Geoff Daniels, Consultant Clinical Scientist and Head of Diagnostics, IBGRL, Bristol Institute for Transfusion Services, NHS Blood and Transplant, Bristol, UKImelda Bromilow, Scientific Consultant, Liverpool, UK

• Abbreviations x1 An introduction to blood groups 1What is a blood group? 1Blood group antibodies 3Clinical importance of blood groups 3Biological importance of blood groups 3Blood group systems 4Blood group terminology and classification 42 Techniques used in blood grouping 8Factors affecting antigen–antibody reactions 8Temperature 8Time and ionic strength 9pH 9Antigen density 9Stages of haemagglutination reactions 10Direct agglutination 11Indirect agglutination 12Enzyme techniques 12Antiglobulin tests 14Elution techniques 18Automation of test procedures 19Flow cytometry 19Molecular blood group genotyping 213 The ABO blood groups 22Introduction 22ABO antigens, antibodies, and inheritance 22A1 and A2 23Antigen, phenotype, and gene frequencies 24ABO antibodies 25Importance of the ABO system to transfusion and transplantation medicine 26Biochemical nature of the ABO antigens 27Biosynthesis of the ABO antigens and ABO molecular genetics 28H, the precursor of A and B 30ABH secretion 31H-deficient red cells 32Further complexities 32Acquired changes 33Associations with disease and functional aspects 344 The Rh blood group system 35Introduction – Rh, not rhesus 35Haplotypes, genotypes, and phenotypes 36Biochemistry and molecular genetics 37D antigen (RH1) 40Molecular basis of the D polymorphism 40D variants 41Clinical significance of anti-D 42D testing 44C, c, E, and e antigens (RH2, RH4, RH3, RH5) 44Clinical significance of CcEe antibodies 45Molecular basis of the C/c and E/e polymorphisms 45Other Rh antigens 45Compound antigens: ce, Ce, CE, cE (RH6, RH7, RH22, RH27), and G (RH12) 46Cw, Cx, and MAR (RH8, RH9, RH51) 46VS and V (RH20, RH10) 46Rh-deficient phenotypes – Rhnull and Rhmod 47Putative function of the Rh proteins and RhAG 475 Other blood groups 49The Kell system 49The Kell glycoprotein and the KEL gene 49Kell system antigens 50Kell system antibodies 51Ko phenotype 51McLeod syndrome, McLeod phenotype, and Kx (XK1) antigen 52The Duffy system 52Fya (FY1) and Fyb (FY2) 52Anti-Fya and -Fyb 53Fy3 and Fy5 53The Duffy-glycoprotein, a receptor for chemokines 53Duffy and malaria 54The Kidd system 54Jka (JK1) and Jkb (JK2); anti-Jka and -Jkb 54Jk(a−b−) and Jk3 55The Kidd-glycoprotein is a urea transporter 55The MNS system 56M (MNS1) and N (MNS2); anti-M and -N 56S (MNS3) and s (MNS4); anti-S and -s 56S− s− U− phenotype and anti-U 57Other MNS antigens and antibodies 57The Diego system 57Band 3, the red cell anion exchanger 57Dia (DI1) and Dib (DI2); anti-Dia and -Dib 58 Wra (DI3) and Wrb (DI4); anti-Wra and -Wrb 58Other Diego-system antigens 59The Lewis System 59Some other blood group systems 61P1PK 61Lutheran 61Yt 61Xg 61Scianna 61Dombrock 62Colton 62Landsteiner–Wiener (LW) 62Chido/Rodgers 62Gerbich 62Cromer 63Knops 63Indian 63I 63JR and Lan 64Vel 64Antigens that do not belong to a blood group system 646 Clinical significance of blood group antibodies 65Antibody production and structure 66Factors affecting the clinical significance of antibodies 69Antibody specificity 69Haemolytic transfusion reactions (HTR) 71Intravascular red cell destruction 72Extravascular red cell destruction 72Haemolytic disease of the fetus and newborn (HDFN) 73Crossmatching for infants under 4 months old 75Autoantibodies 77Tests to assess the potential significance of an antibody 77Decision-making for transfusion 787 Blood grouping from DNA 81Fetal blood grouping 81Blood group typing of patients and donors 82Next generation sequencing 84The future of blood group serology 848 Quality assurance in immunohaematology 85Achieving total quality 85Frequency and specificity of control material 86Quality requirements for safe transfusion practice 88Checklist of critical control points 89Laboratory errors, root cause analysis (RCA), and corrective and preventive action (CAPA) 899 Trouble-shooting and problem-solving in the reference laboratory 92ABO grouping 92Rh grouping 94Problems in antibody screening, identification, and crossmatching 9510 Frequently asked questions 102What is the difference between sensitivity and specificity and how can these be determined? 102Why is anti-A,B no longer obligatory in ABO typing? 102Why are two anti-D reagents often recommended for RhD typing? 103What is the importance of detecting D variant (weak D and partial D) phenotypes? 103How do I control the results for antiglobulin testing? 103Why should RhD positive women be tested more than once during pregnancy? 104How often should transfusion recipients be tested for the presence of antibodies? 104How can passive anti-D be differentiated from anti-D due to alloimmunisation? 104Why do we need to perform antibody screening? Isn’t a crossmatch by IAT at 37°C enough to detectincompatible blood? 105 What is the incidence of alloimmunisation post-transfusion? 105 How do I determine and identify antibodies present in a sample? 105 What is a compound antibody? 105 How can the incidence of compatible donors for a recipient with multiple antibodies be calculated? 106 Why can’t the droppers in bottles of reagents be used instead of a volumetric pipette? 106 What cells should be used when performing an antibody titration? 107 How are the results of titrations reported? 107 What is a Major Obstetric Haemorrhage? 107 What is ‘Massive Transfusion’? 107 When group-specific blood is in short supply, how do I select the ‘next best’ for transfusion? 108 How are high-titre haemagglutinins classified? 108 What is an ‘immediate spin’ crossmatch? 108 What is an ‘electronic crossmatch’? 108 Which patients are not eligible for electronic issue of blood? 108 What is ‘bed-side’ testing? 109 What are signs and symptoms of a suspected transfusion reaction? 109 What action should be taken in the event of a suspected transfusion reaction? 109 In haemovigilance, how should ‘near-miss’ events be characterised? 109 Recommended reading and web sites 111 Index 113

Geoff Daniels, Consultant Clinical Scientist and Head of Diagnostics, IBGRL, Bristol Institute for Transfusion Services, NHS Blood and Transplant, Bristol, UKImelda Bromilow, Scientific Consultant, Liverpool, UK

• Abbreviations x1 An introduction to blood groups 1What is a blood group? 1Blood group antibodies 3Clinical importance of blood groups 3Biological importance of blood groups 3Blood group systems 4Blood group terminology and classification 42 Techniques used in blood grouping 8Factors affecting antigen–antibody reactions 8Temperature 8Time and ionic strength 9pH 9Antigen density 9Stages of haemagglutination reactions 10Direct agglutination 11Indirect agglutination 12Enzyme techniques 12Antiglobulin tests 14Elution techniques 18Automation of test procedures 19Flow cytometry 19Molecular blood group genotyping 213 The ABO blood groups 22Introduction 22ABO antigens, antibodies, and inheritance 22A1 and A2 23Antigen, phenotype, and gene frequencies 24ABO antibodies 25Importance of the ABO system to transfusion and transplantation medicine 26Biochemical nature of the ABO antigens 27Biosynthesis of the ABO antigens and ABO molecular genetics 28H, the precursor of A and B 30ABH secretion 31H-deficient red cells 32Further complexities 32Acquired changes 33Associations with disease and functional aspects 344 The Rh blood group system 35Introduction – Rh, not rhesus 35Haplotypes, genotypes, and phenotypes 36Biochemistry and molecular genetics 37D antigen (RH1) 40Molecular basis of the D polymorphism 40D variants 41Clinical significance of anti-D 42D testing 44C, c, E, and e antigens (RH2, RH4, RH3, RH5) 44Clinical significance of CcEe antibodies 45Molecular basis of the C/c and E/e polymorphisms 45Other Rh antigens 45Compound antigens: ce, Ce, CE, cE (RH6, RH7, RH22, RH27), and G (RH12) 46Cw, Cx, and MAR (RH8, RH9, RH51) 46VS and V (RH20, RH10) 46Rh-deficient phenotypes – Rhnull and Rhmod 47Putative function of the Rh proteins and RhAG 475 Other blood groups 49The Kell system 49The Kell glycoprotein and the KEL gene 49Kell system antigens 50Kell system antibodies 51Ko phenotype 51McLeod syndrome, McLeod phenotype, and Kx (XK1) antigen 52The Duffy system 52Fya (FY1) and Fyb (FY2) 52Anti-Fya and -Fyb 53Fy3 and Fy5 53The Duffy-glycoprotein, a receptor for chemokines 53Duffy and malaria 54The Kidd system 54Jka (JK1) and Jkb (JK2); anti-Jka and -Jkb 54Jk(a−b−) and Jk3 55The Kidd-glycoprotein is a urea transporter 55The MNS system 56M (MNS1) and N (MNS2); anti-M and -N 56S (MNS3) and s (MNS4); anti-S and -s 56S− s− U− phenotype and anti-U 57Other MNS antigens and antibodies 57The Diego system 57Band 3, the red cell anion exchanger 57Dia (DI1) and Dib (DI2); anti-Dia and -Dib 58 Wra (DI3) and Wrb (DI4); anti-Wra and -Wrb 58Other Diego-system antigens 59The Lewis System 59Some other blood group systems 61P1PK 61Lutheran 61Yt 61Xg 61Scianna 61Dombrock 62Colton 62Landsteiner–Wiener (LW) 62Chido/Rodgers 62Gerbich 62Cromer 63Knops 63Indian 63I 63JR and Lan 64Vel 64Antigens that do not belong to a blood group system 646 Clinical significance of blood group antibodies 65Antibody production and structure 66Factors affecting the clinical significance of antibodies 69Antibody specificity 69Haemolytic transfusion reactions (HTR) 71Intravascular red cell destruction 72Extravascular red cell destruction 72Haemolytic disease of the fetus and newborn (HDFN) 73Crossmatching for infants under 4 months old 75Autoantibodies 77Tests to assess the potential significance of an antibody 77Decision-making for transfusion 787 Blood grouping from DNA 81Fetal blood grouping 81Blood group typing of patients and donors 82Next generation sequencing 84The future of blood group serology 848 Quality assurance in immunohaematology 85Achieving total quality 85Frequency and specificity of control material 86Quality requirements for safe transfusion practice 88Checklist of critical control points 89Laboratory errors, root cause analysis (RCA), and corrective and preventive action (CAPA) 899 Trouble-shooting and problem-solving in the reference laboratory 92ABO grouping 92Rh grouping 94Problems in antibody screening, identification, and crossmatching 9510 Frequently asked questions 102What is the difference between sensitivity and specificity and how can these be determined? 102Why is anti-A,B no longer obligatory in ABO typing? 102Why are two anti-D reagents often recommended for RhD typing? 103What is the importance of detecting D variant (weak D and partial D) phenotypes? 103How do I control the results for antiglobulin testing? 103Why should RhD positive women be tested more than once during pregnancy? 104How often should transfusion recipients be tested for the presence of antibodies? 104How can passive anti-D be differentiated from anti-D due to alloimmunisation? 104Why do we need to perform antibody screening? Isn’t a crossmatch by IAT at 37°C enough to detectincompatible blood? 105 What is the incidence of alloimmunisation post-transfusion? 105 How do I determine and identify antibodies present in a sample? 105 What is a compound antibody? 105 How can the incidence of compatible donors for a recipient with multiple antibodies be calculated? 106 Why can’t the droppers in bottles of reagents be used instead of a volumetric pipette? 106 What cells should be used when performing an antibody titration? 107 How are the results of titrations reported? 107 What is a Major Obstetric Haemorrhage? 107 What is ‘Massive Transfusion’? 107 When group-specific blood is in short supply, how do I select the ‘next best’ for transfusion? 108 How are high-titre haemagglutinins classified? 108 What is an ‘immediate spin’ crossmatch? 108 What is an ‘electronic crossmatch’? 108 Which patients are not eligible for electronic issue of blood? 108 What is ‘bed-side’ testing? 109 What are signs and symptoms of a suspected transfusion reaction? 109 What action should be taken in the event of a suspected transfusion reaction? 109 In haemovigilance, how should ‘near-miss’ events be characterised? 109 Recommended reading and web sites 111 Index 113

Geoff Daniels, Consultant Clinical Scientist and Head of Diagnostics, IBGRL, Bristol Institute for Transfusion Services, NHS Blood and Transplant, Bristol, UKImelda Bromilow, Scientific Consultant, Liverpool, UK

• Abbreviations x1 An introduction to blood groups 1What is a blood group? 1Blood group antibodies 3Clinical importance of blood groups 3Biological importance of blood groups 3Blood group systems 4Blood group terminology and classification 42 Techniques used in blood grouping 8Factors affecting antigen–antibody reactions 8Temperature 8Time and ionic strength 9pH 9Antigen density 9Stages of haemagglutination reactions 10Direct agglutination 11Indirect agglutination 12Enzyme techniques 12Antiglobulin tests 14Elution techniques 18Automation of test procedures 19Flow cytometry 19Molecular blood group genotyping 213 The ABO blood groups 22Introduction 22ABO antigens, antibodies, and inheritance 22A1 and A2 23Antigen, phenotype, and gene frequencies 24ABO antibodies 25Importance of the ABO system to transfusion and transplantation medicine 26Biochemical nature of the ABO antigens 27Biosynthesis of the ABO antigens and ABO molecular genetics 28H, the precursor of A and B 30ABH secretion 31H-deficient red cells 32Further complexities 32Acquired changes 33Associations with disease and functional aspects 344 The Rh blood group system 35Introduction – Rh, not rhesus 35Haplotypes, genotypes, and phenotypes 36Biochemistry and molecular genetics 37D antigen (RH1) 40Molecular basis of the D polymorphism 40D variants 41Clinical significance of anti-D 42D testing 44C, c, E, and e antigens (RH2, RH4, RH3, RH5) 44Clinical significance of CcEe antibodies 45Molecular basis of the C/c and E/e polymorphisms 45Other Rh antigens 45Compound antigens: ce, Ce, CE, cE (RH6, RH7, RH22, RH27), and G (RH12) 46Cw, Cx, and MAR (RH8, RH9, RH51) 46VS and V (RH20, RH10) 46Rh-deficient phenotypes – Rhnull and Rhmod 47Putative function of the Rh proteins and RhAG 475 Other blood groups 49The Kell system 49The Kell glycoprotein and the KEL gene 49Kell system antigens 50Kell system antibodies 51Ko phenotype 51McLeod syndrome, McLeod phenotype, and Kx (XK1) antigen 52The Duffy system 52Fya (FY1) and Fyb (FY2) 52Anti-Fya and -Fyb 53Fy3 and Fy5 53The Duffy-glycoprotein, a receptor for chemokines 53Duffy and malaria 54The Kidd system 54Jka (JK1) and Jkb (JK2); anti-Jka and -Jkb 54Jk(a−b−) and Jk3 55The Kidd-glycoprotein is a urea transporter 55The MNS system 56M (MNS1) and N (MNS2); anti-M and -N 56S (MNS3) and s (MNS4); anti-S and -s 56S− s− U− phenotype and anti-U 57Other MNS antigens and antibodies 57The Diego system 57Band 3, the red cell anion exchanger 57Dia (DI1) and Dib (DI2); anti-Dia and -Dib 58 Wra (DI3) and Wrb (DI4); anti-Wra and -Wrb 58Other Diego-system antigens 59The Lewis System 59Some other blood group systems 61P1PK 61Lutheran 61Yt 61Xg 61Scianna 61Dombrock 62Colton 62Landsteiner–Wiener (LW) 62Chido/Rodgers 62Gerbich 62Cromer 63Knops 63Indian 63I 63JR and Lan 64Vel 64Antigens that do not belong to a blood group system 646 Clinical significance of blood group antibodies 65Antibody production and structure 66Factors affecting the clinical significance of antibodies 69Antibody specificity 69Haemolytic transfusion reactions (HTR) 71Intravascular red cell destruction 72Extravascular red cell destruction 72Haemolytic disease of the fetus and newborn (HDFN) 73Crossmatching for infants under 4 months old 75Autoantibodies 77Tests to assess the potential significance of an antibody 77Decision-making for transfusion 787 Blood grouping from DNA 81Fetal blood grouping 81Blood group typing of patients and donors 82Next generation sequencing 84The future of blood group serology 848 Quality assurance in immunohaematology 85Achieving total quality 85Frequency and specificity of control material 86Quality requirements for safe transfusion practice 88Checklist of critical control points 89Laboratory errors, root cause analysis (RCA), and corrective and preventive action (CAPA) 899 Trouble-shooting and problem-solving in the reference laboratory 92ABO grouping 92Rh grouping 94Problems in antibody screening, identification, and crossmatching 9510 Frequently asked questions 102What is the difference between sensitivity and specificity and how can these be determined? 102Why is anti-A,B no longer obligatory in ABO typing? 102Why are two anti-D reagents often recommended for RhD typing? 103What is the importance of detecting D variant (weak D and partial D) phenotypes? 103How do I control the results for antiglobulin testing? 103Why should RhD positive women be tested more than once during pregnancy? 104How often should transfusion recipients be tested for the presence of antibodies? 104How can passive anti-D be differentiated from anti-D due to alloimmunisation? 104Why do we need to perform antibody screening? Isn’t a crossmatch by IAT at 37°C enough to detectincompatible blood? 105 What is the incidence of alloimmunisation post-transfusion? 105 How do I determine and identify antibodies present in a sample? 105 What is a compound antibody? 105 How can the incidence of compatible donors for a recipient with multiple antibodies be calculated? 106 Why can’t the droppers in bottles of reagents be used instead of a volumetric pipette? 106 What cells should be used when performing an antibody titration? 107 How are the results of titrations reported? 107 What is a Major Obstetric Haemorrhage? 107 What is ‘Massive Transfusion’? 107 When group-specific blood is in short supply, how do I select the ‘next best’ for transfusion? 108 How are high-titre haemagglutinins classified? 108 What is an ‘immediate spin’ crossmatch? 108 What is an ‘electronic crossmatch’? 108 Which patients are not eligible for electronic issue of blood? 108 What is ‘bed-side’ testing? 109 What are signs and symptoms of a suspected transfusion reaction? 109 What action should be taken in the event of a suspected transfusion reaction? 109 In haemovigilance, how should ‘near-miss’ events be characterised? 109 Recommended reading and web sites 111 Index 113