Introduction to Immunohematology. W. John Judd, FIBMS, MIBiol Emeritus Professor University of Michigan

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1 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol Emeritus Professor University of Michigan

2 Objectives After reviewing this presentation you will be able to: Outline methods for detecting IgM and IgG antibodies Discuss the principle of gel technology Discuss the potential applications of blood group genotyping 2 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

3 Immunohematology Pretransfusion testing ABO and Rh typing Antibody detection Crossmatching Investigation of immune hemolysis Perinatal testing Blood group phenotyping/genotyping Leukocyte and platelet serology 3 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

4 Antigens and Antibodies BLOOD GROUP ANTIGENS: Present (predominantly) on red blood cells BLOOD GROUP ANTIBODIES Present in plasma (or serum) 4 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

5 Antigen An antigen is a substance (molecule) that, when introduced into a human (or animal) who lacks that substance, triggers the production of antibody by the body s immune system. The antibody thus produced will react specifically with the antigen in an observable way. 5 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

6 Blood Group Antigens 324 blood group antigens recognized 33 blood group systems 40 unassigned antigens Molecular biology of assigned antigens is known http5:// _and/table_of_blood_group_antigens_within_systems_v2.0_ pdf 6 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

7 Antigen Function CARBOHYDRATES ABO H P1PK I LE FORS GLOB LW XG FY LU OK ADHESION MOLECULES IN SC MER2 JMH TRANSPORTERS AND CHANNELS RH CO RHAG GIL JK XR DI COMPLEMENT REGULATION ENZYMES STRUCTURAL OR UNKNOWN CH/RG CROM KN H I FORS KEL YT MNS GE DO 7 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

8 System Notations Common Name ISBT Name Common Name ISBT Name Rh RH P P1PK Kell KEL Colton CO Duffy FY Dombrock DO Kidd JK Cartwright YT Lewis LE MNS MNS Diego DI Lutheran LU 8 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

9 Antigens, Genes and Phenotypes System Antigens Genes Phenotypes ABO A, B A, B, O A, B, O, AB KEL K, k K, k FY Fy a, Fy b Fy a, Fy b K-k+; K+k+; K+k- or K-1,2; K1,2; K-1,2 Fy(a+b+); Fy(a+b-); Fy(a+b-) P P 1 P 1, P 2 P 1, P 2 9 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

10 Blood Group Antigens Key Points Are present on RBCs as glycolipids, proteins or glycoproteins Are inherited characteristics Have biological function Most are assigned to one of 31 blood group systems 10 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

11 Antibodies An antibody, also known as an immunoglobulin, is a protein produced by the immune system following exposure to foreign antigen. Antibodies, usually found in plasma, react with cells carrying the foreign antigen in a very specific manner. 11 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

12 IgG Antibody 12 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

13 IgG vs. IgM Molecules 13 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

14 IgG vs. IgM Antibodies Protein IgG IgM Characteristic Immune Natural Stimulus Protein Carbohydrate Blood Group weight 150 kda 900 kda Complement binding Rarely Yes Antigen binding sites 2 10 Placental Transfer Yes No Direct agglutinin Rarely Yes Example Anti-Rh Anti-A, -B 14 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

15 Blood Group Antibodies EXPECTED Natural anti-a Natural anti-b UNEXPECTED Alloimmune Autoimmune Passive 15 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

16 Blood Group Antibodies Key Points Are stimulated by exposure to foreign antigens in the environment, or by transfusion or pregnancy Are usually IgM and/or IgG immunoglobulins Anti-A and anti-b are expected antibodies (based on RBC ABO type) All non-abo antibodies are unexpected 16 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

17 Antigen-Antibody Reactions Two Types of Tests Direct agglutination test for IgM antibodies Indirect antiglobulin test (IAT) for IgG antibodies 17 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

18 Direct Tests IgM Antibodies ABO Typing Mix antibody and RBCs Incubate (optional) Centrifuge (1000 x g, 15 seconds) Examine Record results 18 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

19 IgM Agglutination 19 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

20 Agglutination 20 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

21 Indirect Antiglobulin Tests For Detection of Immune (IgG) Antibodies Mix plasma (antibody) and RBCs Incubate at 37 o C (10-60 ) Centrifuge, examine, record (optional) Wash x 3 or 4 to remove unbound IgG Add antihuman globulin Centrifuge, examine, record Validate negative tests with IgG-coated RBCs 21 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

22 Detection of IgG Coating Antihuman Globulin 22 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

23 Antigen-Antibody Reactions Key Points Two types of tests are used to demonstrate blood group antigen-antibody reactions IgM antibodies are used (or detected) by direct agglutination tests IgG antibodies are used (or detected) by indirect antiglobulin tests The indirect antiglobulin test (IAT) utilizes antihuman globulin (AHG) reagent, otherwise known as Coombs serum 23 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

24 Routine Serologic Tests ABO and Rh typing (Type) Detection of unexpected antibodies (Screen) Compatibility testing (Crossmatch) Phenotyping beyond A, B and D Direct antiglobulin tests (DAT) 24 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

25 Blood Bank Reagents ABO Monoclonal anti-a and anti-b Anti-A colored blue, anti-b colored yellow to confirm correct reagent has been added Pooled Rh-negative A 1 and B RBCs suspended in a preservative solution 25 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

26 ABO Typing Traditional Tube Tests RBCs 2 tubes 1 drop anti-a 1 drop anti-b 1 drop 3-5% RBCs to each Mix, centrifuge and examine Plasma 2 tubes 2-3 drops plasma to each 1 drop 3-5% A 1 RBCs 1 drop 3-5% B RBCs Mix, centrifuge and examine 26 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

27 ABO Typing Expected Reactions RBCs + Plasma + Type Anti-A Anti-B A 1 RBCs B RBCs O A B AB Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

28 Anti-D for Rh Typing TUBE TESTS: Monoclonal IgM blended with either monoclonal IgG or human (polyclonal) IgG in a low (6% wt/vol) protein diluent GEL MICROCOLUMNS: IgM monoclonal anti-d 28 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

29 Rh Typing DIRECT TEST 1 drop anti-d 1 drop 3-5% RBCs Mix, spin and read TEST FOR WEAK D Incubate negative direct tests at 37 o C Spin and read Wash x 4 Add antihuman IgG Spin and read Confirm negative tests with IgG-coated RBCs 29 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

30 ABO and Rh Typing Gel 10 L prbcs 100 L plasma + 50 L 0.8% RBCs RBC type A Anti-A Anti-B Anti-D Control A1 RBCs B RBCs Plasma type 30 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

31 Distribution of ABO and Rh O A B AB Rh+ Rh- % US blacks % US whites % British % Asians Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

32 ABO and Rh Typing Key Points Can be done by tube, gel and solid-phase assays Two types of tests for RhD: a direct test, and an IAT to detect weak expression of D Apparent D-negative donors (by direct tests) must be tested for weak D Apparent D-negative patients need not be tested for weak of D Different ethnic groups have disparate blood group phenotype frequencies 32 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

33 Antibody Detection (Screening) AABB Requirements Clinically significant* 37 o C incubation... AHG No pooled RBCs Validate alternative methods IgG-coated RBCs * Capable of causing a shortened RBC survival 33 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

34 Methods for Antibody Detection Plasma:RBCs Time at 37 C AHG SAL >2:1, 3-4% PS/IgG ALB >2:1, 3-4% PS/IgG LISS 2:2, 2% PS/IgG GEL 1:2, 0.8% 15 IgG PEG 2:1, 3-4% IgG LIP 2:1, 1% 1 IgG SPA 1:1, 0.4% 15 IgG 34 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

35 Reagent RBCs For Antibody Detection 2-4 group O RBC samples that, between them, carry C c D E e; K k; Fy a Fy b ; Jk a Jk b ; M N S s; Le a Le b and P 1 antigens DCe/DCe, DcE/DcE, and ce/ce Available commercially in preservative solution; new shipment every 2-4 weeks 35 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

36 Two RBC Sample Set For Detecting Unexpected Antibodies SCREEN D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b I R1R I II R2R II 36 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

37 Factors Affecting the Sensitivity of Ag-Ab Reactions Plasma : RBC ratio Time Temperature ph Antibody valency Antibody concentration Ionic strength RBC surface charge Antigen-site density 37 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

38 Ionic Cloud Steric hindrance of antibody by ionic cloud formed when RBCs are suspended in saline Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

39 LISS Tube Tests 2 drops serum 2 drops LISS 1 drop 3-5% RBCs Wash RBCs x 4 Add anti-igg 10=15 at 37 o C Spin, read, grade Spin, read, grade Confirm negative tests with IgG-coated RBCs 39 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

40 Solid-Phase Assays COAT WITH ANTIGEN ADD ANTIBODY + AHG COATED RED BLOOD CELLS Y Y Y Y NEGATIVE REACTION POSITIVE REACTION 40 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

41 Gel Test 0.8% RBCs in LISS (50 L) + Sample (25 L) Gel with Anti IgG Incubated at 37 o C for 15 min Spin for 10 min Anti-IgG Gel Test Reaction 41 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

42 Antibody Detection Key Points Popular methods for detecting unexpected antibodies include LISS, gel and solid-phase adherence assays Automated platforms exist for both gel and solid-phase methods 42 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

43 Antibody Identification Determining the blood group specificity of antibodies causing a positive antibody screen Done by testing the plasma against a panel of reagent RBCs of known phenotypes 43 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

44 Case Study 63-year-old man Transfused 3 units of prbcs after automobile accident 3-years ago Scheduled for coronary bypass graft tomorrow 2 units of prbcs requested 44 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

45 Case Study Initial Studies RBCs + Plasma + Anti-A Anti-B Anti-D Control A 1 RBCs B RBCs SCREEN D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL I R1R I 0 II R2R II Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

46 Identification Panel PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT AC 46 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

47 Findings PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT AC 0 47 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

48 Crossing Out Cell #1 PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT AC 0 48 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

49 Crossing Out Cell #2 PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT AC 0 49 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

50 Crossing Out Cell #3 PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT AC 0 50 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

51 Reaction Pattern PANEL D C c E e M N S s P 1 Le a Le b K k Jk a Jk b Fy a Fy b GEL 1 r r R1R R1R R2R r r rrv rr rr rr rr Ror PATIENT 0 AC 0 51 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

52 Pretransfusion Testing Goals To provide blood and blood products for transfusion in a timely, cost-efficient manner, such that the transfused product provides optimal clinical benefit to the recipient, but does not cause adverse clinical effects or transmit disease 52 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

53 Pretransfusion Testing Donor Patient Donor/ Patient History Requisition Selection ABO/Rh Identification Crossmatch Antibodies Sample Dispense Disease ABO/Rh Bedside ABO/Rh Antibodies Records 53 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

54 Donor Testing Blood Supplier ABO Rh Antibodies Infectious Disease RBCs with anti-a and anti-b Plasma with A 1 and B RBCs Direct tests with anti-d Confirm nonreactive units by a method that detects weak expression of D 37 C incubation, IAT, may use pooled RBCs HBV, HCV, HIV, HTLV, WNV, Chagas, Syphilis, Bacteria 54 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

55 Transfusion Service Testing Donor Unit Confirmatory Tests ABO Rh Antibodies Infectious Disease Confirm all units (RBC type only) Confirm Rh-negative units (direct tests with anti-d) Not required Not required 55 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

56 Required Testing Patient Samples ABO Rh Antibodies RBCs with anti-a and anti-b Serum with A1 and B RBCs Direct tests with anti-d Control system to recognize falsepositive tests 37 o C incubation Antiglobulin Clinically-significant No pooled RBCs 56 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

57 A Pint (Unit) of Blood 57 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

58 Blood Components Derived from Whole Blood Packed Red Blood Cells (prbcs) Plasma Fresh frozen FFP Cryoprecipitate Platelets Granulocytes 58 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

59 The Crossmatch The final test that must be done before blood is assigned to a patient Done to detect serological incompatibility between donor RBCs and patient s plasma 59 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

60 Selection of Components 60 Patient RBCs* Plasma ț O O Any A A, O A, AB B B, O B, AB AB Any AB * Rh-negative RBCs for premenopausal females ț Including Platelets and Cryoprecipitate 60 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

61 Crossmatch Methods 61 Unexpected Antibodies Absent Immediate-Spin or Electronic Present/History Indirect Antiglobulin Test 61 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

62 Other Important Tests Phenotyping, by direct agglutination or by the IAT for antigens beyond A, B and D Direct antiglobulin test (DAT) on a patient s RBCs (for investigation of immune hemolysis) 62 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

63 Blood Group Genotyping Potential Research Applications Testing for rare donors Patients requiring chronic transfusions Patients with >1 antibody Patients with a positive DAT Recently transfused patients Antisera rare and unavailable Fetal RHD genotyping from maternal plasma Zygosity studies All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 63 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

64 Blood Group Genotyping Potential Research Applications Research Studies to Identify: Frequency of rare donors in geographic locations Frequency of multiple blood group systems in selected donor populations Process improvement for advancing immunohematology Research to develop fundamental scientific knowledge related to human disease conditions All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 64 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

65 Blood Group Genotyping Potential Research Applications Patients Requiring Chronic Transfusions: Sickle-cell anemia patients African Americans Thalassemic patients Of Mediterranean origin, Arabs, Asians Match initial transfusions for Rh and K Complete match after first antibody made Patients with >1 Antibody: To predict additional antibodies that the patient may form 65 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures

66 Blood Group Genotyping Potential Research Applications Patients with a positive DAT: Serologic phenotyping cannot be done readily by the IAT Bound IgG can be removed by incubation with: Chloroquine diphosphate (time consuming; 80% effective) EDTA+glycine+HCl (denatures KEL antigens) All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 66 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

67 Blood Group Genotyping Potential Research Applications Recently Transfused Patients: Serologic phenotyping confounded by presence of transfused RBCs Cell separation methods available High-speed microhematocrit centrifugation (tedious) Hypotonic saline (for sickle-cell disease patients) All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 67 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

68 Blood Group Genotyping Potential Research Applications Antisera Rare and Unavailable: Anti-k very rare Anti-Do a and anti-do b are rare, impure, and require use of enzyme-antiglobulin tests Predicting the extended phenotype of reagent RBCs (DO, CO, YT, etc.) All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 68 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

69 Blood Group Genotyping Potential Research Applications Fetal RHD Genotyping from Maternal Plasma: Plasma from pregnant women contains soluble fetal-dna Done in Denmark and the UK to predict fetal Rh type; RhIG withheld if Rh-negative Fetal DNA disappears soon after delivery SRY (male) and paternal VNTRs (female) used to control for fetal DNA All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 69 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

70 Blood Group Genotyping Potential Research Applications Zygosity Studies: Paternal RHD zygosity determination RHD is flanked by two Rh boxes RHD is deleted in Rh-negative Caucasians, leading to formation of a hybrid Rh box Zygosity determination of reagent RBC donors Dce/Dce vs Dce/ce Jk a /Jk a vs. Jk a /Jk All Blood Group Genotyping tests commercially available in the U.S. and Canada are for Research Use Only. Not for use in diagnostic procedures 70 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

71 Summary Elements of blood Antigens and antibodies Antigen-antibody interactions Pretransfusion testing Antibody identification Phenotyping Potential research applications of blood group genotyping 71 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only

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