20-5).151 However, a sample collected 10 minutes after transfusion yields similar information and may be
easier to obtain routinely.152 The CCI calculation is based on the count increment (count increment =
posttransfusion count - pretransfusion count), platelet content of the unit (expressed as x 10'11), and size of the
patient [expressed as body surface area (BSA) in m2]. For an adult patient with a BSA of 2.0 m2 whose platelet
count rose from 5,000/pL to 25,000/pL after a platelet transfusion containing 4.0 x 1011 platelets, the CCI
would be:
(count increment x BSA)/unit content = (20,000/pL x 2.0 m2)/4.0 = 10,000
Units (m2/pL) are usually omitted when reporting the result. A CCI above 7500 is considered evidence of a
successful transfusion; two transfusions with CCIs below 7500 within an hour after transfusion are evidence of
refractoriness. A similar approach is used to compare the recovery rate of platelets to the expected rate.108
Occasionally, despite diligent efforts, no compatible platelets can be found to transfuse to a patient with
platelet refractoriness. Alternative measures can be tried to stem or forestall hemorrhage, but these are of
unpredictable benefit. Administration of antifibrinolytic agents, such as s-aminocaproic acid (Amicar), either
intravenously or, in the case of gingival bleeding, as a mouthwash may allow the clot that is formed to be
sustained.108
Curiously, administration of intravenous Ig (MG), an effective therapy for autoimmune thrombocytopenia,
appears to be of little benefit in alloimmune refractoriness. MG can yield increased increments shortly after
transfusion, but the durability of the response is limited and, by 24 hours after transfusion, the patient usually
returns to his or her baseline level.108 The claim that platelets are accumulating at sites of bleeding even though
they are not detected in circulation via a platelet count
517
CHAPTER 20 HemotherapyDecisions
increment has not been substantiated and is unlikely to be true.108,153'155 Some experts have advocated
administration of platelets by slow drip, perhaps after making aliquots of a therapeutic dose and administering it
over 4 to 12 hours. This approach has the benefit of allowing the clinician to feel that “something” is being done
while minimizing the demand on the transfusion service inventory; however, the predicted benefit to the patient
is based primarily on anecdotal experience.
Contraindications to Platelet Transfusion
Idiopathic (autoimmune) thrombocytopenia (ITP) can cause profoundly low platelet counts, but patients
with autoimmune ITP (particularly children) rarely suffer hemorrhagic consequences.156,157 The transfusion
of platelets in the stable, nonbleeding patient with ITP offers no benefit because the platelets are rapidly cleared
by the circulating antibodies. In situations where the patient is bleeding, clinicians may feel compelled to take
some action, including using antifibrinolytic agents and platelet transfusion. Success in stemming hemorrhage
with transfusion is not universal, but bleeding may slow down or cease after transfusion in perhaps half or two-
thirds of attempts; this proportion is high enough to warrant trying transfusion, particularly when the bleeding is
serious.158
Thrombotic thrombocytopenic purpura (TTP) has traditionally been regarded as a contraindication to
platelet transfusion.159,160 Thrombocytopenia occurs in TTP as platelets are activated and consumed in
thromboses triggered by abnormally large multimers of vWF that are capable of inciting inappropriate platelet
activation without other cofactors.161 The resulting thrombocytopenia may be protective, then, in slowing the
formation of additional pathologic thromboses. Case reports describing the development of coma in close
temporal relationship to platelet transfusion led to the idea that platelets may “add fuel to the fire” and prompt
further thromboses in critical sites. A recent review of the literature accompanied by a prospective observational
study of 54 patients with TTP found no increased frequency of neurologic events or death in patients who
received platelet transfusions.154 Other, well-designed trials are needed to refute or confirm the belief that
platelet transfusion is contraindicated in TTP.
Platelet transfusion is also usually avoided in patients with heparin-induced thrombocytopenia (HIT),
especially the immunologic (Type II) form, to forestall the development of limb- and life-threatening
thromboses.162
Other Uses of Platelets
 Autologous or allogeneic platelets may also be applied topically to an area of surgical reconstruction. The
presence of platelet-derived growth factor through applications of PRP or platelet gels is thought to stimulate
angiogenesis and promote more rapid tissue repair.163,164 A recent systematic review of RCTs on PRP
concluded that, although the use of PRP may reduce the volume of blood components transfused for an average
savings of 247 mL per patient, the use of PRP in adult elective surgery cannot be justified due to the risk of
adverse events, such as hypotension, and the fact that other blood-sparing techniques (eg, use of a lower
transfusion threshold or antifibrinolytic agents) are supported by stronger evidence.165
PLASMA TRANSFUSION
Indications for Plasma
The current data needed to support evidencebased guidelines for plasma transfusion are surprisingly weak.
A systematic review of the available data for subsequent development of guidelines by an expert panel found
the data to be sparse and of low quality.166 The panel developed a guidance document after taking into account
the potential benefits and harms in the available data.167 Indications for plasma transfusion with reasonable
support included massive transfusion and reversal of warfarin anticoagulation in patients with intracranial
hemorrhage. In other clinical scenarios, such as surgery without massive transfusion or
518
AABB TECHNICAL MANUAL
warfarin reversal in the absence of intracranial hemorrhage, the panel did not develop recommendations due
to insufficient data. The panel recommended against plasma infusion in situations that might result in
prophylactic plasma infusion, such as acute pancreatitis or critically ill nonsurgical noncardiac patients. In these
types of patients where coagulopathy or bleeding is absent, the risk of lung injury and mortality outweigh any
perceived benefit.165 A more recent review echoes the conclusion that prophylactic plasma infusion may not
provide benefit.168
Common Clinical Scenarios and the Limitations of Laboratory Tests to Support Transfusion Decisions
Many studies have shown that abnormal coagulation test results do not predict an increased risk of
hemorrhage.169 Standard coagulation screening tests are poor predictors of hemorrhage risk, in part because
they are inordinately sensitive to mild deficiencies of multiple procoagulants.170 As a result, the test results
become abnormal at factor levels that are not clinically associated with bleeding. Additional confusion arises
from the fact that the critical thresholds in these studies, 1.3 times the upper limit of normal or 1.5 times the
midpoint of the reference range (usually almost the same number), usually fall at about an international
normalized ratio (INR) of 1.5 to 2.0.
Because the usual target for oral anticoagulation therapy is an INR of 2.0 to 3.0, some clinicians assume that
patients with an INR close to 2.0 require correction before surgery. However, although reducing coagulation in
patients with an INR of 2.0 prevents their congenital risk factor for hypercoagulability (eg, Factor V Leiden) or
acquired abnormality (eg, prosthetic cardiac valve) from triggering unwanted clotting, this does not mean that
normal, physiologic triggers of the clotting system will be unable to cause an appropriate thrombotic sequence.
This distinction is important to make when deciding whether a patient requires correction of a slightly abnormal
coagulation level. The guidelines for plasma administration of several professional
associations, including the American Society of Anesthesiologists and the College of American
Pathologists, recommend correction of the overcoagulation by plasma transfusion prior to surgery or to
facilitate thrombosis only for an INR of approximately 1.5 to 2.O.171,172
The British Committee for Standards in Haematology noted, similarly, the limited utility of attempting to
correct mild overcoagulation.173 Their guidelines for correcting excessive anticoagulation and those of the
American College of Chest Physicians174 (Table 20-7) notably avoid calling for the use of plasma in lieu of
administration of vitamin K until very abnormal INRs and bleeding are encountered. Administration of vitamin
K can reverse the effects of warfarin promptly (within 6-24 hours) without complicating the reestablishment of
healthy levels of anticoagulation.175
Prothrombin complex concentrates (PCCs) can also be used to rapidly correct the effects of warfarin.176
Success has been reported from combining the transfusion of 2 units of plasma with three-factor PCCs, or by
using a four-factor PCC.177 This is discussed more fully in the “PCC” section below.
With the approval of new anticoagulants that act at different points of the coagulation system, questions
about reversal have arisen when emergent surgery is required or bleeding occurs. The direct thrombin inhibitors
and Factor Xa inhibitors function as their names indicate and offer the benefits of short halflives and a return to
baseline coagulation levels in approximately 24 hours with adequate renal function upon cessation of the drug.
However, there is currently no approved reversal agent. Case reports indicate that attempted reversal with
plasma in bleeding patients was not successful, and more evidence on the utility of PCCs and prohemostatic
therapies is necessary.178 Laboratory tests to determine the amount of drug circulating are still in development.
Patients with cirrhosis commonly have coagulation abnormalities due to their synthetic difficulties. They
also often experience gastrointestinal bleeding due to increased portal vein pressure. However, much more fre
CHAPTER 20 HemotherapyDecisions
519
TABLE 20-7. Guidelines for Correction of Excessive Oral Anticoagulation
Clinical Situation Guideline
INR > therapeutic
but < 5, no significant Lower anticoagulant dosage.
bleeding
Temporarily
discontinue drug if
necessary.
Omit 1-2 doses; monitor INR. Resume oral anticoagulation when INR is in
INR > 5 but < 9, no
therapeutic range or, if patient is at increased risk of hemorrhage, omit a dose and
significant bleeding
give 1-2.5 mg vitamin K, orally.
For rapid reversal before urgent surgery: 2-4 mg vitamin K, orally; repeat dose
with 1-2 mg at 24 hours if INR remains elevated.
INR > 9, no
Omit warfarin; give 5-10 mg vitamin K, orally.
significant bleeding
Closely monitor INR; give additional vitamin K, if necessary.
Resume warfarin at lower dose when INR is within therapeutic range.
Serious bleeding at
Omit warfarin.
any elevation of INR
Give 10 mg vitamin
K, by slow intravenous
infusion.
Supplement with plasma or prothrombin complex concentrate depending on
urgency of correction.
Vitamin K, infusions can be repeated every 12 hours.
Life-threatening
Omit warfarin.
hemorrhage
Give prothrombin complex concentrate with 10 mg vitamin K1 by slow
intravenous infusion.
Repeat as necessary, depending on INR.
INR = international normalized ratio.
Adapted from guidelines developed by the American College of Chest Physicians.174
quent hemorrhage and an inability to clot might be expected in these patients based on their prothrombin
times (PTs) only. The reason why people with cirrhosis do not bleed abnormally—and why intricate pro- and
antithrombotic balances are inherent in their clotting system—was recently elucidated by demonstration that
thrombin generated in vitro in plasma from healthy individuals and people with cirrhosis was the same, but only
after the addition of thrombomodulin, which activates protein C, to the test system.179
As the liver’s synthetic production declines, the amount of procoagulants circulating in plasma decreases,
but so does the level of certain anticoagulants, such as protein C.
Less activity may be expected in the procoagulant system of a patient with cirrhosis, but, at the same time,
there will be less of an opposing force from the coagulation-control system.179 The PT and partial
thromboplastin time do not reflect thrombin generation in these patients in a way that predicts bleeding. Until a
more accurate predictive test becomes available, one role of the transfusion medicine physician will continue to
be to help other clinicians understand why patients with severe liver disease often do not require heroic efforts
(or heroic amounts of plasma) to completely normalize their coagulation system.
 What happens if an attempt is made to correct the patient’s PT before a procedure?

520
AABB TECHNICAL MANUAL
Often, the answer is “surprisingly little.” The authors of one study noted that the mean reduction in INR was
only 0.03 per unit of plasma transfused.180 Another study showed that the PT decreased to the normal range in
less than 1% of plasma recipients who had mildly abnormal PTs before transfusion, and the difference in the
upper limit of normal was reduced by half in only 14.5%.181 The mean decrease in PT was only 0.2 second,
and there was no correlation between PT abnormality and subsequent RBC transfusion. This study also revealed
that only 1 in 10 patients receiving plasma had their PT rechecked within 8 hours—despite the fact that the
ordering clinician thought that the correction was clinically important and the expected shortening of PT
occurred infrequently. The small corrections in PT/INR may reflect the fact that these reductions have an
exponential relationship, rather than a linear relationship, to the proportion of coagulation factors in circulation
(Fig 20-2).
In cases of rapid bleeding, a more proactive approach may be beneficial. The alterations that occur in a
massive transfusion situation are a combination of dilutional coagulopathy, injury-driven factor consumption,
and activation of fibrinolysis.182 Correction of a true coagulopathy after it becomes clinically
manifest can be much more difficult, and increased transfusion of other components may be needed.
Although the early use of non-RBC components reduces mortality risk,16 this approach fails to take into
consideration each patient’s situation; direct involvement of a transfusion medicine specialist can best guide
hemotherapy in these complex, rapidly evolving situations. Such consultation also takes into account other
important factors, such as reduced patient temperature and the presence of acidosis that decrease the in-vivo
activity of the coagulation system significantly.183
Rapid whole-blood testing techniques have become integrated into many centers’ transfusion protocols for
massively bleeding patients. Common viscoelastic coagulation testing (VCT) platforms include
thromboelastography and rotational thromboelastometry. These tests measure the speed and strength of clot
formation. The benefits of these tests include that they generate initial results within 15-20 minutes and can be
used to assess fibrinolysis. Current challenges, however, involve lack of standardization of results, correlation
with standard coagulation tests, or training in interpreting the results.184
A recent meta-analysis of RCTs on TEGbased treatment in massive transfusion included nine studies of
cardiac surgery and one

FIGURE 20-2. Exponential relationship of international normalized ratio (INR) to % factor levels. (Used
with permission from Wayne L. Chandler, MD, Houston Methodist Hospital.)
 521
CHAPTER 20 HemotherapyDecisions
liver transplant study in 776 patients.185 The authors found no difference in mortality between TEG-guided
therapy and conventional practice. There were moderate decreases in bleeding and numbers of patients
transfused with both FFP and platelets in patients undergoing TEG-guided treatment; however, the
heterogeneity of the studies limited the ability to draw definitive conclusions. VCT use in trauma appears to
predict the need for massive transfusion and risk of mortality; however, most data come from observational
studies, and this issue would benefit from additional RCT evidence.186
Dose and Timing of Plasma Transfusion
Although the level of coagulation factors normally varies widely between 50% and 150% of the activity of
circulating clotting factors, numerous publications show that people have the ability to form clots with
significantly lower levels of clotting factors.187 For single factor deficiencies, such as deficiencies of Factors
VIII or IX, 30% activity is often needed for hemostasis. In patients with multiple factor deficiencies, such as
after trauma, factor levels closer to 40% may be needed for hemostasis (see Fig 20-2 and Table 20-8). In
addition, the further the patient’s procoagulant activity is from the normal range, the easier it is to effect
a change in the PT because the relationship between factor activity and PT is more exponential than
linear.179,188
A usual dose of plasma is 10-20 mL/kg. This dose is expected to increase the level of coagulation factors by
20% immediately after infusion. Precise prediction of the amount of plasma needed to be transfused to correct a
particular coagulopathy is not currently possible. Thus, posttransfusion repetition of the coagulation test that
prompted the transfusion is warranted.179,181
When attempting to correct a coagulopathy with plasma transfusion, the biological half-life of
procoagulants must also be considered. Factor VII has the shortest half-life in vivo (approximately 5 hours). If a
transfusion raises the patient’s activity of this factor from 30% to 45% 5 hours later, for example, the activity
level will be halfway back to the steady-state level for that patient (ie, to 37%). Additional correction attempts
must now change the factor concentration in an enlarged plasma volume, and multiple plasma transfusions can
produce pulmonary edema through fluid overload. In addition, if correction is truly required before a hemostatic
challenge, such as major surgery, the plasma should be infused shortly before the procedure for the benefit to be
incurred at the time of the hemostatic challenge.
TABLE 20-8. Coagulation Factor Half-Lives
Factor In-Vivo Half-Life % Needed for Hemostasis
1 3-6 days 12-50
II 2-5 days 10-25
V 5-36 hours 10-30
VII 2-5 hours >10
VIII 8-12 hours 30-40
IX 18-24 hours 15-40
X 20-42 hours 10-40
XI 40-80 hours 20-30
XIII 12 days <5
 522
AABB TECHNICAL MANUAL
Types of Plasma
Several types of plasma may be available for transfusion, and, for most situations, they can be used
interchangeably. To be called “FFP,” the unit must be frozen within 8 hours of collection and transfused within
24 hours of thawing, thus optimizing the levels of the most labile procoagulants, Factors V and VIII. However,
congenital Factor V deficiency is rare. Most patients requiring plasma transfusion already have an elevated
Factor VIII level because it is an acute phase reactant. Therefore, Plasma Frozen within 24 Hours After
Phlebotomy (PF24) has procoagulant contents that are as capable as FFP of correcting most clinical
coagulopathies.189,190
Thawed Plasma made from PF24 or FFP and kept refrigerated after thawing can be effectively used
throughout its 5-day shelf life because most coagulation factors remain at hemostatic levels.191,192 Thawed
Plasma can be part of an effective strategy aimed at reducing wastage of plasma in combination with the
implementation of appropriate ordering practices, as described above.
Transfusion of Plasma for Other Indications
There are several other clinical situations where the transfusion of plasma is indicated based on slightly
different rationales. Therapeutic plasmapheresis may call for partial or complete replacement of the removed
volume of patient plasma. This may occasionally be necessitated by frequent (ie, daily) procedures that can
deplete procoagulant levels more quickly than they can be replenished. However, most patients do not require
such an aggressive plasmapheresis schedule, and the need to use plasma for factor replenishment is very
unusual. TTP presents a different situation, however, because the plasma’s content of ADAMTS13 is
therapeutic, replenishing the patient’s supply that has been reduced by autoantibody or congenital deficiency,
for example.161
 Congenital deficiencies of procoagulants may require prophylactic or therapeutic re
plenishment via plasma transfusion, but patients with these deficiencies are rare. Cl esterase inhibitor
deficiency causes hereditary angioedema due to inappropriate activation of complement. Its management may
include administration of a Cl inhibitor derived from human plasma.191 Factor XI deficiency, although not a
cause of spontaneous bleeding, often results in excessive postoperative bleeding.191 In emergent situations
where specific concentrates are not available, transfused plasma can be used as a source of the deficient
factor.193,194
CRYOPRECIPITATE
TRANSFUSION
The discovery that certain proteins critical to the coagulation system could be concentrated by thawing FFP
at 4 C and centrifugally separating the plasma from the supernatant provided the first truly effective treatment
for patients with hemophilia A, the congenital deficiency of Factor VIII. Cryoprecipitated antihemophilic factor,
known as “cryoprecipitate,” is rarely if ever used today for its original purpose, having been supplanted by other
simpler and safer means of providing the deficient factor. Nevertheless, cryoprecipitate is an essential
component in modern hemotherapy.
Although the US regulatory requirements for the content of cryoprecipitate are based on Factor VIII content
(minimum = 80 U/unit), it is for its fibrinogen content that cryoprecipitate is most commonly used. When
fibrinogen consumption (eg, in DIC) and/or loss (eg, due to massive hemorrhage) occur, exogenous replacement
may be necessary to maintain plasma’s coagulation potential. The fibrinogen concentration necessary to
maintain hemostasis is in the range of 50-100 mg/dL. However, coagulation test results that are used to follow
patients with such conditions usually become abnormal due to hypofibrinogenemia when the fibrinogen
concentration drops below 100 mg/dL. Therefore, maintaining the fibrinogen concentration above this level aids
both the patient and those attempting to understand the situation through laboratory testing. When fibrinogen
levels drop as a result of
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CHAPTER 20 HemotherapyDecisions
DIC or ongoing, high-volume hemorrhage, it may be wise to initiate cryoprecipitate transfusion (or at least
prepare the component) as the critical point of 100 mg/dL is approached (eg, at approximately 120 mg/dL).
Although the dosage of cryoprecipitate is often stated in tens of units (eg, 10, 20, or 30 units), the dosage
required to achieve the desired effect is readily calculated. This calculation is based on the difference between
the current and desired (usually 200 mg/dL) concentration of fibrinogen, a projection of the patient’s plasma
volume [as (1 - hematocrit) x 0.7 dL/kg x body mass, or 30 dL if the patient’s weight is unknown], and the usual
fibrinogen content of cryoprecipitate (250 mg/unit):
Dose (units) = [desired fibrinogen increment (mg/dL) x plasma volume]/250 mg/unit
Although the small volume (5-15 mL) of each cryoprecipitate unit facilitates rapid thawing, the pooling of
units (usually including rinsing the bags to maximize recovery) can be time consuming. Planning ahead for
rapid use later in case a patient needs a massive transfusion, for example, can certainly facilitate patient care.
Some facilities have chosen to produce pools of cryoprecipitate using sterile techniques before freezing the
component. This product has a higher fibrinogen content and a shorter time to issue when needed.
Dysfibrinogenemias are rare congenital abnormalities that result in dysfunctional fibrinogen molecules.
These molecules can lead to an increased risk of thrombosis, bleeding, or both, although they can also have no
clinical manifestations.195 Patients may require administration of cryoprecipitate to correct the deficiency.
Some degree of dysfunctional fibrinogen is also produced in damaged or regenerating livers and in neonates,
but this rarely results in the need to transfuse normal fibrinogen.196
States of localized fibrinolysis can be induced during surgeries that disrupt the prostatic bed, urothelium, or
salivary gland tissue because plasminogen activators are produced by these tissues. In these cases,
cryoprecipitate infusions may be warranted to stop bleeding
with or without the use of antifibrinolytic therapy. Note that bleeding from the urinary tract is a relative
contraindication to antifibrinolytic therapy. States of systemic fibrinolysis can occur with medical conditions
such as widespread amyloidosis or as a result of chemotherapy with L-asparaginase, typically used to treat acute
lymphoblastic leukemia.
Although no longer considered for routine use due to the availability of concentrated plasma derivatives of
copurified Factor VIII and vWF, cryoprecipitate can be used as a source of vWF for patients with von
Willebrand disease (vWD). The most common form of vWD is Type 1, which results in a deficiency of a
normal protein. Type 1 vWD most often can be managed with administration of desmopressin. Patients with
Types 2 and 3 vWD need to have their bleeding managed with an exogenous source of vWF. See Table 20-9 for
general factor replacement guidelines for the treatment of vWD.
Cryoprecipitate can also be applied topically on surfaces where suturing is ineffective to achieve hemostasis
and that are slow to generate clot, such as the raw surface of traumatically injured liver.199 When applied
simultaneously with calcium and thrombin (usually using two syringes), a thick, gelatinous mass quickly forms
over the area and quells the bleeding. This approach can also be used to bind together structures, such as in
surgery in the inner ear. Cryoprecipitate is not typically requested from the transfusion service in current
practice because products that have both lyophilized plasma and thrombin are available commercially.200
Similarly, virus-inactivated fibrinogen concentrates are available that are more readily stored (lyophilized) and
packaged for simple use (discussed below).201
GRANULOCYTE TRANSFUSION
The development of apheresis technology several decades ago allowed the collection of granulocytes
(neutrophils) to transfuse to patients with neutropenia and serious infections. Most of these patients were not
able to mount an appropriate response because of marrow hypoplasia (eg, due to chemotherapy). The
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AABB TECHNICAL MANUAL
TABLE 20-9. General Factor Replacement Guidelines for Treatment of Hemophilia A and von Willebrand
Disease
Minimum Desired Factor VIII Dose Factor IX Duration
Indication
Factor Level (%) (lU/kg) Dose (lU/kg) (days)
Hemophilia*
Severe epistaxis, oral
20-30 10-15 20-30 1-2
mucosal
bleeding’
Hemarthrosis,
30-50 15-25 30-50 1-3
hematoma, persistent
hematuria,’
gastrointestinal bleed
ing, retroperitoneal
bleeding
Trauma without signs
40-50 20-25 40-50 2-4
of bleeding,
tongue/retropharyngeal
bleeding’
Trauma with bleeding,
100 50 100 10-14
surgery,
intracranial bleeding5
von Willebrand
Disease”
Major surgery 50 40-60 daily
30-50 daily or
Minor surgery 30
every other day
Dental extractions 30 20-30, single dose 12 hours
Spontaneous bleeding 30 20-30, single dose
*Data from US Pharmacopeia.197 Dosing intervals based on a half-life of Factor VIII over 8-12 hours (2-3
doses/day) and half-life of Factor IX over 18-24 hours (1-2 doses/day). Maintenance doses of one-half the
initial dose (as shown) may be given at these intervals. The dosing frequency depends on the severity of
bleeding, with more frequent dosing used for serious bleeding.
’In addition to antifibrinolytics.
♦ Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are
necessary to maintain renal output.
 §Factor may be administered continuously. Following the initial loading dose, a continuous infusion at a
dose of 3 lU/kg per hour is given. Subsequent doses are adjusted according to measured plasma factor levels.
’Concentrates labeled in terms of the ratio of von Willebrand factor to ristocetin cofactor. The recommended
doses for adults, number of infusions, and target plasma levels are the same as those for Factor VIII.198
therapy was also administered to patients with congenital neutrophil dysfunction [eg, chronic granulomatous
disease (CGD)]. Not all patients benefited from this approach, however, and only modest improvements in
outcome were noted in a majority of—but not all— trials.202’206 The published literature offers reviews of this
and other topics related to granulocyte transfusion therapy.207,208
With the development of more potent antimicrobial drugs and the availability of cytokines that promote
more rapid marrow recov
ery of granulocyte production, differentiation, and function [such as granulocyte colonystimulating factor
(G-CSF) and granulocyte/ macrophage colony-stimulating factor], the frequency of granulocyte transfusion fell
to very low levels.209,210 This low utilization was also prompted by the recognition that the amount of
granulocytes that could be collected from a donor, even one mobilized with prior administration of a
corticosteroid, on the order of 1 x 1010, was only 1% to 10% of what a healthy marrow would produce each day
in re

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CHAPTER 20 HemotherapyDecisions
sponse to a serious infection. Trials whose authors reported success in using granulocyte transfusion therapy
generally transfused higher doses of granulocytes, but these higher yields were very difficult to obtain.
More recently, the concept of granulocyte transfusion therapy using components with higher, “more
therapeutic” content has generated renewed attention to this component. Despite the availability of newer
antimicrobial agents, infection remains a common problem in patients undergoing rigorous chemotherapy
regimens, especially those culminating in hematopoietic stem cell transplantation, and more rigorous T-cell
depletion schemes in allogeneic hematopoietic transplantation that lead to an increased frequency of serious and
fatal infections, usually due to yeasts and fungi.211,212 Case reports of success in treating patients with CGD
with fungal infections during transplantation have also rekindled interest in granulocyte transfusion.213,214
With the administration to donors of 8 mg dexamethasone orally and 5 pg/kg G-CSF subcutaneously 8 to 16
hours before apheresis, yields of up to 1011 granulocytes (or more) have been reported.215 A clinical trial to
document the effectiveness of these more potent components is under way through the Transfusion Medicine
Hemostasis Clinical Trials Network of the National Heart, Lung and Blood Institute. This use of G-CSF is not
approved by the FDA, and donor informed consent should be obtained before using this approach. In addition,
the suggestion that corticosteroid administration may lead to posterior subcapsular cataracts has prompted some
clinicians to consider avoiding the dexamethasone administration to donors even though this decreases yield by
one-quarter.216 The potential utility of prophylactic granulocyte transfusions remains unclear at present, again
owing to dosage considerations.217
Neonates born preterm or after prolonged premature rupture of membranes are at increased risk of bacterial
sepsis. Their neutropenia is related to a temporary limitation in the production of neutrophils from a marrow
primed to produce red cells. Some of these patients have been treated with transfusions of
granulocytes generated either through an apheresis procedure or from whole-bloodderived huffy coats. The
latter can provide only a limited number of cells and has not been found to be as clinically efficacious as
apheresis.218 Today, potent antibiotics are used much more commonly than granulocyte transfusions in
neonates. MG may also be given because premature infants may have hypogammaglobulinemia,219 although
MG may increase these infants’ susceptibility to other potentially fatal infections.220
Patients with severe neutropenia (absolute neutrophil count <500/pL) are considered for granulocyte therapy
if the infection cannot be controlled with appropriate bactericidal antimicrobials and the neutropenia is
temporary, meaning that recovery of endogenous production in a few days is likely. Patients with CGD are
usually considered for granulocyte transfusions if they have deep-seated abscesses and/or fungal infections that
are not responding to antimicrobial therapy.
Components must be ABO compatible if the granulocyte collection contains 2 mL or more of red cells, as is
often the case. If the patient requires CMV-reduced-risk transfusions, the donor should be CMV seronegative
because leukocyte reduction cannot be used on these components. If the recipient is alloimmunized to HLA
antigens, HLA-compatible donors need to be found or the transfused cells could be cleared rapidly and possibly
cause untoward reactions without achieving their intended purpose. If patients are at risk of transfusion-
associated graft-vs-host disease, as is usually the case for patients with neutropenia, units maybe gamma
irradiated.
Donors often undergo infectious disease testing when they present for predonation stimulation to facilitate
rapid release of the collected component following documentation of ABO/Rh type. Granulocyte units should
be stored for the shortest period possible at room temperature without agitation and administered through
regular blood component filters (eg, 170 microns). Drugs known to interact with granulocytes, such as
amphotericin, are best given at times remote from the granulocyte transfusions (ie, 12 hours before or
526
AABB TECHNICAL MANUAL
after). Transfusions are usually given daily, and sometimes more frequently if yields are low, until the
patient recovers from the infection and/or neutrophil production returns.
PLASMA-DERIVATIVE
TRANSFUSION
Plasma proteins are derived from both source plasma, collected for the purpose of manufacturing plasma
derivatives, and recovered plasma that has been separated from whole-blood donations. In the United States,
approximately 6 million liters of source plasma and 2.5 million units of recovered plasma are produced each
year. The final products are subjected to one or more virus-inactivation techniques. Additional testing (eg, for
hepatitis A virus or parvovirus) may be performed by nucleic acid amplification techniques to add an additional
margin of safety.
The prions associated with variant Creutzfeldt-Jakob disease (vCJD) appear to be successively removed
through multiple steps in the separation process to levels that are beyond the limit of detection. Neither classical
CJD nor vCJD cases have reportedly been attributed to the transfusion of plasma derivatives.221
The pathogen-inactivation techniques described above have an excellent record of not transmitting
infectious diseases. However, most patients with hemophilia in the United States prefer transfusions of Factor
VIII or Factor IX concentrates produced via recombinant DNA technology and, whenever possible, that have
not been manufactured using any human proteins.
Immunoglobin products have often been regarded as free of infectious risks because of their production
method and/or neutralizing antibodies. However, multiple examples of hepatitis C transmission have raised
doubts regarding this belief, although the units involved in these cases were usually prepared through
nonstandard donor-selection or production methods.222'225
Albumin
Albumin was the first of the modern plasma proteins to be developed for transfusion. It serves as a colloid,
expanding plasma volume by approximately the volume administered at the 5% concentration with a half-life of
approximately 16 hours.226 Unless the container has been damaged, albumin’s pasteurization at 60 C for 10
hours prevents the transmission of viruses or bacteria.
There are several obvious and accepted applications for albumin, including volume replacement in patients
who are unresponsive to crystalloids, postparacentesis volume management in patients who are unresponsive to
colloids, volume replacement in patients with severe necrotizing pancreatitis, patients with diarrhea (>2 L/d) or
hypoalbuminemia (<2.0 d/dL) on enteral feedings who do not respond to short-chain peptide supplementation,
and plasmapheresis.227 Use of albumin infusions simply to raise the albumin concentration in patients with
cachexia or other chronic hypoalbuminemia is ineffective and inappropriate but may be helpful in treating the
complication of acute hypoalbuminemia if a concentration of at least 3.0 g/dL can be achieved.228
In most cases of hypovolemia, crystalloids are used as the primary means of volume replacement because of
their effectiveness, availability, and cost. A meta-analysis of recent RCTs found no decrease in mortality
associated with the use of colloids compared to crystalloids in critically ill patients, with no benefit from
albumin vs crystalloid specifically.229 When colloid support is required, other, nonhuman sources, such as
hydroxyethyl starch and dextran, may also be useful for both clinical use and apheresis, although the volume
that can be administered in a day without affecting coagulation function may be limited.230 Synthetic
substitutes may prompt an anaphylactic reaction in rare cases and have been associated with a syndrome of
pruritus beginning several weeks after administration.231
Clotting Factor Concentrates
A complete discussion of the treatment of hemophilia A (Factor VIII deficiency) and hemo
527
CHAPTER 20 HemotherapyDecisions
philia B (Factor IX) deficiency is beyond the scope of this text. Although the first effective treatment for
hemophilia A was cryoprecipitate transfusion, patients with moderate (1%5% Factor VIII activity) or severe
(<1% activity) disease are now treated exclusively with a lyophilized concentrate. The same treatment has
evolved for patients deficient in Factor IX, also anX-linked recessive condition.
For both conditions, the dose of the factor to be administered can be easily calculated based on the patient’s
plasma volume [body mass x 70 mL/kg x (1 - hematocrit)] multiplied by the desired increase in activity level.
Thus, a 70-kg male (hematocrit = 40%) with 1% activity who needs his activity level raised by 99% (0.99
IU/mL) to 100% (1 IU/mL) would require approximately 2900IU of Factor VIII. Because the half-life of Factor
VIII is 8 to 12 hours, repeat doses will be required to ensure hemostasis (ie, >30% activity) through surgery or a
bleeding episode (often a period of at least 10 days); see Table 20-9.
Because recovery of activity may vary by product and patient, close follow-up of patients using repetitive
assays of factor activity is advised. Patients may also be maintained on regular prophylactic doses of Factor
VIII to reduce the risk of spontaneous hemorrhage and preserve joint function, although the value of this
approach has been difficult to prove because of the small sizes of most trials.232,233 Nevertheless, prophylaxis
is a widely recommended course of action.
About one-third of patients with severe hemophilia A develop antibodies against Factor VIII because they
do not produce it or they produce a defective form. These patients can be very difficult to support because it is
impractical to overwhelm the inhibitor (antibody) with higher and higher doses of Factor VIII to stem
hemorrhage. A variety of treatments have been tried, including a high-dose desensitization program,
immunosuppression, and, when rapid reduction in inhibitor titers is needed, plasmapheresis. The advent of
recombinant Factor Vila to bypass the inhibition of Factor VIII activity has improved the management of
bleeding in this dreaded complication of hemophilia therapy.
Prothrombin Complex Concentrates
Procoagulants that require vitamin K for appropriate carboxylation and normal activity— Factors II
(prothrombin), VII, IX and X—are called the “vitamin K-dependent factors.” They have long been known to be
separable from plasma by simple chemical means and have been available as Factor IX complex concentrate or
PCC. This product should not be confused with a Factor IX concentrate used to treat hemophilia B. The latter,
now also available as a recombinant product, contains only Factor IX.
The first technique to produce PCC resulted in activation of a substantial proportion of some procoagulants
and sometimes led to unwanted thromboses. This complication precluded its routine use, but the product was
beneficial in bypassing the effect of Factor VIII inhibitors in some patients with hemophilia A. This activated
form of PCC concentrate was known for its Factor VIII inhibitor bypassing activity. Subsequent development of
PCC with minimal amounts of activated factors has allowed its reconsideration for broader use, particularly as
an antidote to warfarin overdosage.175
Four-factor PCCs have been available in Europe for several years and have demonstrated rapid correction of
coagulation status. In 2013, a four-factor PCC was approved by the FDA for reversal of acute major bleeding
for patients taking vitamin K antagonists.234 Because the product (Kcentra, CSL Behring, Kankakee, IL)
contains adequate concentrations of coagulation Factors II, VII, IX, and X, plasma infusion should not be
necessary. Experience with this product is limited at the time of this writing and its value for reversal of direct
thrombin inhibitors or anti-Xa inhibitors is not established.
The administration of PCCs in other offlabel settings (eg, massive hemorrhage in the absence of warfarin or
intracranial pressure monitor placement in fulminant liver failure) should be weighed against the risk of
thrombosis from giving, in the case of PCCs, a nonactivated but supraphysiologic concentration of clotting
factors. The clinical trials preceding
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AABB TECHNICAL MANUAL
FDA approval did not include patients who had experienced acute thromboembolic events, myocardial
infarction, DIC, stroke, unstable angina, or severe peripheral vascular disease within 3 months before
administration. For this reason, the package insert indicates that the product may not be suitable in patients who
have experienced thromboembolic events in the prior 3 months. In patients with a history of HIT who are
subsequently shown to be negative for HIT on enzymelinked immunosorbent assay, exposure is likely to be safe
because the heparin will be gone by the time the HIT antibody is recalled. However, use is contraindicated in
patients with known HIT. The reduced capacity of a cirrhotic liver to clear the circulating byproducts of the
coagulation cascade, such as D-dimers, may lead to DIC or other derangements of the coagulation system.
Therefore, the use of PCC in patients with liver disease remains risky.173
Recombinant Factor Vila
The production of the activated form of coagulation Factor VII via recombinant Factor Vila (rFVIIa) has led
to the exploration of this drug as a means of addressing a wide variety of hemorrhagic conditions. The drug was
developed and is approved for use in bypassing antibodies against Factor VIII in patients with hemophilia A
and achieving hemostasis without needing to achieve hemostatic levels of Factor VIII in the face of potent
antibodies. It can also be used to address congenital deficiency of Factor VII.235
Beyond these applications, rFVIIa been considered for use in a wide variety of conditions that are not
related to hemophilia or Factor VII deficiency in which bleeding is difficult to control or is threatening the
patient’s life. For example, rFVIIa has been used to counteract the effect of warfarin by replacing the inactive
factor (produced in the presence of the vitamin K antagonist) with active rFVIIa that can immediately stimulate
the downstream procoagulants and produce fibrin.236 The use of rFVIIa has also received much attention in
trauma treatment, liver transplantation, and massive transfusion, where correction of pro
coagulant deficiency and achievement of hemostasis can be challenging.237,238 However, rFVIIa is less
effective in patients with significant acidosis or hypothermia.
In a registry that collected reports of adverse events after use of rFVIIa, the drug was listed as a contributing
cause of death for a large proportion of patients who died after its administration.239 However, in a review of
35 placebo-control trials involving 4468 people, a significant increase in arterial thrombosis, but not venous
thrombosis, was attributed to rFVIIa use.240 A recent Cochrane analysis identified 29 RCTs with 4290 patients
who were treated off label with rFVIIa in a variety of clinical situations. The meta-analysis included separate
analyses of 16 studies of prophylactic use and 13 of therapeutic use in addition to an analysis of all of the trials.
The authors found no effect on mortality in the prophylactic group and a trend toward decreased mortality in the
therapeutic group. Bleeding and transfusion rate outcomes favored rFVIIa use, but these data were only
reported in the smaller studies, so these findings are of uncertain value. Of concern was a trend toward
increased thromboembolic events in the therapeutic group. When all studies were analyzed together, a
statistically significant increase in arterial thromboembolic events was found (RR 1.45; 95% Cl 1.02 to
2.05).241 Given the open question of whether any adverse events are related to unwanted clotting and the
exclusion of patients with a propensity to clot, including those with atherosclerotic disease, from many of the
trials, it may be prudent to refrain from employing rFVIIa in patients who might be at increased risk of
unwanted thrombosis.
A substantial stumbling block in the use of rFVIIa has been its cost. If it is effective in substantially
reducing hemorrhage or morbidity, then its use might be cost-effective. Another approach is to adopt a
standardized rather than weight-based dosing practice, which reduces consumption of the drug and appears to
provide similar clinical results.242 Clinical consultation by a transfusion medicine specialist may be very
helpful in guiding the use of this product.243
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CHAPTER 20 HemotherapyDecisions
Fibrinogen Concentrate
Originally, fibrinogen concentrate, which delivers a small volume of purified plasma-derived factor, was
approved by the FDA for treatment of dysfibrinogenemia or deficiency. As the pathogenesis of coagulopathy of
trauma and surgery and the importance of fibrinolysis in these situations is better understood, the use of
fibrinogen concentrate in these situations is being explored.182 Although cryoprecipitate can also replace
fibrinogen, fibrinogen concentrate can be prepared in advance without the storage and thawing issues that arise
with cryoprecipitate.
A systematic review of the use of fibrinogen concentrate and plasma in patients at risk of bleeding found 1)
decreased bleeding, 2) reduced transfusion requirements, and 3) a low rate of adverse events in patients treated
with fibrinogen concentrate. Results for bleeding and transfusion requirements were inconsistent in plasma-
treated patients.201 The heterogeneity among the studies precluded any true meta-analysis, but the overall trend
favored use of fibrinogen concentrate early in care.
Intravenous Immune Globulin
Concentrates of plasma immune globulins were developed to treat congenital immunodeficiencies and treat
or provide prophylaxis against certain viral exposures. Because immune globulins tend to polymerize during
Cohn fractionation and purification, initial preparations were given intramuscularly to avoid severe hypotensive
and/or anaphylactoid reactions. Subsequent development of a variety of chemical modifications has allowed the
development of MG preparations that have low proportions of aggregates. These products allow the
administration of larger quantities over shorter intervals to achieve more pronounced clinical effects.
Although the initial intended use of MG was to reduce infections in immunodeficient patients, the optimal
dose for this indication remains under discussion. The clinical application of MG has extended far beyond this
indication (Table 20-10).244 For a thorough review of the indications for MG, see the report
by Durabi and colleagues.245 When used for the treatment of hypogammaglobulinemia, an MG dose of 600
mg/kg in adults or 800 mg/kg in children every 4 weeks rather than the usual 300 mg/kg in adults or 400 mg/kg
in children slightly reduces the frequency and duration of infections in patients with primary immune
deficiencies.246 However, the cost-effectiveness of increased doses may be poor, and these doses accelerate
usage rates.
The ever-increasing off-label use of MG poses an additional challenge to the adequacy of the supply for
treating the 50,000 patients with congenital hypogammaglobulinemia in the United States. The FDA-approved
indications for MG account for only 30% of its usage. The other situations in which MG is administered may
have greater or lesser scientific support, but the usage rate in developed countries appears to be increasing at a
rate of approximately 10% per year. The wide variability of doses used (from < 0.03 g per person in Australia to
more than 0.06 g per person in the United States and Canada) without concomitant variations in outcomes,
however, raises the question of the true utility of some of these applications.
The administration of MG can be associated with a wide variety of adverse events (Table 20-11). Some of
these effects appear to result from the use of idiosyncratic combinations of a particular product with a particular
patient. Therefore, advantage should be taken of the wide variety of MG products in the marketplace to find the
form of the medication that is well tolerated by a given patient.247 Slow rates of infusion and pretreatment with
antihistamines and/or steroids usually prevent reactions. Clinically dramatic reactions usually occur only in
patients with agammaglobulinemia who have not previously been treated and who have an infection. IgA may
be present in sufficient concentration in some preparations to prompt an anti-IgA reaction in sensitized patients.
Although the antibody specificities in MG products represent the range and relative concentrations of the
donors’ immune globulin responses and, thus, may convey varying degrees of protection against particular
patho
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AABB TECHNICAL MANUAL
TABLE 20-10. Applications of Intravenous Immune Globulin Primary Immune Deficiencies
Hypo/agammaglobulinemia Selective antibody deficiency Class/subclass deficiency with recurrent infection
Secondary (Acquired) Immune Deficiencies Chronic lymphocytic leukemia Multiple myeloma
 Prevention of cytomegalovirus pneumonitis after hematopoietic stem cell transplantation Reduction of
bacterial infection in children with acquired immunodeficiency syndrome
Immune Gytopenias
(Auto)immune thrombocytopenic purpura [idiopathic thrombocytopenia (ITP)]*
Pure red cell aplasia
Other cytopenias: neonatal alloimmune thrombocytopenia, posttransfusion purpura, and warm autoimmune
hemolytic anemia
Human immunodeficiency virus-related idiopathic thrombocytopenia (Presumed) Autoimmune Disorders
Kawasaki disease Guillain-Barre syndrome Multiple sclerosis Myasthenia gravis Dermatomyositis Systemic
vasculitides
Factor VIII inhibitor deficiency (congenital/acquired hemophilia)
Prevention/treatment of infections
Post-hematopoietic stem cell transplantation Post-solid-organ transplantation and immunosuppression
Parvovirus infection Neonatal sepsis
Other (Presumed) Immunologic Conditions Recurrent spontaneous abortion Graft-vs-host disease Asthma
Myocarditis

531
TABLE 20-10. Applications of Intravenous Immune Globulin (Continued)
Inflammatory bowel disease Stevens-Johnson syndrome Other Conditions Alzheimer’s disease
Predisposition to atherosclerosis Autism
Chronic fatigue syndrome Multifocal motor neuropathy
Rh prophylaxis (if patient cannot receive intramuscular product)
Approved indications shown in bold. Commonly accepted indications shown in italics.
‘Usually used only in chronic UP because most acute UP cases are self-limited. (Many other agents
potentially effective in UP are being developed.244
gens, the large pool sizes used to manufacture these products reduces this variability. Hyperimmune
globulins are manufactured from samples provided by donors chosen for their higher titers of activity against
selected agents, such as hepatitis A or B virus, tetanus, rabies, varicella-zoster virus, or CMV, but are usually
available only in the intramuscular form.
The products may, on occasion, convey sufficient isoagglutinins or red cell alloantibodies of a particular
specificity (usually antiD) to cause a positive direct antiglobulin test result in the recipient, but clinically
detectable or significant hemolysis following use of these products is rare.248 Administration of hyperimmune
anti-D to an Rh-positive patient for treatment of ITP may induce a life-threatening or fatal acute hemolysis, and
the product carries a black-box warning to remind physicians of this potential outcome.
Antithrombin
Antithrombin circulates in plasma as a serine protease inhibitor that inactivates the serine proteases of the
coagulation system, including thrombin and Factors IXa, Xa, XIa, and Xlla, by covalent binding at their serine
active site.249 The ability of antithrombin to accomplish this
inhibition is greatly accelerated by heparin, which induces a change in the polypeptide’s conformation; this
is why antithrombin is known as “heparin cofactor.”
Patients who are congenitally deficient in antithrombin have an increased risk of developing thrombosis.250
Acquired deficiency of antithrombin can also occur due to reduced antithrombin synthesis (as in hepatic
disease), increased antithrombin loss (as in a nephrotic syndrome), increased antithrombin breakdown (eg, due
to L-asparaginase treatment), or increased antithrombin consumption (eg, in DIC, trauma, or surgery). The
administration of heparin also accelerates the metabolism of antithrombin and can lead to a relative resistance to
heparin.250
Although antithrombin is stable in frozen or thawed plasma, antithrombin deficiencies are usually addressed
through the infusion of antithrombin concentrate. Congenital antithrombin deficiency is rare. However,
antithrombin has been used in the treatment of sepsis and DIC to stem thrombotic complications and in patients
being heparinized for extracorporeal circulation who do not experience the expected effects of heparin
treatment.
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AABB TECHNICAL MANUAL
TABLE 20-11. Adverse Events Associated with Intravenous Immune Globulin Use
Infusion Associated
Fever Chest tightness
Chills Dyspnea
Facial flushing Wheezing
Tachycardia Hypotension
Palpitations Anxiety
Abdominal pain Nausea
Headache Vomiting
Lumbar pain Urticarial rash
Other Possible Adverse
Events
Volume overload
Arterial thrombosis
(myocardial infarction,
stroke)
Venous thromboembolism
Disseminated intravascular
coagulation
Hemolysis (due to alloanti
bodies/isoagglutinins)
Nephrotoxicity
Neutropenia
Activated Protein C
Protein C is a serine protease that acts with protein S to hydrolyze and thus inactivate Factors Va and Villa.
It therefore serves an antithrombotic regulatory function. Protein C is normally activated along the
prothrombotic cascade, which leads to an innate regulation of the clotting system. When given as an infusion
over 96 hours to patients with sepsis, recombinant activated protein C concentrate reduced mortality by 19%,
although the risk of serious bleeding almost doubled.251
04-Antitrypsin
a,-Antitrypsin (also known as a,-proteinase inhibitor) is a natural inhibitor of the elastase elaborated by
activated polymorphonuclear leukocytes. If the activity of elastase is left unchecked, as in congenital
deficiencies of 04antitrypsin, excessive damage to pulmonary parenchyma occurs after even minor infections,
leading to development of fatal emphysema at an early age in the 100,000 patients in the United States with this
deficiency. Hepatic cirrhosis can also occur.
 Until the development of several plasmaderived replacements,252 congenitally deficient patients could stem
the advance of tissue destruction only by attempting to avoid and rapidly treat respiratory infections. Periodic
(usually weekly) infusions of 04-antitrypsin can be administered prophylactically to prevent additional lung
damage in these patients.
KEY POINTS
1. Transfusion in the presence of an autoantibody that precludes a negative crossmatch is safe and could be
life-saving, provided that alloantibodies can be excluded.
2. The data needed to support evidence-based guidelines for RBC transfusion have become stronger over
the past several years. The data needed to support plasma transfusion remain surprisingly weak.
3. Compared to amounts of circulating platelets and coagulation factors in healthy people, the amount
required to achieve hemostasis is surprisingly small. When component therapy is used, the aim is to achieve
hemostatic levels rather than normal amounts.
4. The laboratory test results that are currently available to aid in component therapy decisions do not
correlate with an individual patient’s physiologic and hemostatic state.

CHAPTER 20 HemotherapyDecisions
533
5. Data on using blood components to reverse the effects of newer antiplatelet agents and anticoagulants are
very limited, as are the data on using clotting factor concentrates.
6. In the setting of hemorrhagic shock, the current data support the transfusion of platelets, plasma, and
cryoprecipitate early in the resuscitation effort. An increased ratio of these products to RBCs, often referred to
as a 1:1:1 protocol, has been shown to decrease mortality in one large prospective cohort study and a series of
retrospective studies.
 7. For patients without underlying cardiac disease, the data support using an RBC transfusion threshold of 7
to 8 g/dL of hemoglobin. For patients with underlying cardiac disease, current guidelines recommend a
threshold of 8 g/dL of hemoglobin or less. Data are lacking on the appropriate transfusion threshold in patients
with acute coronary syndrome; therefore, a transfusion trigger cannot be established in this setting at this time.
The decision to transfuse RBCs should also take into account the patient’s clinical situation and response to
anemia.
8. Although prophylactic platelet transfusions during aplasia are common practice, it is uncertain whether a
prophylactic vs a therapeutic transfusion approach is optimal.
9. The most common prophylactic transfusion threshold is 10,000 platelets/pL. The current standard
prophylactic dose of platelets of 3 to 4 x 1011 for an adult may be halved without risk of bleeding.
10. When platelets bearing ABO antigens that are foreign to the recipient are transfused, the effect on the
platelet count may be blunted. When plasma in platelet units contains antibodies against A and/or B antigens
expressed on the recipient red cells, hemolysis may occur, with a 1:3000 to 1:10,000 risk from apheresis
platelets. Steps are often taken to limit the amount of incompatible plasma transfused or to avoid high-titer
units, especially for pediatric patients, when time permits.
11. Alio immunized platelet refractory patients may be supported by transfusion of platelets from donors
lacking the targeted epitopes by either phenotyping the donor (unit) or providing HLA-matched units to patients
with HLA alloimmunization.
12. There are very limited data to suggest a benefit in transfusing plasma in settings other than intracranial
hemorrhage after anticoagulation with vitamin K antagonists or massive transfusion.
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190. O’Neill EM, Rowley J, Hannson-Wicher M, et al. Effect of 24-hour whole-blood storage on clotting
factors. Transfusion 1999;39:488-91.
Hemotherapy Decisions
541
191. Scott E, Puca K, Hearly J, et al. Evaluation and comparison of coagulation factor activity in fresh-
frozen plasma and 24-hour plasma at thaw and after 120 hours of 1 to 6 degrees C storage. Transfusion
2009;49:1584-91.
192. Sidhu RS, Le T, Brimhall B, Thompson H. Study of coagulation factor activities in apheresed thawed
fresh frozen plasma at 1-6 degrees C for five days. J Clin Apher 2006;21:2246.
193. Craig T, Pursun EA, Bork K, et al. WAO guideline for the management of hereditary angioedema.
World Allergy Organ J 2012;5:182-99.
194. Bolton-Maggs PHB. The rare inherited coagulation disorder. Pediatr Blood Cancer 2013;60: S37-40.
195. Cunningham MT, Brandt JT, Laposata M, Olson JD. Laboratory diagnosis of dysfibrinoginemia. Arch
Pathol Lab Med 2002;126:149-55.
196. Martinez J. Disorders of fibrinogen. In: Hoffman R, Benz EJ, Shattil SJ, et al, eds. Hematology: Basic
principles and practice. 3rd ed. New York: Churchill Livingston, 2000:1924-36.
197. United States Pharmacopoeial Convention. Hemophilia managment. Transfus Med Rev 1998;12:128-
40.
198. Mannucci PM. How I treat patients with von Willebrand disease. Blood 2001;97:1915-19.
199. Lupinetti FM, Stoney WS, Alford WC Jr, et al. Cryoprecipitate-topical thrombin glue. Initial
experience in patients undergoing cardiac operations. J Thorac Cardiovasc Surg 1985;90: 502-5.
200. Tisseel package insert. Westlake Village, CA: Baxter Healthcare Corporation, 2013.
201. Kozek-Langeneker S, Sorensen B, Hess J, Spahn DR. Clinical effectiveness of fresh frozen plasma
compared with fibrinogen concentrate: A systematic review. Crit Care 2011; 15:R239.
202. Graw RG Jr, Herzig G, Perry S, Henderson IS. Normal granulocyte transfusion therapy. N Engl J Med
1972;287:367-76.
203. Fortuny IE, Bloomfield CD, Hadlock DC, et al. Granulocyte transfusion: A controlled study in patients
with acute non-lymphocytic leukemia. Transfusion 1975;15:548-58.
204. Higby DJ, Yates JW, Henderson ES, Holland JF. Filtration leukapheresis for granulocyte transfusion
therapy. N Engl J Med 1975;292:761-7.
205. Vogler WR, Winton EF. A controlled study of the efficacy of granulocyte transfusions in patients with
neutropenia. Am J Med 1977;63: 548-55.
206. Herzig RH, Herzig GP, Graw RG Jr, et al. Successful granulocyte therapy for gram-negative
septicemia. N Engl J Med 1977;296:701-5.
207. Strauss RG. Granulocyte (neutrophil) transfusion therapy. In: Mintz PD, ed. Transfusion therapy:
Clinical principles and practice. 3rd ed. Bethesda, MD: AABB Press, 2011:433-47.
208. Price TH. Granulocyte transfusion therapy. Transfusion 2006;46:1-5.
209. Gabrilove J. The development of granulocyte colony-stimulating factor and its various clinical
applications. Blood 1992;80:1382-7.
210. Anderlini P, Przepiorka D, Champlin R, Korbling M. Biologic and clinical effects of granulocyte
colony-stimulating factor in normal individuals. Blood 1996;88:2819-25.
211. Pirsch JD, Maki DG. Infectious complications in adults with bone marrow transplantation and T-cell
depletion of donor marrow. Ann Intern Med 1986;104:619-32.
212. Morrison VA, Hakke RJ, Weisdorf DJ. NonCandida fungal infections after bone marrow
transplantation: Risk factors and outcomes. Am J Med 1994;96:497-503.
213. Ozsahin H, von Planta M, Muller I, et al. Successful treatment of invasive aspergillosis in chronic
granulomatous disease by bone marrow transplantation, granulocyte colony-stimulating factor-mobilized
granulocytes, and liposomal amphotericin-B. Blood 1998;92:271924.
214. Bielorai B, Toren A, Wolach B, et al. Successful treatment of invasive aspergillosis in chronic
granulomatous disease by granulocyte transfusions followed by peripheral blood stem cell transplantation. Bone
Marrow Transplant 2000;26:1025-8.
215. Liles WC, Rodger E, Dale DC. Combined administration of G-CSF and dexamethasone for the
mobilization of granulocytes in normal donors: Optimization of dosing. Transfusion 2000;40:642-4.
216. Ghodsi Z, Straus RG. Cataracts in neutrophil donors stimulated with adrenal corticosteroids.
Transfusion 2001;41:1464-8.
217. Vamvakas EC, Pineda AA. Determinants of the efficacy of prophylactic granulocyte transfusions: A
meta-analysis. J Clin Apher 1997; 12: 74-81.
218. Vamvakas EC, Pineda AA. Meta-analysis of clinical studies of the efficacy of granulocyte transfusions
in the treatment of bacterial sepsis. J Clin Apheresis 1996;11:1-9.
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219. Jenson HB, Pollock BH. The role of intravenous immunoglobulin for the prevention and treatment of
neonatal sepsis. Semin Perinatol 1998;22:50-63.
220. Weisman LE, Weisman E, Lorenzetti PM. High intravenous doses of human immune globulin suppress
neonatal Group B streptococcal immunity in rats. J Pediatr 1989;115:445-9.
221. Foster PR. Prions and blood products. Ann Med 2000;32:501-13.
222. Berger A, Doerr HW, Scharrer I, Weber B. Follow-up of four HIV-infected individuals after
administration of hepatitis C virus and GBVC/hepatitis G virus-contaminated intravenous immunoglobulin:
evidence for HCV but not GBH-C.HGV transmission. J Med Virol 1997; 53:25-30.
223. Report of the tribunal of inquiry into the Blood Transfusion Service Board. Ireland: Government
Publications, 1996.
224. Power JR Davidson F, O’Riordan J, et al. Hepatitis C infection from anti-D immunoglobulin. Lancet
1995;346:372-3.
225. Yap PL. Intravenous immunoglobulin and hepatitis C virus: An overview of transmission episodes with
emphasis on manufacturing data. ClinTher 1996;18(SupplB):43:58.
226. Doweiko JR Nompleggi DJ. The role of albumin in human physiology and pathophysiology. Part III:
Albumin and disease states. J Parenter Enteral Nutr 1991;15:476-83.
227. Vermeulen LC Jr, Ratko TA, Erstad BL, et al. A paradigm for consensus: The University Hospital
consortium guidelines for the use of albumin, nonprotein colloid and crystalloid solutions. Arch Intern Med
1995;155:373-9.
228. Vincent JL, Dubois MJ, Navickis RJ, Wilkes MM. Hypoalbuminemia in acute illness: Is there a
rationale for intervention? A metaanalysis of cohort controlled trials. Ann Surg 2003;237:319-34.
229. Perel P, Roberts I, Ker K. Colloids versus crystalloids for fluid resuscitation in critically ill patients.
Cochrane Database Syst Rev 2012;6: CD000567.
230. Brecher ME, Owen HG, Bandarenko N. Alternatives to albumin: Starch replacements for plasma
exchange. J ClinApher 1997;12:146-53.
231. Bork K. Pruritis precipitated by hydroxyethyl starch: A review. Br J Dermatol 2005;152:3-12.
232. Manco-Johnson MJ, Abshire TC, Brown D, et al. Initial results of a randomized, prospective trial of
prophylaxis to prevent joint disease in
young children with factor VIII (FVIII) deficiency (abstract). Blood 2005; 106(Suppl) :6a.
233. Stobart K, Iorio A, Wu JK. Clotting factor concentrates given to prevent bleeding and bleeding-related
complications in people with hemophilia A or B. Cochrane Database Syst Rev 2006;19:CD003429.
234. KCentra package insert. Kankakee, IL: CSL Behring GmbH, 2013.
235. Mariana G, Testa MG, Di Paolantonio T, et al, ad hoc study group. Use of recombinant, activated factor
VII in the treatment of congenital factor VII deficiencies. Vox Sang 1999;77:131-6.
236. Deveras RA, Kessler CM. Reversal of warfarininduced excessive anticoagulation with recombinant
human factor Vila concentrate. Ann Intern Med 2002;137:884-8.
237. Levy M, Peters M, Buller HR. Efficacy and safety of recombinant factor Vila for treatment of severe
bleeding. Crit Care Med 2005;33:883-90.
238. Niemann CU, Behrends M, Quan D, et al. Recombinant factor Vila reduces the transfusion
requirements in liver transplant patients with high MELD scores. Transfus Med 2006; 16:93100.
 239. O’Connell KA, Wood SS, Wise RP, et al. Thromboembolic adverse events after use of recombinant
human factor Vila. JAMA 2006;295:293
8.
240. Levi M, Levy JH, Andersen HF, Truloff D. Safety of recombinant activated factor VII in randomized
clinical trials. N Engl I Med 2010; 363:1791-800.
241. Simpson E, Lin Y, Stanworth S, et al. Recombinant factor Vila for the prevention and treatment of
bleeding in patients without haemophilia. Cochrane Database Syst Rev 2012;3: CD005011.
242. Goodnough LT, Lublin DM, Zhang L, et al. Transfusion medicine service policies for recombinant
factor Vila administration. Transfusion 2004;44:1325-31.
243. Mathew P, Simon TL, Hunt KE, Crookston KR How we manage requests for recombinant factor Vila
(NovoSeven). Transfusion 2007;47:8
14.
244. Bromberg ME. Immune thrombocytopenic purpura: The changing therapeutic landscape. N Engl I Med
2006;355:164-5.
245. Durabi K, Abdel-Wahab O, Dzik WH. Current usage of intravenous immune globulin and the rationale
behind it: Massachusetts General Hospital data and a review of the literature. Transfusion 2006;46:741-53.
CHAPTER 20
Hemotherapy Decisions
543
246. Eijkhout HW, van Der Meer JW, Kallenberg CG, et al for the Inter-University Working Party for the
Study of Immune Deficiencies. The effect of two different dosages of intravenous immunoglobulin on the
incidence of recurrent infections in patients with primary hypogammaglobulinemia: A randomized,
doubleblind, multicenter crossover trial. Ann Intern Med 2001;135:165-74.
247. Nydegger UE. Immunoglobulins. In: Simon TL, Snyder EL, Solheim BG, et al, eds. Rossi’s principles
of transfusion medicine. 4th ed. Bethesda, MD: AABB Press, 2009:260-72.
248. Schwartz J, Spitalnik S, Grima KM. Severe hemolysis following administration of Rho(D) immune
globulin in an ITP patient associated with anti-C. Blood 2006; 107:2585.
249. Griffin JH. Control of coagulation reactions. In: Beutler E, Lichtman MA, Coller BS, et al, eds.
Williams’ hematology. 6th ed. New York: McGraw-Hill, 2001:1435-49.
250. Bucur SJ, Levy JH, Despotis GJ, et al. Uses of antithrombin III concentrate in congenital and acquired
deficiency states. Transfusion 1998;38:481-98.
251. White B, Perry D. Acquired antithrombin deficiency in sepsis. Br J Haematol 2001; 112:26-31.
252. Kolarich D, Turecek PL, Weber A, et al. Biochemical, molecular characterization, and glycoproteomic
analyses of oq-proteinase inhibitor products used for replacement therapy. Transfusion 2006;46:1959-77.
 Chapter 21
Administration of Blood Components
Kim Maynard, BSN, RN, OCN
a»THE SAFE ADMINISTRATION of ^(3 blood and its components requires multidisciplinary
collaboration among clinical and ancillary services and clinicians. Policies and procedures should be developed
with input from transfusionists, the transfusion service, surgeons, anesthesiology care providers, primary-care
physicians, and transport personnel. The transfusionist typically provides the last line of defense in the detection
of errors before the transfusion commences. All personnel involved in the chain of preparing, delivering, and
administering a transfusion should be given appropriate training to ensure the provision of the safest transfusion
possible for patients.
EVENTS AND CONSIDERATIONS BEFORE DISPENSING COMPONENTS
Before a transfusion begins, thoughtful consideration, planning, and preparation are required.
Recipient Consent
The AABB Standards for Blood Banks and Transfusion Services states, “The blood bank or transfusion
service medical director shall participate in the development of policies, processes, and procedures regarding
recipient consent for transfusion.”1[p44) Recipient informed consent should address indications for; risks,
benefits, and possible side effects of; and alternatives to transfusions of allogeneic blood components. Some
state laws require certain additional elements in the patient consent.
 The patient has the right to choose or refuse a transfusion and must have an opportunity to ask questions of
a learned professional before providing consent. Documentation of the consent process must be entered into the
patient’s medical record. Some facilities require an institution-approved signed consent form to document that
the consent process has occurred and the risks/benefits of transfusion were discussed with the patient or legal
Kim Maynard, BSN, RN, OCN, Breast Coordinator (former Transfusion Safety Officer, Transfusion
Medicine Service), Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire The author has disclosed
no conflicts of interest.
545

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AABB TECHNICAL MANUAL
representative. Each institution needs to have a process for recording a patient’s refusal to receive blood or
blood components in the patient’s medical record. Institutional policies should identify health-care providers
who are allowed to obtain consent and the length of time for which that consent remains valid.
Consent for transfusion must be obtained from patients who have the requisite capacity to make such
decisions. If a patient is unable to give consent, a legally authorized representative or surrogate may do so
(depending on local and state laws). If no one is available to provide consent and the need for transfusion is
considered a medical emergency, the blood component may be administered based on the doctrine of implied
consent. Individual state and local laws governing requirements for implied consent may vary, but the emergent
need for transfusion should be carefully documented in the medical record.2
Patient Education and History
The transfusionist should educate the patient about reporting any symptoms that may be indicative of a
reaction and how long the transfusion will take. The patient’s questions should be answered before the
transfusion is started.
It is important to collect a history from the patient before the component is ordered to assess whether the
patient is at increased risk of a transfusion reaction. This history includes previous transfusions and any adverse
reactions. If the patient has had previous reactions to transfusions, the medical team should determine whether
the patient needs to receive medications before the transfusion or whether special processing of the component
is indicated to mitigate the risk of an adverse reaction.
Baseline Assessment of Recipient
A baseline physical assessment should include vital signs. Many institutions also routinely measure oxygen
saturation using oximetry. The assessment should include pretransfusion symptoms, such as shortness of breath,
rash, pruritus, wheezing, and chills, as a basis for comparison after the transfusion starts. A pa
tient with renal or cardiopulmonary disease may require a slower infusion rate to prevent fluid overload, for
example.
A patient with an elevated temperature may destroy cellular components at an increased rate.3 Moreover, if
the patient presents with an elevated temperature before the transfusion, it may be difficult to determine later
whether this was caused by a transfusion reaction. Administration of an antipyretic should be considered in such
cases.
Medical Order
Blood Component
A licensed provider writes two orders for the components to be administered. The first order requests that
compatible crossmatch (if appropriate) unit(s) be prepared for the patient and notes special processing
requirements. The second order explains to the transfusionist how to administer the component, including the
transfusion rate. Both of these orders should specify:
■ Patient name and other independent identifier (eg, date of birth or medical record number).
■ Component [eg, Red Blood Cells (RBCs) or apheresis platelets] to prepare or administer.
■ Special processing required (eg, leukocyte reduction, irradiation, or washing).
■ Number of units or volume to administer.
■ Date and time for the infusion.
■ Flow rate or period for administering the component.
 Note: It is appropriate for the rate and duration of the transfusion (eg, not to exceed 4 hours from the time
that the container is entered until completion)4 to be described in a policy approved by the hospital’s medical
staff.
Laboratory Testing
After receiving an order from a licensed provider, the transfusion service ensures that compatible
components are provided. To issue blood components for a specific patient (ex
547
cept in the case of an emergency transfusion), the laboratory must collect a pretransfusion blood sample.
Typically, the sample is obtained within 3 days of transfusion, with the draw date considered to be day o.ltp36)
Institutional policies may vary regarding the sample outdate. If the patient has not had a transfusion or been
pregnant in the prior 3 months, the sample may be acceptable for longer than 3 days for testing purposes.
According to The Joint Commission, containers used for blood specimens must be labeled in the presence of
the patient.5 The sample must be labeled at the patient’s side with at least two unique identifiers (eg, the
patient’s name, date of birth, or identification number). The identification of the person collecting the sample
and the date on which the sample was collected must be traceable.1(p35)
Pretransfusion testing of the recipient’s blood sample, including for unexpected antibodies to red cell
antigens, is described in detail in Chapters 15 and 16. The time interval from sample receipt to availability of
the requested component can vary greatly. One reason is that availability depends on the inventory of
components maintained by the transfusing facility, and some components may need to be obtained from an
external supplier. Furthermore, if the test results from the pretransfusion sample show that the patient has
clinically significant red cell antibodies, obtaining corresponding antigen-negative or crossmatch-compatible
units may require additional time. Moreover, some components require thawing, pooling, relabeling, or other
preparation before release. All of these factors necessitate timely communication between laboratory and
transfusing personnel. For example, components that are pooled or require thawing may have a shortened shelf
life after being prepared (4-24 hours); transfusionists should be aware of the decreased time available to
complete a transfusion of such components.1(pp51'59)
Venous Access
The transfusionist must determine whether the patient’s venous access [with a central line,
peripheral intravenous line, implanted port, or peripherally inserted central catheter (PICC)] is acceptable
for the infusion of blood components.
Acceptable intravenous catheter sizes for use in transfusing cellular blood components range from 22 to 14
gauge.6 A 20- to 18-gauge intravenous catheter is suitable for the general adult population and provides
adequate flow rates without excessive discomfort to the patient. When an infant or a toddler is transfused, a 24-
to 22-gauge intravenous catheter may be suitable but requires infusion through a syringe (see Chapter 23).7 The
flow rate may need to be adjusted depending on the catheter size.
There is an increased possibility of hemolysis when red cells are transfused rapidly using handheld syringes
through 23-gauge or smaller needles.8 With the use of smaller catheters, blood dilution and a pump are helpful
to administer the unit to prevent slow flow rates that lead to clogging of the intravenous catheter. Units
suspended in a preservative solution, such as Adsol, usually do not require additional dilution. It is important
that the infusion line be patent before the component is received at the patient’s bedside.
Prophylactic Medications Given Before Transfusion
Although antipyretics (eg, acetaminophen) were commonly ordered in the past to reduce the risk of febrile
nonhemolytic transfusion reactions, indications for their use are controversial.9 Some providers use antipyretics
in a prophylactic manner, some wait to use them until the patient has experienced at least one febrile transfusion
reaction, and others believe that prophylactic use of antipyretics may mask the elevated temperature that results
from a transfusion reaction. Evidence supporting any of these approaches is limited.9'12 Some experts
recommend that “in the absence of definitive evidence-based studies, pretransfusion medication to prevent
transfusion reactions should not be encouraged.”13
In a Cochrane review14 of studies on premedication to prevent allergic reactions and
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AABB TECHNICAL MANUAL
febrile nonhemolytic transfusion reactions (FNHTRs), the authors stated that current evidence from three
randomized controlled trials (RCTs) involving 462 patients indicate that no pretransfusion medication regimen
reduces the risk of allergic reaction or FNHTR. They further state that no evidence shows that pretransfusion
medications prevent NHTR. However, the conclusion is based on the evidence from a review of three trials with
a moderate risk of bias and low to moderate quality. A better-powered RCT is necessary to evaluate the role of
pretransfusion medication in the prevention of NHTR.
Antihistamines (diphenhydramine and/ or H2 blockers) may be ordered as premedication for individuals
who have had allergic reactions to transfusions in the past. Meperidine or corticosteroids are occasionally
ordered for patients who have experienced severe rigors during transfusion.15 Corticosteroids have also been
used to premedicate patients who have had prior anaphylactoid reactions or recurrent febrile nonhemolytic
transfusion reactions. The onset of corticosteroid immunosuppression takes a long time. The efficacy of
premedication with corticosteroids has not been adequately assessed, and the optimal timing and efficacy of
corticosteroids have not been established in the transfusion setting. However, corticosteroids have been used to
prevent anaphylactoid reactions to iodinated contrast media in the radiology setting in patients who have had
prior reactions, a situation that is somewhat analogous to that of transfusion. The authors of a number of
publications recommend repeat corticosteroid dosing in the radiology setting, often in combination with HI and
H2 blockers over 13 to 24 hours before the procedure.16,17
If premedication is required, it should be administered in advance of the component’s arrival. If the
premedication is given orally, the transfusionist should wait 30 minutes before initiating the transfusion. If the
premedication is given intravenously, a 10-minute waiting period before initiating the transfusion is suggested.
Equipment
Blood Warmers
Infusions of cold components can cause hypothermia and cardiac complications, increasing morbidity and
mortality.18 The likelihood of clinically important hypothermia is increased when blood is transfused through a
central venous device directly into the right atrium.
Blood warmers are rarely needed during routine transfusions. However, they are used when rapid
transfusion of components is required, especially in trauma or surgery settings. Blood warmers are also
advantageous during transfusions to neonates, where hypothermia can cause serious adverse effects. Opinions
vary on the utility of blood warmers in patients with cold agglutinins.19,20
AABB Standardsltp6) states that warming devices shall be equipped with a temperaturesensing device and a
warning system to detect malfunctions and prevent hemolysis or other damage to blood or blood components.
Warming blood to temperatures >42 C may cause hemolysis.21 The transfusion service should work with
departments that use blood warmers to make sure that the devices are approved by the Food and Drug
Administration (FDA) for infusion of components. Warming devices should be validated and maintenance and
testing of alarms should be performed according to the manufacturer’s suggestions. Blood components should
not be warmed by placing them in a microwave, on a heat source, or in hot water, or by using devices that are
not approved by the FDA specifically for blood warming.
Infusion Systems
Infusion pumps or systems are used to administer fluids, medications, blood, and blood components through
clinically accepted routes of administration. These devices allow for a controlled infusion rate and, thus,
controlled administration over a desired period; they also provide an alarm system to notify clinicians of
problems with the infusion. Consequentiy, the use of infusion pumps or systems may be preferred over simple
gravity administration.
549
However, there is a potential for hemolysis of the cellular components infused through these pumps. The
manufacturer of the pump should be consulted to determine whether the pump is approved for the infusion of
blood components. If it is not approved, the institution should establish a validation plan to confirm that the
pump will not damage components before their use. Many of the electromechanical pump devices that are
approved for blood administration require use of administration sets with standard in-line filters.
Using infusion pumps to deliver transfusions via PICC lines may lead to pressure in the catheter that
exceeds the allowed pounds per square inch. The institution should ensure that the infusion pump used does not
generate pressure that exceeds the recommended limits of the catheter.22
Syringe Infusion Pumps
A syringe infusion pump may be used for small-volume transfusions to neonatal or pediatric patients. Using
this pump requires drawing the blood component through a filter into the syringe. For more details, see Chapter
23.
Pressure Devices
 The use of an externally applied pneumatic pressure device may achieve flow rates of 70 to 300 mL per
minute, depending on the pressure applied. The device should have a gauge to monitor the pressure, which
should be applied evenly over the entire bag. Pressure in excess of 300 mmHg may cause the seams of the
blood component bag to leak or rupture. When a pressure device is used, a large-gauge cannula should be
employed to prevent hemolysis.
The application of an external pressure device to the blood bag to expedite the transfusion of RBC units
causes minimal damage to the red cells and is a safe practice in the majority of patients.23 However, the use of
pressure devices has been reported to provide only a small increase in component flow rates. When rapid
infusion is desired, an increase in cannula size typically provides better results.
Availability of Emergency Equipment
The transfusionist should be prepared to obtain and administer emergency interventions when needed. Items
used to respond to a transfusion reaction include the following:
■ A 0.9% sodium chloride intravenous (IV) solution and administration set to keep an IV line open.
■ Medications to treat a reaction along with a mechanism to obtain emergency medications prescribed to
treat the sequelae of transfusion reactions.
■ A mechanism to activate emergency resuscitation measures in the event of a severe reaction.
■ Ventilatory assistance and an oxygen source.
BLOOD COMPONENT TRANSPORTATION AND DISPENSING
Delivery of Components to the Patient Area
Each institution must have policies and procedures for issuing components and ensuring that they are
delivered in a timely manner to the receiving transfusionist. Components should not leave the controlled
environment until the patient has been properly prepared, including insertion of a patent intravenous catheter,
and the transfusionist is ready to begin the infusion.
Transfusion service personnel should inspect the unit before issuing it for abnormal appearance (significant
color change, cloudiness, clots, clumps, or loss of bag integrity). The component must not be used if any of
these are noted.4 There must be a mechanism to correctly identify the intended recipient and component at the
time of the request to issue the component. Institutions should train staff to request components so that they
arrive in an expedited manner and are not left outside a controlled environment.
550
AABB TECHNICAL MANUAL
Institutions may use dedicated personnel or automated delivery systems (eg, pneumatic tube systems,
automated blood delivery robots, or blood dispensing kiosks in remote sites) to facilitate the delivery of
components to their final destination. Provision of RBCs via automated blood vending machines [remote,
automated, computerized controlled blood storage and dispensing refrigerators, such as BloodTrack HemoSafe
(Neoteric Technology, Vancouver, BC, Canada)] at the point of care may help circumvent delays in
transportation. This solution employs an electronic issuance process, requiring infrastructure and bidirectional
informatics connectivity with the central blood bank to safely issue and return blood units to/from remote
sites.24Staff education in the electronic issuance process employed by vending machines is required to use
these devices. The use of automated and remote delivery systems requires validation prior to implementation to
ensure a safe and effective system for blood delivery and remote blood issue.25
Transfusion services generally allow the issue of only 1 unit at a time unless it is an emergent or large-
volume transfusion.
1. At the time a unit is issued, AABB Standards requires a final clerical check of transfusion service records
with each unit or component. Verification must include1(p42) the intended recipient’s two independent
identifiers (name, date of birth, or patient identification number and/or unique identifier given at the time the
crossmatch sample is drawn), ABO group, and Rh type.
2. The donation identification number, donor ABO group, and, if required, donor Rh type.
3. The interpretation of results of crossmatch tests, if performed.
4. Special transfusion requirements.
5. The expiration date and, if applicable, time.
6. The date and time of issue.
AABB Standards requires that there be a process to confirm agreement among identifying information,
records, the blood or blood component, and the request. Discrepancies must be resolved before a unit is
issued.1(p42)
 Delays in Starting Transfusion
If an unexpected occurrence does not allow for the transfusion to start immediately, the blood component
unit should be promptly returned to the transfusion service for proper storage.
The transfusion service may set limits, based on validation, on the amount of time that a unit may be out of
controlled storage before it is considered unsuitable for reissue. Components may be suitable for reissue if the
appropriate temperature has been maintained.1(p40) They should never be stored or held in a patient care unit
unless there is a controlled and monitored environment for storing components. Trauma areas and surgical units
may have arrangements with the transfusion service to provide controlled storage of units. If the temperature of
a transported component falls outside of 1-10 C, reissue is not permissible.1(p47)
If a unit has been entered (spiked for transfusion), it may not be returned to the transfusion service for
reissue. It must either be infused within 4 hours of the time it was spiked, or it must be discarded.
EVENTS AND CONSIDERATIONS BEFORE COMPONENT ADMINISTRATION
Identification of the Recipient and Correct Component
Once the unit is received, a qualified transfusionist who will administer the blood or blood component to the
patient verifies the component at the patient’s side with another healthcare provider who is qualified in
performing identification verification, as determined by the hospital.5 Alternatively, a one-person verification
process using automated identification technology, such as bar coding, may be used.
The following items should be checked immediately before transfusion:
■ Appearance of the unit. Units should be returned to the transfusion service if there is discoloration,
abnormal cloudiness, pres
551
ence of clots or clumps, or loss of bag integrity.
■ Identification of patient and unit. The patient’s two independent identifiers (eg, name and identification
number) must match the information on both the label on the unit or attached tag and the medical order. The
requirements of the institution for patient identification must be satisfied. Institutions often require, for example,
an additional check to verify that the unit was selected for the patient whose sample was collected for
crossmatch.
■ Medical order. The transfusionist should verify that the component matches the unit called for in the
medical order and that any special processing in the order was performed. In particular, the transfusionist should
ensure that leukocyte reduction or irradiation was performed if ordered.
■ Blood type. The patient’s ABO group (and Rh type if required) should be compatible with that of the unit.
Interpretation of crossmatch tests (if performed) is also verified.
■ Donation identification number.
■ Expiration date (and time, if applicable).
The unit should not be transfused if the expiration date or time has passed.
The transfusion should be withheld if any discrepancy or abnormality is found.
Proper bedside identification of the recipient is the final step to prevent the administration of an incorrect
blood component to a patient. Although individuals often worry about the possibility of exposure to infectious
agents from transfusion, equal concern should focus on the inadvertent transfusion of incompatible blood.
Approximately 1 in every 19,000 units of RBCs is transfused to the wrong patient each year; 1 in 76,000
transfusions results in an acute hemolytic transfusion reaction, and 1 in 1.8 million units of transfused RBCs
results in death from an acute hemolytic transfusion reaction.26
To prevent the potentially fatal consequences of misidentification, specific systems have been developed
and marketed, including identification bracelets with bar codes and/or
radiofrequency identification devices, biometric scanning, mechanical or electronic locks that prevent
access to bags assigned to other patients, and handheld computers suitable for transferring blood request and
administration data from the patient’s bedside to the transfusion service information system in real time. Each
system provides a method to bring staff toward self-correction during the procedure.27,28 Studies show that
rates of positive recipient identification can be increased by such systems. However, none of these systems
negates the need for good quality management, such as adequate standard operating procedures, regular
training, periodic competency assessment, and system monitoring.
Infusion Sets
Components must be administered through special IV tubing with a filter designed to remove blood clots
and particles that are potentially harmful to the patient. Standard blood administration tubing typically has a
170- to 260-micron (macroaggregate) filter, but this micron size is not mandated or required. The tubing can be
primed with either 0.9% sodium chloride or the component itself. The manufacturer’s instructions should be
reviewed for proper use. The IV setup should have a mechanism that allows bypass of the blood IV
administration tubing to start 0.9% sodium chloride in the event of a reaction. A suggested mechanism is to
have a “Y” port or three-way stopcock close to the infusion site that allows for the administration of 0.9%
sodium chloride.
Microaggregate Filters
Microaggregate filters are not used for routine blood administration. These second-generation filters were
originally developed to remove leukocytes and to complement or replace the clot screen in the 1970s.29 They
have since been replaced by more efficient leukocyte reduction filters.30 Microaggregate filters have a screen
filter depth of 20 to 40 microns and retain fibrin strands and clumps of dead cells. Red cells, which are 8
microns in diameter, can flow through the filters. Microaggregate filters are typically used for the
552
AABB TECHNICAL MANUAL
reinfusion of shed autologous blood collected during or after surgery.
Leukocyte Reduction Filters
Leukocyte reduction filters are designed to reduce the number of leukocytes to less than 5 x 106 per RBC
unit (so that more than 99.9% of the leukocytes are removed from the unit). Leukocyte reduction decreases the
incidence of febrile transfusion reactions, risk of HLA alloimmunization, and transmission of cytomegalovirus
by cellular blood components (see Chapter 6).29,30 These filters are provided by various manufacturers for
prestorage use shortly after collection of the units or for poststorage use at the patient’s bedside.
Prestorage leukocyte reduction is usually preferred for a number of reasons, one of which is that it allows
monitoring of leukocyte reduction efficacy. Furthermore, the use of bedside leukocyte reduction filters has been
associated with dramatic hypotension in some individuals, often in the absence of other symptoms. This
happens more frequently with patients taking angiotensin-converting enzyme inhibitors. The use of components
that were filtered in the blood center or transfusion service before storage decreases the incidence of such
reactions.31 If a precipitous drop in blood pressure is noted, the transfusion should be stopped immediately.
It is important to verily that the filter is intended for use with the component being transfused (RBCs or
platelets) and to note the maximum number of units that can be administered through one filter. Filters designed
for RBCs or platelets may not be used interchangeably. The manufacturer’s instructions should be followed for
priming and administering blood components through the filter. Otherwise, leukocyte removal may be
ineffective or an air lock may develop, preventing passage of the component through the filter. Leukocyte filters
should never be used to administer granulocytes or hematopoietic progenitor cells.
Compatible IV Solutions
No medications or solutions other than 0.9% sodium chloride injection, USP, should be administered with
blood components through the same tubing. Solutions containing dextrose alone may cause red cells to swell
and lyse. Lactated Ringer’s solution or other solutions containing high levels of calcium may overcome the
buffering capacity of the citrate anticoagulant in the blood preservative solution and cause clotting of the
component.32
AABB Standards allows exceptions to the above restrictions when 1) the drug or solution has been
approved by the FDA for use with blood administration, or 2) there is documentation available to show that the
addition is safe and does not adversely affect the blood or component.1(p45) Acceptable solutions according to
these criteria include ABO-compatible plasma, 5% albumin, or plasma protein fraction. Certain solutions are
compatible with blood or blood components as noted in the package inserts reviewed by the FDA, including
Normosol-R pH 7.4 (Hospira, Lake Forest, IL), Plasma-Lyte-A injection pH 7.4 (Baxter Healthcare, Deerfield,
IL), and Plasma-Lyte 148 injection (Multiple Electrolytes Injection, Type 1, USP, Baxter Healthcare). There are
several formulations of Plasma-Lyte that are not isotonic or that contain calcium; package inserts must be
checked to confirm their compatibility with components.
MANUAL ADMINISTRATION
Starting the Transfusion
Once the identification of the unit and the recipient is verified, the unit is spiked using an aseptic technique.
At institutions that use Joint Commission hospital accreditation, Joint Commission requirements for the
transfusionist (HR.01.02.01) apply: “If blood transfusions and intravenous medications are administered by
staff other than doctors of medicine or osteopathy, the staff members must have special training for this duty.”33
 553
The blood administration tubing should be filled with either 0.9% sodium chloride or the contents of the
blood component. If any solution or medication other than 0.9% sodium chloride is infused before component
administration, the tubing should be flushed with 0.9% sodium chloride immediately before the blood infusion.
The infusion should start slowly, at approximately 2 mL per minute, for the first 15 minutes while the
transfusionist remains near the patient. Severe reactions may occur after as little as 10 mL has been transfused.
Potentially life-threatening reactions most commonly occur within 10 to 15 minutes of the start of a
transfusion.34
The rate of transfusion should be increased after 15 minutes to ensure the unit is administered within the 4-
hour window. The advantages of using relatively rapid transfusion rates (eg, 240 mL/hour) include correction of
deficiency as rapidly as possible as well as reduced patient and nursing time dedicated to transfusion.
Disadvantages include the potential to cause reactions (eg, volume overload) or to make reactions more severe
(eg, FNHTR, septic reactions, or allergic reactions). More rapid infusion of leukocytes (with use of
nonleukoreduced cellular components) can result in a more rapid rise in body temperature, a FNHTR, or the
transmission of bacteria and/or allergenic substances.35 Many FNHTRs as well as septic, allergic, and even
some hemolytic reactions may not be evident within the first 15 minutes.
If there is no sign of a reaction after the first 15 minutes, the flow rate can be increased to the designated
infusion rate, taking into consideration the patient’s size, blood volume, and hemodynamic condition in
determining the flow rate (see Table 21-1). Careful attention should be paid to avoid transfusing the unit too
rapidly in relation to the patient’s cardiac and/or and respiratory status.
Monitoring the Transfusion
The unit identifiers should never be removed during the transfusion.
The transfusionist should continue to monitor the patient throughout the infusion and check the IV site and
flow rate. If the IV rate has slowed down, the transfusionist should take one or more of the following actions: 1)
check to make sure that the IV is patent and there is no swelling at the site; 2) attempt to administer the
component through an infusion pump; 3) raise or elevate the unit; 4) examine the filter for air, excessive debris,
or clots; or 5) consider the addition of 0.9% sodium chloride as a diluent if the unit is too viscous. Frequent
patient monitoring during the infusion helps alert the transfusionist to a possible transfusion reaction and allows
for early interdiction.
Vital signs should be taken within 5 to 15 minutes of beginning the transfusion and then according to
institutional policy. There is little evidence to support a best practice related to the frequency of vital-sign
monitoring other than at baseline, soon after the start of the transfusion, and after transfusion.36 AABB
Standards requires that the medical record must include pre- and posttransfusion vital signs.1(p45) Vital signs
should be taken at once if there is a suspected transfusion reaction.
The transfusionist should be knowledgeable about signs and symptoms indicative of an adverse reaction and
able to act quickly (see Chapter 27). Visual observation and patient reporting of any changes should be utilized
to determine that a reaction has occurred because the patient may experience symptoms before changes occur in
vital signs. If a transfusion reaction is suspected, the transfusion should be stopped and 0.9% sodium chloride
administered. The 0.9% sodium chloride should be infused near the IV insertion site to avoid flushing the
tubing with the residual component. The unit identification information should be rechecked. The transfusionist
should notify the patient’s care provider of any suspected transfusion reaction and obtain emergency medication
orders as needed to treat the suspected reaction. It is helpful for institutions to summarize and have readily
available to the transfusionist descriptions of common reactions and immediate steps to be taken in the event of
certain symptoms.
TABLE 21-1. Blood Component Transfusions in Nonemergency Settings
554
AABB TECHNICAL MANUAL
CD
C\J
CD
o
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o
o
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to CO
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O
o
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o.
oo
^ 03 O o
C\J CM
cb
o
o
Q. ■
° -9
CQ 2 <C 03
_Q
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s: cq
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o
o
CO
— CD -Q O
H1
sI
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<§ 1 E
■O
CD
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CP
.2 O
^o
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o
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■O
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o
|— _Q LL_ O
-o —1
 03 F
2 CD CD CD -F CO
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Q. Q. CO .
E CD
Up CO
CM cb
CM
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SS
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e5
Cryoprecipitated AHF As rapidly as tolerated Infuse as soon as possi- Crossmatch and ABO com- In-line
(170-260 micron)
ble after thawing; pooling patibility not required is preferred.
555
Once the patient is stable, the transfusion service should be notified of a suspected transfusion reaction, and
institutional policy should be followed for returning the component bag and/or to order the laboratory studies
needed to evaluate the reaction.
Completing the Transfusion
The patient is assessed at the completion of the transfusion, and his or her vital signs are obtained. The bag
and tubing are discarded in a biohazard container if the transfusion was uneventful.
Because patients can experience transfusion reactions several hours to days after the transfusion is complete,
clinical staff should continue to monitor the patient periodically for 4 to 6 hours after the end of the transfusion
to detect febrile or pulmonary reactions that may be associated with blood administration. If the patient is not
under direct clinical supervision after a transfusion, clinical staff should provide written instructions to the
patient and caregiver regarding signs and symptoms to report and a phone number to call or a person to contact
should a reaction occur later.
The transfusion should be documented in the patient’s medical record. At a minimum, AABB Standards
requires documentation of the following1'11451:
1. Transfusion order.
2. Recipient consent.
3. Component name.
4. Donation identification number.
5. Date and time of transfusion.
6. Pre- and posttransfusion vital signs.
7. Volume transfused.
 8. Identification of the transfusionist.
9. Transfusion-related adverse events.
If additional units are transfused, the institution’s guidelines and/or manufacturer’s recommendations should
be consulted to determine whether the same blood administration tubing may be used for subsequent units. If
there are no contraindications from the manufacturer, institutions frequently allow the tubing to be reused as
long as subsequent
units are transfused within 4 hours of the start of the initial transfusion. Therefore, if more than 1 unit can be
infused in 4 hours, blood administration tubing sets may be used for more than one component.
UNIQUE TRANSFUSION SETTINGS
See Chapter 23 for information about transfusion in pediatric and neonatal patients.
Operating Room and Trauma: Rapid Infusions
If components need to be administered rapidly, the use of pressure infusion, large-bore administration
tubing, and 8-Fr intravenous catheters can decrease the infusion time without inducing hemolysis.37,38 Specific
tubing sets are designed for rapid blood administration with appropriate filters. Flow rates as fast as 10 to 25
mL/second (600-1500 mL/minute) have been reported with such tubing. However, at such rapid infusion rates,
steps should be taken to avoid hypothermia. Furthermore, when multiple units are infused through the same
tubing, the flow rate may decrease appreciably.
Hypocalcemia has been noted with rapid transfusions. This is usually transient and dependent on the amount
and rate of citrate infused. Calcium replacement may be administered based on the patient’s ionized serum
calcium level and the rate of citrate administration.39
Transfusion-associated hyperkalemic cardiac arrest has been reported with rapid administration of RBCs. It
may develop with rapid RBC administration even with a modest transfusion volume of between 1 (in a neonate)
and 54 units. Contributing factors are acidosis, hypoglycemia, hypocalcemia, and hypothermia at the time of
cardiac arrest.40
If components are urgently needed and a delay in transfusion could be detrimental to the patient, the
transfusion service should have a process to provide components before all pretransfusion compatibility testing
is completed. In such cases, uncrossmatched
556
AABB TECHNICAL MANUAL
units are released with a signed statement from the requesting physician indicating that the clinical situation
requires urgent release before the completion of testing. If components in the transfusion service inventory are
not immediately accessible to a trauma unit or operating room, a supply of group 0 red cells may be maintained
at these sites. The transfusion service must ensure proper storage of components at these satellite storage sites.
Out-of-Hospital Transfusion
Transfusing blood in a nonhospital setting requires a well-planned program that incorporates all the relevant
aspects of the hospital setting and emphasizes safety considerations.41,42 An outpatient surgery center,
oncology clinic, or dialysis center is likely to be able to provide medical assistance in a timely manner in the
event of an adverse reaction. Medical staff availability, medications, and equipment to handle adverse reactions
must be arranged for in advance. Staff should be competent in performing blood administration procedures and
patient monitoring. Blood administration outside the hospital should be performed by personnel with substantial
experience in blood administration in this setting.
Transfusion in the home generally allows close monitoring of the transfusion event because the personnel-
to-patient ratio is one to
one. The disadvantage is that there is no trained assistant available in the event of a severe adverse reaction.
Issues to consider when preparing for a transfusion in the home include the following:
■ Availability of a competent adult in the home to assist in patient identification and to summon medical
assistance if needed.
■ A mechanism to obtain immediate physician consultation.
■ A telephone to contact emergency personnel and easy access for emergency vehicles.
■ Documentation of prior transfusions unaccompanied by adverse reactions.
■ The ability to properly dispose of medical waste.
CONCLUSIONS
Transfusion of blood components and the creation of blood administration procedures and policies should
be patient centric. Following these policies helps the transfusionist quickly recognize and report suspected
transfusion reactions. Close monitoring and early intervention can make a critical difference in patient
outcomes. The transfusion service should periodically audit the blood administration process to identify
instances of nonconformance, analyze their causes, and institute corrective actions.
KEY POINTS
1. Blood administration involves the process of informed consent, preparation of the recipient,
administration of the appropriate product to the correct recipient, and careful observation of the recipient during
and after the transfusion for any adverse reaction. All steps must be appropriately documented in the patient’s
medical record.
2. A licensed care provider should initiate requests for blood administration with an order for the
appropriate blood component and an order for the administration of the component(s).
3. The recipient should be informed of and educated about the upcoming administration of the blood
component so that he or she can give informed consent for the transfusion.
4. A baseline assessment of the recipient should be performed for subsequent comparison.
5. Just before the planned administration, the transfusionist must verify appropriate venous access,
administration of any prophylactic medications required, and availability of required equipment (eg, blood
warmer, infusion pump, pressure devices, and emergency equipment).

557
6. Institutions should identify appropriate blood and blood component issue and delivery mechanisms to
ensure that the transfusionist receives the components in a timely manner.
7. Transfusion services should ensure that other departments are aware of the requirement for returning
components if a transfusion is delayed.
 8. Before a transfusion is initiated, verification of recipient and component identification should be
performed. The following items should be verified: 1) the appearance of the unit, 2) identity of the recipient and
unit, 3) medical order, 4) blood type, and 5) expiration date/ time of the component.
9. Components must be administered through the appropriate infusion sets and filter if indicated. No
solution other than 0.9% sodium chloride injection, USP, should be administered through the same tubing
unless the tubing has been flushed with 0.9% sodium chloride, USP, immediately before and after the
transfusion.
10. The infusion should start slowly at approximately 2 mL per minute for the first 15 minutes. During this
time, the transfusionist should remain near the patient. If no sign of reaction appears, the infusion rate can be
increased. The transfusionist monitors the patient throughout the infusion and stops the infusion in the event of
an adverse reaction.
11. Infusions must be completed within 4 hours. After completion, the transfusionist takes the patient’s vital
signs. If the patient will not be under direct clinical supervision after the transfusion, the patient and caregiver
should receive instructions regarding signs and symptoms to report and whom to report these reactions to.
12. The following information, at a minimum, about the transfusion must be documented in the patient’s
medical record: 1) the transfusion order, 2) patient consent for transfusion, 3) name of component, 4) donation
identification number, 5) date and time of infusion, 6) pre- and posttransfusion vital signs, 7) volume transfused,
8) identity of the transfusionist, and 9) any adverse reaction.
REFERENCES
1. Levitt I, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
2. Stowell CP Sazama, K, eds. Informed consent in blood transfusion and cellular therapies: Patients,
donors, and research subjects. Bethesda, MD: AABB Press, 2007.
3. Klein H, Anstee D. Mollison’s blood transfusion in clinical medicine. 12th ed. Oxford: Wiley-Blackwell,
2014.
4. AABB, the American Red Cross, America's Blood Centers, and the Armed Services Blood Program.
Circular of information for the use of human blood and blood components. Bethesda, MD: AABB, 2013.
[Available at http:// www.aabb.org (accessed November 26,2013).]
5. The foint Commission. National patient safety goals effective January 1, 2013. Oakbrook Terrace, IL:
The Joint Commission, 2013. [Available at http://www.jointcommission.org/as
sets/l/18/NPSG_Chapter_Jan2013_HAP.pdf (accessed November 26,2013).]
6. De la Roche MR, Gauthier L. Rapid transfusion of packed red blood cells: Effects of dilution, pressure,
and catheter size. Ann Emerg Med 1993;22:1551-5.
7. Barcelona SL, Vilich F, Cote CJ. A comparison of flow rates and warming capabilities of the level 1 and
rapid infusion system with varioussize intravenous catheters. Anesth Analg 2003; 97:358-63.
8. Miller MA, Schlueter AJ. Transfusions via hand-held syringes and small-gauge needles as risk factors for
hyperkalemia, Transfusion 2004;44:373-81.
9. Kennedy LD, Case LD, Hurd DD, et al. A prospective, randomized, double-blind controlled trial of
acetaminophen and diphenhydramine pretransfusion medication versus placebo for the prevention of
transfusion reactions. Transfusion 2008;48:2285-91.
10. Ezidiegwu CN, Lauenstein KJ, Rosales LG, et al. Febrile nonhemolytic transfusion reactions:
Management by premedication and cost
 558
AABB TECHNICAL MANUAL
implications in adult patients. Arch Pathol Lab Med 2004;128:991-5.
11. Geiger TL, Howard SC. Acetaminophen and diphenhydramine premedication for allergic and febrile
nonhemolytic transfusion reactions: Good prophylaxis or bad practice? Transfus Med Rev 2007;21:1-12.
12. Wang SE, Lara PN Jr, Lee-Ow A, et al. Acetaminophen and diphenhydramine as premedication for
platelet transfusions: A prospective randomized double-blind placebo-controlled trial. Am JHematol
2002;70:191-4.
13. Tobian AR, King K, Ness PM. Prevention of febrile nonhemolytic and allergic transfusion reactions
with pretransfusion medication: Is this evidence-based medicine? Transfusion 2008; 48:2274-6.
14. Marti-Carvajal AJ, Sola I, Gonzalez LE, et al. Pharmacological interventions for the prevention of
allergic and febrile non-haemolytic transfusion reactions. Cochrane Database Syst Rev2010;(6):CD007539.
15. Patterson BJ, Freedman J, Blanchette V et al. Effect of premedication guidelines and leukoreduction on
the rate of febrile nonhaemolytic platelet transfusion reactions. Transfus Med 2000;10:199-206.
16. Goss JE, Chambers CE, Heupler FA, et al. Systemic anaphylactoid reactions to iodinated contrast media
during cardiac catheterization procedures: Guidelines for prevention, diagnosis, and treatment. Cath Cardiovasc
Diagn 1995;34:99-104.
17. Tramer MR, von Elm E, Loubeyre P, Hauser C. Pharmacological prevention of serious anaphylactic
reactions due to iodinated contrast media: Systematic review. Br Med J 2006;333: 675-81.
18. Boyan CP, Howland WS. Cardiac arrest and temperature of bank blood. JAMA 1963; 183: 58-60.
19. Donham JA, Denning V. Cold agglutinin syndrome: Nursing management. Heart Lung 1985;14:59-67.
 20. Iserson KV Huestis DW. Blood warming: Current applications and techniques. Transfusion
1991;31:558-71.
21. Hirsch J, Menzebach A, Welters ID, et al. Indicators of erythrocyte damage after microwave warming of
packed red blood cells. Clin Chem 2003;49:792-9.
22. Houck D, Whiteford J. Improving patient outcomes: Transfusion with infusion pump for peripherally
inserted central catheters and
other vascular access devices. J Infus Nurs 2007;30:341-4.
23. Frelich R, Ellis MH. The effect of external pressure, catheter gauge, and storage time on hemolysis in
RBC transfusion. Transfusion 2001;41:799-802.
24. Wong KE Virtual blood bank. J Pathol Inform 2011;2:6.
25. Lum G, D'Amarino MJ. Use of Laboratory robots for transport and delivery of blood products. Lab Med
2009;40:517-22.
26. Vamvakas EC, Blajchman MA. Transfusion related mortality: The ongoing risks of allogeneic blood
transfusion and the available strategies for their prevention. Blood 2009; 113:340617.
27. Pagliaro R Rebulla P Transfusion recipient identification. Vox Sang 2006;91:97-101.
28. Koshy R. Navigating the information technology highway: Computer solutions to reduce errors and
enhance patient safety. Transfusion 2005;45(Suppl 4):189S-205S.
29. Wortham ST, Ortolano GA, Wenz B. A brief history of blood filtration: Clot screens, microaggregate
removal, and leukocyte reduction. Transfus Med Rev 2003; 17:216-22.
30. Lane TA. Leukocyte reduction of cellular blood components: Effectiveness, benefits, quality control,
and costs. Arch Pathol Lab Med 1994; 118:392-404.
31. Zoon KC, Jacobson ED, Woodcock J. Hypotension and bedside leukocyte reduction filters. Int J Trauma
Nurs 1999;5:121-2.
32. Dickson DN, Gregory MA. Compatibility of blood with solutions containing calcium. S Afr Med J
1980;57:785-7.
33. The Joint Commission. Comprehensive accreditation manual for hospitals. Oakbrook Terrace, IL: The
Joint Commission, 2013.
34. Bradbury M, Cruickshank JP. Blood transfusion: Crucial steps in maintaining safe practice. Br J Nurs
2000;9:134-8.
35. Perkins HA, Payne R, Ferguson J, Wood M. Nonhemolytic febrile transfusion reactions: Quantitative
effects of blood components with emphasis on isoantigenic incompatibility of leukocytes. Vox Sang
1966;11:578-600.
36. Oldham J, Sinclair L, Hendry C. Right patient, right blood, right care: Safe transfusion practice. Br J
Nurs 2009;18:312,314,316-20.
37. Iserson KV Knauf MA. Confirmation of high blood flow rates through 150 micron filter/ highflow
tubing. J EmergMed 1990;8:689-91.
CHAPTER 21 Administration of Blood Components
559
38. Floccare DJ, Kelen GD, Altman RS, et al. Rapid infusion of additive red blood cells: Alternative
techniques for massive hemorrhage. Ann EmergMed 1990;19:129-33.
39. Denlinger JK, Nahrwold ML, Gibbs PS, Lecky JH. Hypocalcaemia during rapid blood transfusion in
anaesthetized man. Br J Anaesth 1976;48:995-1000.
40. Smith HM, Farrow SJ, Ackerman ID, et al. Cardiac arrests associated with hyperkalemia during red
blood cell transfusion: A case series. Anesth Analg 2008; 106:1062-9.
41. Fridey JL. Practical aspects of out-of-hospital transfusion. Am J Clin Pathol 1997; 107 (Suppl 1):S64-
71.
42. Evans CS. Out-of-hospital transfusion. Transfusion 1997;37:756-67.
 Chapter 22
Perinatal Issues in Transfusion
Practice

Melanie S. Kennedy, MD; Meghan Delaney, DO, MPH; and Scott Scrape, MD
HEMOLYTIC DISEASE OF the fetUS |Qj01 and newborn (HDFN), fetal/neonatal alloimmune
thrombocytopenia (FNAIT), and immune thrombocytopenia (ITP; previously known as “immune
thrombocytopenic purpura”) affect pregnant women and their fetuses and newborns. The blood bank and
transfusion service play critical roles in supporting the diagnosis and treatment of these conditions, including
the appropriate provision of Rh Immune Globulin (RhIG).
HDFN
HDFN is the destruction of fetal and newborn red cells by maternal red cell alloantibodies that are specific
for inherited paternal red cell alloantigen(s). The maternal IgG antibody is transported across the placenta into
the fetal circulation, where it binds to the correspond
ing red cell antigen, causing the antibodycoated red cells to be destroyed by macrophages in the fetal spleen.
The fetal hematopoietic tissue initially responds by increasing erythropoiesis and releasing many of the newly
produced red cells into the circulation prematurely as nucleated precursors, a condition known as
“erythroblastosis fetalis." With worsening anemia, excess erythropoiesis occurs in the liver and spleen, causing
organ enlargement and portal hypertension. A resulting decrease in liver production of albumin leads to reduced
plasma colloid osmotic pressure, generalized edema, ascites, and effusions known as “hydrops fetalis.”
Untreated, hydrops fetalis, with its associated high-output cardiovascular failure, can lead to fetal death. Severe
disease can occur as early as 18 to 20 weeks’ gestation; severity usually increases in subsequent pregnancies.
Melanie S. Kennedy, MD, Clinical Associate Professor Emeritus, Attending Physician, Transfusion
Medicine, Wexner Medical Center, Department of Pathology, The Ohio State University, Columbus, Ohio;
Meghan Delaney, DO, MPH, Medical Director, Red Cell Genomics, Puget Sound Blood Center, Medical
Director, Blood Bank, Seattle Children’s Hospital, Assistant Professor, University of Washington, Seattle,
Washington; Scott Scrape, MD, Assistant Professor, Director, Transfusion Medicine, Wexner Medical Center,
Department of Pathology, The Ohio State University, Columbus, Ohio The authors have disclosed no conflicts
of interest.
561

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AABB TECHNICAL MANUAL
Maternal Alloimmunization
Females can be alloimmunized to red cell antigens by previous transfusion or transplantation and/or
previous or current pregnancy. Fetomaternal hemorrhage (FMH) occurs spontaneously during pregnancy and its
likelihood increases with gestational age (from 3% in trimester I to 12% and 45% in trimesters II and III,
respectively).1,2 Because exposure to fetal red cells and resulting maternal alloimmunization typically occur
late during pregnancy and at delivery, the fetus and newborn of the first pregnancy are rarely affected. The risk
of FMH is higher in women who have experienced trauma or undergone amniocentesis, cordocentesis, abortion,
or other procedures.2
Complex factors influence the ability of individuals to immunologically respond to red cell antigens. Rh(D)
is the most potent immunogen, in that a 200-mL transfusion of Red Blood Cells (RBCs) stimulates anti-D in
about 85% of D-negative individuals, except those who are immunosuppressed. About half of the remaining
15% will never become immunized, even after repeated challenges with D-positive red cells. Although as little
as 0.1 to 1 mL of Dpositive red cells can stimulate antibody production, the volumes of FMH are generally
small, which contributes to relatively low alloimmunization rates in pregnancy.3 Before Rh(D)
immunoprophylaxis, 16% of ABO-compatible, D-negative mothers with D-positive infants became immunized;
the rate was <2% in ABO-incompatible, D-negative mothers.3'5
Antibodies targeted to blood group antigens in addition to RhD can cause HDFN. The most common of
these antigens are K and c.6 Unlike HDFN caused by anti-D, HDFN caused by anti-K uniquely results in
destruction of fetal erythropoietic precursors in addition to hemolysis.7,8(p527) Other antibodies that have been
less commonly reported to cause moderate or severe disease include E, k, Kpa, Kpb, Ku, Jsa, Jsb, Jka, Fya, Fyb,
S, s, and U.8 In rare cases, anti-Ge and anti-M have been reported to cause destruction of fetal erythropoietic
precursors.
Pathophysiology of HDFN
Hemolysis occurs when the maternal antibody binds to a fetal red cell antigen, causing attachment to the Fc
receptor of macrophages in the spleen of the fetus. The rate of hemolysis and severity of disease are determined
by the IgG subclass, amount of antibody, and number of antigenic sites on the red cells.9 The subclasses IgGl
and IgG3 are more efficient in causing hemolysis than IgG2 or IgG4. Transportation of IgGl and IgG3 across
the placenta is mediated by the Fc receptor beginning in the second trimester and continuing until birth.10
Because IgGl is transported across the placenta earlier and in larger amounts than IgG3, IgGl is associated with
more severe disease. After delivery, antibody persistence can cause continuing destruction of red cells,
progressive anemia, and hyperbilirubinemia. However, the amount of maternal antibody present continues to
decrease over the next 12 weeks, with a half-life of about 25 days.
During gestation, fetal red cell destruction releases hemoglobin. The hemoglobin is broken down into
bilirubin, which passes into the maternal circulation and is conjugated by the maternal liver, preventing the
bilirubin’s return to the fetus. Birth severs the connection with the mother, causing the bilirubin (resulting from
red cell destruction) to remain in the neonatal circulation. Because of the infant’s immature liver enzymatic
pathways, the amount of unconjugated bilirubin can increase to dangerous levels and cause permanent damage
to the brain, known as “kernicterus.”11
Diagnosis
The diagnosis and management of HDFN involve the close cooperation of the patient, obstetrician, and
blood bank/laboratory personnel. During the first trimester, the patient’s obstetric and transfusion history should
be obtained. A previously affected pregnancy alerts the obstetrician to possible problems in the current
pregnancy.12
563
Laboratory Testing
During the first prenatal visit, maternal ABO and Rh should be typed and an antibody screen should be
performed. If an RhD-negative woman has an initial negative antibody screening result, she is a candidate for
RhIG administration (see “RhIG” section below). The antibody screen should be conducted using techniques
that detect IgG antibodies that are reactive at 37 C and in which separate screening cells represent all clinically
important specificities. A positive antibody screening result requires antibody identification. Antibodies such as
anti-I, -PI, -Lea, and -Leb, whether IgM or IgG, may be ignored because these antigens are poorly developed at
birth. Treatment of the mother’s plasma with dithiothreitol (DTT) can help distinguish IgG from IgM
antibodies.13
After identifying an antibody known to cause HDFN, the next step for fetal risk stratification is to test the
father for the presence of the corresponding red cell antigen. If the father is homozygous for the corresponding
antigen, 100% of the fathers’ offspring will be at risk of HDFN. If the father is heterozygous, 50% of the
father’s offspring will be at risk.
For pregnancies sensitized with anti-D, no serologic methods can determine paternal zygosity. In these
situations, paternal DNA testing is indicated.14 Using fetal amniocytes, direct testing of the fetal red cell
genotype can predict the red cell phenotype. The reported sensitivity and specificity of DNA testing by
polymerase chain reaction are 98.7% and 100%, respectively, with a low false-negative rate (1-3%).14
Molecular typing of fetal DNA can also be performed on maternal plasma early in the second trimester.15
During a sensitized pregnancy, antibody titers can assist with management decisions. The AABB-
recommended method is the use of saline antihuman globulin (AHG) incubated for 60-minutes at 37 C (Method
5-3). Other methods, such as using albumin AHG or gel, may result in higher titers than the recommended
method and should be validated with clinical findings and laboratory data to ensure appropriate interpretation
by the obstetrician
and avoid inappropriate referral of patients for high-risk obstetric care. The critical titer for anti-D (the level
below which HDFN and hydrops fetalis are unlikely and no invasive procedures are needed) is 16 in the AHG
phase. As long as the titer is 8 or lower, except in the case of anti-K, the pregnancy can be followed by titers.
Kell-system antigens are present on early red cell precursors, so even a maternal anti-K titer that is relatively
low can cause erythropoietic failure and severe anemia. A critical titer of 8 is generally accepted for Kell system
antibodies.7 Due to the poor reproducibility of titers, individual blood banks should validate their testing
internally and keep previous specimens for subsequent comparison. Flow cytometric quantitation of antibody
mass has proven to be more precise than antibody titers.16
Pregnancy Monitoring
Obstetric care usually involves monitoring affected fetuses with a combination of maternal antibody titers
and fetal ultrasound of the middle cerebral artery to assess fetal anemia.12 When a critical antibody titer is
reached at >16 weeks of gestation, ultrasound and color Doppler ultrasonography are performed to establish
disease severity. Decisions about how and when to treat an affected fetus are based on the degree of fetal
anemia and gestational age.
Treatment
In cases of severe fetal anemia from hemolytic disease, fetal transfusion is indicated to treat the anemia and
suppress fetal erythropoiesis. Intrauterine transfusion (IUT) is performed by inserting a needle into the
umbilical vein using color Doppler enhancement of high-resolution sonography to guide the procedure. A
pretransfusion sample of fetal blood is obtained at the same time to determine the fetus’s blood type, direct
antiglobulin test (DAT) result, antigen type, hemoglobin level, hematocrit, platelet count, and bilirubin level.
DNA typing may be performed as well. Cordocentesis is associated with a 1% to 2% risk of fetal infection,
bleeding, bradycardia, and/or premature
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 AABB TECHNICAL MANUAL
membrane rupture. Because cordocentesis may cause FMH, RhIG immunoprophylaxis should be
administered after the procedure to D-negative women who do not have anti-D.
Blood Product Selection and Administration
For IUT, the blood should be 1) irradiated to prevent transfusion-associated graft-vs-host disease in the
immunologically immature fetus, 2) cytomegalovirus (CMV) reduced-risk (through leukocyte reduction and/or
by being CMV seronegative), and 3) known to lack hemoglobin S to prevent sickling under low oxygen tension.
Donor group 0 RBCs that are antigen negative for the mother’s corresponding antibodies and crossmatch
compatible with maternal plasma are selected. Generally, RBC units that were collected within 7 days before
transfusion are preferred if they are available. Centers have reported that using RBCs that are antigen matched
to the mother decreases the risk of further maternal sensitization from IUT.17
In rare cases, the mother’s antibody is directed at a high-prevalence antigen and no compatible blood is
available. In these situations, the mother’s washed or frozen and deglycerolized RBCs can be used for fetal IUT.
Testing the mother’s siblings or searching rare donor registries may provide additional sources of compatible
units.
The volume of blood to be transfused can be calculated, as shown in the following example, by 1)
determining the fetal and placental total blood volume by multiplying the ultrasound-estimated fetal weight in
grams (eg, 1000 g) by 0.14 mL/g, 2) multiplying this amount (eg, 140 mL) by the difference in posttransfusion
(desired) and pretransfusion hematocrit (eg, 0.40 - 0.15 = 0.25), and 3) dividing the resulting amount by the
hematocrit of the RBC unit (eg, 0.85). In this example, the result is 41.2 mL.
Example:
[(1000 g) x (0.14 mL/g) x (0.40-0.15)1/85 = 41.2 mL
The blood volume transfused by cordocentesis and the rate of transfusion should be adjusted to
accommodate the clinical status of the fetus.18 Transfusion is repeated if needed according to the severity of the
disease or based on an estimated decline in hematocrit of 1% per day to maintain the fetal hematocrit at about
30%.
Other Treatments
Maternal plasma exchange and the administration of intravenous immune globulin (MG) have been used
early in gestation before IUT can be accomplished or as alternatives to intrauterine transfusion.4 Plasma
exchange can temporarily reduce antibody levels by as much as 75%. Because its efficacy was established
before the use of IUT was widespread, its use today is reserved for treatment failures on a case-by-case basis.
The American Society for Apheresis classifies plasma exchange as a Category II treatment based on weak
(Grade 2C) evidence for this indication.19
MG infusion has been shown to stabilize anti-D titers, and results were best when the procedure was started
before 28 weeks’ gestation in a case series of 24 patients.20
Neonatal Management
During the first days after birth, close monitoring of the bilirubin level is necessary because of the threat of
kernicterus, especially in premature neonates.21 The infant may require blue-green light therapy, which
oxidizes the elevated unconjugated bilirubin, allowing the oxidation products to be excreted in the urine. In
addition, MG may be given to the infant to help control hemolysis and, thus, elevated bilirubin. In neonates who
are unresponsive to phototherapy and MG, a double-volumeexchange transfusion removes approximately 90%
of the fetal red cells and 50% of the bilirubin. Exchange transfusion is generally unnecessary if the infant
received IUTs. See Chapter 23 for a discussion of exchange transfusion in the newborn.
565
RhIG
RhIG is available in 300-pg and 50-pg doses to prevent alloimmunization to the D antigen. The risk of a D-
negative mother becoming immunized by a D-positive fetus can be reduced from about 16% to less than 0.1%
by the appropriate administration of RhIG.3,4
Screening and Dosing for RhIG
Antepartum Administration
When a pregnant mother is D negative and the father is D positive, the fetus may be D positive and the
mother may be at risk of D alloimmunization. Such women are candidates for RhIG prophylaxis to prevent
alloimmunization. Dnegative females whose infants are D negative, D-negative females who have been
previously immunized to D, and D-positive females are not candidates for RhIG. Whether women with variants
of D should be considered to be D positive is controversial. Maternal red cell genotyping can assist with RhIG
treatment decisions because some D variants have been found to make anti-D.22,23
The American College of Obstetricians and Gynecologists (ACOG) recommends RhIG administration at 28
weeks’ gestation because 92% of women who develop anti-D during pregnancy do so at or after 28 weeks.3,24
Antenatal RhIG administration reduces alloimmunization to 0.1% compared to 1.5% with postpartum
administration only. In addition, RhIG is indicated after amniocentesis, cordocentesis, version, abortion, or
abdominal trauma.
Postpartum Administration
D-negative mothers without anti-D should receive RhIG after delivery of a D-positive infant. A postpartum
blood sample should be screened for FMH to determine the RhIG dose. The rosette test is 99.5% sensitive to an
FMH of 10 mL or more. After incubation with anti-D, indicator D-positive red cells form agglutinates (rosettes)
with the fetal D-positive red cells. Weak D phenotypes in the mother or fetus can cause false-positive or -
negative test results. Therefore, D-negative mothers with
positive results on a test for fetal blood in the maternal circulation should undergo a serologic weak D test or
molecular RHD testing. If the rosette test result is negative, a dose of 300 pg RhIG (100 pg in the United
Kingdom) is given, which is sufficient to prevent immunization by 15 mL of red cells (5 mL in the United
Kingdom) or 30 mL of whole blood. The presence of residual anti-D from antepartum RhIG does not indicate
ongoing protection.
A positive rosette test result indicates a large FMH. About 0.3% of deliveries have an FMH larger than 30
mL. When a rosette test result is positive, a quantitative test, such as the Kleihauer-Betke (acid/elution) test, or
flow cytometry is needed to calculate the dose of RhIG. Flow cytometry can precisely measure fetal
hemoglobin and/ or D-positive red cells. The use of both markers avoids false-positive results from maternal red
cells containing fetal hemoglobin.25,26
The Kleihauer-Betke test has been shown to be far less precise than flow cytometry because of its technical
difficulty. The KleihauerBetke test is based on the resistance of fetal hemoglobin to acid treatment (Method 5-
2). A thin smear of maternal blood is placed on a slide, treated with acid, rinsed, counterstained, and read
microscopically by counting 2000 cells. The maternal cells appear as ghosts, and the fetal cells are pink. The
formula below is used to calculate the fetal bleeding:
(Fetal cells /total cell counted) x maternal blood volume (mL) = fetal hemorrhage (mL)
Example:
(6 cells/2000 cells) x 5000 mL =
15 mL fetal whole blood
According to the results for this example, a 300-pg vial of RhIG will suppress alloimmunization by 30 mL
of fetal whole blood. Fetal hemorrhage is 15 mL, so the number of RhIG vials is 15 mL/30 mL/vial = 0.5 vial.
Because of the inherently wide estimate generated by the test, if the calculated dose to the right of the decimal
point is >0.5 vial, it should be rounded up to the next whole number plus one vial; if
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AABB TECHNICAL MANUAL
the calculated dose to the right of the decimal point is <0.5 of a vial, it should be rounded down to the next
whole number plus one vial (Table 22-1). In the above example, the dose to be given is two vials. Additional
examples are as follows:
Examples:
1.6 vials calculated =
2 (round up) + 1 (add 1) = 3
1.4 vials calculated =
1 (round down) + 1 (add 1) = 2
Postpartum RhIG should be given to the mother within 72 hours of delivery. If prophylaxis is delayed, the
ACOG recommends that treatment still be administered. If the D type of the newborn is unknown or
undetermined (eg, for a stillborn infant), RhIG should be administered.
Depending on the preparation, RhIG can be given by intramuscular (IM) or intravenous (IV) injection. If IM
preparations are used, multiple doses are given in different sites or at different times within 72 hours. Multiple
doses of the IV preparation may be administered according to the instructions in the package insert.
Serology and Mechanism
Administration of RhIG during pregnancy may produce a positive antibody screening result in
 the mother, but the titer is rarely greater than 4 and thus poses no risk to the fetus. Occasionally, the DAT
result may be positive in a newborn with no evidence of hemolysis. About 10% of the 28-week gestation dose
will be present at delivery (the half-life of IgG is 25 days). This anti-D is not active immunization, so
postpartum RhIG should be given if the newborn is D positive.
RhIG is entirely IgG, whereas active immunization has an IgM component. Thus, new anti-D produced by
the mother can often be detected in the saline phase and can be completely or partially inactivated by 2-
mercaptoethanol or DTT treatment, whereas RhIG cannot. In addition, passively acquired anti-D rarely achieves
a titer above 4. Antibody titers do not correlate with the effectiveness of the RhIG or the amount of FMH.
The mechanism of action of RhIG has not been completely elucidated. Current evidence shows that D-
positive red cells are opsonized by RhIG and removed by macrophages, which release cytokines that result in
immunomodulation.27 The number of IgG molecules known to prevent immunization is much smaller than the
D antigen sites on red cells.
ABO HEMOLYTIC DISEASE
Because of the use of RhIG, ABO incompatibility is now the most common cause of HDFN. HDFN is
triggered when naturally occurring
TABLE 22-1. Amount of RhIG to Administer Based on Amount of Fetomaternal Hemorrhage
Dose
% Fetal Cells Vials to Inject ng (meg) IU
0.3-0.8 2 600 3000
0.9-1.4 3 900 4500
1.5-2.0 4 1200 6000
2.1-2.6 5 1500 7500
Notes:
1. Based on a maternal blood volume of 5000 mL.
2.1 vial of 300 ng (1500 III) is needed for each 15 mL of fetal red cells or 30 mL of fetal whole blood.
 567
IgG anti-A,B in a group 0 mother is transported across the placenta and bound to fetal red cells expressing
A or B antigens. Destruction of fetal red cells rarely leads to severe anemia because fetal ABO antigens are
poorly developed and antibody is neutralized by tissue and soluble antigens. Also, ABO HDFN has no
complement-mediated hemolytic mechanism. If an umbilical cord DAT result is negative, ABO HDFN is
unlikely even if the mother has the corresponding ABO antibody(ies). After birth, hyperbilirubinemia can be
successfully treated with phototherapy in most cases; in rare situations, exchange transfusions may be required.
Group A and B infants of group 0 mothers are more severely affected by ABO HDFN. In populations of
European or Asian ancestry, group A infants are most commonly affected; in populations of African ancestry,
group B infants are most likely to be affected. The overall incidence of ABO HDFN is higher in people of
African than European ancestry.8(p529) In patients with severe disease, the DAT result is nearly always
positive.28
If ABO HDFN is ruled out, antibodies against low-prevalence red-cell antigens inherited from the father
should be suspected. Testing the eluate from cord blood or maternal serum (if ABO compatible) against the
father’s red cells with an antiglobulin technique is often diagnostic.
IMMUNE THROMBOCYTOPENIA
Maternal IgG antibodies to platelets can cross the placenta and cause severe thrombocytopenia. Two
categories of immune thrombocytopenia are recognized: FNAIT and ITE The diagnostic distinction between
them is important for therapy selection.
FNAIT
FNAIT is caused by antibodies that are specific for platelet antigens inherited from the father that are are
absent in the mother. Platelet antigens represent specific polymorphisms in platelet membrane glycoproteins.
Approximately 80% of FNAIT cases are caused by hu
man platelet antigen HPA-la, which is present in about 98% of the US population.29 About 10% of cases
are caused by anti-HPA-5b, 4% by anti-HPA-lb, 2% by anti-HPA-3a, and 6% by other antibodies. The incidence
of affected pregnancies is approximately 1 per 1500 to 2000.30
In about 25% of FNAIT cases, the platelet antibody develops during the first pregnancy and that fetus is
affected. The maternal antibody has been detected as early as 17 weeks’ gestation and the fetus may develop
thrombocytopenia as early as 20 weeks’ gestation. However, the disease is often not discovered until birth,
when the newborn presents with petechiae, ecchymoses, or intracranial hemorrhage. Intracranial hemorrhage
occurs in 10% to 30% of infants and 50% of fetuses with FNAIT.29 The greatest risk of hemorrhage occurs
when the fetal platelet count is less than 50,000/pL. The response of the fetal hematopoietic system to FNAIT is
variable and may include compensatory extramedullary hematopoiesis. In rare cases, hydrops fetalis develops.
Fetal anemia without red cell incompatibility can also occur.
A history of giving birth to a newborn with thrombocytopenia can alert the obstetrician to a potentially
affected pregnancy. The pregnant woman and the father should be typed for platelet antigens, and the woman
should be screened for the alloantibody. DNA testing of the father can determine the zygosity of the antigen
involved.31
The DNA genotype of the fetus can be determined as early as 11 to 13 weeks’ gestation. Assessment of the
fetus should begin at or before 20 weeks’ gestation, when severe thrombocytopenia and hemorrhage can occur.
When cordocentesis is used to determine the platelet count, irradiated, CMV-reduced-risk, antigen-negative
platelets should be used.
If needed, platelet transfusion should be given to the fetus to treat thrombocytopenia and avoid hemorrhage.
Many blood suppliers have identified donors who are negative for human platelet alloantigen la, the most
widely implicated platelet antibody, and can prepare suitable platelets for IUT. The mother is negative for the
implicated alloantigen and
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AABB TECHNICAL MANUAL
 can be a donor if her platelets are washed. When platelet transfusion is needed urgently and maternal
platelets are unavailable, unselected platelets may be used.32
The mother is given MG after the fetus is determined to be affected. The dose is usually 1 g/kg each week.
In a retrospective review, MG treatment alone appeared to be as safe and effective as cordocentesis with platelet
transfusion.33 However, MG treatment failures may occur and can necessitate fetal platelet count monitoring by
cordocentesis and require platelet transfusions.34 The goal of both transfusion and MG treatment is to avoid
hemorrhage. Ultrasound monitoring of the fetus to detect hemorrhage is not recommended because intracranial
hemorrhage usually indicates permanent brain damage. Before vaginal delivery, the fetal platelet count should
be >50,000/pL.
After birth, the infant’s platelet count may decrease in the first few hours to days. In 2 to 3 weeks, when the
antibody has been cleared, the infant’s platelet count returns to normal. The presence or absence of severe
thrombocytopenia and intracranial hemorrhage in the firstborn correlates with the outcomes of subsequent
pregnancies.35
ITP
Autoantibodies against platelets, which are reactive with the mother’s own platelets as well as donor or fetal
platelets, are another cause of fetal and neonatal thrombocytopenia. Throm
bocytopenia in a pregnant woman is common and is rarely associated with ITP. However, ITP may cause
thrombocytopenia in both the mother and fetus. Fortunately, only about 10% of newborns with ITP have platelet
counts <50,000/pL, and only 1% to 2% have a high risk of hemorrhage.36
A pregnant woman with thrombocytopenia or a preexisting diagnosis of ITP should be tested for serum
platelet autoantibody. A negative result generally indicates that the thrombocytopenia was caused by other
conditions and suggests that the fetus or neonate is not at risk. In contrast, a pregnant woman with significant
thrombocytopenia and petechiae or other evidence of hemorrhage caused by autoantibody should undergo
similar treatment to that used in nonpregnant women with ITP
Prednisone at a dose of 1 to 2 mg/kg is usually effective. However, with persistent maternal
thrombocytopenia, MG 1 g/kg/day for 2 to 5 days is indicated. Although the maternal platelet count is often
monitored in these cases, it does not correlate with the newborn’s platelet count.36 Fetal blood sampling to
determine platelet count is not usually recommended because the risk of morbidity and mortality from
cordocentesis is greater than or equal to the risk of severe bleeding in utero or at delivery. However, if fetal
sampling is performed, a platelet transfusion should be administered at the same time. Platelet transfusions may
be needed in about 15% of newborns.36
KEY POINTS
1. HDFN is caused by maternal antibodies that are specific to a paternal red cell antigen. The maternal IgG
antibody is transported across the placenta, where it destroys the fetal red cells.
2. Some antibodies, such as anti-I, -PI, -Lea and -Leb, can be ignored. The most common clinically
significant antibodies are anti-D, -K, and -c.
3. Molecular typing of fetal DNA can be performed on maternal plasma early in the second trimester.
4. The recommended titer method is saline AHG with 60-minute incubation at 37 C. Other methods, such as
those using albumin AHG or gel, may result in higher titers that may lead to misinterpretation by the
obstetrician.
5. For IUT, the blood should be irradiated, CMV reduced-risk, hemoglobin S negative, group 0 (in most
cases), and less than 7 days old.
 CHAPTER 22 Perinatal Issues in Transfusion Practice
569
6. The rosette test is a sensitive method for detecting fetomaternal hemorrhage of approximately 10 mL or
more. Flow cytometry can precisely measure hemoglobin F and/ or D-positive red cells.
7. The calculated RhIG dose should be rounded up if the number to the right of the decimal point is >0.5 or
rounded down if the number is <0.5. In either case, a vial should be added to the result.
8. Despite the prevalence of ABO FIDFN, severe anemia rarely occurs. After birth, hyperbilirubinemia can
usually be treated with phototherapy alone.
9. In fetal/neonatal alloimmune thrombocytopenia, the platelet antibody may develop at around 17 weeks of
gestation in the first pregnancy and fetal thrombocytopenia may develop as early as 20 weeks. Irradiated, CMV-
reduced-risk, antigen-negative platelets should be given to treat thrombocytopenia and avoid hemorrhage.
REFERENCES
1. Bowman IM, Pollock IM, Penston LE. Fetomaternal transplacental hemorrhage during pregnancy and
after delivery. Vox Sang 1986; 51:117-21.
2. Sebring ES, Polesky HF. Fetomaternal hemorrhage: Incidence, risk factors, time of occurrence, and
clinical effects. Transfusion 1990; 30:344-57.
3. Bowman JM. The prevention of Rh immunization. Transfus Med Rev 1988;2:129-50.
4. Bowman IM. Controversies in Rh prophylaxis: Who needs Rh immune globulin and when should it be
given? Am J Obstet Gynecol 1985; 151:289-94.
5. Klein HG, Anstee DJ. The Rh blood group system. In: Mollison’s blood transfusion in clinical medicine.
12th ed. Oxford: Wiley-Blackwell, 2014:167-213.
6. van der Schoot CE, Martine Tax GH, et al. Prenatal typing of Rh and Kell blood group system antigens:
The edge of a watershed. Transfus Med Rev 2003;17:31-44.
 7. Vaughan JI, Manning M, Warwick RM, et al. Inhibition of erythroid progenitor cells by antiKell
antibodies in fetal alloimmune anemia. N Engl I Med 1998;338:798-803.
8. Klein HG, Anstee DJ. Haemolytic disease of the fetus and newborn. In: Mollison’s blood transfusion in
clinical medicine. 12th ed. Oxford: Wiley-Blackwell, 2014:499-548.
9. Pollock JM, Bowman JM. Anti-Rh(D) subclasses and severity of Rh hemolytic disease of the newborn.
Vox Sang 1990;59:176-9.
10. Firan M, Rawdon R, Radu C, et al. The MHC class I-related receptor, FcRn, plays an essential role in
the maternal transfer of gamma
globulin in humans. Int Immunol 2001;13:9931002.
11. Dennery PA, SeidmanDS, Stevenson DK. Neonatal hyperbilirubinemia. N Engl J Med 2001; 344:581-
90.
12. Moise KJ Jr. Management of rhesus alloimmunization in pregnancy. Obstet Gynecol 2008; 112:164-76.
13. Kama T, Erdem G, Tekinalp G, et al. Further hemolytic disease of the newborn caused by anti-M.
AmJHematol 1996;53:280-1.
14. Wagner FF, Flegel WA. RHD deletion occurred in the Rhesus box. Blood 2000;95:3662-8.
15. Akolekar K, Finning K, Kuppusamy R, et al, Fetal RHD genotyping in maternal plasma at Ills weeks of
gestation. Fetal Diagn Ther 2011; 29:301-6.
16. Hilden JO, Backleman K, Nilsson J, Ernerudh J. Flow-cytometric quantitation of anti-D antibodies. Vox
Sang 1997;72:172-6.
17. Schonewille H, Klumper FJCM, Watering LMG, et al. High additional maternal red cell
alloimmunization after Rhesus- and Kmatched intrauterine intravascular transfusions for hemolytic disease of
the fetus. Am J Obstet Gynecol 2007;196:143.el-6.
18. Rodunovic N, Lockwood CJ, Alvarez M, et al. The severely anemic and hydropic isoimmune fetus:
Changes in hematocrit associated with intrauterine death. Obstet Gynecol 1992;79: 390-3.
19. Schwartz J, Winters JL, Padmanabhan A, et al. Guidelines on the use of therapeutic apheresis in clinical
practice—Evidence-based approach from the Writing Committee of the American Society for Apheresis. The
Sixth Special Issue. J Clin Apher 2013;28:145-284.

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20. Margulies M, Voto LS, Mathet E. High dose intravenous IgG for the treatment of severe Rhesus
alloimmunization. Vox Sang 1991;61:1819.
21. American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of
hyperbilirubinemia in the newborn 35 or more weeks gestation. Pediatrics 2004;114:297-316.
22. Domen RE. Policies and procedures related to weak D phenotype testing and Rh immune globulin
administration. Arch Pathol Lab Med 2000;124:1118-21.
 23. Flegel, WA. How I manage patients and donors with weak D phenotype. Curr Opin Hematol
2006;13:476-83.
24. Prevention of Rh D alloimmunization. ACOG practice bulletin #4. Washington, DC: American College
of Obstetricians and Gynecologists, 1999.
25. Radel DJ, Penz CS, Dietz AB, Gastineau DA. A combined flow cytometry-based method for
fetomaternal hemorrhage and maternal D. Transfusion 2008;48:1886-91.
26. Sandler SG, Delaney M, Gottschall JL, College of American Pathologists Transfusion Medicine
Resource Committee. Proficiency tests reveal the need to improve laboratory assays for fetomaternal
hemorrhage for Rh immunoprophylaxis. Transfusion 2013;53:2098-102.
27. Branch DR, Shabani F, Lund N, Denomme GA. Antenatal administration of Rh-immune globulin causes
significant increases in the immunomodulary cytokines transforming growth factor-p and prostaglandin E2.
Transfusion 2006;48:1316-22.
28. Herschel M, Karrison T, Wen M, et al. Isoimmunization is unlikely to be the cause of hemolysis in
ABO-incompatible but direct anti
globulin test-negative neonates. Pediatrics 2002;110:127-30.
29. Davoren A, Curtis BR, Aster RH, McFarland JG. Human platelet antigen-specific alloantibodies
implicated in 1162 cases of neonatal alloimmune thrombocytopenia. Transfusion 2004;44:1220-5.
30. Williamson LM, Hackett G, Rennie J, et al. The natural history of fetomaternal alloimmunization in the
platelet-specific antigen HPA-la (P1A1, Zw3) as determined by antenatal screening. Blood 1998;92:2280-7.
31. Radder CM, Brand A, Kanhai HH. A less invasive treatment strategy to prevent intracranial hemorrhage
in fetal and neonatal alloimmune thrombocytopenia. Am J Obstet Gynecol 2001; 185:683-8.
32. Kiefel V Bassler D, Kroll H, et al. Antigen-positive platelet transfusion in neonatal alloimmune
thrombocytopenia (NAIT). Blood 2006; 107:3761-3.
33. Van den Akker ESA, Oepkes D, Lopriore E, et al. Noninvasive antenatal management of fetal and
neonatal alloimmune thrombocytopenia: Safe and effective. Br J Obstet Gynaecol 2007; 114:469-73.
34. Silver RM, Porter TF, Branch DW, et al. Neonatal alloimmune thrombocytopenia: Antenatal
management. Am J Obstet Gynecol 2000; 182: 1233-8.
35. Birchall JE, Murphy MF, Kroll H. European collaborative study of the antenatal management of feto-
maternal alloimmune thrombocytopenia. Br J Haematol 2003;122:275-88.
36. Webert KE, Mittal R, Sigouin C, et al. A retrospective 11-year analysis of patients with idiopathic
thrombocytopenic purpura. Blood 2003;102:4306-11.
Chapter 23
Neonatal and Pediatric Transfusion Practice
Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH

|RK| TRANSFUSION PRACTICE IN neoM natal and pediatric patients differs from that in adults.1 The
differences are related to physiologic changes occurring during the transition from fetus to adolescent. Blood
volume, hematologic values, immune system maturity, and physiologic responses to hypovolemia and hypoxia
are variable in this heterogeneous population, contributing to the complexity and intricacies of pediatric
transfusion practice.
This chapter discusses neonatal and pediatric transfusion practice during two distinct periods: infancy from
birth to 4 months, and infancy after 4 months and childhood. The pediatric practices addressed include 1)
smallvolume component preparation, 2) transfusion indications for blood components, 3) transfusion
administration and exchange transfusion, 4) transfusion support in specific diseases, 5) rationale for special
processing of blood components, and 6) pediatric massive transfusion protocols (MTPs).
TRANSFUSION IN INFANTS YOUNGER THAN 4 MONTHS
Considerations for Component Preparation and Therapy
The fact that patients younger than 4 months have small blood/plasma volumes and immature organ system
functions necessitates special approaches to component therapy. This is especially important for very-low-
birthweight (VLBW) infants (<1500 g) and extremely lowbirthweight infants (<1000 g).
Fetal and Neonatal Physiology Affecting Transfusion Practice
Healthy full-term neonates have a mean cord blood hemoglobin level of 16.9 ± 1.6 g/dL, whereas that of
preterm neonates is 15.9 ± 2.4 g/dL. The hemoglobin concentration normally declines during the first few
weeks of life, resulting in the physiologic anemia of infancy in newborns and physiologic anemia of
 Cassandra D. Josephson, MD, Director of Transfusion, Tissue, and Apheresis Services, Children’s
Healthcare of Atlanta, and Associate Professor, Pathology and Pediatrics, Emory University School of
Medicine, and Erin Meyer, DO, MPH, Associate Director of Transfusion, Tissue, and Apheresis Services,
Children’s Healthcare of Atlanta, and Assistant Professor of Pathology and Laboratory Medicine, Emory
University School of Medicine, Atlanta, Georgia
C. Josephson has disclosed financial relationships with Immucor and Octapharma. E. Meyer has disclosed
no conflicts of interest.
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AABB TECHNICAL MANUAL
prematurity in preterm infants.2 Both anemias are considered self-limited and are usually tolerated without
harmful effects.
The rate of decline in hemoglobin levels is a function of gestational age at birth. At 4 to 8 weeks after birth,
hemoglobin decreases to as low as 8.0 g/dL in preterm infants weighing 1000 to 1500 g and 7.0 g/dL in
neonates weighing less than 1000 g at birth.3 The physiologic decrease in hemoglobin concentration is due to
several factors: 1) a decrease in erythropoietin (EPO) resulting in diminished red cell production, 2) a decrease
in survival of fetal red cells, and 3) an increasing blood volume due to rapid growth. Reduced EPO production
results from increased oxygen delivery to tissues because of increased pulmonary blood flow, elevated arterial
p02 levels, and increased red cell 2,3-diphosphoglycerate (2,3 DPG) and hemoglobin A levels.
Clinical Considerations Related to Infant Size and Blood Volume
Blood volumes of pediatric patients vary with body weight. A full-term newborn has a blood volume of
approximately 85 mL/kg compared to 100 mL/kg in a preterm newborn. Due to the small total blood volumes
of preterm infants (100 mL or less), blood banks must be capable of providing appropriately sized blood
components.
Many factors, including iatrogenic blood loss from repeated phlebotomy, lead to frequent transfusions.
Hypovolemia is not tolerated well in newborns because their left ventricular stroke volume decreases without an
increase in heart rate when >10% of their blood volume is lost. Thus, newborns must physiologically increase
their peripheral vascular resistance with decreasing cardiac output to maintain systemic blood pressure.
Ultimately, poor tissue perfusion and oxygenation occur, resulting in metabolic acidosis.4
Although transfusion may be required, it is neither necessary nor acceptable to replace the amount of blood
lost mL for mL; rather, transfusions can be administered to maintain a target hemoglobin level in certain clinical
situations.5 When ill preterm and full-term neo
nates receive multiple transfusions, they have proportionately lower levels of circulating fetal hemoglobin
and an increase in adult hemoglobin levels.
Erythropoietic Response
The EPO response in newborns differs from that in adults and older children. In the latter groups, oxygen
sensors in the kidney recognize decreases in oxygen delivery, resulting in the release of EPO into the
circulation. In contrast, in the fetus, this sensor is located in the liver and is less sensitive, resulting in reduced
EPO production in the face of hypoxia (hyporesponsiveness). This response likely occurs to prevent
polycythemia of the fetus in the hypoxic intrauterine environment.
Although EPO production eventually shifts from the liver to the kidney, most premature infants produce the
smallest amount of EPO for any degree of anemia.6 As an alternative to transfusion, the use of recombinant
human EPO has been shown to reduce donor exposures in premature infants and minimize the severity of
anemia.7 As compliance with strict transfusion threshold criteria has increased, clinicians have decreased their
phlebotomy rates in VLBW infants, reducing the rates of iatrogenically induced anemia and numbers of
transfusions. Thus, in most cases, this approach achieves the same goal as EPO therapy (ie, decreases the use of
transfusions and the number of donor exposures) without expensive pharmacotherapy and its risk of adverse
effects.
Cold Stress
Hypothermia in the neonate can trigger or exaggerate a number of responses, including 1) an increase in
metabolic rate; 2) hypoglycemia; 3) metabolic acidosis; and 4) potential apneic events that may lead to hypoxia,
hypotension, and cardiac arrest. In-line blood warmers are required for all Red Blood Cell (RBC) exchange
transfusions to combat the effects of hypothermia. A radiant heater should never be used to warm the blood
being transfused due to the risk of hemolysis. Furthermore, to prevent hemolysis in neonates under
573
going phototherapy, the blood-administration tubing should be positioned to minimize exposure to
phototherapy light.8
Immunologic Status
Their immature immune system predisposes infants to infectious and noninfectious hazards of transfusion.
In fact, much of the special processing of products for preterm infants and neonates is directly related to their
underdeveloped immune function. Most of their humoral immunity (antibody protection) is provided by the
mother starting early in pregnancy (approximately 12 weeks) through placental transfer of immunoglobulins.
Between 20 and 33 weeks of gestation, fetal IgG levels rise significantly because of selective transport system
maturation in the placenta. The breakdown of IgG occurs at a slower rate in the fetus than in the mother,
enabling conservation of the transplacental maternal antibody during the neonatal period. Unexpected red cell
alloantibodies of either IgM or IgG class are rarely produced by the infant during the neonatal period. This lack
of red cell alloantibody production is not well understood but has been postulated to be due to deficient T-
helper-cell function, enhanced T-suppressor-cell activity, and poor antigen-presentingcell function.9
Cellular immune responses are also incompletely developed during this period and may make infants
susceptible to transfusionassociated graft-vs-host disease (TA-GVHD). TA-GVHD has been reported most
frequently in newborns with confirmed or suspected congenital immunodeficiency. The majority of TA-GVHD
cases reported in nonimmunocompromised infants have occurred after intrauterine transfusion and subsequent
postnatal exchange transfusion.10,11 There have also been rare cases of TA-GVHD associated with extreme
prematurity, neonatal alloimmune thrombocytopenia, or extracorporeal membrane oxygenation (ECMO).10,12
Once an infant develops TA-GVHD, the chance of associated mortality is higher than 90%. TA-GVHD can be
prevented by pretransfusion irradiation of cellular blood components.12,13
Metabolic Problems
In infants younger than 4 months, large-volume transfusions of reconstituted whole blood or plasma may
result in acidosis and/or hypocalcemia because of the immature liver’s inability to effectively metabolize citrate.
The immature kidneys also contribute to these complications because they have lower glomerular filtration rates
and concentrating ability than older infants and children, leading to difficulties in excreting excess potassium,
acid, and/or calcium.
POTASSIUM. Small-volume, simple transfusions administered slowly have been shown to have little effect
on serum potassium concentrations in infants younger than 4 months despite the high potassium levels in the
plasma of stored RBCs. In calculating levels of infused potassium, Strauss13,14 determined that an RBC unit
(80% hematocrit) stored in an extended storage medium for 42 days would deliver 2 mL of plasma containing
only 0.1 mmol/L of potassium when transfused at 10 mL/kg. This amount of potassium is much less than the
daily requirement of 2 to 3 mmol/L for a patient weighing 1 kg. It must be stressed that this calculation does not
apply to the transfusion of large volumes of RBCs (>20 mL/kg). Serum potassium can rise rapidly in these
small patients—particularly during surgery, exchange transfusion, or ECMO—and is dependent on the plasma
potassium levels in the blood and manipulation of the blood components.15,16
The type of anticoagulant-preservative solution used to store RBCs at collection determines the amount of
potassium leakage. For instance, a unit of RBCs preserved in an additive solution (AS), such as AS-1, AS-3, or
AS-5, delivers less extracellular potassium than RBCs stored in citrate-phosphate-dextroseadenine (CPDA)-
l.13,17 In addition, special component processing, such as irradiation, can potentiate potassium leaks. If such
components are stored for more than 24 hours, washing may be required to remove the excess potassium before
transfusion.12 This washing practice is supported by several reports of infants who received via central line or
intra
574
AABB TECHNICAL MANUAL
cardiac line either older RBC units or units that had been irradiated (>1 day before transfusion) and had
severe adverse effects, including cardiac arrest and death.1819 This washing practice is controversial, however,
and several institutions accept AS units for low-volume neonatal transfusions without washing as long as these
units do not exceed a certain storage age or time after irradiation.20
2,3-DPG. Levels of 2,3-DPG in red cells are known to decline rapidly after 1 to 2 weeks of storage. This
deficit does not affect older children and adult recipients negatively because of their ability to replenish the
missing 2,3DPG in vivo and to compensate for hypoxia by increasing their heart rate. Infants younger than 4
months are not able to do this as effectively as a result of their low levels of intracellular 2,3-DPG that reach
even lower levels with respiratory distress syndrome or septic shock. Thus, if a large proportion of the neonate’s
blood volume is composed of transfused 2,3DPG-depleted blood, the resulting shift in the hemoglobin oxygen
dissociation curve further increases oxygen affinity for hemoglobin and reduces oxygen availability to the
tissues.
Therefore, the recommended therapy for newborns is an exchange transfusion with RBCs that are usually
less than 14 days old, although this practice is variable and dependent on institutional standard operating
procedures. However, the medical need for fresh RBC units for small-volume transfusions has not been
established and these transfusions have even been characterized as unnecessary.5,13,16 A prospective
randomized controlled trial to assess the outcomes of longer vs shorter storage times of RBCs is necessary in
this population (see “RBC Age” section below).21
RBC Transfusion Support
111 neonates are more likely to receive RBC transfusions than any other patient age group, and RBCs are
the component most often transfused during the neonatal period.5 RBC replacement is considered for sick
neonates when approximately 10% of blood volume has been lost or they have symptomatic anemia.
Indications
Several guidelines have been published over the past 15 years regarding the indications for RBC
transfusions in neonates.22'26 Most of the recommendations are based on experience acquired in clinical
practice rather than published evidence. To this end, a critical need exists for clinical studies in this area.27
Table 23-1 lists the most recently published guidelines.23,26
Compatibility Testing
AABB Standards allows limited pretransfusion serologic testing for infants younger than 4 months.28(p39)
Initial patient testing must include ABO and D typing of the patients’ red cells and screening for unexpected red
cell antibodies using either plasma or serum from the infant or mother. During any hospitalization, crossmatch-
compatibility testing and repeat ABO and D typing need not be conducted as long as all of the following criteria
are met: 1) the antibody screening result is negative; 2) transfused RBCs are group 0, ABO identical, or ABO
compatible; and 3) transfused cells are either D negative or the same D type as the patient. Testing the infant’s
reverse type for anti-A and/or anti-B is not necessary. However, before non-group 0 RBCs can be issued, testing
of the infant’s plasma or serum is required to detect passively acquired maternal anti-A or anti-B and should
include antiglobulin phase. If the antibody is present, ABO-compatible RBCs must be transfused until the
acquired antibody is no longer detected.
If an unexpected non-ABO alloantibody is detected in the infant’s or mother’s specimen, the infant must be
transfused with RBC units lacking the corresponding antigen(s) or units that are compatible by antiglobulin
crossmatch. This regimen should continue until the maternal antibody is no longer detected in the infant’s
plasma or serum. The policy of the hospital transfusion service determines the frequency for reevaluating the
patient’s antibodies. Once a negative antibody screening result is obtained, crossmatches and use of antigen-
negative blood are no longer required in
575
TABLE 23-1. Transfusion Guidelines for RBCs in Infants Younger than 4 Months2326
Hematocrit <20% with low
reticulocyte count and symptomatic
1.
anemia (tachycardia, tachypnea, poor
feeding).
Hematocrit <30% and any of the
2.
following:
a. On <35% oxygen hood.
b. On oxygen by nasal cannula.
On continuous positive airway pressure and/or intermittent
c. mandatory ventilation on mechanical ventilation with mean
airway pressure <6 cm of water.
d. With significant tachycardia or tachypnea (heart rate >180
beats/minute for 24 hours, respiratory rate >80 beats/minute for
 24 hours).
With significant apnea or bradycardia (>6 episodes in 12
hours or 2 episodes in 24 hours requiring bag and mask
e.
ventilation while receiving therapeutic doses of
methylxanthines).
With low weight gain (<10 g/day observed over 4 days
f.
while receiving >100 kcal/kg/day).
Hematocrit <35% and either of the
3.
following:
a. On >35% oxygen hood.
On continuous positive airway pressure/intermittent
b. mandatory ventilation with mean airway pressure >6-8 cm of
water.
Hematocrit <45% and either of the
4.
following:
a. On extracorporeal membrane oxygenation.
b. With congenital cyanotic heart disease.
infants younger than 4 months because of their immature immunologic status.
Multiple observational studies have shown that alloimmunization to red cell antigens is rare during the
neonatal period.9,29,30 For this reason, repeated typing and screening, which are required for adults and
children older than 4 months, is unnecessary in younger infants and contributes to significant iatrogenic blood
loss. Also, the transfusion service should avoid transfusing any components that may passively transfer
unexpected alloantibodies or ABO-incompatible antibodies to recipients.31
Components for Neonatal Transfusion
Advances in neonatology now permit the survival of extremely premature infants with sur
factant therapy, nitric oxide therapy, highfrequency ventilators, and compliance with transfusion practice
guidelines. These advances have substantially decreased the number of RBC transfusions administered in this
population. Most neonatal transfusions are now given to VLBW infants.32
Aliquotingfor Small-Volume Transfusion
The purpose of creating small-volume aliquots is to limit donor exposures, prevent circulatory overload, and
potentially decrease donorrelated risks.33'37 Several technical approaches can be used to accomplish these
goals and minimize blood wastage.38
Small-volume RBC transfusion aliquots are commonly made with a multiple-pack
 576
AABB TECHNICAL MANUAL
system.38,39 Quad packs, employed mostly by blood centers, are produced from a single unit of whole
blood that is diverted into a primary bag with three integrally attached smaller bags. The plasma is then
separated and diverted into one bag during component preparation. The remaining red cells are drawn into the
smaller bags as needed for transfusion. Each of the smaller units has the same expiration date as the original
unit because the system’s original seal has remained intact and a “closed system” is maintained.
A hospital transfusion service can then remove (either by heat sealer or metal clips) each aliquot as needed.
For hospital transfusion services without a sterile connection device (Fig 23-1), this method provides three
aliquots
I
SCD 312

sterile
tubing
welder
i_. .
FIGURE 23-1. A sterile tubing welder (reproduced with permission from Terumo Medical Corp., Somerset,
NJ).
from a single unit.38 However, blood components may still be wasted when used in aliquots that are larger
than the dose selected for each patient based on body weight.
Hospital transfusion services that have a sterile connection device have multiple additional options to
produce aliquots, such as transfer packs [eg, PEDI-PAK system (Genesis BPS, Hackensack, NJ) Fig 23-2],
small-volume bags, or tubing with integrally attached syringes.38 Syringe sets (Fig 23-3) offer the greatest
accuracy for obtaining the desired volume to be transfused based on volume-per-weight calculations.38 Some
syringe sets have an attached 150-micron in-line filter for use during the aliquoting process so that, when issued
by the blood bank, the cells are ready to be placed on a syringe pump without further manipulation of the
component at the bedside. This process eliminates the need for the nurse to transfer blood from the pack to a
syringe at the bedside for delivery by a syringe pump. Removing this additional step reduces the risk of
contamination, mislabeling, or damage to the unit that results in blood loss or spillage.38
Reducing donor exposures is more readily accomplished by this technique, which enables a recipient to
receive multiple small-volume transfusions from a single unit until it reaches its expiration date.40,41 Many
hospital transfusion services assign a single unit of RBCs to one or more infants based on their weight.34'40
Once an aliquot is produced at either the blood center or hospital blood bank, it must be labeled with the
expiration date and the origin and disposition of each smaller unit must be recorded. Aliquot expiration dates
vary from institution to institution, and local standard operating procedures should always be followed.
RBC Additive Solutions
Historically, transfused RBCs for children contained CPDA-1 anticoagulant-preservative solution.40
However, as ASs (see Chapter 6) evolved to extend the shelf life of RBCs, many experts began to question their
safety in neonates. One concern is the large amount of ade
577
Notch for
 FIGURE 23-2. Diagram of PEDI-PAK (reproduced with permission of Genesis BPS, Hackensack, NJ).
nine and mannitol in AS and its relation to renal toxicity. Moreover, mannitol is a potent diuretic with
effects on fluid dynamics that can result in fluctuations in the cerebral blood flow of preterm infants.
Most evidence suggests that small-volume transfusions (5 to 15 mL/kg) containing AS are safe for this
patient population. Specifically, when AS-1 and AS-3 were compared, no harmful effects were observed in
neonates receiving small-volume, simple transfusions.40'42 These transfusions were as effective as CPDA-1
RBCs in increasing hemoglobin levels in recipients. Luban and colleagues used theoretical calculations in a
variety of clinical settings to demonstrate that red cells preserved in
extended-storage media present no major risk when used for small-volume transfusions.43
However, for infants with renal or hepatic insufficiency, it is recommended that AS solution be removed
from RBC units, particularly if multiple transfusions from the same unit are expected. The safety of AS-
preserved RBCs in trauma-related massive transfusions, cardiac surgery, or exchange transfusions is unknown
in this population. Therefore, AS-preserved RBC units should be used with caution in these settings.21,42'45
RBC Age
The age of RBC units and its impact on patient outcomes has become a concern, although

FIGURE 23-3. Syringe with filter (reproduced with permission from Charter Medical, Ltd, Winston-Salem,
NC).

578
AABB TECHNICAL MANUAL
clinical confirmation of the basis for this concern is controversial. A randomized controlled trial conducted
in Canada, Age of Red Blood Cells in Premature Infants (ARIPI), randomly assigned low birthweight infants to
be transfused with RBCs that were 7 days old or less (mean = 5.1 days, n = 188) or with standardissue RBCs
divided into aliquots and stored for 2 to 42 days (mean = 14.6 days, n = 189).46 The primary composite
endpoints included necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH), and bronchopulmonary
dysplasia. The ARIPI trial found no differences in the primary endpoints between infants in the two arms,
suggesting that in the study population, the age of RBCs does not affect these common morbidities of
prematurity.
ARIPI’s external validity has been questioned because of the study's liberal transfusion strategy, use of
SAG-M units, and average duration of blood storage. These practices do not reflect the transfusion practices or
storage solution and ages of RBCs used in many centers in the United States.47 Thus, it has not been firmly
established that there is a causal relationship between morbidities and transfusion of older RBC units in
premature infants.47
Specific Indications for RBCs
 In neonates, symptomatic anemia is the major indication for simple transfusion. Specifically, a venous
hemoglobin of <13 g/dL in the first 24 hours of life necessitates clinical consideration of an RBC transfusion.48
RBC transfusions are also considered when approximately 10% of a sick neonate’s blood volume has been
removed or lost. When 10 mL/kg of RBCs with a hematocrit of >80% are transfused, the expected increase in
hemoglobin concentration in a neonate is approximately 3 g/dL. A similar volume of RBCs with AS usually has
a hematocrit of 65%, and its transfusion results in a projected posttransfusion hemoglobin increase of <3 g/dL
(see Table 23-2 for blood component dosing recommendations and expected results).49
Two randomized controlled trials, the Premature Infants in Need of Transfusion (PINT) and its follow up
study [PINT Followup Outcomes Study (PINTOS)] as well as a University of Iowa study compared the
outcomes of restrictive (Hb = 7 g/dL) vs liberal RBC transfusion triggers (Hb = 10 g/dL) in VLBW
infants.50'52 The Iowa trial revealed a lower rate of transfusion events (3.3 vs 5.2; p=0.025) with the restrictive
strategy com
TABLE 23-2. Blood Components and Dosing of Small Volumes in Neonatal and Pediatric Patients49
Component Dose Expected Increment
Red Blood Cells 10-15 mL/kg Hemoglobin increase 2-3 g/dL*
15%-20% rise in factor levels
Fresh Frozen Plasma 10-15 mL/kg
(assuming 100% recovery)
Platelets [whole-blood-derived 5-10 mL/kg or 1 WBD unit/10 50,000/pL rise in platelet count
(WBD) or apheresis] kg (patients >10 kg) (assuming 100% recovery)1
60-100 mg/dL rise in fibrinogen
Cryoprecipitated AHF 1-2 units/10 kg
(assuming 100% recovery)
‘Dependent on anticoagulant-preservative solution: with 3 g/dL increment for CPD and CPDA-1 and 2 g/dL
for AS-1, AS-3, and AS-5.
Assumes >5.5 x 1010 platelets in 50 mL of plasma (whole-blood-derived) and >3.0 x 10" platelets in 250-
300 mL plasma (apheresis).
CPD = citrate-phosphate-dextrose: CPDA-1 = citrate-phosphate-dextrose-adenine-1; AS = additive solution.
 579
pared to the liberal strategy.52 However, rates of periventricular leukomalacia and death were higher in the
restrictive arm. The PINT study found no significant difference between the two arms, which had the same
thresholds as the Iowa study, in the composite endpoint of death or any of bronchopulmonary dysplasia,
retinopathy of prematurity (Stage >3), or brain injury (periventricular leukomalacia, intracranial hemorrhage
Grade 4, or ventriculomegaly).50,52 The PINTOS study revealed that at 18 to 24 months after birth, infants in
the PINT study’s restrictive arm had more neurodevelopmental impairments than those in the liberal arm.50,51
In summary, these studies indicated that maintenance of higher hemoglobin levels in low birthweight infants
may provide longterm neurologic protection.50-52 Therefore, whether a restrictive or liberal RBC transfusion
strategy should be adopted in this population is not clear and requires further prospective randomized controlled
trial data. The Transfusion of Premature Infants Study is currently being conducted in the United States.53
Exchange Transfusion for Hyperbilirubinemia
Exchange transfusion in neonates involves replacement of one or two whole-blood volumes. The primary
purpose of this therapy is to treat excessively high levels of unconjugated bilirubin (hyperbilirubinemia). In
high concentrations, bilirubin may cross the bloodbrain barrier; concentrate in the basal ganglia and cerebellum
of preterm and full-term infants; and cause irreversible damage, known as “kernicterus,” to the central nervous
system. Preterm and full-term infants are susceptible to hyperbilirubinemia because their immature liver
conjugates bilirubin poorly and their incompletely developed blood-brain barrier allows bilirubin transit.
Phototherapy (use of fluorescent ultraviolet lights) is the current treatment of choice for hyperbilirubinemia;
exchange transfusion is reserved for patients who fail phototherapy.
Two critical objectives of exchange transfusions are the removal of unconjugated bilirubin and
maximization of albumin binding of
residual bilirubin. In addition, in antibodymediated hemolytic processes, exchange therapy removes both
free antibody and antibody-coated red cells, replacing them with antigen-negative red cells.
Exchange transfusion needs to be performed before the development of kernicterus. In full-term infants,
kernicterus rarely develops at bilirubin levels less than 25 mg/dL. However, in ill VLBW infants, kernicterus
can occur at bilirubin levels as low as 8 to 12 mg/dL.54
A double-volume exchange transfusion (two 85-mL/kg transfusions for full-term infants and two 100-
mL/kg transfusions for VLBW infants) removes approximately 70% to 90% of the circulating red cells and
approximately 50% of the total bilirubin.55 However, after the first exchange transfusion, bilirubin levels may
rise again because of a re-equilibration of the extravascular tissue and plasma bilirubin, which may necessitate
another exchange transfusion.56
Occasionally, exchange transfusion is used to eliminate toxins, drugs, or chemicals administered to the
mother near the time of delivery. Exchange transfusion is also used when toxic doses have been administered to
the infant or accumulate at high levels in the infant as a result of prematurity and/or an inborn error of
metabolism.57,58
Exchange Transfusion
COMPONENT CHOICE AND PHYSIOLOGIC EFFECTS. Typically, RBCs are resuspended in ABO-
compatible thawed Fresh Frozen Plasma (FFP) for an exchange transfusion. No single method of combining
components has been shown to be superior to another. Most often, RBCs <5 to 7 days old and stored in CPDA-
1 are used to avoid high levels of potassium and to maximize red cell survival.59 When using ASRBC units,
some blood banks elect to remove the additive-containing plasma to reduce the volume transfused.
Most transfusion services provide RBC units that are hemoglobin S negative, cytomegalovirus (CMV)
reduced-risk (leukocyte reduced and/or CMV seronegative), and irradiated. Irradiation should be performed just
580
AABB TECHNICAL MANUAL
before the exchange to prevent potentiation of the potassium storage lesion. Some experts recommend
washing or removing the supernatant of red cells that have been irradiated to avoid the complications of
hyperkalemic cardiac arrhythmias.60
 The glucose load administered during exchange transfusion can be high in some cases, which stimulates the
infant’s pancreas to release insulin and results in rebound hypoglycemia. Therefore, infant plasma glucose
levels should be monitored during the first few hours following exchange transfusion.
VOLUME AND HEMATOCRIT CONSIDERATIONS. A double-volume exchange in neonates rarely
necessitates the infusion of more than 1 RBC unit. The unit’s hematocrit should be approximately 45% to 60%,
and the unit should have sufficient plasma (based on estimated blood volume) to provide clotting factors.60 If
the neonate’s condition requires a higher postexchange transfusion hematocrit, a small-volume RBC transfusion
may be given or a unit with a higher hematocrit can be used for the initial exchange transfusion. The
reconstituted blood should be well mixed to sustain the intended hematocrit throughout the exchange. The
infant’s hematocrit and bilirubin can be measured by removing the last aliquot of the exchange unit.
VASCULAR ACCESS. Umbilical venous catheters are used for exchange transfusions in preterm and full-
term infants just after birth. If umbilical venous catheters are not available, small saphenous catheters maybe
used.
TECHNIQUES. Two exchange-transfusion techniques are commonly employed: isovolumetric and manual
push-pull. In isovolumetric exchange transfusion, two catheters of identical size provide vascular access. The
catheters allow simultaneous withdrawal and infusion of blood and are regulated by a single peristaltic pump.
The umbilical vein is typically used for infusion, and the umbilical artery is used for withdrawal. The manual
push-pull technique uses a single vascular access portal with a three-way stopcock attached to the unit of blood,
the patient, and a graduated discard
container. A standard filter and in-line blood warmer are recommended.
With both techniques, the absolute maximum volume of each withdrawal and infusion depend on the
infant’s body weight and hemodynamic status. Usually, no more than 5 mL/ kg body weight or 5% of the
infant’s blood volume is removed and replaced during a 3- to 5minute cycle.59 The exchange transfusion
should not be performed rapidly because sudden hemodynamic changes may affect cerebral blood flow and
shift intracranial pressure, contributing to IVH.61 A total double-volume exchange transfusion typically takes
90 to 120 minutes.59
Platelet Transfusion Support
Mild-to-moderate thrombocytopenia (platelet count <150,000/pL) is the most common hemostatic
abnormality in ill preterm and fullterm infants, and it affects approximately 20% of infants in neonatal intensive
care units.62 The causes of thrombocytopenia include impaired platelet production, increased platelet
destruction, abnormal platelet distribution, and/or platelet dilution secondary to massive transfusion. The most
common cause is increased destruction of platelets that is generally associated with a variety of self-limited
conditions.
Indications
Most platelet transfusions in preterm and fullterm infants are performed to treat platelet counts less than
50,000/pL in the presence of active bleeding.63
Prophylactic platelet transfusions in this population are controversial (see Table 23-3 for transfusion
indications and thresholds).27,64 Unlike adult patients who rarely have severe bleeding complications until
platelet counts decline to less than 10,000/pL, preterm infants with other complicating illnesses may bleed at
higher platelet counts. This increased risk may be attributable to 1) lower concentrations of plasma coagulation
factors, 2) circulation of an anticoagulant that potentiates thrombin inhibition, 3) intrinsic or extrinsic platelet
581
TABLE 23-3. Transfusion Guidelines for Platelets in Neonates and Older Children23,26
With Thrombocytopenia
1. Platelet count 5,000 to 10,000/pL with failure of platelet production.
2. Platelet count <30,000/pL in neonate with failure of platelet production.
3. Platelet count <50,000/pL in stable premature infant:
a. With active bleeding, or
b. Before an invasive procedure, with failure of platelet production.
4. Platelet count <100,000/pL in sick premature infant:
a. With active bleeding, or
b. Before an invasive procedure in patient with DIC.
Without Thrombocytopenia
1. Active bleeding in association with qualitative platelet defect.
 2. Unexplained excessive bleeding in a patient undergoing cardiopulmonary bypass.
3. Patient undergoing ECMO with:
a. A platelet count of <100,000/pL, or
b. Higher platelet counts and bleeding.
DIC = disseminated intravascular coagulation; ECMO = extracorporeal membrane oxygenation.
dysfunction/hyperreactivity, or 4) increased vascular fragility.63
A severe complication of prematurity is IVH, which occurs in approximately 40% of preterm neonates in
the first 72 hours after birth. Although prophylactic platelet transfusions may increase platelet counts and
shorten bleeding times, this approach has not been shown to reduce the incidence of IVH, and the severity of
thrombocytopenia appears to be independent of the risk if IVH >Grade 2.62,65 Hence, the use of platelets in
this situation and the appropriate platelet dose remain controversial.66 Posttransfusion platelet counts 15 to 60
minutes after transfusion can help when platelet survival is evaluated; however, these counts are not good
predictors of hemostatic efficacy.
Components and Dose
The use of whole-blood-derived platelets at doses of 5 to 10 mL/kg body weight have been
demonstrated to raise the platelet count of an average full-term newborn by 50,000 to 100,000/pL,
depending on the concentration of the platelet component used.14,62 A similar dosing regimen is typically used
with apheresis platelets. For larger children (>10 kg), transfusion of 1 platelet unit per 10 kg should increase the
platelet count by approximately 50,000 pL.14,67
When possible, the platelet component should be ABO group specific/compatible and should not contain
clinically significant and unexpected red cell antibodies. Transfusion of ABO-incompatible plasma should be
avoided in children, and especially in infants, because of their small blood and plasma volumes.31 If it becomes
necessary to administer ABO-incompatible platelets in an infant, plasma may be removed either by volume
reduction or washing (see Method 6-14). The platelets may then be resuspended in saline or compatible plasma.
However, routine centrifugation to remove plasma from platelets should be avoided

582
AABB TECHNICAL MANUAL
because it is unnecessary and harmful to the platelets.62,63
In addition, when platelets are stored in a syringe, the pH has been shown to decrease rapidly, a potential
problem for an already ill and acidotic recipient.68'70 Therefore, when volume reduction in an open system is
used and the product is placed in a syringe, the processing should be done as close to the time of issuance as
possible and the product should be infused within 4 hours of processing.
Plasma Transfusion Support to Enhance Hemostasis
Infants must synthesize their own coagulation factors because significant amounts are not transplacentally
transferred from the mother. Furthermore, infants are unable to produce normal levels of these proteins in the
early postnatal period.
Physiologically, low levels of vitamin Independent factors (Factors II, VII, IX, and X) and contact factors
(Factor XI, Factor XII, prekallikrein, and high-molecular-weight kininogen) contribute to altered coagulation
test results (see Table 23-4).71,72 Also, the naturally occurring vitamin K-dependent anticoagu
lants (proteins C and S) and the non-vitaminK-dependent antithrombin protein are at low levels at birth. In
spite of these issues, the procoagulant and anticoagulant systems are usually in balance in healthy newborns, so
spontaneous bleeding and thrombosis are rare (see Table 23-4).72 However, the reserve capacity for both
systems is limited. Therefore, serious bleeding may occur in sick premature infants during the first week of life.
Cryoprecipitate and FFP can be transfused to treat bleeding or clotting complications, such as disseminated
intravascular coagulation (DIC).73,74
FFP
FFP is frequently used to replace coagulation factors in preterm and full-term infants, particularly if
multiple factor deficiencies are present, such as hemorrhagic disease of the newborn or vitamin K deficiency
(Table 23-5). The usual dose of FFP is 10 to 15 mL/kg, which is expected to increase all factor activity levels by
15% to 20% unless there is a marked consumptive coagulopathy.64,68
To limit donor exposure for each recipient while minimizing plasma wastage, blood can be collected into a
system with multiple, inte
TABLE 23-4. Screening Laboratory Tests for Hemostasis: Neonates vs Adults (reproduced with
permission)71
Preterm Neonates vs Full- Neonates vs Older Approximate Age Adult
Term Neonates Children/Adults Values are Reached
aPTT Longer Longer 16 years
Prothrombin
Longer Same or longer 16 years
time
INR Higher Same or higher 16 years
Thrombin
Longer Same or longer 5 years
time
Bleeding time Longer Shorter 1 months
PFA-100 Longer Shorter 1 months
ROTEM/TEG
Clotting time Same Shorter 3 months
Clot
Same Shorter 3 months
formation time
Maximal clot
Stronger Stronger 3 months
firmness
aPTT = activated partial thromboplastin time, INR = international normalized ratio.
 583
TABLE 23-5. Transfusion Guidelines for Plasma Products in Neonates and Older Children23'26
Fresh Frozen Plasma (FFP)
Support during treatment of disseminated
1.
intravascular dissemination.
2. Replacement therapy:
a. When specific factor concentrates are not
available, including, but not limited to, antithrombin;
protein C or S deficiency; and Factor II, Factor V, Factor
X, and Factor XI deficiencies.
b. During therapeutic plasma exchange when FFP is
indicated (cryopoor plasma, plasma from which the
cryoprecipitate has been removed).
Reversal of warfarin in an emergency situation,
3. such as before an invasive procedure with active
bleeding.
Note: FFP is not indicated for volume expansion or
enhancement of wound healing.
Cryoprecipitated AHF
Hypofibrinogenemia or dysfibrinogenemia with
1.
active bleeding.
 2. Hypofibrinogenemia or dysfibrinogenemia while
undergoing an invasive procedure.
Factor XIII deficiency with active bleeding or
3. while undergoing an invasive procedure in the
absence of Factor XIII concentrate.
Limited directed-donor cryoprecipitate for
bleeding episodes in small children with hemophilia
4.
A (when recombinant and plasma-derived Factor VIII
products are not available).
5. In the preparation of fibrin sealant.
von Willebrand disease with active bleeding, but
6.
only when both of the following are true:
a. Deamino-D-arginine vasopressin (DDAVP) is
contraindicated, not available, or does not elicit
response.
b. Virus-inactivated plasma-derived Factor VIII
concentrate (which contains von Willebrand factor) is
not available.
grally attached bags that create ready-tofreeze aliquots.26 Once thawed, the aliquots can be subdivided
further for several patients if they can be used within a 24-hour period.
FFP for infants must be ABO compatible and free of clinically significant and unexpected antibodies.
Transfused antibodies can reach high concentrations in infants and children with very small plasma volumes. A
common practice at some institutions is to use group AB FFP because a single unit can provide multiple small-
volume doses for several neonates.
Cryoprecipitated Antihemophilic Factor
Cryoprecipitate transfusions are primarily used to treat conditions resulting from de
creased or dysfunctional fibrinogen (congenital or acquired) or Factor XIII deficiency. Cryoprecipitate is
usually given in conjunction with platelets and FFP to treat DIC in newborns. Typically, 1 unit is sufficient to
achieve hemostatic levels in an infant.
ABO-compatible cryoprecipitate is preferred because transfusion of a large volume of ABO-incompatible
cryoprecipitate may result in a positive DAT result and, in very rare cases, a mild hemolysis.75,76
Cryoprecipitate transfusion is not recommended for patients with Factor VIII deficiency because the
standard therapy is to treat this condition with recombinant Factor VIII products or virus-inactivated,
monoclonal-antibody-purified, plasma-derived products.73,77

584
AABB TECHNICAL MANUAL
 Furthermore, cryoprecipitate should be used only to treat von Willebrand disease if plasmaderived, virus-
inactivated concentrates containing von Willebrand factor are not available. See Table 23-5 for other guidelines
regarding cryoprecipitate use.64
Granulocyte Transfusion Support
Indications
The role of granulocyte transfusion for sepsis in neonates is unclear, and this treatment is rarely used. It is
important to establish the following factors before the transfusion of granulocytes: 1) strong evidence of
bacterial or fungal septicemia; 2) absolute neutrophil count less than 500/pL, chronic granulomatous disease, or
leukocyte adhesion deficiency; and 3) diminishing storage pool (such that 7% of nucleated cells in the marrow
are granulocytes that are metamyelocytes or more mature).15,78,79 (See Table 23-6 for guidelines on
granulocyte transfusion support.)
Components and Dose
Granulocyte concentrates are produced by standard apheresis techniques or by pooling huffy coats from
whole blood. A typical dose for infants is 10 to 15 mL/kg body weight, which is approximately 1 x 109 to 2 x
109 polymorphonuclear cells/kg.15,78 Treatment should be administered daily until an adequate neutrophil
count is achieved and/or the patient shows clinical improvement.
According to AABB Standards, these components must be irradiated when the patient is
TABLE 23-6. Transfusion Guidelines for Granulocytes in Neonates and Older Children23,26
1. Neonates or children with neutropenia or granulocyte dysfunction with bacterial sepsis and lack of
responsiveness to standard therapy.
2. Neutropenic neonates or children with fungal disease not responsive to standard therapy.
at risk of TA-GVHD, and the components must be obtained from CMV-seronegative donors to prevent virus
transmission if the recipient is CMV seronegative.28(p40) Granulocytes must also be ABO compatible with the
red cells of the recipient infant because of the significant red cell content in these components.28(p37) Many
institutions also provide D-compatible components to decrease Rh alloimmunization.
Intravenous immune globulin (MG) in the treatment of early neonatal sepsis has also been studied, but there
is a lack of agreement on its routine application to this patient population.78,80"85
Transfusion Administration
Vascular Access
Vascular access is the most difficult aspect of transfusion administration in patients younger than 4 months,
particularly in preterm infants who require long-term or continuous intravenous infusions. The umbilical vein is
most frequently cannulated after birth to administer fluids and transfusions and to monitor central venous
pressure.86 Vascular catheters (24-gauge) and small needles (25-gauge) generally can be safely used for RBC
transfusions without causing hemolysis if constant flow rates are applied. The outcomes of transfusions using
smaller-gauge needles and catheters have not been evaluated.
Pumps and Warming
When administered slowly, small-volume transfusions typically do not require a blood warmer; however,
control of the rate and volume transfused is important. Electromechanical syringe delivery pumps administer
the blood at a constant rate and are able to provide adequate control. These pumps cause minimal hemolysis and
may even be used with inline leukocyte-reduction filters.87,88 Although there are several devices that can be
used to transfuse blood components, it is important to test and validate the mechanical system chosen for blood
component administration.
585
Filters and Transfusion Sets
All blood component transfusions require a standard filter (150 to 260 microns), even if the components
have undergone leukocyte reduction before storage or at the bedside.75 Microaggregate filters (20 to 40
microns) are occasionally used for simple transfusions because of their small priming volume. However,
hemolysis may occur when stored RBCs are administered through these filters using negative pressure.89
The plastic tubing in the administration sets can add significant amounts of deadspace volume to the
transfusion and may need to be accounted for when preparing a transfusion dose. Pediatric infusion sets created
for platelets and other small-volume components have less dead space than standard sets.
Administration Rates
A lack of clinical studies and evidence-based practices related to blood transfusions in neonates has led to
variability both in rates of transfusion and in devices used among institutions. The rate of RBC and other blood
component administration is dictated by the clinical needs of the pediatric patient. Despite concerns from
neonatologists that rapid blood infusion rates may adversely affect intravascular volume and electrolyte levels,
an increased risk of IVH in these small and fragile patients has not been clearly demonstrated. Therefore,
administering a simple transfusion over 2 to 4 hours is adequate in nonemergent situations. However, in states
of shock or severe bleeding, a rapid infusion is often required.
Unique Therapies and Situations in Neonates
Polycythemia
Neonatal polycythemia is defined as a venous hematocrit >65% or a hemoblogin >22 g/dL at any time
during the first week after birth. Approximately 5% of all newborns develop polycythemia, and this risk may be
higher in smallfor-gestational-age neonates and infants of diabetic mothers. Once the hematocrit rises
above 65%, viscosity increases and oxygen transport decreases. However, in neonates, the exponential rise
in viscosity can occur at a hematocrit as low as 40%.9° Congestive heart failure can result because infants have
limited capability to increase their cardiac output to compensate for hyperviscosity. Central nervous system
abnormalities, pulmonary and renal failure, and NEC can occur from the resultant decreased blood flow.
A partial exchange is used to normalize the hematocrit to between 55% and 60% and improve tissue
perfusion while maintaining blood volume. This exchange is accomplished by removing whole blood and
replacing it with normal saline or other crystalloid solutions. Plasma is not used to replace whole blood because
NEC has been reported as a complication of plasma transfusion.91
The formula below can be used to approximate the volume of replacement fluid required and the volume of
whole blood that must be withdrawn for the partial exchange:
(Blood (Observed Hct Volumeof volume) x Desired Hct)
replacement =
Observed Hct
ECMO
ECMO is a prolonged treatment where blood is removed from the patient’s venous circulation, circulated
through a machine to remove C02 and replenish 02, and then returned to the patient. ECMO has been
successfully used since the early 1980s to provide gas exchange independendy of a patient’s lungs. ECMO
allows patients to recover without exposure to aggressive ventilator support that can cause barotrauma and
permanent lung damage and to maintain the circulation of oxygenated blood during cardiac surgery or in
patients with disease when other forms of treatment fail.92,93 In neonates and children, ECMO has become a
lifesaving advance treatment of meconium aspiration syndrome, persistent pulmonary hypertension of the
newborn, congenital diaphragmatic hernia, and respiratory failure due
586
AABB TECHNICAL MANUAL
to sepsis. It is also used for postoperative support following cardiac surgery.
Because standardized guidelines for transfusion practice in ECMO have not been established, centers
typically establish their own criteria. Table 23-7 provides some guidelines for ECMO.94 Bleeding
complications are frequent during ECMO treatment and may be caused by 1) systemic heparinization, 2)
platelet dysfunction, 3) thrombocytopenia, 4) other coagulation defects, or 5) the nonendothelial ECMO circuit.
Hospital blood banks and transfusion services must be in close communication with the ECMO staff and
observe local protocols to ensure safe, efficient, and consistent care.
ECMO typically requires 1 to 2 units of ABO- and Rh group-specific and crossmatchcompatible RBCs for
blood priming. In addition, 1 unit of group-specific FFP should be allocated to the ECMO patient. RBC units
are usually negative for hemoglobin S, relatively fresh (<5 to 7 days old), irradiated, and CMV seronegative
and/or leukocyte reduced.43 Because the ECMO circuit consumes platelets, higher platelet counts are often
maintained.
TRANSFUSION IN INFANTS OLDER THAN 4 MONTHS AND CHILDREN
RBC Transfusion Support
RBC transfusions in infants older than 4 months and children are similar to transfusions in adults. The most
significant differences between this young group and adults are 1) blood volume, 2) the ability to tolerate blood
loss, and 3) age-appropriate hemoglobin and hematocrit levels. In these infants and children, the most common
indication for RBC transfusion is to treat or prevent tissue hypoxia caused by decreased red cell mass, typically
because of surgery, anemia of chronic disease, or hematologic malignancies. Chronic RBC transfusions are the
treatment of choice for children with sickle cell disease (SCD) or thalassemia. These transfusions are
administered to combat tissue hypoxia and suppress endogenous hemoglobin production. Table 23-8 can help
guide transfusion decisions in patients older than 4 months.
TABLE 23-7. Blood Component Protocols for ECMO94
Clinical Scenario Urgency Components Blood Groups Storage
5-10 <14 days,
Cardiac arrest 2 units RBCs 0-neg RBCs
min AS
5-10 <14 days,
ECMO circuit disruption 2 units RBCs 0-neg RBCs
min AS
Progressive septic shock 0-neg RBCs <10 days,
30 min 2 units RBCs
(nonneonate) or type specific any preservative
1-2 2 units RBCs 1 unit 0-neg RBCs <10 days,
Neonate transferred for ECMO
hours FFP 1 unit platelets AB plasma CPDorCPDA
30-60
Cardiac ICU 2 units RBCs Type specific <7 days, AS
min
Gradual respiratory or cardiac Flours <10 days,
2 units RBCs Type specific
failure on conventional support to days CPD
ECMO = extracorporeal membrane oxygenation; RBCs = Red Blood Cells; AS = additive solution; FFP =
Fresh Frozen Plasma; CPD = citrate-phosphate-dextrose; CPDA = citrate-phosphate-dextrose-adenine; ICU =
intensive care unit.

587
TABLE 23-8. Transfusion Guidelines for RBCs in Patients Older than 4 Months2326
1. Emergency surgical procedure in patient with significant postoperative anemia.
 2. Preoperative anemia when other corrective therapy is not available.
3. Intraoperative blood loss >15% total blood volume.
4. Hematocrit <24% and:
a. In perioperative period, with signs and symptoms of anemia.
b. While on chemotherapy/radiotherapy.
c. Chronic congenital or acquired symptomatic anemia.
5. Acute blood loss with hypovolemia not responsive to other therapy.
6. Hematocrit <40% and:
a. With severe pulmonary disease.
b. On extracorporeal membrane oxygenation.
7. Sickle cell disease and:
a. Cerebrovascular accident.
b. Acute chest syndrome.
c. Splenic sequestration.
d. Aplastic crisis.
e. Recurrent priapism.
f. Preoperatively when general anesthesia is planned (target hemoglobin 10 mg/dL).
8. Chronic transfusion programs for disorders of red cell production (eg, (3-thalassemia major and
DiamondBlackfan syndrome unresponsive to therapy).
Before receiving any RBC transfusion, all pediatric patients older than 4 months require ABO and Rh
testing and screening for the presence of clinically significant antibodies. Compatibility testing should be
performed according to AABB Standards.28(p39)
SCD
Chronic transfusion therapy has been shown to reduce the risk of stroke in patients with SCD by decreasing
the proportion of RBCs containing hemoglobin S, reducing sickling, and preventing blood viscosity
increases.95'98 Chronic transfusions can reduce the risk of recurrent stroke to <10% if hemoglobin levels are
maintained at between 8 and 9 g/dL with a hemoglobin S level <30%.
Simple or partial-exchange transfusions can be administered every 3 to 4 weeks. Erythrocytapheresis has
also been used to prevent iron overload in patients with SCD. See Table 23-8 for a list of other complications of
SCD necessitating either simple or chronic RBC transfusions.
Chronic transfusion therapy must be provided indefinitely because its cessation can lead to a stroke.95,96
The Transcranial Dopplers with Transfusions Changing to Hydroxyurea trial is currently evaluating the role of
hydroxyurea in stroke prevention compared to chronic transfusions in patients with SCD.99 Of note, RBCs for
patients with SCD should ideally be screened for hemoglobin S and leukocyte reduced to prevent HLA
alloimmunization and
 588
AABB TECHNICAL MANUAL
decrease platelet refractoriness in preparation for possible stem cell transplantation.
RBC ALLOIMMUNIZATION IN SCD. Patients with SCD have the highest rates of alloimmunization of
any patient group.100'102 These antibodies are produced against common Rh, Kell, Duffy, and Kidd system
antigens. Many SCD treatment centers perform thorough phenotype analysis of a patient’s red cells before
beginning transfusion therapy. This testing helps reduce the rate of alloimmunization by allowing preferential
selection of phenotypically similar units.96,103,104 However, particularly for patients who are not yet
alloimmunized, this process remains controversial because phenotypically compatible units may be difficult to
obtain.95,104
In academic institutions in the United States and Canada, the most common alloimmunization protocol for
nonalloimmunized patients with SCD is pretransfusion phenotypic matching for C, E, and K antigens.105 Once
patients have developed a red cell antibody, extension of matching to additional red cell antigens (Fy, Jk, S) is
often used to prevent further alloimmunization.106 In a retrospective study, children with SCD undergoing
matched-sibling-donor marrow transplantation demonstrated a decrease in RBC transfusion requirements
during transplantation when they received phenotypically minor RBC antigen-matched units.107 A recent
retrospective review of patients with SCD at Children’s Hospital of Philadelphia showed that the children
developed alloimmunization to Rh antigens despite being transfused with units from Rh-matched minority
donors, and 87% of alloimmunized patients were identified with RH genotyping as having RH variant
alleles.108 This study’s results suggest that the role of genotyping of patients with SCD and minority donors in
alloimmunization rates needs to be studied.108 Method 2-23 can be used to perform phenotyping on autologous
red cells of recently transfused patients with SCD.
Another strategy aimed at preventing alloimmunization in patients with SCD is to develop recruitment
programs specifically for donors of African ethnicity to decrease the
number of antigenically different RBC units from donors of European ethnicity that are transfused.
Leukocyte reduction to prevent alloimmunization to red cell antigens remains controversial.109,110 However,
HLA alloimmunization has recently been demonstrated to occur more frequently in red cell alloimmunized
patients with SCD.111
OTHER COMPLICATIONS OF RBC TRANSFUSIONS IN SCD. The benefits of transfusion therapy in
patients with SCD should be weighed against the complications of transfusion, such as iron overload and minor
red cell antigen alloimmunization, as well as the risks of increased donor exposure during erythrocytapheresis.
Some practitioners have proposed that a clinically successful course of transfusions that maintains the
hemoglobin S level at <30% could, after several years, be transitioned to a strategy of more limited transfusions
with a hemoglobin S target of 40% to 50% to reduce the risk of iron overload.97 Patients with SCD may also be
at risk of life-threatening, delayed hemolytic transfusion reactions.
If a patient’s hemoglobin level decreases after transfusion, the patient might have developed
“hyperhemolytic” syndrome. This poorly understood phenomenon is characterized by destruction of the
patient’s own red cells along with transfused cells. If hyperhemolytic syndrome is suspected, case reports have
shown that stopping transfusion and administering corticosteroids in combination with MG is
beneficial.112,113 These patients should also be monitored closely for the formation of autoantibodies.114
Thalassemia
Thalassemia with severe anemia must be treated with transfusion to improve tissue oxygenation and
suppress extramedullary erythropoiesis in the liver, spleen, and marrow. This approach also decreases many
thalassemia complications. Maintaining target hemoglobin levels of 8 to 9 g/dL allows normal growth and
development in these patients. Supertransfusion protocols aim for higher target hemoglobin levels (11-12 g/dL).
Iron overload is a potential nonpreventable complication of
589
this RBC transfusion protocol that must be treated with chelation therapy beginning early in
childhood.115,116
Platelet and Plasma Support
 The indications for platelet and plasma transfusion support are similar for older infants, children, and adults.
Tables 23-3 and 23-5 provide the indications for the transfusion of platelets, FFR and cryoprecipitate. A recent
study showed that plasma transfusions were independently associated with an increased risk of new or
progressive multiple organ dysfunction, nosocomial infections, and prolonged length of stay in 831 children
admitted to one institution’s pediatric critical care unit.117 Plasma transfusions should be considered with
caution in this population.
In older infants and children, platelets are most often prophylactically administered during chemotherapy.
The transfusion threshold for these patients is usually between 10,000 and 20,000/pL, although the platelet
count should not be the sole determinant for transfusion. In the Prophylactic PLAtelet DOse (PLADO) study,
Slichter et al randomly assigned 1351 patients with hypoproliferative thrombocytopenia to receive one of three
prophylactic platelet transfusion doses (low, medium, or high) with the primary endpoint of World Health
Organization >Grade 2 bleeding.118 The dose of prophylactic platelets administered had no effect on the
incidence of >Grade 2 bleeding.118 A subgroup analysis of the 198 children aged 0-18 years in the PLADO
study revealed that children were at a higher risk of bleeding over a wider range of platelet counts than
adults.119 Therefore, prophylactic platelet transfusions with a threshold of 10,000/pL in concert with different
doses of platelets do not seem to affect the bleeding rates of children or adults.118,119 However, when children
are transfused, the use of ABO-compatible platelets is associated with better clinical outcomes than use of non-
ABO matched platelets.31,120'122
Granulocyte Support
For children older than 4 months, the indications for granulocyte transfusions—persistent neutropenia or
granulocyte dysfunction in conjunction with a bacterial and/or fungal infection(s)—are similar to those
previously mentioned for younger (<4 months) infants. The minimum granulocyte dose for older children and
adults is 1 x 1010 cells/kg.23,78
Granulocyte components should be irradiated, ABO compatible, CMV seronegative (if the recipient is CMV
seronegative), crossmatch compatible with the recipient, and, ideally, administered within 24 hours of
collection. To obtain higher doses of granulocytes for larger patients, donors can be mobilized with steroids,
growth factors [eg, granulocyte colony-stimulating factor (G-CSF)], or a combination. This approach can
increase the amount of granulocytes in the collection by three or four times compared to products from donors
who receive steroid stimulation alone.
The National Heart, Lung, and Blood Institute’s High Dose Granulocyte Transfusions for the Treatment of
Infection in Neutropenia: Resolving Infection in Neutropenia with Granulocytes study is examining the effect of
granulocyte transfusions plus standard microbial therapy vs standard microbial therapy alone for patients with
neutropenia and severe infections as well as the combined effect of GCSF and steroids on granulocyte donation
yield.
PREVENTION OF ADVERSE EFFECTS OF TRANSFUSION IN NEONATES, OLDER INFANTS, AND
CHILDREN
CMV Prevention
CMV may be transmitted to neonates transplacentally, during the birth process or breastmilk feeding, as a
result of personal contact with the mother or nursery staff, or from transfusion. With current technologies, the
risk of acquiring CMV from transfusion is between 1% and 3%.123
590
AABB TECHNICAL MANUAL
The manifestation of CMV infection in neonates is variable, ranging from asymptomatic seroconversion to
death. Studies have revealed that the rate of symptomatic transfusion-transmitted CMV infection in infants is
low compared to the high rate of seropositivity in adults. Moreover, symptomatic CMV infection is uncommon
in neonates born to seropositive mothers.124
The risk of transfusion-transmitted CMV infection is higher in multitransfused low birthweight infants
(<1200 g) born to seronegative mothers.13,123,125 For this reason, these infants should receive only CMV-
reduced-risk blood. One approach is to use blood from CMV-seronegative donors.
Leukocyte Reduction
The benefits of transfusions of leukocytereduced components for neonates include reducing the risk of
transfusion-transmitted CMV13,126,127 A recent study in Canada that evaluated clinical outcomes in
premature infants (weighing <1250 g) before and after nationwide implementation of universal leukocyte
reduction revealed no change in mortality or bacteremia rates. Flowever, rates of retinopathy of prematurity and
bronchopulmonary dysplasia decreased as did length of hospitalization.128 Other known benefits of transfusing
leukocyte-reduced components include prevention of febrile nonhemolytic transfusion reactions and HLA
alloimmunization.
Irradiation
Cellular blood components are irradiated to prevent TA-GVHD in immunocompromised recipients (see
Table 23-9). Expert opinions and practices differ on this topic. Therefore, protocols should be based on the
patient populations served, equipment available, and best practices. The processes of irradiation, irradiator
quality control, and quality assurance are beyond the scope of this chapter but are addressed in Chapters 1 and
9.
Volume Reduction and Washing
The plasma volume of the component is usually reduced in transfusions in premature infants who have renal
ischemia or compromised cardiac function. In 1993, the AABB Committee on Pediatric Hemotherapy stated
that volume reduction of platelet concentrates should be reserved for infants who have total body fluid
restrictions.63 Methods for platelet volume reduction have been published (see Method 6-13).129 However,
optimal centrifugation rates and preparation methods remain unclear. As with any platelet modification, the total
number of platelets typically decreases and platelet activation may occur with these procedures.130
Saline-washed RBCs and platelets are administered in an effort to reduce the risk of adverse reactions to
certain components from plasma, anticoagulant preservative solutions, and high levels of potassium.
Transfusion of
TABLE 23-9. Irradiation Guidelines for Neonates and Older Children Who Require Cellular Blood
Components23,26
1. Premature infants weighing <1200 g at birth.
2. Any patient with:
a. Known or suspected cellular immune deficiency.
b. Significant immunosuppression related to chemotherapy or radiation treatment.
3. Any patient receiving:
a. Components from blood relatives.
b. HLA-matched or crossmatched platelet components.
 591
unwashed RBCs or platelet products procured from the mother of an infant is strongly discouraged.26
Maternal cells must be washed for effective treatment of hemolytic disease of the newborn and neonatal
alloimmune thrombocytopenia when maternal RBCs and platelets are transfused.
Massive Transfusion in the Pediatric Setting
Trauma is the leading cause of death in infants, children, and young adults aged 1 to 21 years. Although
trauma rarely leads to hemorrhagic shock and massive transfusion, resuscitation after trauma can be
challenging.
The evidence to support pediatric MTP is limited, but several pediatric institutions use MTPs to improve the
outcomes of patients who have experienced trauma. The appropriate ratios of RBCs to plasma and platelets
have
not been well defined (ie, 1:1:1 or 2:1:1). However, studies indicate that an MTP in the pediatric setting is
feasible not only for providing rapid and balanced blood product support but also for decreasing the risk of
thromboembolic events.131'133 Furthermore, when Hendrickson et al examined the impact of coagulopathy in
102 pediatric trauma patients, they found that abnormal prothrombin time, activated partial thromboplastin
time, and platelet count at emergency room admission were strongly associated with mortality (p = 0.005,
0.001, and <0.0001, respectively).134 These investigators did not examine the effect of MTP resuscitation on
coagulopathy and mortality, which may provide insights into further optimization of MTPs. Although adult
studies have demonstrated benefit from MTP, the role of the MTP and the optimal ratio of blood components
still needs to be defined in the pediatric setting.131'133
KEY POINTS
1. RBCs are the most frequently transfused blood product in children. Frequent blood loss, including
iatrogenic losses from repeated phlebotomy, contribute to the need for RBC transfusions in the neonatal
population.
2. Symptomatic anemia and/or a target hemoglobin level is a preferred strategy for neonatal RBC
transfusion rather than a milliliter-for-milliliter replacement of due to blood losses.
3. A full-term newborn has a blood volume of approximately 85 mL/kg whereas a preterm infant has a total
blood volume of 100 mL/kg.
4. In infants <4 months of age initial patient testing must include ABO and D typing of their red cells and a
screen for unexpected red cell antibodies, using either plasma or serum from the infant or mother. During any
one hospitalization, crossmatch compatibility testing and repeat ABO and D typing may be omitted as long as
all of the following criteria are met: the antibody screen is negative; transfused red cells are group O, ABO-
identical, or ABO-compatible; and transfused cells are either D negative or the same D type as the patient.
Testing the infant’s reverse type for anti-A and/or anti-B is not necessary.
5. Small-volume simple RBC (no matter what the storage solution) transfusions when administered slowly
have been shown to have little effect on serum potassium concentrations in infants less than 4 months of age
despite elevated potassium levels in the plasma of stored RBCs.
6. During component preparation if aliquots are made with a sterile docking device, it is considered a
“closed system" and the original units expiration date can be used for the new aliquot product.
7. Dosing of RBC units with additive solutions such as AS-1, AS-3, and AS-5 should not exceed 10-15
mL/kg for neonates.
 592
AABB TECHNICAL MANUAL
8. Transfusion of ABO-incompatible plasma should be avoided in pediatric patients and especially in infants
because of their small blood and plasma volumes. If ABO out-of-group platelet transfusion becomes necessary,
plasma may be removed either by volume reduction or washing.
9. Chronic RBC transfusion therapy reduces the risk of stroke in patients with sickle cell disease by
decreasing the percentage of red cells containing hemoglobin S in order to reduce sickling and prevent an
increase in blood viscosity. Hemoglobin levels should be maintained around 8 to 9 g/dL with a hemoglobin S
level of <30%.
10. Patients with sickle cell disease have the highest rates of alloimmunization to red cell minor antigens
than any other patient group. The most commonly formed antibodies are to Rh, Kell, Duffy, and Kidd system
antigens. Many sickle cell treatment centers try to prevent red cell alloimmunization by matching for
phenotypically similar antigen profiles on recipients. This strategy is not the same at all centers and is
controversial because obtaining enough phenotypically similar units may be difficult.
11. Currently it is recommended that low-birthweight infants born to CMV-seronegative mothers receive
CMV-reduced-risk blood for transfusion. These units could be from CMV-seronegative donors or CMV-positive
donors whose donation has been leukocyte reduced.
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mass. Transfusion 2009;49:596-601
2. Blanchette V, Doyle J, Schmidt B, Zipursky A. Hematology. In: Avery GB, Fletcher MA, MacDonald
MG, eds. Neonatology: Pathophysiology and management of the newborn. 4th ed. Philadelphia: JB Lippincott,
1994:952-99.
3. Brugnara C, Platt OS. The neonatal erythrocyte and its disorders. In: Nathan DG, Orkin SH, eds. Nathan
and Oski’s hematology of infancy and childhood. 7th ed. Philadelphia: WB Saunders, 2009:21-66
4. Wallgren G, Hanson JS, Lind J. Quantitative studies of the human neonatal circulation. Acta Paediatr
Scand 1967;179(Suppl):43-54.
5. Hume H, Bard H. Small volume red blood cell transfusions for neonatal patients. Transfus Med Rev
1995;9:187-99.
6. Ohls RK. Evaluation and treatment of anemia in the neonate. In: Christensen RD, ed. Hematologic
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8. Luban NLC, Mikesell G, Sacher RA. Techniques for warming red blood cells packaged in different
containers for neonatal use. Clin Pediatr 1985;24:642-5.
9. DePalma L. Red cell antibody formation in the neonate and infant: Considerations for current
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10. Sanders MR, Graeber JE. Posttransfusion graftversus-host disease in infancy. J Pediatr 1990; 117:159-
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11. Heiko R, Bein G, Sachs U. Transfusion-associated graft-versus-host disease. Transfusion 2009;23:62-71.
12. Ohto H, Anderson KC. Posttransfusion graftversus-host disease in Japanese newborns. Transfusion
1996;36:117-23.
13. Strauss RG. Data-driven blood banking practices for neonatal RBC transfusions. Transfusion
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14. Strauss RG. Transfusion therapy in neonates. Am J Dis Child 1991;145:904-11.
15. Strauss RG. Neonatal transfusion. In: Anderson KC, Ness PN, eds. Scientific basis of transfusion
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18. Harris B, Lumadue J, Luban NLC, Pollack M. Transfusion-related hyperkalemic arrest from irradiated
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plasma potassium levels. Transfusion 1993;33:606-9.
20. Fung, MK, Roseff, SD, Vermoch, KL. Blood component preferences of transfusion services supporting
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24. Warwick R, Modi N. Guidelines for the administration of blood products. Arch Dis Child 1995;72:379-
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25. Voak D, Cann R, Finney RD, et al. Guidelines for administration of blood products: Transfusion of
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29. Strauss RG. Selection of white cell-reduced blood components for transfusions during infancy.
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30. Ludvigsen C, Swanson JL, Thompson TR, McCullough J. The failure of neonates to form red cell
alloantibodies in response to multiple transfusions. Am J Clin Pathol 1987;87:250-1.
31. Josephson CD, Castillejo MI, Grima K, Hillyer CD. ABO-mismatched platelet transfusions: Strategies
to mitigate patient exposure to naturally occurring hemolytic antibodies. Transfus Apher Sci 2010;42:83-8.
32. Winess, JA. Treatment and prevention of neonatal anemia. Neoreviews 2008;9:526-33.
33. Strauss RG, Burmeister LF, Johnson K, et al. AS1 red cells for neonatal transfusions: A randomized trial
assessing donor exposure and safety. Transfusion 1996;36:873-8.
34. Wang-Rodriguez J, Mannino FL, Liu E, et al. A novel strategy to limit blood donor exposure and blood
waste in multiply transfused premature infants. Transfusion 1996;36:64-70.
35. Liu EA, Mannino FL, Lane TA. Prospective randomized trial of the safety and efficacy of a limited
donor exposure transfusion program for premature neonates. J Pediatr 1994;125:926.
36. Bednarek FJ, Weisberger S, Richardson DK, et al. Variations in blood transfusions among newborn
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37. Maier RF, Sonntag J, Walka MW, et al. Changing practices of red blood cell transfusions in infants with
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38. Roseff SD. Pediatric blood collection and transfusion technology. In: Herman JK, Manno CS, eds.
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40. Strauss RG, Burmeister LF, Johnson K, et al. Feasibility and safety of AS-3 red blood cells for neonatal
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41. Goodstein MH, Herman JH, Smith JF, et al. Metabolic consequences in very low birth weight infants
transfused with older AS-1 preserved erythrocytes. Pediatr Pathol Lab Med 1999;18:173-85.
42. Rock G, Poon A, Haddad R, et al. Nutricel as an additive solution for neonatal transfusion. Transfus Sci
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43. Luban NLC, Strauss RG, Hume HA. Commentary on the safety of red cells preserved in extended-
storage media for neonatal transfusions. Transfusion 1991;31:229-35.
44. Tuchschid P Mieth D, Burger R, et al. Potential hazard of hypoalbuminemia in newborn babies after
exchange transfusion with Adsol red blood cell concentrates (letter). Pediatrics 1990;85:234-5.
 45. Brecher ME, ed. Collected questions and answers. 6th ed. Bethesda, MD: AABB, 2000:73-5.
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46. Fergusson, DA, Hebert, R Hogan, DL, et al. Effect of fresh red blood cell transfusions on clinical
outcomes in premature, very-lowbirth-weight infants: The ARIPI randomized trial. JAMA 2012;308:1443-51.
47. Patel, R, Josephson, C. Storage of red blood cells for transfusion of premature infants. JAMA
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48. Pridjian G. Fetomaternal interactions: Placental physiology, the in-utero environment, and fetal
determinants of adult disease. In: MacDonald MG, Seshia MM, Mullett MD, eds. Avery’s neonatology:
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49. Wong E, Roseff SD, eds. Pediatric hemotherapy data card. Bethesda, MD: AABB, 2009.
50. Kirpalani H, Whyte RK, Andersen C, et al. The Premature Infants in Need of Transfusion (PINT) study:
A randomized, controlled trial of a restrictive (low) versus liberal (high) transfusion threshold for extremely low
birth weight infants. J Pediatr 2006;149:301-7.
51. Whyte RK, Kirpalani H, Asztalos EV, et al. Neurodevelopmental outcome of extremely low birth weight
infants randomly assigned to restrictive or liberal hemoglobin thresholds for blood transfusion. Pediatrics
2009;123:207-13.
52. Bell EF, Strauss RG, Widness JA, et al. Randomized trial of liberal versus restrictive guidelines for red
blood cell transfusion in preterm infants. Pediatrics 2005;115:1685-91.
53. National Institute of Child Health and Human Development. Transfusion of prematures (TOP) trial.
Bethesda, MD: NICHD, 2012. [Available at https://www.nichd.nih.gov/ about/Documents/TOP_protocal.pdf
(accessed December 12,2013).]
54. Kleigman RM, Behrman RE, Jenson HB, eds. Nelson’s textbook of pediatrics. 18th ed. Philadelphia:
WB Saunders, 2007.
55. Vales T. Bilirubin distribution and dynamics of bilirubin removal by exchange transfusion. Acta Paediatr
Scand 1963;52S:149.
56. Koenig JM. Evaluation and treatment of erythroblastosis fetalis in the neonate. In: Christensen RD, ed.
Hematologic problems of the neonate. Philadelphia: WB Saunders, 2000: 185-207.
57. Ballard RA, Vincour B, Reynolds JW, et al. Transient hyperammonemia of the preterm infant. N Engl J
Med 1978;299:920-5.
58. Leonard JV The early detection and management of inborn errors presenting acutely in
the neonatal period. Eur J Pediatr 1985; 143:253-7.
59. Wong EC, Pisciotto PT. Technical considerations/mechanical devices. In: Hillyer CD, Strauss RG,
Luban NLC, eds. Handbook of pediatric transfusion medicine. London, UK: Elsevier Academic Press,
2004:121-8.
60. Luban NLC. Massive transfusion in the neonate. Transfus Med Rev 1995;9:200-14.
61. Bada HS, Chua C, Salmon JH, Hajjar W. Changes in intracranial pressure during exchange transfusion. J
Pediatr 1979;94:129-32.
62. Blanchette VS, Kuhne T, Hume H, Heilman J. Platelet transfusion therapy in newborn infants. Transfus
Med Rev 1995;9:215-30.
63. Andrew M, Vegh R Caco C, et al. A randomized, controlled trial of platelet transfusions in
thrombocytopenic premature infants. J Pediatr 1993;123:285-91.
64. Poterjoy BS and Josephson CD. Platelets, frozen plasma, cryoprecipitate: What is the clinical evidence
for their use in the neonatal intensive care unit? Semin Perionatol 2009;33: 66-74.
65. von Lindern JS, van den Bruele T, Lopriore E, Walther FJ. Thrombocytopenia in neonates and the risk
of intraventricular hemorrhage: A retrospective cohort study. BMC Pediatrics 2011;11:16.
66. Josephson C, Su L, Christensen R, et al. Platelet transfusion practices among neonatologists in the
United States and Canada: Results of a survey. Pediatrics 2009;123:278-85.
67. Roseff, SD, Luban, NL, Manno, CS. Guidelines for assessing appropriateness of pediatric transfusion.
Transfusion 2002;42:1398-413.
68. Strauss RG, Levy GJ, Sotelo-Avila C, et al. National survey of neonatal transfusion practices: II. Blood
component therapy. Pediatrics 1993;91:530-6.
 69. Pisciotto P, Snyder EL, Snyder JA, et al. In vitro characteristics of leukocyte-reduced single unit platelet
concentrates stored in syringes. Transfusion 1994;34:407-11.
70. Diab Y, Wong E, Criss VR, et al. Storage of aliquots of apheresis platelets for neonatal use in syringes
with and without agitation. Transfusion2011;51:2642-6.
71. Revel-Vilk S. The conundrum of neonatal coagulopathy. Hematology Am Soc Hematol Educ Program
2012;2012:450-4.
72. Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and
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young infant. Am J Pediatr Hematol Oncol 1990;12:95-104.
73. Andrew M. Transfusion in the newborn: Plasma products. In: Kennedy M, Wilson S, Kelton J, eds.
Perinatal transfusion medicine. Arlington, VA:AABB, 1990:145-77.
74. Goldenberg NA, Manco-Johnson MJ. Pediatric hemostasis and use of plasma components. Best Pract
Res Clin Haematol 2006; 19:143-55.
75. AABB, America’s Blood Centers, American Red Cross, Armed Services Blood Program. Circular of
information for the use of human blood and blood components. Bethesda, MD: AABB, 2013.
76. Bandarenko N, King K, eds. Blood transfusion therapy: A physician’s handbook. 11th ed. Bethesda,
MD: AABB, 2014 (inpress).
77. PresseyJG, Manno CS. Therapy for hemophilia and von Willebrand disease. In: Herman JK, Manno CS,
eds. Pediatric transfusion therapy. Bethesda, MD: AABB Press, 2002:355-82.
78. Price TH. The current prospects for neutrophil transfusion for the treatment of granulocytopenic infected
patients. Transfus Med Rev 2000;14:2-11.
79. Marfin AA, Price TH. Granulocyte transfusion therapy. J Intensive Care Med 2013 (in press).
80. Christensen RD, Bradley PP, Rothstein G. The leukocyte left shift in clinical and experimental neonatal
sepsis. J Pediatr 1981;98:101-5.
81. Sweetman RW, Cairo MS. Blood component and immunotherapy in neonatal sepsis. Transfus Med Rev
1995;9:251-8.
82. Rosenthal J, Cairo MS. Neonatal myelopoiesis and immunomodulation of host defenses. In: Petz LD,
Swisher SN, Kleinman S, et al, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill
Livingstone, 1996:685703.
83. Jenson HB, Pollack BH. The role of intravenous immunoglobulins for the prevention and treatment of
neonatal sepsis. Semin Perinatol 1998:22:5063.
84. Sandberg K, Fasth A, Berger A, et al. Preterm infants with low immunoglobulin G levels have increased
risk of neonatal sepsis but do not benefit from prophylactic immunoglobulin G. J Pediatr 2000; 137:623-8.
85. Hill HR. Additional confirmation of the lack of effect of intravenous immunoglobulin in prevention of
neonatal infection (editorial). J Pediatr 2000;137:595-7.
86. Ehrenkranz RA. The newborn intensive care unit. In: Oski FA, ed. Principles and practice of
pediatrics. 2nd ed. Philadelphia: J.B. Lippincott, 1994;19:322.
87. Burch KJ, Phelps SJ, Constance TD. Effect of an infusion device on the integrity of whole blood and
packed red blood cells. Am J Hosp Pharm 1991;48:92-7.
88. Criss VR, DePalma L, Luban NLC. Analysis of a linear peristaltic infusion device for the transfusion of
red cells to pediatric patients. Transfusion 1993;33:842-4.
89. Longhurst DM, Gooch W, Castillo RA. In vitro evaluation of a pediatric microaggregate blood filter.
Transfusion 1983;23:170-2.
90. Lindemann R, Haga P. Evaluation and treatment of polycythemia in the neonate. In: Christiansen RD,
ed. Hematologic problems of the neonate. Philadelphia: WB Saunders, 2000: 171-83.
91. Black VD, Rumack CM, Lubchenco LD, Koops BL. Gastrointestinal injury in polycythemic infants.
Pediatrics 1985;76:225-31.
92. Kevy SV. Extracorporeal therapy for infants and children. In: Petz LD, Swisher SN, Kleinman S, et al,
eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill Livingstone, 1996:733-55.
93. Bartlett RH, Andrews AF, Toomasian JM, et al. Extracorporeal membrane oxygenation for newborn
respiratory failure: Forty-five cases. Surgery 1982;92:425-33.
94. Friedman DF, Montenegro LM. Extracorporeal membrane oxygenation and cardiopulmonary bypass. In:
Hillyer CD, Strauss RG, Luban NLC. Handbook of pediatric transfusion medicine. London, United Kingdom:
Elsevier Academic Press, 2004:181-9.
 95. Sharon BI. Management of congenital hemolytic anemias. In: Simon TL, Dzik WH, Snyder ES, et al,
eds. Rossi’s principles of transfusion medicine. 4th ed. Bethesda, MD: AABB/WileyBlackwell, 2009:448-69.
96. Cohen AR, Norris CF, Smith-Whitley K. Transfusion therapy for sickle cell disease. In: Capon SM,
Chambers LA, eds. New directions in pediatric hematology. Bethesda, MD: AABB, 1996:39-88.
97. Adams DM, Schultz WH, Ware RF, Kinney TR. Erythrocytapheresis can reduce iron overload and
prevent the need for chelation therapy in chronically transfused pediatric patients. J Pediatr Hematol Oncol
1996;18:3-7.
98. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first stroke by transfusions in children with sickle
cell anemia and abnormal results on
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transcranial Doppler ultrasonography. N Engl J Med 1998;339:5-11.
99. Aygun, B, Wruck, LM, Schultz, WH, et al. Chronic transfusion practices for prevention of primary
stroke in children with sickle cell anemia and abnormal TCD velocities. Am J Hematol 2012;87:428-30.
100. Rosse WF, Gallagher D, Kinney TR, et al. Transfusion and alloimmunization in sickle cell disease.
Blood 1990;76:1431-7.
101. Spanos T, Karageorge M, Ladis V et al. Red cell alloantibodies in patients with thalassemia. Vox Sang
1990;58:50-5.
102. Rosse WF, Telen M, Ware RE. Transfusion support for patients with sickle cell disease. Bethesda, MD:
AABB Press, 1998.
103. Smith-Whitley K. Alloimmunization in patients with sickle cell disease. In: Herman JK, Manno CS,
eds. Pediatric transfusion therapy. Bethesda, MD: AABB Press, 2002:240-82.
104. Hillyer KL, Hare VW, Josephson CD, et al. Partners for Life: The transfusion program for patients with
sickle cell disease. Immunohematol 2006;22:108-11.
105. Afenyi-Annan A, Brecher ME. Pre-transfusion phenotype matching for sickle cell disease patients
(letter). Transfusion 2004;44:619-20.
106. Tahan HR, Holbrook CT, Braddy LR, et al. Antigen-matched donor blood in the transfusion
management of patients with sickle cell disease. Transfusion 1994;34:562-9.
107. McPherson M, Anderson AR, Haight AE, et al. Transfusion management of HLA-matched sibling
bone marrow transplant (BMT) recipients with sickle cell disease (SCD). Transfusion 2009;49:1977-86.
108. Chou ST, Jackson,T, Vege S, et al. High prevalence of red blood cell alloimmunization in sickle cell
disease despite transfusion from Rhmatched minority donors. Blood 2013;122: 1062-71.
109. Blumberg N, Heal JM, Gettings KF. Leukoreduction of red cell transfusions is associated with a
decreased incidence of red cell alloimmunization. Transfusion 2003;43:945-52.
110. Van de Watering L, Jermans J, Witvliet M, et al. HLA and RBC immunization after filtered and buffy
coat-dependent blood transfusion in cardiac surgery: A randomized controlled trial. Transfusion 2003;43:765-
71.
111. McPherson M, Anderson A, Jessup P, et al. HIA Alloimmunization in pediatric sickle cell disease
patients: Potential impact for transfusion
management. Pediatr Blood Cancer 2010;54: 552-8.
112. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell hemolytic transfusion reaction syndrome.
Transfusion 1997;37:382-8.
113. Win N, Doughty H, Telfer P, et al. Hyperhemolytic transfusion reaction in sickle cell disease.
Transfusion 2001;41:323-8.
114. Garratty G. Autoantibodies induced by blood transfusion (editorial). Transfusion 2004;44:59.
115. Hoffbrand AV Al-Refaie F, Davis B, et al. Longterm trial of deferiprone in 51 transfusiondependent
iron overload patients. Blood 1998; 91:295-300.
116. Oliveri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood
1997;89:739-61.
117. Karan O, Lacroix J, Robitaille N, et al. Association between plasma transfusions and clinical outcome
in critically ill children: A prospective observational study. Vox Sang 2013;104:342-9.
118. Slichter SJ, Kaufman RM, Assmann SF, et al. Dose of prophylactic platelet transfusions and prevention
of hemorrhage. New Engl J Med 2010;362:600-13.
 119. Josephson CD, Granger S, Assmann SF, et al. Bleeding risks are higher in children versus adults given
prophylactic platelet transfusions for treatment-induced hyoproliferative thrombocytopenia. Blood
2011;120:748-60.
120. Larsson LG, Welsh VJ, Ladd DJ. Acute intravascular hemolysis to out-of-group platelet transfusion.
Transfusion 2000;40:902-6.
121. Heal JM, Blumberg N. The second century of ABO: And now for something completely different.
Transfusion 1999;39:1155-9.
122. Blumberg N, Heal JM, Hicks GL, Risher WH. Association of ABO-mismatch platelet transfusions
with morbidity and mortality in cardiac surgery. Transfusion 2001;41:790-3.
123. Bowden RA, Slichter SJ, Sayers M, et al. A comparison of filtered leukocyte-reduced and
cytomegalovirus (CMV) seronegative blood products for the prevention of transfusion-associated CMV
infection after marrow transplant. Blood 1995;86:3598-603.
124. Mussi-Pinhata MM, Yamamoto AY, Rego MAC, et al. Perinatal or early-postnatal cytomegalovirus
infection in preterm infants under 34 weeks gestation born to CMV-seropositive mothers within a high-
seroprevalence population. J Pediatr 2004;145:685-8.
CHAPTER 23 Neonatal and Pediatric Transfusion Practice
597
125. Bradley MT, Milam JD, Anderson DC. Use of deglycerolized red blood cells to prevent posttransfusion
infection with cytomegalovirus in neonates. J Infect Dis 1984;150:334-9.
126. Gilbert GL, Hayes K, Hudson H, et al. Prevention of transfusion-associated cytomegalovirus infection
in infants by blood filtration to remove leukocytes. Lancet 1989;i:1228-31.
127. Strauss RG. Selection of white cell-reduced blood components for transfusions during early infancy.
Transfusion 1993;33:352-7.
128. Fergusson D, Hebert PC, Lee SK, et al. Clinical outcomes following institution of universal
leukoreduction of blood transfusions in premature infants. JAMA 2003;289:1950-6.
129. Moroff G, Friedman A, Robkin-Kline L, et al. Reduction of the volume of stored platelet concentrates
for use in neonatal patients. Transfusion 1984;24:144-6.
130. Schoenfeld H, Muhn M, Doepfmer UR, et al. The functional integrity of platelets in volumereduced
platelet concentrates. Anesth Analg 2005;100:78-81.
131. Dehmer JJ, Adamson MD. Massive transfusion and blood product use in the pediatric trauma patient.
Semin Pediatr Surg 2010;19:286-91.
132. Hendrickson JE, Shaz BH, Pereira G, et al. Implementation of a pediatric trauma massive transfusion
protocol: One institution’s experience. Transfusion 2011 ;52:1228-36.
133. Chidester SJ, Williams N, Wang W, Groner JI. A pediatric massive transfusion protocol. J Trauma
Acute Care Surg;73:1273-7.
134. Hendrickson JE, Shaz BH, Pereira G, et al. Coagulopathy is prevalent and associated with adverse
outcomes in transfused pediatric trauma patients. J Pediatr 2012;160:204-9.
 Chapter 24
Patient Blood Management
Mary Ghiglione, RN, MSN, MHA, and Kathleen E. Puca, MD, MT(ASCP)SBB
^^TRANSFUSION OF BLOOD COmpOB181 nents plays a critical role in clinical care. More than 13.5
million Red Blood Cell (RBC) transfusions were administered in US hospitals in 2011 at an estimated cost of
$10 billion.1,2 Transfusion is the single most commonly billed procedure for patients receiving hospital care.3
Blood transfusions can be life-saving, but they can also be associated with complications. Transfusion-
transmitted infections, although rare, are of concern. In addition, a growing body of literature demonstrates an
association between transfusions and poorer patient outcomes.4'8
The need to better understand the benefits and risks associated with transfusion and to control health-care
costs has led to a growing interest by many organizations in initiatives to improve the quality, stewardship, and
utilization of blood components. Identifying best practices—including provision of quality health care without
transfusion—is being addressed by professional societies, federal agencies, hospital systems, and other
stakeholders.
DEFINITION AND SCOPE OF PATIENT BLOOD MANAGEMENT

Patient blood management (PBM) can be defined as an evidence-based, multidisciplinary approach to

optimizing the care of patients who might need transfusion. Evidence-based guidelines for the use of blood
components and education for health-care providers on best practices in blood transfusion are vital elements of
PBM. Employing a combination of pharmacologic, surgical, and medical modalities to conserve blood also
plays an important role in the overall care of patients.
Although the term “patient blood management” may be fairly new, the concepts have developed over time
in parallel with medical advances. A full discussion of this history is beyond the scope of this chapter. However,
two examples illustrate the shared “drivers" of blood transfusion and PBM.
One example is the influence of wartime injuries. The discoveries of blood groups, crossmatching, and
blood preservation in the early years of the 20th century enabled the use
Mary Ghiglione, RN, MSN, MHA, National Director, Patient Blood Management, Accumen, Inc, San
Diego, California, and Kathleen E. Puca, MD, MT(ASCP)SBB, Medical Director, BloodCenter of Wisconsin,
Milwaukee, Wisconsin
The authors have disclosed no conflicts of interest.
599

600
AABB TECHNICAL MANUAL
of banked blood in the treatment of exsanguinating wounds suffered in the armed conflicts of the same
period. Yet transporting the blood was difficult, and battlefield surgeons developed techniques to treat casualties
when no transfusion was possible.
A second example is the influence of Jehovah’s Witnesses, who cite Biblical passages as the foundation for
their refusal of blood transfusion. By the middle of the 20th century, blood transfusion had become universally
accepted as a medical treatment for a wide range of indications. Jehovah’s Witness patients had to seek out
those few physicians and hospitals that would provide medical and surgical care without blood transfusion.
These surgeons and hospitals became increasingly utilized by Jehovah’s Witnesses and other patients, and blood
conservation programs began to flourish. With an emphasis on an individualized, patientcentric approach, these
programs led the way toward the PBM movement seen today.
Although the focus is frequently on surgery, PBM encompasses the entire scope of a patient’s hospital
experience. It includes:
1. Efforts to identify and manage anemia and bleeding risks before any treatment begins.
2. Use of blood-sparing surgical techniques and intraoperative blood recovery methods.
3. Adjunctive strategies during intensive care unit (ICU) and postoperative care that decrease the need for
transfusion.
4. Blood utilization review and feedback to ordering physicians.
5. PBM education of all health-care providers involved in patient care.
The scope of PBM activities is discussed in detail in the section on Basic Elements of a Patient Blood
Management Program.
THE RATIONALE FOR PATIENT BLOOD MANAGEMENT
Blood transfusion became universally accepted as a mainstream medical therapy long before all the risks
and complications associated with it were recognized. The compelling rea
sons for PBM include the risk of clerical errors, errors in patient identification, the threat of a new
transfusion-transmitted pathogen, and the mounting evidence that transfusions are associated with adverse
patient outcomes. Other potential drivers of PBM include increasing patient demand for improved quality in
health care, the projected shrinking of the eligible donor base, and increasing health-care costs.9,10
Risks of Transfusion
The infectious risks of transfusion that became so widely known in the late 20th century, which are now
rare, as well as unknown emerging pathogens continue to be a concern. Improvements in donor screening,
development of effective tests, and research into pathogen inactivation technology have all played a role in
reducing the known infectious disease risks of transfusion. Currently, the risk of either hepatitis C virus
transmission or human immunodeficiency virus transmission is less than 1 in 1,000,000 donations, and the risk
of hepatitis B is less than one in 300,000.11
The noninfectious risks of transfusion, including hemolytic transfusion reactions, transfusion-related acute
lung injury, transfusion-associated circulatory overload, and allergic reactions are more common than infectious
adverse events and often go unrecognized. Chapter 27, Noninfectious Complications of Blood Transfusion,
discusses the diagnosis, etiology, treatment, and prevention of these risks in detail.
An emerging body of evidence indicates that transfusion may not provide the expected therapeutic outcomes
long assumed by both clinicians and patients. This is especially the case in stable, nonbleeding patients.12
Randomized controlled trials assessing the efficacy of RBC transfusion have shown that restrictive transfusion
strategies for nonbleeding patients have no negative effect, and may even improve outcomes for some
patients.8,13'16
Recent research is identifying a link between allogeneic transfusion and worse patient outcomes. Although
many of these studies on blood transfusion are observational,
601
they have enrolled large numbers of patients across many different specialties (eg, cardiology, surgery, ICU,
gastroenterology). Such studies have repeatedly concluded that a strong association exists between RBC
transfusion and worse patient outcomes.5,8,9,17
Several studies have shown the risk of nosocomial infection in ICU, surgical, and trauma patients to be two
to four times higher in transfused patients than in nontransfused patients.5 In addition, the risk is dose-
dependent (ie, the more blood the patient receives, the greater the risk of adverse events).18,19 RBC transfusion
has been associated, in a doserelated fashion, with higher rates of cancer recurrence, stroke, myocardial
infarctions, renal complications, acute lung injury and respiratory arrest, longer ICU and hospital stays, and
mortality both in the hospital and the long term (5 years).5,17,20"24
These deleterious effects on clinical outcomes are not solely related to allogeneic RBC transfusions. One
single-center study of more than 800 critically ill patients demonstrated that 1) transfusion was independently
associated with the risk of acute lung injury and 2) the risk was higher with the transfusion of plasma-rich blood
components (platelets and plasma) than with transfusion of RBC units.25 Plasma transfusion in critically ill
patients has shown little benefit and has been associated with worse patient outcomes.26,27
Thus, a growing body of literature strongly suggests that for the nonbleeding patient the benefit of
transfusion is outweighed by unexpected risks and worse outcomes. The risks can be reduced only by a shift
from the more conventional transfusion practice (where transfusion is often a default decision) to a PBM
approach to transfusion practice (where the decision to transfuse is an option considered after evaluation of the
risks, benefits, and alternatives specific to each patient).
The Patient’s Perspective
Despite advances in safety of the blood supply, patients are concerned about needing a blood transfusion
during their treatment.28,29 Whether due to their underlying illness, inadequate
communication, or their physician’s demeanor, some patients trust that the provider who orders the
transfusion is acting in their best interest. Too often, patients do not ask for, or are not provided with,
information on alternative treatment options before receiving a transfusion.28
Allogeneic transfusion is considered a therapeutic intervention and, as with other medical interventions,
requires the patient to provide informed consent.30,31 In order for the patient to be adequately “informed,” the
consent discussion needs to include information about the risks and benefits not only of the transfusion option,
but of the alternatives to transfusion and the refusal of treatment as well.32 The PBM approach, which
encompasses all the options, provides an excellent avenue for open communication, joint consideration of
treatments, and the potential for improved patient outcomes and satisfaction.
PBM strategies are a necessity for patients for whom blood transfusion is not an option due to medical
status, cultural influences, religious beliefs, or unavailability of compatible blood. From the patient’s
perspective, the rationale for PBM is the ability to make a more informed choice, having access to a higher
quality of care, and potentially experiencing less risk.
The Physician’s Perspective
After “first do no harm,” a physician’s primary responsibility is to ensure effective treatment with minimal
risk. Clinicians intend to do what is best for their patients. But too often they take blood transfusion for granted,
assuming that benefits will be achieved and perceiving the risks to be minimal.
Medical school curricula devote a bare minimum of time for education on the risks of, indications for, and
alternatives to the use of blood.33,34 Physicians often learn transfusion practices based on the norms and
standards developed years ago. It is difficult enough for them to keep up to date with current research and best
practices in their own specialty, let alone to keep abreast of developments in transfusion medicine.
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 AABB TECHNICAL MANUAL
Studies show a wide variation in transfusion practice. Whether or not a patient receives a blood transfusion
is often influenced by the hospital where the patient is admitted or by the physician providing the clinical care,
rather than by the patient’s clinical condition.3,35-38
Transfusion based on physician behavior can be improved with the implementation of evidence-based
clinical practice guidelines and computer “dashboards” identifying best practices linked to blood utilization and
patient outcomes. Providing individual physicians with their usage data in comparison to that of their colleagues
can be a powerful tool for process improvement.
Audits and evaluation of transfusion practices, along with analysis of blood utilization data, are tools
hospitals can apply to identify best practices. From these tools, guidelines, algorithms, and protocols can be
developed and implemented across a service line in an effort to assist individual physicians in meeting quality
standards and improving patient care.
Thus, a PBM approach benefits not only patients, but the physicians who treat them. Physicians who use the
patient-centric approach of PBM will gain a reputation for better patient outcomes, shorter lengths of stay, and
reduced costs. In some facilities, blood utilization data are included on a physician “scorecard,” acknowledging
those physicians whose transfusion practice complies with the hospital’s guidelines.
The Hospital’s Perspective
Economic Pressures
Due to the economic pressures facing hospitals today, it is estimated that by 2020 one in three hospitals will
close or reorganize into a different type of health-care service.39 Opportunities to reduce costs are eagerly
sought by hospital administrators, and reducing blood usage is no exception.
The acquisition cost of an RBC unit is only a fraction of the total cost of that unit to the hospital. Using
activity cost-based modeling, Shander et al2 reported that the total cost for
blood- and transfusion-related activities for surgical patients ranged from $522 to $1183 per unit transfused.
They also found that total annual blood expenditures correlated not with the volume of surgeries performed, but
with the transfusion rate. The authors concluded that reducing the proportion of patients who receive
transfusions, the number of RBC units transfused per patient, or both can be an important cost-saving strategy
for hospitals.
In a model simulation, Zilberberg and Shorr40 showed that universal adoption of a restrictive transfusion
therapy (hemoglobin less than 7 g/dL) in all ICU patients in US hospitals could potentially result in cost savings
of nearly $1 billion annually. Even more important, the simulation indicated that this strategy could improve
quality of care and patient outcomes by preventing nearly 40,000 transfusion-attributable complications.
A recent blood utilization project involving 464 US hospitals identified opportunities for significant
reductions in blood utilization and costs.3 Several elements of a PBM program were noted as important steps
for hospital leaders to consider.
Appropriate reimbursement for healthcare supplies and services, including blood and transfusion, is a
growing concern for hospitals. Medicare reimbursements have failed to keep pace with increased costs, which
have exacerbated the revenue pressures. The Centers for Medicare and Medicaid Services (CMS) structure for
outpatient reimbursement identified that in 2013, payments for the more frequently transfused blood
components would decrease.41 An understanding of all the contributing factors to the cost of transfusion
(including reimbursement) is vital for hospitals to develop. The economic pressures can be an additional
incentive for reducing unnecessary transfusions and improving blood utilization.
Requirements for Accreditation
The Joint Commission promotes several standards related to hospital blood administration42 and requires
hospitals to review the use of blood and blood components in an effort to monitor and improve practices. The
Joint
603
Commission also has developed PBM Performance Measures, which include seven areas for
improvement.43 Although they are not universally practiced, the measures can help hospitals to monitor blood
usage.
Both AABB and the College of American Pathologists (CAP) promote standards for transfusion service
laboratories. These requirements address more specific, technical issues in quality testing and patient safety
related to blood administration.44,45 Often, it is the responsibility of the transfusion service to ensure that
requirements related to transfusion are met—even when the requirements involve activities outside the purview
of the laboratory staff.
For instance, the CAP requirement TRM.41025-Transfusionist Training Phase II requires all personnel who
administer blood components to be trained in proper identification of transfusion recipients. AABB Standards
for Blood Banks and Transfusion Services requires a peer-review program for monitoring and addressing
transfusion practices in all categories of blood and blood components.45'11911 Both require collaboration of
other hospital departments for compliance. With the multidisciplinary, team-oriented approach of PBM, an
additional benefit to hospitals may be increased ease of compliance, as well as more readily accepted and
implemented corrective actions and process improvements.
The Community’s Perspective
Initiatives to limit the use of blood also benefit the community by supporting a safe and adequate blood
supply for those who need it. The increasing age of the population and the diminishing donor pool may have
significant impacts on blood availability. In the next 10 to 20 years, it is expected that the number of individuals
in the United States who are more than 65 years of age will increase at a faster rate than those aged 16 to 64,
and make up 20% of the population.46,47
A recent review of data from the Health and Retirement Study reported that 31% of Americans over the age
of 65 received at least
one transfusion in a 10-year period.48 Although data are limited, it is estimated that patients who are 65 or
older receive 55% to 60% of RBC transfusions.47 Similar population demographics are expected in Germany,
where the predicted shortfall in the blood supply could be 47% by 2020.49
Blood availability may also be affected by changes in donor eligibility and storage of blood units. A better
understanding of iron depletion in frequent blood donors has prompted a discussion of increased donor
hemoglobin requirements and prolonged donation intervals.50,51 If ongoing trials confirm a relationship
between the age of stored blood units and increased morbidity and mortality following transfusion in acutely ill
adult patients, blood inventory management will change.52'56
Any shortening of the shelf-life of the RBC unit could put more pressure on diminishing blood supplies and
donor recruitment efforts may not be able to make up for the loss.57 Implementing PBM strategies to reduce the
need for allogeneic blood and making sure only appropriate transfusions of the correct dose are given are two
ways to support the available blood supply for a community.
BASIC ELEMENTS OF A PBM PROGRAM
As noted earlier in the section on Definition and Scope of PBM, several PBM strategies can be implemented
to decrease the use of allogeneic blood transfusions. They encompass all aspects of the patient’s evaluation and
clinical management. A PBM approach is typically implemented in elective surgical patients in the
preoperative, intraoperative, and postoperative phases of care as outlined in Table 24-158 and in the following
sections. PBM strategies are also relevant to medical patients. Although any one of the strategies used
individually will be successful in decreasing the use of allogeneic blood transfusion, they are most effective
when combined and individualized for the specific patient.59
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TABLE 24-1. Scope of Patient Blood Management
Nonsurgical or ICU and Blood Utilization Medical
Intraoperative
Preoperative Postoperative Review Education
■ Anemia screening ■ Replacement
■ Replacement ■ Clinical prac ■ In-service
and fluids
- grand
management - crystalloid fluids tice guidelines
rounds
- iron therapy - colloid - crystalloid - indications - conferences
■ Surgical
- cautious use of EPO - colloid - thresholds - reminders
techniques
■ Screen for bleeding - meticulous ■ Limit phlebot - restrictive (posters, data
risks and optimize suturing omy strategies cards, etc)
- harmonic
coagulation ■ Tolerate low ■ Blood Utiliza - journal club
scalpel
 - discontinue antico - suction control hemoglobin tion orTransfu ■ Continuing
edu
agulants and anti - rinsing swabs ■ Monitor bleed sion Committee cation
■ Acute
platelet drugs ing ■ Audits - certification
normovole
mic - CME/CE
- discontinue herbal ■ Wound drain - prospective
hemodilution credit
supplements, some ■ Intraoperative age - concurrent - online tools
vitamins blood recovery ■ Iron therapy - retrospective (webinars,
- address genetic ■ Hemostatics ■ Hyperbaric ■ Patient Blood interactive
- topical
coagulation abnor oxygen Management modules)
thrombin
■ Medical
malities - fibrin sealant ■ Point-of-care Coordinator or
and
■ Minimize nursing
- platelet gel testing Transfusion
crystalloids school
in acute bleeding ■ Point-of-care Safety Officer curricula
■ Limit phlebotomy testing
■ Preoperative autolo ■ Monitor acute
bleeding and
gous donation
man
age coagulation
■ Tolerate low
hemo
globin
■ Avoid
hypothermia
■ Tolerate low
blood
pressure
■ Positioning
Modified with permission from Becker J, Shaz B.58
ICU = intensive care unit; EPO = erythropoietin; CME = continuing medical education; CE = continuing
education.
Preoperative Strategies
Patient evaluation and planning are essential first steps of a PBM program. A detailed medical history and
physical examination, with special attention to family and personal history of spontaneous or postoperative
bleeding and anemia, are vital to identifying and supporting high-risk patients. Medications that may affect
coagulation should also be assessed. Structured questions can help with the evaluation, which should be
performed sufficiently ahead of the planned surgery (eg, 30 days) to allow for diagnostic testing and therapeutic
interventions, as needed.60
Anemia Assessment and Management
Recognition and management of anemia in the medical and preoperative patient is a core principle of PBM.
The World Health Organization (WHO) defines anemia as a hemoglobin level less than 13 g/dL in men and less
than 12 g/dL in premenopausal, nonpregnant women.61 Clinical signs and symptoms of anemia (tachycardia,
chest pain, shortness of breath, dizziness, headache, depression, cold extremities, pale skin, fatigue, and
decreased cognitive function) occur when the oxygencarrying capacities of the red cells are unable to meet the
oxygen demand of the tissues.
 605
Anemia is a common condition and is estimated to be as high as 75% depending on the patient’s underlying
conditions, reason for surgery, and definition of anemia used.62,63 Anemia has been independently associated
with increased perioperative mortality and morbidity in patients undergoing joint replacement and cardiac
surgery.62,64 In a retrospective study of noncardiac surgery patients 65 years or older, 30-day postoperative
mortality and cardiac event rates increased by 1.6% for every percentage-point decrease from the normal
hematocrit range.65 Even mild anemia has been shown to be an independent risk factor for adverse outcomes in
patients undergoing colon surgery.66
If anemia is detected, the initial test results may suggest possible etiologies.67 Additional laboratory testing
such as creatinine, reticulocyte count, iron and iron-binding capacity, ferritin, vitamin B12, and folate may help
in the diagnosis. Several published guidelines can assist the PBM program in developing algorithms for
identifying and treating anemia in medical and preoperative patients.67'70 Whenever possible, an elective,
high-blood-loss surgery should be delayed until the anemia is explained and appropriately treated.
Pharmacologic agents for anemia may include iron (both oral and intravenous preparations), folic acid,
vitamin B12, or erythropoiesis-simulating agents (see Appendix24-1). The choice of therapeutic agent should
be guided by the underlying etiology of the anemia, the patient’s other medical conditions, and available time to
treat before any surgery.
Intravenous (IV) iron replacement using iron sucrose, ferric gluconate, or iron dextran has been shown to
quickly correct iron deficiency anemia and may be the preferred route over oral preparations in patients
scheduled for surgery or in the postoperative period. The efficacy of IV iron in treating anemia has been
consistently proven in reducing the need for allogeneic blood transfusions in surgical patients.
Erythropoietic stimulating agents (ESAs) are also effective in increasing the hemoglobin level if sufficient
iron stores are present.
The use of ESAs to lower transfusion rates in patients undergoing elective, orthopedic surgery is well
established.71 However, due to the risk of thromboembolic events, tumor growth, and death associated with
ESAs, the Food and Drug Administration (FDA) has restricted its use, recommending more conservative dosing
and strict monitoring.72
Addressing Bleeding Risk
A second important aspect of PBM is minimizing the risk of bleeding. Assessing a patient’s risk for
bleeding in relation to the type and complexity of surgery and the presence of any pre-existing anemia and/or
coagulopathy can help formulate an individualized plan. Establishing protocols for discontinuation or dose
adjustment of antiplatelet and anticoagulant drugs is an important function of a PBM program and an essential
preoperative planning tool.
Published guidelines such as those developed by the Society for Thoracic Surgery and the American
College of Chest Physicians can provide a basis for development of such protocols.73'75 Protocols help
practitioners to optimize the patient’s hemostasis before an elective surgery while minimizing risk for
thrombotic events.
Herbal supplements such as garlic, ginkgo, and ginseng, have also been known to increase bleeding risk.
Patients should be asked about their use and ingestion should be discontinued before surgery. Readers are
directed to more focused reviews on the effect of herbal supplements on coagulation.76,77
Preoperative Autologous Blood Donation
Historically, preoperative autologous blood donation (PAD) had been the primary means to reduce the use
of allogeneic blood. With PAD the patient donates his/her own blood, ideally 4 to 6 weeks, before surgery. The
goal of the preoperative donation is for regeneration of the patient’s red cell mass and return of the patient’s
hemoglobin to the precollection value before surgery.
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With increasing safety of the blood supply, the number of autologous units collected in the United States has
declined.1,78 Other factors contributing to the decline include high wastage of PAD blood (45% or more is
discarded),79,80 higher risk for preoperative anemia after donation,80,81 errors related to production and
handling of these units,83,84 and increasing acquisition costs that may not be reimbursable.
In addition, a systematic review showed that patients in a PAD program had a higher likelihood of receiving
transfusions (allogeneic and/or autologous) compared to those patients who did not participate in PAD because
their hemoglobin did not completely recover before surgery.82 Therefore, patients in PAD programs incur a
higher overall risk to their health with a greater likelihood of being transfused.
Despite these concerns, PAD may be a reasonable option for patients with rare blood types or multiple red
cell alloantibodies or for patients who refuse allogeneic blood. In these situations, advanced planning and
evaluation of the patient are crucial before PAD is attempted. In order to mitigate the anemia induced by PAD,
the collection needs to occur at least 4 to 6 weeks before surgery (earlier if there are freezing capabilities for the
units) to allow sufficient time for recovery of the patient’s red cell mass.85
Intraoperative Strategies
Acute Normovolemic Hemodilution
Acute normovolemic hemodilution (ANH) is the removal of whole blood from the patient immediately
before or shortly after the beginning of the surgical procedure into a standard blood bag containing
anticoagulant. Crystalloid or colloid solutions or both are reinfused to maintain adequate circulatory volume.
ANH lowers the patient’s hematocrit, improves blood fluidity, and increases cardiac output and organ blood
flow, offsetting the decline in oxygen capacity of the diluted blood.86 Any blood lost from the patient during
the surgery is then diluted and has a reduced red cell content.
The ANH-collected whole blood is typically stored at room temperature (for up to 8 hours) and is often
reinfused near the end of the procedure or whenever transfusion is needed.87(p22] An added value of ANH is
that platelets and coagulation factors remain viable when the product is stored at room temperature. Although
ANH is considered a relatively safe procedure, reports on the clinical efficacy and benefits are mixed.88 In
some studies, the use of ANH resulted in lower allogeneic transfusion rates, fewer units transfused, and
decreased complications,89,90 while others failed to show any benefit.91,92
ANH is more likely to be of benefit in patients undergoing high-blood-loss procedures (1500 mL or greater)
who have preoperative hemoglobin levels of 12 g/dL or higher. Potential contraindications for ANH include
active cardiac ischemia, restrictive or obstructive pulmonary disease, renal failure, hemoglobinopathy
associated with hemolysis, and known coagulation abnormalities.93 Planning and coordination by the surgical
team are important for ANH to be effective.
Intraoperative Blood Recovery
Intraoperative blood recovery is another common PBM strategy. Use of recovered shed blood is efficacious
in several procedures, such as cardiothoracic, orthopedic, neurologic, vascular, and trauma surgery. Reduction
in allogeneic RBC transfusions by 38%, with an average savings of 0.68 unit of allogeneic RBCs per patient has
been reported.94
 Intraoperative blood recovery involves collecting shed blood from the surgical site, centrifuging it, and
washing it with normal saline. During the centrifugation and washing process, plasma, platelets, red cell stroma,
contaminants, and anticoagulant are removed. The washed red cells are transferred to a separate bag, and then
administered to the same patient. Ideally, washed recovered blood should have a hematocrit of at least 45% to
55%.
Concerns regarding the safety of blood recovery often surround its use in cancer and obstetric surgeries. The
concern is that unwanted material and cells (eg, tumor cells,
607
bacteria, and amniotic fluid) from the surgical field may end up in the washed product and be re-infused to
the patient. However, the use of double suction setup and leukocyte reduction filters has been shown to reduce
these risks.95,96 In addition, reviews do not support the theoretical concern for increased risk of either amniotic
fluid emboli or cancer recurrence, although the quality of the studies has been questioned.97,98 Prospective,
appropriately powered studies are needed to define the risks and benefits of recovered blood in these
controversial settings.
A recent novel approach for blood recovery in surgeries involving cardiopulmonary bypass (CPB) is the
Hemobag modified ultrafiltration system (Global Blood Resources, LLC, Somers, CT). After completion of
CPB and decannulation, residual whole blood from the CPB circuit, which can be up to 2 liters, is ultrafiltered
and hemoconcentrated. Excess water and inflammatory mediators are removed, resulting in a whole blood
product with hematocrit of approximately 50%. In contrast to washed, recovered blood, blood processed with
the ultrafiltration system contains platelets, plasma proteins, and coagulation factors that have been preserved
and concentrated.99,100 The use of this device should theoretically reduce exposure to allogeneic blood more
than washed, recovered blood, especially exposure to plasma and platelet transfusions; however, the clinically
relevant impact of this newer technique remains unknown.
Quality control and quality management programs for autologous blood products prepared by intraoperative
blood recovery devices are not yet as well developed and accepted among hospitals as other aspects of
transfusion medicine. Significant opportunity exists in hospitals for collaboration between the transfusion
service, surgery, perfusion, anesthesia, and nursing departments in the development of blood recovery
programs. Guidelines and regulatory requirements for establishing, enhancing, and maintaining quality and
safety for these particular transfusion practices are described in the AABB Standards for Perioperative
Autologous Blood Col
lection and Administration and other AABB publications.58,87,101'103
Anesthesia and Surgical Techniques
Minimizing intraoperative bleeding is critical for reducing the need for allogeneic transfusion. Obvious
measures include meticulous surgical technique and rapid and rigorous control of bleeding. Proper positioning
of the patient can reduce blood loss and is guided by two basic principles—elevating the surgical site above the
level of the heart and avoiding compression of venous drainage of the surgical field.104 The deliberate
reduction of mean arterial pressure, known as “controlled or deliberate hypotension,” in selected patients can
reduce blood flow to the surgical site and thereby decrease blood loss. However, the benefits must be balanced
against potential risk for organ ischemia.59,105
Maintaining normothermia is important for optimal coagulation and hemostasis. Efforts to keep the patient
warm by using warm IV fluids, air warming blankets and by keeping the surgical suite warmer can help to
avoid hypothermia and thus decrease surgical blood loss. Optimal fluid management, including choice of
infusion fluids, volume, and timing of administration, can also affect surgical blood loss and transfusion
requirements. Modern devices for tissue dissection, such as harmonic scalpels, water jet dissectors, and
electrocautery with argon beam coagulation can minimize tissue damage and provide better hemostasis at the
incision site. Other measures influencing surgical blood loss include use of tourniquets, direct control of
bleeding, infiltration of vasoconstrictors into the surgical wound, choice of ventilation patterns, and choice of
anesthesia.59,104'106
Point-of-Care Testing and Transfusion Algorithms
Point-of-care testing (POCT) has several advantages over conventional laboratory testing in that it can 1) be
utilized whenever necessary, 2) provide more rapid turnaround time, and 3) use minimal sample volumes for
testing. Use
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AABB TECHNICAL MANUAL
 of POCT can provide information for timely transfusion decisions and help to avoid iatrogenic anemia.
Several POCT devices are available that provide information on coagulation, platelet function, and clot
stability; they are used in the surgical and critical care settings.107’108
Transfusion based on clinical observation of bleeding and blood loss is highly empirical and variable.
Transfusion algorithms based on POCT can enhance management of bleeding by rapidly distinguishing between
bleeding due to coagulopathy and bleeding of surgical origin and can provide goal-directed transfusion therapy.
In cardiovascular surgical patients, the use of thromboelastography-based transfusion algorithms has
generally shown a reduction in transfusion requirements, particularly plasma and platelets, and a decrease in
blood loss compared to clinician-directed and/or standard laboratory-based transfusion algorithms.109111 In a
single center, small prospective trial, a therapy-guided algorithm based on viscoelastic and aggregometric
POCT was superior to that based on conventional laboratory testing for reducing allogeneic RBC transfusions
in coagulopathic, complex cardiac surgery patients.112 The authors attributed faster turnaround time and the
functional assessment of coagulation by the POCT as an advantage over the conventional quantitative
coagulation tests. It is important to note that institutions need to establish their own algorithmic decisions based
on available coagulation assays and therapeutic options.
Thromboelastography-guided algorithms are also routinely used in liver transplant surgery; however, the
evidence to date is limited in showing reduction in transfusion requirements.113 The use of
thromboelastography in trauma patients is emerging. Although recent studies have shown its potential for early
detection of trauma-induced coagulopathy and fibrinolysis for facilitating goal-directed therapy over
conventional laboratory tests,114,115 ongoing research is needed to assess its effectiveness in reducing
transfusion requirements.
Pharmacologic Agents
Hemostatic agents, such as antifibrinolytics, desmopressin, and topical agents, can assist coagulation during
surgery in an effort to reduce the need for blood transfusion. Aminocaproic acid and tranexamic acid inhibit
fibrinolytic activity, preventing premature breakdown of the blood clot. Systematic reviews have shown both
agents to be safe and effective in reducing surgical blood loss and the need for RBC transfusion.116 Topical
agents such as hemostats, sealants, and adhesives are gaining importance as useful tools during surgery and can
reduce bleeding by causing blood to clot, sealing vessels, or gluing tissues. More focused reviews of these
topical agents and other pharmacologic agents are available.117'119 Brief descriptions of several pharmacologic
therapies that may help control hemorrhage and/or prevent transfusion are found in Appendix 24-1.
POSTOPERATIVE STRATEGIES
Postoperative Blood Recovery
Postoperative blood recovery involves the collection and reinfusion of blood from surgical drains and/or
wounds. An adequate amount of blood needs to be collected and processed for this technique to be effective.
Thus, it is used mainly in cardiac and orthopedic surgical cases where the volume of shed blood can be
significant (500 mL or more).
Postoperative recovered blood can be unwashed or washed. For the unwashed product, shed blood is
collected and filtered in a device until sufficient volume is reached; then the blood is transferred to an infusion
bag for reinfusion. For the washed product, once sufficient shed blood is collected, it is further processed by
washing and then transferred to a bag for reinfusion.
In the past, reinfusion of unwashed shed blood was popular as a blood conservation technique in joint
replacement surgery. More recently, the increasing use of other blood management strategies, particularly
antifibrinolytics, has led to a decline in surgical bleed
609
ing, making postoperative blood recovery unnecessary.116 In addition, unwashed shed blood is less
desirable because it has a hematocrit ranging from 20% to 30% and contains activated clotting and complement
factors, inflammatory mediators, cytokines, and fat particles that can increase the risk for febrile
reactions.120,121
For patients with substantial postoperative blood loss, improved product quality and safety—hematocrit
ranging from 60% to 80% with removal of contaminants—can be achieved by using devices that wash and
concentrate the postoperative wound drainage blood. Maintaining competency of nursing staff and higher cost
for these devices may be disadvantages for some hospitals. However, when compared to allogeneic blood
transfusions, use of postoperative washed products in joint replacement surgery is potentially comparable in
cost.122
 Limiting Phlebotomy-Related Blood Loss
Minimizing the impact of phlebotomy on the development of iatrogenic anemia is a key strategy for
reducing transfusion requirement. Iatrogenic anemia related to routine laboratory testing is common. In a study
of 145 Western European ICUs, blood loss from phlebotomy averaged 41.1 mL per day per patient,123 which
could result in the loss of nearly a unit of blood for an ICU stay of 1-2 weeks. A US retrospective database
study of over 17,000 patients with acute myocardial infarction who were not anemic on admission showed that
patients who developed moderate to severe anemia had experienced nearly 100 mL higher mean blood losses
from phlebotomy over the course of their hospitalization than those who did not develop anemia.124 In a
single-center retrospective study of patients with prolonged ICU stays, after day 21 the odds of being transfused
was independently associated with phlebotomy volume.125
Reducing the quantity and frequency of laboratory testing is a logical measure to reduce the amount of
blood loss related to phle
botomy. Routine, standing orders are a common practice. Laboratory testing should be ordered when
clinically justified and the results are likely to change clinical management. When ordered, collection for
laboratory testing should be consolidated to minimize the number of tubes collected. Another approach is the
use of pediatric or small volume tubes for sample collection. This strategy can reduce blood loss from
laboratory testing by up to 70%.126,127 Smaller sample volumes, often less than 0.5 mL, can also be obtained
with the use of POCT devices.
Even the process of drawing the blood can be a significant cause of blood loss. Patients with invasive lines
(eg, central venous catheters or arterial catheters) have a threefold increase in phlebotomy volume compared to
patients without such catheters. The ease with which samples can be obtained and the added requirement to
discard the first few mLs (up to 10 mL) each time the line is accessed can contribute to greater blood loss.128
Such catheters should be discontinued as soon as they are no longer required. Orders for specimens drawn
from these catheters should be consolidated to minimize the amount of blood otherwise discarded. Use of a
device for blood sampling whereby the initial volume of blood can be reinfused into the patient instead of
discarded has shown reduction in mean phlebotomy volume by 50%, higher hemoglobin levels, and fewer RBC
transfusions even when a restrictive transfusion practice is implemented.127,128
Increased Tolerance of Anemia and Transfusion Thresholds
A patient’s response to anemia is highly individualized and dependent on his or her ability to maintain
adequate oxygen delivery to the tissues. Tolerance is dependent on the patient’s volume status, his or her
physiologic reserve (including cardiac, pulmonary, and renal function), and the dynamics of the anemia. The
response becomes one of the most important factors in determining the need for transfusion.129,130
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Patients with chronic anemia due to chronic renal failure or slow gastrointestinal bleeding often have
physiologically adapted to a lower hemoglobin level by increasing their cardiac output, heart rate, or stroke
volume. Rapid blood loss from surgical bleeding or trauma often results in patients displaying hemodynamic
instability, shock, and other symptoms that require more rapid volume replacement.
Increasing a patient’s tolerance for lower hemoglobin includes increasing oxygen delivery or decreasing
oxygen consumption, which can limit the need for RBC transfusion. Strategies to optimize the patient’s
hemodynamic status and oxygenation may include maintaining normovolemia with intravenous fluids, use of
appropriate vasopressor agents, use of supplemental oxygen or mechanical ventilation, adequate pain control
and/or sedation, maintaining normothermia, and avoidance and prompt treatment of any infections.131
Incorporating evidence-based transfusion protocols may further aid in reducing unnecessary transfusions.
Historically, hemoglobin levels of 10 g/dL or less have been used as the threshold for transfusion. Recent
randomized control trials involving nonbleeding critically ill patients, post-cardiac-surgery patients, elderly hip
fracture patients with cardiac disease, and patients with upper gastrointestinal bleeding have established
evidence-based transfusion thresholds. In all these trials, the use of a more restrictive transfusion threshold (eg,
hemoglobin of 7 to 8 g/dL) has been shown to decrease RBC transfusions without increasing morbidity or
mortality.8,132 In addition, an arbitrary hemoglobin level or “threshold” should not be the sole driver for
transfusion. Transfusion decisions should be individualized and based not only on the hemoglobin level but also
on the patient’s clinical signs and symptoms of anemia and their
ability to tolerate and compensate for the ane
• ns
 mia.
Traditionally, physicians have ordered 2 units for RBC transfusion, a practice that evolved without clear
rationale. A unit of blood can have varying effects on hemoglobin and hematocrit, depending on the patient’s
total
blood volume and shifts in fluid between fluid compartments. In the nonbleeding patient, single-unit RBC
transfusion followed with clinical reassessment is advocated.134,135 Often a single RBC unit provides an
adequate increase in hemoglobin and relieves the patient’s symptoms. The potential impact of implementing a
single-unit transfusion policy can be significant. Based on a transfusion threshold of 8 g/dL of hemoglobin and
adoption of a single-unit strategy, one center predicted that half an RBC unit or more could be saved per
patient.136 When combined with strict adherence to a restrictive transfusion threshold, a single-unit transfusion
policy can have an even greater impact.
BLOOD UTILIZATION REVIEW AND CHANGING PHYSICIAN BEHAVIOR
Changing the behavior of physicians whose transfusion practices are embedded in tradition and habit can be
challenging. The elements required to create a culture change in physician transfusion practice include: 1) a
desire for change, 2) the provision of a new behavior practice, 3) a perception of the new practice as safe and
straightforward, and 4) a presentation of change that is nonthreatening to autonomy.119
More and more hospital administrators are recognizing that the use of blood products may worsen patient
outcomes and that the cost of allogeneic blood is significant. Thus, a desire for change is already under way.
Strategies focused on education of appropriate transfusion practice, providing sound alternatives to transfusion,
and communication and feedback by respected colleagues or “champions” are fundamental elements of change
in transfusion practices. Building a team of dedicated stakeholders and champions as the network for change
management is crucial for success of any change and a key element for a successful PBM program.
Studies have demonstrated the ability of several interventions for changing transfusion
611
practice. Although a systematic review did not find one intervention more effective than another, the authors
concluded that even simple interventions appear to be effective.137 Interventions can be broadly grouped into:
1) education, 2) adoption of guidelines, 3) reminders, and 4) audits with feedback.
Education can be in the form of scheduled conferences or one-on-one teaching. One-on-one education with
physicians, although more time consuming, is likely to have a more sustained effect. The educational content
needs to be evidence-based, provided by respected colleagues or opinion leaders, and be ongoing. Providing
repeated sessions and easy access to educational information, such as on the hospital website, can help support
physicians as they consider changing their practices.
Evidence-based transfusion guidelines are the basis for improving appropriateness in transfusion.
Introduction of the guidelines needs to be combined with education and reminders such as posters and pocket
guidelines. Issuing a memo of the transfusion guidelines to physicians without follow-up typically fails to
reduce blood usage.138 Transfusion guidelines can be reinforced when they are used with computerized
provider order entry (CPOE) systems. Incorporating decision support tools into CPOE transfusion order sets
and requiring physicians to document the indication when outside of the guidelines can be effective in
improving practice.139,140
Interventions to change transfusion practice go hand in hand with auditing or monitoring of transfusion
practice. Audits that occur at the time of the transfusion order or during the 24 hours after the transfusion and
are accompanied with education provide a clear opportunity for changing behavior.141 Prospective audits
(approval before issuing the product) performed manually require resources and can be time consuming. The
implementation of CPOE can provide a more practical approach to monitoring each transfusion order. Chapter
28 provides a detailed discussion of blood utilization auditing.
Blood utilization reports that incorporate benchmarking with the goal of continuous
quality improvement are powerful tools to change transfusion practice. Providing physicians with outcome
data in relationship to their transfusion rates for a surgical procedure or specific treatment as well as comparison
to their peers and best practices are likely to motivate changes. Providing physicians with regular reports helps
to maintain awareness and encourages physicians’ interest in looking for additional strategies to reduce
avoidable transfusions.142
A recent initiative to improve transfusion practice has been the employment of transfusion safety officers or
patient blood management coordinators. As change agents they can provide regular education to all staff
involved in transfusion practice, increase awareness of and access to transfusion alternatives, develop a network
of caregivers and “champions” for the promotion of optimal transfusions, and provide utilization reports for
continuous improvement. Working with the local “champion,” these coordinators can be instrumental in
changing the culture and reducing allogeneic transfusions.143
Medical Education
Education is an integral part of any PBM initiative. Because behavioral change is sometimes difficult to
achieve, repetition and reinforcement over time are typically required for success. Because PBM is
multidisciplinary, different audiences may respond to different approaches.
Educational delivery options may include journal club, grand rounds, webinars, online courses, guest
lecturers, conference attendance, one-to-one instruction, self-study, and blood usage review feedback. Provision
of continuing education credits for educational activities can be an effective motivator for participation.
Although ordering physicians and nursing staff need the greatest understanding of PBM, other groups of
hospital personnel also benefit from educational opportunities. For instance, information technology and
financial personnel may not need to know the most
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AABB TECHNICAL MANUAL
technical details, but they do need to understand the intent, benefits, and outcomes of PBM efforts so they
can effectively support those efforts.
PROGRAM DEVELOPMENT
Successful implementation of a PBM program requires planning, education, and teamwork. It is important
to first analyze the current transfusion practices within the hospital. A needs assessment can help to evaluate the
current behaviors and understand the hospital culture and readiness for change.
Demonstrating the clinical benefits as well as potential cost savings with a PBM program is key to ensuring
support and buy-in at all levels. Care must be taken to ensure that clinical staff understand that the primary
objective of a PBM program is to improve patient outcomes, even though hospital administrators may also
focus on the cost savings.
The specific activities that are carried out as part of PBM program implementation can vary by institution,
and are defined by the executive management team of the facility. Guidance is available from a variety of
professional organizations. The Society for the Advancement of Blood Management offers Administrative and
Clinical Standards for Patient Blood Management Programs, which outlines 12 standards related to the
activities of a formal, comprehensive, organization-wide PBM program.144 A PBM program can be designated
as an activity level 1, 2, or 3 program as outlined in AABB Standards for a Patient Blood Management
Program.145^2'® The responsibilities for oversight and monitoring at each activity level are described in
Appendix 24-2.
An effective PBM program involves a multidisciplinary effort with input and participation from
administration, physician and nursing leadership, laboratory, transfusion medicine, ethics, registration, surgery,
finance, and technology personnel. Identifying champions who are willing to be spokespersons and drivers of
the program is essential when starting a program. A general strategy for imple
TABLE 24-2. General Strategy for Implementing a PBM Program
1. Education
a. A knowledgeable advocate
b. Executives
c. Core group (pharmacy, nursing, blood bank)
d. Any department involved in PBM activities (ordering clinicians, finance, information technology)
2. Buy-in from executives
a. “C” suite (CEO, COO, CFO, CMO)
b. Medical executive committee
c. Blood utilization/transfusion committee
d. Surgical services committee
3. Business proposal
a. Background/current environment
b. Program description
c. Financial aspects
d. Risk/benefit analysis
e. Summary/conclusion
 4. Teamwork
a. Broad-based stakeholder group of those affected by PBM program (multidisciplinary emphasis,
identification of concerns, details to be addressed, enhanced buy-in)
b. Smaller, more focused steering committee (baseline usage data, relationship to strategic plan,
implementation steps, timeline, policy review, monitor progress checks)
c. Effective meetings
d. Milestone celebrations
5. Evaluation
a. Possible improvements/lessons learned
b. Study of metrics/outcome data
c. Analysis/report of program success
d. Possible program expansion
meriting a PBM program is summarized in Table 24-2. More detailed descriptions of PBM program
implementation are available.146,147
613
KEY POINTS
1. Patient blood management (PBM) is an evidence-based, multidisciplinary approach to optimizing the care
of patients who might need transfusion.
2. Several factors have been drivers of PBM, including transfusion-associated risks, demand for improved
quality of care, promotion of evidence-based practice, economic pressures, overutilization of blood components,
and a projected shrinking of the blood supply.
3. Elements of PBM include: 1) managing anemia and bleeding risks before treatment begins, 2)
intraoperative blood recovery and blood-sparing surgical techniques, 3) ICU and postoperative strategies to
reduce the need for transfusion, 4) blood utilization review, and 5) education of health-care providers.
4. PBM can provide benefits to medical as well as surgical patients.
5. Preoperative anemia is common. Identifying and treating preoperative anemia is one of the fundamental
elements of PBM.
6. PBM involves more than just transfusion avoidance. It involves use of pharmaceutical agents, blood
recovery techniques, surgical tools to limit blood loss, limiting phlebotomy for laboratory testing, adherence to
transfusion guidelines, and medical education.
7. A multidisciplinary team with “champions” is critical for success of the PBM program.
8. Ongoing blood utilization reviews with benchmarking and emphasis on patient outcomes are key
elements of a successful program.
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119. Spotnitz W, Burks S. State ofthe art review: Hemostats, sealants, and adhesives II: Update as well as
how and when to use the components of the surgical toolbox. Clin Appl Thromb Hemost 2010;16:497-514.
120. Munoz M, Garcia-Vallejo JJ, Ruiz MD, et al. Transfusion of postoperative shed blood: Laboratory
characteristics and clinical utility. Eur Spine J 2004; 13 (Suppl 1) :5107-13.
121. Sinardi D, Marino A, Chillemi S, et al. Composition of the blood sampled from surgical drainage after
joint arthroplasty: Quality of return. Transfusion 2005;45:202-7.
122. Rao VK, Dyga R, Bartels C, Waters JH. A cost study of postoperative cell salvage in the setting of
elective primary hip and knee arthroplasty. Transfusion 2012;52:1750-60.
123. Vincent JL, Baron JF, Reinhart K, et al. Anemia and blood transfusion in critically ill patients. JAMA
2002;288:1499-507.
124. Salisbury AC, Reid KJ, Alexander KR et al. Diagnostic blood loss from phlebotomy and hospital-
acquired anemia during acute myocardial infarction. Arch Intern Med2011;171:1646-53.
125. Chant C, Wilson G, Friedrich JO. Anemia, transfusion, and phlebotomy practices in critically ill
patients with prolonged ICU length of stay: A cohort study. Crit Care 2006;10:R140.
126. Sanchez-Giron F, Alvarez-Mora F. Reduction of blood loss from laboratory testing in hospitalized adult
patients using small-volume (pediatric) tubes. Arch Pathol Lab Med 2008; 132: 1916-19.
127. Fowler RA, Berenson M. Blood conservation in the intensive care unit. Crit Care Med 2003;
31(Suppl):S715-S720.
128. Mukhopadhyay A, Yip HS, Prabhuswamy D, et al. The use of a blood conservation device to reduce
red blood cell transfusion requirements: A before and after study. Crit Care 2010;14:R7.
129. Lelubre C, Vincet JL. Red blood cell transfusion in the critically ill patient. Ann Intensive Care
2011;1:43.
130. Madjdpour C, Sphan DR, Weiskopf RB. Anemia and perioperative red blood cell transfusion: A matter
of tolerance. Crit Care Med 2006;34(Suppl):S102-S108.
131. Physiology of anemia and oxygen transport. In: Seeber P, Shander A. Basics of blood management. 1st
ed. Malden, MA: Wiley-Blackwell, 2007:9-20.
132. Carson JL, Carless PA, Hebert PC. Transfusion thresholds and other strategies for guiding allogeneic
red blood cell transfusion. Cochrane Database Syst Rev 2012; (4) :CD002042.
133. Carson J, Grossman BJ, Kleinman S, et al. Red blood cell transfusion: A clinical practice guideline
from the AABB. Ann Intern Med 2012;157:49-58.
134. Administrative and clinical standards for patient blood management programs. Englewood, NJ: Society
for the Advancement of Blood Management, 2010.
135. Napolitano LM, Kurek S, Luchette FA, et al. Clinical practice guideline: Red cell transfusion in adult
trauma and critical care. Crit Care Med 2009;37:3124-57.
136. Ma M, Eckert K, Ralley F, Chin-Yee I. A retrospective study evaluating single-unit red blood cell
transfusions in reducing allogeneic blood exposure. Transfus Med 2005;15:307-12.
137. Tinmouth A. Reducing the amount of blood transfused by changing clinicians’ transfusion practices.
Transfusion 2007;47:132S-136S.
138. Tinmouth A, MacDougall L, Fergusson D, et al. Reducing the amount of blood transfused. A
systematic review of behavioral interventions to change physicians’ transfusion practices. Arch Intern Med
2005; 165:845-52.
139. Lam H-TC, Schweitzer SO, Petz MH, et al. Effectiveness of a prospective physician self-audit
transfusion-monitoring system. Transfusion 1997;37:577-84.
140. Fernandez Perez ER, Winters JL, Gajic O. The addition of decision support into computerized
physician order entry reduces red blood cell transfusion resource utilization in the intensive care unit. Am J
Hematol 2007;82:631-3.
141. Damiani G, Pinnarelli L, Sommella L, et al. Appropriateness of fresh-frozen plasma usage in hospital
settings: A meta-analysis of the impact of organization interventions. Transfusion 2010;50:139-44.
619
CHAPTER 24 Patient Blood Management
142. Toy E Effectiveness of transfusion audits and practice guidelines. Arch Pathol Lab Med 1994; 118:435-
7.
 143. Brevig J, McDonald J, Zelinka ES, et al. Blood transfusion reduction in cardiac surgery:
Multidisciplinary approach at community hospital. Ann Thorac Surg 2009;87:532-9.
144. Freedman J, Luke K, Escobar M, et al. Experience of a network of transfusion coordinators for blood
conservation [Ontario Transfusion Coordinators (OnTraC)]. Transfusion 2008; 48:237-50.
145. Holcomb J, ed. Standards for a patient blood management program. Bethesda, MD: AABB, 2014.
146. Thomas J. Building a business case. In: Puca K, Johnson ST, eds. Transfusion medicine’s emerging
positions: Transfusion safety officers and patient blood management coordinators. Bethesda, MD: AABB Press,
2013:77-90.
147. Ozawa S, Thorpe E, Valenti J, Waters JH. Development of a blood management program. In: Waters
JH, ed. Blood management: Options for better patient care. Bethesda, MD: AABB Press, 2008:33-82.
Pharmacologic Therapies for Supporting Patient Blood Management
620
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CHAPTER 24 Patient Blood Management
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CHAPTER 24 Patient Blood Management
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AABB TECHNICAL MANUAL
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CHAPTER 24 Patient Blood Management
629
■ APPENDIX 24-2
Responsibilities for Activity Levels 1,2, and 3 PBM Programs*
Activity Activity Activity
Item Responsibility
Level 1 Level 2 Level 3
Evidence of institutional support for the patient blood
1 X X X
management program at the executive level.
2 Patient outcomes related to transfusion. X X X
Budgeting to the level of care required by the
3 X X X
implementation of these PBM Standards.
4 Pretransfusion patient testing and evaluation. X X X
5 Assessment of potential need for blood usage. X X X
Ordering of blood, including completion of typing and
6 antibody testing before procedure start time with a plan for X X X
antibody-positive patients.
Identification and management of presurgical anemia before
7 elective procedures for which type and screen or type and X X X
crossmatch is recommended.
Preprocedure optimization of patient coagulation function
8 including discontinuation of medications and herbal supplements X X X
that impair coagulation function.
Percentage of blood components wasted by component type
(such as general red cells, rare unit red cells, general platelets,
9 matched platelets, plasma, AB plasma, cryoprecipitate, and X X X
granulocytes) and cause (misordering, mishandling, not released
in a timely manner, outdating in stock, etc).
10 Minimize blood loss due to laboratory testing. X X X
Process for identifying patients lacking identification.
11 X X X
Standard 6.2.3 applies.
Processes to identify, prior to or upon admission, patients
12 X X X
who may refuse transfusion under any circumstances.
13 Adverse events and incidents related to transfusion. X X X
Processes and/or equipment to facilitate rapid decision
14 X X N/A
making with regard to anemia and coagulation management.
A plan by each service line to reduce perioperative blood
15 X X N/A
loss.
16 Strategies to reduce blood loss and manage anemia and X X N/A
coagulopathy in nonsurgical patients.
 Treatment of massive blood loss (massive transfusion)
17 X X N/A
including timely delivery of proper ratios of blood components.
Use of perioperative techniques consistent with current
18 AABB Standards for Perioperative Autologous Blood Collection X N/A N/A
and Administration.
An active program with evidence-based metrics and clinician
19 X N/A N/A
feedback to ensure compliance with transfusion guidelines.
A formal program to care for patients who decline the use of
20 X N/A N/A
blood or blood-derived products.
‘Holcomb J, ed. Standards for a patient blood management program. Bethesda, MD: AABB, 2014:2-3.
 Chapter 25
Transfusion Support for Hematopoietic Stem Cell Transplant Recipients
Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD
|RK|ALLOGENEIC HEMATOPOIETIC STEM BSicell transplantation (HSCT) recipients present a distinct
set of challenges for blood banks and transfusion services. When considering transfusion for an HSCT recipient,
one has to take into account not only the complexities associated with the patient’s underlying condition, but
also potential problems associated with recipient alloantibodies, donor passenger lymphocytes, and different
blood group systems. Over the past two decades, HSCT has become significantly more common. Moreover,
many patients now receive posttransplant care in community hospitals. Thus, issues pertaining to the
complexity of
transfusion support for HSCT recipients are no longer relegated solely to academic medical centers.
Given the increasing numbers of patients undergoing HSCT from related and unrelated donors and the wide
variety of clinical conditions that are currently treated with this approach, transfusion medicine specialists must
be prepared to address the challenges associated with transfusion support for these patients.1 This chapter
provides an up-to-date summary of the most common and important issues faced by blood bank physicians and
other medical practitioners at transfusion services in their treatment of HSCT recipients.
 Christopher A. Tormey, MD, Assistant Professor of Laboratory Medicine, Yale University School of
Medicine, New Haven, and Medical Director, Transfusion Service, VA Connecticut Healthcare System, West
Haven, Connecticut and Jeanne E. Hendrickson, MD, Assistant Professor of Laboratory Medicine, Yale
University School of Medicine, and Associate Medical Director, Transfusion Medicine/Apheresis Service, Yale-
New Haven Hospital, New Haven, Connecticut
C. Tormey has disclosed no conflicts of interest. J. Hendrickson has disclosed a financial relationship with
Terumo BCT.
The views expressed in this article are those of the authors and do not necessarily reflect the position or
policy of the Department of Veterans Affairs or the United States government.
631

632
AABB TECHNICAL MANUAL
IMPLICATIONS OF ABO- AND NON-ABO-ANTI GENINCOMPATIBLE RED BLOOD CELL
TRANSPLANTATION FOR TRANSFUSION
Incompatibilities in ABO blood group antigens are not necessarily a barrier to successful HSCT. In solid
organ transplantation, ABO compatibility may be essential. However, pluripotent and early committed
hematopoietic progenitor cells (HPCs) lack ABO antigens; thus, engraftment of HPCs is uninhibited even in the
presence of circulating ABO antibodies. Nonetheless, discrepancies in ABO and nonABO antigens between a
donor and a recipient do play an important role in transplantation and can become a major complicating factor
in the transfusion support of HSCT recipients.
In allogeneic transplantation, the relationships between the ABO types of the donor and recipient fall into
four categories: compatible, incompatible in the major crossmatch, incompatible in the minor crossmatch, and
bidirectionally incompatible.2'4 Table 25-1 lists potential ABO combinations between donors and recipients and
indicates the associated compatibility or incompatibility. ABO incompatibilities are present in 25% to 50% of
donor/ recipient pairs, and, therefore, careful atten
tion to blood component selection for transfusion is necessary.2,4'6 Although the terms “major,” “minor,”
and “bidirectional incompatibility” are most frequently used in the context of the ABO system, they are also
used to describe the presence of other red cell alloantibodies, such as anti-K or anti-D, in the plasma of the
donor and/or recipient.
Major ABO Incompatibility
Major ABO incompatibility creates two challenges: 1) the potential for acute intravascular hemolysis when
ABO-incompatible donor red cells within the graft are infused to a recipient who has high ABO antibody titers
and 2) the ongoing production of ABO antibodies by host immune cells directed against erythroid progenitors
and mature red cells produced by the engrafting HPCs.
The first challenge is typically addressed during the collection and/or processing of HPCs. Techniques, such
as red cell reduction of a marrow graft product, minimize the risk of hemolysis during infusion. Some HPC
products, including all umbilical cord blood cells, are cryopreserved before administration, and incompatible
donor red cells may be lysed during the freeze/thaw process. There is no general consensus on the threshold
level of
TABLE 25-1. Compatibility for HSCT by ABO Blood Group of the Donor and the Recipient
Recipient Donor ABO Group
ABO Group 0 A B AB
0 Identical Major* Major* Major*
Bidirectional
A Minor Identical Major*
(major/minor)*
Bidirectional
B Minor1 Identical Major*
(Major/minor)*
AB Minor1 Minor1 Minor1 Identical
*Due to a naturally occurring antibody (or antibodies) in the recipient (eg, the donor is group A and the
recipient is group 0 and has naturally occurring anti-A).
 +Due to a naturally occurring antibody (or antibodies) in the donor graft product (eg, the donor is group 0
and has naturally occurring anti-A, and the recipient is group A).
♦ Due to naturally occurring antibodies in both the donor and recipient (eg, the donor is group A and the
recipient is group B).

CHAPTER 25 HSCT Recipient Transfusion Support

633
incompatible red cells that may be safely infused, with currently acceptable volumes ranging from 10 to 20
mL. As suggested by some, the recipient’s antibody titer may also be used in guiding facility policy.2 In the
absence of red cell depletion or cryopreservation, plasmapheresis of the recipient immediately before graft
infusion may be warranted to reduce the circulating titer of ABO antibodies.
The second challenge, the continuous production of antibodies against the A and/or B antigens of engrafted
donor red cells and erythroid precursors, can continue for up to 3 to 4 months after HPC infusion. As a result,
erythropoiesis is often delayed, with red cell engraftment potentially occurring >40 days after transplantation.
Time to red cell engraftment may be even further prolonged with the use of reduced-intensity or
nonmyeloablative conditioning regimens.5,7 In severe cases, pure red cell aplasia (PRCA) can develop.5
Therefore, HSCT involving major ABO incompatibility may render some recipients transfusion dependent for
several months following transplantation. Fortunately, major incompatibility tends not to affect engraftment or
the production of other myeloid-lineage cells.
Minor ABO Incompatibility
Analogous to red cell depletion in major incompatibility, plasma depletion of the graft product can
significantly decrease donor alloantibodies in minor ABO incompatibility. However, even if isoagglutinins are
not completely removed, the ensuing hemolysis is typically mild and self-limited.4 A more significant problem
with minor-incompatible HSCT results from the rapid generation of anti-A and/ or -B by donor lymphocytes
against residual recipient red cells. This so-called “passenger lymphocyte syndrome” occurs approximately 5 to
16 days after infusion of HPCs. The patient experiences acute, immune-mediated hemolysis that can result in
morbidity and even mortality. Fortunately, in most cases, the hemolysis is not severe and eventually subsides
with the clearance of recipient red cells.4 If the hemolysis is severe and potentially life threatening, therapeutic
erythrocytapheresis can be
used to exchange incompatible recipient red cells with donor-compatible red cells. In patients believed to be
at particularly high risk of the passenger lymphocyte syndrome (usually based on the type of posttransplant
immunosuppression), prophylactic erythrocytapheresis may be warranted before transplantation.8
Bidirectional ABO Incompatibility
In bidirectional ABO incompatibility, complications arising from both major and minor ABO-incompatible
HSCT can occur in the recipient. To prevent these complications, the processing of HPC grafts might include
the reduction of both red cell and plasma content. The posttransplant period can be complicated by the onset of
hemolysis within days or weeks (as occurs in minor incompatibility), delayed engraftment of red cells, and/or,
in some cases, PRCA (as occurs in major incompatibility).
Incompatibility Related to Non-ABO Antigens
Red cell antigens of other blood group systems can present challenges similar to the ones described for
ABO-incompatible transplants. Overall, these incompatibilities are generally less frequently encountered, but
the presence of red cell antibodies in the patient (more common) and/or donor (less common) requires attention
and approaches that are similar to those outlined above for ABO incompatibility. Special consideration of graft
donor selection may also be needed when reducedintensity or nonmyeloablative conditioning regimens are used
in alloimmunized recipients, and cognate graft donor antigen/recipient alloantibody pairs should be avoided if at
all possible.
BLOOD COMPONENT CONSIDERATIONS
Selection of Blood Components
The selection of appropriate blood components for recipients of allogeneic HSCT is particularly
problematic. When determining
634
AABB TECHNICAL MANUAL
transfusion requirements for an allogeneic HSCT recipient, it is vital that the blood bank or transfusion
service keep detailed records of the patient’s pretransplant ABO group and ABO antibody titers and the donor’s
ABO group. It is also imperative to determine the stage of the transplant (ie, preparative period, immediate
posttransplant period, or postengraftment period with absence of recipient red cells and/or antibodies).9
Recommendations for optimal component selection at each point in the transplant process are shown in Table
25-2.
With regard to Rh(D), the recipient can continue to receive the type of components transfused before the
transplantation as long as the HSCT donor and recipient match. However, if the recipient is Rh negative and the
donor is Rh positive or vice versa, only Rh-negative Red Blood Cell (RBC) units should be transfused to
prevent alloimmunization to the highly immunogenic D-antigen. Given the small risk of D alloimmunization
from red cells contained in Rh-positive platelet units, Rh-negative platelets may be preferred if the blood supply
allows this. However, it has recently been suggested that this practice be reevaluated.10
RBC Support
The majority of HSCT recipients require transfusion support in the peritransplant period, regardless of
incompatibilities or blood group antigen mismatches. Symptomatic anemia is one of the most common
indications for RBC transfusion in HSCT recipients. In the absence of symptomatic anemia, a patient’s status
and underlying comorbid conditions can be used to determine whether RBC transfusion may be warranted.
Because specific RBC transfusion thresholds in HSCT populations are lacking, clinicians can rely on general
RBC transfusion guidelines. For instance, a threshold hemoglobin level of 7 g/dL is likely appropriate for most
stable, nonpostoperative adult HSCT recipients. However, a slightly higher threshold of 8 g/ dL is likely
appropriate for adults with preexisting heart disease, those at risk of end
stage organ damage, or HSCT recipients in the postoperative stage.1112
The mechanisms underlying anemia and RBC transfusion dependence in HSCT are sufficiently exceptional
to warrant brief further discussion. After intensive chemotherapy, serum erythropoietin (EPO) levels increase
rapidly and peak in the first week after treatment.13'15 Although recipients of autologous HSCT maintain
adequate EPO levels throughout the posttransplantation period, allogeneic recipients do not.16'20 Allogeneic
HSCT recipients experience a prolonged period of inappropriately low endogenous EPO levels, which often
necessitate prolonged RBC transfusion support. Interestingly, mixed results have been obtained by the various
researchers who have examined recombinant EPO dosing after allogeneic HSCT.13'20 Larger studies are
necessary to determine whether EPO is capable of reducing the RBC transfusion burden and preventing the
complications of HSCT, such as PRCA. It is noteworthy that the US Food and Drug Administration has recently
strengthened its warnings about EPO use after some studies showed that EPO treatment had adverse effects
(decreased survival and/or tumor progression) in some patients with cancer.16 Although no studies have
specifically linked EPO use to poor outcomes in HSCT patients, clinicians should be mindful of the potential
risks of EPO usage.
Platelet Support
Platelet recovery after allogeneic HSCT has been studied extensively. Several factors are strongly associated
with the rate at which patients become platelet transfusion independent, including: 1) the relationship between
the donor and the recipient (ie, whether they are related vs unrelated), 2) conditioning regimen used, 3) presence
of graft-vs-host disease (GVHD) or cytomegalovirus (CMV) infection, and 4) HPC CD34+ cellular
source/dose.17'23 To summarize broadly, the findings of several studies suggest that slower platelet engraftment
tends to be more common in patients
TABLE 25-2. Transfusion Support for Patients Undergoing HSCT by Type of ABO Incompatibility and
Stage of Transplant
CHAPTER 25 HSCT Recipient Transfusion Support
635

blood banks and transfusion services may consider providing ABO-mismatched platelets that have been
volume reduced to diminish their plasma content. RBCs = Red Blood Cells.
 636
AABB TECHNICAL MANUAL
receiving grafts from unrelated donors, who have higher-grade GVHD, or who have pretransfusion viral
infections.17'20 There is also increasing evidence to suggest that the source of an allogeneic transplant—such
as cord blood HPCs [HPCs(C)] vs HPCs from apheresis [HPC(A)]—is predictive of time to platelet
engraftment. Several studies have shown that the median time to platelet engraftment for HPC(C) transplants is,
on average, significantly longer than for recipients of HPC(A) or HPCs from marrow.20'23 Thus, longer-term
dependence on platelet transfusion is more likely for individuals in any of the above categories.
One major consideration for platelet transfusion in HSCT recipients is compatibility. Plasma (including the
significant volume of plasma in platelets) contains variable amounts of isoagglutinins. Although transfusion of
limited quantities of ABO-incompatible plasma and platelets is common in routine transfusion, this practice
cannot be readily applied to the recipients of allogeneic HSCT without careful consideration.24 In ABO-
incompatible HSCT, the plasma-containing components should be compatible with both the donor and the
recipient whenever possible. Recommendations for component selection are more fully described in Table 25-2.
In addition to the risk of hemolysis, some researchers have found other morbidities associated with ABO-
incompatible platelet transfusions. For instance, one pediatric study showed that such transfusions, when
combined with the use of melphalan, correlated with the development of hepatic venoocclusive disease,
possibly resulting from the binding of A- and/or B-antigens expressed on the surface of hepatic endothelial
cells.25 Therefore, some centers have an institutional policy requiring the transfusion of ABO-identical RBCs
and platelets only to HSCT recipients; at least one group has noted that this policy may be associated with
improved patient survival.26 There is an obvious need for additional prospective studies to address this issue.
In the context of platelet transfusion support in HSCT recipients, the following additional areas are
discussed more extensively below: 1) prophylactic vs therapeutic transfu
sions, 2) the transfusion “threshold,” 3) the appropriate transfusion dose, and 4) HLA or platelet antibodies.
Prophylactic vs Therapeutic Platelet Transfusions
Platelet transfusions are frequently administered for prophylaxis of bleeding, rather than to treat acute
hemorrhage, in HSCT recipients. Although this has been a traditional management approach for many years,
several recent studies have questioned the utility and clinical benefit of this practice. Two studies, both
published by Wandt and colleagues,27,28 examined the assumption that prophylactic platelet transfusion could
be replaced by therapeutic transfusion in recipients of autologous transplants. The researchers found that
although patients assigned to a therapeutic (rather than prophylactic) transfusion regimen had some bleeding,
there was no evidence of life-threatening hemorrhage. Moreover, therapeutic interventions resulted in dramatic
reductions in platelet usage. However, Stanworth and colleagues29 concluded, based on a recently published
trial, that prophylactic platelet transfusions were more effective in preventing hemorrhage in patients with
hematologic malignancies than in a nontransfused control population. Therefore, questions still remain
regarding the nature of appropriate, evidencebased prophylactic transfusion strategies for patients with
thrombocytopenia. The results of the studies conducted to date and future trials will help shape prophylactic
platelet transfusion practice for HSCT recipients.30
Platelet Transfusion Threshold
The acceptable threshold for prophylactic transfusion of platelets has been studied extensively for >20
years. One of the earliest established thresholds to prevent spontaneous hemorrhage was a platelet count of
<20,000/pL for patients undergoing chemotherapy/HSCT.31 Over time and as clinical trial data have accrued,
studies have consistently revealed that a platelet count <10,000/pL in uncomplicated thrombocytopenia (ie, in
patients without co
637
existing conditions such as fever, bleeding, or bacteremia/sepsis) is a reasonable threshold to prevent
increased bleeding or hemorrhagerelated morbidity.30,32'35 However, a study by Nevo and colleagues36 raises
questions about this threshold. In this study, HSCT recipients who received transfusions at a platelet count
<10,000/pL had significantly increased nonhemorrhagic mortality rates and reduced survival compared to those
infused at counts <20,000/pL. Although changes in the 10,000/ pL threshold should not be based on this study
alone, it is imperative that data continue to be collected to examine the appropriateness of this target platelet
count and, as noted previously, whether prophylactic platelet transfusions are necessary at any platelet count.30
Platelet Dose
Another question that frequendy arises with platelet transfusion is what the optimal platelet dose is per
transfusion episode. To date, three large-scale clinical trials have examined the question of platelet transfusion
dosing.37'39 A meta-analysis of aggregate data from these three studies showed no increased risk of significant
bleeding between low and standard platelet doses.30 It is important to note that, as best illustrated in the
PLADO trial, selecting platelet doses for patients based on body surface area may be a critical factor in the
provision of lower platelet doses. Drawbacks to a lower dosing regimen may include a lower platelet increment
after infusion and the potential for a greater number of platelet transfusions over time.30,39 Clearly, as more
data are collected, lower platelet transfusion doses may become accepted as routine clinical practice for HSCT
recipients.
HSCT Recipients with HLA orHPA Antibodies
Some patients scheduled to undergo HSCT possess antibodies against HLA and/or human platelet antigen
(HPA); both of these types of antibodies may reduce the efficacy of platelet transfusion, resulting in lower
corrected count increments.40 For more thorough discussions
of the evaluation and treatment of platelet refractoriness driven by HLA and HPA antibodies, see Chapters
18 and 19.
In addition to leading to potential problems with platelet transfusion, recipient alloantibodies against HPA or
HLA could delay platelet or white cell engraftment in some transplant settings. This is analogous to what occurs
in the red cell lineage with major ABOmismatched transplants. The issue of HPA and HLA matching in HSCT
donor-recipient pairs has also been raised by several groups because of concerns that mismatches between
donors/ recipients for HPA (which may serve as minor histocompatibility antigens) may increase the risk of
GVHD, although two studies have found otherwise.41,42
Plasma, Cryoprecipitated AHF, and Factor Concentrate Support
There are no specific recommendations regarding the transfusion of plasma, cryoprecipitated antihemophilic
factor, or factor concentrates in HSCT patients; therefore, adherence to general guidelines and/or expert
recommendations for the transfusion of these components is advised.43 As discussed above and outlined in
Table 25-2, the most important issue for plasma transfusion is the ABO group of the recipient and engrafting
cells.
 For HSCT complicated by GVHD, there has been interest in determining whether recombinant factor
concentrates could be used to treat bleeding complications. Dysregulation of hemostasis is a well-known
complication of GVHD. The utility of recombinant activated Factor VII (NovoSeven, Novo Nordisk, Zurich,
Switzerland), a potent procoagulant, has been investigated. A multicenter, randomized controlled trial found no
differences in bleeding score for any of three doses (40, 80, or 160 pg/kg) of Factor Vila compared to control
treatment for hemorrhage associated with GVHD in the setting of HSCT.44 The results of this trial suggest that
recombinant Factor Vila is likely to be ineffective as a first-line therapy for GVHD-associated bleeding.44
However, Factor Vila may still be useful as a last-resort
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AABB TECHNICAL MANUAL
treatment for patients with intractable bleeding.
Another possible hemostasis therapy arises from the fact that GVHD often results in the depletion of Factor
XIII, increasing the risk and severity of gastrointestinal hemorrhage.45 The high concentration of Factor XIII in
cryoprecipitate makes cryoprecipitate a potentially useful therapy for GVHD-related bleeding. Formal studies
are required to determine the efficacy of this approach.
Transfusion Support for Autologous Transplant Recipients
Because recipients of autologous transplants are not exposed to foreign graft products from donors, virtually
all of the concerns regarding major/minor incompatibility (and their impact on transfusion support) are not
applicable to this patient group. Before, during, and after HSCT, autologous recipients should be supported by
transfusions of blood components as needed and in compliance with standard protocols that are applicable to
any transfusion recipient. However, because of their underlying immunosuppression, autologous recipients may
require specialized products or components involving additional processing steps. Such issues and concerns
(applicable to both autologous and allogeneic transplant recipients) are discussed in greater detail below.
PATIENTS WITH NEUTROPENIA AND INFECTION THAT IS UNRESPONSIVE TO
ANTIMICROBIAL THERAPY
Traditionally, infusions of fresh granulocytes have been used to treat severe, antibioticrefractory bacterial or
fungal infections in patients with absolute neutrophil counts less than 500/pL.46,47 (For a more in-depth
discussion of granulocyte therapy, see Chapter 18.) To date, one study has demonstrated that neutrophil
transfusions can be efficacious in treating infection in HSCT recipients with neutropenia; however, this Phase
I/II study included only 11 participants.48 To more fully establish
the clinical efficacy of transfused granulocytes in the setting of HSCT, a multicenter randomized controlled
trial is currently under way.49 The results of this study will be helpful in understanding the potential benefit of
granulocyte infusion for severely ill HSCT recipients.
SPECIAL PROCESSING OF BLOOD COMPONENTS FOR HSCT RECIPIENTS
The irradiation of cellular blood products (eg, RBCs, platelets, and granulocytes) is intended to inhibit the
proliferation of donor lymphocytes, thereby preventing transfusion-associated GVHD, a reaction that is almost
uniformly fatal.9,50 Although it is generally accepted that HSCT recipients require irradiated components
during, and for at least 1 year after, transplantation, it is unclear whether these patients require irradiated
components after this time. Despite the absence of evidence indicating that irradiation is essential after this time
has elapsed, many institutions provide irradiated products indefinitely to HSCT recipients. This is likely a
prudent policy given the potential for lifelong immunosuppression associated with HSCT and the possibility of
relapse of the malignancy for which the patient initially underwent the HSCT.
Blood banks and transfusion services must rigorously ensure irradiation of components transfused to HSCT
recipients. Part of this vigilance is the development of systems or policies to identify patients who require
irradiated products. One approach to help reduce the possibility of inadvertently releasing a nonirradiated unit
for transfusion to an HSCT recipient is to require universal irradiation of selected blood components. In some
institutions, all platelet products are irradiated upon receipt from the regional blood center. Although this
strategy may not be practical for other components, such as RBCs, additional approaches may be developed,
such as irradiation of all components issued to in- and outpatient hematology/oncology wards.
639
The situation is equally complex with regard to CMV a pathogen linked to high morbidity and mortality
rates in HSCT recipients. It has been proposed that transfusions of components that were leukocyte reduced
before storage are as efficacious in the prevention of CMV spread as components collected from donors lacking
antibodies to CMV (CMV-seronegative donors).9 However, a meta-analysis of 829 recipients of CMV-
seronegative components and 878 recipients of leukocyte-reduced components revealed that the risks of CMV
infection were 1.63% and 3.01%, respectively, following transfusion.51 Because CMV is also acquired in the
community, conducting studies addressing this issue definitively remains logistically difficult.
The major shortcomings of CMV-seronegative products include their limited availability and the fact that
seronegative donors may have recently acquired CMV with viremia that is not detected by serologic testing. In
the absence of a large supply of CMV-seronegative products or antigen testing, leukocytereduced units can
reasonably be considered to be “CMV reduced risk.” To better triage requests for CMV-seronegative
components, the CMV status of recipients and donors should also be considered. Some hospitals provide CMV-
seronegative units to HSCT recipients who are CMV seronegative and received a transplant from a CMV-
seronegative donor, although leukocyte-reduced components may be equally efficacious.52
Special processing of transfusion units may also be needed for HSCT recipients who repeatedly experience
common transfusion reactions.50 For instance, HSCT recipients may develop allergic symptoms, such as
pruritis, urticaria, or wheezing; the severity of such symptoms may increase over time. One approach to prevent
repeated allergic reactions is to wash components to remove supernatant plasma using automated methods for
RBCs or by centrifugation and saline resuspension for platelets. These modifications, in conjunction with
timely premedication regimens, can help reduce the risk or severity of recurrent allergic or febrile transfusion
reactions.
SPECIAL CONSIDERATIONS FOR TRANSFUSIONS IN PEDIATRIC HSCT RECIPIENTS
In general, the considerations regarding transfusion support of pediatric HSCT recipients are similar to those
for adults.53 However, indications for transplant, stem cell source, and donor selection may be subtly different
in these patient populations. For example, children with inherited diseases (such as sickle cell disease or
thalassemia major) may be more likely to be treated with HSCT than adults. Furthermore, high-dose
chemotherapy with autologous stem cell rescue is more often utilized to treat some childhood malignancies
(including advanced stage neuroblastoma) than malignancies in adults. In addition, cord blood may be utilized
for transfusions to treat some diseases in pediatric recipients. For example, HPC(C) transfusion often provides a
sufficient dose for a child, but not for an adult, because of a child’s small size.
Special considerations are necessary for transplantation in children (or adults) with sickle cell disease during
both the pre- and peritransplant periods. Red cell phenotyping of the stem cell donor is recommended to prevent
alloimmunizaton in the recipient against the donated cells by guiding both donor selection and product
processing. If possible, stem cell donors who are negative for cognate red cell antigens against which recipients
have red cell alloantibodies should be considered.54 This is particularly important for reducedintensity or
nonmyeloablative conditioning regimens, which can induce long-term mixed chimerism.54 If antigen-positive
donors are utilized, then red cell reduction of the stem cell product is recommended (even in the absence of
ABO incompatibility) to avoid a transfusion reaction during stem cell infusion.
Because of the association between RBC components and HLA/HPA alloimmunization, consideration
should be given to pretransplant HLA/HPA antibody screening.55'57 The results of this screening should be
taken into consideration in planning posttransplant platelet transfusions to comply with the general
recommendations to keep platelet counts
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AABB TECHNICAL MANUAL
above 50,000/pL (to prevent cerebrovascular bleeding).58,59 Such results may also be important in HPC
donor selection for non-HLAmatched, nonmyeloablative transplants. An additional consideration is to perform
red cell exchange transfusion before transplantation with a goal of <30% to 45% hemoglobin S to prevent
vasoocclusive events resulting from granulocyte colony-stimulating factor administration.60
Beta-thalassemia major and severe aplastic anemia may be cured by matched sibling HSCT in childhood,
yet prior transfusion exposure may adversely influence engraftment and outcomes in subsequent
transfusions.61'63 Potential reasons for this association include humoral or cellular immunization to transfused
antigens (whether they are defined or other minor histocompatibility antigens) or iron overload. Early
transplantation for eligible patients may decrease the risk of graft rejection, and careful attention to iron status at
the time of transplantation is also recommended. The intensity of conditioning regimens is also likely a key
factor in outcomes of HSCT in patients with beta-thalassemia major or severe aplastic anemia, and their long-
term risk/benefit ratios deserve careful consideration.
INFORMATION PORTABILITY FOR HSCT RECIPIENTS
 Many patients undergo HSCT far from their usual medical institution. Before transplantation procedures, all
of the patient’s transfusion history (including red cell, platelet, or leukocyte alloantibody testing history) should
be communicated to the transplanting facility. Eventually, these patients may return for follow-up care to their
primary care physicians,
and supporting such patients can be a significant challenge for the transfusion service.
The flow of information from the transplant center to local providers is rarely perfect; with increased
complexity of care, critical data can be overlooked. Some institutions have created a process to deal with such
situations. For example, the nationally networked Department of Veterans Affairs medical center database can
be consulted to detect any ABO or Rh discrepancies and determine whether a particular patient has ever had an
alloantibody detected at another network hospital. If a transfusion service or blood bank does have an
interconnected hospital laboratory information system, such databases can be useful for gathering information
on transplant recipients.
Regardless of the availability of an interconnected information system, the transfusion service should
consider developing a policy with its hematology-oncology colleagues that clearly outlines the approach to
patients who receive their HSCT at an outside facility. This policy should call for communicating the following
important information to a patient’s usual health-care facility after that patient returns from an off-site transplant
facility: 1) whether the transplant was autologous or allogeneic; and, 2) if the transplant was allogeneic, the
ABO type of the donor, and any auto- or alloantibodies developed by the patient during care at that facility. In
some cases, if such information is not completely communicated, the transfusion service can contact the
transplant center and obtain the required information at the time of the patient’s first posttransplant visit. A
portable worksheet with these data should also be made available to patients.
KEY POINTS
1. Allogeneic HSCT recipients face distinct transfusion challenges because of their immunosuppression,
preexisting diseases, and potential for changes in their expressed blood group systems.
2. ABO compatibility is not critical in the selection of potential HSCT donors because pluripotent and early-
committed HPCs lack ABO antigens. ABO incompatibility does, however, influence transfusion decisions
during the peritransplant period.
 641
3. ABO incompatibilities, which are present in 20% to 40% of donor/recipient pairs, fall into three
categories: 1) incompatible in the major crossmatch, 2) incompatible in the minor crossmatch, and 3)
bidirectionally incompatible. Such incompatibilities have the potential to produce acute and, in the case of
major incompatibility, ongoing intravascular hemolysis and pure red cell aplasia. They can be partially
mitigated by red cell and/or plasma depletion of the graft before infusion.
4. Management approaches to transfusion are similar for adult and pediatric HSCT recipients; however,
indications for transplantation, stem cell source, and donor selection may be subdy different.
5. When transfusion requirements for allogeneic HSCT recipients are determined, it is vital that the blood
bank or transfusion service keep detailed records of the recipient’s pretransplant ABO group and the donor’s
ABO group. It is also imperative to determine the stage of the transplant.
6. Platelet concentrates are most frequently transfused to HSCT recipients to prevent an acute hemorrhage.
A platelet count threshold of 10,000/pL in uncomplicated thrombocytopenia is widely accepted as safe for
allogeneic recipients.
7. Cellular blood components are irradiated to inhibit the proliferation of donor lymphocytes, thereby
preventing transfusion-associated GVHD. Many services provide irradiated components indefinitely to HSCT
recipients.
8. RBC and platelet components that have been leukocyte reduced before storage are generally considered
to be equivalent to CMV-seronegative units in terms of CMV transmission risk. Some studies, however, suggest
that seronegative units may be marginally safer.
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transfusion-dependent thalassemia patients. Transfusion 2005;45:761-5.
58. Walters MC, Sullivan KM, Bernaudin F, et al. Neurologic complications after allogeneic marrow
transplantation for sickle cell anemia. Blood 1995;85:879-84.
59. McPherson ME, Anderson AR, Haight AE, et al. Transfusion management of sickle cell patients during
bone marrow transplantation
with matched sibling donor. Transfusion 2009; 49:1977-86.
60. Tormey CA, Snyder EL, Cooper DL. Mobilization, collection, and transplantation of peripheral blood
hematopoietic progenitor cells in a patient with multiple myeloma and hemoglobin SC disease. Transfusion
2008;48:1930-3.
 61. Lucarelli G, Gaziev J. Advances in the allogeneic transplantation for thalassemia. Blood Rev
2008;22:53-63.
62. Lucarelli G, Clift RA, Galimberti M, et al. Marrow transplantation for patients with thalassemia: Results
in class 3 patients. Blood 1996; 87:2082-8.
63. McCann SR, Bacigalupo A, Gluckman E, et al. Graft rejection and second bone marrow transplants for
acquired aplastic anaemia: A report from the Aplastic Anaemia Working Party of the European Bone Marrow
Transplant Group. Bone Marrow Transplant 1994; 13:233-7.
Chapter 26
Therapeutic Apheresis
*
Robertson D. Davenport, MD
a»THERAPEUTIC apheresis is the ^01 treatment of diseases through removal or extracorporeal
manipulation of blood components or specific blood substances. It is distinct from blood component collection
by apheresis, which is covered in Chapter 7. Standards and guidelines for therapeutic apheresis, clinical
privileging, and documentation are provided by AABB and The American Society for Apheresis (ASFA).1'4
PRINCIPLES AND MODALITIES
The goal of therapeutic apheresis is to remove a pathologic element from blood or to modulate cellular
function by manipulation such as through extracorporeal photopheresis (Table 26-1), with or without
replacement of the removed element. Apheresis can be performed manually, but automated techniques are faster
and more efficient. In therapeutic plasma exchange (TPE), 1.0 or 1.5 plasma volumes are typically processed.
Larger volume procedures can increase the risk of coagulopathy, citrate toxicity, or electrolyte imbalance,
depending on the replacement fluid. The effectiveness of apheresis in removing pathologic substances depends
on the concentration of those sub
stances in the blood, the volume of blood processed, and the equilibrium between blood and the substance’s
extravascular volume of distribution. The procedure is most efficient at the beginning because the amount of the
component removed decreases exponentially with time (Fig 26-1). Continued production or mobilization from
the extravascular space will result in a less-than-expected reduction. A 1.0 plasma volume exchange typically
removes approximately two-thirds of a substance if it does not move significantly from the extravascular to the
intravascular space.
Continuous flow centrifuge apheresis devices have a rotating channel designed to introduce whole blood at
one site, and the blood elements subsequently separate by density as blood flows through the channel. The
resulting layers of plasma, platelets, leukocytes, or red cells can be removed selectively. The component to be
removed is diverted into a collection bag, while the remaining blood components are mixed with the
replacement fluid and returned to the patient. The device controls the flow rates of blood withdrawal,
anticoagulant solution and replacement fluid infusion, as well as centrifuge speed to achieve optimal separation.

Robertson D. Davenport, MD, Medical Director, Blood Bank and Transfusion Service, and Associate
Professor, University of Michigan Health System, Ann Arbor, Michigan The author has disclosed no conflicts
of interest.
645

646
AABB TECHNICAL MANUAL
TABLE 26-1. Apheresis Modalities
Blood Component Replacement
Procedure Typical Indication
Removed Fluid
Therapeutic plasma Reduction of an abnormal plasma Albumin or
Plasma
exchange protein, eg, autoantibody plasma
Sickle cell disease-related
Red cell exchange Red cells Red cells
complications
Leukapheresis Butty coat Leukemia with leukostasis As needed
Thrombocytapheresis Platelet-rich plasma Thrombocytosis As needed
 Erythrocytapheresis Red cells Erythrocytosis None
Extracorporeal
Butty coat (reinfused) Chronic graft-vs-host disease None
photopheresis
Selective adsorption Specific plasma protein Hypercholesterolemia None
High-molecular-weight
Rheopheresis Age-related macular degeneration None
plasma proteins

Blood volumes processed

FIGURE 26-1. Theoretical removal of a substance by apheresis. For a substance that is strictly intravascular,
36.8% of the initial concentration remains after a single blood volume exchange. Flowever, if the substance is
also present in the extravascular space with a total volume of distribution equal to twice the blood volume,
60.6% will remain after a single blood volume has been processed.

647
Intermittent centrifugation devices use an alternative method in which a specified volume of whole blood is
first withdrawn into a centrifuge bowl. Blood withdrawal is then stopped, and the extracorporeal blood product
is processed. The rest of the procedure is similar to the continuous flow process in that the blood is centrifuged,
the selected component is diverted into a waste bag, and the remainder of the blood is returned to the patient
with appropriate replacement fluid. The process can be repeated for several cycles. Intermittent centrifugation is
most commonly used for extracorporeal photopheresis (ECP), although a continuous flow ECP device has been
introduced.5
Filtration devices also operate by continuous flow. Anticoagulated whole blood is passed through a
microporous filter that allows plasma to pass through but retains the blood cells. The separated plasma can then
be diverted into a waste bag, or, as in the case of selective adsorption, further processed and returned to the
patient. This type of device is not suitable for cytapheresis. A variant of this technique is rheopheresis, or
double-filtration TPE, in which high-molecular-weight molecules— primarily fibrinogen, low-density
lipoprotein (LDL), fibronectin, and von Willebrand factor (vWF)—are removed, reducing plasma viscosity.
In selective adsorption, blood or plasma is passed over a column that has a high affinity for a specific
component, such as immune globulin G (IgG) or LDL, and the effluent is returned to the patient. This has the
advantage of highly specific removal of the element of interest. However, it is restricted in the United States to
only a few conditions for which affinity adsorbers are available. Such techniques are more widely employed
outside of the United States.
INDICATIONS
Although there are many case reports of successful treatment of a variety of diseases and conditions by
apheresis, there have been few high-quality clinical trials. ASFA has published
clinical guidelines categorizing the indications for apheresis in 78 disease states.2
1. Category I: Disorders for which apheresis is accepted as first-line therapy, either as a primary stand-alone
treatment or in conjunction with other modes of treatment.
2. Category II: Disorders for which apheresis is accepted as second-line therapy, either as a stand-alone
treatment or in conjunction with other modes of treatment.
3. Category III: Disorders in which the optimal role of apheresis therapy is not established. Decision making
for patients should be individualized.
4. Category IV: Disorders in which published evidence demonstrates or suggests that apheresis is ineffective
or harmful. Institutional review board approval is desirable if apheresis treatment is undertaken in these
circumstances.
TPE
The goal of TPE is to remove a pathogenic molecule, protein, or high-molecular-weight complex from
plasma. In addition, TPE may be used to provide a deficient normal substance, such as an enzyme or
coagulation factor. Indications for TPE with the recommended treatment course are listed in Table 26-2. The
frequency and duration of treatment are guided by clinical judgment, although laboratory testing may be helpful
for some indications.
Replacement fluids for TPE are compared in Table 26-3. For most indications, albumin is the preferred
replacement fluid, because it is isosmotic with blood and has a smaller risk of adverse reactions and infectious
disease transmission than plasma. Plasma is indicated for thrombotic thrombocytopenic purpura (TTP) or if
coagulopathy is a concern. Red Blood Cells (RBCs) may also be used to prime the apheresis circuit when
treating small patients (ie, <10-20 kg) in whom the extracorporeal volume may exceed 10% to 15% of the
patient’s blood volume.
Diseases in which a pathogenic autoantibody is targeted include acute and chronic inflammatory
demyelinating polyradiculo
648
AABB TECHNICAL MANUAL
TABLE 26-2. Indications for Therapeutic Plasma Exchange
Typical
Course
Indication Modifying Conditions Category
(Number of
treatments)
QOD (3-
Acute disseminated encephalomyelitis II
6)
Acute inflammatory demyelinating QOD (5-
1
polyneuropathy (Guillain-Barre syndrome) 6)
After IVIG III
Daily
Acute liver failure III
(variable)
Amyloidosis, systemic IV
Amyotrophic lateral sclerosis IV
AlMCA-associated rapidly progressive Daily or
glomerulonephritis (Granulomatosis with Dialysis dependence 1 QOD ' (6-9)
polyangiitis; Wegener granulomatosis)
 DAH 1
Dialysis independence III
Antiglomerular basement membrane disease Dialysis dependent and no Daily or
III
(Goodpasture syndrome) DAH QOD ' (7-10)
DAH 1
Dialysis independence 1
Daily or
Aplastic anemia; pure red cell aplasia Aplastic anemia III QOD ■
(variable)
Pure red cell aplasia III
Daily or
Autoimmunic hemolytic anemia: WAIHA;
Severe WAIHA III QOD ■
cold agglutinin disease
(variable)
Severe cold agglutinin disease II
Burn shock resuscitation III Once
Desensitization, positive Daily or
Cardiac transplantation crossmatch due to donor III QOD
specific HLA antibody (variable)
Antibody-mediated
III
rejection
Daily (3-
Catastrophic antiphospholipid syndrome II
5)
Chronic focal encephalitis (Rasmussen QOD (3-
III
encephalitis) 6)
Chronic inflammatory demyelinating 2-3/week
1
polyradiculoneuropathy (variable)
Coagulation factor inhibitors Alloantibody IV
Daily
Autoantibody III
(variable)
QOD (3-
Cryoglobulinemia Symptomatic/severe 1
8)
Dermatomyositis or polymyositis IV
Dilated cardiomyopathy, idiopathic NYHAII-IV III QOD (5)
 649
TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)
Typical Course
Indication Modifying Conditions Category
Number of treatments)
Homozygotes with small Weekly
Familial hypercholesterolemia II
blood volume (indefinite)
Recurrent in transplanted Daily or GOD
Focal segmental glomerulosclerosis 1
kidney (variable)
HSCT, ABO incompatible Major HPC, Marrow II Daily (1-3)
Major HPC, Apheresis II
Complement gene
Hemolytic uremic syndrome, atypical II Daily (variable)
mutations
Factor H antibodies 1
MCP mutations IV
Hemolytic uremic syndrome,
Shiga toxin associated IV Daily (variable)
infection-associated
S. pneumonae associated III
Henoch-Schonlein purpura Crescentric III QOD (4-11)
Severe extrarenal disease III
Precardiopulmonary Daily or COD ■
Heparin-induced thrombocytopenia III
bypass (variable)
Thrombosis III
Hypertriglyceridemic pancreatitis III Daily (1-3)
Hyperviscosity in monoclonal
Symptomatic 1 Daily (1-3)
gammopathies
Prophylaxis for rituximab 1
Immune complex rapidly progressive
III GOD (3-6)
glomerulonephritis
Immune thrombocytopenia Refractory IV
Immunoglobin A nephropathy Crescentic III QOD (6-9)
Chronic progressive III
Inclusion body myositis IV
 Lambert-Eaton myasthenic syndrome II Daily or QOD
(variable)
Liver transplantation, ABO Desensitization, live Daily or QOD ■
1
incompatible donor (variable)
Desensitization, deceased donor III
Antibody-mediated
III
rejection
Antibody-mediated Daily or QOD
Lung allograft rejection III
rejection (variable)
(Continued)

650
AABB TECHNICAL MANUAL
TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)
Typical
Indication Modifying Conditions Category Course (Number
of treatments)
Acute CNS inflammatory demyelinating
Multiple sclerosis II QOD (5-7)
disease
Weekly
Chronic progressive III
(variable)
Myasthenia gravis Moderate-severe 1 QOD (3-7)
Before thymectomy 1
Myeloma cast Daily or
II
nephropathy QOD (5-7)
Nephrogenic systemic Daily or
III
fibrosis QOD (10-14)
Neuromyelitis optica Daily or
Acute II
(Devic syndrome) QOD (variable)
Weekly
Maintenance III
(variable)
Overdose, Daily
Mushroom poisoning II
envenomation, and (variable)
poisoning Envenomation III
Natalizumab and PML III
Paraneoplastic
III QOD (5-6)
neurologic syndromes
Paraproteinemic
demyelinating igG/igA 1 QOD (5-6)
polyneuropathies
igM 1
Multiple myeloma III
PANDAS; Sydenham
PANDAS exacerbation 1 QOD (5-6)
chorea
 Sydenham chorea 1
Daily or
Pemphigus vulgaris Severe III
QOD (variable)
Phytanic acid storage Daily or
II
disease (Refsum disease) QOD (variable)
POEMS syndrome IV
Daily
Posttransfusion purpura III
(variable)
Psoriasis IV
Red cell
QOD
alloimmunization in Before IUT availability III
(variable)
pregnancy
Renal transplantation, Daily or
Antibody-mediated rejection 1
ABO compatible QOD ' (5-6)
Desensitization, living donor, positive
1
crossmatch due to donor-specific HLA antibody
Desensitization, high PRA deceased donor III

651
TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)
Typical Course (Number of
Indication Modifying Conditions Category treatments)
Daily of
Desensitization, live
Renal transplantation, ABO incompatible 1 QOD
donor
(variable)
Antibody-mediated rejection II
Group A2/A2B into B,
IV
deceased donor
Schizophrenia IV
Scleroderma (Progressive systemic sclerosis) III 2/week (6)
Daily
Sepsis with multiorgan failure III
(variable)
Stiff-person syndrome III QOD (4-5)
Sudden sensorineural hearing loss III QOD (3)
Daily or
Systemic lupus erythematosus Severe II
QOD
Nephritis IV (3-6)
Daily
Thrombotic microangiopathy, drug associated Ticlopidine 1
(variable)
Clopidogrei III
Cyclosporine/tacrolimus III
Gemcitabine IV
Quinine IV
 Thrombotic microangiopathy, HSCT associated Refractory III Daily
(variable)
Daily
Thrombotic thrombocytopenic purpura 1
(variable)
Thyroid storm III Daily (2-3)
Daily or
Toxic epidermal necrolysis Refractory III QOD
(variable)
Voltage-gated potassium channel antibodies II QOD (5-7)
Daily or
Wilson disease Fulminant 1
QOD (3-5)
QOD = every other day; IVIG = intravenous
immunoglobulin; DAH = diffuse alveolar
hemorrhage; WAIHA = warm autoim
mune hemolytic anemia; NYHA = New York hematopoietic stem cell
Heart Association class; HSCT = transplantation;
MCP = membrane cofactor protein; CNS = central nervous system; PML = progressive multifocal
leukoencephalopathy; PANDAS = pediatric autoimmune neuropsychiatric disorders associated with
streptococcal infections; HIT = intrauterine transfusion.
neuropathy (AIDP and CIDP), antiglomerular elude renal transplantation with presensitizabasement
membrane antibody disease, and tion, and antibody-mediated organ transplant myasthenia gravis. Examples of
conditions in rejection, which the goal is to remove an alloantibody in

652
AABB TECHNICAL MANUAL
TABLE 26-3. Comparison of Replacement Fluids
 Replacement Advantages Disadvantages
Solution
2-3 volumes required Hypo-
oncotic
Crystalloids Low cost Nonallergenic l\lo viral risk
Lacks coagulation factors and
immunoglobulins
Higher cost
Albumin Iso-oncotic Low risk of reactions Lacks coagulation factors and
immunoglobulins
Viral transmission risk Citrate
Iso-oncotic
load
Plasma Normal levels of coagulation factors,
ABO compatibility required
immunoglobulins and other plasma proteins
Risk of allergic reactions
Iso-oncotic
Cryoprecipitate- Reduced high-molecular-weight von Willebrand
Same as plasma
reduced plasma factor and fibrinogen
Normal levels of most other plasma proteins
In myeloma with hyperviscosity, the goal is to remove an excessive paraprotein (M protein). Measurement
of plasma viscosity may not be useful in guiding therapy in some patients because plasma viscosity may not
correlate with symptoms. Normal plasma viscosity ranges from 1.4 to 1.8 centipoise (cP). Because most
patients are not symptomatic until the plasma viscosity is more than 4.0 or 5.0 ch patients with mild elevations
may not require treatment. In general, hyperviscosity becomes a concern when M protein concentrations reach 3
g/ dL for IgM, 4 g/ dL for IgG, and 6 g/ dL for IgA.6 Patients receiving rituximab (antiCD20) therapy for IgM
myeloma may experience a transient increase in M protein levels. Patients with pretreatment IgM greater than
5g/dL are at particular risk of developing symptomatic hyperviscosity.7
There are conflicting data regarding the efficacy of TPE for treatment of acute renal failure in myeloma. A
randomized controlled trial of TPE vs. conventional care showed no difference in mortality or renal function at
6 months.8 However, among dialysis-dependent
patients, 43% in the TPE group and none in the control group recovered renal function. Another randomized
clinical trial showed no benefit of TPE in a composite outcome measure of death, dialysis dependence, and
glomerular filtration rate.9 However, biopsy confirmation of the renal diagnosis was not required in this trial.
Similarly, a retrospective cohort study showed no benefit of TPE in either reducing mortality or preserving renal
function.10 If TPE is to be undertaken, biopsy confirmation of cast nephropathy may be advisable.
Diseases in which immune complexes may be pathogenic and can be removed by apheresis include rapidly
progressive glomerulonephritis, cryoglobulinemia, and vasculitis. Other indications include conditions treated
by removal of protein-bound drugs or toxins or high-concentration lipoproteins.
In TTP, a deficiency of the vWF-cleaving metalloprotease ADAMTS-13 results in accumulation of high-
molecular-weight vWF multimers with subsequent intravascular platelet activation and platelet-rich thrombi in
the
 653
microvasculature.11 In many cases, an inhibitor of ADAMTS-13 can be demonstrated. Plasma exchange is
first-line treatment for TTP, with the goal of removing both the inhibitor and large vWF multimers while
simultaneously replacing the deficient enzyme. Secondary forms of microangiopathic hemolytic anemia
associated with systemic lupus erythematosus, hematopoietic progenitor cell transplantation, chemotherapy, or
immunosuppressive medications may be clinically indistinguishable from idiopathic TTP. However, in many
cases, ADAMTS-13 activity has been shown to be normal or only moderately reduced, and the response to
plasma exchange is typically poor in these secondary forms of microangiopathic hemolytic anemias.
Transplant-associated microangiopathic hemolysis rarely responds to apheresis and probably represents a
different disease process.12
TPE to treat TTP is typically performed daily until the platelet count and lactate dehydrogenase level are in
the normal range, but the intensity and duration of treatment should be guided by the individual patient’s course.
After response has been achieved, intermittent apheresis or plasma infusion taper may be instituted, but the
efficacy of this approach in preventing relapse has not been established.13 TPE therapy has greatly improved
the survival rate in TTP; however, treatment failures do occur and cause major morbidity or death.14
Hemolytic uremic syndrome (HUSJ is a similar condition that occurs more commonly in children than
adults. HUS may follow diarrheal infections with verotoxin-secreting strains of Escherichia coli (strain
0157:H7) or Shigella. Compared to patients who have classic TTP, those with HUS have more renal
dysfunction and less prominent neurologic and hematologic findings. Most patients with HUS do not have
antibody to ADAMTS-13 and have normal activity of this protease. Although diarrhea-associated HUS rarely
responds to TPE, atypical HUS (aHUS) caused by complement factor deficiencies or autoantibodies to Factor H
may respond; however, patients with aHUS caused by membrane cofactor protein mutations may not respond to
TPE. A terminal complement inhibitor, eculizumab, may be ef
fective in the treatment of individuals with aHUS.15
TPE is increasingly being used in the treatment of central nervous system acute disseminated
encephalomyelitis. Experience in treating chronic progressive multiple sclerosis with TPE has been
discouraging. However, a randomized clinical trial in acute CNS inflammatory demyelinating diseases
unresponsive to steroids showed that TPE was beneficial.16 Early initiation of TPE is predictive of response,
and some clinical responses may not manifest until later in follow-up.17 TPE may be effective in neuromyelitis
optica, even in the absence of NMO antibodies.18
In focal segmental glomerulosclerosis (FSGS), a circulating factor that increases glomerular permeability
with resultant proteinuria has been demonstrated.19 A recent study has suggested that circulating urokinase
receptor may be involved in the pathogenesis of this disease.20 FSGS frequently recurs after renal
transplantation and can result in allograft failure. TPE may effectively remove the permeability factor and
induce remission in recurrent FSGS following renal transplantation. Response to TPE in the primary form of the
disease has not been well studied.
TPE may be an adjunct to immunosuppression in the treatment or prevention of antibody-mediated rejection
(AMR) of solid organ transplants. AMR presenting in the early posttransplantation period may respond better to
TPE than later AMR.21 TPE before renal transplantation of an ABO-incompatible kidney may be used to
prevent hyperacute rejection, and posttransplantation TPE is often used to treat AMR that occurs in this
setting.22,23 TPE in conjunction with immunomodulatory therapies, such as intravenous immune globulin
(MG), before transplantation can reduce the risk of rejection in HLA-alloimmunized patients.24
Cytapheresis
 The goal of cytapheresis is to remove excessive or pathogenic leukocytes, platelets, or red cells. In addition,
in red cell exchange, donor red cells are used to restore oxygen-carrying
654
AABB TECHNICAL MANUAL
capacity. Indications for cytapheresis are listed in Table 26-4.
In acute leukemia, high blast counts (typically >100,000/pL) can result in microvascular stasis with
headache, mental status changes, visual disturbances, or dyspnea. The leukocyte count at which a patient may
become symptomatic is variable. Typically, patients with acute or chronic lymphocytic leukemia tolerate higher
cell counts than patients with myelogenous leukemia, and cytapheresis may not be required. Cytapheresis
commonly results in a less-than-predicted reduction in leukocyte count, despite excellent collection, because of
mobilization and re-equilibration of cells from extravascular sites. Myelogenous leukemia cells commonly have
a higher density than lymphocytic cells and can be difficult to separate from red cells. Use of hydroxyethyl
starch enhances red cell sedimentation by rouleaux formation and can improve the efficiency of cytapheresis in
acute myelogenous leukemia. Massive thrombocytosis, typically >1,000,000/pL, can occur in essential
thrombocythemia, polycythemia vera, or as a reactive phenomenon. Such patients may be at risk of thrombosis
or hemorrhage. Reduction in platelet count is commonly less than predicted because of mobilization of platelets
to the peripheral blood, primarily from the spleen.
Red cell exchange is most commonly performed in the setting of sickle cell disease (SCD). The goal is both
to reduce the burden of hemoglobin S and to provide donor red cells containing hemoglobin A. Acute chest
syndrome is a serious complication of SCD, presenting as dyspnea, chest pain, and cough, often accompanied
by fever, leukocytosis, decreasing hematocrit, and pulmonary infiltrates. Respiratory failure can develop, and
death occurs in about 3% of cases.25 Red cell exchange is indicated for progressive infiltrates and hypoxemia
refractory to conventional therapy and simple transfusion.26,27 A common goal is to reduce hemoglobin S to
less than 30% with a final hematocrit not to exceed approximately 30%. Red cell exchange may also be
indicated for prevention of stroke in sickle cell anemia. For patients with elevated cerebral blood flow velocity
determined by
transcranial Doppler imaging, transfusion reduces the risk of stroke.28,29 Chronic red cell exchange,
typically every 4 to 6 weeks, can be effective in normalizing cerebral blood flow while minimizing iron
overload associated with simple transfusions. Red cell exchange may have a role in other sickle cell syndromes,
including priapism, multiorgan failure, hepatic/splenic sequestration, intrahepatic cholestasis. In addition, the
technique may be used for prevention of iron overload, and prevention or management of vaso-occlusive pain
crisis.
RBCs for replacement must be ABO compatible and negative for known clinically significant
alloantibodies. For sickle cell disease, the RBCs should be matched for C, E, and K, if possible.30 It is desirable
to use relatively fresh units to maximize posttransfusion red cell survival. Units containing either citrate-
phosphate-dextrose-adenine (CPDA)-1 or additive solutions (AS) may be used. It is desirable that all units used
in a given procedure contain the same anticoagulant solution so that they have similar hematocrits. Chronic red
cell exchange may carry a lower risk of alloimmunization than simple transfusion in sickle cell disease.31
ECP
ECP is a specialized procedure in which the buffy-coat layer is collected from peripheral blood, treated with
8-methoxypsoralen and ultraviolet A light, and re-infused into the patient. The treatment causes cross-linking of
leukocyte DNA, which prevents replication and induces apoptosis. The procedure was developed for the
treatment of cutaneous T-cell lymphoma, although it is increasingly used for other indications (Table 26-5).
ECP has complex immunomodulatory effects, including induction of monocyte differentiation to dendritic cells,
alteration of T-cell subsets, and changes in cytokine production profiles.32 ECP may be effective in acute and
chronic skin graft-vs-host disease (GVHD), although the role in non-skin GVHD is less well defined.33
ECP for solid organ transplant rejection has been best studied in cardiac and lung
655
TABLE 26-4. Indications for Cytapheresis
Indication Modifying Conditions Procedure Category
Babesiosis Severe RCE 1
High-risk population RCE II
Dermatomyositis or
Leukocytapheresis IV
polymyositis
 HSCT, ABO Minor HPC, Apheresis RCE III
incompatible
Hereditary
Erythrocytapheresis 1
hemochromatosis
Hyperleukocytosis Leukostasis Leukocytapheresis 1
Prophylaxis Leukocytapheresis III
Inclusion body
Leukocytapheresis IV
myositis
Inflammatory bowel Adsorptive
Ulcerative colitis Ill/ll
disease cytapheresis
Crohn’s disease Adsorptive cytapheresis III
Malaria Severe RCE II
Overdose Tacrolimus RCE III
Polycythemia vera and
Polycythemia vera Erythrocytapheresis 1
erythrocytosis
Secondary
Erythrocytapheresis III
erythrocytosis
Adsorptive
Psoriasis Disseminated pustular III
cytapheresis
Lymphocytapheresis III
Sickle cell disease,
Acute stroke RCE 1
acute
Acute chest syndrome, severe RCE II
Priapism RCE III
Multi-organ failure RCE III
Splenic sequestration; hepatic
RCE III
sequestration; intrahepatic cholestasis
Sickle cell disease, Stroke prophylaxis/ iron overload RCE II
Non-acute prevention
Vaso-occlusive pain crisis RCE III
Preoperative management RCE III
Thrombocytosis Symptomatic Thrombocytapheresis II
Prophylactic or secondary Thrombocytapheresis III
RCE = red cell exchange; HSCT = hematopoietic stem cell transplantation; HPC = hematopoietic progenitor
cells.
 656
AABB TECHNICAL MANUAL
TABLE 26-5. Indications for Photopheresis
Indication Modifying Conditions Category
Cardiac transplantation Rejection prophylaxis II
Cellular or recurrent rejection II
Cutaneous T-cell lymphoma; mycosis fungoides; Sezary
Erythrodermic 1
syndrome
Nonerythrodermic III
Graft-vs-host disease Skin (chronic) II
Skin (acute) II
Non-skin (acute/chronic) III
Inflammatory bowel disease Crohn's disease III
Bronchiolitis obliterans
Lung allograft rejection II
syndrome
Nephrogenic sytemic fibrosis III
Pemphigus vulgaris Severe III
Psoriasis III
Scleroderma (Progressive systemic sclerosis) III
transplantation. In a randomized clinical trial, prophylactic photopheresis in conjunction with previous
generation immunosuppression (not including calcinurin inhibitors or mycophenolate) resulted in fewer
rejection episodes, decreased HLA antibodies, and reduced coronary artery intimal thickness, but no difference
in time to first episode, incidence of hemodynamic compromise, or survival at 6 or 12 months.34,35 In
recurrent cardiac rejection, photopheresis may decrease severity of rejection and allow for reduced dosage of
immunosuppressives.36 ECP may have a role in the setting of cardiac rejection with hemodynamic compromise
and for bronchiolitis obliterans syndrome status after lung transplantation.37,38
Selective Adsorption
There are currently few established indications for selective adsorption of plasma proteins (Table 26-6) and
few devices have been
approved by the Food and Drug Administration (FDA).
In the two FDA-approved LDL apheresis devices, selective removal of LDL can be accomplished by
passing heparinized plasma over a dextran sulfate column or beads coated with anionic polyacrylate ligands, or
by precipitation of heparin-LDL complexes in acidified plasma. Because LDL production continues, LDL
apheresis treatments must be repeated, typically at 2-week intervals, indefinitely. There is evidence that LDL
apheresis reduces the incidence of major coronary events and stroke.39 In addition, atherosclerotic plaque
regression can occur in some patients.40 Secondary effects of LDL apheresis that may be beneficial include
reduction in levels of C-reactive protein, fibrinogen, tissue factor, and soluble adhesion molecules.41,42 LDL
apheresis may also be used in the treatment of primary or recurrent FSGS, although the mechanism of action
has not been well defined.43

657
TABLE 26-6. Indications for Selective Adsorption
Treatment
Indication Modifying Conditions Category
Modality
Age-related macular degeneration,
Rheopheresis 1
dry
Chronic focal encephalitis
IA III
(Rasmussen encephalitis)
Coagulation factor inhibitors Alloantibody IA III
Autoantibody IA III
Cryoglobulinemia Symptomatic/severe IA II
Dilated cardiomyopathy, idiopathic NYHA ll-IV IA II
LDL
Familial hypercholesterolemia Homozygotes 1
apheresis
LDL
Heterozygotes II
apheresis
LDL
Focal segmental glomerulosclerosis Primary, treatment resistant *
apheresis
Recurrent after renal LDL
transplantation apheresis
Immune thrombocytopenia Refractory IA III
LDL
Liprotein (a) hyperlipoproteinemia II
apheresis
Acute CNS inflammatory
Multiple sclerosis IA III
demyelinating disease
Paraneoplastic neurologic
IA III
syndromes
Paraproteinemic demyelinating
IgG/lgA/IgM IA III
polyneuropathies
 Pemphigus vulgaris Severe IA III
LDL
Peripheral vascular diseases III
apheresis
Phytanic acid storage disease LDL
II
(Refsum disease) apheresis
LDL
Sudden sensorineural hearing loss III
apheresis
Rheopheresis III
*FDA approved but ASFA category not yet assigned.
IA = immunoadsorption; LDL = low-density lipoprotein; NYHA = New York Heart Association class; CNS
= central nervous system.
IgG can be selectively removed by passing an alternative mechanism has been proposed
plasma over a column of staphylococcal pro- in ITP.44 Staphylococcal protein A adsorption
tein A bound to silica. The putative mecha- treatment can be performed manually or in
nism of action is the removal of pathogenic au- conjunction with automated TPE. Affinity col
toantibodies or immune complexes, although umns containing anti-IgG or ABO blood group

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AABB TECHNICAL MANUAL
substances have been tested in clinical trials, but are not currently approved for use in the United States.
ANTICOAGULATION
Acid-citrate-dextrose solution A (ACD-A), is the most commonly used anticoagulant, although heparin in
combination with ACD-A is also used, particularly in the setting of largevolume leukapheresis for
hematopoietic progenitor cell collection. Current automated apheresis devices control the administration rate of
citrate to achieve anticoagulation while minimizing the risk for hypocalcemia. Heparin anticoagulation is
necessary for LDL apheresis and may be desirable for selected patients undergoing TPE who are particularly
susceptible to hypocalcemia, such as small children, or in the setting of severe metabolic alkalosis or renal
failure. With citrate anticoagulation, coagulation monitoring is not generally necessary, although monitoring of
ionized calcium may be helpful for selected patients. The infused citrate in the returned blood is rapidly
metabolized and rarely causes systemic anticoagulation.
ADVERSE EFFECTS
Although therapeutic apheresis is very safe, complications do occur. An adverse event occurs in about 4%
of procedures (Table 26-7), but the majority are mild.45,46 Symptomatic hypocalcemia from infusion of citrate
with the returned blood is the most common adverse effect of apheresis. Perioral and digital paresthesias are the
most common symptoms. Nausea may also occur. Tetany is rare. Cardiac arrhythmia is very rare, but patients
with preexisting hypocalcemia or significant prolongation of the QT interval should be monitored carefully.
Calcium supplementation may alleviate the symptoms of citrate toxicity. A typical supplementary dose is 10 mL
of 10% calcium gluconate per liter of albumin infused. Citrate also chelates ionized magnesium, so it is possible
that hypomagnesemia contributes to the symptoms of citrate toxicity.
TABLE 26-7. Reported Frequency of Adverse Reactions to Apheresis*
Reaction Frequency (%)
Paresthesia 1.30
Hypotension 0.91
Urticaria 0.63
Nausea 0.39
Shivering 0.29
Flushing 0.16
Dyspnea 0.15
Vertigo 0.17
Arrhythmia 0.11
Abdominal pain 0.12
Anaphylaxis 0.02
Total 4.25
‘Adapted from Matsuzaki.40
However, one randomized clinical trial showed no benefit to adding magnesium during leukapheresis with
continuous IV calcium supplementation.47 Metabolism of citrate leads to a mild metabolic alkalosis, which can
exacerbate hypocalcemia, and may cause hypokalemia.48
Allergic reactions are most common with plasma replacement, although they may occur with albumin as
well. Most reactions are mild, characterized by urticaria or cutaneous flushing. More severe reactions can
involve the airways with dyspnea, wheezing, and (rarely) stridor. Most allergic reactions respond quickly to
intravenous diphenhydramine. Anaphylaxis is very rare but can occur. Patients with TTP who receive large
volumes of plasma are most at risk of allergic reactions. Premedication with an antihistamine, or possibly
steroids, is not necessary for routine apheresis but may be indicated for patients with repeated or previous
severe reactions.
Respiratory difficulty during or immediately following apheresis can have many

659
causes, such as pulmonary edema, pulmonary embolism, air embolism, obstruction of the pulmonary
microvasculature, anaphylactic reactions, and transfusion-related acute lung injury (TRALI).49 Hemothorax or
hemopericardium resulting from vascular erosion due to a central venous catheter is rare, but when it occurs it is
typically unsuspected, and may be fatal.50 Pulmonary edema that results from volume overload or cardiac
failure is usually associated with dyspnea, an increase in diastolic blood pressure, and characteristic chest
radiograph findings. Acute pulmonary edema may also arise from damage to alveolar capillary membranes
secondary to an immune reaction or to vasoactive substances in plasma or colloid solutions prepared from
human plasma. Predominantly ocular reactions (periorbital edema, conjunctival swelling, and tearing) have
occurred in donors sensitized to the ethylene oxide gas used to sterilize disposable plastic apheresis kits.51,52
Hypotension during apheresis can be a sign of citrate toxicity; hypovolemia; or a vasovagal, allergic, drug,
or transfusion reaction. Hypovolemia can occur early in a treatment of a small patient when the return fluid
consists of the saline used to prime the apheresis circuit. Vasovagal reactions are characterized by bradycardia
and hypotension. Such reactions usually respond well to a fluid bolus and placing the patient in the
Trendelenburg position.
When hypotension occurs during plasma or red cell exchange, a transfusion reaction such as TRALI, acute
hemolysis, bacterial contamination, or anti-IgA-related anaphylaxis should be considered. Hypotension is more
frequent in children, the elderly, neurology patients, anemic patients, and those treated with intermittent-flow
devices that have large extracorporeal volumes. Continuous-flow devices typically do not have large
extracorporeal volumes but can produce hypovolemia if return flow is inadvertently diverted to a waste
collection bag, either through operator oversight or device malfunction. Hypovolemia may also be secondary to
inadequate volume or protein replacement. During all procedures, it is essential to maintain careful and
continuous records of the volumes removed and returned.
When plasma is exposed to foreign surfaces of plastic tubing or filtration devices, the kinin system can be
activated, resulting in production of bradykinin. Infusion of plasma containing bradykinin can cause abrupt
hypotension. Patients taking angiotensin-converting enzyme (ACE) inhibitors are more susceptible to the
hypotensive reactions because the drugs block enzymatic degradation of bradykinin.53 Hypotensive reactions
are more likely during selective adsorption procedures because the devices expose plasma to a very large
surface area. Because some ACE inhibitors have a long duration of action, stopping the drug only the day
before the procedure may not be sufficient to prevent a reaction.
Intensive TPE without plasma replacement causes depletion of coagulation factors. A 1.0 plasma volume
exchange will typically reduce coagulation factor levels by 25% to 50%, though Factor VIII levels are less
affected.54 Levels of fibrinogen, a large molecule without extravascular distribution, are reduced by about 66%.
If the patient has normal hepatic synthetic function, coagulation factor levels typically return to near normal
within 2 days. Thus, many patients can tolerate TPE every other day for 1 or 2 weeks without developing
significant coagulopathies that require plasma replacement.
Bleeding as a consequence of coagulation factor depletion is rare but has been reported. For patients at risk,
plasma may be used for replacement at the end of the procedure. Apheresis can also cause thrombocytopenia,
particularly with HPC collection. Intensive TPE can cause hypogammaglobulinemia. Serum levels of IgG and
IgM recover to about 40% to 50% of the preapheresis level at 48 hours.54 The absolute immunoglobulin level
at which a patient becomes at risk of infections has not been established.
Albumin-bound drugs are removed by TPE. This can result in subtherapeutic levels of medications unless
dosage adjustments are made. High-molecular-weight biologicals such as MG, antithymocyte globulin, and
monoclonal antibodies have a long intravascular half-life and are readily removed by apheresis. TPE shortly
after administration of
660
AABB TECHNICAL MANUAL
such drugs should be avoided because it may significantly impair their effectiveness. In addition, intensive
apheresis reduces the concentration of potentially diagnostic plasma constituents, so blood for testing should be
drawn before initiating a course of treatment.
Collapsed or kinked tubing, malfunctioning pinch valves, or improper threading of tubing may damage red
cells in the extracorporeal circuit. Instrument-related hemolysis has been reported in 0.06% of therapeutic
apheresis procedures.45 Hemolysis can also occur with the use of incompatible replacement fluids such as 5%
dextrose solution (eg, D5W used to dilute 25% albumin) or ABO-incompatible plasma. The operator should
carefully observe plasma collection lines for pink discoloration suggestive of hemolysis. Other types of
equipment failure such as problems with the rotating seal, leaks in the plastic, and roller pump failure are rare.
Fatalities during apheresis are rare. Death has been reported in 0.006% to 0.09% of therapeutic
procedures.45,55 Most fatalities are attributable to underlying medical conditions.
VASCULAR ACCESS
Therapeutic apheresis requires good vascular access to achieve adequate flow rates. Peripheral access
generally requires at least a 17gauge needle for blood withdrawal and at least an 18-gauge catheter for return.
Patients without adequate peripheral veins or who require multiple frequent procedures may require central
venous catheters. Central venous catheters for apheresis must have rigid walls to accommodate the negative
pressure generated in the withdrawal line. Peripherally inserted central catheters (PICC lines) typically do not
support the high flow rates used in apheresis and should not be used. At least two lumens are required for
continuous-flow procedures, although single-lumen catheters can be used for intermittent-flow procedures. An
implanted subcutaneous port may be an option for some patients requiring long-term treatment, such as chronic
red cell exchange.
The choice of placement site for a central catheter is influenced by the expected duration of treatment.
Subclavian or internal jugular access is generally preferable for treatments lasting up to several weeks. Femoral
access should be used only temporarily because of the higher risk of infection. Patients requiring long-term
treatment usually have a tunneled catheter. With proper care, tunneled catheters can be used for prolonged
periods.
Good catheter care is very important to maintain patency and prevent complications. Catheters need to be
flushed regularly. Heparin or 4% trisodium citrate is usually placed in each lumen after each use to prevent
occlusion by clots. If a port becomes clotted, instillation of a fibrinolytic agent such as urokinase or
recombinant tissue plasminogen activator may restore patency. Routine dressing care is essential to prevent
insertion site infections. An arteriovenous (AV) fistula may also be used for apheresis, but apheresis personnel
should be suitably trained before attempting to access an AV fistula.
Venous access devices may cause further vascular damage, sometimes resulting in thrombosis. Infrequently,
they may result in severe complications such as pneumothorax or perforation of the heart or great vessels. Other
complications include arterial puncture, deep hematomas, and arteriovenous fistula formation. Bacterial
colonization often complicates long-term placement and may lead to catheter-associated sepsis, especially in
patients who are receiving immunosuppressants. Inadvertent disconnection of catheters may produce
hemorrhage or air embolism.
PATIENT EVALUATION
All patients should be evaluated by a physician familiar with apheresis before treatment begins. The
indication, type of procedure, frequency and number of treatments, and the goal or endpoint should be
documented in the patient’s medical record. The nature of the procedure, the expected benefits, the possible
risks, and the available alternatives must be explained to the patient, and the patient’s con
CHAPTER 26 TherapeuticApheresis
661
sent must be documented. The procedure should be performed only in a setting where there is ready access
to care for untoward reactions, including equipment, medications, and personnel trained in managing serious
adverse events such as anaphylaxis.
The assessment should include evaluation of the indication, medical conditions that may affect the patient’s
ability to tolerate apheresis, vascular access, and medications. Some points to consider include the following:
■ Transfusion history: History of transfusion reactions and special blood component requirements.
■ Neurologic status: Mental status and the ability to consent and cooperate.
■ Cardiorespiratoiy status: Adequate ventilation and oxygenation, hyper- or hypovolemia, and cardiac
arrhythmia.
■ Renal and metabolic status: Fluid balance, alkalosis, and electrolyte abnormalities, in
cluding hypocalcemia, hypokalemia, and hypomagnesemia.
■ Hematologic status: Significant anemia or thrombocytopenia, coagulopathy, bleeding, or thrombosis.
■ Medications: Drugs with high albumin binding, immunoglobulins, and biologies.
Appropriate laboratory monitoring is dictated by the indication, type and frequency of procedures, and
concomitant medical conditions. In general, it is wise to obtain a complete blood cell count, type and screen,
and electrolytes assessment before starting treatment. If at all possible, diagnostic studies such as tests for
infectious disease, pregnancy, AD AMTS-13 activity, glomerular basement membrane antibody, or
acetylcholine receptor antibody should be completed before the first treatment, particularly if plasma is used for
replacement. Coagulation monitoring may be appropriate when albumin is used for replacement during
frequently repeated procedures.
KEY POINTS
1. Therapeutic apheresis treats disease by removal or extracorporeal manipulation of pathologic plasma
substances, white cells, platelets, or red cells, and may be accomplished by continuous centrifugation, filtration,
selective adsorption, or photopheresis.
2. The American Society for Apheresis (ASFA) has published guidelines and recommendations for the use
of therapeutic apheresis in clinical practice.
3. Anticoagulation is usually accomplished with citrate, although heparin may be used, particularly for
selective adsorption, hematopoietic progenitor cell collection, and photopheresis.
 4. Albumin is the most commonly used replacement fluid for therapeutic plasma exchange, but plasma may
be indicated for patients with TTP or coagulopathy.
5. Adverse effects of apheresis are usually mild but may include symptomatic hypocalcemia, hypotension,
urticaria, and nausea. Complications of apheresis can include coagulopathy, hypogammaglobulinemia, and
removal of certain drugs and biologicals.
6. Vascular access for apheresis may be accomplished through peripheral veins, but a central venous
catheter or an AV fistula are required for some patients.
7. Medical evaluation of the apheresis patient should focus on the indication, type of procedure, frequency
and number of treatments, therapeutic goal, ability of the patient to tolerate apheresis, vascular access, and
medications. Appropriate laboratory monitoring is dictated by the indication, type and frequency of procedures,
and concomitant medical conditions.

662
AABB TECHNICAL MANUAL
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12. George JN, Li X, McMinn JR, et al. Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome
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13. Bandarenko N, Brecher ME. United States thrombotic thrombocytopenic purpura apheresis study group
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14. Howard MA, Williams IA, Terrell DR, et al. Complications of plasma exchange in patients treated for
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15. Legendre CM, Licht C, Muus R et al. Terminal complement inhibitor eculizumab in atypical hemolytic-
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16. Weinshenker BG, O’Brien PC, Petterson TM, et al. A randomized trial of plasma exchange in acute
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17. Llufriu S, Castillo J, Blanco Y, et al. Plasma exchange for acute attacks of CNS demyelination:
Predictors of improvement. Neurology 2009;73:949-53.
18. Bonnan M, Valentino R, Olindo S, et al. Plasma exchange in severe spinal attacks associated with
neuromyelitis optica spectrum disorder. Mult Scler 2009;15:487-92.
19. Savin VJ, Sharma R, Sharma M, et al. Circulating factor associated with increased glomerular
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20. Wei C, El Hindi S, Li J, Fornoni A, et al. Circulating urokinase receptor as a cause of focal segmental
glomerulosclerosis. Nat Med 2011; 17:952-60
21. Al-Badr W, Kallogjeri D, Madaraty K, et al. A retrospective review of the outcome of plasma exchange
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22. Sivakumaran P Vo AA, Villicana R, et al. Therapeutic plasma exchange for desensitization before
transplantation in ABO-incompatible renal allografts. J ClinApher 2009;24:155-60.
23. Tobian AAR, Shirey RS, Montgomery RA, et al. Therapeutic plasma exchange reduces ABO ti

CHAPTER 26
Therapeutic Apheresis
663
ters to permit ABO incompatible renal transplantation. Transfusion 2009;49:1248-54.
 24. Padmanabhan A, Ratner LE, Jhang JS, et al. Comparative outcome analysis of ABO-incompatible and
positive crossmatch renal transplantation: A single-center experience. Transplantation 2009;87:1889-96.
25. Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and outcomes of the acute chest syndrome in
sickle cell disease. National Acute Chest Syndrome Study Group. N Engl J Med 2000;342:1855-65.
26. Lawson SE, Oakley S, Smith NA, Bareford D. Red cell exchange in sickle cell disease. Clin Lab
Haematol 1999;21:99-102.
27. Stuart MJ, Setty BN. Sickle cell acute chest syndrome: Pathogenesis and rationale for treatment. Blood
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28. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first stroke by transfusions in children with sickle
cell anemia and abnormal results on transcranial Doppler ultrasonography. N Engl JMed 1998;339:5-11.
29. Lee MT, Piomelli S, Granger S, et al. Stroke Prevention Trial in Sickle Cell Anemia (STOP): Extended
follow-up and final results. Blood 2006; 108:847-52.
30. Vichinsky ER Luban NLC, Wright E, et al. Prospective RBC phenotype matching in a strokeprevention
trial in sickle cell anemia: A multicenter transfusion trial. Transfusion 2001;41: 1086-92.
31. Wahl SK, Garcia A, Hagar W, et al. Lower alloimmunization rates in pediatric sickle cell patients on
chronic erythrocytapheresis compared to chronic simple transfusions. Transfusion 2012;52:2671-6.
32. Bladon J, Taylor PC. Extracorporeal photopheresis: A focus on apoptosis and cytokines. J Dermatol Sci
2006;43:85-94.
33. Del Fante C, Scudeller L, Viarengo G, et al. Response and survival of patients with chronic graft-versus-
host disease treated by extracorporeal photochemotherapy: A retrospective study according to classical and
National Institutes of Health classifications. Transfusion 2012;52:2007-15.
34. Barr ML, Baker CJ, Schenkel FA, et al. Prophylactic photopheresis and chronic rejection: Effects on
graft intimal hyperplasia in cardiac transplantation. Clin Transplant 2000;14:1626.
35. Barr ML, Meiser BM, Eisen HJ, et al. Photopheresis for the prevention of rejection in car
diac transplantation. Photopheresis Transplantation Study Group. N Engl J Med 1998; 339:1744-51.
36. Dall’Amico R, Montini G, Murer L, et al. Extracorporeal photochemotherapy after cardiac
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37. Kirklin JK, Brown RN, Huang ST, et al. Rejection with hemodynamic compromise: Objective evidence
for efficacy of photopheresis. J Heart Lung Transplant 2006;25:283-8.
38. Jaksch P, Scheed A, Keplinger M, et al. A prospective interventional study on the use of extracorporeal
photopheresis in patients with bronchiolitis obliterans syndrome after lung transplantation. J Heart Lung
Transplant 2012; 31:950-7.
39. Masaki N, Tatami R, Kumamoto T, et al. Tenyear follow-up of familial hypercholesterolemia patients
after intensive cholesterol-lowering therapy. Int Heart J 2005;46:833-43.
40. Matsuzaki M, Hiramori K, Imaizumi T, et al. Intravascular ultrasound evaluation of coronary plaque
regression by low density lipoprotein-apheresis in familial hypercholesterolemia: The Low Density
Lipoprotein-Apheresis Coronary Morphology and Reserve Trial (LACMART). J Am Coll Cardiol 2002;40:220-
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41. Wang Y, Blessing F, Walli AK, et al. Effects of heparin-mediated extracorporeal low-density lipoprotein
precipitation beyond lowering proatherogenic lipoproteins—reduction of circulating proinflammatory and
procoagulatory markers. Atherosclerosis 2004;175:145-50.
42. Kobayashi S, Oka M, Moriya H, et al. LDLapheresis reduces P-Selectin, CRP and fibrinogen—possible
important implications for improving atherosclerosis. Ther Apher Dial 2006; 10:219-23.
43. Muso E, Mune M, Yorioka N, et al. Beneficial effect of low-density lipoprotein apheresis (LDL-A) on
refractory nephrotic syndrome (NS) due to focal glomerulosclerosis (FGS). Clin Nephrol 2007;67:341-4.
44. Silverman GJ, Goodyear CS, Siegel DL. On the mechanism of staphylococcal protein A
immunomodulation. Transfusion 2005;45:27480.
45. McLeod BC, Sniecinski I, Ciavarella D, et al. Frequency of immediate adverse effects associated with
therapeutic apheresis. Transfusion 1999;39:282-8.
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AABB TECHNICAL MANUAL
46. Norda R, Berseus 0, Stegmayr B. Adverse events and problems in therapeutic hemapheresis. A report
from the Swedish registry. Transfus Apher Sci 2001;25:33-41.
 47. Haddad S, Leitman SF, Wesley RA, et al. Placebo-controlled study of intravenous magnesium
supplementation during large-volume leukapheresis in healthy allogeneic donors. Transfusion 2005;45:934-44.
48. Marques MB, Huang ST. Patients with thrombotic thrombocytopenic purpura commonly develop
metabolic alkalosis during therapeutic plasma exchange. J Clin Apher 2001;16:1204.
49. Askari S, Nollet K, Debol SM, et al. Transfusion-related acute lung injury during plasma exchange:
Suspecting the unsuspected. J Clin Apher 2002;17:93-6.
50. Duntley P, Siever J, Korwes ML, et al. Vascular erosion by central venous catheters. Clinical features
and outcome. Chest 1992;101:1633-8.
51. Leitman SF, Boltansky H, Alter HJ, et al. Allergic reactions in healthy plateletpheresis donors caused by
sensitization to ethylene oxide gas. N Engl J Med 1986;315:1192-6.
52. Purello D’Ambrosio F, Savica V Gangemi S, et al. Ethylene oxide allergy in dialysis patients. Nephrol
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53. Owen HG, Brecher ME. Atypical reactions associated with use of angiotensin-converting enzyme
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54. Orlin JB, Berkman EM. Partial plasma exchange using albumin replacement: Removal and recovery of
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Analysis of 17,940 procedures. J Clin Apher 2001;16:130-3.
Chapter 27
Noninfectious Complications of Blood Transfusion
Catherine A. Mazzei, MD; Mark A. Popovslcy, MD; and Patricia M. Kopko, MD
MR STATISTICALLY, THE GREATEST BSirisk of morbidity and mortality from transfusion is from a
transfusion reaction or the noninfectious complications of blood transfusion. In fact, transfusion-related acute
lung injury (TRALI), hemolytic transfusion reactions (HTRs), and transfusion-associated circulatory overload
(TACO) are the three most commonly reported causes of transfusionrelated mortality.1 It is these noninfectious
complications that are addressed in this chapter.
HEMOVIGILANCE
Hemovigilance may be defined as the collection of information on the complications of transfusion, analysis
of these data, and subsequent data-driven improvements in transfu
sion practices. One of the main purposes of developing a hemovigilance program is to improve reporting of
transfusion-related adverse events. It is widely believed that the major noninfectious complications of
transfusion are both underrecognized and underreported.
The US Biovigilance Network is a national collaboration between government and nongovernment
organizations. The network develops and enhances surveillance systems designed to track adverse reactions and
incidents associated with blood collection; blood transfusion; and the transplantation of cells, tissues, and
organs. Transfusion safety was the first issue that the network addressed.
Definitions and classification schemes are outlined in detail in the appendices of the National Healthcare
Safety Network Biovigilance Component protocol with the goal of

Catherine A. Mazzei, MD, Medical Director, American Red Cross, Northern California Blood Services
Region, Oakland, California; Mark A. Popovsky, MD, Associate Clinical Professor, Harvard Medical School,
Boston, Massachusetts, and Vice President and Chief Medical Officer, Haemonetics Corporation, Braintree,
Massachusetts; and Patricia M. Kopko, MD, Clinical Professor of Pathology, University of California, San
Diego, California
C. Mazzei and P. Kopko have disclosed no conflicts of interest. M. Popovsky has disclosed a financial
relationship with Haemonetics Corporation.
665

666
AABB TECHNICAL MANUAL
improving the quality of national surveillance data.2 The Centers for Disease Control and Prevention
intends to publish the results of system participation to allow benchmarks and best practices to be established.
RECOGNITION AND EVALUATION OF A SUSPECTED TRANSFUSION REACTION
 Identification of a Transfusion Reaction
As with many necessary medical therapies, adverse effects cannot always be accurately predicted or
avoided. The transfusing physician should be aware of such risks when discussing the need for transfusion with
a patient. Informed consent for transfusion may include a discussion of the risks of infectious disease and
serious noninfectious complications, such as TRALI, and HTRs.3 Furthermore, medical staff administering
blood components should be well aware of the signs and symptoms of possible reactions. These staff should be
prepared to mitigate the current episode and prevent future similar reactions when possible.
Many common clinical signs and symptoms are associated with more than one type of adverse reaction (see
Table 27-1). Early recognition, prompt cessation of the transfusion, and further evaluation are key to a
successful outcome. The signs and symptoms that may be indicators of a transfusion reaction include the
following:
■ Fever, generally defined as a >1 C rise in temperature above 37 C [the most common sign of an acute
HTR (AHTR)].4(p907)
■ Chills with or without rigors.
■ Respiratory distress, including wheezing, coughing, and dyspnea.
■ Hyper- or hypotension.
■ Abdominal, chest, flank, or back pain.
■ Pain at the infusion site.
■ Skin manifestations, including urticaria, rash, flushing, pruritus, and localized edema.
■ Jaundice or hemoglobinuria.
■ Nausea/vomiting.
■ Abnormal bleeding.
■ Oliguria/anuria.
Clinical Evaluation and Management of a Transfusion Reaction
The evaluation of a suspected transfusion reaction involves a two-pronged investigation combining clinical
evaluation of the patient with laboratory verification and testing. The nurse or transfusionist must treat the
patient by administering supportive care at the direction of the physician, discontinue the transfusion of the
implicated component, and contact the blood bank for directions on the investigation. When an AHTR is
suspected, several steps must be taken immediately.
Patient-focused steps are as follows:
1. Stop the transfusion immediately but keep the line open with saline.
2. Document the clerical recheck between the patient and the component. The labels on the component,
patient records, and patient identification should be examined to detect any identification errors. Transfusing
facilities may require repeat ABO and Rh typing of the patient using a new sample.5(p88) (See “Standard
Laboratory Investigation of a Transfusion Reaction” section below.)
3. Contact the treating physician immediately for instructions on patient care.
Component-focused steps are as follows:
1. Contact the transfusion service for directions on investigating the cause of the AHTR. Most transfusion
services use a standardized form to document all of the information available on both the patient and the
component.
2. Obtain instructions concerning the return of any remaining component, associated intravenous fluid bags,
and tubing.
3. Identify appropriate samples (blood and urine) to send to the laboratory.
The transfusion service determines whether the blood donor center should be notified of the AHTR.
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management
CHAPTER 27 Noninfectious Complications of Blood Transfusion
667
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Transfusion- Varies by component Bacterial contamina- Fever, chills, hypotension Gram’s stain Broad
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CHAPTER 27 Noninfectious Complications of Blood Transfusion
669
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670
AABB TECHNICAL MANUAL
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CHAPTER 27 Noninfectious Complications of Blood Transfusion
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 672
AABB TECHNICAL MANUAL
Standard Laboratory Investigation of a Transfusion Reaction
When the laboratory receives notice of a possible transfusion reaction, several steps are performed by
technologists:
1. Clerical check of the component bag, label, paper work, and patient sample.
2. Repeat ABO testing on the posttransfusion sample.
3. Visual check of pre- and posttransfusion samples to look for evidence of hemolysis (which may not be
visible if <50 mg/dL of hemoglobin is present).
4. Direct antiglobulin test (DAT) on a posttransfusion sample.
5. Report of findings to the blood bank supervisor or medical director, who may request additional studies,
tests, or quarantine of cocomponents generated from the same donor collection.
The transfusion service must retain any patient records that are related to transfusion reactions, clinically
significant antibodies, or special transfusion requirements.5(pp74'77) Additional information regarding special
products, such as irradiated or washed components required for a particular patient, may be retained by the
transfusion service as well.
In an increasingly mobile society, patients often present for treatment at a facility that has no ready access to
the patient’s medical records. When the patient is able to give a thorough history, transfusion services are able to
share medical information in a timely manner. When this is not possible, medical warning bracelets or wallet
identification cards may benefit patients with multiple alloantibodies whose strength may decrease over time.
Specialized Laboratory Investigations for Selected Reactions
Additional laboratory evaluation may be required for investigations of some nonhemolytic transfusion
reactions, such as anaphylaxis, sepsis, or TRALI, as described in their respective sections.
Acute or Immediate Transfusion Reactions
Acute or immediate transfusion reactions occur within 24 hours of the administration of a component and
often during the transfusion. Acute transfusion reactions include hemolysis, both immune and nonimmune
mediated; transfusion-related sepsis; TRALI; severe allergic reactions, including anaphylaxis; TACO;
complications of massive transfusion; air embolism; febrile nonhemolytic transfusion reactions (FNHTRs); and
mild allergic reactions, such as urticaria or rash. The clinical significance of an acute transfusion reaction often
cannot be determined by the patient’s clinical history or signs and symptoms alone but requires laboratory
evaluation.
AHTRs
Presentation
Rapid hemolysis of as little as 10 mL of incompatible blood can produce symptoms of AHTR. The most
common presenting symptom is fever with or without accompanying chills or rigors. A patient with a mild
reaction may have abdominal, chest, flank, or back pain. If a patient has a severe AHTR, hypotension, dyspnea,
and flank pain may be present and, in some cases, progress to shock with or without accompanying
disseminated intravascular coagulation (DIC). Red or dark urine may be the first sign of intravascular
hemolysis, particularly in the anesthetized or unconscious patient, who may also present with oliguria or, in rare
cases, DIC. The severity of the symptoms of this reaction is related to the amount of incompatible blood
transfused. Prompt recognition of the reaction and immediate cessation of the transfusion can prevent grave
consequences.
Differential Diagnosis
Many of the signs and symptoms of an immune-mediated AHTR also occur in other acute transfusion
reactions. Fever with or without chills and accompanied by hypotension may also develop in transfusion-related
673
sepsis and TRALI. Hemolysis may not be detected immediately and does not occur in sepsis and TRALI.
Respiratory difficulty is not typically a symptom of an AHTR. Immediate treatment requirements for acute
hemolysis are identical to those for sepsis—stop the transfusion and maintain hemodynamic stability—and can
be instituted while the diagnosis is being determined.
The patient’s underlying disease process can make the diagnosis of any AHTR extremely difficult. Patients
with glucose-6-phosphate dehydrogenase (G6PD) deficiency, autoimmune hemolytic anemia, or sickle cell
disease present particularly complicated situations when symptoms such as fever and hypotension occur. In
these patients, autoantibodies and multiple alloantibodies delay the serologic diagnosis of an AHTR and make
identification of the responsible antibody a challenge. Acute hemolysis may also result from nonimmune
mechanisms, as described in the “Nonimmune-Mediated Hemolysis” section below.
Pathophysiology
The interaction of preformed antibodies with red cell antigens is the immunologic basis for AHTRs. The
most severe reactions are associated with transfusions of red cells that are ABO incompatible with the recipient,
resulting in acute intravascular destruction of the transfused cells. Transfusion of ABO-incompatible plasma, as
can occur with apheresis platelets, may also cause hemolysis of the patient’s red cells. Although this form of
acute hemolysis is not usually clinically significant or characterized by typical hemolytic symptoms, it can be
severe if donors have high titers of ABO antibodies. Although rare, the most common circumstance is when
group 0 platelets from donors with high titers of anti-A are transfused to group A recipients.6,7
When preformed immunoglobulin M (IgM) or IgG antibodies recognize corresponding red cell antigens,
complement activation may occur, resulting in intravascular hemolysis, hemoglobinemia, and, eventually,
hemoglobinuria. IgM antibodies are strong activa
tors of complement, and IgG antibodies, when present at sufficient concentrations and of the relevant
subclass, may activate complement as well.
Complement activation involves C3 cleavage with the ensuing production of C3a, an anaphylatoxin, which
is released into the plasma, and C3b, which coats the red cells. If complement activation proceeds to
completion, which is characteristic of ABO antibodies, a membrane attack complex is assembled on the red cell
surface, and intravascular lysis occurs. C5a, an anaphylatoxin that is 100 times more potent than C3a, is
produced as part of this hemolysis. C3a and C5a promote the release of histamine and serotonin from mast
cells, leading to vasodilation and smoothmuscle contraction, particularly of bronchial and intestinal muscles.
C3a and C5a are recognized by many other cell types as well, including monocytes, macrophages, endothelial
cells, platelets, and smooth muscle, and are involved in the production and release of cytokines, leukotrienes,
free radicals, and nitric oxide. The end result may include wheezing, flushing, chest pain or tightness, and
gastrointestinal symptoms. These symptoms may also be caused by release of bradykinin and norepinephrine
caused by antigen-antibody complex stimulation.
Phagocytosis of IgG-coated red cells leads to cytokine release, which plays a role in producing the effects of
acute hemolysis.8 Interleukin-8 (IL-8), which activates neutrophils, and tumor necrosis factor alpha (TNFa),
which activates the coagulation cascade, have also been found after in-vitro incubation of incompatible group 0
whole blood and group A or B red cells.9 Other cytokines involved in the pathogenesis of AHTRs include IL-
ip, IL-6, and monocyte chemoattractant protein 1 (MCP-1). In a mouse model of HTR, transfusion of
incompatible red cells resulted in very high plasma levels of MCP-1 and IL-6 and lower levels of TNFa.10
If complement activation does not proceed to completion, which typically happens with non-ABO
antibodies, the red cells can undergo extravascular hemolysis where cells coated with C3b and/or IgG are
rapidly
674
AABB TECHNICAL MANUAL
removed from the circulation by phagocytes. In extravascular hemolysis, the consequences of complement
activation, including release of anaphylatoxins and opsonization of red cells, may still have adverse effects.
Coagulation abnormalities associated with AHTRs may be caused by various mechanisms. The “intrinsic”
pathway of the clotting cascade may be activated by antigen-antibody interaction, resulting in activation of
Factor XII, also known as “Hageman factor.” Activation of the Hageman factor can result in hypotension
through its effect on the kinin system. The kinin system produces bradykinin, which in turn increases vascular
permeability and causes vasodilation. Activated complement, TNFa, and IL-1 may increase the expression of
tissue factor. Tissue factor, expressed by leukocytes and endothelial cells, can activate the “extrinsic” pathway
and is associated with the development of DIC. DIC is an often lifethreatening consumptive coagulopathy. Its
characteristics include microvascular thrombi formation with ischemic organ and tissue damage; consumption
of platelets, fibrinogen, and coagulation factors; and activation of fibrinolysis with resultant production of
fibrindegradation products. The end result of these activations can vary from generalized oozing to uncontrolled
bleeding.
Shock may be a component of DIC. Hypotension, caused by the release of vasoactive amines, kinins, and
other mediators, produces a compensatory vasoconstrictive response that further aggravates organ and tissue
damage. Renal failure may occur as well. Free hemoglobin impairs renal function, but compromised renal
cortical supply is thought to be the major contributing factor in renal failure. In addition, antigen-antibody
complex deposition, vasoconstriction, and thrombi formation contribute to the development of renal vascular
compromise.
Frequency
The frequency of AHTRs is not easy to determine. The authors of a review article based on data from
several surveillance systems estimated the risk of clinical or laboratory evi
dence of ABO HTR to be 1:80,000 and the risk of a fatal ABO HTR to be 1 in 1.8 million.11 Of the
transfusion-related fatalities reported to the Food and Drug Administration (FDA) from 2007 to 2011, 23% (in
50 patients) were caused byHTRs.1
Treatment
Prompt recognition of an AHTR and immediate cessation of the transfusion are crucial. The unit of blood
should be returned to the blood bank for a transfusion reaction investigation. The infusion of saline should be
continued to maintain venous access, treat hypotension, and maintain renal blood flow, with a goal of a urine
flow rate >1 mL/kg/hour. Saline infusion alone may not be adequate therapy, and the urine output must be
carefully monitored so as not to cause volume overload in the patient. Studies have concluded that low-dose
dopamine may improve renal function initially, but no solid evidence exists that it can prevent renal failure.12
However, these studies, which did not include patients with AHTRs, did show a 25% increase in urine output in
the acute setting. Thus, low-dose dopamine (1-5 pg/kg/minute) may still have a role in treating the
complications of an AHTR, but no recommendations regarding its use may be made based on published
evidence at this time.
The addition of the diuretic furosemide (40-80 mg intravenously in adults, 1-2 mg/kg in children) promotes
increased urine output and further enhances renal cortical blood flow.13 If urine output remains diminished after
a liter of saline has been infused, acute tubular necrosis may have occurred, and the patient may be at risk of
developing pulmonary edema. At this point, a nephrologist should be consulted for further management of the
patient. Oliguric renal failure may be complicated by hyperkalemia and subsequent cardiac arrest; therefore,
these patients should be monitored with telemetry. Metabolic acidosis and uremia often necessitate the
institution of dialysis.
DIC is an equally serious component of an AHTR. DIC is extremely difficult to treat and may be the first
indication that hemolysis
675
has occurred in an anuric or anesthetized patient. Traditional therapy for DIC includes treating or removing
the underlying cause and providing supportive care via the administration of platelets, frozen plasma, and
cryoprecipitated antihemophilic factor (AHF).
The administration of heparin to treat DIC associated with HTRs is controversial. First, the underlying
condition for which the patient required transfusion may be a contraindication to heparin administration. For
example, in patients with recent surgery or active bleeding, heparin can exacerbate bleeding. Second, there is
some thought that because the precipitating event is limited to hemolysis of the volume of blood transfused, the
risks of administering heparin do not justify its use. Those in favor of heparin point out, however, that the most
unstable patients (those with the most severe reactions) are those who received larger volumes of incompatible
blood and are thus the most likely to develop DIC.14 In the worst cases, DIC can become a self-sustaining
vicious cycle of inflammation and consumptive coagulopathy.
Recent studies of optimal treatment for DIC have evaluated the targeting of various components of the
inflammatory and coagulation cascades. Activated protein C has shown some benefit in patients with sepsis and
DIC; however, no new therapeutic agents have been added to the arsenal to treat DIC associated with AHTRs in
recent years.15,16
Unconscious or anesthetized patients may receive multiple units of incompatible blood before acute
hemolysis is recognized. Because the severity of an AHTR is related to the amount of incompatible red cells
transfused, red cell exchange transfusion may be considered. Some severe reactions to a single unit of strongly
incompatible blood may require exchange transfusion as well. Antigennegative blood must be used for the red
cell exchange. In the case of acute hemolysis caused by a non-ABO antibody, the blood bank must be given
adequate time to identify the appropriate units for further simple red cell transfusion or planned red cell
exchange. Likewise, plasma and platelets that will not contribute to hemolysis should be chosen.
Prompt initiation of therapy to aggressively manage hypotension, renal blood flow, and DIC provides the
greatest chance of a successful outcome. Furthermore, consultation with appropriate medical specialists early in
the course of treatment will ensure that the patient receives hemodialysis, cardiac monitoring, and mechanical
ventilation when needed.
Prevention
The most common events leading to the transfusion of a component in error are the very things that the
laboratory evaluates when an AHTR is suspected. Clerical and human errors involving patient, sample, and
blood unit identification are the most common causes of mistransfusion and, therefore, AHTRs. The risk of a
near miss is 1:1,000, wrong blood given is 1:15,000, and ABO-incompatible transfusion is 1:40,000.11
Institutional policies and procedures should be in place to minimize the likelihood of such errors, and corrective
and preventive action programs should target continual reduction of the numbers of such errors. However, no
one method for reducing the number of errors is foolproof. Products available to increase patient safety include
technology-based solutions, such as radiofrequency identification chips, handheld bar-code scanners, and
“smart” refrigerators similar to systems used for pharmacologic agents.
The prevention of potential hemolysis from the administration of minor ABO-incompatible platelets
remains a challenge in patients with HLA antibodies. A number of options, including anti-A or anti-B titration
of the product, limitations in total amount of incompatible plasma transfused from platelets, and volume
reduction, may offer some benefit. The use of platelet additive solutions is also promising because these
solutions replace plasma, which contains ABO antibodies and plasma proteins.17
Nonimmune-Mediated Hemolysis
Transfusion-associated hemolysis can also be due to several nonimmune-mediated causes.
676
AABB TECHNICAL MANUAL
Before issue, improper shipping or storage temperatures as well as incomplete deglycerolization of frozen
red cells can lead to hemolysis. At the time of transfusion, using a needle with an inappropriately small bore
size or employing a rapid pressure infuser can cause mechanical hemolysis, which may result from the use of
roller pumps as well. Improper use of blood warmers or the use of microwave ovens or hot waterbaths can
cause temperaturerelated hemolysis. Few fluids are approved by FDA for transfusion with Red Blood Cells
(RBCs).5(p45) Infusion of RBCs simultaneously through the same tubing with hypotonic solutions or some
pharmacologic agents may cause osmotic hemolysis; for safe administration, RBCs and these solutions or
agents should be given via alternate venous access locations. In rare cases, hemolysis may be caused by
bacterial contamination of the RBC unit. Not infrequently, patients may also experience hemolysis as part of
their underlying disease process. Of note is that although a negative DAT result usually indicates no evidence of
an immune-mediated cause of hemolysis, complete destruction of incompatible transfused red cells may be
associated with a negative DAT result.
When both immune and nonimmune causes of hemolysis have been excluded, the possibility of an intrinsic
red cell membrane defect in the recipient or even in the transfused cells should be considered. Cells with these
defects, such as G6PD deficiency, have increased fragility when challenged with particular stressors and may
undergo coincidental hemolysis.
Treatment
Hemolysis of nonimmune etiology may cause symptoms whose severity depends on the degree of
hemolysis and amount of component transfused. In all cases, the transfusion should be discontinued and
appropriate care should be administered. (See the earlier section on the treatment of AHTRs for details on
managing hypotension and declining renal function.)
Prevention
As is true for the mitigation of any type of transfusion reaction, written procedures for all aspects of the
manufacture and transfusion of blood and components should always be followed. Prompt recognition of
nonimmune hemolysis and robust root cause analysis may prevent additional occurrences.18
Transfusion-Related Sepsis
Presentation
Fever (particularly a temperature of >38.5 C or 101 F) and shaking chills and hypotension during or shortly
after transfusion are the most frequent presenting symptoms of transfusionrelated sepsis. In severe cases, the
patient may develop shock with accompanying renal failure and DIC.
Differential Diagnosis
The abruptness of onset and severity of the signs and symptoms associated with transfusion-related sepsis
may be very similar to those of AHTRs. Mild cases may be confused with FNHTRs. The key to diagnosing
transfusion-related sepsis is culturing the same organism from both the patient and the remainder of the
component. The returned component should be visually examined in suspected cases of posttransfusion sepsis.
Particular attention should be paid to any color changes, especially brown or purple discoloration in an RBC
component and bubbles/ frothiness in a platelet component. A Gram’s stain should be performed on the returned
component.
Treatment
If transfusion-related sepsis is suspected, the transfusion should be stopped immediately and supportive care
of the patient should be initiated. Broad-spectrum antibiotics may be indicated. (See Chapter 8 for a more
detailed discussion of bacterial contamination of transfused blood, including frequency data and prevention
strategies.)
677
FNHTRs
Presentation
An FNHTR is usually defined as the occurrence of >1 C rise in temperature above 37 C that is associated
with transfusion and for which no other cause is identifiable. Accompanying symptoms may include shaking,
chills, an increased respiratory rate, a change in blood pressure, and anxiety. In some instances, the patient may
be afebrile but have the remaining constellation of symptoms. Symptoms usually occur during transfusion but
may occur up to 4 hours after. Most FNHTRs are benign, although they may cause significant discomfort and
even hemodynamic or respiratory effects.
Differential Diagnosis
The symptoms associated with an FNHTR may be present in several other types of transfusion reactions, the
most serious of which are HTRs, sepsis, and TRALI. Each of these other reactions has signs, symptoms, and
associated laboratory results that help distinguish them from an FNHTR once an investigation is begun;
recognition of FNHTR requires diagnosis by exclusion.
Fever may commonly occur as a component of a patient’s underlying illness. In a patient who has been
experiencing spiking fevers during the course of admission, it may be difficult to rule out an FNHTR.
Hemolysis along with any other signs or symptoms of a serious reaction must be ruled out in a patient who
experiences fever associated with transfusion.
Pathophysiology
Recipient leukocyte antibodies may cause febrile transfusion reactions. As few as 0.25 x 109 residual
leukocytes in a blood component can produce a temperature elevation in the recipient. FNHTRs may also be the
result of accumulated cytokines in a cellular blood component.19 This mechanism may be particularly relevant
in reactions that occur after the transfusion of platelets. Some FNHTRs are attributable to recipient antibodies,
particularly HLA
antibodies that react with antigens on transfused lymphocytes, granulocytes, or platelets. Cytokine release in
the recipient in response to these antigen-antibody reactions may contribute to the severity of the reaction.
Whatever the initiating cause, cytokine release is the common event leading to symptoms of FNHTR.
Treatment
When an FNHTR is suspected, the transfusion should be discontinued and a transfusion reaction work-up
initiated. Antipyretics (ie, acetaminophen) should be administered, and the patient may be safely transfused
once the symptoms subside. For more severe reactions that include rigors, meperidine may be necessary where
it is not contraindicated.
In general, the remainder of the implicated component and donor-related co-components should not be
transfused. Many times, an FNHTR does not develop until after the transfusion has been completed. If a portion
of the component remains, the laboratory workup to exclude hemolysis must be completed before the
transfusion is resumed. This may be difficult to accomplish within an acceptable amount of time. Among the
few valid situations in which transfusion of the remainder of the component should be considered is when the
component is a medically indicated rare unit and a significant volume remains untransfused. In that
circumstance, the blood bank’s medical director should be consulted before proceeding with caution because
bacterial contamination of the component might have been the underlying cause of the reaction.20,21
Prevention
Prestorage leukocyte reduction, especially if performed at the time of collection, significantly decreases the
frequency of FNHTRs.22 Premedication with acetaminophen may be beneficial in further reducing the residual
rate of FNHTRs and has not been shown to impair the ability to detect serious complications of transfusion.21
 678
AABB TECHNICAL MANUAL
Allergic Reactions
Presentation
Most allergic transfusion reactions (ATRs) are mild, but their spectrum can range from a simple allergic
reaction (urticaria) to life-threatening anaphylaxis. Symptoms generally occur within seconds or minutes of the
start of the transfusion. In rare cases, the symptoms may take several hours to develop. If symptoms do not
appear until more than 4 hours later, they may represent an allergic reaction that is unrelated to the blood
transfusion. An ATR is diagnosed in the same way as any other allergic reaction.
The mildest form of ATR is urticaria, or hives. An outbreak of swollen, raised, red areas (wheals) on the
skin appears suddenly as a result of the body’s adverse reaction to an allergen. Hives usually cause itching
(pruritus) but may also burn or sting. Hives can appear anywhere on the body and vary greatly in size. They can
last from hours to several days before fading but often respond quickly to treatment with antihistamines. More
extensive cases may be accompanied by angioedema, where the swelling is caused by fluid accumulation
beneath the skin instead of on the surface. It is a deep swelling, often around the eyes and lips, and generally
lasts longer than urticaria. Angioedema can, rarely, involve the throat, tongue, or lungs, causing respiratory
distress.
More serious are anaphylactic transfusion reactions, which include the symptoms of urticaria and
angioedema in the majority of cases.23 Severe hypotension, shock, and loss of consciousness may also occur. In
addition, the respiratory system is often involved, with patients experiencing dyspnea, wheezing, and stridor.
Gastrointestinal disturbances such as nausea, vomiting, diarrhea, and cramping, affect approximately 30% of
these patients. Cardiovascular manifestations in addition to hypotension may include tachycardia, arrhythmia,
or cardiac arrest.
Differential Diagnosis
It is important to distinguish anaphylaxis from other reactions characterized by hypotension,
dyspnea, and/or loss of consciousness. The most common reaction that may be mistaken for anaphylaxis is a
vasovagal reaction, which is characterized by hypotension, diaphoresis, nausea/vomiting, weakness,
bradycardia, and sometimes loss of consciousness. Urticaria, angioedema, pruritus, and respiratory symptoms,
such as wheezing or stridor, are symptoms of anaphylaxis but do not occur in vasovagal reactions. The
respiratory symptoms of anaphylaxis may be suggestive of an acute asthma attack or TRALI. However, the
classic symptoms of allergy, including urticaria, angioedema, and pruritus, do not occur in asthma attacks or
TRALI. Fever, a prominent symptom of HTR and bacterial contamination, is not a feature of anaphylaxis.
Patients who take angiotensin-converting enzyme (ACE) inhibitors and undergo plasma exchange
sometimes develop hypotensive reactions that mimic anaphylaxis when albumin is used as a replacement fluid.
Pathophysiology
ATRs are attributed to hypersensitivity reactions to allergens in the component caused by preformed IgE
antibody in the recipient. In most cases, the causative plasma protein or antigen is not identified. Mast cell
activation (Type I hypersensitivity), resulting in degranulation, with the release of allergic mediators occurs.
Secondary mediators—including cytokines and lipid mediators—are also generated and released. Recent studies
suggest that the mechanisms underlying most ATRs have not been fully elucidated. A combination of recipient,
donor, and component factors is likely involved, which is supported by the incidence of these reactions
compared to the prevalence of IgA, haptoglobin, or C4 deficiency. Recipients with an atopic predisposition
appear to have a higher rate of ATRs, and certain donors’ platelets are associated with an increased risk.
Increased understanding of the underlying mechanisms of ATRs is crucial to improving prevention.24,25
ATRs that progress beyond urticaria may occur in IgA-deficient patients. These anaphylactic reactions are
caused by anti-IgA in the
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recipient. Although IgA deficiency is present in approximately 1:700 people of European ancestry, only a
small percentage of these people ever make antibodies against IgA. Those who do are divided into two groups
based on IgA levels and the type of antibody formed. People with absolute IgA deficiency (<0.05 mg/dL) may
form class-specific antibodies that are often associated with anaphylactic reactions. Those with decreased but
detectable amounts of IgA, or relative IgA deficiency, can form subclass-specific antibodies (eg, anti-IgAl or
antiIgA2) that typically result in less severe reactions.26
 Although precautions should be taken when transfusing an IgA-deficient patient, it must be kept in mind
that the majority of ATRs are caused by allergens to substances other than IgA.26 Other well-known triggers
include patient antibodies against haptoglobin,27 penicillin, and the C4 determinant of complement.28 Patients
taking ACE inhibitors can experience transfusion reactions that are thought to result from dual actions on
bradykinin: inhibition 1) of its catabolism by the ACE inhibitor and 2) of bradykinin by low levels of
prekallikrein activity in plasma protein fraction.
Frequency
ATRs are quite common, with an overall frequency of approximately 1% to 3% of transfusions. ATRs also
represent 12% to 33% of all transfusion reactions.29,30 Urticaria is relatively common, whereas anaphylactic
reactions occur much less often. As with any ATR, anaphylaxis occurs most commonly during the transfusion
of plasma or platelets; it has an incidence of 1:20,000 to 1:50,000 transfusions.29,31 Of the transfusion-
associated fatalities reported to the FDA from 2007 to 2011,6% (in 12 patients) were caused by anaphylaxis.1
Treatment
Urticaria is the only transfusion reaction in which the administration of the component may be routinely
resumed after prompt treatment. When a patient develops symptoms, the transfusion should be paused so that
an antihistamine, typically 25 to 50 mg diphenhydr
amine, may be administered. Once the symptoms have dissipated, the transfusion may be resumed and a
laboratory work-up need not be initiated.30
If the symptoms do not subside—or, in the case of severe urticaria or urticaria accompanied by hypotension
if dyspnea, significant edema, or gastrointestinal symptoms do not subside—the transfusion must be stopped
and the reaction promptly treated. Severe urticarial reactions may require treatment with methylprednisolone
(125 mg intravenously) or prednisone (50 mg orally). Once a severe reaction or developing anaphylaxis is
identified, action should be promptly initiated to maintain oxygenation and stabilize hypotension. Epinephrine
(1:1000) maybe administered intramuscularly or subcutaneously at an adult dose ranging from 0.2 to 0.5 mL or
a pediatric dose of 0.01 mL/kg. If symptoms persist, the dose may be repeated every 5 to 15 minutes up to three
times, unless palpitations, extreme anxiousness, or tremors occur. If the patient is unconscious or in shock,
epinephrine may be given intravenously at a dilution of 1:10,000 (100 pg/mL) and an initial rate of 1 pg/minute.
Such patients ideally receive cardiac monitoring because of the epinephrine’s arrhythmic potential.
Supplemental oxygen should be administered, and the airway should be maintained. Hypotensive patients
should be placed in the Trendelenburg position and supported with crystalloids. If bronchospasm is present,
respiratory symptoms may not respond to the epinephrine, and addition of a beta-2 agonist or aminophylline
may be required. Patients who do not respond, possibly because of the use of a beta-adrenergic blocker or an
ACE inhibitor, may respond to the addition of glucagon as a 1-mg bolus intravenously or by continuous
infusion.23,32
Prevention
Premedication with an antihistamine (25 to 50 mg diphenhydramine) 30 minutes before transfusion may be
helpful in patients with a history of multiple or severe urticarial transfusion reactions. Routine prophylaxis may
also
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be beneficial for patients undergoing multiple transfusions, as in plasma exchange; however, routine
antihistamine premedication of all patients receiving a transfusion has not been shown to decrease the risk of
ATR.33 If antihistamines are not sufficient, prednisone (20-50 mg orally) or parenteral steroids may be of
benefit. For patients whose reactions are severe, unrelenting, and unresponsive to premedication, washing red
cells or platelets may be considered. Plasma reduction or replacement in platelet products may also be
beneficial. Alternatively, the administration of deglycerolized RBCs has met with some success when reactions
have occurred even after RBCs were washed with 2 L of saline.
The plasma used for transfusions for patients with diagnosed IgA deficiency who produce anti-IgA should
be from IgA-deficient donors (<0.05 mg/dL). If the local blood center cannot provide these components, they
may be available through the American Rare Donor Program (see Method 3-13). Other cellular components
(RBCs and platelets) can be depleted of plasma proteins through washing. IgA deficiency without the presence
of anti-IgA or without a history of an anaphylactoid/ anaphylactic reaction does not warrant the use of IgA-
deficient or plasma-depleted components. Intravenous immune globulin (MG) products from various
manufacturers have different IgA amounts. The manufacturer should be contacted to obtain detailed information
about a product and lot number.34 Autologous donation programs may also be considered for patients with IgA
deficiency once they have recovered.
TRALI
Presentation
Clinical signs and symptoms of TRALI typically include fever, chills, dyspnea, cyanosis, hypotension, and
new-onset bilateral pulmonary edema.35 An increase in blood pressure followed by hypotension is not
uncommon. TRALI can be life threatening or fatal. Symptoms arise within 6 hours of transfusion, with most
cases becoming evident within 1 to 2 hours after the end of transfusion. In addition
to the signs and symptoms traditionally associated with TRALI, there is a growing appreciation that the
disorder can be associated with a dramatic transient neutropenia or leukopenia.36
All plasma-containing components, including whole blood, RBCs, platelets, cryoprecipitated AHF, and
fresh frozen plasma (FFP), have been implicated in TRALI. Transfusion volumes as small as 15 mL have led to
TRALI.
TRALI is a form of acute lung injury (ALI). The American-European Consensus Conference37 defined ALI
as acute hypoxemia with a PaO,/ Fi02 ratio of <300 mm Hg and bilateral pulmonary edema on frontal chest
radiograph. The Canadian Consensus Conference38 relied on this definition of ALI when creating its diagnostic
criteria for TRALI: 1) ALI with hypoxemia and Pa02/Fi02 <300 or Sp02 <90% on room air, 2) no preexisting
ALI before transfusion, 3) onset of symptoms within 6 hours of transfusion, and 4) no temporal relationship
with an alternative risk factor for ALL The panel also defined “possible TRALI” using the same criteria as for
TRALI, with the exception that possible TRALI occurs in the setting of an alternative risk factor for ALL
Although the lung injury in ALI is usually irreversible, the lung injury in TRALI is most often transient.
Approximately 80% of patients with TRALI improve within 48 to 96 hours. The remaining 20% of patients
who do not improve rapidly have either a protracted clinical course or a fatal outcome. In one of the largest
studies of TRALI, 100% of the patients required oxygen support and 72% required mechanical ventilation.39
Differential Diagnosis
The three main conditions that need to be distinguished from TRALI are 1) anaphylactic transfusion
reactions, 2) TACO, and 3) transfusion-related sepsis. In anaphylactic transfusion reactions, bronchospasm,
laryngeal edema, severe hypotension, erythema (often confluent), and urticaria are prominent symptoms. Fever
and pulmonary edema are not associated with anaphylactic reactions. The clinical presentation of TACO is very
similar to that
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of TRALI, with respiratory distress, tachypnea, and cyanosis as the most prominent features. The key
distinction between TACO and TRALI is that the pulmonary edema in TACO is cardiogenic, whereas it is
noncardiogenic in TRALI. High fever with hypotension and vascular collapse are prominent features of
transfusion-related sepsis. Respiratory distress is infrequently associated with these reactions. With rapid onset
of respiratory distress, in addition to TACO and TRALI, coincident myocardial infarction and pulmonary
embolus as well as other possible causes of ALI should be considered.
Pathophysiology
The precise mechanism of lung injury in TRALI has not been determined. TRALI has been associated with
the infusion of antibodies to leukocyte antigens and of biologic response modifiers (BRMs).39 Infusions of
these antibodies or BRMs are thought to initiate a sequence of events that results in cellular activation and
damage of the basement membrane. Pulmonary edema occurs secondary to leakage of protein-rich fluid into the
alveolar space.
BRMs accumulate in some cellular components during storage. BRMs enhance the polymorphonuclear cell
oxidative burst, are soluble in chloroform, and consist of a mixture of lysophosphatidylcholines.40
Antibodies to HLA Class I antigens, HLA Class II antigens, and human neutrophil antigens (HNAs) have
been associated with TRALI. These antibodies can be formed after exposure to foreign antigens via pregnancy,
transfusion, or transplantation. In the majority of TRALI cases with antibodies, the source of the antibody is the
donor, not the recipient.
A two-event model of the mechanism of TRALI has been hypothesized.40 In the first event, generation of
biologically active compounds activates pulmonary vascular endothelial cells and primes neutrophils, resulting
in sequestration of neutrophils in the pulmonary microvasculature. This first event can result from a variety of
physiologic stressors, including sepsis, surgery, and massive transfu
 sion, and it predisposes the recipient to develop TRALI if a second event is experienced. The infusion of
BRMs or antibodies is the second event. These stimuli, which ordinarily do not activate neutrophils, activate the
primed neutrophils in the pulmonary microvasculature, which results in pulmonary endothelial damage,
capillary leakage, and pulmonary edema.
Frequency
Although the true incidence of TRALI is unknown, TRALI is the leading cause of transfusion-related
mortality reported to the FDA, with more than 34 cases reported in 2007.41 Recent efforts to decrease the
amount of transfusable plasma collected from female donors appear to be decreasing the number of TRALI
fatalities in the United States.1
In 2006, the year before many blood centers implemented measures to reduce the risk of TRALI from
plasma transfusions, 35 fatal cases of TRALI, 22 of which were associated with transfusion of FFR were
reported to the FDA. Since 2008, the year after many blood centers implemented such measures, the numbers of
TRALI fatalities and of TRALI fatalities associated with FFP transfusions reported to the FDA have decreased
each year.
Treatment
Treatment of TRALI consists of respiratory and circulatory support. Treatment should be as intensive as
dictated by the clinical picture. Oxygen supplementation with or without mechanical ventilation is required in
almost all cases. Pressor agents may be needed to support blood pressure. Diuretics are not indicated because
TRALI is not related to volume overload. Administration of corticosteroids has not been shown to improve the
clinical outcome in TRALI or acute respiratory distress syndrome.42
Prevention
Strategies to reduce the risk of TRALI are complicated by a number of factors. Although approximately
10% of blood donations contain HLA and/or HNA antibodies, TRALI
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occurs in fewer than 1:1000 transfusions.39,40 There is no mechanism to identify which patients are at risk
for developing TRALI. In 2013, AABB announced that a new standard in the 29 th edition of Standards for
Blood Banks and Transfusion Services would require that plasma products and whole blood collected for
transfusion be from male donors, never-pregnant female donors, or females who have been tested since their
last pregnancy and found to be negative for HLA antibodies.5
In 2014 the AABB issued Association Bulletin 14-02, providing guidance on how to implement the new
TRALI risk reduction requirement. In this bulletin, AABB outlined a number of considerations for
implementation of TRALI risk reduction requirements in plasma components and whole blood intended for
transfusion.43
Although the measures required by AABB and described in Association Bulletin 14-02 are expected to
reduce the risk of TRALI, it is important to recognize they do not eliminate TRALI because they do not
decrease the risk of TRALI from RBCs, platelet concentrates, or cryoprecipitate. HLA antibody screening
addresses only the risk of TRALI from HLA antibodies. A practical test to screen the blood supply for HNA
antibodies is not available. In addition, none of these risk-reduction measures addresses the risk of TRALI from
BRMs.
TACO
Presentation
It is well known that transfusion can precipitate acute pulmonary edema caused by volume overload, but
only recently has this problem been recognized as an important complication. Patients older than 70 years and
infants are at greatest risk, although all transfusion recipients are susceptible to some degree.44 Whereas large
volumes of components and nonblood fluids are most frequendy implicated, modest volumes can also
precipitate TACO. High flow rates are frequently cofactors.
TACO has no diagnostic signs or symptoms. Within 1 to 2 hours of transfusion, patients may develop any or
all of the following: gallop; jugular venous distension; elevated
central venous pressure; dyspnea; orthopnea; new ST-segment and T-wave changes on electrocardiogram;
elevated serum troponin; and likely elevated brain natriuretic peptide (BNP), a cardiac marker.45 Increased
blood pressure characterized by a widening of the pulse pressure is characteristic of TACO. Radiographs show a
widened cardiothoracic ratio.
Differential Diagnosis
 TACO is frequently confused with TRALI because both types of reactions produce pulmonary edema. It is
possible for TACO and TRALI to occur concurrently in the same patient. The timelines and the clinical
presentation are similar, but hypertension is a constant feature of TACO, whereas it is only an infrequent and
transient manifestation of TRALI. Furthermore, rapid improvement with diuresis or inotropic agents is
consistent with TACO.
In congestive heart failure, BNP levels are elevated. Several studies have shown that a posttransfusion-to-
pretransfusion BNP ratio of 1.5 with a posttransfusion level at least 100 pg/mL as a cutoff yielded a sensitivity
and specificity greater than 80% in TACO.46 However, a recent study in the intensive care setting found that
BNP was only of moderate value for distinguishing TACO from TRALI.47 With rapid onset of respiratory
distress, possible causes of ALI, such as coincident myocardial infarction, pulmonary embolus, and others,
should be considered in addition to TACO and TRALI.
Frequency
The incidence of TACO is unknown, but several studies suggest that TACO is much more common than
previously known. In the general population, one group found TACO in 1:707 recipients of RBCs, and 20% of
the affected patients received a single unit of RBCs. In studies of older orthopedic patients undergoing hip or
knee replacement, TACO was found in 1% and 8%, respectively.48,49 In one report from the Quebec
hemovigilance network, the incidence was 1:5000 components, and 1.3% of the cases resulted in death.50 In
the critical care setting, 1:356 units transfused resulted in TACO.51 From 2007 to 2011, 15% of the transfusion
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associated fatalities reported to the FDA (in 32 patients) were a consequence of TACO.1
Although RBCs are most commonly associated with TACO, a recent prospective surveillance study found
that FFP was a frequent cause of this complication.52 In this study, 4.8% of patients developed TACO with a
prevalence rate of 1.5%. Of the 24 reactions, 14 (58%) occurred in the intensive care unit.
Treatment
As soon as symptoms suggest TACO, the transfusion should be stopped. The symptoms should be treated by
placing the patient in a seated position, providing supplementary oxygen, and reducing the intravascular volume
with diuretics. If symptoms persist in confirmed TACO, administration of additional diuretics or therapeutic
phlebotomy in 250-mL increments is appropriate.
Prevention
In the absence of ongoing and rapid blood loss, components should be administered slowly, particularly in
patients at risk of TACO (ie, pediatric patients, patients with severe anemia, and patients with congestive heart
failure). Rates of 2 to 4 mL/minute and 1 mL/ kg of body weight per hour are the most frequently cited, despite
a paucity of data on appropriate infusion rates. Total fluid input and output must be monitored.
Complications of Massive Transfusion
The potential complications of massive transfusion, usually defined as the receipt of more than 10 RBC
units within 24 hours, include metabolic and hemostatic abnormalities, immune hemolysis, and air embolism.
Metabolic abnormalities can depress ventricular function. Hypothermia from refrigerated blood, citrate toxicity,
and lactic acidosis from underperfusion and tissue ischemia, which are often complicated by hyperkalemia, can
contribute to this effect. Although metabolic alkalosis caused by citrate metabolism may occur, it is not likely to
be clinically significant. Patients
who lose blood rapidly may have preexisting or coexisting hemostatic abnormalities or develop them during
resuscitation. Hemostatic abnormalities may include dilutional coagulopathy, DIC, and liver and platelet
dysfunction.
Citrate Toxicity
PATHOPHYSIOLOGY AND MANIFESTATIONS. Plasma, whole blood, and platelets contain citrate as an
anticoagulant. When large volumes of these components are transfused rapidly, particularly in the presence of
liver disease, plasma citrate levels may rise, binding calcium and ionized calcium and resulting in
hypocalcemia. In patients with a normally functioning liver, citrate is rapidly metabolized; thus, these symptoms
are transient.53 Hypocalcemia is more likely to cause manifestations in patients who are hypothermic or in
shock.
A decrease in ionized calcium levels increases neuronal excitability, which in the conscious patient leads to
symptoms of perioral and peripheral tingling, shivering, and lightheadedness, followed by a diffuse sense of
vibration, muscle cramps, fasciculations, spasm, and nausea. In the central nervous system, hypocalcemia is
thought to increase the respiratory center’s sensitivity to carbon dioxide, causing hyperventilation. Because
myocardial contraction is dependent on the intracellular movement of ionized calcium, hypocalcemia depresses
cardiac function.54
TREATMENT AND PREVENTION. Unless the patient has a predisposing condition that hinders citrate
metabolism, hypocalcemia caused by citrate overload during massive transfusion can usually be treated by
slowing the infusion. Calcium replacement should be considered when the calcium concentration falls to below
50% of its normal value or the symptoms of hypocalcemia are evident.55
Hyperkalemia and Hypokalemia
PATHOPHYSIOLOGY. When RBCs are stored at 1 to 6 C, the intracellular potassium gradually leaks into
the supernatant plasma or addi
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tive solution. Although the concentration in the supernatant may be high (see Chapter 6) because of the
small volume, the total extracellular potassium load is less than 0.5 mEq for fresh RBC units and only 5 to 7
mEq for units at expiration. These potassium concentrations rarely cause problems in the recipient because
rapid dilution, redistribution into cells, and excretion blunt the effect. However, hyperkalemia can be a problem
in patients with renal failure, premature infants, and newborns receiving large transfusions, such as in cardiac
surgery or exchange transfusion; otherwise, hyperkalemia is typically a transient effect during very rapid
transfusions.
Hypokalemia occurs more frequently than hyperkalemia after transfusion because potassium-depleted donor
red cells reaccumulate this ion intracellularly, and citrate metabolism causes further movement of potassium
into the cells in response to the consumption of protons. Catecholamine release and aldosterone-induced urinary
loss can also trigger hypokalemia in the setting of massive transfusion.56
TREATMENT AND PREVENTION. No treatment or preventive strategy for hypokalemia and
hyperkalemia is usually necessary, provided that the patient is adequately resuscitated from the underlying
condition that required massive transfusion.57 For infants receiving small-volume transfusions infused slowly,
units may be used safely until the expiration date.58 Although washing of RBC units results in very low levels
of potassium, there is no evidence that this is indicated for routine RBC transfusions, even in patients with
impaired renal function.59
Hemostatic Abnormalities in Massive Transfusion
PATHOPHYSIOLOGY. Coagulopathy can occur in massive transfusion, particularly when the lost blood is
initially replaced with RBCs and asanguineous fluids. Coagulopathy in massive transfusion is frequently
ascribed to the dilution of platelets and clotting factors as patients lose hemostatically active blood, and
enzymatic activity is reduced as the core body temperature lowers if a blood warmer is not used. Mortality
rates associated with hemostatic abnormalities range from 20% to 50%.60 The high rate of mortality results
from hypothermia, metabolic acidosis, and coagulopathy.61
Studies of military and civilian trauma patients demonstrated a progressive increase in the incidence of
microvascular bleeding (MVB) characteristic of a coagulopathy with increasing transfusion volumes that
typically occurs after replacement of two to three blood volumes (20 to 30 units).62,63 Although platelet
counts, coagulation parameters, and levels of selected clotting parameters correlate with the volume transfused,
contrary to expectations from a simple dilutional model, the relationship is marked by tremendous variability.
Moreover, there is frequently discordance between the laboratory assessment and the clinical evidence of
bleeding. It has been suggested that the platelet deficits play a more important role in causing the bleeding than
the coagulation deficiencies.
MVB typically occurs when the platelet count falls below 50,000 to 60,000/pL. However, no simple
relationship can be determined between a patient’s coagulation test results and the onset of bleeding. The
etiology of bleeding (elective surgery vs massive trauma) may play a role as well.64
Subsequent studies have refined these observations. Significant platelet dysfunction has been demonstrated
in massively transfused patients.65,66 Low fibrinogen and platelet counts are better predictors of hemostatic
failure than elevations of prothrombin time (PT) and partial thromboplastin time (PTT), suggesting that
consumptive coagulopathy is an important factor in MVB in addition to dilution.63 Similarly, Harke and
Rahman67 showed that the degree of platelet and clotting abnormalities correlates with the length of time that
the patient is hypotensive, suggesting that shock is the most important cause of DIC. In aggregate, Collins68
concluded that “coagulopathy in heavily transfused patients was due to hypoperfusion, not transfusion.” More
recently, more powerful hemostatic assays have been
 685
introduced that may be more predictive of blood component requirements.69
These data may not be generalizable to patients undergoing massive transfusion in the controlled setting of
the operating room, where hypotension caused by volume loss is prevented. In this context, coagulation factor
levels may have priority over platelet problems. Murray and colleagues64 documented that excessive bleeding
in elective surgery patients transfused with more than one blood volume (RBCs and crystalloid) corresponded
to a prolongation in PT and PPT compared to patients with normal hemostasis.
TREATMENT AND PREVENTION. The dilutional model of coagulopathy in massive transfusion suggests
that prophylactic replacement of hemostatic components based on the volume of RBCs or whole blood
transfused prevents the development of a bleeding diathesis. No specific regimen has yet been shown to be
superior to any other in prospective studies, and this is a controversial topic.70 According to AABB guidelines,
there are insufficient data to recommend for or against a 1:1 RBC to plasma transfusion ratio in massive
transfusion.71
Previously, the predominant thinking was that the replacement of platelets and coagulation factors in the
massively transfused surgical and trauma patient should be based on the identification of a specific abnormality
using platelet counts, international normalized ratio, activated PTT, and fibrinogen levels. Frequent monitoring
of these laboratory values serves to avoid overuse of platelets and plasma products (FFP and Cryoprecipitated
AHF) by anticipating the specific components needed while avoiding dilutional coagulopathy. It is imperative
that the laboratory provide results of these tests rapidly. Intraoperative and postoperative laboratory testing, such
as thromboelastography, may be useful.
USE OF ADJUNCT THERAPIES IN MASSIVE TRANSFUSIONS. Recombinant Factor Vila (rFVIIa), a
50-kDa analog of Factor Vila, is licensed in the United States for the treatment with inhibitors of bleeding in
patients with he
mophilia A or B. However, the off-label use of rFVIIa is growing in several areas, including to treat
hemorrhagic bleeding in trauma and surgery.72,73 The recombinant product works by targeting the site of tissue
damage, where it binds to tissue factor. This complex activates Factor X to Factor Xa and, ultimately, Factor
IXa. In patients with hemophilia, rFVIIa bypasses the need for Factor VIII or Factor IX through the activation
of the small amounts of Factor X on activated platelet surfaces at the site of injury. Studies of the efficacy of
rFVIIa have produced mixed results.74 In the absence of definitive data, transfusion services should establish
guidelines on the reasonable use of rFVIIa. In contrast, antifibrinlytics may have a role in controlling massive
bleeding from trauma. The 2010 CRASH-2 study concluded that tranexamic acid should be given as early as
possible in the trauma patient.75
Air Embolism
Air embolism can occur if blood in an open system is infused under pressure or if air enters a central
catheter while containers or blood administration sets are being changed. Air embolism has been reported in
association with intraoperative and perioperative blood recovery systems that allow air into the blood infusion
bag. The minimum volume of air embolism that is potentially fatal for an adult is approximately 100 mL.76
Symptoms include cough, dyspnea, chest pain, and shock.
If air embolism is suspected, the patient should be placed on the left side with the head down to displace the
air bubble from the pulmonic valve. Aspiration of the air is sometimes attempted. However, proper use and
inspection of infusion pumps, equipment for blood recovery or apheresis, and tubing couplers are still essential
to prevent this complication.
Hypothermia
Blood warmers may be used to prevent hypothermia. Proper procedures for the use of blood warmers should
be followed because
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overheating may damage or destroy red cells, causing hemolysis and serious transfusion reactions, including
fatalities. Blood warmers must have a temperature monitor and warning system to decrease the risk of
overheating.5(p6)
DELAYED TRANSFUSION REACTIONS
Delayed transfusion reactions occur days to months or even years after transfusion. As with acute
transfusion reactions, the consequences may be severe but are often treatable.
DHTRs
 Presentation
Fever and anemia occurring days to weeks after transfusion of an RBC component are characteristic of a
DffTR. The hemolysis associated with a DHTR is usually not brisk, but some patients may develop jaundice
and leukocytosis. In a DHTR, the hemolysis is primarily extravascular, so although hemoglobinuria may occur
in rare cases, acute renal failure and DIC are not generally present. In some cases, the hemolysis occurs without
causing clinical symptoms. These patients present with unexplained anemia or do not experience an increase in
hemoglobin concentrations following transfusion.
Differential Diagnosis
Fever with hemolysis may also occur well after transfusion when the component has been contaminated
with an intracellular red cell parasite, such as malaria or babesiosis. Fever without hemolysis may be an
indication of graft-vs-host disease (GVHD) [described in the transfusion-associated (TA)-GVHD section below]
or transfusion-transmitted viral disease (see Chapter 8). Hemolysis resulting from antibody production by donor
passenger lymphocytes may occur after transplantation of a minor ABO-incompatible organ (eg, transplantation
of a group 0 liver in a group A patient).
Pathophysiology
After transfusion, transplantation, or pregnancy, a patient may make an antibody to a red cell antigen that he
or she lacks. Red cell antibodies may cause a delayed transfusion reaction if the patient subsequently receives a
unit of blood expressing the corresponding red cell antigen. Primary alloimmunization occurs anywhere from
days to months after a transfusion of antigen-positive RBCs, depending on the immunogenicity and dose of the
antigen.
Approximately 1% to 1.6% of RBC transfusions are associated with antibody formation, excluding
antibodies to antigens in the Rh system. D-negative blood is usually transfused to D-negative patients, so the
frequency attributable to anti-D is relatively low. Newly formed alloantibodies are routinely detected during
pretransfusion screening (see Chapters 15 and 16). Recently transfused or pregnant patients must have samples
drawn for compatibility testing within 3 days of the scheduled transfusion to ensure identification of any
potential new alloantibodies.5(p36) A 5-year retrospective study of alloimmunization showed that 11 of 2932
patients (0.4%) had developed new antibodies, including anti-E, anti-K, and anti-Jka, within 3 days of their
transfusion.77
DHTRs and delayed serologic transfusion reactions (DSTRs; rapid development of alloantibody in the
absence of laboratory evidence of hemolysis) rarely, if ever, occur as a result of primary immunization and are
generally associated with subsequent transfusions. Antibody titers may slowly decrease after the initial immune
response, with as many as 30% to 40% of alloantibodies becoming undetectable over months to years.
Antibodies against antigens of some blood group systems, such as the Kidd system, frequently exhibit this
behavior. Subsequent transfusion of an antigen-positive unit triggers an anamnestic response, with production of
antibody occurring over the next several days to weeks after the transfusion. The rapidity of antibody
production and hemolytic potential of the antibody combine to influence the clinical presentation. Blood group
antibodies associated with DHTRs/DSTRs include those of the Kidd,
687
Duffy, Kell, and MNS systems, in order of decreasing frequency. 78tpp358‘89)
In a DHTR/DSTR, antibodies may be found in the serum, on transfused red cells, or both. Routine antibody
screening and antibody identification should be possible. If transfused red cells are still present, the DAT result
may be positive. When the DAT result is positive, an eluate should be performed and the antibody identified. If
a segment from the unit is available, antigen typing may confirm the diagnosis.
Frequency
As with AHTRs, the estimated rate of DHTRs varies widely from study to study. Some of this variation is
the result of the practice of considering DSTRs and DHTRs as one category. Also, improvements in laboratory
techniques have contributed to the increased number of DSTRs detected. It is known that these reactions occur
much more frequently than AHTRs, with estimates of approximately 1:2500 for either type of delayed reaction
and a frequency that is twice as high for DSTRs.79 These reactions are likely to be greatly underrecognized
because most patients do not undergo red cell antibody screening following transfusion.80
Treatment
The treatment of DHTRs consists of monitoring the patient and providing appropriate supportive care. The
most frequent therapy is correction of the anemia by transfusing antigen-negative RBCs as needed. When a
DSTR is identified by the laboratory, the patient’s physician and the transfusion service director should be
notified so that unrecognized hemolysis may be appropriately identified and treated.
Prevention
DHTRs/DSTRs caused by known antibody specificities can be prevented by the transfusion of antigen-
negative RBCs. It is essential to obtain prior transfusion records for the recipient because alloantibodies may
have been identified that are no longer detectable but re
quire transfusion of antigen-negative RBCs. Many institutions have programs to provide at least partially
phenotypically matched blood for patients with sickle cell disease and some other patients who have developed
multiple alloantibodies. Patients with sickle cell disease may develop a complication known as “sickle cell
HTR,” where autologous and allogeneic cells are destroyed (see Chapter 23).
Refractoriness to Platelet Transfusions
See Chapter 18.
TA-GVHD
Presentation
The clinical manifestations of TA-GVHD typically begin 8 to 10 days after transfusion, although symptoms
can occur as early as 3 days and as late as 30 days. Signs and symptoms include a maculopapular rash, fever,
enterocolitis with watery diarrhea, elevated liver function test results, and pancytopenia. The rash begins on the
trunk and progresses to the extremities. In severe cases, bullae may develop.81
Unlike GVHD after allogeneic marrow transplantation, TA-GVHD leads to profound marrow aplasia, with a
mortality rate higher than 90%. The time course of the reaction is very rapid; death typically occurs within 1 to
3 weeks of the first symptoms.
Differential Diagnosis
Because the clinical manifestations of TAGVHD appear several days after a transfusion, it may be difficult
to associate the patient’s symptoms with the transfusion. The symptoms can easily be attributed to other
conditions, including drug reactions and viral illness. In cases of TA-GVHD, a skin biopsy reveals a superficial
perivascular lymphocytic infiltrate, necrotic keratinocytes, compact orthokeratosis, and bullae formation.
Molecular techniques, including HLA typing, cytogenetics, and chimerism assessment, can be used to make the
diagnosis.82
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AABB TECHNICAL MANUAL
Pathophysiology
Billingham83 has proposed three requirements for GVHD to develop in a patient. First, there must be
differences in the HLA antigens expressed between the donor and the recipient. Second, immunocompetent
cells must be present in the graft. Finally, the host must be incapable of rejecting the immunocompetent cells.
In TA-GVHD, viable transfused lymphocytes mount an immunologic attack against the transfusion
recipient. Consistent with Billingham’s work, the three primary factors that determine the risk of developing
TA-GVHD are the degree of recipient immunodeficiency, number of viable T lymphocytes in the transfusion,
and degree of a population’s genetic diversity. Risk factors for developing TA-GVHD include leukemia,
lymphoma, use of immunosuppressive drugs administered for transplant or myeloablative chemotherapy,
congenital immunodeficiency disorders, and the neonatal state.84,85
The number of viable lymphocytes in a transfusion can be affected by the age, leukocyte-reduction status,
and irradiation status of the component.86 Unfortunately, the minimum number of viable lymphocytes required
to cause TA-GVHD is unknown. Although current leukocyte-reduction technologies significantly reduce the
number of lymphocytes in a component, leukocyte reduction does not eliminate the risk of TA-GVHD. There
are welldocumented cases in which transfusion of leukocyte-reduced blood components have caused TA-
GVHD.87
Although patients who are immunocompromised are at risk of developing TA-GVHD, the disorder has also
been reported in transfusion recipients with an intact immune system.82 TA-GVHD can occur after a
transfusion from a donor who is homozygous for an HLA haplotype to a heterozygous recipient (oneway
haplotype match). In this circumstance, the recipient’s immune system is unable to recognize the HLA-
homozygous transfused lymphocytes as foreign. In contrast, the transfused lymphocytes are able to recognize
the
host cells as foreign and are able to mount an immunologic attack on the host.
 The degree of genetic diversity in a population also affects the risk of developing TAGVHD. For example,
the estimated risk of developing TA-GVHD ranges from 1:874 in Japan to 1:16,835 in France.82 The difference
is the result of the lower diversity in HLA antigen expression in the Japanese population than in the French
population.
Treatment
Treatment of TA-GVHD has been attempted with a variety of immunosuppressive agents. Unfortunately, the
disorder is almost uniformly fatal; only rare cases of successful treatment, many involving some form of stem
cell transplant, have been reported. Therefore, emphasis is placed on prevention of the disorder.
Prevention
The only reliable way to prevent TA-GVHD is by irradiation of cellular blood components. AABB
Standards requires a minimum dose of 25 Gy (2500 cGy) delivered to the central portion of the container and a
minimum of 15 Gy (1500 cGy) elsewhere.5(p25) AABB Standards also requires irradiation of cellular blood
components when 1) the patient is identified as being at risk of TA-GVHD, 2) the donor is a blood relative of
the recipient, and 3) the donor is selected for HLA compatibility by typing or crossmatching.5(p40) These
standards are minimum requirements for irradiation of cellular blood components, and institutions may choose
to administer irradiated components to other categories of patients (see Table 27-2).
Posttransfusion Purpura
Presentation
Posttransfusion purpura (PTP) is a relatively uncommon complication of transfusion, and its true incidence
is therefore difficult to estimate. Nonetheless, more than 200 cases have been reported in the literature, and data
from the Serious Hazards of Transfusion (SHOT) program in the United Kingdom suggest that
689
TABLE 27-2. Clinical Indications for Irradiated Components
Well-documented indications Intrauterine transfusions
Prematurity, low birthweight, or erythroblastosis fetalis in newborns Congenital immunodeficiencies
Hematologic malignancies or solid tumors (neuroblastoma, sarcoma, Hodgkin disease)
Peripheral blood stem cell/marrow transplantation
Components that are crossmatched, HLA matched, or directed donations (from family members or other
related donors)
Fludarabine therapy Granulocyte components
Potential indications
Other malignancies, including those treated with cytotoxic agents Donor-recipient pairs from genetically
homogeneous populations
Usually not indicated
Patients with human immunodeficiency virus Full-term infants Nonimmunosuppressed patients
the disorder may be more common than previously believed.87 Hospitals that participated in SHOT
reported 44 cases of PTP in 8 years.
Patients typically present with wet purpura and thrombocytopenia 9 days (range = 1-24), on average, after a
transfusion.88 The thrombocytopenia is often profound, with platelet counts of <10,000/pL. Bleeding from
mucous membranes and the gastrointestinal and urinary tracts is common. Mortality rates in large case series
range from 0 to 12.8%, primarily due to intracranial hemorrhage.67,68
PTP has most commonly been associated with transfusions of RBCs or whole blood; however, the disorder
has also been associated with transfusions of platelets or plasma.
Differential Diagnosis
The differential diagnosis of PTP includes consideration of other causes of thrombocytopenia, such as
autoimmune thrombocytopenic purpura, thrombotic thrombocytopenic purpura, heparin-induced
thrombocytopenia, DIC, and drug-induced thrombocytopenia. Although the diagnosis of PTP can be obvious in
patients with previously normal platelet counts and no other significant medical abnormalities, it can be a
challenge in patients with multiple medical problems. Platelet serology studies may aid in the diagnosis.
Pathophysiology
The pathogenesis of PTP is related to the presence of platelet-specific alloantibodies in a patient who has
previously been exposed to platelet antigens via pregnancy or transfusion. The female-to-male ratio of affected
patients is 5 to 1. Antibodies against human platelet antigen la (HPA-la), located on glycoprotein Ilia, are
identified in about 70% of PTP cases. Antibodies to HPA-lb, other platelet antigens, and HLA antigens have
also been implicated in PTP88
The reason for the concomitant destruction of autologous platelets in this disorder is unknown; three
theories have been advanced. According to the first theory, immune complexes bind to platelets through the Fc
receptor, causing destruction of platelets.89 The second theory posits that the patient’s platelets
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AABB TECHNICAL MANUAL
absorb a soluble platelet antigen from donor plasma, making them susceptible to immune destruction.
According to the third theory, the platelet alloantibody has autoreactivity that develops when a patient is re-
exposed to a foreign platelet-specific antigen.88 The third theory currently has the most support.
The use of leukocyte reduction filters may decrease the incidence of PTP. In the three years prior to the
implementation of universal leukocyte reduction, in the United Kingdom, there were 10.3 cases of PTP per
year, compared to 2.3 cases per year after universal leukocyte reduction (p<0.001).87
Treatment
Because the duration of thrombocytopenia in untreated patients is about 2 weeks, it can be difficult to assess
the effectiveness of therapies for PTP. Steroids, whole-blood exchange, and plasma exchange have all been used
to treat PTP. The current treatment of choice for PTP is MG.88 Patients respond within 4 days, on average, and
some respond within hours.
Prevention
PTP typically does not recur with subsequent transfusions. However, there are four case reports of PTP
recurrence. Therefore, for patients with previously documented PTP, efforts should be made to obtain
components from antigen-matched donors. Autologous donations and directed donations from antigenmatched
donors and family members may also be appropriate. Because PTP has also occurred after transfusions of
deglycerolized rejuvenated or washed RBCs, such manipulations are not indicated for the prevention of
recurrence.88
Iron Overload
A unit of RBCs contains approximately 250 mg of iron. The average rate of excretion of iron is
approximately 1 mg per day. As red cells are destroyed, the majority of the released iron cannot be excreted and
is stored in the body as
hemosiderin and ferritin. Transferrin becomes saturated after the administration of 10 to 15 RBC units to a
nonbleeding patient.90 As iron accumulates in the reticuloendothelial system, liver, heart, spleen, and endocrine
organs, tissue damage leading to heart failure, liver failure, diabetes, and hypothyroidism may occur. Patients
who are chronically transfused for diseases such as thalassemia, sickle cell disease, and other chronic anemias
are at greatest risk for iron overload. Prevention of the accumulation of these toxic levels of iron by reducing the
body’s iron stores through the use of iron chelators is therefore extremely important. A cumulative dose of 50 to
100 RBC units can cause significantly greater morbidity and mortality than the underlying anemia.91
Iron chelators bind to iron in the body and tissues and help remove it through the urine and/or feces. The
development of ironchelating drugs, such as parenteral deferoxamine and the oral agent deferiprone, greatly
reduced the complications of iron overload in chronically transfused patients, leading to improved quality of
life. Deferiprone is more effective than deferoxamine in reducing myocardial siderosis.92
The newest agent, deferasirox, has a much longer half-life than deferoxamine and may be administered in
one daily oral dose. Although in-vivo studies are in the early stages, deferasirox has similar rapid access to
intracellular iron stores in cultured myocytes as deferiprone.93 Unlike deferiprone, which has occasionally been
associated with agranulocytosis, the most frequent side effects of deferasirox are transient gastrointestinal
distress and mildly increased creatinine levels that are rarely of clinical significance. Deferasirox treatment for a
median of 2.7 years showed efficacy in children and adults with sickle cell disease, resulting in a significant
dose-dependent decline in their serum ferritin levels without producing any new adverse events.94 Lower doses
of deferasirox have also been used to reduce iron overload in non-transfusion-dependent patients with minimal
side effects.94
691
TABLE 27-3. How to Contact the FDA
Method Contact Details
E-mail fatalities2@fda.hhs.gov
Telephone/voicemail 240-402-9160
 Fax 301-827-6748, Attn: CBER Fatality
Program Manger
US Food and Drug Administration
CBER Office of Compliance and
Biologies Quality
Express mail Document Control Center
10903 New Hampshire Avenue
W071.G112
Silver Spring, MD 20993-0002
FDA = Food and Drug Administration; CBER = Center for
Biologies Evaluation and Research.
FATALITY REPORTING REQUIREMENTS
When the death of a patient results from a reaction to or complication of a transfusion, current good
manufacturing practice regulations require that the fatality be reported to the FDA by the facility that performed
the compatibility testing.95 The director of the Office of Compliance and Biologies Quality at the FDA Center
for Biologies Evaluation and Research must be notified as soon as possible by telephone, express mail, or
facsimile or electronic mail, followed by the submission of a written report within 7 days. Table 27-3 lists the
contact in
formation for the FDA. The report should contain the patient’s medical records, including laboratory
reports, and the autopsy results when available. The patient’s underlying illness may make determination of the
cause of death difficult. If there is any clinical suspicion that the transfusion may have contributed to the
patient’s death, that possibility should be investigated. Most transfusion-associated fatalities are caused by acute
hemolysis, TRALI, or TACO. Investigations of these cases must attempt to rule out laboratory, transfusion
service, or blood administration errors.96
KEY POINTS
1. The blood supply is safer today than at any time in history. Hemovigilance and blood management
programs decrease the risk of noninfectious complications of transfusion.
2. Many transfusion reactions have signs or symptoms that may be present in more than one type of
reaction. Early recognition of the reaction, prompt cessation of the transfusion, and further evaluation are key to
the successful resolution of a reaction.
3. Acute intravascular hemolytic reactions are often caused by sample or patient misidentification and are
thus, for the most part, preventable.
4. Allergic reactions range from urticaria (hives) to anaphylaxis. Patients experiencing severe reactions
should be checked for IgA deficiency and transfused accordingly.
5. Recognition of TRALI requires diagnosis by exclusion. Most patients recover from TRALI with
supportive care.
6. TACO can be confused with TRALI because both feature pulmonary edema. TACO should be suspected
in nonbleeding patients and those with congestive heart failure.
 692
AABB TECHNICAL MANUAL
7. The most common complications of massive transfusion are hemostatic abnormalities. Each institution
should develop its own massive transfusion protocol that takes into account the availability of appropriate
laboratory testing.
8. TA-GVHD has a much more acute and severe course than GVHD after marrow or stem cell
transplantation. TA-GVHD is fatal in >90% of cases and can be prevented by irradiation of blood components.
9. PTP is a serious but rare complication in which antibodies to human platelet antigens result in destruction
of autologous and allogeneic platelets.
10. Iron overload is perhaps the longest-lasting long-term complication of transfusion. Oral iron chelators
gready increase compliance with therapy.
11. TRALI is the leading cause of transfusion-related mortality reported to the FDA.
12. Recipient fatalities must be reported to the FDA by the compatibility testing facility as soon as possible
after a fatal complication of transfusion has been confirmed.
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82. Jacobson CA, Anderson KC, Alyea EP. Transfusion-associated graft-versus-host disease. In: Popovsky
MA, ed. Transfusion reactions. 4th ed. Bethesda, MD: AABB Press, 2012:217-37.
83. Billingham R. The biology of graft-versus-host reactions. In: The Harvey lecture series, 19661967. Vol
62. Orlando, FL: Academic Press, 1968;21-78.
84. Anderson KC, Weinstein HJ. Transfusion-associated graft-versus-host disease. N Engl J Med
1990;323:315-21.
85. Leitman SF, Tisdale JF, Bolan CD, et al. Transfusion-associated GVHD after fludarabine therapy in a
patient with systemic lupus erythematosus. Transfusion 2003 ;43:1667-71.
86. Klein HG. Transfusion-associated graft-versus-host disease: Less fresh blood and more gray (Gy) for an
aging population. Transfusion 2006;46:878-80.
87. Williamson LM, Stainsby D, Jones H, et al. The impact of universal leukodepletion of the blood supply
on hemovigilance reports of posttransfusion purpura and transfusion-associated graft-versus-host disease.
Transfusion 2007;47:1455-67.
88. McFarland JG. Posttransfusion purpura. In: Popovsky MA, ed. Transfusion reactions. 4th ed. Bethesda,
MD: AABB Press, 2012:263-87.
89. Shulman NR, Aster RH, Leitner A, et al. Immunoreactions involving platelets. V. Post-transfusion
purpura due to a complement-fixing antibody against a genetically controlled platelet antigen. A proposed
mechanism for thrombocytopenia and its relevance in “autoimmunity.” J Clin Invest 1961;40:1597-620.
90. Ley TJ, Griffith P, Nienhuis AW. Transfusion haemosiderosis and chelation therapy. Clin Haematol
1982;11:437-64.
91. Sharon BI. Management of congenital hemolytic anemias. In: Simon TL, Snyder EL, Solheim BG, et al,
eds. Rossi’s principles of transfusion medicine. 4th ed. Bethesda, MD: AABB Press, 2009:448-69.
92. Cario H, Janka-Schaub G, Jarisch A, et al. Recent developments in iron chelation therapy. Klin Padiatr
2007;219:158-65.
93. Neufeld EJ. Oral chelators deferasirox and deferiprone for transfusional iron overload in thalassemia
major: New data, new questions. Blood 2006:3436-41.
94. Taher AT, Porter J, Viprakasit V et al. Deferasirox reduces iron overload significantly in non-transfusion
dependent thalassemia: 1year results from a prospective, randomized, double-blind, placebo-controlled study.
Blood 2012;120:970-77.
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95. Code of federal regulations. Title 21, CFR Part 606.170(b). Washington, DC: US Government Printing
Office, 2011 (revised annually).
96. Guidance for industry: Notifying FDA of fatalities related to blood collection or transfusion.
(September 22, 2003) Silver Spring, MD: CBER Office of Communication, Outreach, and Development,
2003.
Chapter 28
Approaches to Blood Utilization Auditing
Alan Tinmouth, MD, FRCPC, MSc, and Simon Stanworth, FRCP, FRCPath, DPhil
auditing the use of blood transB181 fusions is a required function for all hospital transfusion services.1 Both
The Joint Commission and AABB require health-care institutions to monitor the use of blood components. The
Joint Commission also requires hospitals to collect data to monitor the performance of processes that involve
risks or may result in sentinel events, including the use of blood and blood components (Standard PI.3.1.1).2
The AABB requires all facilities to have a peer-review program “that monitors and addresses transfusion
practices for all categories of blood and components" including “usage and discard” and “appropriateness of
use.”3(p91) In addition, federal regulations pertaining to Medicare and Medicaid require that hospitals make
recommendations to the med
ical staff regarding improvements in transfusion procedures.1
Other countries also require hospitals to establish processes to audit and monitor transfusion practices. For
example, in Canada, both the Canadian Standard Association and the Canadian Society for Transfusion
Medicine require hospitals to monitor blood transfusion use, and compliance with the Canadian Standard
Association’s requirements will be mandated by federal law. In England, the Department of Health’s Better
Blood Transfusion initiatives from 1997 to 2012 and the subsequent Patient Blood Management program have
promoted and continued to encourage hospital participation in national audits. This includes a specific program
of National Comparative Audits for hospitals in transfusion.

Alan Tinmouth, MD, FRCPC, MSc, Head, General Medicine and Transfusion Medicine, The Ottawa
Hospital, and Scientist, University of Ottawa Centre for Transfusion Research, Clinical Epidemiology Program,
Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, and Simon Stanworth, FRCP, FRCPath, DPhil,
Consultant Haematologist, Oxford University Hospitals NHS Trust, and Honorary Senior Clinical Lecturer,
University of Oxford, Oxford, United Kingdom
A. Tinmouth is supported by a Research Award from the Department of Medicine, The Ottawa Hospital;
and has disclosed a financial relationship with Amgen. S. Stanworth has disclosed no conflicts of interest.
697

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Factors that help ensure high levels of participation in national audits in England are:
1. The audits contribute to “Quality Account” reports required of National Health Service (NHS) hospitals
(Health Act 2009).
2. Data are used by the Care Quality Commission, an independent regulator of all health and social care
services in England.
3. NHS hospitals participating in a national audit may receive a discount from the NHS Litigation Authority,
which manages negligence and other claims against the NHS in England.
4. The National Blood Transfusion Committee oversees, promotes, and supports all national audits.
In a similar vein, The Joint Commission announced a certification program starting in 2014 to further
recognize accredited hospitals that implement system-wide measures to improve blood utilization.
A number of different methods can be employed to meet the requirements to monitor or audit blood
transfusion use. However, the general objective of all blood utilization programs is to ensure the appropriate use
of blood components. Alternative or complementary goals may include reducing inappropriate use and, as a
result, reducing costs. Historically, audits of blood use have concentrated on controlling or reducing the total
number of blood components transfused and/ or individual “overtransfusions.” This focus was based on the
assumption that inappropriate use consisted primarily of overtransfusion. Unnecessary transfusions result in
unnecessary costs and adverse events. However, with increased attention on limiting patients’ exposure to blood
components, undertransfusion may be a concern. In addition, the dose of blood transfusion requests, which may
result in overuse or underuse, might also need to be assessed in audits. However, neither of these issues has
historically been a major focus of the monitoring of blood transfusion utilization.4
The methods used to audit blood use depend on the desired objectives. Monitoring
trends in the total number of blood units transfused, perhaps to control ordering or inventory management,
requires a different process from that required to limit inappropriate transfusion requests. In all instances, the
transfusion service (technologists and/or physicians) and the institutional transfusion medicine or blood
utilization committee must work together to perform the appropriate audios) and follow-up.
RATIONALE FOR MONITORING BLOOD UTILIZATION
The primary motivation for monitoring blood utilization is to identify instances when blood product
utilization is less than optimal. Interventions can then be implemented to change transfusion practice. Therefore,
audits or monitoring must be combined with a recognized process to effectively provide feedback on the
findings to the appropriate health-care professionals.
Improving or ensuring optimal use of blood transfusions is important for several reasons. Blood is a biologic
agent associated with many possible adverse events, including both infectious and noninfectious
complications.5,6 Many of these complications are well known, others are not usually recognized by physicians
or the general population, and some potential complications are still not fully understood. Unnecessary
complications from inappropriate blood transfusions need to be avoided. In addition, blood components are a
scarce and expensive resource. Transfusing a single unit of Red Blood Cells (RBCs) has been estimated to cost
from $400 to $760 when all the associated costs are included, and many regions have experienced shortages.6'8
Through the careful monitoring of blood utilization, instances of inappropriate blood component use can be
identified and corrective actions can be taken. If a “real-time” or prospective audit system is used, then a
member of the transfusion medicine service can intervene, which may result in a change in the transfusion
request before the issue of a blood unit. A retrospective review does not alter the current transfusion episode but
it does identi
CHAPTER 28 Approaches to Blood Utilization Auditing
699
fy issues that can be addressed through interventions designed to change future transfusion practice.
A variety of interventions have been used to change transfusion practice (Table 28-1). One of the most
common interventions for change is audit and feedback, which is defined by the Cochrane Effective Practice
and Organization of Care Group as “any summary of clinical performance of health care over a specified period
of time.” However, there is less understanding of the effective elements of audit and feedback or how the
feedback should be structured to produce changes in behavior in different settings. Following the delivery of an
intervention, ongoing monitoring allows for assessment of the sustained effectiveness of the intervention and
need for ongoing or additional intervention.
In summary, audits serve the dual function of 1) identifying areas of concern in the use of blood products
and 2) monitoring interventions or changes in the identified areas.
TYPES OF TRANSFUSION AUDITS
Audits of transfusion practice can examine individual blood transfusion requests and/or aggregate
transfusion data. Reviews of individual requests can be performed either prospectively in real time or
retrospectively. Reviews of individual transfusions may also be performed (retrospectively) very shortly after
the event
(usually within 24 hours); this has been termed a “concurrent review” because it still affords the ability to
provide timely feedback on individual transfusion episodes.9 More remote retrospective audits allow for the
review of aggregate transfusion data, which can then be analyzed in several ways. Reviews of individual and
aggregate transfusion data are not mutually exclusive. Because they may serve different functions or purposes,
they can, in fact, be complementary.
Prospective (“Real-Time”) Audits
In a prospective audit, individual transfusion requests are reviewed in “real time” (ie, before the issue of the
blood component). An electronic review using a computer-based algorithm and/or a manual review by
technologists is undertaken whereby the request is compared to local transfusion audit criteria. Clinical data (eg,
hematocrit and indication for RBC transfusion) are required to perform the review. Ideally, this information is
obtained from the clinical staff as part of the transfusion request process1011 or pretransfusion blood values are
automatically retrieved from the laboratory information system.12,13 With computer provider/physician order
entry (CPOE), the institutional guidelines can be incorporated into the request process.11 Laboratory staff can
also obtain these data from the laboratory information system,14 although this is more labor intensive and
difficult to implement
TABLE 28-1. Use of Interventions to Change Transfusion Practice with Different Types of Audits
Prospective Concurrent Retrospective
Intervention
Audit Audit Audit
Individual education/feedback ++ ++ +
Group education/feedback/teaching ++
Guideline dissemination + + +
Incorporation of reminders/guidelines into transfusion ++ ++ +
request form or computerized order entry system
 ++ = very complementary to audit type; + = can be complementary to audit type; - = less complementary or
not complementary to audit type.

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AABB TECHNICAL MANUAL
universally.10 Transfusions that do not meet the audit criteria are flagged and the requesting physician is
contacted by the transfusion medicine service before the blood component is issued to discuss the need for the
transfusion.
A prospective audit system must be organized such that undue delays in filling transfusion requests do not
occur. For example, when CPOE is used to screen the appropriateness of a transfusion request that does not
comply with the institution’s guidelines, that request may still be completed after the ordering clinician enters
the rationale into the information system.11 Delays in obtaining laboratory results and the uncertainties of
clinical cases also need to be identified and reflected in the audit process.
Given these issues and constraints, the criteria for undertaking a review as part of a prospective audit may
need to be less rigid than the guidelines for the optimal utilization of blood components because prospective
audits might delay some transfusions that fail to meet audit criteria for appropriateness.13 Therefore, a
mechanism must be in place to ensure that emergency and urgent transfusions are not unduly delayed. Specific
clinical areas, such as emergency departments and operating suites, where delays in filling transfusion requests
might result in adverse clinical events might be excluded from prospective transfusion audits.
Prospective audits also have the potential to generate animosity in the ordering physician when a transfusion
request is questioned. Engaging requesting physician groups and broadly consulting them during the
development of the audit guidelines and process can help reduce friction and ensure the long-term success of a
prospective audit system. In addition, interactions with the requesting physician often require the involvement
of a transfusion medicine physician or, at least, a senior technologist.14,15 To reduce the potentially onerous
time requirements for technologists and physicians in a prospective audit system, selective audits of either
specific clinical areas (eg, obstetrics or orthopedics) and/or specific time periods maybe used.
 The potential benefits of a prospective audit include the ability to intervene directly and to change a
transfusion request before the component is issued. As a result, an unnecessary transfusion may be stopped12 or
a more appropriate component or dose may be ordered.16 Ideally, the immediate intervention stemming from a
prospective audit also results in long-term changes in the transfusion practice of the ordering physician.
However, the benefits of this immediate intervention must be weighed against the additional work required by
the transfusion service, including the transfusion medicine physician.
A number of reports from single institutions have demonstrated the effectiveness of prospective audits in
reducing the total number of units transfused,14,15,17 the number of units transfused per patient,18'20 the
proportion of patients transfused,18,19,21,22 and the number of inappropriate transfusions19,22,23 (Table 28-
2). Many of these studies used prospective audits in conjunction with other interventions to change transfusion
practice.14,15,17'20,22,23 Although these reports attest to the potential utility of prospective audits to change
transfusion practice, the sustainability of the changes is not known. In one study, the initial reduction in
inappropriate transfusions following the implementation of a prospective audit system did not persist when
inappropriate transfusion rates were reexamined 3 years later.27
Concurrent Audits
A concurrent audit is a review of an individual transfusion request that occurs in the 12 to 24 hours
following the transfusion episode.9,13 As such, it involves processes similar to those of the prospective audit.
However, because a concurrent audit is a posttransfusion review, it cannot lead to any alterations in the
individual transfusion event. Therefore, the concurrent audit is designed only to alter future transfusion practice.
However, follow-up with the requesting physician occurs while the transfusion event remains fresh in his or her
memory. It is hoped that this immediate contact im
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion
Practice
CHAPTER 28 Approaches to Blood Utilization Auditing
701

(Continued)
 TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion
Practice (Continued)
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Handler 198336 Retrospective audit, education RBCs
 703
proves the chance of changing the physician’s future transfusion behavior.
As with the prospective audit, the concurrent review can be conducted by a computer algorithm or manually
using transfusion audit criteria. The review may occur at the time of the transfusion or shortly after the request
is filled. Requests that do not meet the audit criteria are flagged for subsequent review by a senior technologist
or transfusion medicine physician. When performing the review, the transfusion medicine physician may have
access to additional laboratory results that help to determine the appropriateness of each request. However, the
reviewer must also consider the fact that these additional results were not available to the ordering physician at
the time he or she made the request. Because concurrent audits do not delay the filling of transfusion requests,
these audits can assess urgent transfusions and use stricter criteria for appropriateness than prospective audits.
Following the identification of an inappropriate transfusion request, the ordering physician may be
contacted by telephone or email to discuss the case. The less immediate nature and ability to use written
communication may reduce any animosity from the ordering physician and result in a more meaningful
dialogue. Shanberge and colleagues35 reported a 77% reduction in the number of Fresh Frozen Plasma units
transfused after the introduction of a concurrent audit system combined with a new guideline and an education
program.
Conducting concurrent audits is time consuming, and a transfusion medicine physician usually needs to
review inappropriate transfusion requests and follow up with the requesting physicians.13 For these reasons,
performing a concurrent audit of all transfusion requests may not be feasible. Using selective audits, as
discussed for prospective audits, is an option to reduce the workload involved. Concurrent audits may be
particularly relevant for certain patient groups, such as patients treated for trauma or others in the emergency
department, where the urgent need for blood precludes a prospective audit, and accurate time lines (key to
determining the appropriate
implementation of massive transfusion protocols) or communication with the transfusion service might not
be readily recalled in retrospective audits.
Retrospective Audits
 Retrospective audits of blood utilization are commonly performed by hospital teams or blood transfusion
services. The results should be reviewed by the hospital’s transfusion medicine committee. The frequency of
such audits varies from one institution to another. Computerized systems may facilitate ongoing utilization
review across many specialties.11 Unlike prospective and concurrent reviews, retrospective audits can be used
to examine aggregate transfusion data and trends in transfusion utilization. The results may be informative for
understanding wider differences in transfusion practice across different specialty groups. Individual transfusion
requests can also be reviewed for appropriateness as part of a retrospective audit, but doing so may be
cumbersome unless the data are collected prospectively or the laboratory information system can link data on
transfusion events and laboratory results.
Retrospective reviews can analyze data in a variety of ways. The simplest analysis is an examination of the
total numbers of blood components transfused and patients transfused. However, these data might show
considerable temporal variability that limits meaningful analysis. Further analysis is required to make
observations regarding differences in blood utilization. The mean or median number of units transfused per
hospitalized patient and/or procedure provides a more meaningful summary of blood component use. Simply
dividing the total number of units transfused by the total number of patients who received a transfusion is not an
appropriate statistical analysis; any observed changes in total blood component use, particularly during a short
period, could be skewed by a small number of patients who required a large number of units. The proportion of
patients transfused should also be determined and can be further examined by procedure or
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AABB TECHNICAL MANUAL
clinical specialty. All of these analyses comprise the minimal set required to monitor differences in blood
utilization. Additional analyses could include assessments of appropriate and inappropriate transfusion rates,
although these analyses require collecting additional clinical data and reviewing individual transfusion requests,
which may be labor-intensive exercises.
Utilization trends by individual product, individual physician, or clinical service can be analyzed. For
health-care institutions with multiple sites, similar clinical services at different sites can also be compared.
These additional layers of analysis may provide important information to identify variations in practice and
detect inappropriate transfusion practices. These areas could then be targeted by interventions to improve
transfusion practice.
In general, a retrospective review is less labor intensive than a prospective or concurrent review, particularly
because it involves less immediate attention from a technologist or transfusion medicine physician. The amount
of time required to perform the review depends on the amount of detail desired.
The use of retrospective audits and the provision of dedicated feedback to clinicians have been effective in
reducing the total number of units transfused,20,24,29-33 number of units transfused per patient,30-32,37,38
proportion of patients transfused,26,28,36 and number of inappropriate transfusions24,29,34 (Table 28-2). The
long-term durability of these changes in transfusion practice following the introduction of a retrospective audit
has not been evaluated.
INTERVENTIONS TO CHANGE TRANSFUSION PRACTICE
As previously described, the results of transfusion audits should ideally be used in conjunction with
interventions to effect change and improve transfusion practice. Thus, audits should be considered a critical
component of a larger process to optimize the utilization of blood components. This process can be more widely
conceived as dissemination, knowledge transfer, or implementation work, where the
goal is to increase the uptake of research results and/or best practices into daily practice.39
The process of closing the gap between knowledge and action involves first distilling knowledge, usually in
the form of guidelines. National or international guidelines can simply be adopted; however, local development
of guidelines with the involvement of local stakeholders may increase adherence to guidelines.40 Audits then
serve the purpose of identifying gaps between the guidelines (best practices) and current practice (action). When
important gaps are identified through audits, interventions can be developed to target practice changes. To
improve the chance of achieving meaningful and lasting changes in practice, interventions should be carefully
chosen and designed.
Unfortunately, the process of identifying the best interventions has not been well defined. Ideally, the
facilitators of and barriers to change are first identified so that any selected intervention targets these identified
factors.41,42 Concurrent,13 retrospective,37 and prospective21 audits have been effective individual
interventions in changing transfusion practice. However, other interventions, including dissemination of
guidelines,11,20,28,30,32-35 education,15,20,24,30,33,36 introduction of new transfusion request forms, and
computer order entry,24,30,32 have been commonly used in conjunction with audits.
EFFECTIVENESS OF MONITORING AND INTERVENTIONS TO CHANGE TRANSFUSION
PRACTICE
Most published studies on auditing efforts have shown that prospective and retrospective audits reduce
either the total amount of blood transfused or the proportion of inappropriate transfusions (Table 28-2). These
reductions occurred in studies that used a concurrent audit alone,13 a prospective audit alone,21 or a
retrospective audit and feedback alone.37 The prospective audit study showed a reduction in the utilization of
RBCs and frozen plasma but
705
not platelets.21 The retrospective audit study showed a reduction in the utilization of RBCs but not frozen
plasma or platelets.37 The results from these two studies might suggest that prospective and retrospective audits
used in conjunction with other interventions are more effective in improving blood utilization. However, the
poor designs of the studies (almost all of which were uncontrolled, single-center, before-and-after studies) that
examined interventions to change transfusion practice and the possibility of publication bias (eg, because the
results of studies in which an intervention did not result in an improvement in transfusion practice were not
published) do not allow the true or relative effectiveness of individual, or combinations of, interventions to be
determined.43,44
SELECTING AN AUDIT PROCESS TO MONITOR TRANSFUSIONS
How transfusions should be monitored depends on a number of factors that are specific to each
institution.45 The first step in determining how to monitor transfusions is to decide which type of audits to use.
The prospective and concurrent audits serve identical purposes and cannot be used jointly to assess an
individual transfusion episode. Prospective audits require the greatest amount of time from laboratory staff and
transfusion medicine physicians, including 24-hour coverage and support from the laboratory information
system. These requirements may present difficulties for smaller hospitals and busy transfusion medicine
services, respectively. In general, concurrent reviews are more practical in smaller hospitals or hospitals with
limited resources for laboratory staff or transfusion medicine physicians because transfusions can be reviewed
in the 12 to 24 hours following the transfusion episode. If a universal prospective audit of all blood components
is not feasible, then the prospective audit could be limited to particular components (eg, intravenous immune
globulin or recombinant Factor Vila) or specific clinical areas. Retrospective reviews supplement prospective or
concurrent audits
of individual transfusions by providing data on trends in the use of transfusions.
Lists of the steps for implementing prospective, concurrent, and retrospective reviews are provided in Tables
28-3, 28-4, and 28-5, respectively. A transfusion request form or order entry system incorporating guidelines
and/or clinical information (see sample in Appendix 28-1) can aid in identifying inappropriate transfusion
requests through either prospective or concurrent audits.46 Similarly laboratory information systems and
computer algorithms can be used to screen transfusions for appropriateness in all types of audits.13,47 Reviews
may be more frequent in larger transfusion services or less frequent in smaller hospitals. The retrospective data
can allow smaller hospitals to compare their transfusion practices (eg, number of units transfused per patient by
diagnosis-related group) with those of other similar institutions.
CONCLUSIONS
Auditing transfusion practice is a required function for all transfusion services. Transfusion audits provide
information on levels of compliance with standards and frequency of unnecessary transfusions. Audits
combined with guidelines are clearly essential to evaluate transfusion practices at individual institutions, and
they permit the identification of suboptimal transfusion practices.
In general, the findings of most audits continue to show unnecessary use of blood outside established
guidelines. The translation of research findings into hospital practice is often slow and haphazard, and this
applies as much to transfusion as to other branches of health care. Many interventions are undertaken by
hospitals to change their transfusion practices, but there are real uncertainties about their effectiveness and
durability.
There is a need for research that defines the determinants of appropriate transfusion behavior to better guide
the design and selection of interventions that can produce optimal changes in transfusion practice. Ideally, the
selection of audits and interventions should be based on an assessment of local
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 AABB TECHNICAL MANUAL
TABLE 28-3. Steps for Implementing a Prospective Audit System to Monitor Blood Component Utilization
1. The extent and frequency of the audit process is determined.
■ The audit may be performed on all blood components or only on specific blood products.
■ Areas with urgent needs for transfusion (eg, emergency or operating rooms) may be excluded from the
audit to avoid delays in transfusion for their patients.
■ To limit resource demands, a selective audit may be used. For example, transfusion requests may be
audited only from a single ward, selected on a rotating basis, because of its high transfusion volume or a
perceived problem in its transfusion practice.
■ Similarly, audits may be performed only during specific hours to limit off-hour work by laboratory staff
and transfusion medicine physicians.
2. The requirements for clinical information at the time the transfusion requests are met.
■ This may be part of the request form (Appendix 28-1) or computer order entry.
3. Criteria for appropriate or inappropriate indications are developed for transfusions.
■ Audit criteria should be less stringent than optimal transfusion guidelines to reduce audit workload and
conflicts with requesting physicians.
■ Audit criteria should be developed in conjunction with a transfusion medicine committee and various
medical specialists who use significant amounts of blood products to increase acceptance of transfusion audits.
4. Transfusion requests are screened by laboratory staff, transfusion nurses, or a computer algorithm to
identify inappropriate requests.
5. For all inappropriate transfusion requests, the requesting physician is contacted by the transfusion
medicine service.
■ The requesting physician is usually contacted by a transfusion medicine physician or senior technologist.
■ A predefined mechanism is developed to override/bypass a review if blood is required urgently and to
avoid unacceptable delays in filling a transfusion request.
6. Any decisions to change the transfusion request are made in conjunction with the ordering physician.
TABLE 28-4. Steps for Implementing a Concurrent Audit System to Monitor Blood Component Utilization
1-4. The same as for the Prospective Audit.
5. All inappropriate transfusion requests are subsequently reviewed by senior laboratory staff or a
transfusion medicine physician who discusses the transfusion request with the requesting physician.
■ Contact with the requesting physician is made within 24 hours of the transfusion so that the physician
remembers the clinical details. This time frame is optimal for providing feedback and potentially changing
future transfusion behavior.
■ Contact can be made by telephone or electronically.
 707
TABLE 28-5. Steps for Implementing a Retrospective Audit System to Monitor Blood Component
Utilization
1. Determine the
extent and frequency of
the audit reviews.
The frequency of the data reviews needs to be based on the volume of transfusions
■ and resources available. Records should not be reviewed more than 6 months after
transfusions were administered.
The components to be reviewed need to be determined and should include all
■
conventional components and frequently transfused blood derivatives.
2. Determine the
outcomes.
Totals for the numbers of transfusions and patients transfused are the simplest data
to collect but provide a limited understanding of, and ability to monitor, changes in
■ transfusion utilization. Providing data on utilization by patient (ie, mean or median
number of units per patient) and proportion of patients transfused provides a greater
understanding of utilization.
The appropriateness of transfusions can also be reported if clinical and/or
laboratory data are available. These can be aggregate data from prospective or
■
concurrent reviews, or the laboratory information system can link laboratory data to
data on transfusion events.
3. Review the audit
data.
Audit data should be reviewed by the transfusion medicine service and the
■
transfusion medicine committee.
■ Data may be analyzed on individual physicians, departments, or diagnoses.
Data may be compared within the institution or with data from similar institutions
■
to evaluate overall utilization rates for blood components.
■ Data should be shared with relevant departments and physicians.
4. Determine
whether any additional
 interventions to
optimize transfusion
practice are warranted.
barriers to and facilitators of change. Further audits then allow the monitoring of any changes in transfusion
practice.
Future studies are required to determine what interventions are likely to be the most ef
fective in certain circumstances or to compare different interventions, including their cost-effectiveness.
Nonetheless, audits remain a critical part of the process to evaluate blood utilization and improve transfusion
practice.
KEY POINTS
1. Auditing the use of blood components is a necessary function for all transfusion medicine services and is
required by AABB and The Joint Commission. The main purpose of auditing blood utilization is to assess the
appropriate use of components and help optimize their use.
2. Different forms of audits (prospective, concurrent, or retrospective) maybe used depending on the
objectives of the audit and the resources available. In all cases, providing feedback to the users, using other
interventions, or both are necessary to effect change in transfusion practice.
3. Prospective audits require the review of transfusion requests before issue of components, which permits
the opportunity to intervene and stop or change an inappropriate transfusion request. Individual transfusion
requests are reviewed using prespecified audit criteria (eg, guidelines).

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 4. Concurrent audits review individual transfusion requests in the 12 to 24 hours following a transfusion
episode and involve similar processes as the prospective audit. The individual transfusion events cannot be
altered, as they have already occurred, but feedback to the physician within a short period after the transfusion
offers the opportunity to change future transfusion practice.
5. Retrospective audits of transfusion offer the opportunity to review aggregate transfusion data. Reviewing
individual transfusions for appropriateness may be more difficult, given the remoteness of the transfusion event.
Feedback to individual clinicians or clinical services should be part of a retrospective audit process to help
ensure the optimal use of components.
6. All forms of audits can be used alone to effect changes in transfusion practice. Audits may also be
combined with other interventions such as dissemination of guidelines, education, and the introduction of new
transfusion request forms. Ideally the selection of interventions includes local stakeholders and an assessment of
local factors that may affect the effectiveness of interventions.
7. Published studies evaluating the effect of prospective and retrospective audits on transfusion practice
have generally demonstrated a reduction in either the total amount of blood transfused or the proportion of
inappropriate transfusions. Because of the poor quality of published studies, the true relative effectiveness of
individual interventions or combinations of interventions cannot be inferred.
REFERENCES
1. Mintz PD. Quality Assessment and improvement of blood transfusion practice. In: Mintz PD, ed.
Transfusion therapy: Clinical principles and practice. 3rd ed. Bethesda, MD: AABB Press, 2011:813-36.
2. Comprehensive accreditation manual for hospitals (CAMH): The official handbook. OakBrook Terrace,
IL: The Joint Commission, 2014.
3. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda: AABB, 2014.
4. Saxena S, Wehrli G, Makarewicz K, et al. Monitoring for underutilization of RBC components and
platelets. Transfusion 2001;41:58790.
5. Popovsky M, ed. Transfusion reactions. 4th ed. Bethesda, MD: AABB Press, 2012.
6. Tinmouth AT, Fergusson DA, Chin-Yee IH, et al. Clinical consequences of red cell storage in the
critically ill. Transfusion 2006;46:2014-27.
7. Amin M, Fergusson D, Wilson K, et al. The societal unit cost of allogenic red blood cells and red blood
cell transfusion in Canada. Transfusion 2004;44:1479-86.
8. Shander A, Hofmann A, Ozawa S, et al. Activity-based costs of blood transfusions in surgical
patients at four hospitals. Transfusion 2010;50: 753-65.
9. Becker J, Shaz B for the Clinical Transfusion Medicine Committee and the Transfusion Medicine Section
Coordinating Committee. Guidelines for patient blood management and blood utilization. Bethesda, MD:
AABB, 2011.
10. Haspel RL, Uhl L. How do I audit hospital blood product utilization? Transfusion 2012; 52:227-30.
11. Baer VL, Henry E, Lambert DK, et al. Implementing a program to improve compliance with neonatal
intensive care unit transfusion guidelines was accompanied by a reduction in transfusion rate: A pre-post
analysis within a multihospital health care system. Transfusion 2011;51:264-9.
12. Yazer MH, Waters JH. How do I implement a hospital-based blood management program? Transfusion
2012;52:1640-5.
13. Cohn CS, Welbig J, Bowman R, et al. A datadriven approach to patient blood management. Transfusion
2014;54:316-22.
14. Sarode R, Refaai MA, Matevosyan K, et al. Prospective monitoring of plasma and platelet transfusions
in a large teaching hospital results in significant cost reduction. Transfusion 2010;50:487-92.
 709
15. Tavares M, DiQuattro P, Nolette N, et al. Reduction in plasma transfusion after enforcement of
transfusion guidelines. Transfusion 2011;51:754-61.
16. Rothschild JM, McGurk S, Honour M, et al. Assessment of education and computerized decision
support interventions for improving transfusion practice. Transfusion 2007;47:22839.
17. Yeh CJ, Wu CF, Hsu WT, et al. Transfusion audit of fresh-frozen plasma in southern Taiwan. Vox Sang
2006;91:270-4.
18. Rehm JP, Otto PS, West WW, et al. Hospitalwide educational program decreases red blood cell
transfusions. J SurgRes 1998;75:1836.
19. Cheng G, Wong HF, Chan A, et al. The effects of a self-educating blood component request form and
enforcements of transfusion guidelines on FFP and platelet usage. Queen Mary Hospital, Hong Kong. British
Committee for Standards in Hematology (BCSH). Clin Lab Haematol 1996;18:83-7.
20. Solomon RR, Clifford JS, Gutman SI. The use of laboratory intervention to stem the flow of fresh-
frozen plasma. Am J Clin Pathol 1988; 89:518-21.
21. Hawkins TE, Carter JM, Hunter PM. Can mandatory pretransfusion approval programmes be improved?
Transfus Med 1994;4:45-50.
22. Littenberg B, Corwin H, Gettinger A, et al. A practice guideline and decision aid for blood transfusion.
Immunohematology 1995;11:8894.
23. Tuckfield A, Haeusler MN, Grigg AP, et al. Reduction of inappropriate use of blood products by
prospective monitoring of transfusion request forms. MedJAust 1997;167:473-6.
24. Arnold DM, Lauzier F, Whittingham H, et al. A multifaceted strategy to reduce inappropriate use of
frozen plasma transfusions in the intensive care unit. J Crit Care 2011;26:636.
25. Rubin GL, Schofield WN, Dean MG, et al. Appropriateness of red blood cell transfusions in major
urban hospitals and effectiveness of an intervention. Med J Aust 2001; 175:354-8.
26. Capraro L, Syrjala M. Advances in cardiac surgical transfusion practices during the 1990s in a Finnish
university hospital. Vox Sang 2001; 81:176-9.
 27. Tobin SN, Campbell DA, Boyce NW. Durability of response to a targeted intervention to modify
clinician transfusion practices in a major teaching hospital. Med J Aust 2001; 174:445-8.
28. Hameedullah, Khan FA, Kamal RS. Improvement in intraoperative fresh frozen plasma transfusion
practice—Impact of medical audits and provider education. J Pak Med Assoc 2000;50:253-6.
29. Joshi G, McCarroll M, O’Rourke P, et al. Role of quality assessment in improving red blood cell
transfusion practice. Ir J Med Sci 1997;166:16
19.
30. Morrison JC, Sumrall DD, Chevalier SP, et al. The effect of provider education on blood utilization
practices. Am J Obstet Gynecol 1993; 169:1240-5.
31. Brandis K, Richards B, Ghent A, et al. A strategy to reduce inappropriate red blood cell transfusion.
MedJAust 1994;160:721-2.
32. Rosen NR, Bates LH, Herod G. Transfusion therapy: Improved patient care and resource utilization.
Transfusion 1993;33:341-7.
33. Ayoub MM, Clark JA. Reduction of fresh frozen plasma use with a simple education program. Am Surg
1989;55:563-5.
34. Giovanetti AM, Parravicini A, Baroni L, et al. Quality assessment of transfusion practice in elective
surgery. Transfusion 1988;28:166-9.
35. Shanberge JN. Reduction of fresh-frozen plasma use through a daily survey and education program.
Transfusion 1987;27:226-7.
36. Handler S. Does continuing medical education affect medical care. A study of improved transfusion
practices. Minn Med 1983;66:16780.
37. Lam HT, Schweitzer SO, Petz L, et al. Are retrospective peer-review transfusion monitoring systems
effective in reducing red blood cell utilization? Arch Pathol Lab Med 1996; 120: 810-16.
38. Lam HT, Schweitzer SO, Petz L, et al. Effectiveness of a prospective physician self-audit transfusion-
monitoring system. Transfusion 1997;37:577-84.
39. Graham ID, Logan J, Harrison MB, et al. Lost in knowledge translation: Time for a map? J Contin Educ
Health Prof 2006;26:13-24.
40. Harrison MB, Graham ID, Fervers B. Adapting knowledge to a local context. In: Straus S, Tetroe J,
Graham ID, eds. Knowledge translation in health care. Oxford: Blackwell Publishing, 2009:73-82.
41. Cabana MD, Rand CS, Powe NR, et al. Why don’t physicians follow clinical practice guidelines? A
framework for improvement. JAMA 1999;282:1458-65.
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42. Legare F, O'Connor AM, Graham ID, et al. Primary health care professionals’ views on barriers and
facilitators to the implementation of the Ottawa Decision Support Framework in practice. Patient Educ Couns
2006;63:380-90.
43. Wilson K, MacDougall L, Fergusson D, et al. The effectiveness of interventions to reduce physician’s
levels of inappropriate transfusion: What can be learned from a systematic review of the literature. Transfusion
2002; 42:1224-9.
44. Tinmouth A, MacDougall L, Fergusson D, et al. Reducing the amount of blood transfused: A systematic
review of behavioral interventions to change physicians’ transfusion practices. Arch Intern Med 2005;165:845-
52.
45. Qu L, Kiss JE. Blood utilization review. In: Saxena S, ed. The transfusion committee: Putting patient
safety first. 2nd ed. Bethesda, MD: AABB Press, 2013:75-91.
46. Hannon T, Gross I. Transfusion guidelines: Development and impact on blood management. In: Saxena
S, ed. The transfusion committee: Putting patient safety first. 2nd ed. Bethesda, MD: AABB Press, 2013:121-
44.
47. Audet AM, Goodnough LT, Parvin CA. Evaluating the appropriateness of red blood cell transfusions:
The limitations of retrospective medical record reviews. Int J Qual Health Care 1996;8:41-9.
CHAPTER 28 Approaches to Blood Utilization Auditing
711
■ APPENDIX 28-1
Transfusion Order Form in Use at St. Vincent Indianapolis Hospital Since 2001
ST. VINCENT HOSPITALS AND HEAL TH SER VICES
 • USE THIS FORM FOR ALL BLOOD COMPONENT TRANSFUSION ORDERS.
• Check off at least one indication for each type of blood component order.
• The minimal effective dose of all blood components should be used; SINGLE UNIT transfusions of red
cells are often effective.
• Compliance with transfusion guidelines will be monitored by the transfusion committee.
• The blood bank phone # is 803-0421 (86th Street).
□ Blood Transfusion Consent signed
TRANSFUSION ORDER (indicate type and amount):
Request for special red cell products: Irradiated Washed CMV negative
Patient location (3E, ICU, OR, PACU, etc) Utilization reviewD
INDICATION (check all that apply):
Packed Red Cells Most recent hemoglobin g/dL or hematocrit %
One unit of packed red cells in an. adult, 3 mL/kg pediatric dose, will increase hematocrit by approximately
3 % and hemoglobin by 1 g/dL.
□ Hematocrit < 21% or hemoglobin < 7 g/dL
□ Hematocrit < 24% or hemoglobin-<' Sg/dL in a patient with coronary art ery disease and unstable angina/
myocardial infarction/ cardiogenic shock
□ Rapid blood loss with > 30- 40% of estimated blood volume (>1500- 2000 mL) not responding to
appropriate volume resuscitation, or with ongoing blood loss
□ The patient has been determined to be normovolemic and there is evidence to support the need for
increased oxygen carrying capacity as witnessed by (indicate):
NOTE: these indications will be tracked and may be peer reviewed
□ Tachycardia, hypotension not corrected by adequate volume replacement alone
□ PV02 < 25 toir, extraction ratio > 50%, V 02 < 50% of baseline - specify
□ Other- specify
□ Autologous predonated red cells: same criteria as above
Platelets Most recent platelet count / cc3
A single dose of platelets (adult: one apheresis or 6 concentrates: pediatric dose 1 unit// 0 kg) mil increase
the platel et count by 25,000- 35,000/ cc3
□ Platelet count < 10,000/ cc3 prophylactically in a patient with failure of platelet production
□ Platelet count < 20,000/ ccJ and signs of hemorrhagic diathesis (petechiae, mucosal bleeding)
□ Platelet count < 50,000/ cc3 in a patient with (indicate):
□ Active hemorrhage
□ Invasive procedure (recent, in-progress, planned)
□ Platelet dysfunction as documented by- specify
Fresh Frozen Plasma Most recent coag. studies: PT INR PTT Fibrinogen
A dose of SO- 15 mL/ kg is usually adequate to correct a coagulopathy. Patient, weight. kg
□ Abnormal coagulation studies and significant hemorrhage
□ Prophylactic use for PT/' APTT > 1.5 times the mean of the reference range
□ Emergent reversal of coumadin
Crvoprecipitate Most recent coag. studies: PT INR PTT Fibrinogen
One unit per 10 kg is usually adequate when crvoprecipitate is required.. Patient, weight kg
□ Fibrinogen < 100 mg/ dL
□ Fibrinogen < 150 mg/dL with active hemorrhage
/
Physician’s signature /printedname Pager# Date Time
Used with permission from Hannon T, Gross I. Transfusion guidelines: Development and impact on blood
management. In: Saxena S, ed. The transfusion committee: Putting patient safety first. 2nd ed. Bethesda, MD:
AABB Press, 2013:121-44.
 Chapter 29
The Collection and Processing of Hematopoietic Stem Cells
Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and David H. McKenna Jr, MD
HEMATOPOIETIC STEM CELLS (HSCs) ISjfli are primitive pluripotent cells capable of self-renewal and
differentiation into any cells of hematopoietic lineage (lymphocytes, monocytes, granulocytes, erythrocytes, and
platelets), including committed and lineagerestricted progenitor cells, unless otherwise specified, regardless of
tissue source [eg, marrow, mobilized peripheral blood, or umbilical cord blood (UCB)]Clinically, HSCs are able
to fully reconstitute the functions of marrow when transplanted into susceptible recipients. For this reason, HSC
transplantation has been increasingly utilized to treat a diverse array of hematologic and nonhematologic
diseases and conditions.
In vivo, HSCs are concentrated in the marrow, where mesenchymal elements—such as osteogenic
progenitor cells, osteoblasts, adipocytes, mesenchymal stem/stromal cells, and endothelial cells—interact with
hemato
poietic precursors to generate a niche that supports and regulates hematopoiesis.2
Although the cell-surface antigen CD34 is not specific to HSCs, CD34 is used to identify and quantitate
HSCs by flow cytometry in cellular products intended for use in transplantation. Historically, CD34 quantitation
by flow cytometry suffered from significant interinstrument and interprotocol variability, a limitation that has
been addressed by more rigorous quality control (QC) procedures and assay standardization.3,4
CLINICAL UTILITY
 Myriad indications exist for HSC transplantation that range from nonneoplastic immune disorders to
malignancies. An abbreviated list is presented in Table 29-1. In general, indications vary with patient age
because immunodeficiencies and inborn errors of metabolism
Scott A. Koepsell, MD, PhD, Assistant Professor, Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska; Eapen K. Jacob, MD, Assistant Professor, Department of
Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; and David H. McKenna Jr, MD,
Associate Professor, Department of Laboratory Medicine and Pathology, University of Minnesota Medical
School, Minneapolis, Minnesota
S. Koepsell and E. Jacob have disclosed no conflicts of interest. D. McKenna has disclosed a financial
relationship with Novartis, Inc.

713

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TABLE 29-1. Diseases Treated with Autologous or Allogeneic Transplantation
Hematologic Cancers
Leukemia
Lymphoma
Myeloma
Marrow Failure States/Clonal Disorders of Marrow
Aplastic anemia
Fanconi anemia
Pure red cell aplasia
Amegakaryocytosis
Paroxysmal nocturnal hemoglobinuria
Myelofibrosis
Myelodysplasia
Inborn Errors of Metabolism/Congenital Immunodeficiency
Mucopolysaccharidoses
Leukodystrophies
Osteopetrosis
Severe combined immunodeficiency syndrome Wiskott-Aldrich syndrome
Pediatric Cancers
Wilms tumor Neuroblastoma Ewing sarcoma Medulloblastoma Rhabdomyosarcoma Hemoglobinopathies
Thalassemia Sickle cell disease Autoimmune Diseases Solid Tumors
are more common in a pediatric population, whereas the number of patients with a clonal disorder in their
marrow or a hematologic malignancy is greater in adults. Ultimately, the decision to perform HSC
transplantation requires a complex integration of many variables. These variables include patient goals,
prognosis, disease progression, previous therapy, age, availability of a suitable HSC source (ie, marrow,
mobilized peripheral blood, or UCB), and type of transplant (ie, autologous vs allogeneic, and myeloablative vs
nonmyeloablative).
Autologous Transplantation
In general, autologous HSC transplantation is used for hematopoietic rescue after high-dose antineoplastic
therapy. The antitumor effect of the transplant comes solely from the chemotherapy and radiotherapy used
during the conditioning phase of transplantation. For older patients who are not traditional candidates for
autologous transplantation or patients with other significant morbidities, reduced-intensity induction
chemotherapy can broaden the clinical utility of HSC transplantation.
Donor requirements for autologous transplants are based on the donor’s disease state. The patient must be
healthy enough to undergo mobilization (as described later in this chapter) and procurement of HSCs either by
peripheral blood apheresis collection or marrow aspiration. Significant levels of prior chemotherapy, radiation,
or ongoing marrow disease involvement may make the mobilization and collection of HSCs unfeasible due to
the reduced quality or number of HSCs.
 Eligibility requirements are not mandated by the Food and Drug Administration (FDA) for autologous HSC
transplantation [Title 21, Code of Federal Regulations (CFR) Part 1271.90], so the use of screening
questionnaires to identify relevant communicable disease is not required. However, a general health assessment
is needed per AABB Standards for Cellular Therapy Product Services (CT Standards).1 AABB CT Standards
also requires laboratory testing for human immunodeficiency virus (HIV) 1/2; hepatitis B virus; hepatitis C
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virus; syphilis; human T-cell lymphotropic virus, Types I and Type II (HTLV-I/II); and cytomegalovirus
(CMV) because the autologous products are cryopreserved and stored with other products, so the presence of
these viruses would pose a contamination risk.
Allogeneic Transplantation
Indications for allogeneic HSC transplantation vary. However, in general, allogeneic transplantation is used
to treat malignant conditions only when both the induction antineoplastic therapy and the transplanted cells, due
to the graft-vs-neoplasm (GVN) effect, are therapeutic. For patients with an inborn error of metabolism,
congenital immunodeficiency, or other diseases and conditions in which germline mutations are present in the
patient’s cells, allogeneic HSC transplants offer therapeutic benefit by helping to replace the deficient cellular
machinery.
In the allogeneic setting, screening and infectious disease testing are mandated in the United States to
determine whether the transplantation of mobilized peripheral blood or UCB poses a risk of transmission of a
relevant communicable disease to the recipient [Title 21 CFR 1271.3 (r)]. This screening and testing includes
the administration of a screening questionnaire, a physical examination, a review of the relevant medical
records, and applicable testing [Title 21, CFR Parts 1271.3(s) and 21 CFR 1271 Subpart C]. Although marrow
products are administered under Sections 375 and 379 of the Public Health Services Act, marrow screening and
testing are very similar to mobilized peripheral blood and UCB screening and testing because the administration
of all of these products is governed by the standards of various accrediting bodies, such as the AABB, the
Foundation for the Accreditation of Cellular Therapy (FACT),4 and the National Marrow Donor Program
(NMDP) ,5
For UCB transplantation, the screening and testing process is performed on the mother and her samples (see
Chapter 30). Relevant communicable diseases that can be transmitted by transplanted HSCs to the recipient or
to the people who handle the components and
Hematopoietic Stem Cells
for which there is an FDA licensed screening test [Title 21, CFR Part 1271.3(r)[ include HIV hepatitis B and
C, human transmissible spongiform encephalopathy, Treponema pallidum, HTLV-I/II, and CMV Donor
questionnaires have been developed to assist with screening6 and medical record review for these diseases
using FDA guidance documents.7
In the United States, infectious disease testing for HSC donors must be performed in a Clinical Laboratory
Improvement Act certified laboratory as mandated by the FDA. If screening or testing detects a risk of a
relevant communicable disease, the potential HSC donor is considered ineligible. All parties (the donor, the
recipient, and their physicians) are informed of the donor’s eligibility status, and a risk-benefit analysis is
performed to determine whether the donor’s HSCs should be used. If a decision is made to proceed with a
transplant of the ineligible donor’s HSCs, the urgent medical need, as defined by the FDA [Title 21, CFR Part
1271.3(u)], is documented. Depending on the institution’s accrediting body and the circumstances, such as
when the HSCs are from a related first- or second-degree ineligible donor, the requirement for documentation of
an urgent medical need may vary. Finally, in addition to screening and testing for infectious diseases, the
donor’s medical evaluation is used for determining whether the donor’s health is adequate to allow that donor to
undergo the HSC mobilization and procurement process (describedbelow).
Histocompatibility
In addition to infectious diseases, donor characteristics that may affect transplant outcomes include
histocompatibility with the recipient, gender, age, parity, and ABO compatibility. Of these characteristics,
histocompatibility is the most important. In general, if a healthy HLA-matched related donor is available, that
donor is selected instead of an HLAmatched unrelated donor. However, recent data have shown that in patients
with acute myeloid leukemia or myelodysplastic syndrome, an HSC transplant from an HLA
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 matched, unrelated donor may be as effective as a transplant from a sibling.8,9
Major histocompatibility antigens were first studied in depth in various animal models for skin
transplantation.10(pp97'm) In humans, these are HLA antigens and are categorized into Class I (HLA-A, -B,
and -C) and Class II (HLA-DR, -DQ, and -DP). These highly polymorphic molecules are essential in
determining graft survival as well as the likelihood that the recipient will develop graft-vs-host disease
(GVHD). As molecular techniques have supplanted serologic methods, the resolution has improved and the
number of antigens that can be compared has increased. Matching at the antigen level has been replaced by
allelelevel matching for the final selection of peripheral blood and marrow HSC components for a given
recipient.
HLA matching in HSC transplantation has been reviewed in detail elsewhere and is summarized here only
briefly.11 HLA matching has an important impact on outcomes, especially in low-risk patients. Allogeneic
transplantation using either mobilized peripheral blood or marrow HSCs with high-resolution (allele-level)
mismatching at HLA-A, -B, -C, and -DRpi is associated with a 5% to 10% decrease in survival with each
mismatch.12 The results are similar, although the evidence is a bit less clear, for HSC grafts with mismatches in
Class II HLA-DQ and -DP. For mobilized peripheral blood HSC grafts, allele-level mismatching is probably as
detrimental to survival as antigen-level mismatching, although the data come from smaller studies. Many
centers now match at high resolution for 8 to 10 loci (HLA-A, -B, -C, -DR, and -DQ). High-resolution 8/8 and
10/10 matched transplants of HSCs from unrelated donors are at least in part responsible for the improvement in
outcomes of matched unrelated transplants compared to matched related donors.12
Not surprisingly, HSC transplants in recipients who have antibodies against donor HLA [known as donor-
specific antibodies (DSAs)] have adverse outcomes.13 The level at which DSAs have a significant impact is
less clear, as is the appropriate course of action when DSAs are detected. Despite the difficulty
of predicting graft failure based on the presence of DSAs, screening HSC transplant candidates for DSAs
may become more routine as more data emerge on this topic.14
UCB has several unique characteristics with regard to HLA matching. The level of HLA matching required
for UCB is less stringent than that required for marrow and mobilized peripheral blood HSCs. Data on UCB
indicate that matching at 4 of 6 loci is sufficient for HLA-A and -B at the antigen level and for HLADRBl at the
allele level, provided that a sufficient cell dose is achieved.15 As the number of mismatched alleles increases
(from one to two), a higher total nucleated cell (TNC) dose is needed to overcome their deleterious effect,
including the use of combined double-cord transplants.16 In addition, early evidence indicates that
noninherited, maternal HLA may be permissive when considering donor-recipient mismatches.17 Unit-to-unit
matching of 4 of 6 loci in the setting of double-cord blood transplantation is also performed at various
institutions; however, firm evidence to support this practice is not available at this time.
Other Donor Characteristics
For marrow and mobilized peripheral blood HSC donors, other factors that may have a positive effect on
transplant outcomes include male gender, younger age, nulliparity, ABO and CMV status matching, and greater
size of the donor relative to the recipient. Other than HLA, only donor age appears to be associated with
survival.11 ABO incompatibility has been reviewed extensively. ABH antigens found on red cells, platelets, and
neutrophils would suggest that ABO incompatibility would affect outcomes. However, inconsistent results on
outcomes—such as survival, nonrelapse mortality, GVHD, and graft failure—of both major and minor
incompatible transplants have been observed.18 The risk of delayed red cell engraftment, pure red cell aplasia,
increased transfusion requirements, and both immediate and delayed hemolysis is increased in major ABO-
incompatible allogeneic HSC transplantation.
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Other characteristics that are specific to UCB HSC donation and may affect outcomes include issues related
to maternal history, unit collection, processing, and unit storage.19 Characteristics of the UCB unit have been
utilized in a scoring system, the “Cord Blood Apgar,” to determine the utility of the unit after consideration of
the TNC count, CD34 positivity, colony-forming-unit count, mononuclear cell content, and volume.20
DETERMINATION OF GRAFT SOURCE
The choice of the source of HSCs for allogeneic transplantation is determined by several variables,
including the availability of an adequately matched donor. As described above, marrow and mobilized
peripheral blood products in general need a higher level of HLA matching than UCB units. For this reason, a
UCB transplant may be the best alternative when only 1 and 2 allele-mismatched donor units are available. In
addition, for some centers, the relatively rapid availability of UCB units compared to marrow and peripheral
blood products plays a substantial role in graft choice in patients with an immediate need for transplantation. A
typical unrelated marrow or peripheral blood donor search may take months, whereas a search for UCB takes
several days to a few weeks.21
GVHD and GVN Effect
GVHD and GVN effect, although linked in terms of their cause, have opposite outcomes. In GVHD, donor
lymphocytes attack host-recipient tissues in the skin, lung, liver, gastrointestinal tract, and other organs. GVHD
may be categorized as acute or chronic, and it occurs in more than 40% of individuals who receive an allogeneic
HSC transplant. GVHD is associated with significant morbidity and mortality. Not surprisingly, the risk of
GVHD is related to the graft source. Consequently, GVHD risk increases as the lymphocyte content of the HSC
product increases. Various techniques have been used to decrease the risk of GVHD, in
Hematopoietic Stem Cells
eluding T-cell depletion, use of antithymocyte globulin, and pharmacologic measures.22
The GVN effect is also thought to be driven by donor lymphocytes. In recent years, mobilized peripheral
blood has greatly outpaced marrow as an HSC source due to its easier collection and the belief that GVN effect
may be improved. The first randomized controlled trial comparing marrow and mobilized peripheral blood HSC
grafts in unrelated donor myeloablative transplantations found that chronic GVHD, but not acute GVHD, risk
was higher in patients who received a peripheral blood HSC transplant.23
Kinetics
The kinetics of engraftment are affected by many variables, such as degree of HLA matching, dose of
HSCs, and use of granulocyte colony-stimulating factor (G-CSF). In addition, the characteristics of the stem
cell source may play a role. For instance, stem cells in UCB appear to have a greater regenerative capacity than
those in marrow and peripheral blood.24 In general, the rate of engraftment is predicted by the number of
progenitor cells in each graft. This is greatest in mobilized peripheral blood, followed by marrow, UCB, and
other HSC sources.
In a meta-analysis of studies that compared related transplantation with donor-mobilized peripheral blood
and marrow, the median time to neutrophil engraftment was 14 vs
21 days and to platelet engraftment was 14 vs
22 days, respectively.25 These findings have been corroborated by a more recent metaanalysis of studies of
allogeneic related transplantation in which mobilized peripheral blood vs marrow HSC engraftment was 15 vs
21 days for neutrophils and 13 vs 21 days for platelets, respectively.26 The more rapid engraftment of
peripheral blood compared to marrow HSCs was also seen in a study of unrelated allogeneic transplantation in
which median neutrophil engraftment occurred at 15 and 19 days, respectively, and median platelet engraftment
occurred at 20 and 27 days, respectively.27 These findings were confirmed in the randomized controlled trial of
Anasetti
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AABB TECHNICAL MANUAL
and colleagues23 in which the relative differences in time to engraftment was 5 days shorter for neutrophils
and 7 days shorter for platelets. A comparison of adult unrelated marrow to UCB HSC grafts showed that
engraftment occurred earlier with both neutrophils (median of 18 days for matched marrow, 20 for mismatched
marrow, and 27 for mismatched UCB) and platelets (median of 29 days for matched marrow and mismatched
marrow, and 60 days for mismatched UCB).28 In the setting of reduced-intensity UCB transplantation, median
neutrophil engraftment times approaching those of mobilized peripheral blood and marrow HSC transplants
have been observed.24
Survival
The most important outcome measure for any transplant procedure is patient survival. For recipients of
transplants from matched related donors, mobilized peripheral blood may offer an advantage in overall and
disease-free survival over marrow HSC grafts, at least in recipients with late-stage hematologic malignancies.25
In recipients of myeloablative transplants from unrelated donors for hematologic malignancies, a recent
randomized controlled trial indicated that marrow and mobilized peripheral blood sources have equivalent
effects on survival, with marrow grafts associated with less chronic GVHD but more graft fail
23
ure.
 Based on these findings, the rapid increase in the use of mobilized peripheral blood relative to marrow in
recent years may change. This may not be true, however, in nonmyeloablative patients or transplant recipients at
high risk of infection or graft failure. For pediatric transplant recipients, marrow may actually be preferred over
mobilized peripheral blood, although reports differ.29,30 In addition, when mismatched marrow and
mismatched UCB were compared, there was no difference in the rate of overall mortality in adults with
leukemia; however, recipients of matched marrow HSC grafts did have a lower overall mortality rate.28
Transplant physicians face a complex choice when determining which donor and stem cell source is best for
their patients based on numerous factors, including the patient’s disease, disease stage, age, and comorbidities.
As more data are collected, the choice of HSC graft may evolve.
COLLECTION/SOURCES OF HSCS
Regardless of the source of HSCs, standards of both FACT and AABB require all institutions that collect
HSCs to have a procedure in place to obtain informed consent from the donor or the donor’s representative, as
dictated by local laws.1(ppl6'17,20'21)4 The informed consent process should include providing information to
the donor regarding the risks/benefits of the procedure, the tests performed on the donor that are designed to
protect the recipient, alternative collection methods, and protection of their health information. In addition, the
donor should be given the opportunity to ask questions as well as to refuse donation. The risks that are specific
to each collection procedure are discussed below.
Another requirement for all facilities that collect HSCs is to provide donor access to medical care based on
the risks and clinical situation associated with each type of donation. Specifically, procedures should be in place
to provide medical or emergency care to donors who experience adverse effects. Clearance from the donor’s
physician for HSC collection should be documented.
Marrow HSC Collection
In addition to undergoing relevant donor screening, infectious disease testing, and HLA compatibility
testing, marrow donors must also be physically suitable for donation. Marrow harvest is an invasive procedure
performed under sterile conditions in the operating room under anesthesia. Therefore, the donor must be able to
tolerate the type of anesthesia required to successfully perform the harvest. Another consideration is the donor’s
medical history. Autologous donors and some
719
CHAPTER 29
allogeneic donors may have had previous radiation therapy to the pelvis, which may limit the amount of
marrow available for harvest in the posterior iliac crest. Similarly, previous chemotherapy may limit the number
of nucleated cells that can be aspirated from the marrow space. For autologous donors, a significant tumor
burden in the marrow space is a contraindication for collection of HSCs by marrow harvest because the graft
would be contaminated by tumor cells.
Physically, the donor must be able to tolerate the volume loss associated with marrow harvest, which means
that young or small donors may not be suitable. The Be The Match Registry limits the volume collected from a
marrow donor to 20 mL/kg.5 Typically, the volume of the harvest requested is dictated by the recipient’s
weight, with a minimum of 2.0 to 3.0 x 108 nucleated cells/kg needed to facilitate efficient engraftment. Thus,
during harvest, checking the TNC count midway through the procedure can help with estimating the total
volume needed. Alternatively, CD34 quantitation midway through the procedure or on the final product for QC
may also be performed, depending on the institution’s policies.
The marrow harvest technique varies considerably depending on institutional practice. In general, an 11- to
14-gauge needle on a syringe flushed with anticoagulant is inserted into the posterior iliac crest, and
approximately 5 mL of marrow is aspirated. The needle and syringe are then rotated to a different vector, and
the aspiration is repeated. Vigorous aspiration is avoided to prevent significant peripheral blood contamination
of the product. The aspirated marrow is collected into a large collection bag containing anticoagulant and media
and/or an infusible-grade electrolyte solution. The process is repeated utilizing different bone sites until the
collection volume target, based on TNC count or donor volume limit, is reached.
Serious complications of marrow harvest are rare. However, minor complications, such as pain at the site of
harvest, fatigue, insomnia, nausea, dizziness, and anorexia, occur frequently but resolve in most donors by 1
month after the procedure.31 Marrow donors often
Hematopoietic Stem Cells
have significant decreases in their hemoglobin concentrations after the procedure. As a result, almost all
marrow donors donate autologous Red Blood Cells (RBCs) before the procedure, and 76% of donors receive at
least 1 autologous RBC unit during or shortly after marrow harvest.31 If the donor requires an allogeneic RBC
or platelet transfusion before or during the procedure, the units should be irradiated to prevent viable leukocytes
from these blood products from contaminating the graft. Marrow donors should be made aware that they might
need to undergo a transfusion as part of the informed consent process.
Peripheral Blood HSC Collection
Pharmacologic methods for mobilizing HSCs from the marrow into the peripheral circulation combined
with apheresis technology have made peripheral HSC collection the most common procedure for HSC
donation.32 Because peripheral collection of HSCs requires only vascular access, most apheresis procedures
used to collect HSCs are performed on an outpatient basis, with minimal side effects. However, donors with
poor vascular access or who may need a number of apheresis procedures for the collection of a sufficient
number of HSCs may require the placement of a central line, which imposes an additional risk. AABB CT
Standards requires that the placement of any central line be confirmed before HSC collection is
initiated.1*11481
HSCs can be mobilized into the peripheral circulation using a variety of chemotherapeutic agents,
hematopoietic growth factors, or receptor antagonists. For most healthy allogeneic HSC donors, sufficient
numbers of HSCs can be mobilized with the administration of hematopoietic growth factor, often G-CSF, alone.
G-CSF is administered once per day at a dose of 5 to 20 pg/kg, and doses are often rounded to the nearest vial
size.33 Total white cell count and CD34 percentage can be monitored to determine the optimal collection time,
which is usually 3 to 4 days after initiation of G-CSF treatment. The side effects of G-CSF, which are common
and mild, include bone pain, myalgia, headache, insomnia, flu-like
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AABB TECHNICAL MANUAL
symptoms, sweating, anorexia, fever, chills, and nausea.33 Potentially serious complications, such as splenic
rupture, are rare. Other growth factor preparations are available, including a pegylated form of G-CSF that
offers the advantage of a one-time dose in a majority of donors.
For some autologous donors and rare allogeneic donors, mobilization of HSCs can be challenging,
potentially requiring additional pharmacotherapy to mobilize an adequate number of cells and facilitate efficient
engraftment. The minimum number of cells needed for transplantation is commonly cited as 2 x 106 CD34+
cells/kg, although 5 x 106 CD34+ cells/kg is more desirable.34 In autologous donors, a chemotherapy drug,
such as cyclophosphamide, can be added to the G-CSF regimen. Although the number of HSCs collected can be
increased by using a combination of chemotherapy and G-CSF, complications such as cytopenias and additional
apheresis may outweigh the potential benefits of this strategy.35 If the benefits of adding chemotherapy to G-
CSF to stimulate peripheral HSC mobilization outweigh the risks, this mobilization strategy may be utilized
successfully in patients with significant tumor burden.
Patients who are poor mobilizers may benefit from the addition of plerixafor, a chemokine (C-X-C motif)
receptor 4 antagonist, in combination with G-CSF. Various clinical studies have been published demonstrating
that plerixafor in combination with G-CSF can increase HSC collection yields. A number of clinical scenarios
where plerixafor may be utilized have been published, and patients with multiple myeloma or lymphoma who
have difficulty mobilizing HSCs may benefit from plerixafor therapy to collect a sufficient quantity of HSCs
during donation.34
Peripheral collection of the HSCs is performed using an apheresis device according to the manufacturer’s
instructions. For most allogeneic donors, sufficient numbers of HSCs can be obtained with one to two collection
procedures. Up to 20% of donors experience minor apheresis/collection-related adverse events, such as citrate
toxicity, nausea, fatigue, chills, hypertension, hypotension, allergic reactions,
or syncope.32 Autologous donors experience similar collection-related side effects, which can be
problematic in donors who require multiple apheresis procedures due to poor mobilization. Depending on the
donor scenario, large-volume apheresis techniques may be used to limit the number of total procedures.36
AABB CT Standards requires a complete blood count to be performed within 24 hours before the procedure
begins for all mobilized donors, and this is especially important for autologous donors because the apheresis
procedure can deplete platelets.1(p47)
UCB HSC Collection
UCB collection is discussed in detail in Chapter 30 and is not addressed here.
PROCESSING OF HSCS
 Processing methods for HSCs can be divided into routine methods, which are usually centrifuge based, and
specialized methods that involve a variety of technologies. Routine methods include volume (plasma) reduction,
red cell reduction, huffy coat preparation, thawing/washing, and filtration. Volume reduction is performed in the
settings of minor ABO-mismatched allograft (marrow or peripheral blood) transplantation to reduce the amount
of incompatible plasma and prevent fluid balance/overload issues in small patients and/or patients with renal
disease or cardiac failure. Volume reduction may also be performed before cryopreservation (eg, for UCB
banking where storage space is limited or during cell concentration optimization).
Classically, red cell reduction employs sedimenting agents (eg, hydroxyethyl starch) to reduce red cell
content. These agents are used to prevent hemolytic transfusion reactions when major ABO incompatible
marrow HSC allografts and allografts with other clinically relevant red cell antigens (eg, Kell, Kidd) are
transplanted. Red cell reduction before freezing also limits the amount of lysed red cell fragments and free
hemoglobin on infusion and may be particularly important for patients with renal failure. Red cell reduction
may also
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be useful when storage space is limited. Because apheresis instruments collect mononuclear cells efficiently,
with very little red cell content, peripheral blood-derived HSCs generally do not require red cell depletion.
Buffy coat concentration of marrow involves centrifugation and harvesting of the white cell fraction and can
be performed with an apheresis or cell-washing device. Manual centrifugation may be used when product
volume is too low for apheresis or cell washing devices. Buffy coat preparation is usually used to reduce the
unit volume for cryopreservation or as a method of red cell reduction before further manipulation (eg,
immunomagnetic selection).
The thawing procedure for all HSCs, regardless of source, is similar. Although this procedure is
straightforward, it should be done carefully because frozen plastic containers are prone to break for a variety of
reasons.37 The product should be handled with care while it is verified to determine the product’s identity and
ensure the integrity of the bag. The product is then placed into a clean or sterile plastic bag and submerged in a
37 C waterbath. If the freezing bag breaks, the product may be recovered using this approach, but a risk-benefit
discussion with the patient’s physician should take place to determine the course of the patient’s care.
Gentle kneading allows the thaw procedure to proceed relatively quickly while preventing recrystallization
and consequent cell damage/death. A hemostat should be used to prevent loss of the product if the bag breaks,
and the contents should be aseptically diverted into a transfer bag. A sample should also be sent for culture.
Washing the HSCs removes lysed red cells, hemoglobin, and cryoprotectant [dimethyl sulfoxide (DMSO)].
Although UCB is typically red-cell depleted before cryopreservation, it remains the primary HSC product that
is routinely washed. This practice is changing, however, and Chapter 30 discusses alternative approaches to
UCB preparation for infusion. Historically, most institutions based their UCB processing methodology,
including the thawing/washing procedure, on the proce
Hematopoietic Stem Cells
dure originally described by Pablo Rubinstein of the New York Placental Blood Program.38 Briefly, the
thawing process involves slow, sequential addition of a wash solution (eg, 10% dextran followed by 5%
albumin), transfer into an appropriately sized bag for centrifugation, and resuspension of cell pellet(s) before
delivery to the patient care unit for infusion. Many laboratories perform two centrifugation steps, removing the
supernatant from the first spin and centrifuging that portion a second time before combining the two cell pellets.
This approach optimizes cell recovery.39
Marrow harvest typically involves sequential filtration in the operating room or the laboratory to remove
bone spicules, aggregates, and debris. Opinions regarding the use of standard blood filters upon infusion of
HSCs vary, however. Whether to use a standard blood filter (>170 microns) is up to the individual cell
processing laboratory and/or transplant center. If an institution opts to use a standard blood filter, the laboratory
should validate its filtration process.
SPECIALIZED CELLPROCESSING METHODS
Specialized cell-processing methods are used to optimize product purity and potency beyond levels obtained
through routine methods. Several of these methods, which require unique reagents and instrumentation, are
discussed in other chapters. For this reason, the descriptions of these methods in this chapter are brief and focus
on their application to HSCs.
Elutriation
 Counter-flow centrifugal elutriation is a specialized method that separates cell populations based on two
physical characteristics— size and density (sedimentation coefficient). A centrifuge is used to separate the cell
populations of a cell product based on density alone. However, if fluid/media is passed through the chamber
housing the cells in the direction that is opposite (counterflow) to the centrifugal force, adjustment of flow rate
and/or centrifu
722
AABB TECHNICAL MANUAL
gation speed allows the separation of cell populations based on size as well. Through this process, cells with
“signature” size/density profiles can be separated from the rest of the cells. Historically, this method was used
for T-cell depletion of HSC grafts. In more recent years, the method has been used to enrich monocytes for
preparation of dendritic-cell vaccines.
Cell-Selection Systems
Immunomagnetic cell selection systems incorporating monoclonal-antibody-based technologies to target
cell-surface antigens (eg, CliniMACS system, Miltenyi Biotec Bergisch, Gladbach, Germany) have become a
widely used method of cell depletion/enrichment at many institutions. These methods involve the isolation of
the cell type of interest by either positive selection (target cells retained) or negative selection (target cells
depleted). Monoclonal antibodies (eg, anti-CD34 for HSC isolation) are coupled to 50-nm ferromagnetic
particles. Magnetically labeled target cells are retained in the process as the cell suspension passes through a
column in which a magnetic field is generated. Unlabeled cells pass through the column and are collected in a
negative-fraction bag. Target cells are then released from the column by removal of the magnetic field from the
column, which allows passage of the cells into a separate collection bag.
Cell Expansion
Because the dose of nucleated, CD34+, and colony-forming cells has a positive correlation with patient
outcomes, much effort has been focused on ex-vivo expansion of HSCs and progenitors. It is thought that
successful expansion enhances hematopoietic engraftment while reducing transfusion dependence, risk of
infection, and duration of hospitalization. In recent years, UCB has become the focus of expansion trials due
both to the higher proliferative and self-renewal capacity of HSCs from UCB and the limit on cell quantity in a
UCB collection. Most expansion cultures contain a cytokine cocktail that includes stem cell factor,
FLT-3 ligand, and thrombopoietin along with novel and/or proprietary ingredients. The media, culture
vessels, and culture duration used vary from protocol to protocol.
CRYO PRESERVATION
Methods for cryopreservation must be used because HSCs may need to be stored for weeks to years prior to
being transplanted.40 Most cell-processing laboratories use the cryoprotectant DMSO, usually at 10% final
concentration, and a source of plasma protein for cryopreservation of HSCs. DMSO is a colligative
cryoprotectant; it diffuses rapidly into the cell, reducing the osmotic stress on the cell membrane. DMSO
prevents dehydration injury by moderating the nonpenetrating extracellular solutes that form during ice
formation. It also slows extracellular ice crystal formation. Some laboratories add hydroxyethyl starch (HES),
which allows the use of a decreased concentration of DMSO (eg, 5% DMSO and 6% HES). HES is a
nonpenetrating (extracellular), macromolecular cryoprotectant. This highmolecular-weight polymer likely
protects the cell by forming a glassy shell, or membrane, around the cell, retarding the movement of water out
of the cell and into the extracellular ice crystals.
The HSCs may be frozen at a controlled rate or a noncontrolled rate, in which the HSC product is simply
transferred into a freezer bag and placed in a -80 C mechanical freezer. Controlled-rate freezing is favored in the
clinical laboratory setting, and it utilizes computer programming to incrementally decrease HSC product
temperature in a closely monitored fashion. The controlled rate freezing protocols used vary from institution to
institution.
In general, the HSC product is placed in the chamber and initially cooled at a rate of 1 C/minute. When the
temperature decreases to approximately -14 C to -24 C, the HSC product begins to transition from a liquid to a
solid. At this time, the freezer undergoes a period of supercooling to counteract the latent heat of fusion that is
released by the phase change. Following solidification of the HSC product, cooling proceeds at the rate of 1 Cl
723
CHAPTER 29
minute until the product has reached -60 C. At this point, the product is cooled at a controlled rate
determined by the institution until it reaches -100 C. Following both controlled-rate and noncontrolled-rate
freezing, the HSC product is transferred to a storage freezer. An increasing number of laboratories store HSCs
in the vapor phase of liquid nitrogen (LN,) at temperatures below -150 C; however, some laboratories do store
cells in the liquid phase ofLN2.
QC
QC testing in the clinical cell therapy laboratory serves two purposes: determine the suitability and safety of
the cellular product for the patient and monitor overall laboratory practices. QC testing is aimed at
characterizing the safety, purity, identity, potency, and stability of the cellular product. The extent of QC testing
is primarily dependent on the complexity of manufacturing the product and the nature of the clinical experience
(ie, standard practice vs a clinical trial).
Common QC tests for HSCs include cell count and differential, viability, CD34+ cell enumeration, sterility
testing, and colonyforming unit assays. Cell count and differential are performed on a hematology analyzer. Cell
viability may be determined using a variety of methods, including trypan blue, acridine orange, and 7-
aminoactinomycin D (flow cytometry). Microscope-based methods utilizing vital dyes or fluorescent stains may
be particularly useful for a quick assessment of overall nucleated cell viability. Flow cytometry-based analysis
can be useful when cell populationspecific viability needs to be determined. Most CD34+ cell-enumeration
strategies are based on guidelines of the International Society for Cellular Therapy.41 Sterility testing at most
institutions is performed using an automated microbial detection system.
The clonogenic assay (most commonly used to count colony-forming units) is the only truly functional
assessment of HSCs routinely performed in clinical laboratories. The results of this assay correlate with the
speed and likelihood of engraftment of HSCs from
Hematopoietic Stem Cells
marrow, peripheral blood, or UCB.42'45 However, the similar correlation between results and engraftment
speed and likelihood as well as the more rapid availability of results have made CD34+ cell enumeration the
accepted, albeit surrogate, QC test for graft potency. The clonogenic assay is still useful, despite difficulties in
standardization, especially for HSCs that are stored for a long time (eg, UCB banking).46
SHIPPING AND TRANSPORT OF HSC CELLULAR PRODUCTS
Shipping and transport of cellular therapy products allow the geographic separation of donors and
recipients. The two terms are given specific definitions by accreditation organizations.1(pp35'36),4(ppl3'14)
With shipping, the product leaves the control of trained personnel in the facilities involved in the distribution
and receipt of the product.4lppl3'14) Conversely, with transport the transfer of a product between or within
facilities occurs under the control of trained personnel.
Three issues are particularly important to ensure the safe delivery of HSC products: product integrity, safety
of the personnel involved in the transport, and compliance with applicable regulations and standards. The
necessary conditions for shipping and transport vary depending on the type of product, its state (fresh or
cryopreserved), and the distance involved. These issues are reviewed in depth elsewhere.47
During shipment, the product must be placed in a secondary container that can prevent leakage and is
validated for the temperature range required for the product and the anticipated duration of shipping. This
temperature range may be defined in standards (eg, <-150 C for cryopreserved cord blood) or by an institutional
protocol.4 For fresh products, several studies have shown that shipment at 2 C to 8 C can maintain CD34+ cell
viability more effectively than shipment at room temperature particularly for shipping times of between 24 and
72 hours.48'50 This effect appears to be more pronounced for peripheral
724
AABB TECHNICAL MANUAL
blood products than marrow products and for products with higher concentrations of HSCs.
Cryopreserved products are shipped in dry shippers charged with LN2. These containers maintain
temperatures below -150 C for up to 2 weeks and their temperatures are continuously monitored.1(p35) If the
products are shipped to a noncontiguous facility or on public roads, an appropriately labeled outer container
must be used to further protect the product during transport and shipping.4 Depending on the mode of transport
(eg, air or ground), additional federal government requirements must be met. If products are shipped abroad,
international requirements must be met. If high-dose conditioning therapy has been given to the recipient,
shipment using a qualified courier is required.4 The product should not be x-rayed; instead, it should be
manually inspected, if necessary. Appropriate records must accompany the product.
The receiving institution must have protocols in place for receipt and inspection of the product for
acceptability for transplant.1(pp36'37)’4
 PATIENT CARE
Once an HSC product is ready for infusion, it should be delivered to the patient care unit without delay.
After the physician approves the product for infusion and proper identification procedures are carried out, the
product is infused by intravenous (IV) drip directly into a central line, typically without a needle or pump. Some
institutions use a standard blood filter at the bedside. To maximize cell dose, the product bag and IV tubing may
be flushed with sterile saline after the bag empties. Sterile saline also may be added directly to the bag if the
flow rate becomes too slow.
HSC products are usually infused as quickly as the patient can tolerate, particularly for thawed cells that
have not been washed or diluted, to lessen the DMSO toxicity to the cells. Although Rowley and Anderson51
concluded that DMSO is not toxic to HSCs at clinically relevant concentrations (ie, 5% or 10%) at either 4 C or
37 C for up to 1 hour of incubation, they also noted that the addition of 1%
DMSO to culture dishes suppressed colonyforming units. However, these studies were performed on fresh
cells, and studies of the effects of DMSO on HSCs that have been previously cryopreserved are limited. The
possible functional defect due to DMSO coupled with the not-infrequent need to hold clinical products (because
of patient-care-related issues) raise concern about the possibility of cell injury from the infusion of thawed,
unwashed HSC products.
The patient’s vital signs should be checked before infusion, immediately after infusion, and 1 hour after
infusion, at a minimum. All of the monitoring information should be captured on the accompanying infusion
form. When completed, this form should be returned to the laboratory. If an adverse reaction occurs, more
frequent monitoring is required.
Reactions associated with HSC infusion may be very similar to those that occasionally occur with blood
transfusion (ie, allergic, hemolytic, and febrile reactions and those due to microbial contamination). However,
some reactions may be less likely depending on the cell-processing technique used (eg, red cell reduction,
plasma reduction, postthaw washing, or dilution). Reactions often attributed to DMSO (eg, nausea, vomiting,
cough, and headache) are less common with infusions of smaller volumes and/or washed/diluted products.52,53
HSC products are usually well tolerated, however. Because the possibility of a severe reaction does exist,
aggressive IV hydration (eg, 2 to 6 hours before and 6 hours after infusion, with the use of diuretics as needed)
and use of prophylactic antiemetics, antipyretics, and antihistamines may be warranted.
The transplant physician and the medical director of the cell therapy laboratory should be notified
immediately of an unexpected or moderate-to-severe reaction. An investigation should begin and include
laboratory testing (eg, direct antiglobulin test, antibody titer, Gram’s stain, or culture) that targets the signs/
symptoms of the patient.
Data on clinical outcomes (eg, engraftment) and adverse events should be reviewed regularly and discussed
with the institutional
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CHAPTER 29
quality management group. Quarterly reviews are reasonable for engraftment analysis. The medical
director’s review should include assessment of the HSC product’s quality indicators (eg, dose, viability, and
colony-forming units), associated deviations, and presence of infusion reactions, with a focus on potential
laboratory-related component affecting any less-than-optimal outcome.
OTHER REGULATORY CONSIDERATIONS
The relevant regulations with regard to HSC collection are discussed earlier in this chapter. In general,
HSCs that are minimally manipulated and collected for transplantation in an autologous fashion or transplanted
to a firstor second-degree relative are regulated solely under Section 361 of the Public Health Service Act and
are subject to the jurisdiction of the Center for Biologies Evaluation and Research of the FDA. If HSCs are
manufactured in a way that alters their relevant biological characteristics (eg, if they are genetically modified,
expanded ex vivo, or combined with a drug) or the cells are intended for transplantation into a non-first- or
second-degree relative, then the HSC product is subject to regulation under Tide 21, CFR Part 1271, as a drug
and/or biologic
Hematopoietic Stem Cells
product and requires licensing or an exemption from licensing from the FDA as part of an investigational
new drug (IND) application. HSCs from unrelated donors facilitated through the Be The Match Registry may be
administered under their IND (BB-IND 6821) or an institutionally held IND. Similarly, HSCs can now be
obtained from FDA-licensed UCB sources or administered under an institutional IND.
 CONCLUSION
The indispensable, lifesaving role of HSCs in medicine has been established, especially for patients with
hematologic disorders. As the understanding of HSC biology increases and the ability to engineer HSC grafts
expands, the clinical applications of HSCs will likely continue to grow. Along with the fast-paced growth in the
use of HSCs, emerging novel technologies and approaches to manipulate HSCs are adding to the challenge of
ensuring that HSCs continue to serve as a safe and effective cellular therapy product for patient use. Addressing
this challenge will require regulatory agencies and accrediting bodies to continue to update and modify their
applicable rules, regulations, and standards.
KEY POINTS
1. Hematopoietic stem cells (HSCs) derived from the patient being treated (autologous) or a donor
(allogeneic) can be used to treat a variety of malignant and nonmalignant conditions.
2. Autologous HSCs in general are used to rescue the marrow function of a patient undergoing high-dose
chemotherapy and/or radiotherapy.
3. In addition to rescuing the marrow function of a patient undergoing high-dose chemotherapy and/or
radiotherapy, allogeneic HSCs also have graft-vs-neoplasm effect and/or the ability to replace defective cellular
machinery.
4. Regardless of the HSC donor source, AABB CT Standards requires laboratory testing for human
immunodeficiency virus, Types 1/2; hepatitis B virus; hepatitis C virus; syphilis; human T-cell lymphotrophic
virus, Types I and II; and cytomegalovirus.
5. Screening and testing for infectious diseases in allogeneic HSC donors is mandated by the Food and Drug
Administration (FDA), and when a risk for a communicable disease is discovered, the donor is ineligible (but
may still donate if there is urgent medical need).
6. Allogeneic HSC donors are chosen with regard to their histocompatibility with the recipient. ABO-Rh
compatibility between the donor and recipient is not necessary.
 726
AABB TECHNICAL MANUAL
7. HSCs can be obtained by aspiration of marrow, collection of umbilical cord blood, or by peripheral blood
mobilization followed by collection with an apheresis machine.
8. HSCs usually require minimal manipulation, and can be stored following cryopreservation with the
cryoprotectant, dimethyl sulfoxide.
9. Specialized HSC manipulation techniques can be used to reduce the HSC product volume, lysed cells, red
cells, and cryoprotectant depending on the recipient’s clinical needs.
10. Quality control (QC) is essential for providing a safe and efficacious HSC product. Common QC testing
includes cells counts (CD34+ cell enumeration, total nuclear cell count), microbial contamination testing, and
viability testing.
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donor stem cell transplantation. Blood 2013;121:863-5.
31. Miller JR Perry EH, Price TH, et al. Recovery and safety profile of marrow and PBSC donors:
Experience of the National Marrow Donor Program. Biol Blood Marrow Transplant 2008;14: 29-36.
32. Pulsipher MA, Chitphakdithai P, Miller JR et al. Adverse events among 2408 unrelated donors of
peripheral blood stem cells: Results of a prospective trial from the National Marrow Donor Program. Blood
2009; 113:3604-11.
33. Gertz MA. Review: Current status of stem cell mobilization. Br J Haematol 2010;150:647-62.
34. Keating CM. Plerixafor. Drugs 2011;71:162347.
35. To LB, Haylock DN, Simmons PJ, Juttner CA. The biology and uses of blood stem cells. Blood
1997;89:2233-58.
36. Abrahamsen JF, Stamnesfet S, Liseth K, et al. Large-volume leukapheresis yields more viable CD34+
cells and colony-forming units than normal-volume leukapheresis, especially in patients who mobilize low
numbers of CD34+ cells. Transfusion 2005;45:248-53.
37. Khuu HM, Cowley H, David-Ocampo V; et al. Catastrophic failures of freezing bags for cellular therapy
products: Description, cause, and consequences. Cytotherapy 2002;4:539-49.
38. Rubinstein P, Dobrila L, Rosenfield R, et al. Processing and cryopreservation of placental/ umbilical
cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 1995;92:10119-22.
39. Laroche V, McKenna D, Moroff G, et al. Cell loss and recovery in umbilical cord blood processing: A
comparison of post-thaw and postwash samples. Transfusion 2005;45:1909-16.
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40. Fleming KK, Hubei A. Cryopreservation of hematopoietic and non-hematopoietic stem cells. Transfus
Apher Sci 2006;34:309-15.
41. Sutherland DR, Anderson L, Keeney M, et al. The ISHAGE guidelines for CD34+ cell determination by
flow cytometry. J Hematother 1996;5:213-26.
42. Spitzer G, Verma DS, Fisher R, et al. The myeloid progenitor cell: Its value in predicting hematopoietic
recovery after autologous bone marrow transplantation. Blood 1980;55:31723.
43. Douay L, Gorin NC, Mary JY, et al. Recovery of CFU-GM from cryopreserved marrow and in vivo
evaluation after autologous bone marrow transplantation are predictive of engraftment. Exp Hematol
1986;14:358-65.
44. Schwartzberg L, Birch R, Blanco R, et al. Rapid and sustained hematopoietic reconstitution by
peripheral blood stem cell infusion alone following high-dose chemotherapy. Bone Marrow Transplant
1993;11:360-74.
45. Migliaccio AR, Adamson JW, Stevens CE, et al. Cell dose and speed of engraftment in
placental/umbilical cord blood transplantation: Graft progenitor cell content is a better predictor than nucleated
cell quantity. Blood 2000; 96:2717-22.
46. Pamphilon D, Selogie E, McKenna D, et al. Current practices and prospects for standardization of the
hematopoietic colony-forming unit assay: A report by the cellular therapy
team of the Biomedical Excellence for Safer transfusion (BEST) collaborative. Cytotherapy 2013;15:255-
62.
47. Regan D. Transportation and shipping of cellular therapy products. In: Areman EM, Loper K, eds.
Cellular therapy: Principles, methods and regulations. Bethesda, MD: AABB, 2009: 362-74.
48. Antonenas V) Garvin F, Webb M, et al. Fresh PBSC harvests, but not BM, show temperature-related
loss of CD34 viability during storage and transport. Cytotherapy 2006;11:15865.
49. Jansen J, Nolan P, Reeves M, et al. Transportation of peripheral blood progenitor cell products: Effects
of time, temperature and cell concentration. Cytotherapy 2009;11:79-85.
50. Kao G, Kim H, Daley H, et al. Validation of short-term handling and storage conditions for marrow and
peripheral blood stem cell products. Transfusion 2011;51:137-47.
 51. Rowley SD, Anderson GL. Effect of DMSO exposure without cryopreservation on hematopoietic
progenitor cells. Bone Marrow Transplant 1993;11:389-93.
52. Davis JM, Rowley SD, Braine HG, et al. Clinical toxicity of cryopreserved bone marrow graft infusion.
Blood 1990;75:781-6.
53. Stroncek DF, Fautsch SK, Lasky LC, et al. Adverse reactions in patients transfused with cryopreserved
marrow. Transfusion 1991;31: 521-6.
Chapter 30
Umbilical Cord Blood Banking
Alelcsandar M. Babic, MD, PhD, and Donna M. Regan, MT(ASCP)SBB
|KK| over the past 25 years, umbilical BScord blood (UCB) has evolved into an important source of
hematopoietic stem cells (HSCs) for transplantation. Classified by the Food and Drug Administration (FDA) as
a biologic drug, stem cells derived from UCB are also used in a growing number of large, multiinstitutional
trials in immunotherapy and regenerative medicine. This chapter summarizes the practical aspects of creating an
inventory that provides UCB cells for clinical use, recent research, economic considerations in balancing
inventory size with the potential for unit selection, and the regulatory structure that keeps pace with product
innovation.
BACKGROUND
UCB has become a widely accepted source of HSCs for transplantation in pediatric and adult patients
lacking an HLA-matched donor.1'6 The advantages of UCB-derived HSCs over other HSCs include their higher
proliferative and self-renewal capacity and their association with a lower incidence of graft vs host disease
(GVHD), particularly acute GVHD, de
spite their less stringent HLA-matching requirements.4,5,7'11 In-vitro and in-vivo animal research suggests
that these attributes of UCB transplantation are due to the primitive nature and naive immune system of
UCB.12'14 Transplant candidates, especially those with rare HLA types, are often successful in finding an
acceptable UCB unit. Furthermore, the search time, in general, to find a donor is markedly reduced for UCB
transplant recipients than for recipients of HSCs from other sources because the cellular characteristics, donor
screening/ eligibility, and HLA typing of UCB units are determined at the time of banking.15
The first UCB bank, established by Dr. Hal E. Broxmeyer, provided the donor graft for the historic 1988
UCB transplant as well as four additional HLA-matched sibling transplants.16,17 As UCB gained initial
acceptance as an HSC source in the related transplant setting, the possibilities for the use of unrelated HSC units
became apparent and supported the rationale for the establishment of unrelated cord blood banks (CBBs). The
first unrelated CBB was established by Dr. Pablo Rubinstein at the New York Blood Center in 1992.18
AleksandarM. Babic, MD, PhD, Medical Director, and Donna M. Regan, MT(ASCP)SBB, Executive
Director, St. Louis Cord Blood Bank and Cellular Therapy Laboratory, SSM Cardinal Glennon Children’s
Hospital, St. Louis, Missouri
The authors have disclosed no conflicts of interest.

729

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AABB TECHNICAL MANUAL
CBBs in Dusseldorf and Milan opened shortly thereafter. Recent reports indicate that more than 600,000
unrelated UCB units are banked worldwide for potential clinical use, and nearly 30,000 unrelated UCB
transplants have been performed to date.19 In 2011 alone, the World Marrow Donor Association reported that
4093 UCB products were shipped to hospitals for transplantation to unrelated patients in 47 countries; in
comparison, 3,743 marrow grafts were performed in that year.19 In addition, a number of private CBBs have
been established worldwide for families to bank the UCB of their infants for future use by family members.
Two of the largest private CBBs have recently reported that they have stored UCB stem cells from 700,000
newborns, and each of these CBBs has distributed approximately 250 UCB products for traditional
transplantation and regenerative applications.
The sections of this chapter describe the current generally accepted practices for UCB banking (primarily
from a public CBB’s perspective), including donor-related issues, collection and processing methods, and
storage and shipment as well as recommended transplant center-related activities for product preparation and
infusion. The chapter continues with a brief discussion of UCB banking economics and how inventories can be
created to meet the needs of a diverse patient population and address cell dose limitations while continuing to
serve as cost-effective means of providing access to therapy in an “affordable health care” environment. The
chapter finishes with an overview of standards and regulations.20
DONOR-RELATED ISSUES Recruitment
Most women agree to donate the UCB of their infants when they become aware that the UCB that is
normally discarded as medical waste can be recovered and used to save the life of another individual. However,
a woman’s ability to donate UCB may be limited by the availability of a CBB servicing the area in which she
gives birth.
Engaging pregnant women and making arrangements before they give birth to donate their infant’s UCB is
critical for achieving the desired results. Early education of pregnant women allows for more complete medical
screening, comprehensive donor protection, availability of adequate supplies to collect UCB, and acquisition of
truly informed consent from the woman. Recruitment by a public CBB typically begins with education of the
physician/midwife by the CBB and the distribution of informational materials to obstetrics offices or prenatal
class staff. This approach enlists support for UCB donation while providing information to obstetric staff. This
information empowers staff to introduce the idea of UCB donation to pregnant women and respond to their
initial inquiries while referring more detailed questions to CBB personnel.
The informational materials that CBBs distribute to obstetrics office and prenatal class staff about UCB
donation address basic information, such as the name of the CBB, whether the CBB is public or private, the cost
(or lack thereof) of donation, names of participating hospitals, medical uses of UCB, and how UCB is collected.
The materials also discuss the risks of UCB donation to the mother and infant and whether the donated unit will
be available for the donating family’s use. Within an informational packet, a CBB may include the health
history questionnaire, which is used to solicit information about the pregnancy, risk factors of the parents, and
medical history of first-degree family members.
The CBB explains to potential donors the need for their written permission (informed consent) to collect
and store the UCB for later use in transplantation or research. Participating mothers must understand that their
blood will be drawn and tested for certain infections, such as hepatitis and human immunodeficiency virus, to
reduce the risk of transmission of disease through the transplantation of their infant’s UCB. The CBB
emphasizes that all information and test results obtained during the process are held strictly confidential to
protect the identity and privacy of donors and
731
that the family will not be approached to donate more cells after the infant is delivered.
The role of the pregnant woman’s physician cannot be understated. During pregnancy, a woman trusts her
physician to provide reliable care and advice. Therefore, a woman’s decision to donate or store her UCB can be
as simple as acceptance of a recommendation offered by her physician. In addition, a woman’s decision to
donate may be influenced by recruitment materials designed to appeal to all ethnic groups.
In spite of broad awareness campaigns and recruitment efforts, some women may not be aware of UCB
donation or may not have registered to participate when they arrive at a hospital to give birth. For this reason,
informational material on UCB donation should also be available in the labor and delivery area. The extent to
which women not previously informed about UCB donation can be recruited at this stage (ie, delivery) varies.
However, in most cases, pregnant women have been previously informed of the option of UCB donation but
simply have not registered or completed the necessary documentation.
Pregnant women may also be solicited for UCB donation by private CBBs. For a fee, these banks will
arrange for UCB to be collected and held in long-term storage for use only by that family. These CBBs provide
written or video information to pregnant women that is specific to their program.
Consent
Consent must be obtained from the pregnant woman for UCB collection, processing, testing, storage, and
medical use.21'26 Although the UCB actually belongs to the newborn infant, consent is obtained from the
mother because her infant cannot provide it and testing of her blood for transmissible diseases is required.
Consent from the father is not necessary and does not increase the safety of the UCB for transplantation.27
Although consent to collect UCB must be obtained before delivery of the infant, CBBs use different
approaches to obtain consent for further manufacturing, testing, and use of
UCB. A CBB may use a single consent form covering all activities. Presented in the prenatal period, a
single consent process affords the pregnant woman adequate time to seek information and consider her options
under lowstress circumstances. Other CBBs may use a phased consent process that permits collection and, if the
resulting harvest meets criteria for banking, allows the bank to approach the mother later for permission to
screen, process, test, and store the product. This latter process facilitates collection from mothers who have not
previously been introduced to UCB banking.
Criteria have been suggested that protect the woman’s ability to make an informed decision during
childbirth. The Advisory Council on Blood Stem Cell Transplantation (ACBSCT) has recommended that each
CBB develop a policy on informed consent that considers the stage of labor and stress of the woman, the
amount of counseling on UCB donation that she has received, and the amount of time available for an adequate
discussion of UCB donation.28,29 Final judgment regarding the woman’s ability to give intralabor consent
should rest with the obstetric staff.
In October 2011, the FDA classified UCB banking as a manufacturing rather than an investigational activity
when a CBB distributes products for specified indications. If not licensed, manufactured UCB products may
still be distributed for nonlicensed (clinical research) applications as an investigational new drug (IND).
Health History and Medical Evaluation
It is incumbent on the CBB to provide a safe product by minimizing the transmission of genetic or
infectious diseases. A donor-eligibility determination, based on donor screening and testing for relevant
communicable disease agents and diseases, is required for all donors of cells or tissue, including UCB cells. A
robust medical health history designed to solicit information on exposures to infectious diseases and history of
symptoms indicating genetic disorders is obtained by interviewing the mother and reviewing her medical
record. The
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AABB TECHNICAL MANUAL
father’s medical history may be solicited to identify any issues that might affect the quality of the UCB, but
this is not required. If not secured before delivery of the infant, the history must be obtained no later than 7 days
after delivery.24tp73) The history questionnaire may be self-administered with follow-up by CBB staff, or it
can be completed through direct interview by CBB staff or hospital staff who are adequately trained in, and
capable of, answering the woman’s questions.
The approach to screening and testing the mother is admittedly more conservative than the approach for the
infant donor. It is the mother’s circulation that nourishes the fetus during pregnancy and because of this shared
physiology, her infectious exposures are relevant to determining the eligibility of her infant’s UCB for
transplantation. Also associated with risk are certain maternal conditions during labor and delivery, such as
fever, prolonged time after membrane rupture, or administration of antibiotics—all of which suggest possible
bacterial contamination that could be transmitted through the UCB product.
Medical screening includes an extensive family genetic history because the infant has not had an
opportunity to manifest many inherited diseases. If first-degree relatives have a history of malignancy or parents
have been treated with chemotherapy and/or radiation, the infant’s UCB cannot be donated. In addition, if UCB
is used to compensate for deficiencies associated with specific genetic disorders, the product is tested for the
presence of the targeted enzyme when such testing is available.
Infants are not required to be retested or reexamined at 6 to 12 months of age to evaluate their eligibility to
donate UCB because of the difficulty of locating some families, concerns about parental and infant privacy, the
difficulty of maintaining the records required to trace families, and cost. In spite of the difficulties of
reconnecting with a donor family, however, some CBBs follow up with families at various intervals after birth
to ensure that the infant has not developed any transmissible diseases and instill confidence in the suitability of
the infant’s UCB for transplantation.
Donor Testing
The second step in determining donor eligibility is accomplished through laboratory testing. The strategy for
UCB donation is similar to that used for whole blood donation except that the tests are performed on maternal
blood and not the blood of the infant donor. This approach is used because it is assumed that infectious diseases
present in the UCB originated from the mother and, thus, infectivity is likely to be detected in a maternal blood
sample.
Because testing is conducted on maternal blood, it is necessary to obtain the mother’s consent for testing as
well as for collection of the UCB. It is also important to note that the testing reagents used must be approved by
FDA for use in donor screening rather than diagnostic testing. In addition, the sample types collected must be
cleared for use in this type of testing, and the laboratory must be authorized to perform the test. In the United
States, no donor tests have been approved for UCB samples; therefore, infectious disease testing is performed
on a maternal sample. The samples for infectious disease testing must be collected on the day of UCB collection
or within 7 days before or after the delivery.24(p40)
The CBB may perform testing or issue a contract to a laboratory to conduct testing to rule out collections
from donors with hemoglobinopathies. Certain CBBs store units from donors who have sickle cell trait or alpha
thalassemia trait because these units may have unique HLA types needed for transplantation in patients from
minority populations. Units with homozygous abnormal hemoglobin or compound heterozygous abnormal
hemoglobin are unsuitable for transplant. UCB units selected for transplant in a patient with an inherited disease
should be tested for that disease before being used.30 Therefore, it is important to archive appropriate UCB and
maternal ancillary samples for additional and/ or confirmatory testing.
Regardless of who obtains the medical history or tests maternal or UCB samples, the final donor eligibility
must be determined by
733
the CBB medical director and approved by its quality control unit.22,24(ppl‘2,71‘72)
UCB COLLECTION
The methods to collect UCB consist of cleansing of the cannulation site and aseptic collection. Typically, a
sterile collection bag containing citrate-phosphate-dextrose (CPD) solution anticoagulant is used, although acid-
citratedextrose and lyophilized heparin are also acceptable anticoagulants for UCB collection.
After delivery, the umbilical cord is clamped, cut, and separated from the infant. The clamping must be
timed so as not to interfere with routine delivery practice. UCB can be collected before (in utero) or after (ex
utero) the placenta has been delivered. There are advantages and disadvantages to each method, but neither
appears to be better overall.31 A brief discussion of the two methods is provided below.
Regardless of whether the UCB is collected in vivo vs ex vivo, the process of UCB collections is essentially
identical. As in whole-blood collection, the venipuncture needle is attached to the collection bag. After an
appropriate umbilical vein is identified, the site is cleansed. Typically, this involves wiping the cord with
isopropanol and then scrubbing it with a broad-spectrum topical microbicide (eg, povidone iodine) for at least
30 seconds. Alternatively, a few CBBs have chosen to use a broad-spectrum antimicrobial formulation of 2%
chlorhexidine gluconate/70% isopropyl alcohol in place of traditional iodophors. Subsequendy, a hemostat is
placed on the tubing a few inches from the needle. The hemostat is opened once the vein has been accessed,
allowing blood to flow into the bag. Removal of the hemostat before puncturing the vein may allow air to
contaminate the tubing. While the UCB is being collected (a 3- to 5-minute process), the UCB should be gently
mixed with the anticoagulant to prevent clotting. The umbilical cord appears collapsed when the collection is
complete. Placement of the bag on a laboratory scale during collection allows the collector to monitor the
volume and provides an
additional means to assess completion of collection.
Once the collection is complete, the tubing is “stripped,” forcing the blood inside the tubing into the
collection bag and allowing it to mix with the anticoagulant. This can be accomplished most easily with a
specialized blood-banking tool (ie, a tube stripper), although newer bags include a filtered vent to eliminate the
need for additional equipment. The UCB unit can then be packed, stored, and transported to the cell-processing
laboratory in a temperature-controlled environment.
In-Utero Collection
In-utero collection is generally performed by the obstetrician or nurse/midwife after the newborn has been
delivered and assessed and the umbilical cord has been clamped and cut. If the well-being of the newborn
and/or mother is in question, the collection is not attempted. If a decision to collect the UCB is made, care must
be taken to maintain sanitary conditions. Unless a sterile bag or extension set is used, the traditional supplies are
not sterile and inadvertent misplacement could contaminate the surgical field.
For logistical reasons, it is advisable to have preassembled collection kits available for in-utero collection.
An instructional packet may be included in these kits. This packet might contain, for example, a donor
information form; description of the collection procedure; list of collection kit contents; maternal medical
history form; consent form(s); and packaging, storage, and shipping instructions. Collection kits also include
items such as one or two collection bags, antimicrobial supplies, tubing closures, appropriate sample tubes for
infectious disease testing, sample tube labels, product labels, biohazard and other appropriate stickers (eg, for
indicating temporary storage temperatures), and a secondary specimen and product bag. In some cases, the
hospital’s maternal label may be used to identify maternal tubes and UCB products because these labels include
two forms of identification and precautions are taken to protect donor confidentiality on these labels.
734
AABB TECHNICAL MANUAL
Validated containers and/or processes are used to ship the UCB. Acceptable temperatures during shipping
can range from cold to ambient, depending on the shipping distance and conditions, and are defined based on
stability studies. Temperature during transport may be monitored depending on the risk to the UCB involved in
the shipping process.
The primary advantage of in-utero compared to ex-utero collection is the substantially lower cost because
dedicated CBB collection personnel are not required. As a result, the only costs are for collection and shipping
supplies. In addition, several studies have suggested that in-utero collection methods may result in the collection
of greater volumes of cells and higher counts of nucleated and CD34+ cells and colony-forming units
(CFUs).32,33
In addition, the initial costs and efforts associated with education and training of the physician/midwife
collectors need to be considered. However, once obstetrics practice group members are trained, their hands-on
experience should improve the quality of their collections. In-service and educational sessions can provide
refresher courses and demonstrate appreciation of clinicians’ support for UCB collection. Subsequently, a
program of continued competency assessments, collection-site visits, and regular communication can be utilized
to maintain high-quality UCB collections.
Ex-Utero Collection
Ex-utero collections are often performed by dedicated UCB collection staff. The advantages of this
approach over in-utero collection are its use of standardized collection methods, limited personnel involvement,
and greater control.
After delivery, the cord is clamped at a time that does not interfere with the physician’s routine practice. The
placenta is immediately taken by CBB staff to a suitable location, where it is suspended in a device to allow
collection of blood by gravity. The collection proceeds as described above. The placenta and cord are more
accessible in the ex-utero setting and, therefore, the use of manipula
tion, such as application of pressure to the cord (“milking"), can increase the collection volume. Caution
must be used to prevent maternal contamination and lysis of cells.
Because blood clotting readily occurs during UCB collection, which adversely affects the volume and
number of cells collected, it is important to minimize delays during the collection process. Wong and colleagues
hypothesized that a higher incidence of macroscopic clots in ex-utero collections resulted in the collection of
lower nucleated cell and CFU counts.34
Because it occurs outside the delivery room with all of its activity, ex-utero collection may inherently
provide better process control (ie, a lower risk of microbial contamination and labeling errors), but it raises the
costs associated with collection. In addition, a lack of availability of collectors on all shifts may limit the
opportunity for a facility to participate in UCB collection. Some investigators have questioned these points.35
Because data demonstrate that in-utero and ex-utero collection methods are equivalent, the choice of UCB
collection method should be based on the needs and capabilities of each UCB collection program.31
UCB PROCESSING Methods
In the early 1990s, UCB units were often stored in an unmanipulated state without volume reduction (ie, red
cells and/or excess plasma were not removed before storage) in an effort to conserve the number of stem cells in
the product.18,36 Concern about infusion-related complications associated with red cell incompatibility, free
hemoglobin, dimethyl sulfoxide (DMSO), and logistical issues surrounding limited storage capacity motivated
an evaluation of processing methods to minimize the red cell content and size of the final UCB unit while
limiting stem cell loss.
At present, a variety of processing techniques for volume reduction and removal of red cells are employed,
and the majority of these methods involve sedimentation, centrif
735
ugation, and/or filtration. The most common means of reducing red cell content is the use of sedimenting
agents, such as hydroxyethyl starch (HES), gelatin, polygeline, and dextran.37,39 One of the earliest methods
for processing UCB utilizing HES was developed by Pablo Rubinstein.39 Modifications of his method have
been commonly used by other programs.40 An alternative preparation method involves density separation by
layering UCB cells over Percoll or Ficoll-Hypaque. This method ultimately results in a mononuclear cell-
enriched product essentially devoid of red cells.41 Also, utilization of commercially available leukocyte
reduction filters and semiautomated methods results in similar in-vitro cell recovery to the standard HES
method.37,42
Initially, UCB was processed with other HSC products in laboratories that were part of a research facility,
hospital transfusion service, or blood center. However, the methodology for processing UCB products has
evolved to involve dedicated processing laboratories designed to comply with current good manufacturing
practice (cGMP) by using more standardized processing techniques for volume reduction, removal of red cells,
and cryopreservation.
An important driving force behind most significant recent changes in UCB processing was a set of
recommendations made by FDA as part of its process for the submission of biologies license applications
(BLAs). These recommendations provide guidance on how to provide assurances of the safety, purity, potency,
and effectiveness of UCB products. These recommendations have compelled the industry to standardize its
manufacturing processes by using reagents that have been approved for human use and systems and supplies
that have been cleared by the FDA. To date, the following systems have been approved by the FDA for the
processing of UCB:
■ Automation technology incorporated into the SEPAX Cord Blood Processing System manufactured by
Biosafe SA (Lake Geneva, Switzerland). This technology offers a closed and sterile processing system that
harvests nucleated cells and enriches stem
cells from UCB. The technology is adaptable to a large-scale processing environment. The SEPAX machine,
which is essentially composed of a centrifuge with piston position sensors surrounding the chamber and
pneumatic pump system, is operated using computer-controlled protocols to achieve component separation.
Consumable kits consist of a large syringe-type barrel separation chamber with bags and tubing connected via a
stopcock assembly. Biosafe’s SEPAX system received FDA clearance in January 2007 and a European CE mark
of approval in 2001.
■ The AXP (AutoXpress Cord Blood Processing System), manufactured by ThermoGenesis (Rancho
Cordova, CA), is an automated, functionally closed system that harvests stem-cell-rich huffy coat from UCB.
The system reduces a unit of UCB to a precise volume chosen by the operator and selectively isolates
mononuclear cells. The system includes the AXP device, a docking station, a processing set, and XpressTRAK
software. The AXP system received 510 Ck) clearance from the FDA in October 2007.
■ PrepaCyte-CB, a UCB-processing system manufactured by CytoMedical Design Group (St. Paul, MN),
received FDA 510(k) clearance in January 2009. PrepaCyte-CB is a functionally closed sterile bag system
composed of three integrally attached processing and storage bags containing the PrepaCyte-CB separation
solution. While isolating nucleated cells, PrepaCyte-CB removes most red cells and nucleated red cells from the
final processed UCB unit. The system requires plasma extractors and a standard laboratory centrifuge to
sediment and concentrate desired cells.
Each of these methods has advantages and disadvantages with respect to cell recovery, red cell removal,
processing time, and cost. Each laboratory must therefore validate and determine which processing method is
most appropriate for its situation. Cellular function, product integrity, and safety must be considered when
developing a validation plan. Nucleated and CD34+ cell recovery, viability,
736
AABB TECHNICAL MANUAL
potency (eg, ability to form CFUs), and sterility testing (before and after cryopreservation) are typical
validation performance measures. Factors to consider when choosing a processing method include workload,
processing time, cost, maximization of storage time, and production efficiency (number of UCB units processed
per staff member per day).
Quality Control Testing and Characterization
Quality control (QC) testing to assess the adequacy of the product and process is essential. QC testing is
usually performed on receipt (prior to manipulation) of a UCB unit and before cryopreservation. Typical QC
assays include counts of nucleated cells (white cells plus nucleated red cells), mononuclear cells (monocytes
plus lymphocytes), and CD34+ cells; hematocrit; CFU assay; and viability and sterility (aerobic, anaerobic, and
fungal) testing. All testing methods must be validated for their intended use.
UCB products used for allogeneic transplant must be tested for HLA Class I and Class II antigens, including
HLA-A, -B, and -DRB1 loci; HLA-C and HLA-DQB typing is recommended.22 In this setting, ABO/Rh typing
is performed primarily to assist clinicians with red cell and plasma product support after infusion. Table 30-1
lists these basic tests.
Cryopreservation and Long-Term Storage
Successful outcomes of UCB transplantation hinge on the CBB’s ability to preserve the integrity of UCB
over substantial lengths of time. Freezing and storage methods must be robust enough to ensure that the quality
of the UCB unit is maintained for many years.
The most common method of UCB cryopreservation consists of freezing the cells in a cryogenic bag in the
presence of 10% DMSO as a cryoprotectant.43'45 One of the main advantages of using cryogenic bags over
freezing vials is the ability to perform post-thaw confirmatory HLA typing and QC testing using an integrally
attached tubing segment. Both AABB and the Foundation for the Accreditation of Cellular Therapy (FACT)
mandate the use of cryogenic bags with attached segments for the freezing of UCB.22,24(p53)
It has been well established that slow cooling in a programmable controlled-rate freezing device at a rate of
1 C/minute results in adequate recovery of CD34+ cells/human progenitor cells.44,45 Such instruments
compensate for the heat of fusion phase, mitigate process variability, and limit cell damage. As with all critical
processes involved in collecting and storing UCB, the freezing process must be validated and procedures must
be in place that govern the use and operation of the controlled-rate freezer (or equivalent), expected
TABLE 30-1. Summary of QC Testing for UCB
Test Method(s)
Cell count Hematology analyzer, manual differential
CD34+ cell
Flow cytometry (single or dual platform)
enumeration
Viability assay Dye exclusion (light and/or fluorescence microscopy), flow cytometry
Clonogenic assay CFU (most commonly used in clinical laboratories), LTC-ICs
Sterility testing Aerobic, anaerobic, and fungal culture
HLA typing Molecular
ABO/Rh typing Serology
Hematocrit Standard/routine
QC = quality control, umbilical cord blood, CFU = colony-forming unit, LTC-ICs = Long-term
UCB = culture-initiating cells.
 737
cooling rates and freezing curve parameters, endpoint freezing temperature, addition of cryoprotectant, final
cell and cryoprotectant concentrations, and storage temperature. Because equipment failures can occasionally
occur, simplified passive freeze methods can also be validated to provide satisfactory cryopreservation of
human stem cell products.46,47
Both AABB and FACT require cryopreserved UCB to be stored at <-150 c.22,24(p54) Once frozen, UCB
units are typically transferred into a monitored liquid-nitrogen (LN2) storage container and either overwrapped
and immersed in liquid (at -196 C) or in vapor phase to minimize the potential for crosscontamination during
long-term storage.
To ensure the safety and stability of the stored UCB units, control measures should be established for
product security and segregation, storage container monitoring, inventory control, and duration of storage.
Storage containers must be located in a secure area to prevent unauthorized access. For units in which
transmissible disease screening and testing results are positive or the testing is incomplete, there must be a
designated quarantine storage area or process to prevent accidental release. To ensure that temperature and LN2
levels are continuously maintained, a monitoring system with local and remote alarm capabilities must be in
place. Alarm limits should be set to allow adequate time for staff to respond to notifications and react before
inventory is compromised. All UCB products and reference sample storage locations should be cataloged using
an inventory-management system that allows for rapid retrieval. The duration of storage and product expiration
dates should be defined in operating procedures, even when expiry dates have yet to be determined.
Studies of the long-term effects of ultralow-temperature storage of UCB units have shown that retention of
viability and/or proliferative function of UCB cells frozen in the liquid phase of LN2 remains acceptable for at
least 23 years.48,49 However, each CBB must establish and maintain a written testing program designed to
assess the integrity, potency, safety, and stability of drug products manufactured under its facility-specific
manufacturing
and storage conditions. The CBB’s UCB program must address individual products before distribution (lot
release testing) and demonstrate the stability of the drug’s active ingredients, used in determining expiration
dates.
Post-Thaw Stability and Other Testing
Accreditation organizations require UCB products to have integrally attached segments that are
representative of the UCB product and used to verify the results of HLA typing.24(p78) One of these segments
can be used for confirmatory testing—a process in which product identity is confirmed and potency is evaluated
before the UCB unit is released to a transplant facility. Extended HLA typing, viability, potency, or stability
testing can be performed using other replicate aliquots of the UCB unit (retention samples). Progenitor assays,
CD34+ cell enumeration, viability testing, or others tests performed on segment material before distribution
may be useful in determining the product’s potency. Traditionally performed as an internal QC process, the
results of these tests are not released due to their lack of demonstrated correlation with engraftment. However,
transplant centers, knowing that these results are available, are requesting them from CBBs when determining
which UCB units to select. Accordingly, studies are under way to assist with interpretation of the variable
results obtained when testing is performed on the limited material in these samples.
SHIPMENT
With a number of commercial carriers available, UCB can be routinely transported to processing
laboratories or transplant facilities within hours to a few days. It is important to ensure that UCB units and
products are properly packaged and shipped to prevent damage or deterioration during shipment. This process is
the responsibility of the CBB. Validated packaging and shipping procedures should demonstrate that acceptable
temperatures and unit integrity are maintained.24(p35) Each CBB needs to define the shipping conditions
(temperature, type of shipping container, and
738
 AABB TECHNICAL MANUAL
packaging material) for the transport of its fresh and frozen UCB products.
Shipping methods must also be designed to protect the safety of the personnel involved.22,50 The FDA,
International Air Transport Association (IATA), US Department of Transportation (DOT), AABB, and FACT
have established packaging and labeling requirements for the shipping of biologies.22,24,51'53 IATA requires
that shipping containers be able to withstand extreme external temperature variability and that primary outer
containers be leakproof, constructed to resist breakage, and durable enough to withstand pressure changes and
falls. In addition, CBBs must package UCB in a secondary inner container, such as a plastic resealable bag, with
enough absorbent material to contain the contents of the product in the event of a leak or break.22,52
Shipping Fresh UCB
The requirements for the transport of freshly collected UCB are not well defined, and each facility must
establish transport temperature criteria and acceptable limits that are commensurate with the risks involved.
Available options include transport at room temperature or use of insulated, precooled stabilizing packs. Wada
and colleagues observed a 1% drop in viability for every 4-hour increase in transport time for recently collected
UCB units that were shipped at ambient temperature.54 However, a series of studies has shown that UCB can
be preserved in CPD for up to 48 hours before processing and retains satisfactory cell recovery rates and
progenitor content.55'58
Shipping Cryopreserved Units
An advantage of selecting a frozen UCB product over a fresh marrow or apheresis product is that the frozen
UCB product can be shipped and secured at the transplant facility before the recipient is conditioned for
transplant. Cryopreserved products are shipped to transplant facilities in portable LN2 “dry” shipping containers
to maintain the products in a frozen state. Insulated containers are used that allow LN2 to be absorbed into the
vessel wall,
creating an ultracold environment. FACT requires that LN2 dry shippers be validated to maintain
temperature of <-150 C for at least 48 hours beyond the time of delivery to the transplant facility.22
For a dry shipper to be fully effective, it must be charged or filled with LN2 24 hours before the estimated
time of release from the processing laboratory. This allows for complete absorption of the LN2 into the wall of
the shipping container. Improperly prepared dry shippers present a risk of LN2 leakage, and CBBs may be
subject to civil/criminal penalties from US DOT in the event of a spill.
Dry shippers are vulnerable to incident handling and can be compromised if they are mistreated or
positioned incorrectly. This will result in warm conditions and thawing of the product, rendering it unusable for
the clinical procedure. To minimize the risk of product loss, shipping instructions must be clear and the
conditions of transport, particularly temperature, must be continuously monitored.
Transport Labeling and Record Requirements
AABB and FACT require continuous monitoring of the temperature during shipment, which is
accomplished through the use of a data logger.22,24<p35) According to AABB and FACT, the shipping
container must include the name, address, and telephone number of the shipping and receiving institutions; the
phrases “medical specimen,” “do not irradiate” (if applicable), and “do not x-ray”; and biohazard labels (as
appropriate) .22,24(p68) Biological products must be packaged and shipped in compliance with all applicable
governmental regulations. Transportation records that identify the shipping facility, date and time the unit was
shipped and received, courier, and contents of each shipping container should be maintained.22
RECEIPT OF UCB FOR TRANSPLANTATION
UCB transplantation is a coordinated effort potentially involving several entities—the reg
739
istry, such as the National Marrow Donor Program (NMDP), CBB, and cell-processing laboratory/clinical
team at the transplant center. The process is initiated by the placement of a reservation of or order for a UCB
unit, usually by the clinical program’s transplant coordinator, based on the institutional algorithm or guidelines.
The coordinator may rely on the laboratory or medical director to answer questions related to a prospective
UCB unit, including those associated with technical issues and donor medical history. It is advisable to initiate
involvement of the cell-processing laboratory early in the unit-selection process.59
Once the unit’s quality is confirmed and it is approved for shipment, the coordinator should notify the cell-
processing laboratory of the impending arrival of a UCB unit, including any special handling requirements (eg,
size and dimensions of product canister or indications of risk factors requiring quarantine). When the unit is
received at the laboratory, it should be carefully unpacked and examined to verify its labeling and integrity
before it is placed into the LN2 storage tank. The dry shipper may be weighed to check for excessive LN, loss
(in general, greater than 10 pounds).
Because both AABB and FACT standards require continuous temperature monitoring of cryopreserved
products during shipment, temperature-monitoring devices must be included in the shipment. Upon the unit’s
arrival, the technologist should confirm that the UCB remained within the acceptable temperature range during
shipment. If the temperature-monitoring device displays a digital temperature, the temperature when the unit is
unpacked should be recorded. If the device does not show a temperature or the data cannot be downloaded, the
CBB should forward a copy of the data when they become available.
If the device for continuous temperature monitoring cannot be located, a temperaturemeasuring device from
the transplant center should be inserted for several hours to ensure that the shipper can maintain an acceptable
temperature. The CBB should be notified that no temperature-monitoring device was present in the shipment,
and documentation of
the CBB’s shipper validation should be requested.
The identity of the UCB unit should be verified at the time of inspection, before the unit is placed in storage.
The product label should indicate the appropriate minimum partial label requirements—unique product
identifier (ie, unit number) and proper product name. All other product information should be attached to the
product on a tie tag and/or in the accompanying paper work. Whether affixed or attached, the CBB paper work
that accompanies the UCB and the documents generated by the transplant program/ coordinator should be
compared. Any inconsistencies should be immediately and appropriately investigated.
The unit information, including donor medical history and infectious disease testing results, should again be
reviewed for completeness. If there are any comments not previously communicated to the receiving laboratory,
particularly any related to the donor’s medical history, they should be referred to the receiving laboratory’s
medical director. The medical director should determine whether the information is significant and requires any
further action and/or notification of the transplant physician. If any infectious disease testing is found to be
incomplete or the results are positive, the unit should be placed into quarantine storage, and the appropriate
personnel (eg, laboratory supervisor, medical/laboratory director, coordinator, and/or transplant physician)
should be notified. The product label/tie tag must be updated accordingly (eg, to include a biohazard label) and
a special medical release form may need to be completed as well.
Any further testing deemed necessary by the transplant center (eg, additional HLA or viability testing or
CFU count) should be performed as soon as possible on an integrally attached segment, if one accompanies the
unit. If a unit’s identity is in question and an integrally attached segment is not available, a rapid (Class I
serologic) HLA test may be performed along with the standard product testing on the thawed product (ie, on the
day of the transplant procedure).60
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AABB TECHNICAL MANUAL
Instructions for handling and preparing frozen UCB products are provided by the UCB manufacturer in the
shipment. However, the receiving laboratory may use alternative thawing methods based on its own validated
procedures, clinical protocol, or physician preference.
THAWING AND WASHING OF UCB
Coordination of the transplant event requires consistent communication among all members of the clinical
team. In the days before the planned transplantation, the coordinator should verify the infusion date with the
clinical team, and both the coordinator and laboratory should again review all relevant records. Subsequently,
the patient care unit should be contacted at least 1 day before the day of transplant to schedule an infusion time.
Given the wide variety of types of products received from an increasing number of CBBs, the most appropriate
thawing procedure must be determined based on the transplant center’s validated method or CBB’s
recommendations. There may be considerations if the product was manufactured to reduce red cell and plasma
content (RBC reduced) or to reduce plasma content only (red cell replete) before cryopreservation. The choice
of the thawing procedure may be further affected by the requirements of the clinical protocol in which the
patient is enrolled.
Currently, three different practices for preparing UCB products for infusion are available: the traditional
thaw-and-wash method, the thaw-and-dilution technique, and the bedside thaw method.61,62
Despite the potential advantage of the bedside product preparation method in minimizing potential cell loss
from postthaw manipulation, this approach is not recommended because of the difficulty of rescuing the product
at the bedside if the bag’s integrity is compromised and the adverse effect of prolonged exposure of thawed cells
to DMSO if the infusion is delayed. Furthermore, this method lacks the capacity for process control and product
assessment. Finally, this method
should not be used with red cell-replete products according to recent FACT requirements.
The traditional washing method originally described in 1995 is recommended if the product’s exposure to
DMSO, free hemoglobin, and volume approach critical limits for recipients, particularly pediatric patients or
those with underlying cardiac, pulmonary, or sensitizing conditions.39 More recently, a dilution or simple
reconstitution approach has gained support, primarily due to concerns about cell loss during the wash step.63,64
Both methods are initiated in similar fashion:
■ The UCB product is carefully removed from the storage tank and a thorough inspection is performed to
evaluate the container’s integrity. Verification of the product’s identity on its label is conducted by two
technologists.
■ The unit is sealed in a clean or sterile transparent bag (ie, a plastic zipper bag) and submerged in a 37 C
waterbath of clean or sterile water or saline. Gentle kneading of the UCB bag during thawing helps accelerate
the thawing process while preventing recrystallization and consequential cell damage or death. If a leak is
discovered after initiation of the thaw procedure, the site of the container break is determined and a hemostat is
employed or the product is positioned to prevent further escape of the cellular material. The contents can then
be aseptically transferred into a transfer bag under a biological safety cabinet.40
■ The thaw solution is used for product dilution and should be prepared in advance. It typically contains
10% dextran and a protein source, usually human serum albumin in a final concentration of approximately 2.5%
to 4.2%. The supplementation of the thaw solution with protein (albumin) has been proven to restore osmolarity
and extend cell viability.39 Subsequently, a volume of solution at least equal to the volume of the UCB product
is gradually added to the bag while it is gently mixed. The product and solutions are drained into a labeled
transfer bag and left to equilibrate for 5 minutes. At this stage, if the product is to be
741
reconstituted or diluted only, samples are removed for product testing.
■ If the product is to be washed, the labeled transfer bag is centrifuged at 400-600 x gfor 15 minutes at 10
C. The supernatant is expressed, leaving behind a pellet of washed UCB cells. If it is the policy of the transplant
laboratory to centrifuge the supernatant, the second labeled transfer bag is spun at 400 x gfor 15 minutes at 10
C, the supernatant is again expressed, and the two cell pellets are combined into one labeled bag.
■ The UCB cells are resuspended in a volume of thaw solution that is appropriate for the weight of the
patient while additional consideration is given to the potential for fluid overload.
■ Some laboratories filter the resuspended product with a standard blood filter (at 170260 microns).
■ Samples are removed for QC tests, such as total nucleated cell (TNC), CD34+, and CD3+ cell counts;
microbial culture (bacteria, fungus); confirmatory ABO/Rh typing; CFU assay; and viability testing. Final
volume, cell dose, and cell recovery are determined. After completion of paper work and labeling, the unit can
be released for infusion).58
It is anticipated that thawing procedures based on the type of product processed will be standardized
through the collaboration of UCB bankers and transplanters.
INFUSION OF UCB
To facilitate the product infusion procedure, it is necessary to maintain good communication between the
patient and the physician overseeing the infusion. Hence, it is a good practice to again describe the general
process, including graft selection, cell processing, infusion, potential side effects/adverse reactions, and plans
for premedication. This conversation should take place on, or shortly before, the day of the transplant
procedure. A procedure for infusion is outlined below.50 The general approach and potential adverse reactions
are
similar to those for infusions of other HSC products.65
Once the UCB product has been thawed and washed, it should be delivered to the patient care unit without
delay. Subsequently, a form acknowledging the receipt of the UCB should be signed by a nurse, who then
notifies the patient’s physician of the UCB unit’s arrival. After the physician approves the infusion and proper
identification procedures are followed, the unit is infused by intravenous (IV) drip or syringe push directly into
a central line without a needle or a pump. Some institutions use a standard blood filter at the bedside. If a filter
is used in the laboratory after the thaw/ wash, a second standard blood filter at the bedside is not needed.
The thawed UCB must be transfused as soon as clinically possible. Timely infusion is most likely to be
challenging with UCB that has not been washed or diluted. Although Rowley and Anderson66 concluded that
DMSO is not toxic to HSCs at clinically relevant concentrations (ie, 5% or 10%) at either 4 C or 37 C for up to
1 hour of incubation, they also noted that the addition of 1% DMSO to culture dishes suppresses CFU assay
results. It is important to note that these studies were performed on fresh cells.67 Studies of the effects of
DMSO on HSCs that have been previously cryopreserved are limited. However, some investigators have noted
similar suppression of colony formation when thawed UCB samples are not washed and are immediately placed
into CFU culture.67 Thus, the possible functional defect due to DMSO coupled with the not infrequent need to
hold clinical products raises the concern that cell injury could occur with thawed UCB, particularly when cells
are not washed or diluted.
The unit bag and IV tubing should be flushed with sterile saline after the unit bag empties to maximize cell
dose. Sterile saline may be added direcdy to the unit bag if the flow rate becomes unusually slow. Because the
final volume of a UCB unit is relatively low (roughly 60 to 100 mL with the thaw/wash methods described
above), the infusion rate is slow (5-10 mL/minute) and the infusion process is typically completed within 15 to
30
742
AABB TECHNICAL MANUAL
minutes of the unit’s receipt in the patient care unit.
It is recommended that the patient’s vital signs be checked before infusion, immediately after infusion, and
1 hour after infusion at a minimum. Should an adverse reaction occur, more frequent monitoring is
recommended. The transplant physician and the medical director of the cell therapy laboratory should be
notified immediately of an unexpected or moderate to severe reaction. A prompt investigation should include
confirmation of product identity, a product inspection, and the initiation of any appropriate laboratory testing
(eg, direct antiglobulin test, antibody titers, Gram’s stain, or culture). All of the monitoring results should be
captured on the accompanying infusion form, which should be returned to the laboratory. Serious adverse
reactions must then be communicated as appropriate to the NMDP, distributing CBB, and/or the IND
application or license holder for reporting to the FDA.
Reactions associated with UCB infusion may be very similar to those that occasionally occur with blood
transfusion (ie, allergic, hemolytic, or febrile reactions and those due to microbial contamination). However,
with combinations of the various processing methods for UCB (eg, red cell depletion, plasma reduction, and
postthaw wash step), some types of reactions to UCB (ie, those attributed to red cell antigens and plasma
proteins) may be less likely to occur with UCB than other blood transfusions. Likewise, if the UCB has
undergone red cell depletion and/or wash steps, renal failure due to infusion of red cells and free hemoglobin
should occur less often than with infusions of HSCs from other sources. Reactions (ie, nausea, vomiting,
coughing, and headache) often attributed to DMSO should be less common with UCB infusions as weip68,es
Bacterial contamination, which occurs in up to 5% of collections, is unusual in released UCB units because
CBBs exclude these units from their inventories.70'72
UCB infusions are usually very well tolerated; if reactions occur, they are typically mild and readily
managed by the clinical team.65 However, because the possibility of a severe re
action exists, aggressive IV hydration is recommended (eg, for 2 to 6 hours before and 6 hours after infusion
with diuretics as needed). In addition, administration of prophylactic antiemetics, antipyretics, and
antihistamines is suggested.
ECONOMIC ISSUES
The establishment of a diverse and sustainable inventory from which approximately 1% of products are
distributed for clinical use is an expensive undertaking for a CBB. One of the main reasons for this selectivity in
product utilization is the strong association of successful outcomes of UCB transplantation with the grade of the
HLA match and cell dose.73 As a result, UCB selection has recently been directed toward utilization of
products with high TNC content. This finding further complicates the strategy of banking products from donors
who are not well represented in registries due to the reduced volume and cellular content in the collections from
these populations.
To limit the negative economic impact of these trends, CBBs must expand their collection efforts to increase
the proportion of units with higher TNC counts.74 In addition, expanding the number of collection sites to
allow broader participation of donors could support diversification efforts. Improving the efficiency of the
collection process in established centers could increase the number of products eligible for further manufacture
and their likelihood of selection while offsetting the associated costs related to education, staffing, and
transportation. Collaborating to identify alternative uses for clinical-grade products could also help CBBs
achieve self-sufficiency.
The transition to a full cGMP setting has been accompanied by spending money to expand UCB inventories.
The facility used to manufacture human cells, tissues, and cellular and tissue-based products (HCT/Ps) must be
maintained in a clean, sanitary, and orderly manner to prevent the introduction, transmission, or spread of
communicable diseases, as described in Title 21, CFR Part 1271.190(b). To minimize the risk of product
contamination, manufacturers (CBBs) may upgrade their facil
743
ities to offer International Organization for Standardization Class 5 processing areas, define robust cleaning
and disinfection plans, enhance environmental monitoring to ensure continued control of the manufacturing
areas, and develop comprehensive stability plans to establish expiry dates of products manufactured under these
conditions. Facility retrofitting, process redesign, and hiring of additional staff to accommodate upgraded
activities increase the fixed cost of UCB manufacturing.
Whereas pharmaceutical drug manufacturers can recover the cost of research and development along with a
substantial profit margin from the consumer, it is improbable that CBBs will be able to incorporate increasing
costs of UCB product manufacture into the amounts invoiced to clinical programs for product acquisition.
Doing so may encourage transplant centers to pursue alternative means of treatment for their patients, a strategy
that would be detrimental to the survival of UCB manufacturers. Therefore, public CBBs must be innovative
and resourceful in all aspects of their business strategy.
The Stem Cell Therapeutic and Research Act
The Stem Cell Therapeutic and Research Act of 2005 was passed by Congress and signed by President
George W. Bush in December 2005 as Public Law 109-129. In October 2010, President Obama signed the Stem
Cell Therapeutic and Research Reauthorization Act of 2010 (Public Law 111-264). The implementation of both
acts is managed by the Health Resources and Services Administration (HRSA) of the US Department of Health
and Human Services (DHHS). Provisions in these acts that are specific to UCB include:
■ The CW Bill Young Cell Transplantation Program, intended to increase the numbers of UCB units and
establish an outcomes database to collect data to characterize and refine all aspects of UCB transplantation.
Through the Stem Cell Therapeutic Outcomes Database, the Center for International Blood and Marrow
Transplant Re
search received a contract to collect data on all allogeneic (related and unrelated) HSC transplantations
performed in the United States and those that were completed with products procured through the program but
performed outside the United States.
■ Solicitation and subsequent contractual agreements with CBBs to collect and store at least 150,000 new
UCB units that meet specific criteria comprise the National Cord Blood Inventory (NCBI). CBBs with a
contract from the NCBI also provide UCB units that do not meet these criteria for research studies.
■ Requirement for the US Government Accountability Office to report on efforts to increase UCB unit
collection for the NCBI.
■ Establishment of the ACBSCT to make recommendations to the Secretary of DHHS.
This legislation not only validated UCB as a credible therapeutic option but also provided financial support
to CBBs for the creation of larger inventories. However, federal funding does not cover the costs associated
with manufacturing and is limited in terms of appropriations. In the 2010 reauthorization, Congress challenged
CBBs to expand their collection while lowering costs and improving the efficiency of UCB unit collection
without decreasing the quality of the UCB units collected. In addition, CBBs are required to demonstrate
ongoing measurable progress toward achieving self-sufficiency in their collection and banking operations.
REGULATIONS AND STANDARDS
FDA
In 1997, the FDA announced a novel, riskbased, tiered approach to the regulation of somatic cells and
tissues.75 The approach outlined in the FDA’s proposed approach to regulation of cellular and tissue-based
products (February 27, 1997) was followed by the good tissue practice (GTP) regulations of 2004 “requiring
cells and tissues to be handled according to procedures designed to prevent
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AABB TECHNICAL MANUAL
contamination and preserve [cell and] tissue function and integrity.”51 This historic document has set the
framework for cell therapy laboratories by identifying the agency’s expectations regarding the prevention of
disease transmission and laboratory process controls. The GTP requirements are intended to 1) prevent
unknowing use of contaminated tissue at risk of transmitting infectious disease, 2) prevent improper processing
of tissue in ways that could cause damage or risk of contamination, and 3) ensure the safety and efficacy of
products that are more than minimally manipulated.75,76 Based on these principles, all human cellular and
tissue-based products intended for use in unrelated recipients, regardless of degree of manipulation or poten
tial risk, are required to comply with the requirements for donor testing and screening,76 establishment
registration,77 and the current GTP regulations.51
Since 2004, CBBs have been required to register with the FDA as manufacturers of UCB51 and
demonstrate compliance with GTPs. For CBBs that manufacture and/or store more than minimally manipulated
products (eg, UCB that is activated, expanded, or genetically modified) or combine UCB with nontissue
components, the FDA requires submission of an IND application and adherence to licensure application
requirements. Table 30-2 summarizes the FDA’s regulations governing HCT/Ps, including UCB.
TABLE 30-2. Summary of FDA Regulations Regarding Human Cells, Tissues, and Cellular and Tissue-
Based Products (HCT/Ps) and Hematopoietic Progenitor Cells-Cord (HPC-Cs)75
Products Regulated Specific Product
HCT/Ps that are or will be regu- ■ All allogeneic, unrelated HCT/Ps derived from cord and peripheral blood
lated as biological products ■ HCT/Ps that are more than minimally manipulated (eg, expanded, acti
vated, or genetically modified).
■ HCT/Ps that are combined with a drug, device, or biological product.
■ HCT/Ps that are intended for nonhomologous use (eg, for cardiac repair).
HPC-Cs that are currently subject to biologies license application (BLA) requirements
HPC-C that is currently subject to investigational new drug requirements
Allogeneic, unrelated, and minimally manipulated cells intended for use in unrelated donor HPC
transplantation procedures in conjunction with an appropriate preparative regimen for hematopoietic and
immunologic reconstitution in patients with disorders affecting the hematopoietic system that are inherited,
acquired, or result from myeloablative treatment. FDA intends to grant licensure to products manufactured
according to recommended establishment and process controls and that comply with all applicable regulatory
requirements.
When no satisfactory alternative treatment is available, HPC-Cs that are not FDA licensed and are:
1. Allogeneic, unrelated, and minimally manipulated.
2. Intended for use in unrelated donor HPC transplantation procedures in conjunction with an appropriate
preparative regimen for hematopoietic and immunologic reconstitution in patients with disorders affecting the
hematopoietic system that are inherited, acquired, or result from myeloablative treatment.
3. Manufactured in a US cord blood bank and intended to be used in the United States.
Manufactured in a US cord blood bank before BLA approval and do not meet licensing requirements.
Prospectively manufactured in the United States and for which there is no satisfactory alternative.
FDA = Food and Drug Administration.
745
Until 2011, minimally manipulated UCB intended for use in unrelated recipients was not licensed. However,
the FDA has long considered licensure as a way of improving the safety and quality of UCB by instituting
requirements that would be legally enforceable.51 The FDA believed that compelling clinical safety and
efficacy data existed to support the development of product standards as an approach toward licensure. In 1998,
the FDA issued a request for the public to submit comments regarding establishment controls, process controls,
and product standards for UCB manufacturers.78 After reviewing the submitted information, the FDA
determined that sufficient data did exist to support the development of processing and product standards for
granting licensure.
In October 2009, the FDA published its final guidance, which described ways to assist UCB manufacturers
in submitting a BLA.79 Originally published with indications restricted to “hematopoietic reconstitution in
patients with hematologic malignancies, certain lysosomal storage and peroxisomal enzyme deficiency
disorders, primary immunodeficiency diseases, bone marrow failure, and beta thalassemias,” the guidance was
modified by the FDA in 2011 to broaden the indications to “use in unrelated donor hematopoietic progenitor
cell transplantation procedures in conjunction with an appropriate preparative regimen for hematopoietic and
immunologic reconstitution in patients with disorders affecting the hematopoietic system that are inherited,
acquired, or result from myeloablative treatment.” This guidance applies to hematopoietic progenitor cells
(HPCs), UCB [HPCscord (HPC-Cs)] manufactured by US UCB establishments and non-US UCB
establishments that distribute UCB in the United States. For establishments that manufacture UCB products for
autologous use or use by a first- or second-degree blood relative, the FDA encourages the same manufacturing
recommendations and applicable regulations be followed.20
The FDA recognizes that there will be circumstances in which the best available UCB unit for a patient is
not a licensed unit. There
may be units from UCB establishments in which a BLA is pending; units that do not meet licensing
requirements, or units that come from non-US CBBs that have chosen not to apply for licensure. Under such
circumstances, the FDA requires submission of an IND application. FDA published the guidance for such
applications as companion documents to the BLA.80
In March 2014, FDA updated the 2009 BLA and 2011 IND guidance documents. The BLA guidance
document was updated to expand the indications for use, clarify the type of clinical data required in the
application, and replaced the term “HPC-C” with “HPC, Cord Blood.” Similar revisions were made in the IND
guidance document, in addition to clarifying that a table of contents is required for all IND applications.
In summary, the FDA regulates HPCs, HPC-C blood for unrelated allogeneic use as a drug under the Food
Drug and Cosmetic Act and as biological products under the Public Health Services Act. Therefore, regulations
applicable to HPC-Cs include:
1. Title 21, CFR Part 201 and 610 Subpart G Labeling.
2. Title 21, CFR Part 202: Prescription drug advertising.
3. Title 21, CFR Parts 210 and 211: cGMP.
4. Title 21, CFR Part 600: Biological products, general.
5. Title 21, CFR Part 601: Licensing.
6. Title 21, CFR Part 610: General biological products standards.
7. Title 21, CFR Part 1270 (Section 351 of the Public Health Services Act): HCT/Ps with systemic effect
intended for use in unrelated persons.
8. Title 21, CFR 1271 (Section 361 of the Public Health Services Act): HCT/P registration and listing, donor
eligibility, and GTR
In the event that one regulation is in conflict with another, the one that is more specifically applicable to
UCB supersedes the one that is more general.
In combination, standards regulating biologic drugs are akin to those for pharmaceutical manufacturing, and
their application to a
746
AABB TECHNICAL MANUAL
cellular therapy product, such as UCB, is challenging. In contrast to pharmaceutical drugs, which are
chemicals, are manufactured in tablet lots, and can be terminally sterilized, UCB products are composed of
viable cells, are manufactured in single-unit lots, and must be processed aseptically. Recognizing the value and
unique nature of each UCB product, FDA has been very cautious in regulating UCB to avoid preventing
patients from having access to existing or prospectively manufactured products. At the time of this writing, the
FDA has approved the BLAs of five CBBs in the United States (see Table 30-3).
AABB and FACT
The AABB and FACT are the two professional organizations that have established standards for HPCs,
including UCB, in the United States.22,24 AABB has incorporated the requirements for UCB processing
facilities in its Standards for Cellular Therapy Product Services.24 FACT and NetCord combined their efforts to
establish a unique set of standards for UCB-related activities.22 Both AABB and FACT have created standards
that align with the FDA’s GMP and GTP requirements and center on quality systems essentials. These standards
cover donor suitability, collection, processing controls, document controls, facility manage
ment, materials, records, storage, distribution, quality audits, quality plans, errors/deviations, labeling,
personnel, equipment, validation, outcome reviews, and adverse event reporting. In 2009, the ACBSCT
recommended that both AABB and FACT be recognized as accreditation organizations for the NCBI. Both
organizations incorporated specific standards to ensure that CBBs comply with the contracts held with HRSA.
In a post-licensure era, it is expected that UCB banking will continue to evolve to balance the creation of
large inventories of safely manufactured products that not only meet demands for cell dose and matching from
clinicians, but also target new populations and applications for use of this abundantly available resource.
Currendy, there are several ongoing clinical trials involving HSCs and HPC expansion approaches to overcome
limited per-unit cell doses and the lengthy time required to achieve an absolute neutrophil count of 500/pL;
UCB-derived natural killer, T-regulatory, and dendritic cells for immunotherapeutic treatments; and UCB-
derived mesenchymal stem or stromal cells to enhance engraftment and regenerative applications. However,
optimal methods for production and clinical applicability of these new products have yet to be established.
TABLE 30-3. Approved Cord Blood Biologic License Applications*
Product Name Manufacturer
Hemacord New York Blood Center (New York, NY)
HPC-Cord Blood University of Colorado Cord Blood Bank (Denver, CO)
Ducord Carolinas Cord Blood Bank (Durham, NC)
Allocord St. Louis Cord Blood Bank (St. Louis, M0)
HPC-Cord Blood LifeSouth Community Blood Centers (Gainesville, FL)
As of March 2014.

747
KEY POINTS
1. UCB stem cells have a higher proliferative capacity and immune tolerance than stem cells from adult
sources, as demonstrated by the decreased incidence of GVHD in UCB recipients despite the less stringent
HLA-matching requirements for UCB transfusion. These features allow the use of more flexible matches with
UCB, which provide the ability to support patients with rare HLA types and permit matches across ethnic lines.
2. UCB has evolved from being considered biologic waste to acceptance as a viable source of HSCs. More
than 30,000 unrelated donor UCB transplant procedures have been performed, and there are an estimated
600,000 UCB units banked worldwide for use as an HPC source. In 2008, UCB surpassed marrow as the most
frequently utilized source of donor cells for unrelated transplantation.
 3. Engaging pregnant women and arranging donation of their infant’s UCB in advance is critical for
achieving the best results. Early education allows for more complete medical screening, comprehensive donor
protection, availability of adequate supplies, and acquisition of thorough informed consent.
4. UCB can be collected before (in utero) or after (ex utero) the placenta has been delivered. There are
procedural and economic advantages and disadvantages to each method. When performed properly, both are
acceptable for providing high-quality collections.
5. Current methods of manufacturing UCB products involve reduction of red cell and plasma fractions
before cryopreservation. Methods generally involve sedimentation and/or centrifugation and may be performed
manually or with automated devices that have been cleared for use by the FDA.
6. Transplant centers must determine the most appropriate thawing procedure for the products received
based on the manufacturer’s instructions, red cell volume, patient-specific variables, and their own validated
method. Current practices for preparing UCB for infusion consist of bedside thawing, the traditional thaw-and-
wash method, and a thaw-and-reconstitution technique.
7. Following the announcement of a tiered approach to regulating UCB in 1997, the FDA published a
guidance document in 2009 defining a process by which CBBs can submit a BLA to the FDA for UCB.
Through this process, five public CBBs have been authorized to manufacture HPC products from UCB for
allogeneic use.
8. The clinical utility of several cell types within UCB (eg, mesenchymal stem and immune cells) is being
investigated for nonhematopoeitic applications, immunomodulation, and regenerative medicine.
9. To ensure their economic sustainability, CBBs must adapt to recent trends in product selection, improve
collection efficiency, collaborate to identify alternative uses for clinical-grade products, and successfully
transition to a full cGMP and licensure environment.
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AABB TECHNICAL MANUAL
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32. Surbek DV] Schonfeld B, Tichelli A, et al. Optimizing cord blood mononuclear cell yield: A
randomized comparison of collection before vs after placenta delivery. Bone Marrow Transplant 1998;22:311-
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33. Solves R Moraga R, Saucedo E, et al. Comparison between two strategies for umbilical cord blood
collection. Bone Marrow Transplant 2003;31:269-73.
34. Wong A, Yuen PM, Li K, et al. Cord blood collection before and after placental delivery: Levels of
nucleated cells, haematopoietic progenitor cells, leukocyte subpopulations and macroscopic clots. Bone Marrow
Transplant 2001;27:133-8.
35. Solves P, Moraga R, Mirabet V, et al. In utero or ex utero cord blood collection: An unresolved question.
Transfusion 2003;43:1174-6.
36. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic
stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157-66.
37. Solves P, Mirabet V, Planelles D, et al. Red blood cell depletion with a semiautomated system or
hydroxyethyl starch sedimentation for routine cord blood banking: A comparative study. Transfusion
2005;45:867-73.
38. Tsang KS, Li K, Huang DR et al. Dextran sedimentation in a semi-closed system for the clinical banking
of umbilical cord blood. Transfusion 2001;41:344-52.
39. Rubinstein P Dobrila L, Rosenfield RE, et al. Processing and cryopreservation of placental/ umbilical
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40. Alonso JM III, Regan DM, Johnson CE, et al. A simple and reliable procedure for cord blood banking,
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41. Almici C, Carlo-Stella C, Wagner JE, Rizzoli V Density separation and cryopreservation of umbilical
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42. Takahashi TA, Rebulla P, Armitage S, et al. Multi-laboratory evaluation of procedures for reducing the
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43. Institute of Medicine. Cord blood: Establishing a national hematopoietic stem cell bank program.
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44. Fraser JK, Cairo MS, Wagner EL, et al. Cord Blood Transplantation Study (COBLT): Cord blood bank
standard operating procedures. J Hematother 1998;7:521-61.
45. Donaldson C, Armitage WJ, Denning-Kendall PA, et al. Optimal cryopreservation of human umbilical
cord blood. Bone Marrow Transplant 1996;18:725-31.
46. McCullough J, Haley R, Clay M, et al. Longterm storage of peripheral blood stem cells frozen and
stored with a conventional liquid nitrogen technique compared with cells frozen and stored in a mechanical
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 47. Antoniewicz-Papis J, Lachert E, Wozniak J, et al. Methods of freezing cord blood hematopoietic stem
cells. Transfusion 2014;54:194-202.
48. Kobylka P Ivanyi P Breur-Vriesendorp BS. Preservation of immunological and colonyforming
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cryopreserved cord blood cells. Transplantation 1998;65:1275-8.
49. Broxmeyer HE. Will iPS cells enhance therapeutic applicability of cord blood cells and banking? Cell
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54. Wada RK, Bradford A, Moogk M, et al. Cord blood units collected at a remote site: A collaborative
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58. Solomon M, Wofford J, Johnson C, et al. Factors influencing cord blood viability assessment before
cryopreservation. Transfusion 2009;50:820-30.
59. McCullough J, McKenna D, Kadidlo D, et al. Issues in the quality of umbilical cord blood stem cells for
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60. McCullough J, McKenna D, Kadidlo D, et al. Mislabeled units of umbilical cord blood detected by a
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umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment. Bone Marrow
Transplant 2003;32: 145-50.
63. Barker JN, Abboud M, Rice RD, et al. A “nowash” albumin-dextran dilution strategy for cord blood unit
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64. Regan DM, Wofford JD, Wall DA. Comparison of cord blood thawing methods on cell recovery,
potency, and infusion. Transfusion 2010; 50:2670-5.
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66. Rowley SD, Anderson CL. Effect of DMSO exposure without cryopreservation on hematopoietic
progenitor cells. Bone Marrow Transplant 1993;11:389-93.
67. Laroche V) McKenna DH, Moroff G, et al. Cell loss and recovery in umbilical cord blood processing: A
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68. Davis JM, Rowley SD, Braine HG, et al. Clinical toxicity of cryopreserved bone marrow graft infusion.
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marrow. Transfusion 1991;31: 521-6.
 70. Bornstein R, Flores Al, Montalban MA, et al. A modified cord blood collection method achieves
sufficient cell levels for transplantation in most adult patients. Stem Cells 2005; 23:324-34.
71. M-Reboredo N, Diaz A, Castro A, Villaescusa RG. Collection, processing and cryopreservation of
umbilical cord blood for unrelated transplantation. Bone Marrow Transplant 2000;26:1263-70.
72. Lecchi L, Ratti I, Lazzari L, et al. Reasons for discard of umbilical cord blood units before
cryopreservation. Transfusion 2000;40:122-4.
73. Barker JN, Scaradavou A, Stevens CE. Combined effect of total nucleated cell dose and HLA match on
transplantation outcome in
CHAPTER 30 Umbilical Cord Blood Banking
751
1061 cord blood recipients with hematologic malignancies. Blood 2010;115:1843-9.
74. Bart T, Boo M, Balabanova S, et al. Impact of selection of cord blood units from the United States and
Swiss registries on the cost of banking operations. Transfus Med Hemother 2013; 40:14-20.
75. Food and Drug Administration. Proposed approach to regulation of cellular and tissuebased products.
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and tissue-based products; Final Rule. Fed Regist 2004;69:29786-834.
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establishment registration and listing; final rule. Fed. Regist 2001;66:5447-69.
78. Food and Drug Administration. Request for proposed standards for unrelated allogeneic peripheral and
placental/umbilical cord blood
hematopoietic stem/progenitor cell products; request for comments. Fed Regist 1998;63: 2985.
79. Guidance for industry: Minimally manipulated, unrelated allogeneic placental/umbilical cord blood
intended for hematopoietic reconstitution for specified indications. (October 2014). Silver Spring, MD: CBER
Office of Communication, Outreach, and Development, 2009. [Available at http://www.fda.gov/down
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es/Blood/UCM187144.pdf (accessed December 6,2013).]
80. Food and Drug Administration. Guidance for industry and FDA staff: IND applications for minimally
manipulated, unrelated allogeneic placental/umbilical cord blood intended for hematopoietic and immunologic
reconstitution in patients with disorders affecting the hematopoietic system. (March 2014) Silver Spring, MD:
CBER Office of Communication, Outreach, and Development, 2014. [Available at:
http://www.fda.gov/BiologicsBloodVac cines / GuidanceComplianceRegulatorylnfor
mation/Guidances/CellularandGeneTherapy/ ucm388218.htm (accessed April 8,2014).]
 Chapter 31
Tissue-Derived Non-Hematopoietic Stem Cell Sources for Use in Cell-Based Therapies
Yameena Jawed, MD; Brian Johnstone, PhD; Sreedhar Thirumala, PhD; and Keith March, MD, PhD
gB REGENERATIVE MEDICINE HOLDS
BOi great promise for the treatment of some of the most intractable diseases afflicting humankind. Each
year brings new findings that advance the science of regenerative medicine toward becoming clinical reality. In
fact, achieving this goal seems closer now than ever with the recent success in growing functional human livers
in the laboratory.1 However, the reality is that, although these results are promising, their clinical translation
still will require many years, if not decades, of further research and refinement before patients receive organs
grown in the laboratory.
Nevertheless, the use of stem cells has already shown great promise for treating a wide
variety of diseases in clinical trials. Each month, new successes are reported in treatments of even the most
serious life-threatening diseases, such as amyotrophic lateral sclerosis (Lou Gehrig disease).2 Interestingly,
there is scant evidence that these salutary effects occur through the regeneration of degenerated tissues by the
implanted stem cells. In fact, there is very little evidence in animal disease models or, where possible, human
patients that transplanted cells are capable of longterm engraftment in sufficient numbers to account for the
observed effects. Therefore, alternative mechanisms have been invoked to explain the measurable benefits of
mesenchymal stem cell (MSC) therapies. The prevalent
 Yameena lawed, MD, Postdoctoral (Research) Fellow, Indiana Center for Vascular Biology and Medicine,
Indiana University School of Medicine; Brian lohnstone, PhD, Preclinical Director, Center for Regenerative
Medicine, Director, Cardiovascular Ischemia and Vasculogenesis Core, and Associate Research Professor,
Indiana University School of Medicine; Sreedhar Thirumala, PhD, Senior Scientist, Cook General
BioTechnology, LLC; and Keith March, MD, PhD, Director, Indiana Center for Vascular Biology and Medicine,
Director, Vascular and Cardiac Center for Adult Stem Cell Therapy, and Professor of Medicine, Physiology, and
Biomedical Engineering, Indiana University School of Medicine, Indiana University, Indianapolis, Indiana The
authors have disclosed no conflicts of interest.
753

754
AABB TECHNICAL MANUAL
hypotheses of “paracrine support” are discussed in detail below. This new understanding has led to a
widespread adoption of the more general phrase “cell-based therapy” to encompass the panoply of beneficial
effects of treatment with stem cells from a variety of sources—effects that are independent of stable tissue
engraftment and tissue regeneration. In practice, though, the term “regenerative medicine” is still widely used,
especially by the lay public, to refer to any stem cell-based treatment.
Strictly speaking, regenerative medicine is simply defined as the “process of replacing or regenerating
human cells, tissues or organs to restore or establish normal function.”3 The origins of cellular therapy date
back to 1968 with the first bone marrow transplant of hematopoietic stem cells to restore the hematopoietic
system in dysfunctional or ablated marrow.4 From these origins, there has been great progress in identifying
many different sources of potentially therapeutic stem and progenitor cells in the adult system. These findings
have also been translated to earlystage clinical trials to test the effectiveness of these approaches in a number of
diseases that are not currently addressed by traditional pharmacologic therapies or surgery.
Treatment with both autologous and allogeneic stem and progenitor cells has been evaluated. Autologous
cell therapy has the advantage of involving fewer potential regulatory hurdles and, in some cases, such as
reconstructive surgery, is arguably within the traditional scope of the practice of medicine and, therefore,
outside regulatory oversight. Autologous cell-based therapies, although attractive and perhaps more tractable
than allogeneic cell-based therapies, are practical only when the source of cells is abundant and their extraction
is without significant morbidity or enhanced mortality. For these reasons, autologous therapy has been limited
to cells from marrow and adipose tissues; however, the former is mostly composed of hematopoietic cells,
whereas the latter is made up of approximately 15% to 30% MSCs on isolation.4,5 Allogeneic therapies,
however, usually require the mass production of stem cells originally obtained
from genetically nonidentical donor tissue in carefully controlled environments with established controls to
ensure quality and safety.
Much research has focused on the ability of these regenerative therapies to replace organ transplants. In
2006, an article was published on what was labeled the “first artificially grown organ.”5 In this study, scientists
were able to produce sections of a bladder and provide some degree of urinary continence to patients with spina
bifida. Currently, most research relates to tissue and organ progenitor cells that either increase or support the
functionality of preexisting tissues or induce new organ structures to form in situ, a process technically termed
“organ reconstruction.” The reconstruction of bioengineered organs or even for a regenerative therapy treatment
potentially requires a massive number of stem cells. For many stem-cell-mediated therapies, the number of cells
needed to effectively treat a patient greatly surpasses the number of cells available from donors.
MSC SOURCES
Tissue sources of stem cells used for cell-based therapies are varied and cover nearly every organ system in
the body. Based on the developmental stage of the donor, these cells can be classified as embryonic, fetal, or
adult. Embryonic stem cells are primordial cells isolated from the eight-cell blastula that has the capacity to
form any cell type found in the human body. Stem cells isolated from postnatal or reproductive tissues are
known as “adult stem cells.” Most of these cells were discovered and defined based on such attributes as the
ability to proliferate for many generations in culture as well as their intrinsic property of differentiation in
response to environmental or chemical stimuli. The ability to differentiate along multiple lineages (eg,
mesodermal, ectodermal, and endodermal) is important. The hallmarks of pluripotentiality and multipotentiality
distinguish stem cells from progenitor cells; the latter have undergone partial differentiation in situ and, thus,
are committed to differentiate terminally into a single cell type (eg, endothelial progenitor cells or
preadipocytes,
755
CHAPTER 31
which form endothelial cells and adipocytes, respectively).
The most studied adult multipotent cells are mesoderm-derived MSCs, alternatively termed “multipotent
stromal cells,” which originate from the stromal compartment of skeletal muscle, reproductive organs
(umbilical cord and uterus), amnion, marrow, and adipose tissues. The recent advances in techniques to
reprogram terminally differentiated cells to induce pluripotency (termed “induced pluripotent stem cells") have
opened up a vast source of stem cells from nonembryonic tissues. Induced pluripotent cells present exciting new
opportunities for cell-based therapies and are covered in detail in Chapter 33. Marrow-derived MSCs (BM-
MSCs), which are the nonhematopoietic cells isolated from marrow stroma, are also discussed in detail in
Chapter 33 and are only briefly discussed here, mostly to compare them with MSCs derived from other tissues
and to discuss their current therapeutic uses.
BM-MSCs were the first nonhematopoietic stem cells discovered and, due to having been studied in great
detail, are considered the archetypal MSCs. Because the numbers and quality of MSCs decline with the aging of
donors and due to the invasiveness of the procedures required to procure marrow, other sources of MSCs were
sought. Perhaps the most primitive adult MSCs are those obtained from umbilical cord tissue, umbilical cord
blood, amnion, and amniotic fluid.6'10 The dental pulp of the third molar is another source of MSCs.11 It has
been recently recognized that adipose tissue is a rich and abundant source of cells with stem cell properties.12
Additional sources of MSCs are placenta and uterine endometrial cells shed during menses.1314 Each of these
tissue-specific MSCs is discussed in more detail below.
MSCs from different tissue sources differ with respect to prevalence, proliferative capacity,
immunophenotype, and differentiation potential (Table 31-1), which may be important variables governing the
clinical utility of each MSC type. In the cases of low abundance in source tissues or difficulty of obtaining
tissue specimens, MSCs must be first isolated
Tissue-Derived Stem Cells
from the nondesirable cell types (such as leukocytes and endothelial cells) and then expanded under
conditions that are permissive for maintaining stem-cell-like properties. Because of the cost and time required to
generate sufficient numbers of qualified cells under current good manufacturing practice (cGMP) conditions,
the effort is only economically practical when culture expansion yields sufficient numbers of MSCs at lower
passages to treat many hundreds, if not thousands, of patients.
Most adult tissues contain low percentages of MSCs that are not available in sufficient abundance, with the
exception of adipose tissues. BM-MSCs constitute only a small fraction (approximately 1 in 3.4 x 104 cells) of
cells in adult marrow.28 Although the frequency of MSCs in fetal tissues may be higher, these tissues are
comparatively quite small and limited in availability, and their use is associated with ethical concerns when
their collection involves fetal destruction.29 Examples of MSCs found in low numbers in source tissues but that
are highly expandable are muscle-derived MSCs (M-MSCs), cord blood MSCs (CBMSCs), umbilical cord
MSCs (UC-MSCs), placental MSCs (Pl-MSCs), endometrial lining MSCs (EL-MSCs), and dental pulp MSCs
(DPMSCs).
Adipose-derived stem cells (ASCs), which are phenotypically similar to BM-MSCs, are more than 500-fold
more prevalent per gram of fat than per gram of marrow.30 Most patients have excess fat that can be easily and
safely extracted for processing to isolate ASCs. This property makes adipose tissue an ideal source of MSCs for
either autologous or allogeneic applications. ASCs are perhaps the only type of MSC available in numbers that
are sufficient for immediate or “point-of care” use in therapeutic applications without the need for culture
expansion.31'33 Extraction of adipose tissue is accomplished with minimally invasive techniques, such as
lipoaspiration, with little associated morbidity even on removal of tissue volumes as large as 1-3 liters. Thus,
the procurement and use of ASCs is a uniquely practical approach to point-of-care autologous applications.
TABLE 31-1. Comparison of MSCs Derived from Different Tissue Sources
756
AABB TECHNICAL MANUAL
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Tissue-Derived Stem Cells
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758
AABB TECHNICAL MANUAL
Periadventitial cells (called “pericytes”), which support the integrity of microvessels (capillaries and
arterioles), have been recently discovered to possess multilineage potential.34 Traktuev et al highlighted the
perivascular location and presumptive prepericyte or pericyte function of MSCs within adipose tissue.35 The
extension of this finding to recognize the perivascular location of MSCs throughout the body led to the current
hypothesis that pericytes are the source of all MSCs; this can explain both the high phenotypic similarity of
MSCs from different tissue sources as well as the broad distribution of these cells, in association with blood
vessels, throughout the body.35'37 It has also been proposed that the biological function of perivascular MSCs
is intimately related to systemic circulation in that once activated, they either secrete trophic factors that
promote endogenous repair into the blood or may even mobilize to home to injured tissues, where they provide
structural units for repair through differentiation.38
PROPERTIES OF CLINICAL RELEVANCE
MSCs display several key complementary properties that are of potential clinical use: immunomodulation,
paracrine effects, and differentiation. Immunomodulatory functions include immunosuppression or immune
stimulation, depending on the signals in the microenvironment.39 In the laboratory environment, MSCs can be
induced to differentiate into endothelial cells, neural cells, astrocytes, cardiomyocytes, skeletal myocytes,
chondrocytes, adipocytes, osteocytes, and other cells that are developmentally derived from the endoderm and
exoderm.40'43 The low immunogenicity of MSCs also makes them a relatively safe allogeneic cell source in
comparison with certain other cell types, including embryonic stem cells.44 There is evidence from preclinical
and, in some cases, clinical studies to suggest that each of these properties contributes to the therapeutic
efficacy of MSCs.
The paracrine effects of MSCs are increasingly recognized as an important, if not the primary, mechanism
of action to promote tis
sue rescue and repair as well as to modulate the immune system.45 The demonstrated ability of MSCs to
effect change in disease models and patients in the absence of long-term engraftment and differentiation in
numbers required to account for damaged tissue replacement can be most easily explained by paracrine
mechanisms. Thus, exogenously administered MSCs home to the site of injury or disease through signals that
are primarily inflammatory and reside temporarily at the site where they secrete paracrine factors with
prosurvival, antiinflammatory, and repair-inducing properties before clearance from the system.46
Although the evidence supports the predominant function of secreted factors in the diminution of injury or
disease, there is also evidence that cell-mediated contact may be an important immunomodulator during the
period of residency of MSCs at the target site.47 The concept of paracrine support by MSCs was first directly
demonstrated in diseases of peripheral tissues and the central nervous system by treatment of disease models
with the cocktail of factors present in media conditioned by the growth of BM-MSCs and ASCs.48 In another
early demonstration, ablating production of a single factor (hepatocyte growth factor) by stable small interfering
ribonucleic acid knockdown abolished the salutary effects of ASCs in a mouse hind-limb ischemia model of
peripheral vascular disease.46
ISOLATION AND EXPANSION
Obtaining sufficient numbers of MSCs commonly requires their extensive expansion in cell culture. The
isolation and expansion of MSCs from marrow aspirate is covered in detail in Chapter 33. Liposuction surgery
is a well-tolerated and safe procedure for producing a large amount of lipoaspirate that can be processed to yield
ASCs.
The method of ASC isolation in different laboratories varies subtly, but it usually involves enzymatic
dissociation from adipocytes and extracellular matrix, filtration to remove particulates, and low-speed
centrifugation to separate adipocyte and nonadipocyte cells.
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CHAPTER 31
Following surgical removal of subcutaneous fat, lipoaspirate samples are washed extensively in phosphate-
buffered saline to deplete erythrocytes and then treated by continuous agitation with collagenase Type I solution
for 30 minutes to 1 hour at 37 C. It is important that the collagenase also contain neutral proteases, presumably
to liberate cells from the extracellular matrix fragments; these cells are removed by subsequent filtration. The
solution is neutralized with an equal volume of serum-containing medium and centrifuged at low speed
(approximately 300 x g for 10 minutes). The buoyant adipocytes migrate in opposition to centrifugal force. The
sedimenting cells are a heterogenous mixture termed the “stromal-vascular fraction” (SVF), which contains
variable proportions of endothelial cells, pericytes, ASCs, and resident leukocytes (primarily monocytes and
macrophages).47'50 The cell pellet can then be resuspended in an 160 mM NH4C1 solution (for 10 minutes at
room temperature) to lyse erythrocytes, and the suspension is passed through a 100-micron cell strainer to
remove debris. After an additional low-speed centrifugation, the cells are resuspended in growth media, such as
DMEM/F12 with 10% fetal bovine serum, for counting and are then placed in tissue culture flasks and grown
under typical conditions of 5% C02 at 37 C.
Enrichment of ASCs occurs through plating on uncoated tissue-culture plastic in the presence of a rich
medium that is not permissive for leukocyte and endothelial cell attachment; thus, the latter cells are removed
with a media change after overnight culture. After an initial lag phase, ASCs proliferate with doubling times of
36 to 48 hours, depending on the growth medium used. The cell surface marker profile of ASCs is altered with
their expansion. For example, the CD34 marker expression of cells in culture decreases rapidly and becomes
unmeasurable after two or three passages.34,36 Conversely, the CD105 and CD166 markers, which are
expressed at low levels in fresh isolates, increase during culture.51
The umbilical cord consists of two arteries and a vein supported by a surrounding matrix of gelatinous
connective tissue known as
Tissue-Derived Stem Cells
“Wharton’s jelly.” Stromal cells associated with the matrix include specialized fibroblast-like cells, mast
cells, and MSCs.52,53 Methods for isolation of UC-MSCs are varied and differ between laboratories; however,
in all cases, the vein and arteries are first removed, followed by processing of the remaining cord tissue into
smaller fragments (reviewed by Can and colleagues54). In some protocols, the interior of the cord is scraped to
remove the Wharton’s jelly before mechanical disruption. The pieces are placed in an enzymatic solution of
collagenase (typically Type I) and, depending on the protocol, other proteolytic enzymes to digest the matrix
and release the stromal cells. Various digestion times, from 30 minutes to 16 hours, have been employed,
depending on the enzymes used and their concentration.53
Alternative protocols involve the conduct of explant culture with the cord fragments.56 For protocols
involving enzymatic digestion, the homogenate is filtered through a 100-micron filter to remove debris. UC-
MSCs from the total cell population can be selected by either fluorescence-activated cell sorting or culture at
low density to identify colony-forming cells. Typical mesenchymal cell surface markers, such as CD90, CD105,
and CD73, are expressed by UC-MSCs; however, the level of expression varies depending on the isolation
method used.56
Isolation of amnion-derived Pl-MSCs is accomplished by first removing the chorion, followed by digesting
them at 37 C with either dispase II (2.4 U/mL for 1 hour) or trypsinEDTA (various concentrations and
incubation times have been used) to release the epithelial cells. The Pl-MSCs are then released through
subsequent digestion with collagenase (0.75 mg/mL) and deoxyribonuclease (0.075 mg/ mL).57 Human
chorionic Pl-MSCs are liberated similarly after removal of the surrounding layers and treatment with 2.4 U/mL
dispase II at 37 C, followed by 1 to 3 hours of treatment with collagenase II (270 U/mL) or collagenase A (0.83
mg/mL). For both chorionic and amnionic Pl-MSCs, the liberated cells are centrifuged at 200 x g for 10 minutes
to obtain the cell pellet, which is then resuspended, counted, and cultured.13 An alternative to digesting
760
AABB TECHNICAL MANUAL
 placental tissue is to isolate Pl-MSCs present in the amniotic fluid. This method relies on removal of
amniocytes by an initial culture period and then transferal of nonadhering Pl-MSCs to a new culture dish, where
they are allowed to adhere and proliferate.8 Each method for PlMSC isolation includes culturing to expand the
cells.58
DP-MSCs are isolated from the interior pulp chamber of extracted molars. The pulp tissue is removed from
the crown and root regions and then digested with a combination collagenase Type I (3 mg/mL) and dispase (4
mg/mL) for 1 hour at 37 C.59 Single-cell suspensions are separated from debris by passage through a 70-
micron filter. Cells proliferate well in a-MEM supplemented with 20% fetal calf serum (FCS), L-ascorbic acid
2-phosphate (100 pM), and L-glutamine (2 mM).60,61
Because extraction and digestion of tissues are not needed to isolate cells, EL-MSCs are a convenient
therapeutic cell source. Isolation of EL-MSCs is easily accomplished by culturing menstrual-blood-containing,
densitygradient-purified mononuclear cells on uncoated plastic vessels and selecting adherent cells after a brief
overnight incubation in complete media, such as DMEM supplemented with 20% FCS.14 Adherent EL-MNCs
are expanded in the same medium for multiple generations until sufficient numbers of cells are obtained.
M-MSCs are pluripotent stem cells that are distinct from committed progenitors (such as satellite cells in
skeletal muscle) and can be isolated from skeletal, smooth, and cardiac muscle tissue.62,83 The most common
technique to isolate M-MSCs is based on their adhesion characteristics and uses a process termed the “modified
preplate technique.” In this method, the muscle mass is isolated by a biopsy and dissected into pieces with razor
blades or scalpels. The tissue is dissociated using enzymes, such as collagenase II (1%) and dispase II (2.4
U/mL).84,65 The dissociated cells are then resuspended in culture medium, which is then placed in collagen-
coated flasks. Within minutes to hours of seeding, the first cells, including fibroblastic-like and myoblast cells,
attach. The supernatant containing the
nonadherent fraction is then transferred to a new collagen-coated plate. The slowly adhering cells, which
contain M-MSCs, are allowed to adhere and grow over several days to weeks, after which the proliferating cells
are expanded by passaging.
STANDARDIZATION OF METHODS FOR ISOLATION AND EXPANSION OF CLINICAL PRODUCT
Although many countries have implemented regulations governing the manufacture and use of stem cells
for cell therapy, there is still significant variation in the procedures used for isolation, expansion, preservation,
and shipping, which are critical components of clinical translation.86,67 It is imperative that cGMP
requirements and quality-control systems be used to ensure, to the greatest extent possible, consistency in stem
cell identity and potency.
These practices are established at most companies that are pursuing federal regulatory approval for off-the-
shelf allogeneic stem cell products. This regulatory approval requires careful documentation of manufacturing
processes and product characterization (termed “chemistry, manufacturing, and controls”). These companies
include Mesoblast Ltd. (Melbourne, Australia), Athersys Inc. (Cleveland, OH), Medistem Inc. (San Diego, CA),
and Pluristem Therapeutics Inc. (Haifa, Israel).
Autologous MSC therapies are also typically required to demonstrate consistency of a product for
regulatory approval when the cells are derived from multiple donors. Two complementary models for
autologous cell preparation are currendy employed: the centralized manufacturing facility and point-of-care
devices. The former is typified by Aastrom Biosciences Inc. (Ann Arbor, MI) and RNL Bio (Seoul, South
Korea). Devices for isolation in the operating theater are in development (and, in limited cases, approved for
marketing) by such companies as Cytori Therapeutics (San Diego, CA), Tissue Genesis Inc. (Honolulu, HI),
and Ingeneron (Houston, TX).
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CHAPTER 31
CELL PRODUCT BANKING AND MANAGEMENT OF SUPPLY CHAIN AND END USE
Manufacturing, storage/banking, and distribution are all critical for ensuring a steady supply of cellular
products to clinics around the world. Fortunately, these processes can be modeled on those already established
by the biological drug industry, which has more than two decades of experience with supply-chain optimization.
The biologies model must be adapted to cellular therapeutics to address key logistical considerations, such as
maintaining the viability of cellular therapeutics during shipping as well as after receipt at a clinical facility.
Many biologies are stored as a lyophilized powder or in suspension or solution at normal freezer or refrigeration
temperatures (ie, -20 C). Extended storage of cells requires temperatures below -80 C, and these cells are most
commonly stored in liquid-nitrogen tank facilities.
 Defining optimal and reproducible conditions for long-term storage, transport, and revival of MSCs is
critical for maintaining cell viability and, thus, potency. Defining these conditions is particularly important for
allogeneic MSCs produced in a centralized facility under cGMP controls but also needs to be addressed for
autologous cells that will be used for repetitive treatments and the avoidance of repeated extractions and
isolations of cells.
Good results with long-term storage at -196 C (temperature of liquid nitrogen) or <-150 C (temperature of
vapor-phase nitrogen) have been obtained with typical cell cryopreservation protocols using media containing
serum and dimethyl sulfoxide (DMSO), provided that cooling and thawing rates are carefully controlled.68
Although both DMSO and animal serum present concerns, there are currently few acceptable alternatives to
their use; thus, most protocols require that these media be depleted before the cells are used for the treatment of
patients.69'73
Clinical banking requires strict adherence to cGMP requirements for cell product processing, storage, and
shipment to the end user to ensure preservation of viability and
Tissue-Derived Stem Cells
therapeutic potency. Consequently, closedsystem containers are required to meet pharmaceutical quality
standards for packaging, storage, and distribution of cellular products at low temperatures. An ideal
cryopreservation container is stable despite exposure to a wide range of temperatures for extended periods and
allows selective access to the sample when desired for retrieval. As the demand for cell therapy increases,
commercial-scale cell therapy product manufacturing and banking will require the containers to be compatible
and integrated into the automated fill lines traditionally used in pharmaceutical settings to enable lot sizes of
several hundred to several thousand doses.
Although freezers that maintain cryopreservation temperatures are likely to be available in large hospitals,
especially those affiliated with universities, these freezers are not available within most clinical practices.
Smallerscale solutions, such as liquid-nitrogen Dewar flasks or dry ice in insulated coolers, can be employed for
relatively short-term storage; however, use of these flasks or coolers may be associated with logistical and
safety issues. Alternatively, a just-in-time approach may be taken where therapeutic cells are shipped directly
from centralized facilities in insulated containers that have sufficient dry ice to maintain a steady temperature
for a few days to a week. The cells are thawed immediately before use, washed to remove cryoprotective
agents, and resuspended in diluent for administration.
Another option is to use practices established for the shipment and short-term storage of tissue products. In
general, viable tissues are placed in biocompatible containers in contact with nutrient-rich media and shipped at
ambient temperatures within insulated containers via overnight courier service. The shelf life of certain tissues
packaged and shipped in this manner is approximately 1 week. An example is Apligraf (manufactured and
distributed by Organogenesis, Canton, MA), a living cell skin graft approved by the US Food and Drug
Administration for the treatment of chronic venous leg and diabetic foot ulcers. This product has a shelf life of
10 days after it is shipped.
762
AABB TECHNICAL MANUAL
Regardless of the method used to transfer cellular therapy products to the end user, there is a critical need to
manage the supply chain’s integrity and ensure that the cell transport and clinical site storage run efficiently
while adverse incidents are minimized. Particularly with autologous or type-matched therapeutics, and with
downward cost pressures due to increased competition and falling reimbursement rates, it is critical to avoid
errors and interruptions in the delivery of these therapies to hospitals and private clinics.
THERAPEUTIC APPLICATIONS OF MSCS
Cell therapy using BM-MSCs or ASCs is currently being investigated in clinical trials for a wide variety of
diseases that are not adequately addressed by current pharmacological or surgical approaches. These trials
include some that evaluate culture-expanded autologous and allogeneic MSCs and ASCs and others that involve
autologous ASCs that can be freshly isolated in the context of SVF using any of several point-of-care systems.
The number of clinical studies evaluating these therapies has steadily increased over the last decade, and the
number of publications relating to stem cell preclinical as well as clinical studies over time demonstrates this
trend.
 0 I l/ll II ll/lll III NR
Phase
FIGURE 31-1. Number of registered clinical trials of therapeutic uses of mesenchymal stem cells (as of
March 2014).
NR = not reported.
The majority of these studies are early-phase trials (Fig 31-1). As can be seen in Fig 31-2, the types of
diseases under investigation are wide ranging and affect essentially all organs and systems in the body. The
target populations include both pediatric (65 studies) and adult (286 studies) patients.
Immunomodulation and Skeletal Repair
The most intensively investigated class of disorders treated with MSCs is immune system diseases (58
trials), which include graft-vshost disease (GVHD; 23 trials) and autoimmune disorders, such as multiple
sclerosis

////i
CN CT
n*
& V'
&
* dP
/
Disease Category
FIGURE 31-2. Diseases for which mesenchymal stem cell therapy is under investigation (as of March
2014). CNS = central nervous system.
763
CHAPTER 31
(13 trials), Type 1 diabetes (10 trials), and lupus (4 trials).
The potential of MSC treatment for modifying these diseases is supported by a substantial volume of
literature demonstrating the immunomodulatory effects of these cells in preclinical studies of animal diseases.
Administration of MSCs significantly reduced the incidence and severity of GVHD after transplantation of
major-histocompatibility-complex-mismatched blood and tissues in animal models.74 77 These preclinical
results have been translated into seminal early trials and then successful Phase II and III clinical trials with BM-
MSCs for prevention and treatment of acute and chronic GVHD after allogeneic hematopoietic stem cell
transplantation.78'80 The success of the Phase II and III trials led to regulatory approval of the first licensed
stem cell therapy, Prochymal (Osiris Therapeutics), in North America for treatment of refractory GVHD. This
therapy is also available in Canada.
The discovery in 1998 that MSCs differentiate into cartilage under the influence of transforming growth
factor-beta ushered in a new era of investigations of MSC performance in a multitude of chondrogenic
conditions.81 At the time of publication, 28 clinical studies were evaluating the use of MSCs to treat
osteoarthritis and rheumatoid arthritis. Similarly, clinical trials have demonstrated that MSCs can be used
successfully to repair various bone defects, including critical size defects in the long bone, hard palate
reconstruction, and calvarial defects.82'84
Cardiovascular and Cerebral Diseases
MSCs are being investigated for stand-alone treatment of ischemic heart diseases as well as in combination
with coronary artery bypass surgery and left ventricular device implantation. Early open-label, uncontrolled
trials of MSCs and ASCs have had safety-related primary endpoints. The APOLLO trial demonstrated that the
intracoronary delivery of autologous SVF to patients with acute ST segment elevation myocardial infarction
was safe.85 The complementary PRECISE trial was a
Tissue-Derived Stem Cells
clinical trial of SVF administration in the treatment of symptomatic, nonrevascularizable ischemic
myocardium. The POSEIDON trial evaluated both allogeneic and autologous BMMSCs as a therapy for
ischemic cardiomyopathy and found that both cell types were safe when delivered directly into the
myocardium.86 Two Phase II randomized, double-blind, placebo-controlled trials of intramyocardially injected
autologous BM-MSCs for heart failure have been recently initiated in Europe and the United States to confirm
the positive effects of these cell treatments observed in early openlabel Phase I/II trials.87,88
Diseases of peripheral limb vascular insufficiency are also under clinical investigation as targets for MSC
therapy. In six patients with Buerger disease, 24 weeks of treatment with multiple intramuscular ASC injections
had positive clinical outcomes, including decreased pain.89 Intramuscular injection of ASCs also reduced
symptoms of diabetic foot and atherosclerotic obliterans.90 A randomized, placebo-controlled trial of BM-MSC
injected directly into the affected limb of patients with critical limb ischemia demonstrated safety as well as
indices of efficacy.91
Central nervous system trials involving MSC treatment have been dominated by investigations of the
treatment of ischemic stroke (nine trials).92 Additional diseases for which MSCs are being evaluated include
Alzheimer disease, Parkinson disease, spinal cord injury, amyotrophic lateral sclerosis, and multiple
sclerosis.93'96 The safety and efficacy of intravenous autologous BM-MSC infusion in patients with severe
middle cerebral artery ischemic stroke was evaluated in a 5-year follow-up study in which the mortality rate of
the treated group was lower than that of the control group, and no major side effects were observed during the
follow-up period.97
Inflammation Modulation and Barrier Repair
The antiinflammatory and proendothelial survival activities of MSCs suggest that they might have
therapeutic utility for diseases involving systemic inflammation and associated endo
764
AABB TECHNICAL MANUAL
thelial barrier dysfunction, such as sepsis, acute lung injury, and pancreatitis. Related potentially therapeutic
properties in this context include immunomodulation; differentiation into lung epithelial cells; secretion of
antimicrobial peptides; as well as other cytoprotective factors affecting endothelium, such as keratinocyte
growth factor and angiopoietin.98'101 Preclinical studies in murine models have shown that MSCs can be
beneficial in acute lung injury induced by lipopolysaccharide, pneumonia, or systemic sepsis.102 In one study,
ASC therapy decreased oxidative stress and minimized ischemia/reperfusion-induced damage to the rat
lung.103 Phase I trials of both BMMSCs and ASCs to treat acute respiratory distress syndrome are under way.
Intravenous UC-MSC therapy has also been shown to alleviate fibrosis, improve histologic scores, and decrease
inflammatory cytokine levels in rats with chronic pancreatitis.104
Gastrointestinal Diseases
Intestinal diseases are an additional category of disorders for which MSC treatment is currenffy undergoing
investigation. The largest number of trials (11) address Crohn’s disease. In a Phase I trial of 10 patients with
fistulizing Crohn’s disease, local injections of BM-MSCs resulted in complete fistula closure in seven patients
and partial closure in three.105 In 2012, a Phase III trial investigating the safety and efficacy of allogeneic
ASCs for the treatment of complex perianal fistulas in patients with Crohn’s disease was initiated.106
Treatment of end-stage, chronic liver diseases, primarily cirrhosis, using MSCs is under active investigation.
A Phase I/II clinical study, in which eight patients with end-stage liver disease were treated with
predifferentiated autologous BM-MSCs injected into either the portal vein or peripheral vein, showed
significant improvement in liver function and clinical parameters.107 A placebo-controlled trial of 30 patients
with decompensated liver cirrhosis demonstrated that systemic infusions of UC-MSCs were safe and improved
liver function at 1 year.108 An open-label trial of peripheral venous delivery of UC-MSCs to pa
tients with biliary cirrhosis also showed that the treatment was safe and improved liver function.109
Autologous BM-MSCs were administered to 53 patients with liver failure due to complications of hepatitis B
infection, and patient responses were evaluated at early and late intervals.110 Within 3 weeks of the infusions,
there was a significant improvement in liver function in treated patients compared to the 105 matched patients
admitted in the same time frame. Although indices of safety at the early and late time points (approximately 3.5
years) were no different in the treatment and control groups, the benefit of the treatment did not persist at the
late time point.
CURRENT RESEARCH AND DEVELOPMENT: FOCUS ON CELL CULTURE AND HANDLING
In the context of the positive results and lack of apparent safety concerns from numerous clinical studies
conducted on MSCs to date, there are many issues that must be routinely addressed before regulatory approval
and widespread adoption of MSCs in the clinical setting. For many culture protocols, animalderived
supplements, such as bovine serum (which is also commonly included in preservation media), have historically
been used as a source of growth factors and to facilitate postthaw cell culture. However, the use of bovine serum
and the xenogeneic proteins it contains could stimulate immune reactions in the host. In addition, bovine serum
must be obtained from sources that minimize the risk of transmitting prions and other as-yet-unidentified
zoonotic diseases.111,112 Furthermore, DMSO’s toxicity at concentrations currently used to cryopreserve
MSCs along with the undesirable osmotic shock to the cells during its addition and removal may render DMSO
undesirable to preserve MSCs intended for clinical use.
Considerable effort has been invested in identifying substitutes for bovine serum and DMSO; both of these
agents must be depleted by washing the cells upon thawing and before injection.69'73 For example, there has
been considerable effort directed at developing serum
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CHAPTER 31
free, or at least animal-serum-free, cryopreservation media containing cell protectants other than
DMSO.113,114 Strategies for eliminating or reducing the exposure of MSCs to DMSO and technologies to
remove DMSO from thawed cells have been proposed in the literature but rarely implemented in clinical
settings.
An important consideration that has restrained efforts to alter the growth conditions of MSCs is that such
modifications might alter the phenotype, genetic stability, or subsequent biological properties of MSCs.115'118
Alterations that could influence these properties are not limited to serum components because micronutrient
levels can also influence genomic stability and biological properties.119 A particular concern regarding the
modification of media constituents is that much of the preclinical and clinical data that underlie our knowledge
regarding the potential benefits and safety of MSCs may not be directly relevant to cells grown in medium
containing bovine serum substitutes.120 The absence of continuity between the extensive amount of previous
preclinical data on MSC safety and efficacy may increase the regulatory burden for obtaining licensing to
market MSCs as drugs.
In addition to addressing issues of manufacturing and storage, the adequate demonstration that exogenously
administered MSCs possess an exceedingly low potential to form tumors or promote endogenous tumor
formation has been required to obtain regulatory approval for MSCs. Published reports indicate a lack of
consensus on the tumor-formation potential of multipotent MSCs. Long-term cultures of MSCs have been
reported to be associated with genomic instability, resulting in the accumulation of genetic alterations that have
the potential to become manifest as malignant transformation.121'123 Conversely, other studies have found no
indication of chromosomal aberrations with extended cultures of MSCs and ASCs.124-127 These opposing
findings may be due to subtle differences in the MSC cultures examined, culture conditions, or methods for
detecting genetic abnormalities.
Regardless, tumor formation resulting from MSC or ASC treatment is indeed very rare
Tissue-Derived Stem Cells
and has been calculated to occur at a frequency of 10-9.128 There has been at least one report of MSC
transformation in vivo to form tumors.19 These studies emphasize the need to thoroughly test highly expanded
MSCs to be used in clinical trials for genetic alterations. These results also may suggest that, whenever
possible, lower-passage MSCs, or ASCs and umbilical MSCs, should be used because these cells require less
expansion due to their much greater availability after their initial recovery from tissue.
Tumorigenesis may also be promoted by the recruitment of MSCs to the tumor stroma. There are conflicting
reports regarding whether human MSCs support tumor formation. A number of investigations have shown that
MSCs enhance tumor growth, whereas others have reported that MSCs are associated with tumor
suppression.130-141 These discrepant results may be due to the different experimental systems used to assess
tumor promotion. In addition, the conditions for each MSC preparation might have differed in substantial ways
that influenced the outcomes.
Given this still-evolving understanding, it is important to assess each MSC preparation for tumorigenic
potential and carefully consider the exclusion of patients with active or recent cancer diagnoses in the context of
clinical applications.
CONCLUSIONS AND FUTURE DIRECTIONS
It is evident that the field of cell-based therapies holds great promise and is on the cusp of widespread
commercialization, which will have an extensive impact on the health-care field in the coming years. Today, the
potential market value of stem cell therapy has been estimated at $5.1 billion with adult stem cells holding a
majority share (more than 80%) of the market.142
 An important concern revolves around the potential expense of both autologous and allogeneic cell-based
therapies. Much emphasis over the last several years has focused on off-theshelf allogeneic cell therapy
products, which are considered to be more suitable for clinical
766
AABB TECHNICAL MANUAL
adoption and successful commercialization. However, allogeneic therapies may be challenging to produce
cost-effectively due to the complexities involved in their manufacturing and storage. For allogeneic use, cells
from a single qualified source must be massively expanded and stored for a long time to treat large numbers of
patients. Because successful commercialization is heavily influenced by costs, the manufacturing and banking
of these massive number of cells in a safe, robust, and costeffective manner while preserving key therapeutic
properties will be critical. These parameters are significantly affected by the source of material, isolation and
manufacturing methods, safety, banking, shipping, and tracking.
Finally, the successful use and ultimate potential for widespread adoption of these therapies will be affected
by regulatory requirements for the production and marketing of any cell-based therapies, including issues
ranging from product characterization to safety testing and clinical trial design. As the field continues to mature,
the regulatory pathway and its challenges are becoming progressively refined. It is hoped that this evolution will
continue to lead to the establishment of a betterdefined path for permitting the field to test the promise of cell
therapy more efficiently and meet the expected future demands for these treatments.
KEY POINTS
1. Multipotent mesenchymal stem cells (MSCs) can be harvested from a diverse array of tissues, including
marrow, adipose tissue, reproductive organs, placenta, dental pulp, and skeletal muscle.
2. The hallmarks of pluripotentiality and multipotentiality distinguish stem cells from progenitor cells.
3. The beneficial properties of these therapeutic cells appears to be more related to transitory effects
produced by secreted substances or brief contact with target cells rather than direct tissue replacement.
4. The key complementary properties that are of potential clinical use are immunomodulation, paracrine
effects, and differentiation.
5. The method of MSC isolation usually involves enzymatic dissociation, filtration, and lowspeed
centrifugation.
6. Early clinical experience suggests that cellular therapies are safe and effective; however, longer-term
studies are required to fully assess the durability of these effects and the potential for tumorigenicity.
7. Key to future commercial success will be developing well-characterized and consistent therapeutic cell
products through establishing strict controls for manufacturing, storage, and supply processes.
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pancreatic cancer cell death both in vitro and in vivo. PloS One 2009;4:e6278.
138. Maestroni GJ, Hertens E, Galli P. Factor(s) from nonmacrophage bone marrow stromal cells inhibit
Lewis lung carcinoma and B16 melanoma growth in mice. Cell Mol Life Sci 1999;55: 663-7.
139. Lu YR, Yuan Y, Wang XJ, et al. The growth inhibitory effect of mesenchymal stem cells on tumor cells
in vitro and in vivo. Cancer Biol Ther 2008;7:245-51.
140. Dasari VR, Kaur K, Velpula KK, et al. Upregulation of PTEN in glioma cells by cord blood
mesenchymal stem cells inhibits migration via downregulation of the PI3K/Akt pathway. PloS
0ne2010;5:el0350.
141. Secchiero P, Zorzet S, Tripodo C, et al. Human bone marrow mesenchymal stem cells display anti-
cancer activity in SCID mice bearing disseminated non-Hodgkin’s lymphoma xenografts. PloS One
2010;5:elll40.
142. Mason C, Brindley DA, Culme-Seymour EJ, et al. Cell therapy industry: Billion dollar global business
with unlimited potential. Regen Med 2011;6:265-72.
Chapter 32
Human Allografts and the Hospital Transfusion Service

Lance D. Trainor, MD, and Rita A. Reilc, MD

a»THE surgical use of human tis12Ifll sue allografts continues to expand. Every year, member organizations
of the American Association of Tissue Banks (AATB) recover tissue from more than 30,000 donors and provide
more than 2 million tissue grafts for transplantation.1 Many surgical specialties, including orthopedics, plastic
surgery, urology, neurosurgery, sports medicine, trauma, and reconstructive surgery, use human tissue.
Increasingly sophisticated grafts are being developed by tissue suppliers to meet diverse clinical needs.
There is no regulatory requirement for the activities of a tissue-dispensing service to be managed by any
particular individual or department within a hospital. However, because ordering, receiving, storing, dispensing
(issuing), tracking, tracing, investigating adverse events, and managing recalls are functions performed by both
transfusion and tissue-dispensing services, the AABB recommends a centralized tissue-dispensing model
located within the transfusion service.2
TISSUE TRANSPLANTATION
Allografts are selected for transplantation based on intrinsic qualities that meet the surgeon’s functional
requirements for the patient. Bone, tendons, and corneas are the most frequently implanted human tissues;
others include cartilage, skin, veins, dura mater, fascia, and heart valves. Most tissues are procured from
deceased donors within 24 hours of death after appropriate consent (also known as “authorization”) is obtained
from a designated legal authority (eg, the donor’s next of kin) or by first-person authorization if the donor
registered his or her wishes before death. Strict adherence to aseptic surgical recovery techniques minimizes
contamination of tissues with microorganisms from the donor’s skin and intestinal flora as well as the
surrounding environment.
 Responsible persons at tissue banks determine donor eligibility based on an evaluation of 1) answers
provided by the next of kin or other knowledgeable person to questions
Lance D. Trainor, MD, Divisional Medical Director, LabCorp, Dublin, Ohio, and Rita A. Reik, MD, Chief
Medical Officer, OneBlood, Lauderhill, Florida The authors have disclosed no conflicts of interest.
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about the donor’s travel and medical history and any high-risk behaviors, 2) available medical records, 3)
the autopsy report (if an autopsy was performed), 4) a thorough physical assessment of the donor to identify
evidence of highrisk behaviors or active communicable disease, 5) the suitability of any blood sample collected
for infectious disease testing, and 6) the circumstances of death. A history of high-risk activity that increases the
possibility of transmission of disease leads to the rejection of the donor. Allografts, similar to blood products,
are released for transplantation only after the donor has been tested for relevant communicable diseases and the
results are determined to be acceptable (see Table 32-1).
Background and Definitions
Human tissue banking in the United States started more than 60 years ago at the US Navy Tissue Bank. The
use of tissue allografts, now common, has evolved into a highly innovative and continuously changing field.
Collaboration between tissue bank scientists and enduser surgeons has produced a wide variety of life-saving
and life-enhancing tissue grafts.
Allografts are tissues transferred between individuals of the same species. Allografts may be processed to
remove cells or carefully preserved to maintain cellular viability. They can be derived from a single tissue or
multiple tissues acting as a functional unit. During pro
cessing, human tissue allografts may be combined with other biocompatible agents to achieve desired
handling and functional characteristics. Bone allografts, due to their hard and rigid nature, can be precisely
machined for compatibility with surgical instrumentation.
Autografts are tissues implanted into the individual from whom they were removed. For example, bone that
has been surgically removed from a patient’s ilium can be shaped to desired dimensions and implanted into the
vertebral disk space of the same patient. The autograft bone promotes fusion of adjacent vertebrae, providing
stability and relief from the pain of degenerative disc disease or trauma. Advantages of autografts include the
elimination of communicable disease transmission risk and the ready availability of graft material.
Disadvantages include morbidity associated with the additional surgical procedure for the patient, including
pain and potential surgical-site infection. In addition, the quality (eg, strength) and quantity of autologous tissue
may not be adequate for the intended use, and removal of the patient’s tissue may adversely affect function at
the site from which it was removed.
Isografts, which are tissues transferred between genetically identical individuals, such as identical twins, are
uncommonly used.
TABLE 32-1. Required Infectious Disease Testing of Human Allografts3
Infectious Agent Test Performed
Hepatitis B surface antigen Hepatitis B core antibody (IgM and
Hepatitis B
IgG)
Hepatitis C (HCV) Hepatitis C antibody HCV nucleic acid testing
Human immunodeficiency virus (HIV) HIV-1 and HIV-2 antibodies HIV-1 nucleic acid testing
Human T-cell lymphotropic virus
HTLV-I and HTLV-II antibodies
(HTLV)*
Syphilis Nontreponema- or treponema-specific assay
‘Required for tissues that are rich in viable leukocytes only.
 775
Xenografts are tissues transplanted from one species to another; a growing number of medical products are
being developed from highly processed nonhuman animal tissues.
Tissue Processing
After tissue has been procured from a deceased donor, it is processed using aseptic technique in a controlled
environment in a facility designed to prevent contamination or cross-contamination. Similar to good
manufacturing practice, good tissue practice (GTP) requires the facility to establish and maintain controls over
temperature, humidity, ventilation, and air filtration. Thorough cleaning and disinfection of processing rooms
and equipment are required to ensure a proper environment.
Tissues are debrided of extraneous soft tissue and cut to specification. In some cases, more than 100 grafts
can be produced from a single donor. Precision bone grafts, including a growing array of spinal implants, are
crafted using sophisticated, computer-aided cutting devices to meet the exacting specifications required by
surgical instrumentation; precisely milled allografts allow surgeons to operate more quickly and efficiently.
During processing, various solutions, including antibiotics, alcohols, and surfactants, may be used to reduce
or eliminate bacterial contamination and remove extraneous lipids and other biologic material. Depending on
the type of graft, graft sterilization may or may not be possible; grafts containing viable cells or a fragile matrix
cannot be subjected to sterilization methods without destruction of cellular or matrix integrity. Ionizing
radiation is the most frequently used sterilization technique. Ethylene oxide and proprietary methods of tissue
sterilization may also be used by tissue processors.
Several methods of tissue preservation are available for long-term storage of human allografts, including
freezing and lyophilization (freeze drying). Both processes destroy cell viability. Tissue integrity can be
enhanced through cryopreservation, a process in which tissues are frozen in a protective medium at a
steady, controlled rate. The use of cryoprotectants, such as glycerol or dimethyl sulfoxide, minimizes cell
damage caused by cell shrinkage and intracellular ice formation during freezing.
 Refrigerated storage can preserve cellular viability in osteochondral allografts for which preservation of
living chondrocytes is essential. These grafts, which consist of an intact articular surface with associated soft
tissue and bone, are used for treating traumatic and degenerative joint conditions. Refrigeration is not suitable
for long-term storage of allografts.
Clinical Uses of Allografts
Allografts are used in a variety of surgical procedures. Cadaveric human bone can be used to replace bone
lost to degenerative disease, trauma, or malignancy. Allogeneic human bone has unique healing characteristics,
including osteoconductive and osteoinductive properties. In vivo, it acts as a scaffold that allows recipient
capillary growth into the graft (osteoconductivity) and provides stimulation for the production of new bone
(osteoinductivity) by exposing the patient’s osteogenic progenitor cells to bone morphogenetic proteins (BMPs),
growth factors in bone that induce new bone formation. The result is creeping substitution, in which bone
remodeling occurs through osteoclastic resorption of the implanted tissue and osteoblastic generation of new
bone.
A variety of human tissues are used for transplantation.4 Selected clinical uses are listed in Table 32-2.
Bone-tendon (Achilles tendon) or bone-ligament-bone (patellar-tibia ligament) grafts are routinely used for
anterior cruciate ligament repair. The implanted tendon or ligament spans the joint space, and the bone provides
an anchor into the femur and/ or tibia, restoring joint stability. Crushed bone subjected to acid demineralization
can be used alone or suspended in a biologically compatible carrier and applied to exposed bone surfaces.
BMPs in demineralized bone stimulate osteogenesis, fusion of adjacent bones, and healing. Cadaveric skin can
be used as a temporary wound dressing for severe
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TABLE 32-2. Clinical Uses of Selected Human Allografts
Allograft Tissue Surgical Use
Amnion Wound covering Conjunctiva surface repair Corneal ulcer repair
Skeletal reconstruction
Bone (cortical, corticocancellous,
Spinal fusion
and cancellous)
Dental implant placement
Bone-tendon (Achilles tendon)
Bone-tendon-bone (patellar ligament) Anterior cruciate ligament repair Posterior cruciate ligament
Tendon (semitendinosus, gracilis, repair Rotator cuff restoration Biceps tendon rupture repair
and peroneus longus)
Cardiac valves (aortic and
Valvular insufficiency correction Congenital cardiac defect repair
pulmonary)
Cartilage (costal) Facial reconstruction
Keratoconus repair Traumatic scarring reversal Corneal ulcer
Cornea
excision
Hernia repair
Decellularized skin
Soft tissue reconstruction Gingival restoration
Demineralized bone Dental implant placement Spinal fusion
Dura mater Dural defect/cerebrospinal leak repair
Fascia lata Soft tissue reconstruction Pelvic floor support
Meniscus Meniscus replacement
Osteoarticular/osteochondral graft
Joint restoration
(bone and joint cartilage)
Dura patch
Pericardium Eyelid reconstruction
Soft tissue reconstruction
Sclera Eye enucleation Scleral ulcer repair Eyelid repair
Skin Protection of underlying tissues from severe burns
Coronary artery bypass grafting Tissue revascularization
Veins/arteries
Aneurysm repair Dialysis access shunts
 111
burns, protecting underlying tissues from dehydration and environmental pathogens. Human skin can be
processed to remove cellular elements, producing an acellular collagen matrix that provides a scaffold for
revascularization and cellular incorporation in soft tissue reconstructive surgery. In addition, donor tissues can
be used to treat a variety of ocular conditions. Thinning, scarring, or clouding of the cornea may be treated by
corneal transplantation. Sclera and amnion-derived allografts can be used to treat glaucoma, scleral ulcers, and
traumatic injuries.
Although bone and soft tissue allografts may provoke a recipient immune response, such responses are not
usually clinically significant, presumably due to a lack of residual cellular material in processed grafts.5
Therefore, it is not necessary to match most allografts to the recipient’s HLA or ABO type. Possible exceptions
include cryopreserved veins and arteries, for which some clinicians request ABO compatibility, and frozen,
unprocessed bone allografts containing marrow or red cells. Development of Rh(D), Fy(a), and Jk(b) antibodies
in recipients following transplantation of unprocessed bone allografts has been documented.6 If unprocessed
bone is to be used for an Rh(D)-negative female of childbearing potential, her future offspring may be at risk of
hemolytic disease of the fetus and newborn. Rh Immune Globulin prophylaxis should be considered if the Rh
type of the red cells or marrow contained in the allograft is positive or unknown.
Disease Transmission through Tissue Transplantation
Rare, sporadic transmissions of infectious diseases, including human immunodeficiency virus (HIV),
hepatitis C virus (HCV), hepatitis B virus (HBV), and Creutzfeldt-Jakob disease (CJD), have been documented
following tissue implantation. In addition, bacterial and fungal infections from allografts have resulted in
morbidity and death. Potential sources of contaminating agents include donor flora, premortem infection, and
environmental contaminants in recovery and processing fa
 cilities. Advances in screening, testing, and processing methods continue to improve the safety profiles of
human tissue products.
REGULATIONS AND STANDARDS
The Food and Drug Administration (FDA) regulates the activities of tissue banks and tissue distribution
intermediaries, which are establishments or persons engaged in the recovery, screening, testing, labeling,
processing, storage, and/or distribution of human tissues for clinical use, under Title 21, Parts 1270 and 1271 of
the Code of Federal Regulations (CFR).3 These entities manufacture what the FDA classifies as human cells,
tissues, and cellular and tissue-based products (HCT/Ps). Examples of common tissue products are listed in
Table 32-3.
The three rules of 21 CFR Part 1271 concern 1) registration of tissue bank establishments, 2) donor
eligibility, and 3) GTP related to handling HCT/Ps. Compliance with current GTP regulations is required of
tissue banks and tissue distribution intermediaries to control contamination and cross-contamination that may
lead to transmission of disease. Tissuedispensing institutions, such as hospitals, dental offices, and surgical
centers that provide and use tissue within their own facility are not subject to this oversight except under certain
circumstances (ie, redistribution of allografts or autografts to affiliated institutions located at a different address
or attempts to sterilize autografts).
For a hospital-based tissue-dispensing service, regulatory oversight is governed by voluntary accrediting
organizations. Tide 21, CFR Parts 1270 and 1271 do not apply to facilities that only receive, store, and dispense
tissue, and engage in no further manufacturing or redistribution of the tissue product. However, The Joint
Commission, AABB, College of American Pathologists (CAP), Association of perioperative Registered Nurses
(AORN), AATB, and Eye Bank Association of America (EBAA) all publish standards that apply to the
practices of tissue-dispensing services.8'13 The
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TABLE 32-3. Examples of Common Cell and Tissue Products8,14
Amniotic membrane
Bone and demineralized bone matrix
Bone marrow
Bone paste, powder, or putty Cancellous chips Cardiac (heart) valves Cartilage Cornea
Dermal matrix Dura mater Embryos Fascia
Hematopoietic stem cells
Ligaments
Meniscus
Ocular tissues
Oocytes
Pericardium
Peripheral blood stem cells Sclera
Semen and sperm Skin
Tendons
Umbilical cord blood stem cells Vascular grafts (veins and arteries)
location of a tissue-dispensing service in a hospital or medical facility and the scope of its responsibilities
dictate which standards are applicable. All of these standards are updated regularly; therefore, a periodic review
of the most recent versions is required to guarantee ongoing compliance.
The AATB and EBAA are voluntary accrediting organizations dedicated to ensuring that human tissues
intended for transplantation
are safe, available, and of high quality. AATB’s standards pertain to institutional and quality program
requirements; donor authorization/ informed consent; donor screening and testing; and tissue recovery,
processing, release, and distribution.12 EBAA’s scope encompasses all aspects of eye banking.13 Accreditation
by these organizations is based on verified compliance with established standards and periodic inspections.
Both organizations serve as scientific and educational resources for the donation and transplantation
communities. A tissue-dispensing service may find AATB and EBAA accreditation of a supplier to be valuable
in assessing that supplier’s qualifications.
HOSPITAL TISSUE SERVICES
 There is no requirement for a tissue-dispensing service to be managed in any particular department or by
any specific individual. The AABB supports a tissue-dispensing service within the transfusion service, which
has expertise in providing human-derived products that are perishable, potentially infectious, and sometimes in
short supply and that require temperature-controlled storage. The tasks of ordering, receiving, storing,
distributing (issuing), tracking, and tracing products as well as investigating adverse events, including
complaints, recalls, and look-back investigations, are activities that are common to the transfusion service and
tissue-dispensing service.
Responsibility for Hospital-Based Tissue Services
The Joint Commission requires that organizations assign oversight responsibility for their tissue program,
use standardized procedures in tissue handling, maintain traceability of all tissues, and have a process for
investigating and reporting adverse events. Either a centralized or decentralized process is permitted to manage
these activities. In either model, designated oversight is required to coordinate tissue-related activities and
ensure standardization of practices throughout the organization. The Joint Commission standards apply to
779
human and nonhuman cellular-based transplantable and implantable products, including both tissue
allografts and certain medical devices, as classified by the FDA.
Written Standard Operating Procedures
Hospital tissue services must have written procedures (printed or electronic) for all functions pertaining to
the acquisition, receipt, storage, issuance, and tracing of tissue grafts as well as procedures for investigating
adverse events and handling recalls. Manufacturers’ instructions for handling tissues should be incorporated
into standard operating procedures (SOPs). When the blood bank or transfusion service is responsible for tissue,
the AABB requires the medical director to approve all medical and technical policies and procedures pertaining
to tissue.9(p2)
Tissue Supplier Qualifications and Certification
In contrast to blood banks, tissue processors and distributors often specialize in providing particular types of
products (grafts). As a result, a hospital tissue-dispensing service may need to acquire human tissue products
from more vendors than the number of vendors from which a transfusion service commonly obtains blood
products.
Tissue suppliers should be selected based on their ability to reliably provide high-quality tissues that meet
expectations for availability, safety, and effectiveness. The tissue service should establish minimal criteria for
the qualification of prospective suppliers. According to The Joint Commission, tissue supplier requirements
must include evidence of current FDA registration and state licensure if such licensure is required. A written
process for review and approval of suppliers is expected and should contain the elements listed in Table 324. A
list of approved suppliers that includes documentation of each supplier’s qualifications, certifications, and
appropriate licenses or permits should be developed and maintained. Tissue services should establish proce
dures to receive or monitor evidence of compliance or noncompliance, such as reviewing FDA warning
letters and tissue allograft recalls or market withdrawals. Accreditation by AATB and/or EBAAmaybe
desirable.
Qualification information for each supplier should be reviewed and approved annually by the hospital tissue
service. During these reviews, the performance of suppliers in meeting the transplant facility’s needs should be
evaluated. Each year, the tissue service should determine whether the supplier remains registered with the FDA;
whether AATB and/or EBAA accreditation is current can also be confirmed. FDA web postings should be
reviewed for information related to closures, recalls, or MedWatch reports. FDA inspection reports on tissue
processors can be requested from the FDA through the Freedom of Information Act. Complaints from
transplanting surgeons concerning the supplier’s tissue should be reviewed along with any reports of infections
that might have been caused by transplanted allografts. Further use of a particular tissue supplier should depend
on approval by the tissue service’s director. Hospital management may consider establishing a committee of
internal stakeholders, including transplant physicians, to provide oversight in the approval of tissue suppliers.
Inspection of Incoming Tissue Allografts
Before being placed in storage, tissue allografts must be inspected upon receipt from a tissue supplier to
ensure that the packaging remains intact and the label is complete, appears to be accurate, and is adequately
affixed and legible before acceptance into inventory. The incoming inspection results should be recorded along
with the date, time, and name of the staff person who conducted the inspection.
 The Joint Commission requires that hospitals verify package integrity and ensure that the transport
temperature was controlled and acceptable. Inspection of the shipping container for evidence of residual coolant
(eg, wet ice for refrigerated grafts or dry ice for frozen grafts) may be useful to determine that the re
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TABLE 32-4. Vendor Qualification Criteria for Human Allografts
Criterion Documentation/Performance
FDA registration Current Form FDA 3356 /eHCTERS Query
FDA inspection findings Form 483 findings, if any
Warning letters and responses, if any
Voluntary accreditation, if
Current AATB accreditation for tissues
available
Current EBAA accreditation for ocular tissues
State license and registration,
Copy of state license and registration (to be reviewed annually)
if required by state law
Reliable supply of needed
Adequate notification of tissue shortages
tissues
Ability to meet special requests
Suitable expiration dates for tissue products
Transparency of the Willingness to provide information regarding donorselection and tissue-
organization processing processes
Medical consultation Accessibility of the tissue supplier’s medical director
Quality assurance resources Accessibility of the tissue supplier’s quality assurance staff
New or trial tissue product
Willingness to provide information on newly released tissue products
support
Professionalism of sales Approval sought by representatives through designated channels before
representatives promoting or providing tissue within the hospital
FDA = Food and Drug Administration, AATB = American Association of Tissue Banks, EBAA = Eye Bank
Association of America; eHCTERS = Human cell and tissue establishment registration.
quired tissue-specific storage environment was maintained during transportation.
Many distributors use “validated” shipping containers that are tested to maintain required temperatures for a
specified period. If such a container was used, the receiver of the tissue simply needs to verify that there is no
damage to the container and that it has been received and opened within the specified time frame.
Tissues requiring “ambient temperature” (defined as the temperature of the immediate environment) for
storage and shipping do not need to have the temperature verified upon receipt. However, the temperature of
tissues requiring “room-temperature” storage should be verified and documented if the manufacturer
has specified a temperature storage range in the package insert.
Tissue Storage
As with blood components, tissue grafts are stored under various conditions (see Table 325). The
appropriate storage conditions depend on the nature of the tissue, method of preservation, and type of
packaging.
Hospital tissue services should store tissue allografts according to the processor’s instructions in the package
insert. Storage devices can include “ambient” and/or roomtemperature cabinets, refrigerators, mechanical
freezers, and liquid-nitrogen storage units. Continuous temperature monitoring of refrig
 781
TABLE 32-5. Storage Conditions for Commonly Transplanted Human Tissue12
Human Tissue Storage Conditions Temperature*
Cardiac and
Frozen, cry op reserved -100 C or colder
vascular
Above freezing (0 C) to
Musculoskeletal Refrigerated
10 C
Frozen, cryopreservedand noncryopreserved (temporary
-20 C to -40 C
storage for 6 months or less)
Frozen, cryopreserved and noncryopreserved (long-term
-40 C or colder
storage)
Lyophilized Ambient1
Liquid nitrogen (liquid or
Reproductive Frozen, cryopreserved
vapor phase)
Above freezing (0 C) to
Skin Refrigerated
10 C
Frozen, cryopreserved -40 C or colder
Lyophilized Ambient1
‘Warmest target temperature unless a range is listed.
fAmbient temperature monitoring not required for lyophilized tissue.
erators and freezers is required. Room-temperature storage equipment need only be monitored if this is
required by the allograft’s package insert. Storage equipment should have functional alarms and emergency
backup capability. Lyophilized tissues whose package insert instructions specify ambient temperature or colder
storage can tolerate a very broad range of temperatures and their temperatures do not require monitoring.
Storage SOPs should address steps to be taken in case of excursions from allowable temperature limits or in
the event of equipment or power failure. Emergency backup alternatives, including arrangements for temporary
storage, should be described in written procedures.
Tissue Allograft Traceability and Record Keeping
Proper management of human allografts requires that the hospital tissue service document all of the steps
taken in tissue handling as they occur and maintain comprehensive
records of these steps. The records should be accurate, legible, and indelible. They must identify the staff
who have handled the tissue and the dates when the tissue was handled as well as the time when the tissue was
accepted, prepared, or processed. The records should be detailed and provide a clear history of all actions
performed. Documentation of the tissue supplier, unique numeric or alphanumeric identifier(s) of the allograft,
its expiration date, and the recipient’s name must be maintained for all tissue grafts used.
Tissue service records need to permit bidirectional traceability of all tissues from the donor and tissue
supplier to the recipient(s) or other final disposition, including the discard of tissue because of damage to
package integrity, opening of the tissue package in the operating room without use, or expiration. Records
should be retained for 10 years, or longer if required by state or federal law, after distribution, transplantation,
discard, or expiration (whichever occurs last).

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Tissue usage information cards or other systems supplied with the allograft by the tissue bank must be
completed and returned to the tissue source facility. This information helps maintain the traceability of the
allograft and expedites market withdrawals or recalls when necessary. The tissue supplier may also use this
information to better understand allograft utilization, obtain positive or negative feedback, and better meet the
needs and expectations of customers.
Recognizing and Reporting Adverse Events Possibly Caused by Allografts
Use of human-derived medical products, such as tissue allografts, carries risks that must be balanced with
clinical benefits. Human allografts have been associated with bacterial, viral, fungal, and prion transmissions. In
addition, allografts may have structural defects that sometimes lead to unsuccessful outcomes as a result of
donor selection or processing factors.
Hospital tissue services are required to have procedures to investigate in a timely way any infections
suspected to be caused by a tissue allograft. The Joint Commission requires that allograft-transmitted infections
and other severe adverse events be immediately reported by the hospital to the tissue supplier.
Transplant surgeons play a critical role in the identification of allograft-associated adverse outcomes and
need to immediately notify the hospital tissue service when they suspect such events. Prompt notification of
adverse events enables the hospital tissue service to investigate the cause, report the issue to the tissue supplier,
and institute corrective action, including sequestration of any other suspect allografts. The investigation of
infections and other adverse events requires cooperation between the tissue-dispensing service, clinicians, and
tissue supplier. Consultation with the hospital infection-control department or an infectious disease specialist
may be beneficial. Early notification can prevent an untoward outcome for other recipients of allografts from an
implicated donor.
State health departments have lists of communicable diseases that must be reported when they are newly
diagnosed. A new diagnosis of HIV or viral hepatitis in a tissue allograft recipient where the allograft is
suspected as a possible source must be reported to the state department of health by the patient’s physician or
the director or medical director of the hospital tissue service. An epidemiologic investigation may be needed to
establish whether the tissue allograft was the source of the recipient’s infection. Early notification of the state
department of health can result in timely assistance with the investigation.
Recalls and Look-Back Investigations
A tissue product recall or market withdrawal occurs when a tissue allograft is determined by the tissue
supplier to be compromised or potentially infectious. The supplier may recall all of the tissues from a specific
donor or processing lot, sequester tissues in inventory, and notify hospitals to which the allografts were shipped.
The hospital may be instructed by the supplier to quarantine the allografts in inventory, identify recipients,
and/or notify the transplanting surgeon(s) of the recall. Surgeons should evaluate the circumstances of the recall
and notify the recipient as appropriate that a tissue graft was recalled.
Look-back investigation can be triggered when a tissue donor is subsequently found to have been infected
with HIV human T-cell lymphotropic virus Types I or II, HBV, HCY or other infectious disease known to be
transmitted by tissue grafts. Because most tissues are procured from deceased donors, infectious disease testing
of subsequent donor blood samples is not possible. Look-back investigations involving tissue grafts are
uncommon.
Tissue Autograft Collection, Storage, and Use
Surgical reconstruction using the patient’s own tissues often has advantages over the use of cadaveric tissue.
These advantages include ready availability, faster incorporation, appro
CHAPTER 32 Tissue Services in the Hospital
783
priate size or shape, and relative safety from viral disease transmission.
A bone flap removed during decompressive craniectomy is a commonly used example of tissue autograft
use. In this procedure, a section of skull is excised by the neurosurgeon to reduce intracerebral pressure caused
by brain swelling due to trauma, stroke, or surgery. After removal, the skull fragment is rinsed, packaged,
frozen, and stored for future reimplantation during a procedure known as “cranioplasty.”
Written procedures should address the collection, microbial testing, packaging, storage, and issuance of
tissue autografts for reimplantation. Bacterial testing by obtaining appropriate cultures should be performed
after surgical removal and before packaging. Autografts should not be collected from patients with systemic
infections or if the tissue is in close proximity to an infected area. Autografts may be stored at the medical
facility where they are collected or at an off-site, FDA-registered tissue bank. Procedural recommendations
have been published by the AATB and AORN.
TRANSFUSION SERVICE SUPPORT FOR ORGAN TRANSPLANTATION
Organ transplantation relies on the coordinated activities of organ-procurement organizations (OPOs) and
the hospital transplant team. A successful transplant program requires an interdisciplinary team that has well-
defined policies, procedures, and communication pathways. The transfusion service provides appropriate
compatibility testing and transfu
sion support before, during, and after transplantation.
OPOs evaluate potential donors, obtain authorization (consent), and prepare organs for transportation. The
United Network for Organ Sharing (UNOS) coordinates the US organ transplant system. The evolving UNOS
guidelines for organ allocations are designed to achieve equitable distribution of life-saving organs. Factors
such as severity of recipient illness, geographic proximity, and donor-recipient compatibility are considered.
UNOS provides detailed policies and other relevant information on its website.15
Depending on the organ, ABO and/or HLA compatibility may be important factors for transplantation
success. ABO antigens expressed on the vascular endothelium of organs constitute strong histocompatibility
barriers. Major incompatibility between donor ABO antigens and recipient plasma can result in acute humoral
rejection of the transplanted organ and threaten a successful outcome. As a result, UNOS requires two separate
determinations of ABO type for both 1) transplant candidates prior to listing their needs on the Organ
Procurement and Transplantation Network waiting list and 2) donors prior to making an organ available for
transplant. ABO-incompatible organ transplants are sometimes performed following a conditioning regimen
that may include plasma exchange.
Services that may be required by a transplant program include provision of cytomegalovirus-reduced-risk
components, blood irradiation, massive transfusion support, ABO subgroup typing, and immunohematology
reference testing. Transfusion services should understand and address such expectations of the transplant team.
KEY POINTS
1. Surgical use of human allografts has grown dramatically in recent years, with more than 2 million tissue
grafts used annually in the United States. The majority of these grafts are obtained from deceased human donors
who meet stringent screening requirements similar to those applied to blood donors.
2. Not all tissue allografts are sterile. Depending on the type of allograft, sterilization may not be possible
because it could jeopardize the viability of the cellular elements or fragile matrix
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of the graft and adversely affect in-vivo performance. Methods of sterilization include the use of ionizing
radiation or ethylene oxide and some proprietary processes and techniques.
3. Rare examples of HIM HCM HBV, CJD, and bacterial and fungal disease transmission from allografts
have been documented. Therefore, like blood components, allografts are released for use only after infectious
disease testing has been completed and the results deemed acceptable.
4. In general, bone and soft tissue allografts do not need to be matched for HLA or ABO type.
5. Hospital-based tissue services are not subject to FDA regulatory oversight if their activities are limited to
receiving, storing, and dispensing tissue to a requesting surgeon. However, The Joint Commission, AABB,
CAP, AORN, AATB, and EBAA publish standards that apply to such services.
6. Tissue banks are engaged in the recovery, screening, testing, labeling, processing, storage, and
distribution of human tissues. They are regulated by the FDA under the Title 21, CFR Parts 1270 and 1271 as
manufacturers of HCT/Ps.
7. No regulatory or standard-setting organization requires a hospital tissue-dispensing service to be managed
by a particular department or individual. However the AABB favors a centralized model for managing this
activity within the transfusion service.
REFERENCES
1. American Association of Tissue Banks. About AATB. McLean, VA: AATB, 2013. [Available at
http://www.aatb.org/About-AATB (accessed December 8,2013).]
2. Eastlund DT, Eisenbrey AB for the Tissue Committee. Guidelines for managing tissue allografts in
hospitals. Bethesda, MD: AABB, 2006.
3. Code of federal regulations. Title 21, CFR Parts 1270 and 1271. Washington, DC: US Government
Printing Office, 2014 (revised annually).
4. Woll JE, Smith DM. Bone and connective tissue. Clin Lab Med 2005;25:499-518.
 5. Malinin TI. Preparation and banking of bone and tendon allografts. In: Sherman OH, Minkoff J, eds.
Arthroscopic surgery. Baltimore, MD: Williams and Wilkins, 1990:65-86.
6. Cheek RF, Harmon JV Stowell CP. Red cell alloimmunization after a bone allograft. Transfusion
1995;35:507-9.
7. Eastland T, Warwick, RM. Diseases transmitted by transplantation of tissue and cell allografts. In:
Warwick E, Brubaker SA, eds. Tissue and cell clinical use: An essential guide. West Sussex, United Kingdom:
Blackwell, 2012:72-113.
8. The Joint Commission. Transplant safety. In: Comprehensive accreditation manual for hos
pitals: The official handbook. Oakbrook Terrace, IL: The Joint Commission, 2014:TS1.
9. Levitt J, ed. Standards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB, 2014.
10. College of American Pathologists. Standards for laboratory accreditation. Northfield, IL: CAR 2012.
11. Association of Perioperative Registered Nurses. Perioperative standards and recommended practices.
Denver, CO: AORN, 2013.
12. Dock N, Osborne J, Brubaker S, et al. Standards for tissue banking. 13th ed. McLean, VA: American
Association of Tissue Banks, 2012.
13. Eye Bank Association of America. Medical standards. Washington, DC: EBAA, 2011.
14. Food and Drug Administration. FDA regulation of human cells, tissues, and cellular and tissue based
products (HCT/Ps) product list. Rockville, MD: Food and Drug Administration, 2013. [Available
atwww.fda.gov/Biologics BloodVaccines/TissueTissueProducts/Regula tionofTissues/ucml50485.htm (accessed
November 13,2013).]
15. United Network for Organ Sharing. Richmond, VA: UNOS, 2013. [Available atwww.unos.org (accessed
December 8,2013).]

Chapter 33
Blood and Marrow-Derived Nonhematopoietic Stem Cell Sources and Immune Cells for Clinical
Applications

Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc; and

Garnet Suck, PhD, MSc
 hematopoietic stem cell IBS transplantation (HSCT) is an established modality of treatment for a wide range
of hematologic diseases, including leukemias and lymphomas. One of the most powerful mechanisms for cure
is the presence of the graft-vstumor (GVT) or graft-vs-leukemia (GVL) effect, which is mediated largely by
mature immune effector cells, such as T and natural killer (NK) cells.1 The immune response that is generated
is derived from a complex system of interactions involving antigen recognition and presentation mediated by
dendritic cells (DCs). DCs are also known as “professional antigen
presenting cells” and are key to orchestrating the adaptive immune response in an antigenspecific manner.
The success of HSCT has led to enormous interest in the isolation, characterization, expansion, and
modification of these immune cells to augment the powerful GVT response. Most of these cells form part of the
hematopoietic stem cell (HSC) graft infused into patients and can be derived from blood.
In fact, the discovery of the GVT effect came from the observation that depleting T cells from the graft
resulted in a greater risk of leukemia relapse. Conversely, the occurrence
Mickey B. C. Koh, MD, PhD, Director, Stem Cell Transplantation, St. George’s Hospital and Medical
School, London, United Kingdom, Medical Director, Cell Therapy Facility, Health Sciences Authority,
Singapore; Edward R. Samuel, PhD, MSc, Clinical Scientist and Senior Research Associate, Department of
Haematology, University College London Medical School, Royal Free Campus, London, United Kingdom; and
Garnet Suck, PhD, MSc, Division Director, Productions, Institute for Transfusion Medicine, German Red Cross
Blood Donation Service North-East nonprofit GmbH, Berlin, Germany The authors have disclosed no conflicts
of interest.
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of graft-vs-host disease (GVHD), which is also mediated by T-cells, was protective and reduced the chance
of relapse.1
In addition to HSCs and their differentiated progeny, the marrow contains nonhematopoietic cells, often
referred to as “stromal cells,” which include mesenchymal stem cells (MSCs). In addition to providing stromal
support for hematopoietic cells, MSCs give rise to structural components, such as bone, cartilage, tendon, and
fat. Their wide-ranging and potent differentiation properties have generated considerable clinical interest in
their use for cell therapy. MSCs also appear to exert immunosuppressive effects and are therefore of interest as
immunomodulators.
Self-renewing pluripotent stem cells are rare and difficult to isolate. The demonstration that pluripotent stem
cells can be derived from differentiated cells, including skin fibroblasts and blood, has transformed the field of
stem cell therapy. The use of these cells has the potential to have myriad applications in all areas of medicine.
IMMUNE CELLS FOR CLINICAL THERAPY
Cellular immunotherapy is the exploitation of immune cells to target and treat diseases. This approach is
most established in cancer patients, where these novel treatment modalities are used to overcome the limitations
and toxicides of chemotherapy and radiotherapy.
Considerable progress in cellular processing technology has allowed for advanced and complex immune-
cell manipulations. It is now possible to characterize immune cells with high precision, isolate specific cell
types with high purity, and engineer and amplify them ex vivo in sophisticated culture systems that comply with
good manufacturing practice (CMP) requirements. The adoption of automation has also allowed for more
reproducible immune cell expansion.
Blood banks have been instrumental in these advances in cellular immunotherapy due to their preexisting
infrastructure, which includes the GMP-compliant processing of blood units for transfusion and meticulous
quality-assurance systems embedded into the processes. In addition, the stringent donorselection processes,
widespread use of automation, use of mandatory release criteria for blood units, and requirements of traceability
and hemovigilance of blood banks have been incorporated into cellular immunotherapy.
Donor Lymphocyte Infusion: A PostHSCT Treatment
Although allogeneic HSCT can achieve longlasting cures in many patients, relapse still occurs in some
patients and is often associated with a poor prognosis.2 A second HSCT is possible but is often associated with
high toxicity and morbidity risk, especially if the relapse occurs within a year of the first transplant.
Clinical Efficacy of Donor Lymphocyte Infusions
 The discovery that T cells are one of the main drivers of the GVT or GVL response led to the development
of donor leukocyte (lymphocyte) infusions (DLIs) after HSCT. One of the key observations in the development
of DLIs was that patients with chronic myeloid leukemia (CML) that relapsed after HSCT could achieve
longlasting remissions with the administration of DLIs.3
Lymphocytes for DLI are collected from a donor via leukapheresis using a process similar to peripheral
blood stem cell collections, except that prestimulation with a colony-stimulating factor or mobilizing agent is
not usually required. The leukapheresis product is usually then aliquoted into doses starting from as low as 1 x
106 CD3-positive cells/kg recipient weight, followed by half- or 1-log increments. The initial dose is often
infused fresh, and aliquots for subsequent doses are cryopreserved.
The efficacy of DLIs is dependent on the type and aggressiveness of the underlying disease and the disease
burden at the time of relapse. It has been demonstrated that patients with relapsed CML benefit most from DLIs
with an 80% complete response rate for those in cytogenetic relapse; patients in hematologic relapse respond
less well.3 In comparison, only
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15% to 40% of patients with relapsed acute myeloid leukemia (AML) respond to DLIs, and patients with
acute lymphocytic leukemia (ALL) have virtually no response to DLL The lack of efficacy of DLI in acute
leukemias compared to CML is thought to be related to a lack of antigen expression on tumor cells and the
more rapid proliferative kinetics associated with acute leukemias.4
The starting dose for DLIs is often in the order of 1 x 106 CD3-positive cells/kg.5 Lower starting doses
have been administered, especially in the unrelated transplant setting. Response is usually not immediate and
can take as long as 3 months. For this reason, subsequent doses of DLIs are often administered as far apart as 3
months and in incremental doses half to 1 log apart so that responses can be adequately gauged.3
There is no exact correlation between dose and response. Patient response can be difficult to predict because
it depends on the disease, stage of relapse, patient factors, HLA matching, and donor characteristics.6
Complications of DLIs
The major complication of DLI is GVHD, which is caused by alloreactive donor T cells attacking healthy
host cells. In early studies, up to 50% to 90% of patients developed GVHD after DLL However, more recently,
the rate of GVHD following DLI has declined due to an improved understanding of the biology of DLIs and
predictive risk factors for GVHD in this setting. The GVHD that occurs after DLI can be very severe and
require systemic immune suppression, which can lead to significant morbidity and mortality as a result of
opportunistic infections.
Another major complication of DLI is the development of marrow aplasia, which is thought to be due to the
immune-mediated destruction of host hematopoeisis.6
The variable results in the initial studies of DLIs in patients with frank disease relapse together with the
treatment’s potential toxicides have resulted in crucial modifications to this T-cell therapy to increase its GVT
activity while minimizing its side effects. These modi
fications include the use of CD8-depleted and alloantigen-depleted DLIs and the introduction of a “suicide”
gene, such as the Herpes simplex virus-tyrosine kinase gene, into the T cells to selectively eliminate them if
GVHD develops.4
A group of researchers at University College London Hospital has also pioneered a strategy of using a dose-
escalating DLI regimen to treat residual or progressive disease or mixed donor chimerism after
reducedintensity, T-cell-depleted transplantation.5 This strategy has resulted in an impressive 70% response rate
in patients with low-grade lymphomas or Hodgkin disease.
New Directions in DLI Applications: Targeted and Antigen-Specific T-Cell Therapy
Adoptive immunotherapeutic approaches employing more targeted or antigen-specific Tcell populations
have been used successfully in the treatment of hematologic malignancies and solid tumors as well as viral
infections after HSCT. To develop immunotherapies using cytotoxic T lymphocytes (CTLs; T cells expressing
CD8), potential target antigens on tumor cells or viruses must first be identified. These antigens must be capable
of providing epitopes for specific immune responses and be present in sufficient quantity and duration to engage
responder T cells.4
A step forward in efficiently isolating T cells was achieved with the development of the major
histocompatibility complex (MHC) multimer method. In this method, high-affinity binding of MHC molecules
to the T-cell receptor is achieved through oligomerization of MHC molecules (multimers) as ligands.7 This
method allows specific detection and isolation of antigen-specific T cells through fluorescence-activated cell
sorting in a flow cytometer or the use of closed magnetic selection systems, such as the automated CliniMACS
instrument (Miltenyi Biotec, Bergisch Gladbach, Germany). The multimer method has recently been improved
with the invention of the novel streptamer technology, which has entered clinical testing.8,9
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Adoptive transfer of donor-derived, virusspecific CTLs constitutes an important novel treatment of life-
threatening viral infections after HSCT and an alternative to traditional antiviral drug therapy, such as
ganciclovir. Cytomegalovirus (CMV) reactivation is a common complication of HSCT. Infusions of CTLs
generated against the CMV pp65 antigen have achieved good clearance of the virus with few side effects and
limited GVHD. Similarly, donor-derived Epstein-Barr virus (EBV)-specific CTLs have been successfully used
for both prophylaxis and treatment of EBV-associated lymphoproliferative disease (LPD). In one study, more
than 60 patients were prophylactically treated with these CTLs and none developed LPD, compared to 11.5% of
patients who developed LPD in a historical, nontreated control group.4 Significandy, gene-marked donor CTLs
have been demonstrated to persist in DLI recipients for as long as 7 years.4
A recent major breakthrough has been achieved with the development of engineered T cells bearing
chimeric antigen receptors (CARs) composed of antibody-binding domains connected to domains that activate
T cells. This strategy was developed to overcome T-cell tolerance, which has been a limitation of CTL
populations that have been isolated and expanded based on recognition of tumor-associated antigens. One such
CAR construct utilizes an antibody-binding domain with specificity for the B-cell antigen CD19 coupled with
CD137 (a costimulatory receptor in T cells) and the CD3-zeta signaling domain.10 The construct’s efficacy was
recently demonstrated in a clinical pilot trial in which autologous CAR-T cells directed against CD19-bearing
advanced chronic lymphocytic leukemia (CLL) with more than 1000-fold expansion in vivo and demonstration
of trafficking to the marrow. Significantly, memory CAR-T cells were generated, and two out of three patients
with advanced CLL achieved complete remission with persistence of T cells for more than 6 months.10 This
proof of concept has just been extended into the treatment of B-cell ALT, with impressive results and the
attainment of complete remissions in some treated patients.11
NK Cells
Particularly promising candidates for cellular immunotherapy are NK cells. These large granular
lymphocytes of innate immunity are characterized by a CD3~CD56+ phenotype in humans and are natural
defenders against malignancies and infectious diseases. Their cytotoxicity is immediate and they do not require
antigen priming. Autologous NK cell attack is prevented via recognition of HLA Class I molecules by cognate
NK-cell inhibitory killer immunoglobulin-like receptors (KIRs).
In-Vivo Expansion
Early cellular immunotherapy protocols involving NK cells employed short-term stimulation in vitro with
high-dose interleukin (IL)-2 for 1 to 5 days, generating a heterogeneous lymphokine-activated killer (LAK) cell
product with a relatively low representation of cytotoxic NK cells. A better understanding of NK cell biology
and recent advances in cell selection, monitoring, and expansion technologies have enabled the development of
more promising new NK-cell-therapy approaches.12
An important key finding was the observation that alloreactive NK cells in the graft exerted a potent
antileukemic effect in the context of T-cell-depleted, HLA-haploidentical HSCT.13 Significantly, there were no
relapses in a group of patients with high-risk AML when NK cells exerted their effects due to donorpatient KIR
mismatching, compared to a 75% relapse rate when no NK alloreactivity was present.14
NK cells have also been administered in a non-HSCT setting. Patients with poor-prognosis AML, Hodgkin
disease, metastatic melanoma, or renal carcinoma were given haploidentical NK cells.15 In-vivo expansion of
these cells occurred with assistance from the lymphopenia induced by a lympho-depleting preparative regimen,
such as cyclophosphamide and fludarabine. This regimen induced a marked increase in endogenous IL-15
levels, which is essential for donor NK cell expansion and survival in the patient. Significantly, these infused
and in-vivo-expanded NK cells did not induce GVHD but did produce complete remissions
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in 5 of 19 patients with AML. These results offer another proof of the ability of NK cells to separate GVL
from GVHD with the aim of exploiting the former while minimizing the latter. As in the haploidentical
transplant setting, the effect was more marked when KIR-ligand mismatched (indicating alloreactive) donors
were used.
 In clinical protocols involving NK cells, effectors are enriched by magnetic CD3 depletion followed by
CD56 selection or through combined CD3 (T-cell) and CD19 (B-cell) depletion using the CliniMACS (Miltenyi
Biotech) to reduce the risk of T-cell-mediated GVHD and EBV infections.16
Ex-Vivo Expansion
An alternative method to in-vivo expansion of NK cells is expansion of NK cells in long-term, GMP-
compatible culture systems that generate large numbers of highly cytotoxic effectors. This method has been
established for clinical “off-the-shelf” production of the highly cytotoxic NK cell line NK-92 [Conkwest (Del
Mar, CA) proprietary line].17,18 In contrast, GMPcompatible expansion of primary NK cells has remained
challenging, although promising protocols are under development.
New cell-culture media, including serumfree media, are now available. Furthermore, novel growth factors,
such as IL-15, are being used instead of the traditional IL-2 to further optimize culture conditions with efficient
biasing of proliferation of the NK-cell population within LAK cultures after long-term expansion.19
Highly purified NK cells are more challenging to expand. However, purified NK cells can be grown on
feeder cell sources of either autologous or allogeneic origin. An elegant way to obtain autologous feeder cells is
to retain the nonselected, NK-cell-negative fraction after a magnetic isolation procedure. Following irradiation
of this by-product to inhibit unwanted cell proliferation during culture, these cells can be seeded as a feeder
layer to support NK-cell stimulation and proliferation. Other protocols have been established that involve the
use of allogeneic cell sources. Expo
nential NK-cell expansion, for example, was achieved with the EBV-transformed lymphoblastoid cell line or
the leukemic cell line K562, engineered to present both IL-15 and the costimulatory molecule CD137 (4-IBB)
on the surface (K562-CD137/IL-15). These protocols are undergoing clinical testing.16,19
Automated cell processing within bioreactors, such as Wave1” (GE Healthcare Life Sciences, Freiburg,
Germany) or G-Rex (Wilson Wolf Manufacturing, New Brighton, MN), provides a developing platform for the
mass production of NK cells in less time with better reproducibility.16 These methods are also used for other
immune effector therapies. Furthermore, shipment of fresh NK cells under IL-2 protection is feasible without
the need for a controlled incubator environment (37 C and 5% C02) and facilitates international distribution of
such products.20
NK-cell therapy products are generally safe and well tolerated, especially when invivo injections of IL-2 are
not required. However, NK-cell products are not entirely risk free. Two cases of significant hemolytic anemia
after treatment with minor ABO-mismatched NK-cell-enriched products have been reported that were
presumably due to the presence of isoagglutinin-producing passenger B lymphocytes in the NK-cell product.21
Cytokine-Induced Killer Cells: Polyclonal T Cells with NK-Cell Features
The traditional LAK cell culture protocol has been further developed to generate a population of rapidly
expanding polyclonal T cells known as “cytokine-induced killer” (CIK) cells.22 Culture conditions for CIK
expansion are designed to obtain an increased proportion of superior cytotoxic CD56+CD3+, doublepositive T
cells or NK-like T cells. The generation of such CD56+ T cells is promoted in this procedure through the initial
addition of gamma interferon (IFNy; 1000 U/mL). The T-cellactivating, anti-CD3 monoclonal antibody OKT3
(50 ng/mL) and IL-2 (300 U/mL) are added 24 hours later, and the cultures are maintained under permanent IL-
2-stimulation for 21 to 28 days.23 The heterogeneous CIK
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AABB TECHNICAL MANUAL
population at the end of culture comprises a very small proportion (about 2%) of NK cells. Most of the cells
(>90%) in the culture are T cells, of which about 35% express the doublepositive phenotype CD3+CD56L
Characteristics ofCIK Cells
CIK cells combine features of NK-cell (CD56+CD3') and T-cell (CD56”CD3+) effectors. The different
pathways involved in CIK cytotoxicity are under active investigation. Similar to NK cells, CIK cells have
cytolytic potential that is immediate and independent of antigen priming.24 However, studies have shown that
target cell recognition requires direct MHC Class I engagement, a mechanism that is not yet fully understood.
As with NK cells, the critical role of the activating receptor NKG2D in tumor lysis has been demonstrated for
targets, such as myeloma cells, that express its ligands MIC A/B or ULBPs.
Clinical Trials
Results from clinical trials using autologous or allogeneic CIK cells in a variety of hematologic
malignancies and solid tumors have been reported. Overall, CIK cell therapy was well tolerated and associated
with a low risk of GVHD.25 Results for cancer clearance and increased survival varied. Patients with a lower
tumor burden, such as those who had undergone HSCT, are probably the most likely to benefit from CIK
effectors. In this sense, CIK cells may be used as an alternative to DLI. Strategies to increase the cytolytic
potential of CIK cells, including co-administration of IL-2 and engineered CARs, are currently being
investigated.
DCs in Immunotherapy
DCs play a critical role in the regulation of the adaptive immune response. DCs have been called “sentinels
of the immune system,” and they possess the ability to elicit a primary immune response in resting naive T
lymphocytes. Naive CD8+ T cells, for example, differentiate into CTLs through interaction with cognate
antigens presented by mature DCs on their MHC Class I receptors.26 DCs are capable
of eliciting full T-cell activation upon engagement of costimulatory molecules (eg, B7:CD28 engagement)
and mediate cytotoxic responses against a broad range of malignancies. Particularly attractive is the intrinsic
potential of DCs to gain immunologic memory for combating subsequent tumor invasions.27
Defining DCs and Their Subsets
Unlike T cells, no single cell surface marker is exclusively expressed on DCs that can be used to define
these cells. Instead, a combination of morphology and various surface markers is used, including the expression
of MHC Class II antigens and absence of markers that define other lineages, such as CD3 and CD19. DCs also
express a variety of adhesion molecules, including CDlla (lymphocyte function-associated antigen 1); the
intercellular adhesion molecule 1 family; and costimulatory molecules, such as CD80 and CD86. Two
additional markers of mature DCs in humans are CD83 and CMRF-44.28
In the blood, DCs are present in different subsets that are distinguished by CD303 (BDCA2, CLEC4C;
plasmacytoid DC), CD1C (or BDCA1; myeloid DC), and CD141 (BDCA3 or thrombomodulin; myeloid DC)
markers. Different functions could be attributed to these subsets, with CD141 -positive myeloid DCs playing a
role in eliciting CD8 T-cell responses.29
The rarity of DCs has made it difficult to study them, although they were first described more than 30 years
ago.26 The ability to generate large numbers of DCs from CD34+ marrow precursors or CD14+ monocytes in
vitro has expanded the field of DC-based cellular immunotherapy.
Expansion Protocols
Initial tissue-culture protocols for the generation of DCs (the first-generation DC vaccines) used a
combination of granulocyte macrophage colony-stimulating factor (GM-CSF) with IL-4 for the stimulation of
monocytes in peripheral blood mononuclear cells (PBMCs) to differentiate into DCs. However, the resulting
DC vaccines usually contained a mixture of
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immature or only partially mature DCs and often other additional cell types. Since then, refinements of
protocols (known as “secondgeneration” DC expansion protocols) have been described using several new
cytokine/ GM-CSF combinations that include IFNa, tumor necrosis factor (TNF), or IL-15.29 One such
cytokine cocktail consists of TNFa, IL-ip, IL-6, and prostaglandin 2 for additional DC stimulation. More
recently, “third-generation” DC vaccines have been developed that are polarized toward a cytotoxic immune
response (innate and Type 1 immunity) and are currently being tested in clinical trials.29,30
In-vitro manipulation of DCs includes loading (pulsing) with tumor recognition molecules, such as tumor-
antigen-derived peptides, recombinant tumor antigens, or tumorderived ribonucleic acid or deoxyribonucleic
acid. Furthermore, DC tumor-hybrid vaccines have been created with the potential to activate a combined CD4+
and CD8+ T-cell response through simultaneous presentation of several tumor antigens, including a direct
fusion of DCs to leukemic blasts, which are easily obtainable from patients with leukemia.31,32 However,
release of suppressive factors, such as TGFp, by the tumor cell fused in the hybrid may limit the vaccine’s
efficacy.31
Cancer Vaccine Approved by Food and Drug Administration
One of the successes of cellular therapy has been in the treatment of prostate cancer. A first-generation DC-
based vaccine, sipuleucelT (Provenge; APC 8015; Dendreon Corporation, Seattle, WA), was approved by the
US Food and Drug Administration (FDA) based on a 4-month improvement in survival compared to placebo in
the pivotal randomized clinical trial of men with castration-resistant metastatic prostate cancer.33 This is an
autologous cellular immunotherapy designed to stimulate a patient’s own immune system to respond against the
prostate cancer. Sipuleucel-T consists of autologous PBMCs that are enriched for antigen-presenting DCs that
have been activated ex vivo with a recombinant fusion protein, prostatic acid phosphatase (PAP), fused
 to GM-CSF. PAP is an immunogenic prostate antigen whereas GM-CSF is an immune-cell activator. Each
dose is manufactured by obtaining a patient’s T cells via leukapheresis and shipping them to the company to
manufacture the vaccine. After this process, the patient’s own cells are reinfused into the patient to treat the
prostate cancer. Provenge is administered intravenously in a three-dose schedule given at about 2-week
intervals.
The effectiveness of Provenge was studied in 512 patients with metastatic prostate cancer that was
refractory to hormone treatment in a randomized, double-blind, placebocontrolled, multicenter trial.33 The
median survival duration for patients receiving Provenge treatments was 25.8 months compared to 21.7 months
for those who did not receive the treatment.
MSCs
MSCs are a rare subset of cells of nonhematopoietic origin that were first discovered in 1968 by
Friedenstein as an adherent fibroblast-like population after isolation from the marrow, where they represent
0.001% to 0.01% of total cells.34 It was subsequently shown that MSCs could be isolated from various tissues,
including amniotic fluid, adipose tissue, umbilical cord blood, dental pulp, muscle connective tissue, and
placenta, and could be rapidly expanded in vitro, offering the potential for use in clinical cellular therapy.35 The
rarity of MSCs has meant that the majority of research to date has relied on isolating them from the marrow or
adipose tissue before expansion in culture, a prerequisite for clinical-scale therapies. MSCs are often referred to
as “multipotent” due to their capacity for differentiation into cells of mesodermal lineage, including osteoblasts,
chondrocytes, adipocytes, and, under specific conditions, several nonmesodermal cell types, including
neurons.36 It is this capacity for multipotency that has generated a great deal of interest in using MSCs for
tissue engineering and regenerative medicine.
Expanded MSC cultures do not all possess the same level of multipotency. Although a low frequency of
self-renewing progenitors has
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AABB TECHNICAL MANUAL
been identified among marrow-derived MSCs, it remains unclear whether MSCs from other tissues share
these properties. This has resulted in the use of the term “multipotent MSCs” as an alternative to “MSCs”
because “multipotent MSCs” does not imply that the cells have “stem-cell-like” properties or self-renewal
ability without differentiation (Table 33-1).37
The defining characteristics of MSCs have often differed among investigators, which prompted the
publication of the minimal criteria for the definition of the phenotypic and functional properties of MSCs by the
International Society of Cellular Therapy (ISCT).38 The ISCT guidelines for defining MSCs are listed in Table
33-2.
MSCs have been identified as modulators of several effector functions and immune responses through their
interaction with both the innate and adaptive immune systems. They play a key role in the support of
hematopoiesis, contributing to the maintenance of the highly specialized microenvironment of the marrow
through the regulation of HSC numbers and control of their maturation and differentiation.39 MSCs have been
shown to be capable of exerting a profound immunosuppressive effect on both polyclonal and antigen-specific
T-cell responses through induction by cellular or humoral stimuli. They largely have no immunologic
restriction, meaning that they can be used without HLA matching.40 The immunoregulatory functions of
MSCs, including the suppression and inhibition of T-, B-, and NK-cell functions and DC activity, therefore offer
a promising option for the treatment of immune-mediated disorders,
including GVHD, autoimmunity, and solid-organ-transplant rejection. The suppressive properties displayed
by culture-expanded MSCs are promoted through the expression of indolamine 2,3-dioxygenase and other
effector molecules, many of which are augmented by IFN-y stimulation.41,42
Isolation and Expansion of MSCs
The isolation and initial expansion of MSCs is performed in three distinct phases over a period of 3 to 4
weeks:
1. Isolation and seeding.
2. Cell passaging.
3. Final harvest.
The clinical production of MSCs for therapeutic use is carried out in large tissue-culture flasks, although
MSC production can be industrialized in 25,000-cm2 culture chambers or CellSTACKs™ (Sigma-Aldrich
Corning, New York, NY). Marrow aspirates/harvests remain the most common starting material for the isolation
of MSCs. Following harvest, marrow is collected into preservative-free heparin before transportation to the
laboratory, where the first step is the isolation of bone-marrow mononuclear-cell (BMMNC) fraction by
lightdensity centrifugation. At this stage, MSCs are isolated either by their ability to adhere to tissue culture
plastic, as described below, or are directly purified using monoclonal antibodies to CD105, CD146, or CD271
by immunomagnetic or flow-cytometric sorting.43
TABLE 33-1. The Multipotentiality of MSCs
Self-Renewal Differentiation Transdifferentiation
Marrow cavity Mesodermal lineage Ectodermal lineage Endodermal lineage
Connective stromal cells Epithelial cells Muscle cells
Cartilage cells Neurons Gut epithelial cells
Fat cells Lung cells
Bone cells
MSCs = mesenchymal stem cells.

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TABLE 33-2. Criteria for Identification of MSCs ■ Adherence to tissue culture plastic
■ Phenotype (levels of expression)
Positive (>95% +) Negative (<2% +)
■ CD73 ■ CD45
■ CD90 ■ CD34
■ CD105 ■ CD14
■ CDIIb
■ CD79a
■ CD19
 ■ HLA-DR
■ Demonstration of in-vitro differentiation into
osteoblasts, adipocytes, chondrocytes MSCs = mesenchymal stem cells.
For isolation by plastic adherence, BMMNCs are seeded onto tissue-culture flasks at a density ranging from
0.5 x 105 to 5 x 105 BMMNCs per cm.2 The BMMNCs are cultured in alpha-modified minimum essential
medium supplemented with 10% fetal bovine serum (FBS). Alternative MSC expansion protocols have also
been described using human platelet lysate autologous serum as an alternative source of serum and growth-
factor-supplemented, serum-free media.35
Despite the prescreening of FBS for use in clinical studies, its application in clinicalgrade cellular therapies
still raises concerns because, theoretically, it is a putative source of prions and virus transmission. These
concerns make the future use of serum-free media in all clinical products an attractive option.
The propagation of adherent cells is maintained by the removal of nonadherent cells on the third day and
their subsequent feeding every 3 to 5 days with fresh culture medium. Cultures are reviewed daily for:
■ Adherence of cells to flasks.
■ Shape of adherent cells (round vs fibrocytic appearance).
■ Confluence of cells.
■ Amount of debris in medium as an indication of medium change.
Adherent cells consistently achieve 80% confluence after 9 to 14 days, at which point the cells are passaged
following enzymatic dissociation from tissue culture flasks with trypsin. Passaging of cell cultures once they
have achieved confluence is often a prerequisite when an attempt is made to propagate cells in sufficient
quantity for therapeutic use. However, continual passage of MSCs can lead to replicative exhaustion and
alterations in phenotype, which may play a role in modifying their regenerative and immune-suppressive
properties or potentially limiting their survival and function in vivo.44 The malignant transformation potential
of MSCs can be eliminated by reducing cell passaging, although it has also been reported that MSCs can be
safely expanded in vitro until the 25th passage.45 MSCs are harvested and can be cryopreserved at the
recommended therapeutic dose or administered “fresh.”
Immunophenotyping by flow cytometry is an essential part of the quality assurance/ quality control process
of manufacturing using the cell-surface markers CD73, CD90, and CD105. Differentiation of MSCs into a
specific lineage often requires the addition of a complex array of growth factors that are selected based on the
desired mature cell phenotype.
Standardization of MSC isolation and expansion is now gathering pace among the cell therapy community
as MSCs are increasingly used in clinical trials and manufactured under GMP conditions. The technology for
culturing large numbers of expanded MSCs for clinical use has also undergone significant development.
Devices, such as The Quantum Cell Expansion System (Terumo BCT, Lakewood, CO), a closed automated
hollow fiber bioreactor culture system with disposable sets, are now commercially available and designed to
reproducibly grow adherent cells in GMP environments while minimizing both space and operator variability.
Clinical Applications of MSCs
The main indications for use of MSCs are in the treatment of GVHD, Crohn disease, cardiovascular
disorders, multiple sclerosis, spinal
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AABB TECHNICAL MANUAL
cord injury, liver disease, diabetes mellitus, and various bone and cartilage defects. MSCs can be used
without HLA matching because they do not induce GVHD and are not rejected by recipient T cells. These
characteristics favor banking of cryopreserved MSCs from thirdparty donors. Such banked MSCs can be
released and shipped as required for multiple clinical applications as “off-the-shelf” products.
One of the earliest studies that illustrated the regenerative properties of MSCs involved their use in treating
three children with osteogenesis imperfecta.46 This disease is caused by a deficiency in the production of Type
I collagen and results in defective connective tissue formation, leading to multiple fractures, bony deformities,
and shortened stature. Following the infusion of unmanipulated allogeneic marrow, MSCs from within the graft
migrated to the bone and gave rise to osteoblasts, where their presence correlated with an improvement in bone
structure and function. The same group of researchers has since modified their treatment protocol and
demonstrated its safety and efficacy after administering two doses of marrow-derived, ex-vivo-expanded MSCs
following standard marrow transplantation in patients with this disease.47
 The therapeutic role of MSCs in HSCT has been largely targeted toward alleviation of GVHD following
transplantation. However, the codelivery of MSCs at the time of transplantation has received attention because
of their potential role in graft tolerance and engraftment. Marrow-derived MSCs have been used successfully
for the treatment of steroid-refractory, severe, acute GVHD, for which there is currently no established
definitive therapy. The clinical benefits of infusing MSCs to treat GVHD were first demonstrated when
haploidentical MSCs were administered in two transfusions to a 9-year-old boy with treatment-resistant, Grade
IV acute GVHD of the gut.48 This has triggered great interest over the past decade, and subsequent Phase I/II
trials have demonstrated both the safety and efficacy of the use of MSCs in HSCT in the setting of steroid-
refractory GVHD.49
Optimal therapeutic doses have not been defined, although clinical responses have been documented with
infusions of MSCs as low as 0.8 x 105/kg. However, a Phase III, industry-led clinical trial examining the
transfusion of high doses of MSCs (2 x 106/kg) for the treatment of steroid-refractory GVHD showed no
increase in overall complete response rate.44
The clinical use of MSCs for tissue regeneration has received the most interest for cardiovascular repair.50
Many trials have had encouraging results in the improvement of cardiac function after injection of
autologousmarrow-derived MSCs. Adipose-tissue-derived MSCs are also currently being investigated as part of
two industry-led clinical trials, APOLLO and PRECISE, exploring their safety, feasibility, and efficacy in
patients with either acute or chronic myocardial infarction.51
Although the therapeutic effects of MSCs in cardiac repair have been acknowledged in the scientific
community, several issues remain unresolved, including the optimal dose and the mechanism of improvement in
cardiac function. The differentiation of MSCs into cardiomyocytes remains controversial, and the literature in
this field is still unclear and contradictory. There have been a number of recent publications providing evidence
that in vitro, MSCs may be induced to exhibit cardiomyocyte-like features, but the exact culture conditions are
not clear. A number of compounds are involved in their generation through modulation of signaling pathways.
There has been no direct evidence of cardiomyocyte differentiation in vivo following administration of MSCs
for cardiac repair, but functional improvement has been observed. These observations in preclinical and human
trials have led to the hypothesis that the improvements are related to the paracrine effects of transplanted cells,
which induce myocardial repair by releasing signals, including cytokines and chemokines, into the surrounding
tissue rather than generation of de-novo cardiomyocytes.50'52
The multipotency of MSCs has also become attractive to industry, and two commercially driven clinical
trials involve third-party MSCs manufactured by Osiris Therapeutics
795
Inc. (Columbia, MD) for use in patients with GVHD or Crohn’s disease and mesenchymal precursor cells
manufactured by Mesoblast Ltd. (New York, NY) for the treatment of cardiovascular, orthopedic, and
neurologic disease. Although the clinical efficacy of such MSC products has yet to be fully evaluated in many
of the proposed disease indications, their application as cellular therapies continues to evolve rapidly.
MSCs and Tissue Engineering
The use of MSCs in tissue engineering has developed rapidly over the last few years with the advent of
scaffolds to improve the outcomes of stem cell applications by offering more precise targeting of MSCs
combined with the possibility of making them biodegradable, depending on the therapy. Scaffolds can be used
in a number of ways: 1) seeding of MSCs onto scaffolds in vitro and implantation after a short incubation
period, 2) MSC seeding and subsequent differentiation into lineage-specific cell types in short-term culture (1-2
weeks) before implantation, and 3) induction of differentiation in culture before seeding onto scaffolds and
implantation shortly afterwards.
In 2008, the first fully tissue-engineered bronchial transplant was reported.53 In this case, a
nonimmunogenic, decellularized piece of human donor trachea was reseeded with autologous marrow-derived
MSCs. The MSCs were differentiated into chondrocytes in vitro before surgical implantation to replace a
bronchus that had been previously damaged by tuberculosis infection. The paucity of donor organs has limited
the use of this approach. As a result, the use of biosynthetic nanocomposite material seeded with MSCs has
emerged as a therapeutic option for tracheal replacement.54
INDUCED PLURIPOTENT STEM CELLS
The archetypical pluripotent cell is the embryonic stem cell (ESC), which is derived from embryos and is
capable of long-term selfrenewal and differentiation into all cells and tissues in the human body. The
uniqueness of
 these capabilities was called into question when Gurdon55 demonstrated that the nuclei of differentiated
cells actually retain all of the genetic information in pluripotent stem cells. The cloning of Dolly the sheep, for
example, showed that the genome of even fully specialized cells remains genetically totipotent; that is, the
genome can support the development of an entire organism.
The next key finding was that manipulation of transcription factor expression can change a cell’s fate.56
Experiments revealed that lineage-associated transcription factors can change cell fate when these factors are
ectopically expressed in certain heterologous cells. Lineage-associated transcription factors help establish and
maintain cellular identity during development by driving the expression of cell-type-specific genes while
suppressing lineage-inappropriate genes.
The third contributory result was obtained from the Nobel Prize-winning work of Yamanaka,57 who
screened a pool of 24 pluripotency-associated candidate genes for factors. He determined that a core set of four
transcription factors comprising Klf4, Sox2, c-Myc, and Oct4 was sufficient to produce induced pluripotent
stem cells (iPSCs). This research was initially conducted in murine models, but the approach was also
successful with adult human skin fibroblasts. Many other investigators since then have demonstrated that this
cocktail of genes can be used to derive iPSCs from other somatic cell populations, including keratinocytes,
neural cells, and stomach and liver cells.
Now that iPSCs can be generated, the primary question is whether iPSCs and ESCs are molecularly and
functionally equivalent. Comparisons of their genome-wide-expression patterns and global histone
modifications have demonstrated a high degree of similarity between ESCs and iPSCs. However, unlike ESCs,
iPSCs are derived from somatic tissues and therefore do not raise the ethical issues associated with ESCs.58
The ability to obtain starting material from skin, blood, and umbilical cord immediately opens up the horizon
for translational clinical applicability.59
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AABB TECHNICAL MANUAL
Tumorigenicity
The use of integrating viruses in the generation of iPSCs raises concern about the potential for oncogenesis
and teratoma formation. iPSC-derived cells do indeed have a propensity to form tumors after transplantation,
and human iPSC-derived blood progenitor cells also appear to undergo premature senescence.60 Their
translation to human application will need to overcome this safety concern. Recent approaches to generate
iPSCs free of potentially harmful mutagenic effects have used either vectors that do not integrate into the host
cell genome or site-specific integrating vectors to direct the integration away from known oncogenic sites.
Finally, these techniques have been refined so that iPSC generation no longer requires such integrating
vectors.60
Therapeutic Applications
The scope for iPSC-based clinical cell therapy in the future is bewilderingly vast. The last few years have
seen continual refinements in techniques to allow for large-scale GMP manufacturing. At the time of this
writing, a new trial has just received conditional approval in Japan to use iPSCs to treat age-related macular
degeneration.
In-Vitro Generation of Hematopoietic Cells
One of the most exciting prospects for using iPSCs in cellular therapy is that of in-vitro generation of
hematopoietic cells, including erythrocytes. Current blood transfusion practice is very safe, but its past has been
fraught with infectious disease transmission. Blood banks continue to contend with issues of red cell
compatibility and the need to have sufficient donor pools and adequate inventory systems. These issues can
potentially be resolved with large-scale manufacturing processes using iPSCs.61
Disease Modeling
One important application of iPSCs is the modeling of human diseases by generating specific tissue types of
interest followed by the induction of necessary pathologic changes. This process essentially replicates the
disease in a Petri dish. However, this approach may be limited to single-gene disorders, such as sickle cell
disease. For now, it remains unclear whether multigenic diseases, such as diabetes or Alzheimer’s disease, are
equally amenable to in-vitro modeling using iPSCs.62
Drug Testing
If disease modeling using iPSCs is successful and reproducible, these ex-vivo-derived pathologic tissues can
be subjected to pharmaceutical testing to identify potential new drugs that are more potent and targeted than
existing drugs.62
 In conclusion, iPSCs hold great promise for regenerative medicine and pharmaceutical evaluation. They
represent a potentially viable source of non-HSCs to treat a wide variety of diseases. However, the path to final
fruition of their promise is laden with several challenging hurdles, and work is still necessary to overcome these
difficulties.
REGULATORY AND OVERSIGHT ACTIVITIES
The cell therapy field has developed along the same lines as blood banking. Both fields share the strengths
of multidisciplinary teams (physicians, nurses, laboratory officers, quality managers, infectious disease experts,
and immunologists). Both fields also share an emphasis on large-scale manufacture, automation, product release
criteria, computer systems for tracking and inventory, cold chains for transportation, product traceability, and
monitoring of side effects.
Thus far, the majority of cell therapy products have been manufactured from sources from a mixture of
blood banks, academic
797
GMP facilities, and commercial companies. Harmonization in the terminology of cell-therapy products is
being undertaken by the Cellular Therapy Coding and Labeling Group, a technical subgroup of ICCBBA that
promotes the use of ISBT 128 (the information technology standard for transfusion medicine and
transplantation) labeling. This harmonization will, of course, facilitate the transportation of such products across
national borders. In addition, the Alliance for Harmonization of Cell Therapy Accreditation consists of
representatives from a variety of organizations involved in HSCT and cell therapy. Its primary aim is to create a
single set of quality, safety, and professional requirements for cellular therapy.
Cell therapy products need to be governed under suitable regulatory frameworks, including national ones
that cover cells, tissues, and organs. Such national regulatory frameworks for cell-therapy products are not in
existence worldwide but will be important as the field develops and the need grows for cross-border
transportation of cells.
Cellular products can be classified as either substantially or minimally manipulated. Substantially
manipulated cell-therapy products are treated as biologies. In the United States, these are known as “351”
human cells, tissues, and cellular and tissue-based products and are regulated by the Center for Biologies
Evaluation and Research of the FDA. In Europe, substantially manipulated products are known as advanced
therapeutic medicinal products (ATMPs). They are manufactured in accordance with national legislation and
oversight by the European Medicines Agency.
The question that arises next is how the issuance and inventories of these biologies will be managed in the
local hospital environment. The hospital pharmacy is the natural domain for medicinal products oversight.
However, the unique qualities of cell therapy products render them more suitable for the sphere of activity and
concern of the blood bank and GMP processing facility.
Most importantly, the maturation of the cell-therapy field and its clinical applicability in many fields of
medicine will mean that more patients will be treated with this novel therapeutic option. Because living cells are
often administered to patients, short-term as well as longer-term side effects and potential toxicides should be
systematically monitored. Such monitoring systems have been in place for pharmaceuticals (pharmacovigilance
programs) and blood (hemovigilance programs), and these programs could be extended to cellular therapy
products (cellulo-vigilance programs).
CONCLUSION
The history of cell therapy has seen great advances of late with the entrance into the market of the first
FDA-approved therapeutic cancer vaccine, Provenge, as well as the dramatic successes of antigen-specific T-
cell therapy in hematologic diseases. These successes will certainly spur continuing interest in the development
of clinical trials. The attraction of an “off-the shelf,” third-party product is that it will increase the number of
patients who can be treated and heighten interest in the international transportation of such products.
KEY POINTS
1. Hematopoietic stem cell transplantation (HSCT) is well established for the treatment of hematologic
diseases. A graft-vs-tumor (GVT) effect is largely mediated by cytotoxic immune effector cells, such as T cells
and natural killer (NK) cells.
2. Recent technological advances allow good manufacturing practice (GMP)-compliant isolation,
characterization, expansion, and modification of immune effector cells for application as cellular therapeutics in
clinics.
 3. Donor lymphocyte infusion (DLI) has demonstrated considerable benefit as post-HSCT treatment in
relapsed patients, particularly in chronic myeloid leukemia (CML). These are

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AABB TECHNICAL MANUAL
obtained from leukapheresis and constitute mainly T cells. A major side effect is graft-vshost disease
(GVHD) as a consequence of a healthy host cell attack by alloreactive T cells.
4. NK cells are innate immune cells, do not require antigen priming, and do not induce GVHD. Current
promising developments to improve clinical outcome include treatment with alloreactive NK cells, mismatched
for their inhibitory receptors, as well as in-vivo or ex-vivo activation and expansion of NK cells.
5. Cytokine-induced killer cells (CIK) cells or NK-like T cells are expanded in long-term cultures. Trials
with CIK cell therapy have thus far demonstrated good tolerability with a low risk of GVHD and some efficacy.
They may be applied as an alternative to DLI.
6. Dendritic cells (DCs) play a key role in regulating the adaptive immune response to an antigen-specific
manner. A DC-based vaccine, sipuleucel-T (Provenge) is approved by the US Food and Drug Administration as
an autologous immunotherapy against prostate cancer.
7. Mesenchymal stem cells (MSCs) possess potent differentiation properties (multipotency) and have
sparked considerable clinical interest. They are generally isolated from marrow or adipose tissue and expanded
in vitro. They can give rise to structural components, such as bone, cartilage, tendon, and fat, and appear to
exert immunosuppressive functions. Indications for applications of MSCs include GVHD, Crohn disease,
cardiovascular disorders, cord injury, liver disease, diabetes mellitus and bone and cartilage defects.
8. Promising therapeutic effects of autologous MSCs in regenerative cardiac repair have been reported
although the potential for differentiation of MSCs into cardiomyocytes remains controversial and more results
are needed.
9. Third-party MSCs can be applied “off-the-shelf” without HLA matching and are not rejected by recipient
T cells.
 10. The field of stem cell therapy has been transformed with the discovery that self-induced pluripotent stem
cells (iPSCs) can be generated from differentiated cells, including keratinocytes, neural cells, and stomach and
liver cells. The use of iPSCs involves considerably fewer ethical issues compared to that of embryonic stem
cells.
11. iPSCs can potentially be applied in a wide range of medical conditions and for in-vitro generation of
hematopoietic cells. Techniques for large-scale GMP-compliant manufacturing of iPSCs are being developed.
12. Cellular products are classified as either minimally or substantially manipulated, with a more stringent
regulatory framework for production of the latter in the US and Europe.
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 Index
Page numbers in italics refer to figures or tables
A
A antigen, 292-293, 295, 302, 457 A(B) phenotype, 296, 298 ABO compatibility
of apheresis platelets, 169 of Cryoprecipitated AHF, 375, 554, 583 of granulocytes, 173, 375, 525, 554, 584
in hemolytic transfusion reactions, 673 of HSC transplants, 632-633, 635, 716 of organ transplants, 491,492,
783 of plasma, 375, 379, 554, 583, 635 of platelets, 375, 457, 513-514, 554 after HSCT transplantation, 635,
636 in hemolytic transfusion reactions, 675 in pediatric patients, 581, 589 of RBCs, 375, 378-379, 504, 554,
635 of tissue transplants, 111 of Whole Blood, 375 ABO discrepancies, 296, 298-301 ABO hemolytic disease of
the fetus and newborn, 566-567 ABO system, 291-301
acquired B phenotype, 296-297, 298 antibodies of
anti-A and anti-B, 293, 297 anti-Aj, 297, 301 anti-A,B, 297
antigens of, 291, 292-293, 457 B(A) and A(B) phenotypes, 296, 298 biochemistry of, 292-293, 294, 302 in
development and aging, 293 genetics of, 259, 266, 273, 293-295 O alleles, 295 phenotypes of, 292
subgroups of, 292-293, 295-296, 298, 300 ABO testing
of blood components, 225-226, 297-298, 378 with cold autoagglutinins, 300, 301,438 comparison with
previous records, 378 discrepancies in, 296, 298-301 hemolysis in, 297 interpretation of, 292, 296 for organ
transplantation, 783 in pediatric patients, 385, 574, 587
 in prenatal studies, 563 reagents for, 298 of recipients, 297-298, 375 in transfusion reaction evaluation, 672
of umbilical cord blood, 736 with warm autoagglutinins, 432 ABTI antigen, 361 Accidents, 45
Accreditation, 90, 91, 603-604, 698 ACE inhibitors. See Angiotensin-converting enzyme inhibitors
Acid-elution stain (Kleihauer-Betke), 565-566 Acid elutions, 429
Acquired B phenotype, 296-297, 298 Activated partial thromboplastin time, 519 Activated protein C, 532
Acute disseminated encephalomyelitis, 653 Acute lung injury, 680. See also Transfusionrelated acute lung
injury Acute normovolemic hemodilution, 606 ADAMTS-13, 652-653 Additive solutions, 136-137,138, 576-
577 Additives, in serologic testing, 376-377 Adipose-derived stem cells, 755, 758-759, 762, 794
Adsorption, 407-408 allogeneic, 433-434 autologous cold, 438-439 autologous warm, 432-433 and elution,
409 Adverse reactions
to apheresis, 169, 170, 658-660, 720 in blood donors, 20, 22, 142-146 to donor lymphocyte infusions, 787 to
G-CSF, 719-720 to HSC infusions, 724-725, 742 to IVIG, 529, 532 management of, 20-23 related to medical
devices, 97 related to tissue allografts, 782 to transfusion (See Transfusion reactions) transfusion-transmitted
diseases (See Transfusion-transmitted diseases)
AET. See 2-aminoethylisothiouronium bromide Age
of blood samples, 370-371, 410-411, 417

803

804
AABB TECHNICAL MANUAL
of donors, 119,120
effect on antigens and antibodies, 293, 300, 410
of RBC units, 574, 577-578 Agglutination
in antiglobulin testing, 372
assays utilizing, 241-242, 279-280
interpreting and grading reactions, 372
mixed-field, 278, 298
principles of, 371
spontaneous
in ABO testing, 298, 300, 301 dispersing with sulfhydryl reagents, 432, 438
in Rh testing, 332 Agreements, 6-7, 9-10,108 AHG serum, 372 AIDS, 124, 125, 182 Air embolism, 669,
685 Alarm systems, 36, 214 Albumin, bovine (reagent), 376, 417 Albumin solutions (colloid), 526, 647, 652
Aliquoting components, 224, 575-576, 583 Alleles, 262-264 defined, 256 frequencies of, 274, 275 polymorphic,
264 terminology for, 279, 280 Allergic reactions to apheresis, 658 in HSCT patients, 639 to latex, 45
to transfusions, 547-548, 667-668, 678-680 Alloantibodies, 391-392. See also Antibodies Allogeneic
adsorption, 433-434 Allogeneic HSC transplantation, 715-718. See also Hematopoietic stem cell transplantation
Allografts, 774. See also Tissue Alloimmunization. See also Antibodies
in delayed hemolytic transfusion reactions, 686
in hemolytic disease of the fetus and newborn, 562 HLA
in HSC transplant recipients, 637, 639640, 716
management of, 670 in organ transplant recipients, 488-489 and plasma transfusions, 151 in platelet
refractoriness, 458, 459-461, 488-489, 515-516 in TRALI, 489, 681, 682 in transfusion reactions, 489, 490, 677
to platelet antigens, 515-516, 567-568, 637, 689-690
prevention of, 329-330, 515 red cell, 391, 670
in sickle cell disease, 283,329-330, 379, 588, 639
cq-antitrypsin, 532 cq-proteinase inhibitor, 532 American Rare Donor Program, 421 Aminocaproic acid,
608, 621-622 2-aminoethylisothiouronium bromide (AET), 301, 405, 407
Amniotic fluid-derived MSCs, 759-760 Amotosalen-UV treated plasma, 153,205 Anaphylactic reactions,
658, 668, 678-679, 680 Anemia
acute, 500-502
 and bleeding prevention, 508-509 chronic, 502, 610 defined, 604
in delayed transfusion reactions, 686 fetal (See Hemolytic disease of the fetus and newborn)
hemolytic (See Hemolytic anemia) in HSC transplantation, 634 iatrogenic, 609 in infants, 571-572, 578-579
iron deficiency, 605, 620 management of, 605 preoperative, 604-605 RBC transfusions in, 500-502 screening
donors for, 121 signs and symptoms of, 604 tolerance, 609-610 Anesthesia, 607 Angioedema, 678
Angiotensin-converting enzyme inhibitors, 659, 669, 678, 679 Ankylosing spondylitis, 493-494 Antibiotics,
in donors, 122,129 Antibodies
autoreactive (See Autoantibodies) detection of (See Antibody detection) drug-induced, 392, 440-444, 465-
466, 516 to Factor VIII, 527 granulocyte, 466-469 HLA (See HLA antibodies) identification of (See Antibody
identification)
platelet (See Platelet antibodies) to reagent components, 298, 300, 417 red cell (See Red cell antibodies)
structure of, 246-248
Antibody-dependent cellular cytotoxicity, 419
805
Antibody detection, 375-377
with auto antibodies, 432-435, 438-439 by ELISA, 243-244 frequency of testing, 419 of granulocyte
antibodies, 469 of HLA antibodies, 460, 487 interpreting results of, 381, 382 methods for, 376-378 in pediatric
patients, 385, 574-575, 587 of platelet antibodies, 243, 456, 463-464, 465-466
in prenatal evaluations, 563 reagents for, 376-377, 393, 396 with rouleaux present, 416 solid-phase assays
for, 243 specimen requirements for, 370-371 Antibody identification
anomalous reactions in, 416-417 of antibodies to high-prevalence antigens, 394-395, 415-416
of antibodies to low-prevalence antigens,
416
with antibodies to reagent components,
417
with autoantibodies, 432-435 autologous control in, 400, 403-404, 417419
complex problems, 402, 403-404 exclusion (“rule-out”) in, 398 factors affecting, 410-411 frequency of
testing, 419 immunohematology reference labs for, 419 interpreting results of, 397 with multiple antibodies,
408, 410, 412, 414 with no discernible specificity, 411-412 positive and negative reactions in, 397-398 with
positive DAT, 401, 404, 415-416, 417419
preanalytical considerations for, 392-393 in prenatal evaluations, 563 probability values in, 400 reagents for,
393, 396 and selection of blood, 274, 379, 419-421 specimen requirements for, 393 test methods for, 396
adsorption, 407-408 alteration in pH, 406 combined adsorption-elution, 409 dispersing autoagglutination, 438
elution, 408-409 enzymes, 402, 405 flowcharts for, 403-404 identification panels, 393, 396-400 inactivation of
antigens, 405, 406-407 increased incubation time, 405
increased serum-to-cell ratio, 405 inhibition techniques, 406 LISS and PEG, 402
phenotyping autologous red cells, 401402
selected cells, 397, 398 temperature reduction, 405 titration studies, 409-410, 563 variations in antigen
expression in, 410, 411-412
Antibody screen. See Antibody detection Antibody specificity prediction method, 460 Anticoagulant
medications in blood donors, 129 reversal of, 517-518, 519, 622-623 Anticoagulant-preservative solutions,
136,137 Anticoagulation, in apheresis, 658 Antifibrinolytic agents, 522, 608, 621-622 Antigen capture assays,
464-465 Antigen-matching, 281, 329-330, 379, 588, 654 Antigens. See also specific blood groups acquired,
296-297, 298 antithetical, 263-264 of blood group collections, 361-362 of blood group systems, 278-279
chromosomal location of, 259-261 granulocyte, 466-468 high-prevalence, 361-362, 415-416, 421 HLA, 475-
476, 480-483 inactivation of, 405, 406-407 low-prevalence, 362, 416 methods for detecting, 241-243, 244-246,
279-285
platelet, 243, 453-458 prevalence of, 274 terminology for, 278-279, 280 variations in expression of, 410
Antiglobulin test, 371-372 in crossmatching, 380 direct, 372, 425-430 indirect, 372 reagents for, 372, 396
sources of error in, 373-374 use of IgG-coated cells, 376 Antihistamines, 548, 679-680 Antiplatelet agents,
122,129,169, 509 Antipyretics, 547 Antithrombin, 531 Apheresis
complications of, 169, 658-660, 720 component collection by, 167-176 adverse donor reactions in, 169,170
donation requirements for, 121,122,
123, 168-169, 171
 granulocytes, 168, 172-173, 175-176
806
AABB TECHNICAL MANUAL
instrumentation for, 168,173-176 multicomponent donations, 171-172,174 plasma, 168, 170, 173-174
platelets, 168-170, 174-175 quality control of, 171 RBCs, 168, 171-172, 175 HSCs collected by (See Peripheral
blood HSCs)
records of, 170,172
therapeutic (See Therapeutic apheresis) Aplastic anemia, 640 Arterial puncture, in donors, 145 Arthritis,
763 Aspirin, 122, 169, 509 Assessments
of blood utilization, 24-25, 611, 697-707 competency, 7-8 external, 25, 86-87 internal, 24
management of, 13, 23-26 proficiency testing, 25-26, 91 quality indicators, 24 risk, 98-99, 102 Audits,
transfusion, 25
concurrent, 699, 700, 703, 706 prospective, 611, 699-700, 701-702, 706 retrospective, 699, 701-702, 703-
704, 707 selecting a process for, 705 Auto control. See Autologous control Autoadsorption. See Adsorption
Autoagglutination
in ABO typing, 298, 299, 300, 301 dispersing with sulfhydryl reagents, 432, 438
in Rh testing, 332 Autoantibodies cold
in ABO testing, 300, 301, 438 adsorption of, 438-439 antibody identification with, 418 in cold agglutinin
disease, 308-309, 437439
in mixed AIHA, 439
in paroxysmal cold hemoglobinuria, 440 in Rh testing, 332,438 use of sulfhydryl reagents with, 438 in
warm autoimmune hemolytic anemia, 431 defined, 391
disease associations with, 392 distinguishing from alloantibodies, 283 drug-induced, 443 low-affinity, 437
in phenotyping problems, 401 platelet, 461, 465, 568
warm
ABO testing with, 432 adsorption of, 432-435 with alloantibodies, 418-419, 432-435, 438-439
antibody identification with, 418-419 disease associations with, 392 mimicking alloantibodies, 434-435 in
mixed-type AIHA, 439 in phenotyping problems, 401 Rh testing with, 332,432 specificity of, 435 transfusion
with, 436-437, 506 in warm AIHA, 431 -435 Autoclaving biohazardous waste, 55 Autografts, 774, 782-783.
See also Tissue Autoimmune hemolytic anemia classification of, 430 cold agglutinin disease, 437-439 DAT-
negative, 437 mixed-type, 439-440 paroxysmal cold hemoglobinuria, 440 serologic findings in, 431 transfusion
in, 436-437, 439-440, 506 warm, 430-437
Autoimmune neutropenia, 468 Autoimmune thrombocytopenic purpura, 463, 465, 517, 568
Autologous adsorption, 432-433,438-439 Autologous blood collection of
by acute normovolemic hemodilution, 606
by intraoperative blood recovery, 606607
low-volume units, 141 by preoperative blood donation, 605-606 donor selection for, 120, 131 infectious
disease testing of, 190-191 for rare phenotypes, 421 separation from transfused cells, 401 Autologous control
in antibody identification, 400, 403-404 positive, 404, 417-419 in pretransfusion testing, 377, 382
Autologous HSC transplantation, 638, 714715, 718-719. See also Hematopoietic stem cell transplantation
Automated testing systems, 378 Autosomal inheritance, 265-267
B
B antigen, 292-293, 302 acquired, 296-297, 298 on platelets, 457
807
B(A) phenotype, 296, 298 Babesiosis, 126, 201 Bacterial contamination, 198-199 detecting, 198-199
inspecting components for, 149, 224 during phlebotomy, 141,198 of platelets, 150, 198-199, 221 of RBCs, 198
reactive testing results for, 188 of tissue allografts, 777, 782 in transfusion-associated sepsis, 669,
676
Band 3 glycoprotein, 352-353 Bernard Soulier syndrome, 456 Bg (Bennett-Goodspeed) antigens, 362, 480,
490
Bilirubin, 562, 564, 579 Bioassays, for radiation monitoring, 61 Biocontamination control system, 41
Biohazardous waste, 53-55,108 Biological product deviations, 22, 89, 90 Biological products, regulations for,
84, 85 Biological response modifiers, 681 Biological safety cabinets, 49, 50-51 Biologies license applications,
735, 744, 745, 746
 Biosafety, 47-55
biological safety cabinets for, 49, 50-51 biosafety levels, 48, 73 Blood-borne Pathogens Standard, 47
decontamination for, 49, 52 in donor room, 53 emergency response plan for, 53 engineering controls for, 49-52
hazard identification and communication for, 48-49 in laboratory, 53
personal protective equipment for, 52 safe work practices for, 52-53 standard precautions, 48 storage of
biohazards, 52, 54-55 training for, 48 waste management, 53-55 Biovigilance. See Hemovigilance Bleeding
acute, transfusion for, 501-502 and anemia, 508-509 assessing risk of, 518-521, 605 microvascular, 684
Bleeding disorders. See Coagulopathy Blood administration
baseline assessment of recipient, 546 blood warmers for, 548, 572, 584, 685-686 delays in starting
transfusion, 550 delivering blood to patient area, 549-550
emergency equipment for, 549 emergency release, 158, 227-228, 385-386, 506, 555-556 errors in, 551, 675
filters for, 551-552, 554, 585 identification procedures in, 550-551 infusion pumps for, 548-549, 584 infusion
rates for, 553, 554, 585 infusion sets for, 551-552, 585 IV solutions for, 552 medical history in, 546 medical
order for, 546-547, 551, 699-700, 703-704
monitoring patient in, 553, 555 in neonates, 584-585 out-of-hospital, 556 patient consent in, 545-546, 601
patient education in, 546 post-administration events, 555 premedications for, 547-548, 677, 679-680 pressure
devices for, 549 starting the transfusion, 552-553 for surgery and trauma, 555-556 syringe infusion pumps for,
549 transfusion reactions in, 553, 555, 666 venous access for, 547, 580, 584 Blood-borne Pathogens Standard,
47, 53 Blood clots, in RBCs, 149 Blood collection
additive solutions for, 136-137,138, 576577
adverse donor reactions in, 20, 22,142146
anticoagulant-preservative solutions for, 136,137
autologous, 605-606 blood containers for, 136-139,140 of blood samples, 141, 368, 370, 384 of components
by apheresis, 167-176 in disaster planning, 100-102 donation intervals for, 120,122 donor and component
identification in, 139-140
donor care after, 142 equipment quality control, 38 mobile sites for, 41 phlebotomy in, 140-141 process of,
141 safety of, 41, 53
of umbilical cord blood, 733-734 volume collected, 120, 141-142, 143 Blood component selection
ABO/Rh compatibility in, 375, 378-379, 504, 513-515, 554, 635
after non-group-specific transfusions, 396
808
AABB TECHNICAL MANUAL
with clinically significant antibodies, 274, 379,419-421
for exchange transfusions, 574, 579-580
for HSC transplantation, 633-634
for intrauterine transfusions, 564
for massive transfusions, 386
of platelets, 375, 459-461, 513-514, 581-582
rare types, 421
of red cell components, 375, 378-379, 504 for red cell exchange, 654 in urgent situations, 228, 385-386 in
warm autoimmune hemolytic anemia, 435-436
Blood components. See also specific components
administration of (See Blood administration) aliquoting, 224, 575-576, 583 bacterial contamination of, 198-
199, 224 CMV-reduced-risk, 190, 525, 564, 639 costs of, 602 cryopreservation of, 155 density of, 143
expiration of, 213, 215-220, 551 identification of, 139-140, 226, 384-385, 549, 550, 551
inspection of, 148, 149, 224, 226, 385, 549, 550-551
irradiated, 155-156, 222, 590, 688, 689 issuing, 226, 384-385, 549-550 labeling, 139, 158-159, 226, 384-
385 leukocyte reduction of, 154-155, 222-223, 504-505, 590
monitoring use of, 697-707 ordering, 226, 367-368, 546, 699-700, 703704
pooling, 156-157, 223-224 preparation and processing of, 146-148, 221-224
quality control of, 159-160 quarantine of, 157-158, 189, 224 rare, 421
receiving into inventory, 225 records of, 384-385 regulations regarding, 83 retrieval of prior donations, 187-
188, 189190
return and reissue of, 226-227, 550 selection of (See Blood component selection)
 shortages of, 101-102
storage of, 213-214, 215-220, 221, 503-504
testing
ABO and Rh, 225-226, 297-298, 378 infectious disease, 182-191, 192-204
phenotyping, 281, 329-330, 379, 420, 588, 654
traceability of, 225
transporting, 100-101, 146-147, 213-214, 215-220, 225
volume reduction of, 157, 223, 513-514, 581-582, 590
washed, 223, 590-591, 639 Blood containers
additive solutions in, 136-137,138 for aliquots, 576
anticoagulant-preservative solutions in, 136,137
configurations of, 136-137,139 diversion pouch on, 139,141 modification by sterile connecting device,
139,140
properties of, 136
Blood donation. See Blood collection; Donors Blood exposure, 44-45, 47-48, 49, 53,124 Blood group
genomics, 278-287
for antigen-negative blood donors, 285 to confirm D type of donors, 285 discrepancies between phenotype
and genotype, 240, 285-287, 402 to distinguish alloantibody from autoantibody, 283
to predict phenotype of recently transfused patients, 240, 281
to predict phenotype when red cells are coated with IgG, 283, 401-402, 436 in prenatal practice, 283-285
Blood group systems. See also specific blood groups
clinical significance of antibodies in, 338340, 413-414 defined, 279 genetics of, 255-279 chimerism, 278
gene mapping, 259-261, 277-278 inheritance patterns, 265-273 population genetics, 274-275 principles of,
256-258, 261-264 relationship testing, 276-277 terminology for, 278-279, 280 Blood loss
phlebotomy-related, 609 signs and symptoms of, 501 transfusion for, 501-502 Blood management. See
Patient blood management
Blood order schedules, 227 Blood pressure of donors, 120 hypotension
809
during apheresis, 659 associated with ACE inhibitors, 659, 669, 678
deliberate, 607 in recipients, 674, 679 Blood pressure cuffs, 38 Blood recovery, 606-607, 608-609 Blood
samples
age of, 370-371, 410-411, 417 from arterial or central lines, 609 collection of, 141, 368, 370, 384
confirming linkage with blood requests,
370
for hemoglobin/hematocrit screening, 121
hemolyzed, 370
incompletely clotted, 370
labeling, 370, 547
lipemic, 370
for PCR, 235, 236
for pretransfusion testing, 368, 370-371,
393, 547
retention and storage of, 371 transportation and shipment of, 63 Blood spills, 53, 54 Blood transfusions. See
Transfusions Blood utilization review, 25, 697-707 interventions to change transfusion practice, 610-611, 699,
704-705 rationale for, 698-699 selecting audit process, 705 types of audits
concurrent, 699, 700, 703, 706 prospective, 611, 699-700, 701-702, 706 retrospective, 699, 701-702, 703-
704, 707 Blood volume, of pediatric patients, 572 Blood warmers, 37, 548, 572, 584, 685-686 Bombay
phenotype, 292, 293, 303 Bone grafts, 123, 775 Bone marrow. See Marrow Bovine serum, 764-765, 793 Brain
natriuretic peptide, 682 Bruising, in donors, 145 Buerger disease, 763 Buffy-coat concentration of marrow, 721
Buffy-coat method of platelet preparation,
146, 147, 150, 156-157
C
Cl-esterase inhibitor deficiency, 522 Calcium supplementation, 658, 670, 683 Calibration, equipment, 10,11,
33 Cancer
 in blood donors, 126,128 leukemia
cytapheresis in, 654
donor lymphocyte infusions for, 786787, 788
natural killer cells in, 788 platelet transfusions in, 507 prostate, 791 Carboprost, 626 Cardiac patients
platelet transfusions in, 509 red cell transfusions in, 500-501 treatment with MSCs, 763, 794 Carriers, of
traits, 261 Cash reserves, 104 Catheters
for apheresis, 660 drawing specimens from, 609 for neonates, 580, 584 for transfusions, 547 Cause-and-
effect diagrams, 27, 28 CCI. See Corrected platelet count increment CDlla/CDllb, 468 CD34 cells, 723, 736
CD36, 458 CD59, 361 CD109, 456-457 CD177, 466-467, 469 Cefotetan, 442 Ceftriaxone, 442 Cell counters,
38 Cell counts, of HSCs, 723, 736 Cell division, 258, 262, 263 Cell washers, 36 Cellular immunotherapy
with cytokine-induced killer cells, 789-790 with dendritic cells, 790-791 donor lymphocyte infusions, 786-
788 with induced pluripotent stem cells, 795-796 with natural killer cells, 788-789 regulation and oversight of,
796-797 Centrifugation, in component preparation, 147-148
Centrifuges, 36, 38
Centromere, 257
Cephalosporins, 442,443
Ceppellini effect, 321, 323
Cerebral diseases, 763
cGMP. See Good manufacturing practice
cGTP. See Good tissue practice
Chagas’ disease, 126, 200-201
Change control, 33
Charts
Pareto, 27, 29 pedigree, 265, 266 process flow, 27 run and control, 24, 33 Check cells, 376
810
AABB TECHNICAL MANUAL
Chemical hygiene plan, 55 Chemical/organic solvent elutions, 429 Chemical safety, 55-60
chemical categories, 57, 76-77 chemicals found in blood banks, 74-75 emergency response plan for, 59, 78-
82 engineering controls for, 58 hazard identification and communication, 56-58
personal protective equipment for, 58 safe work practices for, 58-59 training for, 56 waste disposal, 60
Chemiluminescence assays, 419 Chemotherapy, 720
Chido/Rodgers system, 260, 338, 356-357, 406
Chido substance, 406
Chikungunya virus, 202
Children. See Neonates; Pediatric patients
Chimeric antigen receptors, 788
Chimerism, 278, 298, 489-490
Chlamydia trachomatis, 191
Chloroquine diphosphate, 407
Choline transporter-like protein 2, 467-468
Chromatids, 257, 258
Chromosomes, 256-258, 259-261, 277-278. See also Genes
Chronic granulomatous disease, 348-349, 524, 525
Circular of Information for the Use of Human Blood and Blood Components, 158-159 Circulatory overload,
transfusion-associated, 669, 682-683 Cirrhosis, 518-519, 764 Cis position, 272, 329 Citrate toxicity, 658, 670,
683 CJD. See Creutzfeldt-Jakob disease Clean rooms, 40-41 Cleaning and decontamination, 49, 52 Clinical
Laboratory Improvement Amendments (CLIA), 89-90 Clocks, 37
Clonogenic assays, 723, 736 Clotting factors. See Coagulation factors CMV. See Cytomegalovirus
Coagulation factors
concentrates, 124, 126, 526-527, 637-638 in Cryoprecipitated AHF, 153-154, 522-523 half-lives of, 521 and
INR, 520 in neonates, 582 replacement of, 524 in Thawed Plasma, 221 Coagulopathy in apheresis, 659
in blood donors, 126, 128-129 in hemolytic transfusion reactions, 674 in massive transfusion, 591, 684-685
in neonates, 582-583 plasma transfusions for, 517-522 Codominant inheritance, 265 Cold agglutinin disease,
437-439 antibody detection in, 438-439 serologic findings in, 431, 437-438 specificity of autoantibodies in,
308-309, 392,439
Cold autoadsorption, 438-439 Cold autoantibodies
ABO typing with, 300, 301, 438 adsorption of, 438-439 antibody identification with, 418 in cold agglutinin
syndrome, 308-309, 437439
in mixed AIHA, 439
in paroxysmal cold hemoglobinuria, 440 phenotyping with, 401 in Rh testing, 332, 438 use of sulfhydryl
reagents with, 438 in warm autoimmune hemolytic anemia, 431
Cold-reactive alloantibodies, 300, 301 Cold stress. See Hypothermia Collections, blood group, 361-362
Colloid solutions, 526 Colony-forming cell assays, 723, 736 Colton system, 260, 339, 355 Column
agglutination technology, 377, 396 Committee, for transfusion oversight, 24-25 Communications, emergency
plans for, 105107
Compatibility testing. See Pretransfusion testing
Competency assessments, 7-8 Complement
in acute transfusion reactions, 673-674 in autoimmune hemolytic anemia, 431, 437
role in immunity, 248-250 Complications. See Adverse reactions Complotype, 478 Computer crossmatch,
380-381 Computer systems
alternative systems for, 20 backup of, 18, 19-20 management of, 19-20 security of, 19 validation of, 15-16
Computerized provider order entry, 611, 699, 700
Concurrent audits, 699, 700, 703, 706
811
Confidentiality of information, 19 Consanguineous mating, 266 Consent
for apheresis, 170 for blood donation, 119 for cord blood donation, 731 for donation of HSCs, 718 for
transfusion, 545-546, 601 Contact lists, in disaster planning, 105 Containers
for blood collection, 136-139,140 for MSC storage, 761 for shipping
dry shippers, 724, 738, 739 HSCs, 724-725, 738 quality control intervals for, 38 temperature of, 724-725,
734, 738, 739, 779-780
validation of, 147, 225 Contamination bacterial, 198-199 detecting, 198-199 inspecting components for,
149, 224 during phlebotomy, 141,198 of platelets, 150, 198-199, 221 of RBCs, 198
reactive testing results for, 188 of tissue allografts, 777, 782 in transfusion-associated sepsis, 669, 676
fungal, 777, 782 in PCR, 236
Continuity of Operations Plan, 102-109 alternate facilities in, 103-104 cash reserves in, 104 decision trees
in, 103 elements of, 103
emergency communications plan in, 105107
essential functions in, 103 insurance in, 104 logistics in, 107-108 security and safety in, 104 staffing in, 108-
109 utilities in, 108 vital records in, 104 Contracts, 9-10, 108 Control charts, 24, 33 Control process, 3 Controls
autologous, 377, 382, 400, 403-404, 417-419 IgG-coated cells, 376 for Rh typing reagents, 331-332 Copper
sulfate, 38, 64,121 Cord blood. See Umbilical cord blood Cord blood banks, 729-730, 742-746
Cordocentesis, 563-564, 567, 568 Corrected platelet count increment, 458, 512, 516
Corrective action, 26, 27 Corticosteroids, 172, 548 Cost collection, 361 Costs, health-care, 602 Counter-
flow centrifugal elutriation, 721-722 CPOE. See Computerized provider order entry Creutzfeldt-Jakob disease,
202-203 and plasma derivatives, 526 screening donors for, 125,126 transmission through transplantation, 777
variant, 125, 202-203 Crohn’s disease, 764 Cromer system, 260, 339, 357-358 Cross-reactive groups, 482
Crossing-over, gene, 270-271, 479 Crossmatch-to-transfusion ratios, 227, 381 Crossmatching, 379-381
with alloantibodies, 420-421 antiglobulin test in, 380 computer, 380-381 HLA (lymphocyte), 487, 491-492
immediate-spin, 380 "in-vivo,” 419, 506-507 interpretation of results, 381,382 in pediatric recipients, 385, 574-
575 platelet components, 460-461, 489, 516 Cryoprecipitate Reduced Plasma, 152, 219220, 652
Cryoprecipitated AHF, 153-154
ABO compatibility of, 375, 554, 583 coagulation factors in, 153-154, 522-523 dose of, 523
expiration of, 157,218, 222 in HSC transplantation, 637 indications for, 522-523 infusion of, 554
for pediatric patients, 578, 583-584 pooled, 157, 218, 222, 224, 523 preparation of, 153 storage of, 153, 218,
222 thawing, 222
topical application of, 523 transportation and shipping of, 218 Cryopreservation agents for, 155, 722 of
HSCs, 722-723 of platelets, 155 of RBCs, 155 of tissue allografts, 775 Crystalloids, 501-502, 526, 652 CTL2,
467-468 Cytapheresis, 653-654, 655
812
AABB TECHNICAL MANUAL
Cytokine-induced killer cells, 789-790 Cytomegalovirus, 190 and CTL infusions, 788 in neonates, 589-590
preventing with leukocyte reduction, 190, 590, 639
screening donors for, 191 Cytomegalovirus-reduced-risk products, 190, 525, 564, 590, 639 Cytotoxic T
lymphocytes, 787-788
D
D antigen, 323-328 antibody to (anti-D)
clinical significance of, 338 in HDFN, 338, 562 and partial D, 328 passively acquired, 566 and platelet
transfusions, 515 in tissue transplantation, 111 titrations of, 563 and weak D, 328 clinical considerations for,
328 D epitopes on RHCE, 325, 326 D-negative, 321, 327 D-positive, 323-325, 327 elevated D, 325, 327 in
fetus, 283-284 nonfunctional RHD, 325 partial D, 324, 326, 327, 328, 333 in recipients, 327-328, 375 testing
for (See Rh testing) weak D, 323-324
in donors, 285, 327, 328 in fetus, 565
in prenatal patients, 565 in recipients, 327-328, 375 DARC glycoprotein, 349-351 DAT. See Direct
antiglobulin test DAT-negative autoimmune hemolytic anemia, 437
DDAVP. See Desmopressin Decontamination procedures, 49, 52,
55
Deep vein thrombosis, in donors, 145 Deferasirox, 690 Deferiprone, 690 Deferoxamine, 690 Deferrals. See
Donor deferrals Deglycerolization, 155, 222 Delayed transfusion reactions hemolytic, 250, 588, 670, 686-687
management of, 670-671 serologic, 686, 687 Dendritic cells, 785, 790-791
Dengue virus, 202
Density, of blood cells and components, 143 Derivatives. See Plasma derivatives Design output, 3, 33
Designated donations, 130 Desmopressin, 624 Dexamethasone, 525 DIC. See Disseminated intravascular
coagulation
Diego system, 259, 338, 352-354 Dimethyl sulfoxide (DMSO)
for cryopreservation of products, 155, 722, 761
functional effect on HSCs, 741 toxicity to, 724, 764-765 2,3-Diphosphoglycerate (2,3-DPG), 574 Diploid,
defined, 258 Direct antiglobulin test, 425-430
false positive/negative results in, 373-374 method for, 426-427 positive test
after HSC transplantation, 428 in antibody identification, 401, 404, 415416,417-419 causes of, 426
in cold agglutinin syndrome, 431 drug-induced, 428, 440-444, 448-451 elution with, 428-430 evaluation of,
427-430 medical history in, 427-428 in mixed-type AIHA, 431, 439 in paroxysmal cold hemoglobinuria,
431, 440
predicting phenotype with, 283 in warm autoimmune hemolytic anemia, 431
in pretransfusion testing, 372, 377 principles of, 426-427 reagents for, 372, 426-427 specimens for, 427
in transfusion reaction evaluation, 426, 672 Direct thrombin inhibitors, 518 Directed donations, 131 Disaster
management
for blood and transfusion services, 100-102 business operations planning in, 102-109 cycle of, 98
emergency management agencies in, 99-100 lessons learned from recent disasters, 112 overview of, 97
planning team in, 102 regulatory issues in, 109-111 risk assessment in, 98-99 testing disaster plans, 111-112
Disease modeling, 796
813
Disinfectants
to clean work surfaces, 49, 52 for venipuncture, 140-141,198 Disposal, waste, 54-55, 60, 63, 108
Disseminated intravascular coagulation, 674675
Dithiothreitol (DTT)
to disperse autoagglutination, 301, 432,438 for inactivating blood group antigens, 405, 407, 413-414
Diversion pouches, 139,141,198 DMSO. See Dimethyl sulfoxide DNA, 232-233, 237
DNA-based assays. See Nucleic acid analysis Documents, 13,16-18. See also Records Dolichos biflorus
lectin, 295, 301 Dombrock system, 260, 339, 354-355 Donath-Landsteiner test, 312,440 Donation identification
number, 135,139-140, 159, 551
Donor deferrals
 for allogeneic donors, 122-126 for bleeding conditions or blood diseases, 126, 128-129
blood donation intervals for, 122,123,169,170 for blood exposure, 124 for blood transfusions, 123,125 for
bone, skin or dura mater grafts, 123,126 for cancer, 126, 128 for clotting factor concentrates, 126 developing
criteria for, 127-130 during disasters, 101,110 for drugs taken by donor, 122,125,129-130, 169
for heart and lung conditions, 126, 129
for hemoglobin/hematocrit, 121
for incarceration, 125
for infectious diseases, 124-126
for needlesticks, 124
for piercings, 124
for pregnancy, 122
for reactive infectious screening tests, 187188, 189
records of, 118,119 regarding sexual contacts, 124,125 for tattoos, 124 for transplants, 123
for travel outside the US or Canada, 125,
126
for vaccinations or shots, 123 Donor History Questionnaire, 121-127,130 Donor lymphocyte infusion, 786-
788 Donor room, biosafety in, 53 Donor selection
for autologous donations, 118,131
blood-center-defined criteria for, 127-130 consent of donor in, 119-120 for directed donations, 131 Donor
History Questionnaire in, 121-127 education of donor in, 119-120 for exceptional medical need, 130 for
frequent donors, 130 hemoglobin or hematocrit in, 120, 121 for HSC transplantation, 714-717 identification of
donor, 118-119 overview of, 117-118 physical examination in, 120 registration process in, 118-119 Donor-
specific antibodies, 716 Donor testing ABO, 297-298
for blood group antigens, 285, 379, 420 for cord blood donation, 732-733 for HSC transplantation, 714-716
for infectious diseases, 182-191, 192-204 Rh, 285, 327, 328
silenced or nonexpressed genes in, 286 weak D, 327 Donors
adverse reactions in, 20, 22,142-146 age of, 119,120 of apheresis RBCs, 123,171 autologous, 131, 190-191,
605-606 with bleeding conditions or blood diseases, 126, 128-129 with cancer, 126, 128 consent of, 119, 170,
718, 731 deferral of (See Donor deferrals) designated or directed, 130-131 donation intervals for,
120,122,123,169, 170
educational materials for, 119-120,122 effect of disasters on, 101,110 family members as, 421 frequent or
repeat, 130 general health of, 122 hearing- or vision-impaired, 120 with heart and lung conditions, 126, 129
hemoglobin/hematocrit in, 120, 121 for HLA-matched platelets, 488-489 for HSC transplantation, 714-717
identification of, 118-119,139 legally incompetent, 119 leukapheresis, 122
medications taken by, 122, 129-130,169 non-English-speaking, 120 notification of abnormal test results,
189 phlebotomy of, 140-146 physical examination of, 120 plasmapheresis, 122, 170
814
AABB TECHNICAL MANUAL
plateletpheresis, 122, 168-169 postphlebotomy care of, 142 pregnancy in, 122
qualification requirements for, 121-127
with reactive screening tests, 186-190
records of, 119
registration of, 118-119
sexual contacts of, 124,125,126
temperature of, 120
of tissue allografts, 773-774
and TRALI, 682
of umbilical cord blood, 730-733 Dosage effect, 264, 393, 410 Dosimeters, 61,156 2,3-DPG, 574
Drug-induced immune hemolytic anemia, 440-444
antibodies in, 441-443 classification of, 441-443 drugs associated with, 448-451 laboratory investigation of,
443-444 mechanisms of, 441
Drug-induced thrombocytopenia, 462, 465466
Drug testing, with induced pluripotent stem cells, 796 Drugs
 administered for leukapheresis, 172 antiplatelet agents, 122, 129,169, 509 causing positive DAT, 428, 440-
444, 448451
hemostatic agents, 608, 621 -622 intravenous use of, 125 platelet antibodies induced by, 465-466, 516
pretransfusion medications, 547-548, 677, 679-680
removal of, in apheresis, 659-660 taken by donors, 122,129-130, 169 Dry ice, 225
Dry shippers, 724, 738, 739 DTT. See Dithiothreitol Duffy system, 349-351 antibodies of, 338, 350
antigens, 259, 349-350 Duffy glycoprotein, 350-351 and malaria, 351 phenotypes and genotypes, 349 silenced
alleles in, 287 Dura mater transplants, 126 Dysfibrinogenemias, 523
E
ECMO. See Extracorporeal membrane oxygenation Education
for donors, 119-120,122 for patients, 546 for physicians, 611-612 for umbilical cord blood donation, 730-
731 Electrical safety, 46-47
ELISA. See Enzyme-linked immunosorbent assay Elutions, 408-409, 428-430 Elutriation, 721-722
Embolism, air, 669, 685 Emergency Communications Plan, 105-107 Emergency equipment, 549 Emergency
management. See Disaster management
Emergency Management Agencies, 99, 100, 106-107, 111-112
Emergency release of blood, 158, 227-228, 385386, 506, 555-556 Emergency response plans, 44 for
biohazards, 53 for chemical spills, 59, 78-82 for electrical emergency, 47 for fires, 46
for radiation safety, 62 Emergency showers, 58 Employees. See Personnel End-product test and inspection,
33 Endothelial barrier dysfunction, 763-764 Engineering controls for biosafety, 49-52 for chemical safety, 58
for electrical safety, 47 for fire prevention, 46 general guidelines for, 44, 72 for radiation safety, 62
Engraftment, in HSC transplantation, 717-718 Enhancement media, 376-377, 396, 417 Enzyme-linked
immunosorbent assay, 243245, 464, 465-466 Enzymes, 377, 402, 405, 413-414 Epinephrine, 679
EPO. See Erythropoietic stimulating agents Epsilon-aminocaproic acid (EACA), 608, 621622
Equipment
apheresis, 168, 173-176, 645-646 critical, 10-11
decontamination of, 49, 52 emergency, 549 management of, 10-11,13 manufacturers directions for, 11
personal protective, 44, 52-53, 70-71 quality control of, 159 alarm systems, 36 performance intervals for, 36-38
thermometers, 37
815
regulations for, 83-84, 87 for transfusions, 548-549 validation of, 15 Er antigens, 361 Ergonomics, 41-42
Errors
in ABO and Rh typing, 300, 332 fatalities due to, 551 identification, 551, 675 mislabeled specimens, 370
quarantine release, 192, 195-196 sources of, in antiglobulin test, 373-374 wrong blood in tube, 378, 384
Erythroblastosis fetalis, 561 Erythrocytapheresis, 646, 655 Erythroid phenotypes, 345, 362-363 Erythropoiesis,
in neonates, 572 Erythropoietic stimulating agents, 572, 605, 620-621, 634
Estrogen, conjugated, 625 Ethnic groups, differences in
in antibody identification, 392-393, 394395, 415
in Duffy system, 349 in Kell system, 347 in Kidd system, 351-352 in MNS system, 341
in Rh system, 318-319, 320, 321, 323, 324, 325, 327
Exchange transfusions blood warmer for, 572 component choice for, 574, 579-580 for hemolytic disease of
the fetus and newborn, 564
for hyperbilirubinemia, 579 techniques for, 580 vascular access for, 580 volume of, 580 Executive
management, 5 Expiration, of components, 215-220, 551 Exposure control plan, 47, 48 Extracorporeal
membrane oxygenation, 585586
Extracorporeal photopheresis, 646, 654, 656 Eyewashes, 72
F
Face shields, 71 Facilities
alternate, during disasters, 103-104 clean rooms in, 40-41 design and workflow of, 40 ergonomic design of,
41-42 housekeeping in, 40 licensure and registration of, 84, 86
mobile sites, 41
quality management of, 5-6, 7,12 regulatory agencies for, 39-40, 68-69 restricted areas in, 41 safety program
of, 7,12, 42-64 Factor Vila, recombinant, 528, 623, 637-638, 685
Factor VIII
 antibodies to, 527 concentrates, 524, 526-527 in Cryoprecipitated AHF, 153,154, 522 Factor IX complex
concentrate, 518, 527-528, 623
Factor IX concentrates, 524, 527 Factor Xa inhibitors, 518 Factor XIII, 638
Failure modes and effects analysis, 28 False positive/negative test results, 332,373374
Fatalities
during apheresis, 660 of blood donors, 20, 145-146 due to identification errors, 551 due to TRALI, 681 due
to transfusions, 20, 691 of employees, 45 related to medical devices, 87 reporting, 20, 45, 87, 145, 691 Fatigue,
in donors, 145 Fc receptors, 248, 249, 250, 466 FDA. See Food and Drug Administration Febrile nonhemolytic
transfusion reactions, 489, 548, 667, 677
Fenwal apheresis systems, 168, 173,174,175 Fetal and neonatal alloimmune thrombocytopenia, 461, 567-
568 Fetal bovine serum, 764-765, 793 Fetal hemoglobin, hereditary persistence of, 362
Fetomaternal hemorrhage, 565-566 Fetus
genotyping, 283-284, 330, 563, 567 hemolytic disease in, 561-564, 566-567 pretransfusion testing of, 563
thrombocytopenia in, 461, 567-568 transfusions in, 563-564 weak D in, 565 Fever, 489, 667, 676, 677, 686 FFP.
See Fresh Frozen Plasma Fibrin clots, 300 Fibrinogen
abnormalities of, 522, 523 in Cryoprecipitated AHF, 153-154, 522 Fibrinogen concentrate, 529, 624 Ficin,
402
816
AABB TECHNICAL MANUAL
Filters
Leukocyte reduction, 154, 223, 552, 554 microaggregate, 551-552, 585 standard in-line, 551, 554, 585 Fire
prevention, 45-46 First aid, 44-45 Fish-bone diagrams, 27,28 Flow cytometry, 246
in HLA antibody testing, 487, 491 to measure fetomaternal hemorrhage, 565 to measure residual
leukocytes, 154-155 in platelet antibody detection, 460,464,465 in red cell survival studies, 419 Fludarabine,
443
Fluids, replacement, in apheresis, 522, 647,
652
Fluorescent methods for nucleic acid analysis, 238-240
Focal segmental glomerulosclerosis, 653 Food and Drug Administration inspections by, 86-87 regulations
and guidance
for biological products, 83, 84, 85 cGMP, 7, 8, 20, 213, 742, 760, 761 cGTP, 8, 20, 87, 743-744, 775, 111
for HPCs, 87-89, 743-746 for infectious disease testing, 182, 184, 187-188
for licensure and registration, 84, 86 for medical devices, 83-84, 87 for recalls and withdrawals, 89 for
tissues, 777
reporting adverse events to, 20, 22, 87 reporting fatalities to, 20, 87, 145, 691 variances to requirements, 11
Forensic testing, 493
Forms, 17,18. See also Documents; Records Forssman system, 261, 340, 360 Freeze-thaw elutions, 429
Freezers, 36, 214 Freezing
cryoprotective agents for, 155, 722, 761 Fresh Frozen Plasma, 151 hematopoietic progenitor cells, 722-723,
736-737 platelets, 155 RBCs, 155
Frequency, allele (gene), 274, 275-276 Fresh Frozen Plasma, 151
ABO compatibility of, 375, 554, 583, 635 aliquoting, 583
expiration of, 151, 218-219, 221 leukocyte content of, 505 in massive transfusion, 502, 685 for pediatric
patients, 578, 582-583, 589
preparation of, 147, 151 quarantine, 152
as replacement fluid in apheresis, 522, 647, 652
storage of, 151, 218-219 thawing, 151, 221 transfusion of, 522, 554 transportation and shipping of, 218-219
Fume hoods, 58 Fungal contamination, 777, 782
G
G-CSF. See Granulocyte colony-stimulating factor
Gastrointestinal diseases, 764 Gel-column agglutination technology, 377,
396
Gene locus, 256 Genes, 256-258
 of blood group systems, 259-261, 277-278 frequencies of, 275-276 of major histocompatibility complex,
476480
mapping, 259-261, 277-278 mutation of, 264
nucleic acid structure in, 232-233 position effect, 272-273 silent or amorphic, 264, 267, 286-287 suppressor
or modifier, 273 syntenic, 271 Genetic principles
alleles, 262, 264, 275, 276 blood group gene mapping, 259-261, 277278
cell division, 258, 262, 263 chimerism, 278
genes and chromosomes, 256-258 genotype and phenotype, 261-262 inheritance patterns, 265-273
autosomal, 265-267 gene interaction and position effect, 272-273
independent segregation and independent assortment, 270 linkage and crossing-over, 270-271, 272, 479
linkage disequilibrium, 271, 479-480 pedigrees, 265,266 sex-linked, 267-269
of major histocompatibility complex, 476480
polymorphism, 264 population genetics, 274-276 relationship testing, 276-277 X chromosome inactivation,
258, 261
817
Genotype
defined, 261-262 DNA-based assays for, 282
for antigen-negative blood donors, 282, 285
to distinguish alloantibody from autoantibody, 282, 283 of fetus, 283-284, 330, 563, 567 platelet
genotyping, 465 in prenatal practice, 283-285, 330 in recently transfused patients, 240, 281, 282, 330
of Rh system, 285, 330 in sickle cell disease patients, 330 when red cells are coated with IgG, 282, 283,401-
402,436 frequencies of, 275-276 nomenclature for, 280 and phenotypes, 240, 261-262, 285-287, 402 Gerbich
system, 260, 339, 357 Gill system, 261, 340, 359-360 Glanzmann thrombasthenia, 455 Globoside collection,
260, 309, 310, 311, 340 Gloves, 70-71
and latex allergy, 45 use in donor room, 53, 140 Glycerolization of red cells, 155 Glycine-HCl/EDTA, 407
Glycoproteins, platelet, 453, 454-455, 455-457, 458
GMP. See Good manufacturing practice Goggles, safety, 71 Gonorrhea, in donors, 124 Good manufacturing
practice
for component manufacturing, 213 for HCT/Ps, 742 for MSCs, 760, 761 for nonconforming events, 20 for
training personnel, 8 for work environment, 7 Good tissue practice
for infectious disease testing, 87 for nonconforming events, 20 for tissue processing, 775, 777 for training
personnel, 8 for umbilical cord blood, 743-744 Grading test results, 372 Graft-vs-host disease
and donor lymphocyte infusions, 787 in HSC transplantation, 638-639, 717 transfusion-associated, 671,
687-688 HLA system in, 489-490, 688 in neonates, 573
treatment with MSCs, 762, 763, 794 Graft-vs-tumor effect, 715, 717, 785
Granulocyte agglutination test, 469 Granulocyte colony-stimulating factor (GCSF)
for HSC mobilization, 719-720 in leukapheresis, 172-173, 524, 525 side effects of, 719-720 Granulocyte
immunofluorescence test, 469 Granulocytes
ABO compatibility of, 173, 375, 525, 554, 584
antigens and antibodies, 466-469, 681, 682 apheresis collection of, 168, 172-173 CMV-reduced-risk, 525
dose of, 584 expiration of, 217 HLA-matched, 525 in HSC transplantation patients, 638 increasing yields of,
172-172, 525 indications for, 525, 584, 589 infusion of, 173, 523-526, 554 irradiation of, 173, 217, 525
laboratory testing of, 173 in pediatric patients, 584 storage of, 173, 217, 221, 525 transportation and shipping
of, 217 Growth factors, recombinant, 172-173, 719720
Growth hormone, in donors, 129 GTP. See Good tissue practice GVHD. See Graft-vs-host disease
H
H system, 301-304 alloanti-H, 303, 339 autoanti-H, 303 autoanti-HI, 303, 339
biochemistry and genetics of, 260, 292-293, 301-303
Bombay phenotype, 292, 293, 303 H antigen, 292, 293, 301 Para-Bombay phenotype, 303 transfusion
practice for, 304 Hand washing, 72 Haploid, defined, 258 Haplotype
ancestral, 480,493 defined, 271
of HLA system, 478, 479, 480 of Rh system, 319, 320, 321-322 Hardy-Weinberg equilibrium, 275-276
Hazardous areas of facilities, 41 Hazardous materials biohazards, 47-55 chemicals, 55-60 classification of, 56
818
 AABB TECHNICAL MANUAL
identification and communication of, 43-44 for biosafety, 48-49 for chemical safety, 56-58 for electrical
safety, 47 for fire safety, 46 radioactive, 60-63 safety plan for, 42 shipping, 63
waste management of, 53-55, 63-64 Hazards, risk assessment of, 98-99,102 HBsAg. See Hepatitis B surface
antigen HBV. See Hepatitis B virus HCT/Ps (human cells, tissue, and cellular and tissue-based products). See
also Stem cells; Tissue
biological product deviations of, 22 infectious disease testing on donors of, 191192,714-715, 774
regulation of, 87-89, 743-746, 777-778, 796797
HCV. See Hepatitis C virus HDFN. See Hemolytic disease of the fetus and newborn
Health history questionnaires. See Donor history questionnaire Heart disease, in blood donors, 126, 129
Heart transplants, 493-494 Heat elutions, 429 Heating blocks, 37
Hemagglutination, 241-242, 279-280, 396 Hematocrit
in blood donors, 121
of blood in exchange transfusions, 580
in neonates, 585
of red cell components, 149
as transfusion threshold, 692
of Whole Blood, 148
Hematologic disorders, in blood donors, 128129
Hematoma, in donors, 145 Hematopoietic growth factors, 172-173, 719-720 Hematopoietic progenitor cells.
See Hematopoietic stem cells Hematopoietic stem cell transplantation ABO typing discrepancies in, 298, 300
adverse reactions to, 724-725, 742 allogeneic, 632-638, 715 autologous, 638, 714-715 and CMV, 639
diseases treated with, 713-714 donor lymphocyte infusion after, 786-788 donor requirements for, 714-717
engraftment kinetics in, 717-718 graft-vs-host disease after, 638-639, 717, 794 graft-vs-neoplasm effect in, 717
histocompatibility in, 491, 715-716 history of, in blood donors, 123 incompatible
bidirectional ABO, 633, 635 major ABO, 632-633, 635, 716 minor ABO, 633, 635, 716 related to non-ABO
antigens, 633 patient care in, 724-725 patient survival after, 718 positive DAT after, 428 products for (See
Hematopoietic stem cells) records of, 640
regulation of, 87, 88, 89, 725, 743-746 standards for, 746 transfusion support for autologous recipients, 638
component processing, 638-639 Cryoprecipitated AHF, 637 factor concentrates, 637-638 incompatible
transplants, 632-633 information portability, 640 in patients with HLA/HPA antibodies, 637, 639-640
in patients with neutropenia and infection, 638
in pediatric patients, 639-640 plasma, 637
platelets, 634, 635, 636-637 RBCs, 634
selection of components, 633-634, 635 transfusion reactions after, 639 Hematopoietic stem cells
collection of, 718-720, 733-734 cryopreservation of, 722-723, 736-737 donors of, 714-717 infusion of, 724-
725 irradiation of, 638 processing, 720-722 quality control of, 723, 736 regulation of, 87, 88, 89, 725 shipping
and transport of, 723-724, 737-738 sources of, 717-720 storage of, 736-737 thawing, 721 washing, 721, 740-
741 Hemizygous, defined, 263, 267 Hemoglobin
in blood donors, 120, 121 decreased, tolerance for, 609-610 in infants, 571-572, 578-579, 585 in Red Blood
Cells, 149 in red cell apheresis donors, 171 testing methods for, 121 as transfusion threshold, 499-501, 505-506,
578-579,610
819
Hemoglobin F, 362 Hemoglobin S, 564, 587, 588 Hemoglobin substitutes, 507 Hemoglobinometers, 38
Hemoglobinopathies, 587-589. See also Sickle cell disease Hemolysis
in ABO testing, 297 in antiglobulin tests, 372 extravascular, 250, 430, 673-674 in hemolytic disease of the
fetus and newborn, 562
intravascular, 251, 430, 672-673 nonimmune, 660, 669, 675-676 passenger lymphocyte syndrome, 633 in
patient samples, 370, 672 Hemolytic anemia autoimmune
classification of, 430 cold agglutinin disease, 437-439 DAT-negative, 437 mixed-type, 439-440 paroxysmal
cold hemoglobinuria, 440 serologic findings in, 431 transfusion in, 436-437, 439-440, 506 warm, 430-437
drug-induced immune, 440-444 neonatal, 561-564, 566-567 positive DAT in, 426, 428 Hemolytic disease of
the fetus and newborn, 561-564 ABO, 566-567
antibodies associated with, 338-340, 347348, 413-414, 562
 blood selection and administration in, 564 diagnosis of, 562-563 maternal alloimmunization in, 562
neonatal management in, 564 pathophysiology of, 562 pregnancy monitoring in, 563 preventing, 565-566
testing in, 563
antibody titers, 409, 563 elutions, 429
Rh testing, 283-285, 332, 563, 565 treatment of, 563-564 Hemolytic transfusion reactions acute, 667, 672-
675
antibodies associated with, 338-340, 413-414
clinical evaluation and management of, 666, 667, 672
due to misidentification errors, 551
elutions in, 429
HLA antibodies in, 490
delayed, 250, 588, 670, 686-687 intravascular hemolysis in, 251 nonimmune, 669, 675-676 Hemolytic
uremic syndrome, 653 Hemophilia, 524, 526-527, 528 Hemorrhage
and anemia, 508-509 assessing risk of, 518-521, 605 fetomaternal, 565-566 intraventricular, 581 transfusion
for, 501-502 Hemostasis
during massive transfusion, 591, 684-685 in neonates, 582-583 tests for, 518-521, 582 Hemostatic agents,
608, 621-627 Hemovigilance, 22, 33, 665-666 Heparin, 462, 465-466, 658, 675 Heparin-induced
thrombocytopenia, 462, 465-466, 517, 528
Hepatitis, non-A,non-B, 179,182,196 Hepatitis B immune globulin, 45 Hepatitis B surface antigen, 179,196
Hepatitis B virus, 196
employee exposure to, 44-45 nucleic acid testing for, 186, 196 prophylaxis for, 44
reactive testing results for, 187,188, 189 residual transfusion risk of, 192,194, 196 screening donors for,
124,126, 179,184, 191, 196
transmission through transplantation, 774, 777, 782
Hepatitis C virus, 196-197 employee exposure to, 44-45 nucleic acid testing for, 185-186,197 reactive
testing results for, 187,188, 189 residual transfusion risk of, 192-193,194, 197
screening donors for, 124,126, 179,184, 191, 197
transmission through transplantation, 774, 777, 782
Hepatitis E virus, 203-204 Herbal supplements, 605 Hereditary hemochromatosis, 476 Hereditary
persistence of fetal hemoglobin syndrome, 363
Heredity, genetics of. See Genetic principles HES. See Hydroxyethyl starch Heterozygous, defined, 263-264
High-prevalence antigens 901 series, 361-362 antibodies to, 415-416, 564 blood selection for, 421
820
AABB TECHNICAL MANUAL
High-titer, low-avidity antibodies, 409-410 Histocompatibility. See HLA system HIV. See Human
immunodeficiency virus Hives, 667, 678, 679-680 HLA alleles, 483-484 HLA antibodies
detection of, 460, 487 donor specific, 716
in HSC transplant recipients, 637, 639-640, 716
management of, 670 and plasma transfusions, 151 in platelet refractoriness, 458, 459-461, 488489, 515-516
in TRALI, 489,681,682 in transfusion reactions, 489, 490, 677 HLA antigens Bg, 362, 480, 490
Class I and Class II, 475, 480-482, 484 configuration of, 481 cross-reactive groups, 482 identification of,
484-487 nomenclature for, 481-482 on platelets, 458 “public,” 482-483 “splits,” 482
HLA-matched platelets, 459-460 HLA Matchmaker program, 460, 488-489 HLA system
biochemistry, tissue distribution and structure of, 480-484 biologic function of, 484 and chimerism, 489-490
disease associations with, 493-494 genetics of, 476-480
absence of antigens, 478-479 crossovers, 479
finding HLA-identical siblings, 478 linkage disequilibrium, 479-480 organization of, 476-478 patterns of
inheritance in, 478-480 in graft-vs-host disease, 489-490, 688 importance of, 475-476 HLA typing
cellular assays for, 486-487 crossmatching, 487, 491-492 DNA-based assays for, 485-486, 493 in forensic
testing, 493 of Granulocytes, 525 lymphotoxicity assays for, 486 of platelets, 459-460, 488, 516 in relationship
testing, 493 in transplantation, 491-493, 715-716 HNA. See Human neutrophil antigens Homolog, defined, 267
Homozygous, defined, 263-264 Hook effect, 245 Hospitals, 91, 602-603 Housekeeping, 40
HPA. See Human platelet alloantigens HSC. See Hematopoietic stem cells HSCT. See Hematopoietic stem
cell transplantation
 HTLV. See Human T-cell lymphotropic virus Human immunodeficiency virus, 194-196 educating donors
about, 119 employee exposure to, 44-45 nucleic acid testing for, 185-186,195 reactive testing results for,
187,188, 189 reporting new cases of, 782 residual transfusion risks for, 192-193,194, 195
screening donors for, 124,125,126,182, 184, 191, 194-196
transmission through transplantation, 774, 777, 782
Human neutrophil antigens, 466-468 Human platelet alloantigens, 453-457 Human resources, 7-9,12, 109
Human T-cell lymphotropic virus, 197 reactive testing results for, 187,188, 189 screening donors for, 184,191,
197 transmission through transplantation, 774, 782
Human urea transporter 11, 352 Hydatid cyst fluid, 312, 406 Hydrops fetalis, 561 Hydroxyethyl starch, 172,
722 Hyperbilirubinemia, 579 Hyperhemolytic syndrome, 588 Hyperkalemia, 555, 683-684 Hyperviscosity, 652
Hypocalcemia, 555, 658, 670, 683 Hypofibrinogenemia, 522-523 Hypogammaglobulinemia, 300, 529, 659
Hypoglycemia, 580 Hypokalemia, 683-684 Hypotension
in apheresis, 659
associated with ACE inhibitors, 659, 669, 678
deliberate, 607
in transfusion recipients, 674, 679 Hypothermia, 548, 572-573, 607, 670, 685-686 Hypovolemia, 526, 659
I
I and i antigens, 306-309 anti-I, 308, 340 anti-i, 308
821
in cold agglutinin disease, 308-309, 392, 439 disease associations with, 307, 392 genetics of, 260, 307-308
phenotypes, 306-307 transfusion practice with, 309 Iatrogenic anemia, 609 I CAM-4, 355-356 Identification
of blood components
before administration, 226, 551 before issue, 226, 549-550 donation identification number, 135, 139-140,
159
labeling, 158-159, 384-385 of donors, 118-119, 139 of equipment, 11 errors in, 551, 675 of personnel, 19 of
persons issuing blood, 385 of phlebotomists, 370, 547 of problems and solutions, 26-29 of recipients, 226, 368-
370, 384-385, 547, 549, 550, 551
of umbilical cord blood, 739 Idiopathic thrombocytopenia, 463, 465, 517, 568
IgA, 247, 248, 678-679, 680 IgE,247, 248 IgG, 247,248, 249,573 IgG-coated cells (check cells), 376 IgM,
246, 248
cold-reactive autoagglutinins, 437-439 in complement activation, 249 dispersing autoagglutination caused
by, 301,438 in infants, 573
in intravascular hemolysis, 251 structure of, 24 7
Immediate-spin crossmatch, 380 Immune complexes, 652 Immune thrombocytopenic purpura, 463,465,
517, 568 Immunity
antibodies in, 246-248 complement in, 248-250 extravascular hemolysis in, 250 Fc receptors in, 248, 249,
250 in infants, 573
intravascular hemolysis in, 251 Immunoglobulin products, 526, 529-531,532 Immunoglobulins, 246-248.
See also specific immunoglobulins
Immunohematology reference laboratories, 419
Immunomagnetic cell separation, 722
Immunomodulation, 504, 758, 762-763, 764 In-vivo testing, 419, 506-507 Incarceration, donor history of,
125 Incidence, defined, 274 Incinerators, hospital/medical/infectious waste, 55
Incubation times, 405 Incubators, platelet, 36, 214 Independent assortment, 270 Independent segregation,
270 Indian system, 260, 339, 358-359 Indirect antiglobulin test, 372, 373-374 Induced pluripotent stem cells,
755, 795-796 Infants. See Neonates; Pediatric patients Infection, in transplant patients, 638, 782 Infectious
disease screening approaches to, 183 of autologous donations, 190-191 for Babesia, 201
for bacterial contamination, 198-199
for chikungunya virus, 202
for CMV, 190
for dengue virus, 202
for HBV, 196
for HCV, 196-197
for HEV, 203-204
 historical overview of, 179-182
for HIV, 194-196
in HSC transplantation donors, 191-192, 714-715 for HTLV, 197
international variations in, 192 logistics of, 183 for malaria, 199-200, 201-202 nucleic acid testing, 185-186
for parvovirus B19, 203 for prions, 202-203 reactive test results in, 186-190 residual infectious risks of
transfusion, 192194
serologic testing, 185 for syphilis, 198 in tissue transplantation, 774 for Trypanosoma cruzi, 200-201 for
umbilical cord blood, 732 in the United States, 184 for West Nile virus, 200 Infectious diseases
emerging agents, 203, 204 safety precautions for, 47-55 screening for (See Infectious disease screening)
transmitted by plasma derivatives, 203 transmitted by tissue transplantation, 111, 782
822
AABB TECHNICAL MANUAL
Infectious waste, 53-55 Inflammation modulation, 763-764 Information management, 13, 19-20 Infusion
pumps, 548-549, 584 Infusion rates, 553, 554, 555, 585 Infusion sets, 551-552, 585 Inheritance patterns
autosomal, 265-267 crossing-over, 270-271, 479 gene interaction and position effect, 272273
independent segregation and independent assortment, 270 linkage, 270-271, 272 linkage disequilibrium,
271, 479-480 of major histocompatibility complex, 478480
pedigrees, 265, 266 sex-linked, 267-269 Inhibition tests, 406 Injuries, 45, 49, 87
INR. See International normalized ratio Inspections
of components
before administration, 550-551 before release, 224, 226, 385, 549 after preparation, 148,149 documentation
of, 224 of incoming supplies, 10 of tissue grafts, 779-780 Insulin, bovine, 129 Insurance, 104 Integrins, 455-456
Internal event reports, 20, 21 -22 International normalized ratio (INR), 518,519, 520
Intraoperative blood recovery, 606-607 Intrauterine transfusions, 563-564 Intravenous immune globulin,
529-531 ABO discrepancies with, 300 adverse effects of, 529, 532 in antibody identification problems, 392
applications of, 530-531 for fetal and neonatal immune thrombocytopenia, 568 in HDFN, 564
in platelet refractoriness, 516 positive DAT with, 428 Intravenous solutions, 552 Intraventricular
hemorrhage, 581 Inventory, blood
during disasters, 101-102,110 management of, 227-228 and patient blood management, 603 receiving
components into, 225
Investigational new drug application, 731,744, 745
Iron, supplemental, 605, 620 Iron chelation therapy, 690 Iron deficiency anemia, 605, 620 Iron overload,
588, 671, 690 Irradiated products, 155-156, 222 expiration of, 215, 217, 222 granulocytes, 173, 217, 525 in
HSC transplantation, 638 indications for, 688, 689 for intrauterine transfusions, 564 for pediatric patients, 590
platelets, 156, 217, 459-460 potassium leak in, 573 quality control of, 156 RBCs, 156,215 storage of, 215, 217
transportation of, 215, 217 Whole Blood, 215 Irradiators, blood, 37, 62, 155-156 Ishikawa diagrams, 27, 28
Isografts, 774 Isohemagglutinins, 292 Issuing components, 226
delivering blood to patient area, 549-550 identification of recipient and component before, 384-385, 549-
550 inspections prior to, 226, 385, 549 reissue, 226-227, 550
in urgent situations, 158, 227-228, 385-386, 506, 555-556
ITP. See Immune thrombocytopenic purpura IVIG. See Intravenous immune globulin
J-K
John Milton Hagen system, 260, 339, 359 JR system, 261, 340, 360 Juran’s Quality Trilogy, 2-3 Karyotype,
256 Kell system, 345-349
allele frequencies in, 275-276 antibodies of, 338, 347-348 antigens of, 259, 346-347 biochemistry and genes
of, 259, 346 functional aspects of, 348 in HDFN, 284, 347-348, 562, 563 Kmod,348
K0 (null) phenotype, 348 phenotypes of, 346 position effect in, 272-273 Kernicterus, 562, 564, 579 Kidd
system, 351-352 antibodies of, 338, 352
823
antigens of, 259, 351-352 genetics of, 259 Kidd glycoprotein, 351, 352 phenotypes of, 351 in transfusion
reactions, 352 Kidney transplantation, 491-492 Kleihauer-Betke acid-elution test, 565-566 Knops system, 260,
339, 358 Kx system, 348-349 antibodies of, 339 genetics of, 258, 260, 261, 269 McLeod phenotype, 258, 261,
269, 348
L
 Labels
for biohazardous materials, 49 for blood components, 139, 158-159, 226, 384-385
for blood samples, 370, 547 control of, 17,18 for hazardous chemicals, 57-58 ISBT 128 system for, 159 for
shipments, 738 for umbilical cord blood, 739 Laboratories
biosafety precautions for, 53 regulations for, 89-91 Laboratory coats, 70 Lan system, 261, 340, 360-361
Landsteiner-Wiener system, 260, 339, 355-356 Latex allergies, 45,136 LDL apheresis, 656
Leadership, organizational, 5-6,12, 105 LEAN, process improvement, 28-29 Lectins, 295, 301
Leukapheresis
collection of granulocytes by, 175-176 donation intervals for, 120,122 indications for, 646, 654, 655
instrumentation for, 168, 175-176 Leukemia
in blood donors, 126, 128 cytapheresis in, 654
donor lymphocyte infusions for, 786-787, 788 natural killer cells in, 788 platelet transfusions in, 507
Leukocyte-reduced components, 504-505 expiration, transportation and storage of, 216,217
leukocyte content in, 154-155, 505 for pediatric patients, 590 platelets, 154, 156, 169-170,217, 505
poststorage filtration, 222-223 prestorage filtration, 154-155, 223, 552 to prevent CMV infection, 190, 590, 639
RBCs, 149, 154,216, 504-505 to reduce alloimmunization, 515 to reduce incidence of PTP, 690 Leukocyte
reduction filters, 154, 223, 552, 554 Leukocytes, in components, 154-155, 505 Lewis substance, 406 Lewis
system, 304-306
antibodies of, 305-306, 338 antigens of, 259, 302, 304 biochemistry and synthesis of, 302, 304 disease
associations with, 306 expression in children, 305 genetics of, 259, 304-305 phenotypes of, 304-305 saliva
testing for, 406 transfusion practice with, 306 Licensure, of facilities, 84, 86 Likelihood ratio, 277 Linkage, 270-
271,272 Linkage disequilibrium, 271,479-480 Lipemic samples, 370 Liquid nitrogen
for shipping, 724, 738 for storage, 723, 737, 761 Liquid Plasma, 152,220 LISS, 376,402,417 Liver,
transplantation of, 493-494 Liver disease, 764 LKE antigen, 309, 310, 313 Look-back investigations, 187-188,
189-190, 782 Low-prevalence antigens, 362,416 Low-volume units, 141,142, 149 Lui freeze thaw elution, 429
Luke antigen, 309, 310, 313 Lung conditions, in blood donors, 126,129 Lung transplants, 493-494 Lutheran
system, 344-345 antibodies of, 338, 345 antigens of, 259, 344-345 genetics of, 259, 267, 272, 273 Lymphocyte
crossmatching, 487 Lymphocytes, T cytotoxic, 787-788 Lymphocytotoxicity assays, 485, 486, 487
Lymphoproliferative disease, 788 Lyonization, 258, 261
M
MACE (modified antigen capture ELISA), 464 MAIGA (monoclonal antibody immobilization of
granulocyte antigens), 469 MAIPA (monoclonal antibody immobilization of platelet antigens assay), 464 Major
histocompatibility complex, 475. See also HLA system
Class I and Class II antigens in, 480-482
824
AABB TECHNICAL MANUAL
genetics of, 476-480 public antigens, 482-483 split and cross-reactive groups in, 482 Major
histocompatibility complex multimer method, 787 Malaria, 201-202
and Duffy glycoprotein, 351 screening donors for, 125,126, 202 Markers, defined, 255 Market withdrawals,
782 Marrow
autologous, 714-715 collection of, 718-719 cryopreservation of, 722-723 donor requirements for, 714-716
engraftment kinetics of, 717-718 histocompatibility of, 715-716, 717 infectious disease testing of, 191, 715
infusion of, 724-725 MSCs derived from, 755, 762, 763 processing, 720-722 quality control of, 723 red cell
reduction in, 720-721 regulations for, 87, 88, 89, 725 shipping and transport of, 723-724 transplantation
outcomes using, 718 Marrow aplasia, 787 Masks, 71
Massive transfusions
complications of, 591, 683-685 component replacement in, 501-502, 685 defined, 228, 386 Factor Vila,
recombinant in, 685 in pediatric patients, 591 pretransfusion testing in, 228, 330, 386 Material safety data
sheets, 57, 58 Materials management, 9-10,12 Maximum surgical blood order schedules, 227 McLeod
phenotype, 258, 261, 269, 348-349 2-ME. See2-mercaptoethanol Mechanical hemolysis, 660, 676 Media,
working with, 107 Medical devices, regulations for, 83-84, 87 Medical history
in antibody identification, 392 in blood donor selection, 121-127 for cord blood donation, 731-732 in
evaluation of positive DAT, 427-428 of recipients, 546 Medical waste, 53-55 Medication Deferral List, 129-130
Medications. See Drugs Meiosis, 258, 263
 Membrane attack complex, 249-250 2-mercaptoethanol (2-ME), 407,432,438
Mesenchymal stem cells
autologous vs allogeneic use, 754 future directions for, 765-766 identification criteria for, 793 isolation and
expansion of, 758-760, 792793
multipotentiality of, 754-755, 791-792 properties of, 756-757, 758, 786, 791-792 research and development
on, 764-765 shipping, 761-762 sources of, 754-758 standardization of methods for, 760 storage and banking,
761 therapeutic applications of, 762-764, 793-795 and tissue engineering, 795 tumor-forming potential of, 765
Message mapping, 107 Messenger RNA, 233
Metabolic abnormalities, in infants, 573-574 Methergine, 625 Methyldopa, 443
Methylene blue-treated plasma, 153,205 Microaggregate filters, 551-552, 585 Microarray assays, 245
Microlymphocytotoxicity tests, 485, 486, 487 Microsatellites, 277 Microvascular bleeding, 684 Minisatellites,
277 Misoprostol, 626 Mitosis, 258, 262
Mixed-field agglutination, 278, 298, 362 Mixed leukocyte culture, 486-487 Mixed-type autoimmune
hemolytic anemia, 431, 439-440
MNS system, 337, 341-344
antibodies of, 338, 343-344, 406 antigens of, 259, 341, 344 effect of enzymes on, 314 genetics of, 259, 341,
343 glycoproteins of, 341, 342, 343 linkage disequilibrium in, 271 phenotypes of, 341 S-s-U- phenotype, 343
Mobilization regimens for granulocytes, 172-173 for HSCs, 719-720
Molecular immunohematology. See Blood group genomics
Monoclonal antibody immobilization of granulocyte antigens, 469 Monoclonal antibody-specific
immobilization of platelet antigen, 464 Monocyte monolayer assay, 419 Mortality. See Fatalities MSC. See
Mesenchymal stem cells
825
Multiple sclerosis, 653 Multiplex nucleic acid amplification, 238 Multipotent stromal cells. See
Mesenchymal stem cells
Mutations, genetic, 264, 362-363 Myeloma, IgM, 652
N
Nageotte hemocytometry, 154-155 Narcolepsy, 494
NAT (nucleic acid testing), 185-186 in HBV testing, 196 in HCV testing, 197 in HIV testing, 195 Natural
killer cells, 788-790 Near-miss events, 26, 33 Needlesticks, 49, 124, 141 Neisseria gonorrhea, 191 Neonatal
alloimmune neutropenia, 468 Neonatal alloimmune thrombocytopenia, 461, 567-568
Neonates (younger than 4 months)
ABO and Rh typing in, 300, 385, 574 ABO antigens and antibodies in, 293, 300 anemia in, 571-572
antibody screening in, 385 antigenic variations in, 305, 410, 411 blood volume in, 572 compatibility testing in,
385, 574-575 ECMO in, 585-586 erythropoietic response in, 572 hemoglobin in, 571-572, 578-579 hemolytic
disease in, 561-564, 566-567 hemostasis in, 582-583 hypothermia in, 572-573 immunologic status of, 573
Lewis antigens in, 305 metabolic problems in, 573-574 neutropenia in, 468, 525 polycythemia in, 585
thrombocytopenia in, 461, 567-568, 580-581 transfusion-associated GVHD in, 573 transfusions in
administration of, 584-585
age of units for, 577-578
aliquots for, 224, 575-576, 583
of Cryoprecipitated AHF, 578, 583-584
dosing for, 578
exchange, 564, 574, 579-580
of FFP, 578, 582-583
of granulocytes, 584
indications for, 574, 575, 578-579
of platelets, 578, 580-582
ofRBCs, 574-580
safety of additive solutions in, 576-577
transfusion thresholds in, 578-579 vascular access for, 547, 580, 584 Nerve injury, in donors, 145
Neutralization techniques, 406 Neutropenia
autoimmune, 468
granulocyte transfusions for, 523-525, 584, 589
 in HSC transplant patients, 638 of infancy, 468 neonatal alloimmune, 468 NMDP registry, 725
Nomenclature
for blood group systems, 259-261, 278-279, 280
for granulocyte antigens, 467 for HLA system, 481-482, 483-484 for human platelet alloantigens, 453-455
of Rh system, 318-319, 319, 320 Nonconformances
biological product deviations, 22, 89, 90 classification of, 22-23 internal event reports, 20, 21-22
management of, 13, 20-23 Nonimmune-mediated hemolysis, 660, 669, 675-676
NOR phenotype, 311 Normovolemic hemodilution, 606 Notifications, of reactive screening tests, 189190
Nucleic acid analysis
detection of amplification products in, 238240
for genotyping, 282
for antigen-negative blood donors, 282, 285
to distinguish alloantibody from autoantibody, 282, 283 of fetus, 283-284, 330, 563, 567 platelets, 465
in prenatal practice, 283-285, 330 in recently transfused patients, 240, 281, 282, 330
of Rh system, 284-285, 287, 330 in sickle cell disease patients, 330 when red cells are coated with IgG, 282,
283,401-402, 436 for granulocyte antigens, 469 in HLA typing, 485-486, 493 hybridization-based methods of,
233 for infectious diseases, 185-186,195, 196, 197 isolation of nucleic acids in, 233 nucleic acid sequence-
based amplification in, 237-238 overview of, 231-232
826
AABB TECHNICAL MANUAL
for platelet antigens, 465 polymerase chain reaction in, 233-237, 280281
in pretransfusion testing, 378 in relationship testing, 277 reverse-transcriptase PCR in, 236-237 of single
nucleotide polymorphisms, 240 transcription-mediated amplification in, 237-238
in West Nile virus testing, 200 Nucleic acid sequence-based amplification, 237-238
Nucleic acids, structure of, 232-233
O
Obstetrics, controlling bleeding in, 625-626 Octreotide, 626-627 Oh (Bombay) phenotype, 292, 293, 303 Ok
system, 260, 339, 359 Oligonucleotide probes, 485 Orders, physician auditing, 697-707
computerized provider order entry, 611, 699, 700
pretransfusion, 381, 383, 384, 546-547 sample form for, 711 surgical blood orders, 227 verifying prior to
transfusion, 551 Organ transplantation
ABO compatibility in, 491, 492, 783 history of, in blood donors, 123 HLA testing in, 491-492, 493 kidney,
491-492 paired donations, 492 positive DAT after, 428 regenerative therapy in, 754 rejection of, 653, 654, 656
transfusion support for, 783 Organizations
for emergency management, 99-100,109110
for quality system regulation, 1-2 for safety, 39-40, 68-69 structure of, 5-6,12 Orientation programs, 7-8
Osteoarthritis, 763 Osteogenesis imperfecta, 794 Outpatients, 368, 556 Oxytocin, 625
P
P blood groups, 309-313 antibodies of, 312, 338 antigens of, 259, 260, 309-312
biochemistry of, 310-311 disease associations with, 312-313, 392, 440 genetics of, 259, 260 molecular
biology of, 311-312 phenotypes of, 309-310 transfusion practice with, 312 PI substance, 312, 406 P1PK system,
259, 309, 338 Panel-reactive antibody, 487, 492 Panels, red cell, 393, 396-400 Papain, 402
Para-Bombay phenotype, 303 Pareto analysis, 27,29 Paroxysmal cold hemoglobinuria, 312, 392, 431, 440
Partial D, 324, 326, 327, 328, 333 Partial thromboplastin time, 519 Parvovirus B19,187, 203, 313 Passenger
lymphocyte syndrome, 633 Paternal samples, testing
DNA-based testing, 284-285, 563 in relationship testing, 276-277 Pathogen reduction technology, 204,205
and emerging infectious agents, 194 for plasma, 153
for plasma derivatives, 203, 526 to reduce risk of septic reactions, 199 Patient blood management activity
levels of, 612, 629 acute normovolemic hemodilution in, 606 anemia assessment in, 604-605 anesthesia in, 607
auditing in, 24-25, 697-707 bleeding risk assessment in, 605 blood recovery in, 606-607, 608-609 blood
utilization review in, 604, 611 changing physician behavior in, 610-611 coordinators for, 611 definition of, 599
increased tolerance of anemia in, 609-610 limiting phlebotomy in, 609 medical education in, 604, 611-612
pharmacologic agents in, 608, 620-627 point-of-care testing in, 607-608 preoperative autologous blood
donation in, 605-606
 program development, 612 rationale for, 600-603 responsibilities for, 629 scope of, 600, 604 surgical blood
orders in, 227 surgical techniques in, 607 transfusion algorithms in, 608 transfusion thresholds in, 610
827
PBSC (peripheral blood stem cells). See Peripheral blood HSCs PCC. See Prothrombin complex
concentrates PCR. See Polymerase chain reaction PEDI-PAK system, 576, 577 Pediatric patients (older than 4
months). See also Neonates
ABO antigens and antibodies in, 293, 300 CMV prevention in, 589-590 Lewis antigens in, 305
pretransfusion testing in, 300, 587 thrombocytopenia in, 581 transfusions in
aliquoting for small volumes, 224, 575576
of CMV-reduced-risk components, 590 of Cryoprecipitated AffF, 578, 583 of FFP, 578, 583, 589 of
granulocytes, 584, 589 in HSC transplantation patients, 639640
of irradiated components, 590 of leukocyte-reduced components, 590 massive, 591
of platelets, 578, 581, 582, 589, 590 ofRBCs, 578, 586-589 with sickle cell disease, 587-588, 639 syringe
infusion pumps for, 549 with thalassemia, 588-589, 640 vascular access for, 547 of volume-reduced
components, 590 of washed components, 590-591 Pedigrees, 265,266 Peer review, 24-25, 697-707 PEG. See
Polyethylene glycol Penicillin, 442
Performance improvement standards, 26 Periadventitial cells, 758 Pericytes, 758 Peripheral blood ffSCs
allogeneic, 715-717 autologous, 714-715 collection of, 718, 719-720 cryopreservation of, 722-723 donor
eligibility of, 714-717 engraftment kinetics of, 717-718 infectious disease testing on donors of, 191, 714-715
infusion of, 724-725 mobilization of, 719-720 patient survival with, 718 processing, 720-722 quality control
of, 723 regulations for, 87, 88, 89, 725 shipping and transport of, 723-724
Personal protective equipment, 44, 70-71 for biosafety, 52-53 for chemical safety, 58 gloves, 45, 53, 70-71
Personnel
accidents and injuries in, 45, 49 blood exposure in, 44-45, 47-48, 49 competency assessment of, 7-8 contact
list of, 105 disaster plans for, 108-109 essential, 105, 109 hepatitis prophylaxis for, 44 identification of, 19 latex
allergies in, 45 orientation program for, 7-8 protective equipment for, 44, 52-53, 70-71 records, 19
safety monitoring programs for, 44 selection of, 7
staffing plan, 9,108-109,110-111 training (See Training)
PF4 ELISA, 465-466 pH, 406,411 pH meters, 37
Phenotype. See also specific blood groups calculations for, 274,421 defined, 261-262 and genetic mutations,
264 and genotypes, 240, 261-262, 285-287, 402 nomenclature for, 280 prevalence of, 274 rare, 394-395, 421
Phenotyping
antigen-matching, 281, 329-330, 379, 588, 654
autologous red cells, 401-402 with DNA-based assays, 280-281, 282 for antigen-negative donors, 282, 285
to confirm D type of donors, 285, 330 to distinguish alloantibody from autoantibody, 282, 283 in prenatal
practice, 283-285, 330 in recently transfused patients, 240, 281, 282, 330
when red cells are coated with IgG, 282, 283,401-402, 436 donor units, 420 solid-phase assays for, 242-243
Phlebotomy. See also Blood collection adverse reactions to, 142-146 blood loss due to, 609 for collection of
blood samples, 368, 370 disinfection methods for, 140-141 of donors, 140-146 vein selection for, 140
828
AABB TECHNICAL MANUAL
Photopheresis, 646, 654, 656 Physical assessment
of apheresis patients, 660-661 of donors, 120 of recipients, 546 Physicians
changing behavior of, 610-611, 704-705 educating, 611-612
perspective on patient blood management, 601-602
Physiologic anemia of infancy, 571-572
Piercings, 124
Piperacillin, 442, 443
Pipettes, recalibration of, 37
P1A1 (HPA-1) antigen, 455, 567
Plasma
ABO compatibility of, 375, 554, 583, 635 aliquoting, 583 coagulation factors in, 221 collection by
apheresis, 168,170 cryoprecipitate reduced, 152, 219-220, 652 donors of, 682
 expiration of, 151, 218-220, 221 fresh frozen (See Fresh Frozen Plasma) frozen within 24 hours after
Phlebotomy, 152
liquid, 152, 220 pathogen-reduced, 153, 205 preparation of, 147-148, 151 for pretransfusion testing, 392,
393 recovered (for manufacture), 152-153, 220, 526
as replacement fluid in apheresis, 522, 647, 652
solvent/detergent-treated, 153, 205 source, 170, 526 storage of, 151, 218-220 thawed, 151, 152, 219, 221,
522 thawing, 221
transfusion of (See Plasma transfusions) transportation and shipping of, 218-220,
225
Plasma derivatives csq-antitrypsin, 532 activated protein C, 532 albumin, 526 antithrombin, 531
clotting factor concentrates, 526-527 fibrinogen concentrate, 529, 624 infectious disease screening for, 203
intravenous immune globulin, 529-531,532 pathogen reduction for, 203, 526 prothrombin complex
concentrates, 527528, 623
recombinant Factor Vila, 528, 623
Plasma exchange. See Therapeutic plasma exchange
Plasma transfusions
to correct PT/INR, 518, 519-520 dose and timing of, 521 and HLA antibodies, 151 indications for, 517-518,
522 in cirrhosis patients, 518-519 in correction of anticoagulation, 518, 519 in HSC transplantation, 635, 637 in
massive transfusion, 502, 685 in pediatric patients, 578, 582-583, 589 infusion of, 554 types of plasma for, 522
Plasmapheresis, 170 consent for, 170
donation intervals for, 120,122, 170 instrumentation for, 168, 173-174 plasma transfusion in, 522 red cell
losses in, 170 therapeutic plasma exchange, 647-653 adverse effects of, 658-660 in hemolytic disease of the
fetus and newborn, 564
indications for, 647, 648-651, 651-653 replacement fluids for, 646, 647, 652 Platelet antagonists, 122,
129,169, 509 Platelet antibodies anti-HPA, 455-457 autoantibodies, 461, 465, 568 in autoimmune
thrombocytopenic purpura, 463, 568
detecting, 243, 456, 463-464, 465-466, 639640
drug-induced, 462, 465-466 in fetal and neonatal alloimmune thrombocytopenia, 455, 456, 461, 567 in HSC
transplant patients, 637 in platelet refractoriness, 515-516 in posttransfusion purpura, 455, 456, 461462, 689-
690 Platelet antigens ABO antigens, 457 GPIV/CD36, 457, 458 GPVI, 457, 458 HLA antigens, 458, 488 HPA,
453-457 Platelet counts
corrected platelet count increment, 458, 512, 516 in infants, 568
in plateletpheresis donors, 169 posttransfusion platelet recovery, 458, 512 as transfusion threshold, 507-509,
581, 589, 636-637
829
Platelet disorders
drug-induced thrombocytopenia, 462, 465466,516
fetal and neonatal alloimmune thrombocytopenia, 461, 567-568 immune thrombocytopenia purpura, 463,
465,517, 568
in massive transfusion, 684 platelet transfusion refractoriness, 458-461 posttransfusion purpura, 461-462,
671, 688690
Platelet factor 4 ELISA, 465-466 Platelet gel, 222,517 Platelet incubators, 36, 214 Platelet-rich plasma,
146,148,150, 517 Platelet transfusions
ABO compatibility of, 375, 457, 513-514, 554 after HSCT transplantation, 635, 636 in hemolytic
transfusion reactions, 675 in pediatric patients, 581, 589 contraindications to, 517 to correct thrombocytopathy,
509-510 dosage of, 510-511, 581, 637 in fetus, 567-568
in HSC transplantation, 634, 635, 636-637 indications for, 507, 509-510, 580-581 infusion of, 554
in massive transfusion, 502, 685 in pediatric patients, 578, 580-582, 589 prophylactic vs therapeutic, 507,
510-511, 636
refractoriness to, 458-461 ABO compatibility in, 457 causes of, 459
HLA antibodies in, 458, 459-461, 488489,515-516, 670
HLA-matched platelets for, 459-460, 488-489
HPA antibodies in, 453-457 managing, 514, 516-517 platelet crossmatching for, 460-461,489, 516
preventing alloimmunization in, 515516
 selection of platelets for, 459-461, 488489
response to, 512 Rh matching, 515
thresholds for, 507-509, 581, 589, 636-637 unit type and age, 511, 512, 513 Plateletpheresis, 168-170
adverse reactions to, 169 donor selection and monitoring in, 120,
122, 168-169
instrumentation for, 168, 174-175
records of, 170 therapeutic, 654 volume collected in, 169 yields from, 510 Platelets, Apheresis
additive solution in, 217
agitation of, 214, 221
bacterial contamination of, 198-199,
221
biochemical changes in storage of, 221,511, 513
collection of, 168-170 compared to Whole-Blood-derived Platelets, 511,512 crossmatching, 460-461, 489,
516 donors of, 168-169 expiration of, 217
HLA matched, 459-460, 488-489, 516 irradiated, 156, 217, 459-460 laboratory testing of, 169-170
leukocytes reduced, 169-170, 217, 505 pathogen reduction for, 205 storage of, 214, 217
transfusion of (See Platelet transfusions) transportation and shipping of, 217 volume-reduced, 157, 223,
513-514 washed, 223
Platelets (Whole-Blood-derived), 150-151 agitation of, 150, 214, 221 bacterial contamination of, 150, 198-
199, 221
biochemical changes in storage of, 221,511, 513
clumping in, 148
compared to Apheresis Platelets, 511,512 cryopreservation of, 155 expiration of, 150, 156, 216-217, 224
irradiated, 156,217 leukocytes-reduced, 154, 156, 218, 505 pathogen reduction for, 205 pooled, 156-157, 217,
223-224, 512 preparation of, 146, 147-148, 150 storage of, 150-151, 214, 216-217, 221 transfusion of (See
Platelet transfusions) transportation and shipping, 150-151,216217
visual inspection of, 148, 199 volume-reduced, 157, 223, 581-582, 590 washed, 223, 590-591 Plerixafor,
720
Point-of-care testing, 607-608 Policies, 11,17, 18 Polyagglutination, 300,332 Polycythemia, 585
Polyethylene glycol (PEG), 376-377, 402
830
AABB TECHNICAL MANUAL
Polymerase chain reaction, 233-237 in genotyping, 280-281 in HLA typing, 485 oligonucleotide probes,
485 problems with, 235-236 real-time, 238-240 reverse transcriptase, 236-237 sequence-based typing, 486
sequence-specific primers, 485-486 Polymorphism, 264, 276-277 Pooled components, 156-157, 223-224
Cryoprecipitated AHF, 157, 218, 224, 523 platelets, 156-157, 217, 223-224, 512 Reconstituted Whole Blood,
224 storage, transportation, and expiration of, 217,218, 223-224 Population genetics, 274-276 Position effect,
272-273 Postoperative blood recovery, 608-609 Posttransfusion platelet recovery, 458, 512 Posttransfusion
purpura, 461-462, 671, 688690
Postzone effect, 242
Potassium, 573-574, 683-684
PPR. See Posttransfusion platelet recovery
Preadmission testing, 368
Pregnancy
in blood donors, 122 in patient history, 370-371, 392 testing during (See Prenatal studies) umbilical cord
blood recruitment in, 730731
Premedication, 547-548, 677, 679-680 Prenatal studies
ABO/Rh testing, 563 antibody detection, 563 antibody identification, 416 antibody titration, 409, 563 DNA-
based testing, 283-285 on fetus, 283-284, 330, 563 of paternal samples, 284-285, 563 in pregnant women, 284,
330 in fetal and neonatal immune thrombocytopenia, 567 Preoperative autologous donation, 131, 605606
Preservatives, antibodies to, 417 Pressure devices, 549 Pretransfusion testing ABO and Rh typing, 375 after
non-group-specific transfusions, 386 antibody detection and identification, 375378, 381
antiglobulin test, 371-372, 373-374 with autoantibodies, 432, 438-439
 autologous control, 377 and blood availability, 384 blood samples for, 368, 370-371, 384, 547 in cold
agglutinin disease, 438-439 comparison with previous records, 378 component selection, 375, 378-379
crossmatching, 379-381 donor unit testing, 378 identification of recipients, 368-370 in massive transfusions,
228, 386 orders for, 381, 383, 384, 546-547 in pediatric recipients, 385, 574-575, 587 reading and interpreting
reactions, 372, 381, 382
requests for transfusion, 367-368, 383, 546 699-700, 703-704
serologic testing, principles of, 371-372 tubeless methods, 377-378 turnaround times, 547 in urgent
situations, 227-228, 385-386 Prevalence, of phenotypes, 274 Preventive action, 26,27 Primed lymphocyte
typing, 486, 487 Primers, PCR, 235-236 Prions, 202-203
Probability values, in antibody identification, 398, 400 Proband, 265
Problem identification and resolution, 26-29
Procedures, 11,17, 18, 23
Process
capability, 33 control, 3, 33 flow charts, 27 improvement, 13, 26-29 management, 3-4,11,13, 14-16
validation, 14 Processes, 11,17,18 Production, principles of, 4 Proficiency testing, 25-26, 91 Propositus, 265
Prospective audits, 699-700, 701-702, 706
Prostate cancer, 791
Protamine, 622
Protein analysis, 240-247
agglutination-based methods for, 241-242 ELISA for, 243-245 flow cytometry for, 246 protein microarrays
for, 245 SPRCA for, 242-243 Western blotting for, 245-246 Protein C, 532 Protein microarrays, 245
Prothrombin complex concentrates, 518, 527 528, 623
831
Prothrombin time, 519-520, 521
Proton pump inhibitors, 626
Provenge, 791
Prozone effect, 241-242
PRP. See Platelet-rich plasma
Pseudogenes, 476, 478
Psoralen-treated plasma, 153,205
PT. See Prothrombin time
PTT. See Partial thromboplastin time
Public antigens, 482-483
Pulmonary disease, in blood donors, 126, 129
Pulmonary edema, 659, 680, 681, 682
Pulse, of donor, 120
Pumps, infusion, 548-549, 584
Q
Quad packs, 576 Qualification defined, 33 of equipment, 15 of personnel, 7 of suppliers, 9, 779, 780 Quality
assurance, 1-2, 34 Quality control, 16
of blood components, 159-160,171 of copper sulfate solution, 38 defined, 2, 34 of equipment, 36, 37
ofHSCs, 723, 736, 739, 741 performance intervals for, 36-38 records of, 16
unacceptable results for, 16 Quality improvement, 3 Quality indicators, 24, 34 Quality management systems
Code of Federal Regulations references, 35 components of, 4-5,12-13 customer focus, 6-7,12 documents
and records, 13, 16-19 equipment management, 10-11,13 facilities, work environment and safety, 7, 12
general concepts of, 1-5 human resources, 7-9,12 information management, 13, 19-20 management of
nonconforming events, 13, 20-23
monitoring and assessment, 13, 23-26 organization and leadership, 5-6,12 process improvement, 13, 26-29
process management, 11,13, 14-16 quality control performance intervals, 36-38 suppliers and materials
management, 9-10, 12
terminology of, 33-34
Quality oversight, 5-6 Quality planning, 2-3 Quality System Essentials, 2 Quarantine
of collected blood, 157-158 of nonconforming products, 224, 225 of repeatedly reactive units, 187-188, 189
Quarantine FFP, 152 Quarantine release errors, 192,195-196
 R
Radiation safety, 60-63 Raph system, 260, 339, 359 RBCs. See Red Blood Cells Reagents
for ABO testing, 298
antibodies to components of, 298, 300,417
antiglobulin, 372, 396, 426-427
bovine albumin, 376
chloroquine diphosphate, 407
contamination of, 332
DTT, 405
for elutions, 429
enhancement media, 396, 417
enzymes, 377, 402, 405
glycine-ffCl/EDTA, 407
LISS, 376, 402
manufacturers directions for, 11
PEG, 376-377, 402
for phenotyping, 420
quality control intervals for, 38
red cells, 376, 393, 396
for Rh testing, 326, 327, 331-332
sulfhydryl, 407
ZZAP, 407
Real-time PCR, 238-240 Recalls, 89, 90, 782 Recipients
ABO and Rh testing, 297-298, 327-328, 375 antibody detection in, 357-377 baseline assessment of, 546
consent of, 545-546, 601 crossmatching in, 379-381 education of, 546
identification of, 226, 368-370, 384-385, 549, 550, 551
immunocompromised, 190 medical history of, 392, 546 monitoring during and after transfusions, 553, 555
pediatric patients (See Neonates; Pediatric patients)
phenotyping, 329-330, 401-402 records of, 378, 672
tracing (look-back), 187-188, 189-190, 782
832
AABB TECHNICAL MANUAL
of unknown identity, 368, 370 weak D in, 327-328, 375 Recombination, 271 Reconstituted Whole Blood,
224 Records
altering or correcting, 18-19 apheresis, 170,172 blood component, 384-385 checking before blood issue,
226, 384-385, 550
comparing testing results to, 378
confidentiality of, 19
donor, 119
electronic, 18,19
HSC transplantation, 640
management of, 13, 16,18-19
personnel, 19
protection of, during disasters, 104, 111 quality control, 16 storage of, 19
of tissue allografts, 781-782 transfusion, 226, 555, 640, 672 Recovered Plasma, 152-153, 220 Red Blood
Cell transfusion, 499-507
ABO/Rh compatibility of, 375, 378-379, 504, 554, 635
in autoimmune hemolytic anemias, 436437, 439-440, 506 in chronic anemia, 502 dose of, 505-506, 610 in
emergency release, 227-228, 385-386, 506, 555-556
exchange transfusion, 579-580 hemoglobin targets in, 499-501, 505-506 in hemorrhagic shock, 501-502 in
HSC transplantation, 634 of incompatible units, 506-507 indications for, 499-502, 574, 575, 578-579 infusion
of, 554, 585 intrauterine, 563-564 leukocyte reduced, 504-505 in massive transfusion, 228, 386, 501-502, 683-
685
 in pediatric patients, 574-580, 586-589 restrictive vs liberal strategies for, 499-501 Red Blood Cells,
Apheresis, 171-172 collection of, 168, 175 donation requirements for, 120,122,123, 171
expiration, storage, and transportation of, 216
leukocytes reduced, 216 quality control of, 171 records of, 172
Red Blood Cells, Deglycerolized
expiration, storage, and transportation of, 215, 222
leukocyte content of, 505 preparation of, 155, 222 quality control of, 222 rejuvenated, 216 Red Blood Cells,
Frozen
expiration, storage, and transportation of,
215
preparation of, 155 rejuvenated, 216
thawing and deglycerolizing, 155, 222 Red cells, in-vitro generation of, 796 Red Blood Cells, Leukocytes
Reduced, 504-505 expiration, storage, and transportation of,
216
leukocyte content of, 149, 505 prestorage filtration, 154 Red Blood Cells (RBCs)
additive solutions for, 136-137, 138, 576577
age of, 574, 577-578 aliquoting, 224, 575-576 anticoagulant-preservative solutions for, 136,137
bacterial contamination of, 198 biochemical changes of storage in, 221, 503-504
collected by apheresis, 171-172 cryopreservation of, 155 deglycerolized, 155, 215, 222, 505 expiration of,
149, 215-216 frozen, 155,215, 222 hemoglobin/hematocrit of, 149 irradiated, 156,215 leukocyte content of, 505
leukocytes reduced, 216, 504-505 low-volume units, 141,142, 149 pathogen reduction of, 205 phenotyping,
420, 588 preparation of, 147-148 rare, 421
in red cell exchange, 654 rejuvenated, 216
storage of, 214, 215-216, 221, 410-411 substitutes for, 507 survival studies of, 419, 506-507 transfusion of
(See Red Blood Cell transfusion)
transportation and shipping of, 215-216, 225
visual inspection of, 149 washed, 216, 223, 505, 590-591
833
Red cell antibodies. See also specific blood groups
with autoantibodies, 432-435 and with HDFN, 338-340, 347-348, 413-414, 562
and with hemolytic transfusion reactions, 338-340, 413-414, 686-687 clinical significance of, 338-340, 347-
348, 376, 392, 413-414, 419 defined, 391
detection of (See Antibody detection) disease associations with, 392 distinguishing alloantibodies from
autoantibodies, 283 dosage effect of, 264, 393, 410 effect of DTT on, 413-414 effect of enzymes on, 413-414 to
high-prevalence antigens, 392-393,394395, 415-416, 564 high-titer, low-avidity, 409-410 identification of (See
Antibody identification) low-affinity, 437 to low-prevalence antigens, 416 multiple, 412, 414 naturally
occurring, 391, 392 nonhemolytic, 251-252 in selection of units, 379, 419-421 serologic reactivity of, 413-414
in sickle cell disease, 329-330, 379, 588 in tissue transplant patients, 111 Red cell exchange, 646, 653-654, 655,
675 Red cell losses, in apheresis, 170,171 Red cell reduction, 720-721 Red cell substitutes, 507 Reference
laboratories, 419 Refrigerators, 36, 214
Regenerative medicine, 753-754. See also Stem cells
Registration
of donors, 118-119 of facilities, 84, 86 Regulatory issues, 83-91
for biological products, 84, 85 for blood-related devices, 83-84, 87 for cellular therapy products, 760,
796797
for emergencies, 109-111 FDA inspections, 86-87 hospital regulations and accreditation, 91, 603-604
for HSCs, 87-89, 725, 743-746 licensure and registration, 84, 86 medical laboratory laws and regulations,
89-91
for quality systems, 1-2 for radioactive materials, 60-61 recalls and withdrawals, 89, 90 safety, 39-40, 60-61,
68-69 for tissue, 777-778
Reissuing blood products, 227-228, 550
Rejuvenated RBCs, 216
Relationship testing, 276-277, 493
Relative risk, 494
 Remedial action, 26,27
Renal failure, 652, 674
Reports
of adverse events related to tissue grafts, 782
facility quality, 27 of fatalities, 20, 45, 87, 145, 691 of injuries, 45, 87 internal event, 20,21 -22 Requests for
transfusion, 367-368, 383, 546, 699-704
Requirement, defined, 34 Respiratory distress, 658-659, 680-681 Restriction fragment length polymorphism
analysis, 238
Retrospective audits, 699, 701-702, 703-704, 707
Reverse transcriptase PCR, 236-237 RFLP. See Restriction fragment length polymorphism analysis Rh
Immune Globulin, 565-566
antepartum administration of, 564, 565 in antibody identification problems, 392 dosage for, 565-566
positive DAT after, 428 postpartum administration of, 565-566 serology and mechanism of, 566 use in platelet
transfusions, 515 Rh system, 317-333 antibodies of, 331, 338 antigens of, 259, 318-319, 321-330 C/ candE/ e,
328-330 D, 323-328 G, 328
characterization of, 317, 319 clinical considerations for, 328 ethnic differences in, 318-319, 320, 321, 325,
326, 327
genes and proteins of, 259, 272, 273, 319, 320, 321,325 genotypes, 321, 322-323 haplotypes in, 319, 320,
321-322 phenotypes, 321-323 RhAG, 261, 331,340, 360 RlW 273, 331
terminology for, 318-319, 319, 320
834
AABB TECHNICAL MANUAL
Rh testing
with autoagglutinins, 332, 432, 438 of blood components, 225-226, 285, 327, 328, 378
for C, c, E, e antigens, 322-323 comparison with previous records, 378 in component selection, 379, 515 for
D antigen, 327-328, 330 discrepancies in, 328, 333 DNA-based assays, 284-285, 287, 330 false-
positive/negative results in, 332 of fetus, 283-284, 330, 563 in hemolytic disease of the fetus and newborn, 332,
563, 565 in HSC transplantation, 634 in multitransfused patients, 330 in pediatric patients, 332, 385, 574, 587
phenotyping, 322-323 in prenatal evaluation, 284, 330, 563 reagents for, 326, 327, 331-332 of recipients, 327-
328, 375 for sickle cell disease patients, 283, 329-330 for weak D, 327, 328, 375, 565 RhAG system, 261, 319,
331, 340, 360 Rheopheresis, 646, 647 Rheumatoid arthritis, 763 Riboflavin-treated plasma, 153,205 Risks
assessment of, 98-99, 102 of bleeding, 605 relative, 494
of transfusion, 192-194, 600-601 RNA, 233
Rodgers blood group. See Chido/Rodgers Rodgers substance, 406 Root cause analysis, 27, 28, 29 Rosette
test, 565
Rotational thromboelastometry, 520 Rouleaux
in ABO testing, 298, 300
in antibody detection/identification, 416
in Rh testing, 332
saline replacement technique for, 301,
417
Run charts, 24
S
Safe work practices for biosafety, 52-53 for chemical safety, 58-59 for electrical safety, 47 for fire
prevention, 47 general guidelines for, 44, 72 for radiation safety, 62 Safety goggles, 71
Safety program
accidents and injuries, 45, 49 biosafety, 47-55, 73 chemical safety, 55-60, 74-82 in disaster management,
104 electrical safety, 46-47 emergency response plan, 44 employee health services, 44-45 engineering controls,
44, 72 fire prevention, 45-46 first aid and follow-up, 44-45 hazard identification and communication, 43-44
hepatitis prophylaxis, 44 latex allergies, 45 management controls, 42-43, 44 personal protective equipment,
44, 70-71 quality management of, 7,12 radiation safety, 60-63 regulations and recommendations for, 3940, 68-
69
safe work practices, 44, 72 safety officers, 42, 56, 61 safety plan, 42
shipping hazardous materials, 63 training, 43
 waste management, 63-64 Saline replacement technique, 301, 417 Samples, blood. See Blood samples
Sandwich ELISA, 244 Scianna system, 259, 339, 354 Scoring reactions, 372 SD (solvent/detergent-treated)
plasma, 153, 205
Sda antigen, 361-362, 372, 406 Sda substance, 406 Secretors
genetics of, 301, 302, 303, 304-305 linkage with Lutheran group, 271,272 Security, 104 Sedimenting agents,
172 Segments, of RBCs, 149 Selective absorption, 646 Sepsis, transfusion-associated, 198, 199, 669, 676
Sequence-based typing, 486 Sequence-specific oligonucleotide probes, 485 Sequence-specific primers, 485-
486
Serotonin release assay, 466 Serum proteins, in typing discrepancies, 298, 301,332
Serum-to-cell ratio, 405 Services, critical, 4, 9-10 Sex-linked inheritance, 267-269
835
Sexual contacts, of blood donors, 124,125,126
Sharps injuries, 49,141
Shipping
blood components, 146-147, 150-151, 213214, 215-220, 225
containers for, 38, 225, 724-725, 738 in disaster planning, 107-108 frozen products, 225, 724, 738
hazardous materials, 63 HSCs, 723-724, 734, 737-738 labeling, 738
monitoring temperature during, 213-214, 724-725, 738, 739, 779-780 MSCs, 761-762 plasma derivatives,
220 samples, 63 tissue, 220, 779-780 Shock, 501-502, 674 Short tandem repeat analysis, 277 Showers,
emergency, 58 Siblings, 266, 478 Sickle cell disease
alloimmunization in, 329-330, 379, 588,
639
blood selection in, 379, 587-588, 654 delayed transfusion reactions in, 588, 687 genotyping for, 283, 330 in
HSCT patients, 639 in pediatric patients, 587-588, 639 red cell exchange in, 587, 654 separation of transfused
from autologous cells in, 401
transfusion in, 379, 587-588 Side effects. See Adverse reactions Signs, safety, 46, 48-49, 57, 58 Silent
mutations, 264, 267, 286-287 Single nucleotide polymorphisms, 240, 264 Sipuleucel-T, 791 Six Sigma, 28-29
Skeletal repair, with MSCs, 763 Skin appearance, in donors, 120 Skin grafts, 123, 775, 111 Solid-phase red cell
adherence testing for detection of HLA antibodies, 487 for detection of platelet antibodies, 463-464 for
phenotyping red cells, 242-243 for platelet crossmatching, 460 for pretransfusion testing, 377, 396 Soluble
substances, 406 Solutions
additive, 136-137,138, 576-577 anticoagulant-preservative, 136,137 intravenous, 552
Solvent/detergent-treated plasma, 153, 203, 204, 205
Source Plasma, 170 Specific gravity, of blood cells and components, 143 Specification, defined, 34 Spills
blood, 53, 54 chemical, 59, 78-82 radioactive, 62 “Splits,” 482
SPRCA. See Solid-phase red cell adherence testing
SSOP. See Sequence-specific oligonucleotide probes
SSP. See Sequence-specific primers Staffing, 9, 108-109, 110-111 Standard Operating Procedures. See
Procedures
Standard precautions, 48 Standards
for biosafety, 47
for performance improvement, 26 for quality management, 1- 2 for tissue transplantation, 778-779
Staphylococcal protein A absorption, 657 Stem Cell Therapeutic and Research Act, 743 Stem cells
adipose-derived, 755, 758-759, 762, 794
embryonic, 795
hematopoietic
collection of, 718-720, 733-734 cryopreservation of, 722-723, 736-737 donors of, 714-717 infusion of, 724-
725 irradiation of, 638 processing, 720-722 quality control of, 723, 736 regulation of, 87, 88, 89, 725 shipping
and transport of, 723-724, 737738
sources of, 717-720 storage of, 736-737 thawing, 721 washing, 721, 740-741 induced pluripotent, 755, 795-
796 mesenchymal
autologous vs allogeneic use, 754 future directions for, 765-766 identification criteria for, 793 isolation and
expansion of, 758-760, 792-793
 multipotentiality of, 754-755, 791-792 properties of, 756-757, 758, 786, 791-792 research and development
on, 764-765 shipping, 761-762 sources of, 754-758
836
AABB TECHNICAL MANUAL
standardization of methods for, 760 storage and banking of, 761 therapeutic applications of, 762-764, 793-
795
and tissue engineering, 795 tumor-forming potential of, 765 Sterile connection devices, 37,139,140, 576
Sterility testing, ofHSCs, 723, 736 Storage
of biohazardous material, 52, 54-55 of blood components, 213-214, 215-220, 221 biochemical changes in,
221, 503-504, 511,513
Cryoprecipitated AHF, 153, 218, 222 granulocytes, 173, 217, 221, 525 plasma, 151,218-220 platelets, 150-
151, 216-217, 511, 513 RBCs, 214, 215-216, 221, 503-504 red cell antigen deterioration with, 410411
Whole Blood, 147, 215 of blood samples, 371 in disaster plan, 108 equipment for, 214 of hazardous
chemicals, 58-59 ofHSCs, 736-737 liquid nitrogen, 723, 737, 761 ofMSCs, 761-762 of plasma derivatives, 220
of records, 19
temperature for, 213-214, 215-220, 221 of tissue grafts, 220, 775, 780-781, 783 Storage lesion, 221, 503-
504, 511, 513 STR. See Short tandem repeat analysis Stroke, 763
Stromal-vascular fraction, 759, 762, 763 Sulfhydryl reagents, 407, 438 Suppliers, 9-10,12, 779, 780
Supplies, critical, 9-10,11,107,108 Surgery
acute normovolemic hemodilution in, 606 anemia assessment before, 604-605 anesthesia in, 607 assessing
bleeding risk in, 605 blood administration in, 555-556 blood ordering practices for, 227 blood recovery in, 606-
607, 608-609 deliberate hypotension in, 607 fluid management in, 607 maintaining normothermia in, 607
pharmacologic agents in, 608, 620-627 point-of-care testing in, 607-608 positioning in, 607
preoperative autologous donation for, 605606
techniques in, 607 temperature regulation in, 607 transfusion algorithms in, 608 Survival studies of red
cells, 419, 506-507 Syncope, 144-145 Syntenic genes, 271 Syphilis, 198
reactive testing results for, 187,188, 189 screening blood donors for, 124,184, 198 screening tissue donors
for, 774 Syringe aliquoting devices, 576 Syringe infusion pumps, 549, 584
T
T-activation, 300
TA-GVHD. See Transfusion-associated graft vs-host disease
TACO. See Transfusion-associated circulatory overload Tattoos, 124 Temperature
of antibody reactivity, 405, 413-414 of collected whole blood, 147 for component storage, 213-214,215-220,
221
of donors, 120 monitoring systems for, 214 of recipients, 546, 676, 677 regulation of, during surgery, 607
for shipping containers, 213-214, 225, 734, 738, 739, 779-780 Teratogens, 122,129
TerumoBCT apheresis systems, 168,174,175 Testing. See also specific testing methods; Pretransfusion
testing grading results of, 372 of incoming supplies, 10 method validation, 14 point-of-care, 607-608
regulations for, 90 Thalassemia, 588-589, 640 Thawed Plasma, 151, 152,219, 221, 522 Thawing
Cryoprecipitated AHF, 222 devices for, 37 frozen RBCs, 222 HSCs, 721 plasma, 151, 221 Therapeutic
apheresis
adverse effects of, 658-660 anticoagulation in, 658 cytapheresis, 653-654, 655 extracorporeal photopheresis,
646, 654, 656 indications for, 646, 647, 648-651, 655, 656, 657
837
modalities of, 645, 646, 647 patient evaluation in, 660-661 principles of, 645, 646, 647 replacement fluids
in, 522, 646, 652 selective absorption, 656-658 therapeutic plasma exchange, 646, 647-653 vascular access in,
660 Therapeutic plasma exchange, 647-653 adverse effects of, 658-660 in hemolytic disease of the fetus and
newborn, 564
indications for, 646, 647, 648-651, 651-653 replacement fluids for, 646, 647, 652 Thermal amplitude
studies, 419, 438 Thermometers, 37 Thrombocytapheresis, 646, 654, 655 Thrombocytopathy, 509-510
Thrombocytopenia
drug-induced, 462, 465-466, 516 fetal and neonatal alloimmune, 461, 567-568 heparin-induced, 462, 465-
466, 517, 528 immune, 463, 465, 517, 568 management of, 514 in pediatric patients, 580-581 in
plasmapheresis, 659 platelet transfusions in, 507-517 posttransfusion purpura, 461-462, 671, 688690
 thrombotic thrombocytopenic purpura, 517, 522, 652-653, 713 Thrombocytosis, 654 Thromboelastography,
520-521, 608 Thrombosis, in blood donors, 145 Thrombotic thrombocytopenic purpura, 517, 522, 652-653, 713
Time, incubation, 405 Timers, 37 Tissue
autografts, 782-783 collecting, 773, 782-783 expiration of, 220
hospital-based services for, 778-783 infectious disease testing on donors of, 191192, 774
oversight responsibility for, 778-779 processing, 775 recall of, 782
receipt and inspection of, 779-780 regulations and standards for, 87, 88, 89, 777-778
standard operating procedures for, 779 storage of, 220, 775, 780-781, 783 suppliers of, 779, 780 traceability
and records of, 781-782 transplantation of, 773-777
ABO compatibility in, 777 adverse events in, 782 antibody development after, 777 background of, 774-775
clinical uses for, 775-777 disease transmission through, 777 donor eligibility for, 773-774 history of, in blood
donors, 123,126 look-back investigations in, 782 and MSCs, 795 organ transplantation, 783 transfusion support
for, 783 types of grafts for, 774 transporting, 220, 779-780 Tissue banks and distributors, 777 Tissue
engineering, 795 Titration of antibodies, 409-410, 563 Topical hemostatic agents, 608, 625 TPE. See
Therapeutic plasma exchange Traceability, 225, 781-782 Training
biosafety, 48 cGMP and cGTP, 8 chemical safety, 56 disaster, 109,111-112 electrical safety, 47 fire safety,
46 general safety, 43 new employees, 7-8 radiation safety, 61-62 Traits, 256, 265
TRALI. See Transfusion-related acute lung injury
Tranexamic acid, 608, 621-622 Trans position, 272
Transcription-mediated amplification, 237238
Transfusion-associated circulatory overload, 669, 682-683
Transfusion-associated graft-vs-host disease, 671, 687-688
HLA system in, 489-490, 688 in neonates, 573
Transfusion-associated sepsis, 198,199, 669, 676
Transfusion committee, 24-25 Transfusion reactions
acute hemolytic, 667, 672-675 air embolus, 669, 685 allergic, 547-548, 667, 678-680 alloimmunization, 670
(See also Alloimmunization) anaphylactic, 658, 668, 678-679, 680 biovigilance programs in monitoring,
665666
838
AABB TECHNICAL MANUAL
clinical evaluation and management of, 666, 667-671
coagulopathy, 591, 684-685 delayed hemolytic, 250, 588, 670, 686-687 febrile nonhemolytic, 489, 548, 667,
677 graft-vs-host disease, 671, 687-688 in HSCT patients, 639 hyperkalemia and hypokalemia, 555, 683684
hypocalcemia, 555, 658, 670, 683 hypotension, 659, 669, 678 hypothermia, 548, 572-573, 670, 685-686
identification of, 553, 555, 666 iron overload, 588, 671, 690 laboratory investigation of, 672 nonimmune
hemolysis, 669, 675-676 platelet refractoriness, 458-461 posttransfusion purpura, 461-462, 671, 688690
records of, 672 reporting, 22
signs and symptoms of, 666, 667-671 TACO, 669, 682-683 TRALI, 668, 680-682
transfusion-related sepsis, 198,199, 669, 676 Transfusion-related acute lung injury, 468, 489, 668, 680-682
Transfusion-related immunomodulation, 504 Transfusion safety officers, 611 Transfusion thresholds
in pediatric patients, 578-579 for platelet transfusions, 507-509, 581, 589, 636-637
for red cell transfusions, 499-501, 578-579, 610, 634
Transfusion-transmitted diseases Babesiosis, 201 chikungunya virus, 202 dengue virus, 202 hepatitis B
virus, 192,194, 196 hepatitis C virus, 192-193,194, 196-197 human immunodeficiency virus, 194-196 human T-
cell lymphotropic virus, 197 malaria, 201-202 parvovirus B19, 203 prions, 202-203 safety precautions for, 47-
55 syphilis, 198
Trypanosoma cruzi, 200-201 West Nile virus, 200 Transfusions
administration procedures for (See Blood administration) algorithms for, 608 auditing, 25, 697-707
in blood donors, 123,125 chimerism after, 489-490 consent for, 545-546, 601 costs of, 602
of Cryoprecipitated AHF, 522-523, 583-584 during disasters, 101 documentation of, 226, 555 exchange,
564, 574, 579-580 fatalities due to, 20, 551, 681, 691 of granulocytes, 173, 523-526, 584 guidelines for, 611,
704 and HLA system, 488-490 in HSC transplantation, 633-640 intrauterine, 563-564 massive, 228, 386, 501-
502, 591, 683-685 in medical history, 123, 370-371, 392, 417418,428, 546
 monitoring appropriateness of, 697-707 non-group-specific, 396 in operating room and trauma, 555-556 in
organ transplantation, 783 out-of-hospital, 556 in pediatric patients, 574-591 of plasma, 517-522, 582-583 of
plasma derivatives, 526-532 of platelets, 507-517, 580-581 of RBCs, 499-507, 574-580 requests for, 367-368,
383, 546, 603-604, 699-700
risks of, 192-194, 600-601 selection of components for, 375, 378-379 in urgent situations, 227-228, 385-
386, 506, 555-556
of Whole Blood, 502-503 Transmissible spongiform encephalopathy, 202-203
Transplantation. See specific types of transplantation Transportation
of blood components, 146-147, 150-151, 213-214, 215-220, 225 containers for, 38, 225, 724-725 in disaster
plan, 107-108 of frozen products, 225, 724, 738 of hazardous materials, 63 of HSCs, 723-724, 734, 737-738
labeling requirements, 738 monitoring temperatures during, 213-214, 738, 739, 779-780 ofMSCs, 761-762 of
plasma derivatives, 220 of samples, 63 of tissue, 220, 779-780 Trauma, blood administration in, 555-556
Travel, by blood donors, 125,126, 202
INDEX
839
Treponema pallidum, 191, 198 TRIM. See Transfusion-related immunomodulation
Trypanosoma cruzi, 184, 187, 190, 192, 200-201 TTP. See Thrombotic thrombocytopenic purpura
Tumorigenicity, of stem cells, 765, 796 Two-unit red cell collection, 120,122,123,168, 171-172
Type and crossmatch, 383, 384 Type and hold, 381,383 Type and screen, 382, 383, 384
U
UCB. See Umbilical cord blood Ulex europaeus lectin, 295 Umbilical cord blood
antigen expression on, 410, 411 DAT testing on, 427 for transplantation advantages of, 729 cell expansion,
722 collection, 733-734 consent for collection, 731 cryopreservation, 736-737 donor recruitment for, 730-731
donor testing, 715, 716, 717, 732-733 economic issues regarding, 742-743 engraftment kinetics, 717-718 health
history and medical evaluation, 731-732
HLA matching, 716, 736 infusion, 741-742 labeling, 738, 739 legislation regarding, 743 patient survival,
718 post-thaw testing, 737 processing, 734-736 quality control testing, 736, 739, 741 receipt of, 738-740
regulations and standards, 87, 88, 89, 743-746
shipping, 734, 737-738 storing, 736-737
thawing and washing, 721, 740-741 Umbilical cord blood banks, 729-730, 742-743 Uniforms, 70
United Network for Organ Sharing, 783 Urgent release of blood, 227-228, 385-386, 506, 555-556
Urine neutralization, 362,406 Urticaria (hives), 667, 678, 679-680 Utilities, in disaster plan, 108 Utilization
of blood. See Blood utilization review
V
Vaccines, 44,123, 791 Validation
of computer systems, 15-16 definition of, 34 of equipment, 15 of processes, 14
of shipping containers, 147, 225 of test methods, 14 validation plans, 14-15 Vapors, hazardous, 59 Variable
number of tandem repeats (VNTR), 277
Vascular access for apheresis, 660 in pediatric patients, 547, 580, 584 for transfusions, 547
Vasovagal reactions, 143-145, 169, 659, 678 Vector-borne diseases, 199-202 Vel system, 261, 340, 361
Venipuncture, 140-141 Verification, defined, 34 Viability assays for HSCs, 723, 736 View boxes, 37 Viruses
donor screening for, 194-197, 200 transmitted by tissue transplants, 774, 782 Viscoelastic coagulation
testing, 520-521, 608 Viscosity, plasma, 652 Vital signs, 546, 553, 555, 724, 742 Vitamin K, 518, 622 Volume
of blood
in exchange transfusions, 580 in intrauterine transfusions, 564 in neonatal transfusions, 578 in pediatric
patients, 572 in whole blood collections, 120, 141-142,143 Volume overload. See Transfusion-associated
circulatory overload Volume-reduction of HSCs, 720
of platelets, 157, 223, 513-514, 581-582, 590 von Willebrand disease, 129, 523, 524, 584 von Willebrand
factor, 154, 523
W
Warfarin, 518, 519, 622 Warm autoantibodies ABO testing with, 432 adsorption of, 432-435 with
alloandbodies, 418-419, 432-435, 438439
antibody identification with, 418-419 disease associations with, 392
840
 AABB TECHNICAL MANUAL
mimicking alloantibodies, 434-435 in mixed-type AIHA, 439 in phenotyping problems, 401 Rh testing with,
332,432 specificity of, 435 transfusion with, 436-437, 506 in warm AIHA, 431 -435 Warm autoimmune
hemolytic anemia, 430437
adsorption testing in, 432-435 autoantibody specificity in, 435 blood selection in, 435-436 serologic
characteristics of, 431-432 serologic problems of, 432 transfusions in, 436-437 Warmers, blood, 37, 548, 572,
584, 685-686 Washed components, 223 HSCs, 721, 740-741
for pediatric patients, 573-574, 590-591 platelets, 223, 590-591, 639 RBCs, 216, 223, 505, 590-591, 639
Waste management, 63-64 biohazardous, 53-55 chemical, 60 disposal, 54-55 radioactive, 63 treating, 55
Waterbaths, 37 WB. See Whole Blood Weak D, 323-324
in donors, 285, 327, 328 in fetus, 565
in prenatal patients, 565 in recipients, 327-328, 375 testing for, 327, 328, 375, 565 West Nile virus, 200
reactive testing results for, 187,188, 190 screening donors for, 184,186,192, 200 Western blotting, 245-246
Whole Blood, 148-149
ABO compatibility of, 375 collection of, 135-146
adverse donor reactions in, 142-146 containers for, 136-139,140 donation intervals for, 120,122 donor and
blood identification in, ISOMO
donor care after, 142 handling after, 146-147 phlebotomy, 140-141 process of, 141
volume collected, 120, 141-142,143 expiration of, 148-149, 215 hematocrit of, 148 indications for, 502-503
irradiated, 215 leukocyte content of, 505 processing, 146, 147-148 reconstituted, 149, 224 storage of, 215
temperature for, 147 transportation of, 146-147, 215, 225 Wipe tests, 61 Withdrawals, 89, 90 WNV. See West
Nile virus Work environment, 7,12, 39-42 Work instructions, 17 Wristbands, patient, 368 Wrong blood in tube,
378, 384
X-Z
X-borne genes, 267-269 X chromosome inactivation, 258, 261 Xenografts, 775 Xg system, 354
antibodies of, 339, 354 genes and antigens of, 259, 354 inheritance pattern of, 258, 268 XK gene, 258, 260,
261, 269, 348 Yt system, 259, 338, 354 Zygosity, 264, 393, 410 ZZAP, 407, 438