Cross transfusion and compatibility
- 2. Ahmad A. Al-Qudah LM755 - ADVANCED DIAGNOSTIC IMMUNOHEMATOLOGY AND BLOOD BANKING Cross transfusion and Compatibility
- 3. Cross transfusion and Compatibility - Pretransfusion Testing . - Pretransfusion testing elements : - ABO & Rh - Antibody Screening - Cross Matching - Antibody Identification . - Special Techniques in Ab Identification : - Elution - Hemagglutination inhibition - Titration
- 4. Pretransfusion Testing History - After Landsteiner described the ABO . - Crossmatch : - Major Crossmatch : Recipient Serum + Donor RBCs. - Minor Crossmatch : Recipient RBCs + Donor Plasma (not required by AABB) - RT ( IS Phase ) IgM , then Rh (IgG) and detection at various Temperatures with or without enhancements .
- 5. Pretransfusion Testing - To select blood components that will not cause harm to the recipient and will have acceptable survival when transfused. - ABO compatibility , Rh compatibility , clinically significant Antibody Screening and Identification .
- 6. Indirect Antiglobulin Test Phases - Immediate Spin ( IS ) Phase at Room Temperature . Detection of ABO incompatibility and Cold Antibody (M,N, Lea+b,P1) - 37° with Enhancement media (LISS) Allows IgG and other complement to bind to RBCs . - Anti Human Globulin Phase ( AHG ) Detection of IgG or complements binds to RBCs - Check Cells : Approve the Results
- 7. ABO & Rh - ABO & Rh typing for (recipient ) sample . - ABO & Rh typing for Donor sample .
- 8. Antibody Screening - Autoantibody : is an antibody manufactured by the immune system that is directed against one or more of the individual's own cells, Causes Many autoimmune disease.
- 9. Antibody Screening - Alloantibody : is an immune response to foreign antigen, Formed from previous transfusion or pregnancy. - Antibody Screening test performed to detect auto-antibodies /allo-antibodies using DAT (Direct Anti-human Globulin Test)
- 10. Antibody Screening - Inclusion of auto control helps to rule out : Auto antibodies Allo antibodies - Auto control : Negative - alloantibody . Positive – autoantibody .
- 11. Antibody Screening - Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serum. - If antibodies are detected, they must be identified .
- 14. Cross Matching - Cross match is done to ensure there are no clinically significant Abs in patient blood ( Serum ) against donor RBCs and so prevent hemolytic reaction during or after transfusion .
- 15. Cross Matching - Major cross-match test, consisting of mixing the patient’s serum with donor RBCs. - Minor cross-match test, consisting of mixing the donor’s plasma with patient’s RBCs. - Minor cross-match test has been completely eliminated in most blood banks according to AABB SOPs since 1960.
- 16. Cross Matching Prepare 5% suspension of donors RBCs
- 17. Cross Matching Patient serum 2 drops Donor RBC 1 drop, 5% Immediate centrifuge ABO incompatibility 22oC • Detects only IgM antibody, reactive at 22oC. • Clinically significant IgG antibody reactive at 37oC not detected
- 18. Cross Matching Patient serum 2 drops Incubation 37oC, 1 hr Donor RBC 1 drop, 5% 3 washes Centrifuge 2 drops AHG Mix properly No agglutination = compatible Detects clinically significant (IgG) antibody Agglutination = incompatible
- 19. Antibody Identification - It is a set of commercially screening cells with different antigenic expression corresponding to the most commonly encountered Abs.
- 20. Antibody Identification - Screening Cells and Panel Cells are the same with minor differences: - Screening cells - Antibody detection - Sets of 2 or 3 vials - Panel cells - Antibody identification - At least 10 vials per set. - Panel test is just an extended version of an antibody screen
- 21. Antibody Identification - 11 panel cells of Group O , last one is auto control (Patient RBCs + Patient serum) .
- 22. Antibody Identification - Each of the panel cells has been antigen typed .
- 23. Antibody Identification - Immediate Spin Technique (IST) Patient serum 2 drops Panel Cell 1 drop Immediate centrifuge incompatibility 22oC
- 25. Antibody Identification Incubation 37oC, 15 min Centrifuge 2 drops LISS Mix properly Agglutination No agglutination Detects clinically significant (IgG) antibody (LISS) 37°C Phase Patient serum 2 drops Panel Cell 1 drop
- 27. Antibody Identification IAT Phase Incubation 37oC Centrifuge 2 drops AHG Mix properly Agglutination No agglutination Patient serum 2 drops Panel Cell 1 drop Wash 3 times saline
- 28. Antibody Identification 2+ 0 0 2+ 0 0 2+ 0 0 2+ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - Record Results , Grade of Reaction . - Add Check cells to any negative AHG .
- 29. Antibody Identification - Read Results , Interpreting Antibody Panels
- 30. Antibody Identification Phase-1: IS , Rx. at room temperature (24°C) - Cold reactive antibodies (Ex. Anti-H, I, i, P) Phase-2: LISS Rx at 37°C: - Rh incompatibility - wide thermal range IgM antibodies (Anti-Le, I, P) Phase-3: AHG Rx. (at 37°C) - will detect the vast majority of IgG antibodies
- 31. Antibody Identification - Basic steps to follow when interpreting panels : • “Ruling out” means crossing out antigens that did not react • Circle the antigens that are not crossed out • Look for a matching pattern • Rule of Three .
- 34. Antibody Identification - The rule of three must be met to confirm the presence of the antibody. - A p-value ≤ 0.05 must be observed. - This gives a 95% confidence interval. - Patient serum MUST be: - Positive with 3 cells with the antigen - Negative with 3 cells without the antigen
- 35. Antibody Identification - Rule of three :
- 36. Antibody Identification - P value = Num# of Positive RBCs! * Num# of Negative RBCs Total Num# of RBCs used P value = 3! * 4! P value = (1*2*3) * (1*2*3*4) (1*2*3*4*5*6*7) P value = 2.85 , P value is ≤ 0.05 7
- 37. Special Techniques in Ab Identification - Elution Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified and measured . Y Y Y Y Sensitized RBC Positive DAT Elution Y Frees antibody Antibody ID
- 38. Special Techniques in Ab Identification - Elution • Acid elution (glycine acid) - Most common - Lowers pH, causing antibody to dissociate • Organic solvents (ether, chloroform) - Dissolve bilipid layer of RBC • Heat (conformational change) • Freeze-Thaw (lyses cells)
- 39. Special Techniques in Ab Identification - Haemagglutination inhibition: based on the neutralization of certain antibodies with its corresponding soluble substances (antigens).
- 40. Special Techniques in Ab Identification - Haemagglutination inhibition :
- 41. Special Techniques in Ab Identification
- 42. Special Techniques in Ab Identification Antibody titration • semiquantitative determination of Ab’s concentrations • serial 2-fold dilutions of tested serum are prepared • serum testing against corresponding antigens • TITER: is the reciprocal of the highest serum dilution that gives macroscopic agglutination
- 43. Special Techniques in Ab Identification Antibody titration