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BASIC & APPLIED CONCEPTS of BLOOD BANKING and TRANSFUSION PRACTICES Third Edition tahir99-VRG & vip.persianss.ir
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BASIC & APPLIED CONCEPTS of TBRLAONOSDFUBSAINOKNINPGRAaCnTdICESKathy D. Blaney, MS, BB(AtSaCPh)SivBrB9ip9.p-&eVrsRiaGnss.iTr hird Edition Tissue Typing Laboratory Florida Hospital Orlando, Florida; LifeSouth Community Blood Centers Gainesville, Florida Paula R. Howard, MS, MPH, MT(ASCP)SBB Community Blood Centers of Florida A Division of OneBlood, Inc. Lauderhill, Florida tahir99-VRG & vip.persianss.ir
3251 Riverport Lane St. Louis, Missouri 63043 BASIC & APPLIED CONCEPTS OF BLOOD BANKING AND ISBN: 978-0-323-08663-9 TRANSFUSION PRACTICES Copyright © 2013 by Mosby, an imprint of Elsevier Inc. Copyright © 2009, 2000 by Mosby, Inc., an affiliate of Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). VRGNotices 9 - .irKnowledge and best practice in this field are constantly changing. As new research and experience ir9 sbroaden our understanding, changes in research methods, professional practices, or medical h & streatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in nevaluating and using any information, methods, compounds, or experiments described herein. In ta iausing such information or methods they should be mindful of their own safety and the safety of rsothers, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the emost current information provided (i) on procedures featured or (ii) by the manufacturer of each .pproduct to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their ipown experience and knowledge of their patients, to make diagnoses, to determine dosages and the vbest treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. International Standard Book Number: 978-0-323-08663-9 Publishing Director: Andrew Allen Working together to grow Content Manager: Ellen Wurm-Cutter libraries in developing countries Publishing Services Manager: Catherine Jackson Senior Project Manager: David Stein www.elsevier.com | www.bookaid.org | www.sabre.org Design Direction: Maggie Reid tahir99-VRG & vip.persianss.ir Printed in the United States Last digit is the print number: 9 8 7 6 5 4 3 2 1
This book is dedicated to my family, Tommy and Sean, for their support. And to all the students and professionals I have worked with throughout my career in immunohematology. KDB This third edition is dedicated in loving memorium to my parents, William and Olga Juda, who encouraged my individuality and desire for continuous learning and to my partner, Jack, for his perpetual belief and support of my professional goals. And as always to all of my former CLS students who energized my personal joy of learning and inspired my desire for excellence in teaching. PRH tahir99-VRG & vip.persianss.ir
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Reviewers Charlotte Bates, MEd, MT(ASCP) Max P. Marschner, MBA, MT(ASCP)SBB, CHS Instructor Manager, Tissue Typing Lab Medical Laboratory Science Department Florida Hospital Medical Center Armstrong Atlantic State University Orlando, Florida Savannah, Georgia Nicole S. Pekarek, MAT, MT(ASCP) Dorothy A. Bergeron, MS, CLS(NCA) Instructor Associate Professor and Program Director Clinical Laboratory Science Instructor Clinical Laboratory Science Program Winston-Salem State University Department of Medical Laboratory Science Winston-Salem, North Carolina University of Massachusetts Dartmouth North Dartmouth, Massachusetts Ellen F. Romani, MHSA, MT(ASCP)DLM, BB Department Chair Kim Boyd, MS, MT(AMT) Medical Laboratory Technology Program Assistant Professor Spartanburg Community College Medical Laboratory Technology Program Spartanburg, South Carolina Amarillo College Amarillo, Texas Judith A Seidel, MT(ASCP)SBB Clinical Instructor, Immunohematology Cara Calvo, MS, MT(ASCP)SH Clinical Laboratory Science Program Program Director and Lecturer Indiana University Health Medical Technology Program Indianapolis, Indiana Department of Laboratory Medicine University of Washington Melissa Volny, MT(ASCP)SBB, MBA Seattle, Washington Coordinator of Transfusion Services Centegra Health System Linda Collins, MS, MT(ASCP) McHenry, Illinois; Instructor Elgin Community College Delaware Technical and Community College Elgin, Illinois Georgetown, Delaware Terry Kotrla, MS, MT(ASCP)BB Department Chair and Professor Medical Laboratory Technology Program Austin Community College Austin, Texas tahir99-VRG & vip.pevrisiianss.ir
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Preface Basic & Applied Concepts of Blood Banking and Trans- • Chapter summaries, in varying formats, to provide a fusion Practices was developed for students in 2- or succinct overview of the chapter’s important points 4-year medical laboratory science programs, laboratory professionals undergoing retraining, and other health • Critical thinking exercises to illustrate the practical care professionals who desire knowledge in routine applications to the clinical environment blood banking practices. Basic didactic concepts are introduced, and the practical application of these theo- • Illustrations and tables designed to reinforce and sum- ries to modern transfusion and blood bank settings is marize the most important information found in the emphasized. chapter The third edition’s presentation of topics was reorga- The third edition includes updates to the ever-chang- ing field of blood banking. Donor criteria and testing nized to improve the overall flow of the information. We have been updated to include the current donor restric- also included additional details on some topics more tions, infectious disease testing methods, and current appropriate for the 4-year medical laboratory science requirements for viral marker testing. A new chapter was programs. added to address automation for the transfusion service. The section on molecular techniques applying to blood The third edition also has an accompanying Evolve banking was expanded, accompanied by an expanded website where the ancillaries are highlighted. For stu- section on HLA. The chapter on blood components and dents, the ancillaries include additional case studies and therapy includes a description of new products such as access to the laboratory manual. The instructor ancillar- leukoreduced components and red cell apheresis. ies include an image collection that features figures found in the text, an extensive collection of test bank questions This textbook provides important features to assist as well as answers to the critical thinking exercises, and both the student and the instructor. Each chapter PowerPoint presentations for each chapter that include features: illustrations appearing in this text. • Chapter outlines listing the important elements in the We are very appreciative of the editors at Elsevier for chapter their patience and professionalism in the manuscript • Learning objectives for use by both the student and review and publication process for this third edition. We are proud of the final product, which is user friendly to the instructor students and instructors. • Study questions for self-assessment • Key words with definitions on the same page Kathy D. Blaney Paula R. Howard tahir99-VRG & vip.peirxsianss.ir
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Contents PART I: FOUNDATIONS: BASIC SCIENCES AND Inheritance and Nomenclature of HLA, 19 REAGENTS Testing and Identification of HLA and Chapter 1 IMMUNOLOGY: Basic Principles and Antibodies, 21 Applications in the Blood Bank, 1 Hematopoietic Progenitor Cell Transplants, 22 SECTION 1 CHARACTERISTICS ASSOCIATED WITH Graft-versus-Host Disease, 23 ANTIGEN-ANTIBODY REACTIONS, 2 Platelet Antigens, 23 General Properties of Antigens, 2 Chapter 2 BLOOD BANKING REAGENTS: Overview General Properties of Antibodies, 3 and Applications, 28 Molecular Structure, 3 SECTION 1 INTRODUCTION TO ROUTINE TESTING IN Fab and Fc Regions, 5 IMMUNOHEMATOLOGY, 29 Comparison of IgM and IgG Antibodies, 5 IgM Antibodies, 6 Sources of Antigen for Testing, 29 IgG Antibodies, 7 Sources of Antibody for Testing, 30 Primary and Secondary Immune Response, 7 Routine Testing Procedures in the Antigen-Antibody Reactions, 8 Properties That Influence Binding, 8 Immunohematology Laboratory, 30 SECTION 2 CHARACTERISTICS ASSOCIATED WITH RED SECTION 2 INTRODUCTION TO BLOOD BANKING CELL ANTIGEN-ANTIBODY REACTIONS, 10 REAGENTS, 31 Red Cell Antigens, 10 Regulation of Reagent Manufacture, 31 Red Cell Antibodies, 12 Reagent Quality Control, 32 Immunohematology: Antigen-Antibody Reactions SECTION 3 COMMERCIAL ANTIBODY REAGENTS, 32 In Vivo, 12 Polyclonal versus Monoclonal Antibody Transfusion, Pregnancy, and the Immune Products, 32 Polyclonal Antibody Reagents, 33 Response, 12 Monoclonal Antibody Reagents, 33 Complement Proteins, 12 Monoclonal and Polyclonal Antibody Clearance of Antigen-Antibody Complexes, 14 Reagents, 34 Immunohematology: Antigen-Antibody Reactions Reagents for ABO Antigen Typing, 34 In Vitro, 14 Reagents for D Antigen Typing, 36 Overview of Agglutination, 14 Low-Protein Reagent Control, 37 Sensitization Stage or Antibody Binding to SECTION 4 REAGENT RED CELLS, 38 Red Cells, 14 A1 and B Red Cells for ABO Serum Testing, 38 Factors Influencing First Stage of Screening Cells, 39 Antibody Identification Panel Cells, 40 Agglutination, 15 Lattice-Formation Stage or Cell-Cell SECTION 5 ANTIGLOBULIN TEST AND REAGENTS, 40 Principles of Antiglobulin Test, 40 Interactions, 16 Direct Antiglobulin Test, 42 Factors Influencing Second Stage of Indirect Antiglobulin Test, 43 Sources of Error in Antiglobulin Testing, 43 Agglutination, 16 Antiglobulin Reagents, 44 Grading Agglutination Reactions, 17 Polyspecific Antihuman Globulin Reagents, 45 Hemolysis as an Indicator of Antigen-Antibody Monospecific Antihuman Globulin Reagents, 45 Reactions, 18 IgG-Sensitized Red Cells, 46 SECTION 3 HUMAN LEUKOCYTE ANTIGEN (HLA) SYSTEM xi AND PLATELET IMMUNOLOGY, 19 Human Leukocyte Antigens, 19 Testing Applications in the Clinical Laboratory, 19
xii CONTENTS SECTION 6 PRINCIPLES OF ANTIBODY POTENTIATORS AND SECTION 2 ABO AND H BLOOD GROUP SYSTEM LECTINS, 47 ANTIGENS, 80 Low-Ionic-Strength Saline (LISS), 47 General Characteristics of ABO Antigens, 80 Polyethylene Glycol, 48 Inheritance and Development of A, B, and Enzymes, 48 Bovine Serum Albumin, 48 H Antigens, 81 Lectins, 49 Common Structure for A, B, and H Antigens, 82 Development of H Antigen, 82 SECTION 7 OTHER METHODS OF DETECTING ANTIGEN- Development of A and B Antigens, 82 ANTIBODY REACTIONS, 49 ABO Subgroups, 84 Comparison of A1 and A2 Phenotypes, 84 Gel Technology Method, 49 Additional Subgroups of A and B, 85 Microplate Testing Methods, 50 Importance of Subgroup Identification in Donor Solid-Phase Red Cell Adherence Methods, 52 Testing, 86 Chapter 3 Genetic Principles in Blood Banking, 59 SECTION 3 GENETIC FEATURES OF ABO BLOOD GROUP SYSTEM, 86 SECTION 1 BLOOD GROUP GENETICS, 60 Genetic Terminology, 60 SECTION 4 ABO BLOOD GROUP SYSTEM ANTIBODIES, 88 Phenotype versus Genotype, 61 Punnett Square, 61 General Characteristics of Human Anti-A and Genes, Alleles, and Polymorphism, 61 Anti-B, 88 Inheritance Patterns, 62 Immunoglobulin Class, 88 Silent Genes, 63 Hemolytic Properties and Clinical Mendelian Principles, 63 Significance, 88 Chromosomal Assignment, 64 In Vitro Serologic Reactions, 89 Heterozygosity and Homozygosity, 64 Genetic Interaction, 65 Human Anti-A,B from Group O Individuals, 89 Linkage and Haplotypes, 65 Anti-A1, 89 Crossing Over, 66 SECTION 5 ABO BLOOD GROUP SYSTEM AND SECTION 2 POPULATION GENETICS, 67 TRANSFUSION, 89 Combined Phenotype Calculations, 67 Gene Frequencies, 68 Routine ABO Phenotyping, 89 Relationship Testing, 68 Selection of ABO-Compatible Red Blood Cells SECTION 3 MOLECULAR GENETICS, 69 and Plasma Products for Transfusion, 90 Application of Molecular Genetics to Blood Banking, 69 SECTION 6 RECOGNITION AND RESOLUTION OF ABO Polymerase Chain Reaction, 70 DISCREPANCIES, 91 Polymerase Chain Reaction–Based Human Leukocyte Antigen Typing Technical Considerations in ABO Phenotyping, 91 Procedures, 70 Sample-Related ABO Discrepancies, 92 Molecular Testing Applications in Red Cell Typing, 72 ABO Discrepancies Associated with Red Cell Polymerase Chain Reaction–Based Red Cell Testing, 92 Typing Procedures, 72 ABO Discrepancies Associated with Serum or PART II: OVERVIEW OF THE MAJOR BLOOD Plasma Testing, 96 GROUPS SECTION 7 SPECIAL TOPICS RELATED TO ABO AND H Chapter 4 ABO and H Blood Group Systems and BLOOD GROUP SYSTEMS, 100 Secretor Status, 77 Classic Bombay Phenotype, 100 SECTION 1 HISTORICAL OVERVIEW OF ABO BLOOD Secretor Status, 101 GROUP SYSTEM, 79 Chapter 5 Rh Blood Group System, 107 SECTION 1 HISTORICAL OVERVIEW OF THE DISCOVERY OF THE D ANTIGEN, 108 SECTION 2 GENETICS, BIOCHEMISTRY, AND TERMINOLOGY, 108 Fisher-Race: CDE Terminology, 110 Wiener: Rh-Hr Terminology, 110 Rosenfield: Numeric Terminology, 111
International Society of Blood Transfusion: CONTENTS xiii Standardized Numeric Terminology, 111 Genetics of Duffy Blood Group System, 135 Determining the Genotype from the Characteristics of Duffy Antibodies, 135 Phenotype, 111 Duffy System and Malaria, 136 SECTION 3 ANTIGENS OF THE Rh BLOOD GROUP SECTION 5 KIDD BLOOD GROUP SYSTEM, 136 SYSTEM, 112 Characteristics and Biochemistry of Kidd D Antigen, 112 Antigens, 136 Weak D, 112 Kidd Antigens Facts, 136 Weak D: Genetic, 115 Biochemistry of Kidd Antigens, 136 Weak D: Position Effect, 115 Weak D: Partial D, 115 Genetics of Kidd Blood Group System, 137 Significance of Testing for Weak D, 116 Characteristics of Kidd Antibodies, 137 Other Rh Blood Group System Antigens, 117 SECTION 6 LUTHERAN BLOOD GROUP SYSTEM, 138 Compound Antigens, 117 G Antigens, 118 Characteristics and Biochemistry of Lutheran Antigens, 138 Unusual Phenotypes, 118 Lutheran Antigens Facts, 138 D-Deletion Phenotype, 118 Biochemistry of Lutheran Antigens, 139 Rhnull Phenotype, 119 Rhmod Phenotype, 119 Genetics of Lutheran Blood Group System, 139 Characteristics of Lutheran Antibodies, 139 SECTION 4 Rh ANTIBODIES, 119 Anti-Lua, 139 General Characteristics, 119 Anti-Lub, 139 Clinical Considerations, 119 SECTION 7 LEWIS BLOOD GROUP SYSTEM, 140 Transfusion Reactions, 119 Hemolytic Disease of the Fetus and Newborn, 120 Characteristics and Biochemistry of Lewis Antigens, 140 SECTION 5 LW BLOOD GROUP SYSTEM, 120 Lewis Antigens Facts, 140 Biochemistry of Lewis Antigens, 140 Relationship to the Rh Blood Group System, 120 Inheritance of Lewis System Antigens, 141 Chapter 6 Other Blood Group Systems, 126 Characteristics of Lewis Antibodies, 142 SECTION 1 WHY STUDY OTHER BLOOD GROUP Serologic Characteristics, 142 SYSTEMS? 127 SECTION 8 I BLOOD GROUP SYSTEM AND i ANTIGEN, 142 Organization of Chapter, 127 I and i Antigens Facts, 143 SECTION 2 KELL BLOOD GROUP SYSTEM, 129 Biochemistry of I and i Antigens, 143 Serologic Characteristics of Autoanti-I, 143 Characteristics and Biochemistry of Kell Antigens, Disease Association, 144 129 Kell Antigens Facts, 129 SECTION 9 P1PK BLOOD GROUP SYSTEM, GLOBOSIDE Biochemistry of Kell Antigens, 129 BLOOD GROUP SYSTEM, AND GLOBOSIDE BLOOD GROUP Immunogenicity of Kell Antigens, 130 COLLECTION, 144 K0 or Kellnull Phenotype, 130 P1 Antigen, 144 Genetics of Kell Blood Group System, 131 P Antigen, 144 Characteristics of Kell Antibodies, 131 P1PK and GLOB Blood Group System Antigens SECTION 3 Kx BLOOD GROUP SYSTEM, 132 Facts, 144 Biochemistry, 145 Kx Antigen and Its Relationship to Kell Blood P1PK and GLOB Blood Group System Group System, 132 Antibodies, 146 McLeod Phenotype, 132 Anti-P1, 146 McLeod Syndrome, 133 Autoanti-P, 146 Anti-PP1Pk, 147 SECTION 4 DUFFY BLOOD GROUP SYSTEM, 134 SECTION 10 MNS BLOOD GROUP SYSTEM, 147 Characteristics and Biochemistry of Duffy Antigens, 134 M and N Antigens, 147 Duffy Antigens Facts, 134 S and s Antigens, 148 Biochemistry of Duffy Antigens, 134 Genetics and Biochemistry, 148 GPA: M and N Antigens, 148 GPB: S, s, and U Antigens, 148
xiv CONTENTS Standards and Regulations Governing the Crossmatch, 191 Antibodies of MNS Blood Group System, 148 Anti-M, 149 Crossmatch Procedures, 191 Anti-N, 150 Serologic Crossmatch, 192 Anti-S, Anti-s, and Anti-U, 150 Computer Crossmatch, 192 SECTION 11 MISCELLANEOUS BLOOD GROUP Limitations of Crossmatch Testing, 193 SYSTEMS, 150 Problem Solving Incompatible Crossmatches, 194 PART III: ESSENTIALS OF PRETRANSFUSION SECTION 2 PRINCIPLES OF COMPATIBILITY TESTING, 194 TESTING Overview of Steps in Compatibility Testing, 194 Chapter 7 Antibody Detection and Recipient Blood Sample, 194 Identification, 158 Comparison with Previous Records, 196 Repeat Testing of Donor Blood, 196 SECTION 1 ANTIBODY DETECTION, 159 Pretransfusion Testing on Recipient Sample, 197 Antibody Screen, 159 Tagging, Inspecting, Issuing, and Transfusing Autocontrol and Direct Antiglobulin Test, 160 Blood Products, 198 Potentiators, 161 Patient History, 161 SECTION 3 SPECIAL TOPICS, 199 SECTION 2 ANTIBODY IDENTIFICATION, 162 Urgent Requirement for Blood and Blood Initial Panel, 162 Components, 199 Panel Interpretation: Single Antibody Specificity, 163 Massive Transfusion, 201 Autocontrol, 165 Maximum Surgical Blood Order Schedule, 201 Phases, 165 Type and Screen Protocols, 201 Reaction Strength, 165 Crossmatching Autologous Blood, 202 Ruling Out, 165 Crossmatching of Infants Younger than 4 Months Matching the Pattern, 166 Rule of Three, 166 Old, 202 Patient’s Phenotype, 166 Pretransfusion Testing for Non–Red Blood Cell Multiple Antibodies, 166 Multiple Antibody Resolution, 168 Products, 203 Additional Techniques, 168 Antibodies to High-Frequency Antigens, 169 Chapter 9 Blood Bank Automation for Transfusion Additional Testing, 170 Services, 208 High-Titer, Low-Avidity Antibodies, 170 Antibodies to Low-Frequency Antigens, 171 SECTION 1 INTRODUCTION TO AUTOMATION IN Enhancing Weak IgG Antibodies, 171 IMMUNOHEMATOLOGY, 208 Cold Alloantibodies, 172 Forces Driving the Change to Automation, 209 SECTION 3 AUTOANTIBODIES, 174 Benefits and Barriers of Automated Cold Autoantibodies, 174 Specificity, 175 Instruments, 209 Avoiding Cold Autoantibody Reactivity, 176 Potential Benefits, 209 Adsorption Techniques, 177 Potential Challenges, 210 Warm Autoantibodies, 177 Characteristics of an Ideal Instrument for the Specificity, 178 Blood Bank, 211 Elution, 178 Adsorption, 180 SECTION 2 SELECTION OF AUTOMATION TO MEET LABORATORY NEEDS, 211 Chapter 8 Compatibility Testing, 188 Vendor Assessment, 211 SECTION 1 PRINCIPLES OF THE CROSSMATCH, 190 Base Technology Assessment, 212 What Is a Crossmatch? 190 Instrument Assessment, 212 Purposes of Crossmatch Testing, 191 SECTION 3 AUTOMATED TESTING SYSTEMS, 213 Automated Systems for Solid Phase Red Cell Adherence Assays, 213 Hemagglutination Assays, 214 Solid Phase Red Cell Adherence Assays, 215 SolidscreenR II Technology, 216 ErytypeR S Technology, 217 Automated System for Gel Technology Assays, 220
CONTENTS xv PART IV: CLINICAL CONSIDERATIONS IN SECTION 3 PREDICTION OF HEMOLYTIC DISEASE OF THE IMMUNOHEMATOLOGY FETUS AND NEWBORN, 250 Chapter 10 Adverse Complications of Maternal History, 250 Transfusions, 226 Antibody Titration, 250 Ultrasound Techniques, 251 SECTION 1 OVERVIEW OF ADVERSE REACTIONS TO Amniocentesis, 252 TRANSFUSION, 227 Cordocentesis, 252 Hemovigilance Model, 227 Fetal Genotyping, 253 Recognition of a Transfusion Reaction, 227 SECTION 4 POSTPARTUM TESTING, 253 SECTION 2 CATEGORIES OF TRANSFUSION REACTIONS, 228 Postpartum Testing of Infants and Mothers, 254 D Testing, 254 Hemolytic Transfusion Reaction, 228 Acute Hemolytic Transfusion Reaction, 228 ABO Testing, 255 Delayed Hemolytic Reaction, 230 Direct Antiglobulin Test, 255 Non–Immune-Mediated Mechanisms of Red Cell Intrauterine Transfusions, 255 Destruction, 231 SECTION 5 PREVENTION OF HEMOLYTIC DISEASE OF THE Delayed Serologic Transfusion Reactions, 232 FETUS AND NEWBORN, 255 Febrile Nonhemolytic Transfusion Reactions, 233 Allergic and Anaphylactic Transfusion Reactions, Antepartum Administration of Rh Immune Globulin, 256 234 Transfusion-Related Acute Lung Injury, 234 Postpartum Administration of Rh Immune Transfusion-Associated Graft-versus-Host Disease, Globulin, 256 Screening for Fetomaternal Hemorrhage, 257 235 Quantifying Fetomaternal Hemorrhage, 258 Bacterial Contamination of Blood Products, 236 Transfusion-Associated Circulatory Overload, 237 SECTION 6 TREATMENT OF HEMOLYTIC DISEASE OF THE Transfusion Hemosiderosis, 237 FETUS AND NEWBORN, 258 Citrate Toxicity, 237 Posttransfusion Purpura, 238 In Utero Treatment, 258 Postpartum Treatment, 259 SECTION 3 EVALUATION AND REPORTING A TRANSFUSION REACTION, 238 Phototherapy, 259 Exchange Transfusion, 259 Initiating a Transfusion Reaction Investigation, Selection of Blood and Compatibility Testing for 238 Additional Laboratory Testing in a Transfusion Exchange Transfusion, 260 Reaction, 240 PART V: BLOOD COLLECTION AND TESTING Records and Reporting of Transfusion Reactions and Fatalities, 241 Chapter 12 Donor Selection and Phlebotomy, 267 Hemovigilance Component, 241 Records, 241 SECTION 1 DONOR SCREENING, 268 FDA Reportable Fatalities, 241 Registration, 268 Chapter 11 Hemolytic Disease of the Fetus and Educational Materials, 268 Newborn, 246 Health History Interview, 270 SECTION 1 ETIOLOGY OF HEMOLYTIC DISEASE OF THE Questions for Protection of the Donor, 270 FETUS AND NEWBORN, 247 Questions for Protection of the Recipient, 272 Physical Examination, 275 SECTION 2 OVERVIEW OF HEMOLYTIC DISEASE OF THE General Appearance, 275 FETUS AND NEWBORN, 247 Hemoglobin or Hematocrit Determination, 275 Temperature, 275 Rh Hemolytic Disease of the Fetus and Blood Pressure, 275 Newborn, 248 Pulse, 275 Weight, 275 ABO Hemolytic Disease of the Fetus and Informed Consent, 276 Newborn, 249 SECTION 2 PHLEBOTOMY, 276 Alloantibodies Causing Hemolytic Disease of the Fetus and Newborn Other than Anti-D, 249 Identification, 276 Bag Labeling, 276
xvi CONTENTS Arm Preparation and Venipuncture, 277 PART VI: BLOOD COMPONENT PREPARATION AND Adverse Donor Reactions, 277 TRANSFUSION THERAPY Postdonation Instructions and Care, 277 Chapter 14 Blood Component Preparation and SECTION 3 SPECIAL BLOOD COLLECTION, 279 Therapy, 304 Autologous Donations, 279 SECTION 1 BLOOD COLLECTION AND STORAGE, 305 Preoperative Collection, 279 Normovolemic Hemodilution, 280 Storage Lesion, 306 Blood Recovery, 280 Types of Anticoagulant-Preservative Directed Donations, 280 Solutions, 307 Apheresis, 281 Additive Solutions, 307 Therapeutic Phlebotomy, 281 Rejuvenation Solution, 308 Chapter 13 Testing of Donor Blood, 286 SECTION 2 BLOOD COMPONENT PREPARATION, 309 SECTION 1 OVERVIEW OF DONOR BLOOD TESTING, 286 Whole Blood, 311 Indications for Use, 311 Required Testing on Allogeneic and Autologous Donor Blood, 287 Red Blood Cell Components, 311 Indications for Use, 311 SECTION 2 IMMUNOHEMATOLOGIC TESTING OF DONOR Red Blood Cells Leukocytes Reduced, 312 UNITS, 287 Apheresis Red Blood Cells, 313 Frozen Red Blood Cells, 314 ABO and D Phenotype, 287 Deglycerolized Red Blood Cells, 314 Antibody Screen, 288 Washed Red Blood Cells, 315 Red Blood Cells Irradiated, 315 SECTION 3 INFECTIOUS DISEASE TESTING OF DONOR UNITS, 288 Platelet Components, 316 Indications for Use, 316 Serologic Tests for Syphilis, 288 Platelets, 317 Rapid Plasma Reagin Test, 288 Pooled Platelets, 317 Hemagglutination Test for Treponema pallidum Apheresis Platelets, 317 Antibodies, 289 Platelets Leukocytes Reduced, 318 Confirmatory Testing for Syphilis, 289 Plasma Components, 318 Principles of Viral Marker Testing, 289 Fresh Frozen Plasma, Plasma Frozen within Enzyme-Linked Immunosorbent Assay, 289 24 Hours of Phlebotomy, 318 Nucleic Acid Testing, 291 Cryoprecipitated Antihemophilic Chemiluminescence, 291 Factor, 319 Controls, 291 Sensitivity and Specificity, 292 Apheresis Granulocytes, 321 Viral Hepatitis, 292 SECTION 3 DISTRIBUTION AND ADMINISTRATION, 321 Hepatitis Viruses, 292 Hepatitis Tests, 294 Labeling, 321 Storage and Transportation, 323 Human Retroviruses, 295 Human Immunodeficiency Virus Types 1 Transportation of Blood Components, 323 and 2, 296 Administration of Blood Components, 324 Nucleic Acid Testing for Ribonucleic Acid of Human Immunodeficiency Virus Chapter 15 Transfusion Therapy in Selected Type 1, 297 Patients, 329 Human T-Lymphotropic Virus Types I and II, 297 SECTION 1 TRANSFUSION PRACTICES, 329 Western Blotting, 297 Urgent and Massive Transfusion, 329 West Nile Virus, 298 Cardiac Surgery, 330 Recipient Tracing (Look-Back), 299 Neonatal and Pediatric Transfusion Issues, 331 Additional Tests Performed on Donor Transplantation, 332 Blood, 299 Organ Transplants, 333 Cytomegalovirus, 299 Hematopoietic Progenitor Cell Transplantation, Chagas Disease, 299 Testing for Bacterial Contamination of Blood 333 Therapeutic Apheresis, 335 Components, 300 Oncology, 336 Chronic Renal Disease, 337
CONTENTS xvii Hemolytic Uremic Syndrome and Thrombotic Change Control, 352 Thrombocytopenic Purpura, 338 Personnel Qualifications, 352 Supplier Qualification, 354 Anemias Requiring Transfusion Support, 339 Error Management, 354 Sickle Cell Anemia, 339 Validation, 355 Thalassemia, 339 Facilities and Equipment, 355 Immune Hemolytic Anemias, 340 Proficiency Testing, 355 Label Control, 356 Hemostatic Disorders, 340 SECTION 3 SAFETY, 356 SECTION 2 ALTERNATIVES TO TRANSFUSION, 341 Standard versus Universal Precautions, 356 Blood Bank Safety Program, 356 PART VII: QUALITY AND SAFETY ISSUES Physical Space, Safety Equipment, Protective Devices, and Warning Signs, 357 Chapter 16 Quality Assurance and Regulation of Decontamination, 359 the Blood Industry and Safety Issues in Chemical Storage and Hazards, 359 the Blood Bank, 345 Radiation Safety, 359 Biohazardous Wastes, 359 SECTION 1 REGULATORY AND ACCREDITING AGENCIES Storage and Transportation of Blood and Blood FOR QUALITY AND SAFETY, 346 Components, 360 Personal Injury and Reporting, 360 Food and Drug Administration, 346 Employee Education, 360 AABB, 347 Other Safety Regulations, 347 APPENDIX A: ANSWERS TO STUDY QUESTIONS, 365 Occupational Safety and Health Act, 347 Environmental Protection Agency, 347 GLOSSARY, 367 SECTION 2 QUALITY ASSURANCE AND GOOD INDEX, 373 MANUFACTURING PRACTICES, 348 Quality Assurance, 348 Quality Assurance Department, 348 Good Manufacturing Practices, 348 Components of a Quality Assurance Program, 348 Records and Documents, 348 Standard Operating Procedures, 351
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FOUNDATIONS: BASIC SCIENCES AND REAGENTS PART I IMMUNOLOGY: Basic Principles and 1 Applications in the Blood Bank CHAPTER OUTLINE Immunohematology: Antigen-Antibody Reactions In Vitro SECTION 1: CHARACTERISTICS ASSOCIATED WITH Overview of Agglutination ANTIGEN-ANTIBODY REACTIONS Sensitization Stage or Antibody Binding to Red Cells Factors Influencing First Stage of Agglutination General Properties of Antigens Lattice-Formation Stage or Cell-Cell Interactions General Properties of Antibodies Factors Influencing Second Stage of Agglutination Molecular Structure Grading Agglutination Reactions Fab and Fc Regions Hemolysis as an Indicator of Antigen-Antibody Comparison of IgM and IgG Antibodies Reactions IgM Antibodies IgG Antibodies SECTION 3: HUMAN LEUKOCYTE ANTIGEN (HLA) Primary and Secondary Immune Response SYSTEM AND PLATELET IMMUNOLOGY Antigen-Antibody Reactions Properties That Influence Binding Human Leukocyte Antigens SECTION 2: CHARACTERISTICS ASSOCIATED WITH RED Testing Applications in the Clinical Laboratory CELL ANTIGEN-ANTIBODY REACTIONS Inheritance and Nomenclature of HLA Red Cell Antigens Testing and Identification of HLA and Red Cell Antibodies Antibodies Immunohematology: Antigen-Antibody Reactions In Vivo Hematopoietic Progenitor Cell Transplants Transfusion, Pregnancy, and the Immune Response Graft-versus-Host Disease Complement Proteins Clearance of Antigen-Antibody Complexes Platelet Antigens LEARNING OBJECTIVES 7. List the variables in the agglutination test that affect sensitization and lattice formation. On completion of this chapter, the reader should be able to: 8. Accurately grade and interpret observed agglutination 1. Define the following terms in relation to red cells and reactions using the agglutination grading scale for transfusion: antigen, immunogen, epitopes, and antigenic antigen-antibody reactions performed in test tubes. determinants. 9. Compare the classical and alternative pathways of 2. Describe the characteristics of antigens that are located complement activation. on red cells, white cells, and platelets. 10. Outline the biological effects mediated by complement 3. Diagram the basic structure of an IgG molecule proteins in the clearance of red cells. and label the following components: heavy and light chains, Fab, and Fc regions, variable region, hinge 11. Recognize hemolysis in an agglutination reaction and region, antigen-binding site, and macrophage-binding explain the significance. site. 12. Outline how the immune system responds to antigen 4. Compare and contrast IgM and IgG antibodies with stimulation through transfusion and pregnancy. Explain regard to structure, function, and detection by the factors that cause variations in these in vivo agglutination reactions. responses. 5. Distinguish the primary and secondary immune response 13. Using the principles of tissue matching, select the best with regard to immunoglobulin class, immune cells potential graft given the HLA typing and antibody involved, level of response, response time, and antibody specificities. affinity. 14. Predict the probable HLA typing results in a family 6. Apply the properties that influence the binding of an study performed for graft selection. antigen and antibody to agglutination tests to achieve optimal results. 1
2 PART I n Foundations: Basic Sciences and Reagents 15. Compare and contrast the class I and II MHC complexes 17. Define graft-versus-host disease (GVHD) and select with regard to antigens, their associated immune cells, methods of prevention in transfusion and and their role in immunity. transplantation. 16. Explain the role of HLA testing in platelet transfusion 18. Outline the serologic test methods used in HLA typing support, organ transplants, and hematopoietic progenitor and antibody identification. cell transplants. Immunohematology: study The science of immunohematology embodies the study of blood group antigens and of blood group antigens and antibodies. Immunohematology is closely related to the field of immunology because it antibodies. involves the immune response to the transfusion of cellular elements. Red cells (erythro- cytes), white cells (leukocytes), and platelets are cellular components that can potentially initiate immune responses after transfusion. To enhance the reader’s understanding of the physiology involved in this immune response, this text begins with an overview of the immune system with an emphasis on the clinical and serologic nature of antibodies and antigens. Antigen: foreign molecules that SECTION 1 bind specifically to an antibody or a T-cell receptor. CHARACTERISTICS ASSOCIATED WITH ANTIGEN-ANTIBODY REACTIONS Allogeneic: cells or tissue from a GENERAL PROPERTIES OF ANTIGENS genetically different individual. An antigen is a molecule that binds to an antibody or T-cell receptor. This binding can Autologous: cells or tissue from occur within the body (in vivo) or in a laboratory test (in vitro). In chemical terms, self. antigens are large-molecular-weight proteins (including conjugated proteins such as gly- coproteins, lipoproteins, and nucleoproteins) and polysaccharides (including lipopolysac- Hapten: small-molecular-weight charides). These protein and polysaccharide antigens may be located on the surfaces of particle that requires a carrier cell membranes or may be an integral portion of the cell membrane. Antigens are located molecule to be recognized by the on viruses, bacteria, fungi, protozoa, blood cells, organs, and tissues. immune system. Transfused red cells contain antigens that may be recognized as foreign to the indi- B lymphocytes (B cells): vidual receiving the blood. These antigens are called allogeneic because they are unfamil- lymphocytes that mature in the iar to the individual being transfused but are derived from the same species. These foreign bone marrow, differentiate into antigens may elicit an immune response in the recipient. The body’s immune system plasma cells when stimulated normally recognizes and tolerates self-antigens. These antigens are termed autologous by an antigen, and produce because they originate from the individual. However, the failure to tolerate self-antigens antibodies. may cause an immune response against cells or tissue from self. This immune response to self may result in various forms of autoimmune disease. In terms of transfusion, an T lymphocytes (T cells): allogeneic transfusion involves the exposure to antigens that are different from the indi- lymphocytes that mature in the vidual receiving a transfusion, whereas an autologous transfusion involves antigens that thymus and produce cytokines originated in the recipient. to activate the immune cells including the B cell. The concept of an antigen having sufficient size to induce an immune response con- trasts with a hapten, which is a small-molecular-weight particle that requires a carrier Cytokines: secreted proteins that molecule to initiate the immune response. Haptens may include medications such as regulate the activity of other cells penicillin and are sometimes referred to as partial antigens. by binding to specific receptors. They can increase or decrease cell The immune response to foreign or potentially pathogenic antigens involves a complex proliferation, antibody production, interaction between several types of leukocytes. In the transfusion setting, immune and inflammation reactions. response is primarily humoral, involving mainly B lymphocytes (B cells). Following a transfusion, the recipient’s B cells may “recognize” these foreign red cell antigens through Memory B cells: B cells B-cell receptors (Fig. 1-1). This recognition causes the B cells to present the antigen to produced after the first exposure the T lymphocytes (T cells). After presentation, the T-cell cytokines signal the B cells to that remain in the circulation and be transformed into plasma cells that produce antibodies with the same specificity as the can recognize and respond to an original B-cell receptors. These antibodies are glycoprotein molecules that continue to antigen faster. circulate and specifically recognize and bind to the foreign antigen that originally created the response. Memory B cells are also made at this time. If there is a reexposure at a later Plasma cells: antibody- date, the memory B cells can respond quickly and change into antibody-producing plasma producing B cells that have cells; memory B cells do not require presentation to the T cell to be activated. Memory reached the end of their B cells allow a fast response to an antigen, an important principle used in vaccination. differentiating pathway.
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 3 Lymphocyte Lymphocyte Mature clones with precursor lymphocyte diverse receptors arise in generative Antigens that exhibit the lymphoid organs greatest degree of foreignness from the host elicit the Clones of mature strongest immune response. lymphocytes Antigen X Antigen Y specific for many antigens enter lymphoid tissues Antigen-specific clones are activated (\"selected\") by antigens Plasma cells Anti-X Anti-Y produce antibody antibody antibodies specific for the antigen Fig. 1-1 B-cell response to an antigen. Mature lymphocytes develop receptors for antigens before they encounter the antigen. The antigen stimulates the lymphocyte that has the receptor with the best fit. These lymphocytes are signaled to produce a B-cell clone, which differentiates into plasma cells that produce an antibody with a single specificity. (From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) Many different antibodies to foreign antigens can be produced in the immune response, Antigenic determinants: sites each binding to a different antigen on the surface. For example, red cells have many dif- on an antigen that are recognized ferent antigens on their surface. When red cells from one donor are transfused to a patient, and bound by a particular several different antibodies may be produced in the immune response to the transfused antibody or T-cell receptor (also red cells. The different antigenic determinants, also called epitopes, on a red cell can elicit called epitopes). the production of different antibodies. Each B cell has a unique specificity, which is “selected” by the antigenic determinant to expand into a clone of identical plasma cells Epitopes: single antigenic making antibodies with the same specificity as the original B-cell receptor. determinants; functionally, they are the parts of the antigen that The term antigen is often inappropriately used as a synonym for an immunogen. An combine with the antibody. immunogen is an antigen that is capable of eliciting an immune response in the body. The immune system’s ability to recognize an antigen and respond to it varies among Clone: family of cells or individuals and can even vary within an individual at a given time. Several important organisms having genetically characteristics of a molecule contribute to its degree of immunogenicity (Table 1-1). For identical constitution. example, different biological materials have varying degrees of immunogenicity. Protein molecules are the most immunogenic, followed by carbohydrates and lipids, which tend Immunogen: antigen in its role to be immunologically inert. In addition, complex compounds, such as a protein- of eliciting an immune response. carbohydrate combination, are more immunogenic than simpler molecules. Antigens on red cells, white cells, and platelets vary in their ability to elicit an immune response. Carbohydrates: simple sugars, such as monosaccharides and GENERAL PROPERTIES OF ANTIBODIES starches (polysaccharides). Molecular Structure Lipids: fatty acids and glycerol compounds. Antibody molecules are glycoproteins composed of four polypeptide chains joined together by disulfide bonds (Fig. 1-2). The terms antibody and immunoglobulin (Ig) are often used An immunogen is an antigen that provokes the immune response. Not all antigens are immunogens.
4 PART I n Foundations: Basic Sciences and Reagents Secreted IgG Antigen- binding site Heavy Light chain chain N VL N N N S S SS SS S S SS S CL VH S S S S CC CH1S Hinge S SSCH2 Fab Fc receptor/ S region complement Fc binding sites region S SSCH3 S Tail piece CC Disulfide bond S S Ig domain S S Fig. 1-2 Basic structure of an IgG molecule. Antigen binds to the variable region of the heavy and light chains. The variable region (VL and VH) is part of Fab (fragment, antigen-binding). The opposite end, composed of the heavy chain, is constant for each type of immunoglobulin. It is called the Fc (fragment, crystallizable) region, which determines the antibody function. The Fc region contains the complement binding region and the cell activation region. (Modified from Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) TABLE 1-1 Factors Contributing to Immunogenicity: Properties of the Antigen Antibody: glycoprotein Chemical composition and Proteins are the best immunogens, followed by complex (immunoglobulin) that recognizes complexity of the antigen carbohydrates a particular epitope on an antigen and facilitates clearance of that Degree of foreignness Immunogen must be identified as nonself; the greater the antigen. difference from self, the greater likelihood of eliciting an immune response Immunoglobulin: antibody; glycoprotein secreted by plasma Size Molecules with a molecular weight >10,000 D are better cells that binds to specific immunogens epitopes on antigenic substances. Dosage and antigen density Number of red cells introduced and the amount of Heavy chains: larger antigen that they carry contribute to the likelihood of polypeptides of an antibody an immune response molecule composed of a variable and constant region; five major Route of administration Manner in which the antigenic stimulus is introduced; classes of heavy chains determine intramuscular or intravenous injections are generally the isotype of an antibody. better routes for eliciting an immune response Light chains: smaller synonymously. Five classifications of antibodies are designated as IgG, IgA, IgM, IgD, polypeptides of an antibody molecule composed of a variable and IgE. The five classes are differentiated on the basis of certain physical, chemical, and and constant region; two major biological characteristics. Each antibody molecule has two identical heavy chains and two types of light chains exist in identical light chains joined by disulfide bond (S-S) bridges. These molecular bridges humans (kappa and lambda). provide flexibility to the molecule to change its three-dimensional shape.
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 5 Variable region is specific for different epitopes Red cell with various epitopes, Each immunoglobulin represented by shapes molecule consists of two identical heavy chains and Fig. 1-3 Variable region of an immunoglobulin. The specificity of an antibody is determined by the unique two identical light chains variable region that “fits” antigenic determinants or epitopes. (either kappa or lambda). The five distinctive heavy-chain molecules distinguish the class or isotype. Each heavy Isotype: one of five types of chain imparts characteristic features, which permit them to have unique biological func- immunoglobulins determined by tions. For example, the IgA family, which possesses alpha heavy chains, is the only anti- the heavy chain: IgM, IgG, IgA, body class capable of residing in mucosal linings. IgE antibodies can activate mast cells IgE, and IgD. causing immediate hypersensitivity reactions. IgD is an antigen receptor on the naive B cell.1 The immunoglobulins most involved in transfusion medicine are IgM and IgG, and Kappa chains: one of the two these are discussed in more detail subsequently. There are two types of light chains: kappa types of light chains that make up chains and lambda chains. Antibodies possess either two kappa or two lambda chains an immunoglobulin. but never one of each. Lambda chains: one of the two Each heavy-chain and light-chain molecule also contains variable regions and constant types of light chains that make up regions (or domains). The constant regions of the heavy-chain domain impart the unique an immunoglobulin. antibody class functions, such as the activation of complement or the attachment to certain cells. The variable regions of both the heavy chains and the light chains are con- Variable regions: amino- cerned with antigen binding and constitute the area of the antibody that contains idiotope terminal portions of (the idiotypic portion). This area is the binding site or pocket into which the antigen fits immunoglobulins and T-cell (Fig. 1-3). T-cell receptors also have specific antigen-binding receptors referred to as the receptor chains that are highly idiotope. The hinge region of the antibody molecule imparts flexibility to the molecule variable and responsible for the for combination with the antigen. antigenic specificity of these molecules. Fab and Fc Regions Constant regions: nonvariable Early experiments to identify antibody structure and function used enzymes to cleave the portions of the heavy and light immunoglobulin molecule. Enzymes such as pepsin and papain can divide the immuno- chains of an immunoglobulin. globulin molecule to produce two fragments known as Fab (fragment, antigen-binding) and Fc (fragment, crystallizable). Fab contains the portion of the molecule that binds to Idiotope: variable part of an the antigenic determinant. Fc consists of the remainder of the constant domains of the antibody or T-cell receptor; the two heavy chains linked by disulfide bonds (see Fig. 1-2). Certain immune cells, such as antigen-binding site. macrophages and neutrophils, possess receptors for the Fc region of an immunoglobulin. These immune cells are able to bind the Fc portion of antibodies that are attached to red Hinge region: portion of the cells or pathogens and assist in their removal by phagocytosis. This mechanism is one immunoglobulin heavy chains way that antibodies facilitate the removal of potential harmful antigens (Fig. 1-4). In between the Fc and Fab region; transfusion medicine, the antibodies attached to red cell antigens can signal clearance in provides flexibility to the molecule the liver and spleen, a process called extravascular hemolysis. to allow two antigen-binding sites to function independently. COMPARISON OF IgM AND IgG ANTIBODIES Extravascular hemolysis: red Because IgM and IgG antibodies have the most significance in immunohematology, the cell destruction by phagocytes following discussion focuses on these two immunoglobulins. Table 1-2 summarizes residing in the liver and spleen important features of IgM and IgG antibodies. usually facilitated by IgG opsonization.
6 PART I n Foundations: Basic Sciences and Reagents Opsonization Binding of Fc receptor Phagocytosis Breakdown of RBC IgG to Fc receptors signals of RBC and removal by IgG of RBC in the on phagocyte activation of liver and spleen phagocyte RBC RBC IgG antibody Signals Phagocyte Fc receptor Fig. 1-4 Antibody attaches to the Fc receptor on a macrophage to signal clearance. The variable portion of the immunoglobulin attaches to the antigen on the red cell, while the macrophage attaches to the Fc portion. The red cell is transported to the spleen and liver for clearance. (Modified from Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) TABLE 1-2 Comparison of IgM and IgG CHARACTERISTIC IgM IgG Heavy-chain composition Mu (µ) Gamma (γ) Light-chain composition Kappa (κ) or lambda (λ) Kappa (κ) or lambda (λ) J chain Yes No Molecular weight (D) 900,000 150,000 Valence 10 2 Total serum concentration (%) 10 70-75 Serum half-life (days) 5 23 Crosses the placenta No Yes Yes; very efficient Yes; not as efficient Activation of classical pathway of complement Intravascular Extravascular Clearance of red cells Detection in laboratory tests Immediate-spin Antiglobulin test From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders. The visible agglutination of IgM Antibodies antigen-positive red cells with IgM antibodies in vitro is also When the B cells initially respond to a foreign antigen, they produce IgM antibodies first. referred to as immediate-spin The IgM molecule consists of five basic immunoglobulin units containing two mu heavy or direct agglutination. chains and two light chains held together by a joining chain (J chain) (Fig. 1-5). Classified as a large pentamer structurally, one IgM molecule contains 10 potential antigen-combining Valency: number of epitopes per sites or has a valency of 10. Because of their large structure and high valency, these mol- molecule of antigen. ecules are capable of visible agglutination of antigen-positive red cells suspended in saline. The agglutination of red cell antigens by IgM antibodies is also referred to as immediate- spin and direct agglutination. IgM antibodies constitute about 10% of the total serum immunoglobulin concentration.1 An important functional feature associated with IgM antibodies is the ability to acti- vate the classical pathway of complement with great efficiency. Only one IgM molecule
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 7 Antigen-binding sites Heavy chain Kappa or lambda light chain Basic immunoglobulin unit J chain (joining chain) Fig. 1-5 Pentamer structure of the IgM molecule. Five basic immunoglobulin units exist with 10 antigen- binding sites. is required for the initiation of the classical pathway in complement activation. Complete Intravascular hemolysis: red activation of the classical pathway of complement results in hemolysis of the red cells cell lyses occurring within the and intravascular destruction (intravascular hemolysis). Antibodies of the ABO blood blood vessels usually by IgM group system are typically IgM and can cause rapid hemolysis of red cells if an incompat- activation of complement. ible unit of blood is transfused. The complement system is discussed later in this chapter. Bivalent: having a combining IgG Antibodies power of two. The IgG antibody molecule consists of a four-chain unit with two gamma heavy chains Hemolytic disease of the and two light chains, either kappa or lambda in structure. This form of an immunoglobu- fetus and newborn: condition lin molecule is known as a monomer. IgG antibodies constitute about 70% to 75% of caused by destruction of fetal or the total immunoglobulin concentration in serum.1 The molecule is bivalent; it possesses neonatal red cells by maternal two antigen-combining sites. Because of the relatively small size and bivalent structure of antibodies. the molecule, most IgG antibodies are not effective in producing a visible agglutinate with antigen-positive red cells suspended in saline. The antigen-antibody complexes can be The antiglobulin test method viewed with the use of the antiglobulin test discussed in Chapter 2. is necessary to detect antigen-antibody complexes Fc receptors on placental cells allow the transfer of IgG antibodies across the placenta involving IgG antibodies during pregnancy. This transfer of IgG antibodies from the mother to the fetus protects in vitro. newborns from infections. The mother’s IgG antibodies may also cause destruction of fetal red cells in a condition called hemolytic disease of the fetus and newborn (HDFN). This condition occurs if the mother makes an antibody to red cell antigens from exposure through transfusions or prior pregnancies. If the fetus has the corresponding antigen, the IgG antibodies target the red cells for destruction. Laboratory testing to detect this process is discussed in subsequent chapters. Four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) exist as a result of minor variations in the gamma heavy chains. The amino acid differences in these heavy chains affect the biological activity of the molecule. Subclasses IgG1 and IgG3 are most effective in activat- ing the complement system.2 Because of their immunoglobulin structure, two molecules of IgG are necessary to initiate the classical pathway of complement activation. PRIMARY AND SECONDARY IMMUNE RESPONSE Primary immune response: immune response induced by Immunologic response after exposure to an antigen is influenced by the host’s previous initial exposure to the antigen. history with the foreign material. There are two types of immune responses: primary and secondary. The primary immune response is elicited on first exposure to the foreign
8 PART I n Foundations: Basic Sciences and Reagents Affinity maturation: process of antigen. The primary response is characterized by a lag phase of approximately 5 to 10 somatic mutations in the days and is influenced by the characteristics of the antigen and immune system of the immunoglobulin gene causing host. Host properties that can contribute to the antigen response include the following: the formation of variations in the • Age affinity of the antibody to the • Route of administration antigen. B cells with the highest • Genetic makeup affinity are “selected” for the best • Overall health—stress, fatigue, disease fit, and the resulting antibody is • Medications (immunosuppressive) stronger. Lag phases may extend for longer periods. During this period, no detectable circulating Secondary immune response: antibody levels exist within the host. After this lag period, antibody concentrations immune response induced after a increase and sustain a plateau before a decline in detectable antibody levels. IgM antibod- second exposure to the antigen, ies are produced first, followed by the production of IgG antibodies. The specificity of which activates the memory the original IgM molecule (determined by the variable region) is the same as the specificity lymphocytes for a quicker of the IgG molecule seen later in the immune response. As a result of a process of gene response. rearrangement in the B cell, the affinity of the antibody produced after each exposure increases. This process is called affinity maturation, and it is the reason why antibodies Anamnestic response: often produce a stronger reaction in laboratory tests if the patient has had repeated secondary immune response. exposure to the antigen. The second contact with the identical antigen initiates a secondary immune response, or anamnestic response, within 1 to 3 days of exposure. Because of the significant pro- duction of memory B cells from the initial exposure, the concentrations of circulating antibody are much higher and sustained for a much longer period. Antibody levels are many times higher because of the larger number of plasma cells. IgM antibodies are also generated in the secondary immune response. However, the principal antibody produced is of the IgG class (Fig. 1-6). In the clinical setting, detecting a higher level of the IgM form of an antibody may indicate an acute or early exposure to a pathogen, whereas finding an increase in the IgG form of an antibody of the same specificity may indicate a chronic or late infection. Multiple stimulations of the ANTIGEN-ANTIBODY REACTIONS immune system with the same antigen produce antibodies Properties That Influence Binding with increased binding strength as a result of affinity The binding of an antigen and antibody follows the law of mass action and is a reversible maturation. process. This union complies with the principles of a chemical reaction that has reached equilibrium. When the antigen and antibody combine, an antigen-antibody complex or Immune complex: complex of immune complex is produced. The amount of antigen-antibody complex formation is one or more antibody molecules determined by the association constant of the reaction. The association constant drives bound to an antigen. the forward reaction rate, whereas the reverse reaction rate is influenced by the dissocia- tion constant. When the forward reaction rate is faster than the reverse reaction rate, Affinity: strength of the binding antigen-antibody complex formation is favored. A higher association constant influences between a single antibody and an greater immune complex formation at equilibrium (Fig. 1-7). epitope of an antigen. Avidity: overall strength of Several properties influence the binding of antigen and antibody. The goodness of fit reaction between several epitopes and the complementary nature of the antibody for its specific epitope contribute to the and antibodies; depends on the strength and rate of the reaction. Factors such as the size, shape, and charge of an affinity of the antibody, valency, antigen determine the binding of the antigen to the complementary antibody. This and noncovalent attractive forces. concept of goodness of fit is most easily seen by viewing the antigen-antibody binding as a lock-and-key fit (Fig. 1-8). If the shape of the antigen is altered, the fit of the antigen for the antibody is changed. Likewise, if the charge of the antigen is altered, the binding properties of the antigen and antibody are affected. The strength of binding between a single combining site of an antibody and the epitope of an antigen is called the affinity. When the immune complex has been generated, the complex is held together by non- covalent attractive forces, including electrostatic forces (ionic bonding), hydrogen bonding, hydrophobic bonding, and van der Waals forces. The influence of these forces on immune complex stability is described further in Table 1-3. The cumulative effect of these forces maintains the union between the antigen and antibody molecules. Avidity is the overall strength of attachment of several antigen-antibody reactions and depends on the affinity
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 9 A Primary Secondary antibody response antibody response First Repeat IgG exposure exposure Plasma IgG cells Plasma cells IgM in peripheral Amount of antibody lymphoid tissues Activated Low-level B cells antibody production Plasma Plasma cells Memory cells in Memory in bone marrow B cell bone B cell marrow Naive B cell 10 >30 0 5 10 >30 05 Days after antigen exposure Days after antigen exposure B Primary response Secondary response Lag after Usually 5-10 days Usually 1-3 days immunization Larger Peak Smaller response Relative increase in IgG and, under certain situations, in IgA or IgE Antibody Usually IgM>IgG (heavy chain isotype switching) isotype Higher average affinity (affinity maturation) Antibody Lower average affinity, affinity more variable Fig. 1-6 Primary and secondary immune responses. The initial exposure to an antigen elicits the formation of IgM, followed by IgG antibodies and memory B cells. The second response to the same antigen causes much greater production of IgG antibodies and less IgM antibody secretion. (From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) Antigen-antibody reactions Applying the are reversible Law of Mass Action Equilibrium constant or Ab Ag AbAg affinity, K, is given by K [AbAg] K [Ab] [Ag] Fig. 1-7 Kinetics of antigen-antibody reactions. The ratio of the forward and reverse reaction rates gives the equilibrium constant. Ab, antibody; Ag, antigen.
10 PART I n Foundations: Basic Sciences and Reagents Good fit: high attraction, low repulsion Poor fit: low attraction, high repulsion Fig. 1-8 Good fit. A good fit between the antigenic determinant and the binding site of the antibody molecule results in high attraction. In a poor fit, the forces of attraction are low. TABLE 1-3 Forces Binding Antigen to Antibody Electrostatic forces Attraction between two molecules on the basis of opposite charge; (ionic bonding) a positively charged region of a molecule is attracted to the negatively charged region of another molecule Hydrogen bonding Attraction of two negatively charged groups (X−) for a H+ atom Hydrophobic bonding Weak bonds formed as a result of the exclusion of water from the antigen-antibody complex van der Waals forces Attraction between the electron cloud (−) of one atom and the protons (+) within the nucleus of another atom of the antibody, valency of the antigen, and noncovalent attractive forces. The goal of laboratory testing procedures in the blood bank is to create optimal conditions for antigen-antibody binding to facilitate the detection and identification of antibodies and antigens. SECTION 2 CHARACTERISTICS ASSOCIATED WITH RED CELL ANTIGEN-ANTIBODY REACTIONS Before discussing red cell antigens and antibodies, the reader must have a solid knowledge of the location of these concepts. When a blood sample undergoes centrifugation, the denser red cells travel to the bottom of the tube. The liquid portion of the sample is known as plasma (if an anticoagulant was added) or serum (no anticoagulant added and sample is allowed to clot). • Red cell antigens are located on the red cells. They are part of the cell membrane or protrude from the cell membrane. • Red cell antibodies are molecules in the plasma or serum (Fig. 1-9). RED CELL ANTIGENS Researchers have defined 30 blood group systems with more than 250 unique red cell antigens.3 A blood group system is composed of antigens that have been grouped accord- ing to the inheritance patterns of many blood group genes. Every individual possesses a unique set of red cell antigens. Because of a diversity of blood group gene inheritance patterns, certain racial populations may possess a greater prevalence of specific red cell antigens. Scientific research has determined the biochemical characteristics of many red cell antigens and their relationship to the red cell membrane. In biochemical terms, these antigens may take the form of proteins, proteins coupled with carbohydrate molecules
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 11 BLOOD SAMPLE Name_Date ID #_Initial Buffy coat has white blood cells and platelets. Antibodies are in plasma or serum. Antigens are on the red blood cell membrane. Fig. 1-9 Blood sample with red cell antigen and antibody locations identified. The serum or plasma contains the antibody, whereas the red cell membrane contains the antigen. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.) MNS Knops XG N-glycan NH2 Gerbich Lutheran OK O-glycan Cromer Indian LW SC GPI-linkage Yt Dombrock ABO NH2 Kell Rh Colton JMH H COOH Kx Kidd EMM* Lea/ Diego GIL Outside Leb NH2 Duffy P1 I P Ii* Pk* COOH NH2 COOH NH2 COOH Inside Single-pass Multi-pass GPI-linked proteins proteins proteins Fig. 1-10 Illustration of multiple epitopes. The red cell has multiple epitopes or antigenic determinants. The unique configuration of the antigenic determinant allows recognition by a corresponding antibody molecule. (From Reid ME, Lomas-Francis C: The blood group antigen facts book, ed 2, San Diego, 2004, Elsevier Academic Press.) (glycoproteins), or carbohydrates coupled with lipids (glycolipids). Generally, the red cell Glycoproteins: compounds antigens protrude from the surface of the red cell membrane in three-dimensional con- containing carbohydrate and figurations (Fig. 1-10). Because of this orientation on the surface of the red cell, the protein molecules. antigens are accessible to antibody molecules for agglutination reactions. Agglutination using red cells is called hemagglutination. Determination of the specificity of the red cell Glycolipids: compounds antigen or antibody in most routine blood banking procedures is performed by hemag- containing carbohydrate and lipid glutination tests that can be done in a tube, in a microplate, or in a microtube filled with molecules. gel particles (the gel test). Solid-phase testing is an alternative method that uses red cell adherence to a solid support rather than hemagglutination. Chapters 2 and 9 discuss these Agglutination: visible clumping methods and the reagents used in these laboratory tests. of particulate antigens caused by interaction with a specific Some red cell antigens are more immunogenic than others and must be matched to the antibody. patient receiving a transfusion. For example, the D antigen within the Rh blood group system is highly immunogenic compared with other red cell antigens. The possibility of stimulating anti-D antibody production is high in an individual lacking D antigens who is transfused with red cells possessing D antigens. Patients who lack the D antigen should receive components containing red cells that also lack the D antigen.
12 PART I n Foundations: Basic Sciences and Reagents IgG antibodies react best at RED CELL ANTIBODIES 37° C, and IgM antibodies react best at room The most significant immunoglobulins in transfusion medicine are IgG and IgM. Most temperature or lower (in vitro). clinically important antibodies react at body temperature (37° C), are IgG, and can cause immune destruction of transfused red cells possessing the corresponding antigen. The destruction of the red cells can cause transfusion reactions, anemia, and HDFN. IgM antibodies react best at room temperature (20° C to 22° C) or lower (to 4° C) and are usually not implicated in the destruction of transfused red cells. The antibodies to ABO antigens are an important exception to this rule. Antibodies to ABO antigens are of the IgM class and react in vitro at room temperature and in vivo at body temperature. A transfusion of the wrong ABO blood group (antigen) would effectively activate the complement system and cause hemolysis of the transfused cells. Alloantibodies: antibodies with IMMUNOHEMATOLOGY: ANTIGEN-ANTIBODY REACTIONS IN VIVO specificities other than self; stimulated by transfusion or Transfusion, Pregnancy, and the Immune Response pregnancy. Antibody screen test: test to During transfusion and pregnancy, a patient is exposed to many potentially foreign determine the presence of antigens on red cells, white cells, and platelets that possess varying degrees of immuno- alloantibodies. genicity. Because of this exposure to foreign antigens, a patient’s immune system may In vivo: referring to a reaction become activated or “sensitized” with the resultant production of circulating antibodies. within the body. The antibodies produced in response to transfusion and pregnancy are classified as alloantibodies. Complement system: group of serum proteins that participate in The antibody screen test is performed on the patient before transfusion to detect any an enzymatic cascade, ultimately existing red cell alloantibodies. If a red cell alloantibody is detected, a test is performed generating the membrane attack to identify the specificity of the antibody. Once the specificity is identified, donor units complex that causes lysis of lacking the red cell antigen are selected for transfusion. Detecting and identifying antibod- cellular elements. ies in the patient before transfusion is important to avoid the formation of antigen- antibody complexes in vivo (within the patient’s body), which would lessen the survival Membrane attack complex: of the transfused cells. C5 to C9 proteins of the complement system that mediate Immunization may also occur during pregnancy as fetal blood cells may enter the cell lysis in the target cell. maternal circulation at delivery. Alloantibody production may be observed as an immune Hemolysis: lysis or rupture of response to red cell, white blood cell, or platelet antigens of fetal origin. Women are erythrocytes. routinely screened during the first trimester of pregnancy for the presence of red cell alloantibodies that can destroy fetal red cells before or after delivery. The red cell destruc- tion may lead to clinical complications of anemia and high levels of bilirubin in the fetus or newborn. Complement Proteins The complement system is a group of serum proteins that have numerous biological roles related to antigen clearance, cell lysis, and vasodilation (Fig. 1-11). These proteins nor- mally circulate in an inactive or proenzyme state. On activation, they are converted into active enzymes that enhance the immunologic processes. Nine components of the complement family are designated C1 through C9. When activation occurs by an antibody in the classical pathway, the C1q, C1r, C1s complex splits C4 and C2 proteins into two parts. Each protein is converted into protein frag- ments and given the distinction a or b (e.g., C4 is converted to C4a and C4b). The smaller fragment is designated a, and the larger one is designated b. Typically, the larger b fragment binds to the cell, and the smaller a fragment enhances the inflammatory response. From the splitting of C4 and C2, C4b and C2a fragments join to form C3 convertase, which splits C3 into C3a and C3b. The C3 convertase joins with C3b to form C5 convertase, which splits C5. The final formation of a membrane attack complex causes lysis of various cells (hemolysis of red cells), bacteria, and viruses by disrupting the cell membrane. The direct attachment of the membrane attack complex, consisting of the complement proteins C5 to C9, to the cell surface produces holes in the cell mem- brane and osmotic lysis. If the membrane attack complex becomes attached to trans- fused red cells, hemolysis occurs with a subsequent release of free hemoglobin into the circulation.
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 13 Effector functions Alternative Classical pathway pathway Target cell Antibody Initiation of complement activation Early steps C3 C3 C3b C3 C3b C3b C3a C3a: Inflammation C3b is C3b C3b: Opsonization deposited on target cell C3b and phagocytosis C5b C5 C5b C5a: C5a Inflammation Late steps Complement C9 proteins form Lysis of target cell membrane attack complex Fig. 1-11 Comparison of the classical and alternative complement systems. The classical pathway is initiated by an antigen-antibody reaction. The alternative pathway is initiated by the membrane property of a microorganism. Following the split of the C3 component, the two pathways are identical. Three major biological activities of the complement system are opsonization, lysis of target cells, and stimulation of inflammatory mediators.4 (Modified from Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) The early steps in the activation of the complement proteins can occur in either of two Classical pathway: activation of complement that is initiated by pathways: antigen-antibody complexes. • The classical pathway is activated by the presence of an antibody bound to an antigen. Alternative pathway: activation Red cell destruction that may result from antibody-coated red cells is caused by the of complement that is initiated by foreign cell-surface constituents. activation of this pathway. • The alternative pathway does not require a specific antibody for activation. Foreign cell-surface constituents, such as bacteria, viruses, and foreign proteins or carbohy- drates, initiate it. Regardless of the activation mode, the final steps involved in cell lysis are common to both pathways. In addition, the consequences of complement activation, which serves as an important amplifier of the immune system, are common to both pathways. The
14 PART I n Foundations: Basic Sciences and Reagents TABLE 1-4 Biological Effects Mediated by Complement Proteins Opsonization Clear immune complexes Anaphylaxis Enhance phagocytosis Lysis Promote release of enzymes from neutrophils Chemotactic Increase smooth muscle contraction and inflammation Kill foreign antigens by membrane lysis Recruit platelets and phagocytes Anaphylatoxins: complement peptides generated during the formation of the membrane attack unit have the following split products (C3a, C4a, and additional functions (Table 1-4): C5a) that mediate degranulation • Anaphylatoxins C3a, C4a, and C5a assist in the recruitment of phagocytic cells and of mast cells and basophils, which results in smooth muscle the promotion of inflammation. These complement proteins attach to mast cells and contraction and increased vascular promote the release of vasoactive amines, which help make blood vessels permeable permeability. for fluid and cells to enter the area. • The C5a protein is chemotactic for neutrophils and attracts these cells to the site of Vasoactive amines: products injury. such as histamines released by • Complement also functions as an opsonin, which is a substance that binds to an antigen basophils, mast cells, and platelets to promote phagocytosis. Phagocytic cells are generally inefficient. If the substance is that act on the endothelium and coated with an opsonin, the process of phagocytosis becomes extremely efficient. smooth muscle of the local Receptors on the surface of the phagocytic cell have a higher affinity for opsonins. vasculature. C3b and antibodies are opsonins, which promote the clearance of bacteria and other cells to which the opsonins are attached. A test to determine whether the red cell is Chemotactic: movement of cells coated with complement components is a useful serologic tool when red cell destruc- in the direction of the antigenic tion is being investigated. C3b and C4b proteins are made up of a “c” and “d” stimulus. complex. The C4d and C3d breakdown products can also be detected on red cells. Opsonin: substance (antibody or Clearance of Antigen-Antibody Complexes complement protein) that binds to an antigen and enhances Antigen-antibody complexes are removed from the body’s circulation through the mono- phagocytosis. nuclear phagocyte system.1 This system acts as a filter to remove microbes and old cells. The system is present in secondary lymphoid organs such as the spleen, lymph nodes, Receptors: molecules on the cell liver, and lungs. The largest lymphoid organ, the spleen, is particularly effective for remov- surface that have a high affinity ing old and damaged red cells from the blood and clearing the body of antigen-antibody for a particular ligand. complexes. Red cells that have bound IgG or complement components are removed by the spleen. Mononuclear phagocyte system: system of mononuclear IMMUNOHEMATOLOGY: ANTIGEN-ANTIBODY REACTIONS IN VITRO phagocytic cells, associated with the liver, spleen, and lymph nodes, Overview of Agglutination that clears microbes and damaged cells. Antigen-antibody reactions occurring in vitro (in laboratory testing) are detected by visible agglutination of red cells or evidence of hemolysis at the completion of testing. In vitro: reaction in an artificial The absence of hemagglutination in immunohematologic testing (a negative reaction) environment, such as in a test implies the lack of antigen-antibody complex formation. A negative reaction is interpreted tube, microplate, or column. to mean that the antibody in the test system is not specific for the antigen. A positive reaction indicates that an antigen-antibody immune complex was formed. The specificity Sensitization: binding of of the antibody matched the antigen in the test system. The agglutination test must be antibody or complement performed correctly to reach the correct conclusion regarding the presence or absence of components to a red cell. the antigen or antibody. Lattice formation: combination The next section describes the factors affecting the hemagglutination reaction, which of antibody and a multivalent occurs in two stages, referred to as the sensitization step and the lattice formation step.4 antigen to form cross-links and These concepts are summarized in Table 1-5. result in visible agglutination. Sensitization Stage or Antibody Binding to Red Cells A positive reaction is indicated by agglutination. A negative In the first stage of red cell agglutination, the antibody binds to an antigen on the red reaction is indicated by no cell membrane. This stage requires an immunologic recognition between the antigen and agglutination.
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 15 TABLE 1-5 Factors Affecting Agglutination STAGE FACTOR DESCRIPTION Sensitization Temperature IgG, 37° C; IgM, ≤22° C Incubation time Immediate-spin or following Lattice formation pH a specific time at 1-8° C, Ionic strength room temperature, or 37° C Zeta potential 7.0 (physiologic is ideal) Can be adjusted with reagents Zone of equivalence Distance between cells caused Centrifugation by charged ions Antigen and antibody concentrations Time and speed of centrifugation to bring cells close together antibody. During this recognition stage, antigenic determinants on the red cell combine Serum-to-cell ratio: ratio of with the antigen-binding site of the antibody molecule. No visible agglutination is observ- antigen on the red cell to able at this stage. antibody in the serum. The physical joining of an antigen and antibody is essentially a random pairing of the two structures determined largely by chance. Antibody concentration and antigen recep- tor accessibility and quantity may influence the probability for this collision. An increase in antibody concentration increases the probability of collision events with the corre- sponding antigen. This concept is referred to as the overall effect of the serum-to-cell ratio or concentration of antigen and antibody in immunohematologic tests. Increasing the amount of serum placed in the test tube increases the concentration of antibodies available for binding to red cell antigens. When a patient’s serum demonstrates weak reactions in agglutination testing, this simple technique may be used to enhance the first stage of the agglutination reaction. Increasing the amount of antigen or red cell concentration does not increase the reaction probability. Factors Influencing First Stage of Agglutination In addition to the serum-to-cell ratio, certain environmental factors may influence the sensitization and lattice formation in the two stages of agglutination reaction. Temperature of Reaction Most antibodies of clinical relevance in transfusion are IgG antibodies reacting at approxi- mately 37° C.2 By combining the sources of antigen and antibody and incubating them at this temperature, the first stage of the agglutination reaction is enhanced. In contrast, IgM antibodies are more reactive at lower temperatures, generally at room temperature (ambient temperature) or lower. Incubation Time Increasing the incubation time may help in weak antibody Allowing adequate time for the combination of antigen and antibody to attain equilibrium investigations. also enhances the first stage of the agglutination reaction. The length of time recom- mended for optimal antigen-antibody reactivity varies with the test procedure and the Immediate-spin: interpretation reagents used in testing. Some test procedures may indicate a predetermined incubation of agglutination reactions period performed at variable temperatures, such as 37° C, room temperature, or 1° C to immediately after centrifugation 8° C. Additionally, the test procedures may indicate an immediate-spin step that indicates and without incubation. a combination of test reagents and sample with no period of incubation. pH The optimal pH for hemagglutination is around 7.0, which is the physiologic pH range. This pH range is adequate for most of the important red cell antibodies.
16 PART I n Foundations: Basic Sciences and Reagents IgM IgM Fig. 1-12 Agglutination. Agglutination refers to red cells clumping together as a result of interactions with specific antibodies. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.) ؊ ؊ ؊؊ ؊ ؊؊ ؊ ؊؊ ؊ ؊ ؊ ؊ ؊؊ ؊؊ ؊ ؊ Fig. 1-13 Effect of the zeta potential on the second stage of agglutination. The zeta potential keeps red cells apart and is less likely to be agglutinated by IgG antibodies. IgM antibodies are more likely to cause direct agglutination. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.) Low-ionic-strength reagents Ionic Strength act by increasing the rate of antibody uptake on the cells. In an isotonic environment, such as physiologic saline, Na+ and Cl− ions are attracted to the oppositely charged groups on antigen and antibody molecules. As a result of this Zeta potential: electrostatic attraction, the combination of antigen and antibody is hindered. If the ionic environment potential measured between the is reduced, this shielding effect is reduced, and the amount of antibody uptake onto the red cell membrane and the red cell is increased. slipping plane of the same cell. Lattice-Formation Stage or Cell-Cell Interactions After the red cells have been sensitized with antibody molecules, random collisions between the antibody-coated red cells are necessary to develop cross-linkages for the visualization of red cell clumping or agglutination within the test tube (Fig. 1-12). Visible agglutinates form when red cells are in close proximity to promote the lattice formation of antibody-binding sites to antigenic determinants on adjacent red cells. Factors Influencing Second Stage of Agglutination Distance Between Red Cells The zeta potential, or the force of repulsion between red cells in a physiologic saline solution, exerts an influence on the agglutination reaction. Red cells possess a net negative charge on the cell surface in a saline suspension. Cations (positively charged ions) from the saline environment are attracted to these negative charges. A stable cationic cloud surrounds each cell and contributes a force of repulsion between molecules of similar charge. As a consequence of this repulsive force, the red cells remain at a distance from each other. This distance between the cells is proportional to the zeta potential (Fig. 1-13).
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 17 Prozone Postzone Zone of Zone of Zone of antibody excess equivalence antigen excess (small complexes) (large complexes) (small complexes) Fig. 1-14 Zone of equivalence. Maximum agglutination is observed when the concentrations of antigens and antibodies fall within the zone of equivalence. (Modified from Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 7, Philadelphia, 2011, Saunders.) Because of the larger size, pentameter shape, and multivalent properties of IgM molecules, agglutination is facilitated between adjacent red cells that have IgM attached to them. In contrast, IgG antibody molecules are smaller and less able to span the distance between adjacent red cells generated by the zeta potential. IgG molecules may attach, but visible agglutination might not occur. Optimal Concentrations of Antigen and Antibody Zone of equivalence: number of binding sites of multivalent Maximum amounts of agglutination are observed when the concentrations of antigens antigen and antibody are (red cells) and antibody (serum) fall within the zone of equivalence (Fig. 1-14). When the approximately equal. concentration of antibody exceeds the concentration of antigen, antibody excess (or prozone) exists, which decreases the amount of agglutinated red cells. In contrast, post- Prozone: excess antibody causing zone occurs when the concentration of antigen exceeds the number of antibodies present. a false-negative reaction. The amount of agglutinates formed under these circumstances is also suboptimal and diminished. Postzone: excess antigen causing a false-negative reaction. Immunohematologic testing is designed to obtain reactions within the zone of equiva- lence. Commercial antibody preparations are diluted to optimal antibody concentrations for testing. Red cell preparations are diluted to a 2% to 5% suspension in saline for optimal antigen concentrations. The use of red cell suspensions greater than 5% may affect the ability of the test reaction to fall within the zone of equivalence and cause a “false-negative” reaction because of postzone. Effect of Centrifugation The time and speed of centrifugation are important factors for the detection of aggluti- nated red cells. Centrifugation helps to facilitate the formation of a latticed network by forcing the red cells closer together in the test environment. Centrifuges are calibrated to define the optimal speed and time for the best reaction. Overcentrifugation or undercen- trifugation and too-high or too-low speeds can cause false-positive or false-negative reactions. Grading Agglutination Reactions In immunohematology, antigen-antibody reactions are measured qualitatively. The pres- ence of an antigen-antibody reaction is detected with red cell agglutination, but the concentration of the immune complex is not determined in a quantitative manner. Red cell antigen and antibody reactions may be performed in test tubes, in microplate wells,
18 PART I n Foundations: Basic Sciences and Reagents 4ϩ Red cell button is a solid agglutinate; clear background 3ϩ Several large agglutinates; clear background 2ϩ Many medium-sized agglutinates; clear background 1ϩ Medium- and small-sized agglutinates; background is turbid with many free red cells 0 No agglutinated red cells are visible; red cells are observed flowing off the red cell button during the process of grading Fig. 1-15 Grading antigen-antibody reactions. Consistency in grading reactions allows for correct interpretation of results in the immunohematology laboratory. (Modified from Gamma Biologicals, Houston.) and in microtubes filled with gel particles. Because of the qualitative nature of the mea- surement, the reading and grading of agglutination reactions are subjective. To standardize this element of subjectivity among personnel performing the testing, a grading system for agglutination reactions has been established. The conventional grading system for tube testing uses a 0 to 4+ scale (Fig. 1-15). Slight variations in this conventional grading system may be established in individual institutions. Agglutination reactions are read by shaking and tilting the test tubes until the red cell button has been removed from the bottom of the tube. Negative agglutination reactions are interpreted after the red cell button has been completely resuspended. An agglutination viewer lamp with a magnifying mirror is usually used to evaluate the agglu- tination reactions. Laboratories using a microscopic reading in some testing have criteria established for grading these reactions. Supernatant: fluid above cells or Hemolysis as an Indicator of Antigen-Antibody Reactions particles after centrifugation. In addition to agglutination as an indicator of an antigen-antibody reaction in the immu- A hemolyzed patient sample is nohematology laboratory, red cell hemolysis observed in the tube is also an indicator of not acceptable for serologic the reactivity of an antigen and antibody in vitro. If the complement system is activated testing in the blood bank by an immune complex, hemolysis of the red cells along with agglutination can occur. because hemolysis is The final steps in the process of complement activation initiate the membrane attack interpreted as a positive complex, causing membrane damage. As a consequence of this damage, intracellular fluid reaction. is released to the reaction environment. The red cell button is often smaller compared with the red cell button present in other tubes. A pinkish to reddish supernatant is observed after the tubes have been centrifuged. For grading a tube with hemolysis, an H is traditionally used when this phenomenon is observed. Some red cell antibodies char- acteristically display hemolysis in vitro, such as antibodies to the Lewis system antigens and anti-Vel, which are discussed in later chapters.3 It is important to recognize hemolysis as an antigen-antibody reaction. Hemolysis is detected in vitro using fresh serum samples. Serum has active complement proteins. Because anticoagulants bind calcium, which is necessary for complement activation, plasma samples do not demonstrate complement activation.
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 19 SECTION 3 HUMAN LEUKOCYTE ANTIGEN (HLA) SYSTEM AND PLATELET IMMUNOLOGY HUMAN LEUKOCYTE ANTIGENS Refractoriness: unresponsiveness to platelet Testing Applications in the Clinical Laboratory transfusions owing to HLA-specific or platelet-specific antibodies or Most nucleated cells such as leukocytes and tissue cells possess inherited antigens on the platelet destruction from fever or cell surface called human leukocyte antigens (HLA). HLA antigens and the antibodies sepsis. Responsiveness is they elicit are involved in transfusion and transplantation medicine. HLA antibodies, measured by posttransfusion similar to red cell antibodies, result from exposure to foreign antigens during transfusions platelet counts. of blood products and from pregnancy. These antibodies can cause poor platelet response, or refractoriness, in patients requir- ing platelet transfusions. To improve platelet response, donor platelets that are HLA matched with the recipient may be necessary. HLA antibodies are also responsible for reactions that cause chills and fever in some patients receiving red cell transfusions. In this situation, blood products that are “leukocyte reduced” to avoid HLA antigens and residual cytokines usually prevent further reactions. HLA testing is not routine in the transfusion service or blood bank setting; however, an understanding of its inheritance, nomenclature, and application is important for optimal patient support. Table 1-6 lists applications of HLA testing. Organ and hematopoietic progenitor cell transplants rely on HLA matching for the best outcome. In addition, HLA testing is used to assess risk factors for disease suscep- tibility. These tests are not diagnostic for the associated diseases but are used to assess relative risk. A growing application of HLA typing has been in pharmacogenomic appli- cations. Certain HLA antigens are associated with optimal drug therapy regimens for certain diseases.2 Inheritance and Nomenclature of HLA Haplotype: set of linked genes inherited together because of The genes encoding the expression of the HLA antigens are part of the major histocom- their close proximity on a patibility complex (MHC) gene system located on chromosome 6. Although the MHC chromosome. system was first recognized and named from experiments in tissue transplantation, it is now known that the role of the MHC is essential in the recognition of self and nonself, Alleles: different forms of a gene the coordination of cellular and humoral immunity, and the immune response to present at a particular antigens. chromosomal locus. The MHC genes are divided into three categories or classes (Fig. 1-16). Class I includes Polymorphic: genetic system the A, B, and C loci; class II includes the DR, DP, and DQ loci; and class III includes that expresses several possible genes coding some complement proteins and cytokines. The genes are closely linked and alleles at specific loci on a inherited as a haplotype. Each person inherits one haplotype from each parent and both chromosome. are expressed. This inheritance pattern is illustrated in the example in Fig. 1-17. There are hundreds of possible variations of each gene, called alleles, at each locus (Table 1-7). Each antigen expression is identified with a unique number that is determined by either serologic (antigen-antibody reactions) or molecular methods, which are discussed further in Chapter 3. The MHC region is the most polymorphic system of genes in humans TABLE 1-6 HLA Testing Applications • Hematopoietic progenitor cell transplants • Solid-organ transplants • Platelet selection for refractory patients • Disease association • Ankylosing spondylitis—B27 • Celiac disease—DQ2 • Optimize certain drug therapy regimens • Abacavir sensitivity (for HIV treatment) and B*57:01 allele9 HIV, Human immunodeficiency virus.
20 PART I n Foundations: Basic Sciences and Reagents Chromosome 6 Class II DP Class III DQ Class I DR Complement proteins: C4, Factor B, C2 Cytokines: TNF-α, LTβ, LT A B C Fig. 1-16 Major histocompatibility complex (MHC). MOTHER A11 FATHER A3 B44 B35 A2 Cw12 A1 Cw5 B7 DR13 B8 DR8 Cw7 DQ8 Cw3 DQ7 DR17 DR4 DQ2 DQ5 Potential offspring: Child 2 Child 3 Child 4 Child 1 A2 A1 A2 A3 A11 A1 A11 A3 B7 B8 B7 B35 B44 B8 B44 B35 Cw7 Cw3 Cw7 Cw5 Cw12 Cw3 Cw12 Cw5 DR17 DR4 DR17 DR8 DR13 DR4 DR13 DR8 DQ2 DQ5 DQ2 DQ7 DQ8 DQ5 DQ8 DQ7 Fig. 1-17 Example of the inheritance pattern of class I and class II HLA antigens. A complete set of alleles located in each parent’s chromosome is inherited as a unit by each child. There is a 25% chance that two children in a family will inherit the same sets and have identical HLA typing. TABLE 1-7 HLA Nomenclature GENETIC ANTIGEN NUMBER OF ALLELE EXAMPLE NUMBER OF LOCUS A1 to A80 ANTIGENS A*01:01 ALLELES A B7 to B82 B*07:02 B Cw1 to Cw10 28 C*01:02 1729 C DR1 to DR18 50 DRB1*01:01 2329 DR DQ1 to DQ9 10 DQB1*05:01 1291 DQ DP1 to DP6 24 DPB1*01:01 1150 DP 9 160 6 150 http://hla.alleles.org/nomenclature/stats.html. Accessed December 2011. because of the many possible alleles at each location. The probability that any two indi- viduals will express the same HLA antigens is extremely low. The naming of the HLA antigens consists of a letter designating the locus, including A, B, C, DR, DQ, and DP, and a number indicating the antigen, for example, A2, B27, Cw7, DR1, DQ5. For the C locus, the “w” is included in the nomenclature to distinguish HLA C-locus antigens from complement components. The HLA nomenclature began when the only test method was the serological lymphocytotoxicity assay. Today, more accurate and specific molecular typing assays are used. As a result of the increased sen- sitivity of the typing methods and increased knowledge of the glycoprotein structure of the HLA antigens, the number of alleles that can be determined continues to increase, whereas the total number of antigens has remained the same. The World Health
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 21 Organization is responsible for standardizing the HLA nomenclature.5 For example, using the correct nomenclature, as of January 2010, A2 is expressed as A2 for antigen level resolution determined by serologic testing, HLA-A*02 for low-resolution typing, and HLA-A*02:01 if high-resolution testing is performed. Allele-level, high-resolution typing is particularly important for hematopoietic graft survival (discussed later). Testing and Identification of HLA and Antibodies Testing to Identify HLA As described earlier in this chapter, red cell antigens and antibodies are primarily identi- fied by agglutination reactions or hemagglutination test methods. Because of the nature of leukocyte antigens and antibodies, agglutination techniques are not effective. Serologic identification of HLA antigens requires the lymphocytotoxicity test method, which is described subsequently. Class I antigens are found on the surface of platelets, leukocytes, and most nucleated cells in the body. Mature red cells lack HLA antigens, although reticulocytes express HLA class I antigens. HLA class II antigens are found on the antigen-presenting cells— macrophages, dendritic cells, and B cells. In the circulation, the number of cells expressing class I antigens is greater than the number of cells with class II antigens. Because class II antigens are not found on platelets, it is not necessary to match Class II antigens when HLA-compatible platelets are requested. In serologic typing methods, a suspension of T or B lymphocytes is added to microtiter plates containing known antibody specificities. After incubation, rabbit complement is added. If the antibody on the plate matches the antigen on the cell, complement is acti- vated, causing cell injury or a positive reaction. Cell damage is detected by the addition of a dye. This injury allows the dye to enter the cell, providing a visual means to distin- guish the positive cells from nonreactive cells. Each well is scored while viewing under the microscope, and a pattern of reactivity determines the class I or class II typing. Serologic typing of the HLA antigens is not always clear because of antibodies that can often react to more than one epitope (cross-reactive). In many cases, specific antibod- ies have not been developed to recognize the many different HLA antigens that can be expressed. For these reasons, the lymphocytotoxicity test (Fig. 1-18) is being replaced or supplemented with more sensitive and specific molecular methods, which are described in Chapter 3. Antibody Detection and Identification ABO compatibility is essential for the success of all solid-organ transplants. Second to ABO compatibility, careful matching of HLA antigens in patients with existing antibodies is important for long-term graft survival for kidney, heart, and lung transplants. Fig. 1-19 shows that the degree of HLA matching for kidney transplants corresponds to the survival of the graft.6 The soluble nature of HLA antigens in the liver allows for a greater flexibility in the selection of donor tissue for liver transplants, even when preformed antibodies exist. Organ transplant candidates are periodically screened for developing HLA antibod- ies so that a suitable match can be determined when an organ becomes available. Cells that are positive will take up the dye and stain dark, indicating a positive reaction Negative Positive Fig. 1-18 Lymphocytotoxicity test for identification of HLA antigens. Complement and a dye are used to determine whether there is antigen-antibody recognition. Complement-mediated cell membrane damage occurs if the antigen and antibody form a complex. The damaged membrane becomes permeable to the dye, which enters the cell, allowing a positive reaction to be observed. Dye exclusion is a negative reaction.
22 PART I n Foundations: Basic Sciences and Reagents Five-year survival (%) of renal allografts 80 75 70 65 60 55 50 12345 6 0 Number of mismatched HLA alleles Fig. 1-19 HLA matching correlates with renal graft survival. Matching HLA antigens between the donor and recipient significantly improves renal allograft survival. The data are shown for deceased (cadaver) grafts. (From Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 7, Philadelphia, 2011, Saunders.) Calculated panel-reactive Patients can become sensitized to HLA antigens by the following types of exposures antibody (CPRA): estimates the to alloantigens7: percentage of donors that would • Pregnancies. About 30% to 50% of women with three or more pregnancies develop be incompatible with a transplant candidate, based on the HLA antibodies. In some women, the antibodies are present for just a short time (weeks candidate’s antibodies to HLA to months), whereas they may persist for many years in other women. antigens. • Blood transfusions. About 50% of patients who receive multiple transfusions develop antibodies. Today, most patients who require blood transfusions receive leukocyte- Unacceptable antigens: reduced blood, which decreases the chances for a patient to become sensitized. antigens that the potential graft • Previous transplant. About 90% of patients develop HLA antibodies within 2 weeks recipient is reacting against; of a failed graft. antibodies to these antigens could The term panel-reactive antibody (PRA) was developed from serologic tests performed reduce the graft survival. on microtiter trays composed of a panel of common antigens. A higher PRA meant that the recipient had many reactions and was less likely to be compatible with available Mixed lymphocyte culture organs. (MLC): In vitro reaction of T cells With the increase in sensitivity and specificity of newer solid-phase methods, PRA from one individual against MHC determined by serologic methods has been replaced with a calculated panel-reactive anti- antigens on leukocytes from body (CPRA).7 This number is based on the antigens that the organ candidate is reactive another individual. The technique against, called unacceptable antigens, and provides a statistical guide for determining measures the response of HLA reactivity within the population. The calculation uses a formula and HLA frequencies class II differences between donor derived from HLA types found in more than 12,000 donors. This number is used as one and recipient cells usually through assessment in determining a candidate’s placement on the waitlist for available organs. radioactive measurements of DNA Crossmatching the recipient serum with the T cells and B cells from the potential donor synthesis. This test was historically is another important procedure to avoid rejection caused by antibodies to the donor used for compatibility and D (class tissue. Crossmatching is possible for kidney and pancreas transplants, when there is suf- II) antigen typing. ficient time; however, it is not always possible with heart, lung, and heart/lung transplants. The mixed lymphocyte culture (MLC), historically used for crossmatching, has been replaced with immunofluorescent flow cytometric techniques.8 HEMATOPOIETIC PROGENITOR CELL TRANSPLANTS Hematopoietic progenitor cells (HPCs) can be obtained from bone marrow, peripheral blood, and cord blood. Diseases treated with HPCs include but are not limited to
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 23 congenital immune deficiencies, aplastic anemia, leukemia, lymphoma, and Hodgkin’s disease. Close allele-level HLA matching between the donor and the recipient of HPC transplants is important to avoid transplant rejection and graft-versus-host disease (GVHD). Preformed HLA antibodies and ABO compatibility are less important in HPC transplants compared with solid-organ transplants. Graft-versus-Host Disease In HPC transplantation, the risk of GVHD increases if HLA matching is not close. Allele- level matching is important for optimal graft survival. GVHD occurs when grafted immunocompetent cells from a donor mount an immune response against the host tissue. Because recipients of HPC transplants are often immunosuppressed to allow for donor cell engraftment, this process is more likely to occur. Clinical symptoms include rash, diarrhea, and jaundice, and GVHD can be fatal if left untreated. GVHD can also occur in immunocompromised patients receiving blood components from related donors. Irradiation of blood components from first-degree relatives is performed to reduce the risk of transfused leukocytes proliferating in the host. Irradia- tion prevents the viable leukocytes within the blood component from replicating. Leu- kocyte reduction of the blood components is insufficient to avoid GVHD because even small amounts of leukocytes can proliferate in a susceptible patient. However, in the case of a bone marrow recipient, irradiation of the progenitor cells is contraindicated because viable stem cells that are able to proliferate are essential. Blood products such as platelets and red cells that are often provided to patients after a bone marrow transplant should be irradiated. Chapter 15 provides a more detailed discussion of transplantation. PLATELET ANTIGENS Neonatal alloimmune thrombocytopenia: antibody Platelets possess inherited membrane proteins that can also elicit an immune response. destruction of a newborn’s Platelet antibodies are less frequently found because there is less antigen variability in the platelets caused by antibodies population. Antibodies to platelet antigens are the major cause of neonatal alloimmune formed from prior pregnancies thrombocytopenia (NAIT), in which maternal alloantibodies against antigen inherited and directed to paternal antigens. from the father can cause fetal platelet destruction. Posttransfusion purpura (PTP), in which the platelets of a transfusion recipient are destroyed after transfusion, is caused by Alloantibodies: antibodies with platelet antibodies. Platelet antibodies can also decrease the expected increment of plate- specificities other than self; lets after a platelet transfusion. stimulated by transfusion or pregnancy. The most common platelet antibody is directed against HPA-1a antigen, also known as PlA1. HPA-1a is present on the platelets of about 98% of the population.2 In cases of Posttransfusion purpura: NAIT, in which the mother’s antibody is directed against the infant’s platelet antigen, antibody destruction of platelets therapy involves either washed maternal platelets or antigen-negative platelets from a after transfusion. donor who is HPA-1a-negative. Platelet recipients who have become refractory, in addi- tion to HLA matching, can be crossmatched with a test method designed to detect platelet antigen incompatibility. CHAPTER SUMMARY Characteristics of Antigens • Antigens are foreign molecules that combine with an antibody or immunoglobulin. The part of the antigen that combines with the antibody is the epitope or antigenic determinant. • Red cells, white cells, and platelets have numerous antigens that can elicit an immune response after exposure from transfusions, pregnancy, and transplantation. Characteristics of Antibodies • The structural components of immunoglobulins consist of heavy chains and light chains, constant regions and variable regions, disulfide bonds, and a hinge region.
24 PART I n Foundations: Basic Sciences and Reagents • The five types of immunoglobulins—IgM, IgG, IgA, IgD, and IgE—differ in their heavy chain, function, and role in the immune response. • IgM molecules are pentameric with 10 antigen-binding sites and are most efficient in the activation of complement proteins. IgM is produced first. IgM antibodies are usually detectable by direct or immediate-spin agglutination in tube testing. • IgG molecules possess two antigen-binding sites and constitute the greatest percent- age of the total immunoglobulin concentration in serum. The antiglobulin test is required to detect IgG. Antigen-Antibody Interactions • An immune complex is an antibody bound to an antigen. Immune complexes are formed in vivo and are eventually cleared by the body. • In laboratory testing, immune complexes are formed in vitro to determine the iden- tity of an antigen or antibody or to predict the reaction after transfusion of a blood product. Typically, the antigen is on the red cell, and the antibody is in the patient’s serum or plasma or in the reagent. • Hemagglutination is a two-step process involving the antibody binding to red cells and the formation of lattices between sensitized red cells. • The fit or the complementary nature of the antibody for its specific epitope deter- mines the strength and rate of the reaction, also called the affinity of the bond. • The immune complex is held together by attractive forces, including electrostatic forces, hydrogen bonding, hydrophobic bonding, and van der Waals forces. The HLA System and Platelet Immunology • Genes encoding the expression of the HLAs are part of the MHC found on chromo- some 6 and code for class I (A, B, C), class II (DR, DQ, DP), and class III comple- ment proteins. • HLA antigens are inherited as a haplotype and are important in recognition of self and nonself, coordination of cellular and humoral immunity, and the immune response to antigens. • A refractory or poor platelet response to transfused platelets can be caused by anti- bodies directed against HLA or platelet antigens, and HLA-matched or HLA- compatible platelets may be considered. • Hematopoietic progenitor cell transplants rely on careful HLA typing to avoid GVHD and transplant rejection. • GVHD is a serious consequence of transfusion of blood products from donors that have been HLA matched or are related to the recipient and can be avoided by irra- diation of the blood product. • HLA antibody detection and compatibility testing for potential organ recipients are important techniques to avoid graft rejection. CRITICAL THINKING EXERCISES EXERCISE 1-1 Draw an IgG molecule and identify the following parts: a. Antigen-binding site b. Complement-binding region c. Macrophage-binding site d. Variable region e. Hinge region EXERCISE 1-2 Two different patients received red cell units during surgery. One patient developed an IgG antibody after transfusion; the other patient did not develop an IgG antibody. What factors contribute to an immune response to transfusion?
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 25 EXERCISE 1-3 A specimen was received in the blood bank for antibody identification. Would the patient’s serum or red cells be used for this test? EXERCISE 1-4 A technologist failed to notice that the centrifuge had not properly centrifuged the test tube set up for antibody identification. What would be the potential error in the inter- pretation of this test? EXERCISE 1-5 A technologist added extra drops of red cells to the test tube she was using for antibody identification because she was concerned that she had gotten a weak reaction in the initial test. Why is this action not recommended? What could be the potential result of this action? EXERCISE 1-6 A patient who is not responding to platelet transfusions requires an HLA-matched platelet transfusion. The patient’s HLA type is performed and is reported as A2, A11, B8, B35. A donor is called with a compatible type to donate for this patient. Why were the class II antigens not considered for matching? EXERCISE 1-7 A red cell unit from a donor was received at the transfusion service to be reserved for a directed donation for the donor’s sister who was undergoing hip replacement surgery. What must be done before transfusion of this unit to prevent graft-versus-host disease? Why is leukocyte reduction insufficient to prevent this disease? EXERCISE 1-8 Parents of a kidney transplant candidate were tested for a potential compatible kidney because the candidate demonstrated HLA antibodies. The patient’s HLA antigens were typed and found to be A1, A2, B27, B50, DR17, and DR11. Antibodies identified were specific for A3, B18, and DR7 antigens. If the mother’s HLA typing is A1, A3, B35, B27, DR4, and DR11, and the father’s typing is A2, A24, B50, B44, DR11, and DR17, what antigens did the father contribute? List the probable HLA types of the candidate’s three siblings. Select the kidney or kidneys with the best match. EXERCISE 1-9 In the investigation of a patient’s serum showing a weak IgM antibody (1+ agglutination reactions), what variables could you change to make the antigen-antibody reactions stronger in vitro? EXERCISE 1-10 Why do IgM antibodies activate the classical pathway of complement more efficiently than IgG antibodies? EXERCISE 1-11 Two students performed agglutination tests using the same antigen and antibody. One student observed a 1+ reaction after the immediate-spin reading, whereas the other student observed a 2+ reaction. What variables could cause the differences between these results? When would these differences have a significant impact on testing? STUDY QUESTIONS c. macrophages d. plasma cells 1. Antibodies are produced by: a. natural killer cells b. T cells
26 PART I n Foundations: Basic Sciences and Reagents 2. Which of the following cells expresses HLA class II antigens? a. B cells c. erythrocytes b. platelets d. T cells 3. Select the term that describes the unique part of the antigen that is recognized by a corresponding antibody. a. immunogen c. avidity b. epitope d. clone 4. _____ molecules are usually not good immunogenic substances. a. protein c. lipid b. carbohydrate d. glycoprotein 5. The chemical composition of an antibody is: a. protein c. carbohydrate b. lipid d. glycoprotein 6. In a hemagglutination test, the antigen is: a. on the red cell membrane c. in the red cell nucleus b. secreted by the red cell d. in the plasma or serum 7. Hemagglutination can be enhanced by increasing: a. the temperature higher than 37° C c. increasing the antigen concentration b. the incubation time d. pH greater than 7 8. Molecules that bind to an antigen to increase phagocytosis are: a. opsonins c. haptens b. cytokines d. isotypes 9. An epitope is also termed a(n): c. antigenic determinant a. binding site d. immunogen b. allotype 10. Agglutination reactions characterized by many small agglutinates in a background of free cells would be graded in tube testing as: a. 1+ c. 3+ b. 2+ d. 4+ 11. An order for blood products for a recent recipient of a bone marrow graft was received in the transfusion service. Because these patients are especially susceptible to GVHD from a transfusion, which blood product would best prevent GVHD? a. leukocyte reduction of the unit c. irradiation of the blood product b. washing the unit with normal d. providing HLA-matched blood saline products 12. Complement activation can result in: d. generation of vasoactive amines a. cell lysis e. all of the above b. enhanced cell clearance c. neutrophil activation 13. The mixed lymphocyte culture (MLC) is an older technique in the HLA laboratory used to determine: a. HLA-A antigens c. HLA antibody identification b. HLA-C antigens d. HLA-D antigens and compatibility
CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 27 For questions 14 through 25, match the characteristic with the correct immunoglobulin class. Class Characteristic 14. contains 10 antigen-binding sites a. IgA 15. produced early in an immune response b. IgG 16. found in mucosal linings c. IgM 17. able to cross the placenta d. IgE 18. highest plasma/serum concentration 19. pentameter shape 20. activates the complement cascade most efficiently 21. can initiate allergic reactions 22. associated with intravascular cell destruction 23. detected with the antiglobulin test 24. detected in the immediate-spin phase of the agglutination test 25. reacts best at room temperature REFERENCES 1. Stevens CD: Clinical immunology and serology, ed 2, Philadelphia, 2003, FA Davis. 2. Brecher ME: Technical manual, ed 17, Bethesda, MD, 2011, AABB. 3. Reid ME, Lomas-Francis C: The blood group antigen facts book, ed 3, London, 2004, Elsevier. 4. Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders. 5. HLA nomenclature. http://hla.alleles.org. Accessed December 2011. 6. Abbas AK, Lichtman AH, Pillai S: Cellular and molecular immunology, ed 7, Philadelphia, 2011, Saunders. 7. Organ Procurement and transplant network; http://optn.transplant.hrsa.gov/. Accessed December 2011. 8. Rodey GE: HLA beyond tears, ed 2, Durango, CO, 2000, Denovo. 9. Mallal S, Phillips E, Carosi G, et al: HLA-B*5701 screening for hypersensitivity to abacavir, N Engl J Med 358:568, 2008.
2 BLOOD BANKING REAGENTS: Overview and Applications CHAPTER OUTLINE SECTION 5: ANTIGLOBULIN TEST AND REAGENTS Principles of Antiglobulin Test SECTION 1: INTRODUCTION TO ROUTINE TESTING IN IMMUNOHEMATOLOGY Direct Antiglobulin Test Sources of Antigen for Testing Indirect Antiglobulin Test Sources of Antibody for Testing Sources of Error in Antiglobulin Testing Routine Testing Procedures in the Immunohematology Antiglobulin Reagents Laboratory Polyspecific Antihuman Globulin Reagents SECTION 2: INTRODUCTION TO BLOOD BANKING REAGENTS Monospecific Antihuman Globulin Reagents Regulation of Reagent Manufacture IgG-Sensitized Red Cells Reagent Quality Control SECTION 3: COMMERCIAL ANTIBODY REAGENTS SECTION 6: PRINCIPLES OF ANTIBODY POTENTIATORS Polyclonal versus Monoclonal Antibody Products AND LECTINS Polyclonal Antibody Reagents Low-Ionic-Strength Saline (LISS) Monoclonal Antibody Reagents Polyethylene Glycol Monoclonal and Polyclonal Antibody Reagents Enzymes Reagents for ABO Antigen Typing Bovine Serum Albumin Reagents for D Antigen Typing Lectins Low-Protein Reagent Control SECTION 4: REAGENT RED CELLS SECTION 7: OTHER METHODS OF DETECTING ANTIGEN- A1 and B Red Cells for ABO Serum Testing Screening Cells ANTIBODY REACTIONS Antibody Identification Panel Cells Gel Technology Method Microplate Testing Methods Solid-Phase Red Cell Adherence Methods LEARNING OBJECTIVES 13. Discuss the different sources of possible errors in the performance of antiglobulin testing. On completion of this chapter, the reader should be able to: 14. Compare and contrast the composition and appropriate 1. Describe the basic principles of routine testing in the uses of polyspecific and monospecific antiglobulin immunohematology laboratory. reagents. 2. Identify sources of antigen and antibody used in testing. 15. Discuss the role of potentiators in routine testing. 3. List several routine tests performed in the 16. Describe and differentiate the mechanism of action for immunohematology laboratory. the following potentiators: low-ionic-strength saline, 4. Describe the relationship of potency and specificity to bovine serum albumin, polyethylene glycol, and proteolytic enzymes. blood banking reagents. 17. Define and identify common lectins used in blood banking. 5. Compare and contrast polyclonal and monoclonal 18. Compare and contrast the principles of gel technology, microplate techniques, and solid-phase red cell adherence antibodies. techniques. 6. Describe the reagents available for ABO typing. 19. Analyze quality control data for performance criteria 7. Describe the reagents available for D typing. and acceptability. 8. Define the low-protein reagent control and describe its 20. Research product inserts and quality control procedures for product requirements. purpose. 21. Apply critical thinking skills to solve issues associated 9. Describe the different types and purposes of reagent red with reagent performance. cells. 10. Describe the basic principles of antiglobulin testing. 11. Distinguish between direct and indirect antiglobulin tests. 12. Identify the indications for implementing direct and indirect antiglobulin tests. 28
CHAPTER 2 n Blood Banking Reagents: Overview and Applications 29 This chapter demonstrates how the basic principles of antigen-antibody (Ag-Ab) reactions in vitro are applied in the immunohematology laboratory. Basic laboratory procedures and the major reagents used in the testing of patient and donor samples are discussed. A section on gel technology and solid-phase red cell adherence is included to introduce technologies used in the detection of Ag-Ab reactions that are different from traditional tube testing. SECTION 1 For antigen testing, antigens are on the red cell; antibodies INTRODUCTION TO ROUTINE TESTING IN IMMUNOHEMATOLOGY are in the antisera (commercial antibodies). The basic procedures in immunohematology are derived from the principle of placing a source of antigen and a source of antibody into a testing environment to detect an Ag-Ab reaction (Fig. 2-1). The evidence for the formation of an Ag-Ab reaction in-vitro has traditionally been the visualization of agglutinates or the presence of hemolysis within the test tube. Alternative techniques for the detection of Ag-Ab reactions are available based on detection systems using gel and solid-phase adherence technology. These methods all share common denominators: a source of antigen and a source of antibody added to a testing environment. Selection of the appropriate source of either antigen or antibody for inclusion in the procedure depends on the purpose or intent of the test. Is testing directed toward detect- ing the presence or absence of a particular red cell antigen? Or is testing directed toward detecting the presence or absence of a particular red cell antibody? In either situation, no matter what variable is unknown in testing, a known source is used for the other variable. If the unknown variable is the antigen, the unknown antigen source combines with a known source of antibody to enable Ag-Ab reactions. In the procedure to detect the pres- ence of the B antigen on a patient’s red cells, the red cells are combined with a commercial source of antibody, anti-B reagent. If the B antigen is present on the red cells, agglutina- tion is observed in the test tube. If the B antigen is absent on the red cells, no agglutination is observed in the test tube (Fig. 2-2). SOURCES OF ANTIGEN FOR TESTING Sources of antigen for immunohematologic testing include reagent red cells or red cells from patient or donor blood samples. Reagent red cells are commercially prepared cell suspensions. The manufacturer of reagent red cells has previously identified many of the red cell antigens. Therefore these reagent red cells provide known sources of red cell antigens. By using a known antigen, an unknown antibody can be detected or identified based on a positive or negative reaction. When using red cell suspensions from a patient’s sample, the red cell antigens are the unknown factor. The patient’s red cells are tested with a known antibody to determine ϩϭ Antigen Antibody Agglutination Fig. 2-1 Routine testing in the immunohematology laboratory. Sources of antigen from red cells and sources of antibody from serum are added. A positive result is the Ag-Ab reaction shown as agglutination. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.)
30 PART I n Foundations: Basic Sciences and Reagents Testing patient RBCs for B antigen with reagent anti-B ϩϭ Reagent Anti-B Patient RBCs Fig. 2-2 Example of a routine test. Patient red cells are tested for the presence of B antigen with a commercial antibody reagent, anti-B. The presence of agglutination indicates that the patient’s red cells contain B antigens. (Modified from Immunobase, Bio-Rad Laboratories, Inc., Hercules, CA.) TABLE 2-1 Sources of Antigen and Antibody in Agglutination Reactions for Blood Bank Tests TESTING FOR KNOWN SOURCE UNKNOWN SOURCE Antigen Reagent red cells Patient or donor red cells Antibody Commercial antisera (anti-A, anti-B) Patient or donor serum/plasma the antigen identity. When a known antibody is used, the unknown antigens on the patient’s red cells can be detected, based on agglutination reaction. If antigen is present, agglutination is visible. If antigen is absent, no agglutination is visible (see Fig. 2-2). SOURCES OF ANTIBODY FOR TESTING Sources of antibody for immunohematologic testing include commercial antisera or serum or plasma from the patient. Commercial antisera contain known red cell antibodies and are used to identify unknown antigens. Antibodies may also be present in the serum or plasma of blood samples from patients or donors. Similar to patient antigens, the antibodies made by patients are usually unknown. Patient serum or plasma samples are tested for the presence of red cell antibod- ies using a known antigen source for identification or detection (Table 2-1). Antibody identification: ROUTINE TESTING PROCEDURES IN THE IMMUNOHEMATOLOGY LABORATORY procedure that determines the identity of a red cell antibody Several universal procedures in immunohematology apply these principles in the testing detected in the antibody screen by of patient samples before transfusion with blood products containing red cells or in the reacting serum with commercial testing of donor samples. Table 2-2 lists the sources of antigen and antibody in these panel cells. procedures. • ABO and D typing for the detection of the A, B, and D antigens • ABO serum/plasma testing for the detection of ABO antibodies, anti-A and anti-B • Determination of the presence or absence of red cell antigens from other blood group systems in patient and donor samples (e.g., testing a donor unit for the E antigen of the Rh blood group system) • Antibody screen (antibody detection) for the detection of preformed antibodies to red cell antigens as a result of previous exposure to red cells through transfusion and pregnancy • Antibody identification for determination of the red cell antibody specificity after detection with the antibody screen
CHAPTER 2 n Blood Banking Reagents: Overview and Applications 31 TABLE 2-2 Routine Procedures in the Immunohematology Laboratory PROCEDURE PURPOSE SOURCE OF ANTIGEN SOURCE OF ANTIBODY ABO/D typing Patient’s red cells Commercial anti-A, Detects A, B, and D (forward grouping) antigens Reverse grouping anti-B, and anti-D ABO serum testing cells (A1 and B) Patient’s serum or Detects ABO (reverse grouping) antibodies Screening cells plasma Antibody screen Patient’s serum or Detects antibodies Panel cells Antibody with specificity to plasma identification red cell antigens Donor’s red cells Patient’s serum or Crossmatch Identifies specificity plasma of red cell antibodies Patient’s serum or plasma Determines serologic compatibility between donor and patient before transfusion • Crossmatch for the serologic check of the donor unit and patient compatibility before Crossmatch: procedure that transfusion combines donor’s red cells and patient’s serum to determine the SECTION 2 serologic compatibility between INTRODUCTION TO BLOOD BANKING REAGENTS donor and patient. As introduced in the previous section, reagent red cells and commercial antibodies are Reagents categories include: routinely used in the blood bank to detect Ag-Ab reactions. These reagents are the tools • Reagent red cells: known of blood banking, allowing the provision of safe and viable blood products. Technologists using these tools require a technical knowledge of correct reagent use and the limitations red cell antigens of each reagent to interpret the results from patient and donor testing rapidly. A discus- • Antisera: known red cell sion of the composition, sources, uses, and limitations of reagents is presented following an overview of the regulatory aspects of reagent manufacturing. antibodies • Antiglobulin reagents: anti-IgG or anti-C3d or combination of anti-IgG and anti-C3d • Potentiators to enhance antibodies REGULATION OF REAGENT MANUFACTURE Food and Drug Administration (FDA): U.S. agency responsible for Commercial antisera and reagent red cell products are licensed by the Center for Biologics the regulation of the blood Evaluation and Research of the U.S. Food and Drug Administration (FDA). The pub banking industry and other lication, Code of Federal Regulations, outlines the FDA criteria for the licensure of manufacturers of products reagents in conjunction with other regulations for the manufacture of blood and blood consumed by humans. components.1 Code of Federal Regulations: The FDA has established minimum standards relating to product specificity and FDA publication outlining the potency for use in blood banks and transfusion services before a license is assigned to a legal requirements of blood commercial reagent. Specificity reflects the unique recognition of the antigenic determi- banking facilities. nant and its corresponding antibody molecule. For example, commercial anti-D reacts with red cells possessing D antigens and does not react with red cells lacking D antigens. Specificity: unique recognition of Potency addresses the strength of the Ag-Ab reaction. For example, commercial anti-A an antigenic determinant and its reagents are manufactured to agglutinate strongly (3+ to 4+) with red cells possessing the corresponding antibody molecule. A antigen. Potency: strength of an Ag-Ab When a manufacturer has shown that a product has met the FDA specificity and reaction. potency requirements, the reagent is assigned a product license number that is displayed on the product’s label. Each product is also labeled with a manufacturer’s expiration date. According to FDA regulations, routine blood banking reagents cannot be used in testing after the expiration date. Exceptions to this rule can be made for rare antisera and red cells if the reagent has acceptable quality control results. If reagents are produced for tahir99-VRG & vip.persianss.ir
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