Introduction to Immunohematology W. John Judd, FIBMS, MIBiol Emeritus Professor University of Michigan
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
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
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
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
Blood Group Antigens 324 blood group antigens recognized 33 blood group systems 40 unassigned antigens Molecular biology of assigned antigens is known http5://www.isbtweb.org/fileadmin/user_upload/wp_on_red_cell_immunogenetics _and/table_of_blood_group_antigens_within_systems_v2.0_110914.pdf 6 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
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
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
IgG Antibody 12 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
IgG vs. IgM Molecules 13 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
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
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
IgM Agglutination 19 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
Agglutination 20 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
Detection of IgG Coating Antihuman Globulin 22 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
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
ABO Typing Expected Reactions RBCs + Plasma + Type Anti-A Anti-B A 1 RBCs B RBCs O 0 0 + + A + 0 0 + B 0 + + 0 AB + + 0 0 27 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
Distribution of ABO and Rh O A B AB Rh+ Rh- % US blacks 49 27 20 4 94 6 % US whites 45 40 11 4 83 17 % British 47 42 8 3 85 15 % Asians 33 30 27 10 99.5 0.5 31 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
Methods for Antibody Detection Plasma:RBCs Time at 37 C AHG SAL >2:1, 3-4% 30-60 PS/IgG ALB >2:1, 3-4% 15-30 PS/IgG LISS 2:2, 2% 10-15 PS/IgG GEL 1:2, 0.8% 15 IgG PEG 2:1, 3-4% 15-30 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
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
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 R1R1 + + 0 0 + 0 + + + + + 0 0 + + 0 + + I II R2R2 + 0 + + 0 + + 0 + + 0 + + + 0 + + + II 36 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
Ionic Cloud Steric hindrance of antibody by ionic cloud formed when RBCs are suspended in saline +-+-+-+- 38 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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 4+ 3+ 2+ 1+ 0 Anti-IgG Gel Test Reaction 41 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
Case Study Initial Studies RBCs + Plasma + Anti-A Anti-B Anti-D Control A 1 RBCs B RBCs 0 0 4+ 0 4+ 4+ 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 R1R1 + + 0 0 + 0 + + + + + 0 0 + + 0 + + I 0 II R2R2 + 0 + + 0 + + 0 + + 0 + + + 0 + + + II 4+ 45 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 1 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + 2 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 3 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 PATIENT AC 46 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 1 0 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + 2 0 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 3 0 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 4+ 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 2+ 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 0 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 0 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 0 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 0 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 0 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 0 PATIENT AC 0 47 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 1 0 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + 2 0 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 3 0 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 4+ 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 2+ 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 0 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 0 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 0 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 0 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 0 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 0 PATIENT AC 0 48 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 1 0 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + 2 0 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 3 0 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 4+ 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 2+ 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 0 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 0 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 0 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 0 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 0 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 0 PATIENT AC 0 49 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 1 0 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + 2 0 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 3 0 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 4+ 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 2+ 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 0 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 0 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 0 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 0 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 0 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 0 PATIENT AC 0 50 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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 0 + + 0 + + + + 0 0 0 + 0 + + + 0 + 0 0 2 R1R1 + + 0 0 + 0 + + + + + 0 0 + + + + + + 0 3 R1R1 + + 0 0 + + 0 0 + + 0 + + + + + + 0 + 0 4 R2R2 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + + 4 4+ 5 r r 0 0 + + + + 0 + 0 + 0 + 0 + + + 0 + 5 2+ 6 rrv 0 0 + 0 + + + 0 + + 0 0 0 + 0 + 0 + 6 0 7 rr 0 0 + 0 + 0 + 0 + + 0 + + 0 0 + + 0 7 0 8 rr 0 0 + 0 + + + + + + + 0 0 + + 0 + + 8 0 9 rr 0 0 + 0 + + 0 + 0 + 0 + + + + 0 0 + 9 0 10 rr 0 0 + 0 + + + + + 0 + 0 0 + + + + 0 10 0 11 Ror + 0 + 0 + + + 0 0 + 0 + 0 + + + 0 0 11 0 PATIENT 0 AC 0 51 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
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
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
A Pint (Unit) of Blood 57 Introduction to Immunohematology W. John Judd, FIBMS, MIBiol February 2012 Business Use Only
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
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
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
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
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
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
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
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
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
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
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
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
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
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