Measures in the difficult cross matching and individual compatibility

1. Measures in the difficult cross matching and individual compatibility Ziyan Zhu Shanghai Blood Center

2. Problem Solving in Antibody Identification • Investigation of a broadly reacting serum antibodies to high incidence antigens antibody mixtures • A cautionary tale!! highlighting importance of antibody exclusion • Useful hints for antibody investigations My Talk

3. Problem Solving in Antibody Identification • Not specifying a particular IAT method • Not describing methodology • Autoantibody investigation Not in my My Talk!

4. 实验前几个容易忽略的问题 • 血样：血清还是血浆 • 肝素对Polybrene 的影响、 • 定型试剂：ABO/Rh是否知道所针对的表位 • ABO反定型是否需O细胞，是否需Rh阴性细胞；所用的抗D会漏检哪些D表位 • 直抗、抗筛与抗体鉴定系统是否有对照 • 是否有自身对照、阴性对照、阳性对照 (直接阴、阳性对照;BSA、IgG致敏的RBC )

5. Tools of our trade

6. Where do the problems start? Antibody Screen Negative Positive One or more cells All cells No problem! Problem Problem

7. Positive reactions What are they due to? Are they significant? ‘Wanted’ ‘Unwanted’ Routine techniques should not be ‘over sensitive’

8. Before we start • Serum or plasma ? • Clinical and serological history of patient • Full phenotype • Ethnic background of patient Useful information

9. Asian In(a+b-) Black S-s-U- Js(a+b-) hrS-, hrB- Cr(a-) White K+k- Kp(a+b-) Yt(a-) Co(a-) etc Chinese D, Fya Ethnic Background of Patient Lack of a high incidence antigen

10. Black RoS- P1- Le(a-b-) K- Jk(a+b-) Fy(a-b-) Ethnic Background of Patient Combination of ‘routine’ antigens

11. Investigation of any serum Choosing a panel of cells • Select according to reactions observed in antibody screen • Select according to phenotype of patient, if known • Include enzyme panel (papain) • Room temperature might be useful • Be aware - all panel cells are group O!

12. Investigation of a broadly reacting serum A B IAT IAT IAT IAT Cells untreated papain untreated papain 1 4 4 3 0 2 4 4 3 0 3 3 0 4 4 4 3 0 4 4 5 4 4 3 0 6 4 4 3 0 7 4 4 3 0 8 4 4 3 0 9 4 4 3 0 Patient 0 0 0 0 eg Rh,Kell,Jk,Colton,Sc eg JMH, Inb, Ge2, Yta

13. Investigation of a broadly reacting serum C D IAT IAT IAT IAT 180C (RT) Cells untreated papain untreated papain 1 1 0 4 4 1 2 3 0 4 4 2 3 0 2 4 3 1 4 0 4 0 0 2 5 1 0 4 4 2 6 2 0 3 0 1 7 1 0 2 4 0 8 3 0 4 4 3 9 2 0 3 0 2 Patient 0 0 0 0 0 eg Ch, Rg, Kna/McCa, Yka eg anti-S+Jka+Fya+P1

14. What to do next?

15. Investigation of a broadly reacting serum

16. Investigation of a broadly reacting serum

17. Options ! Papain resistant/sensitive 1 • Type patient cells for (selected) high incidence antigens • Match (selected) rare + null cells against patient serum 2

18. Option 1 Cell typing • If negative found - check with another example of antibody • Match appropriate phenotype cells against serum (fresh or frozen) • If negative, use further examples (if possible) to exclude additional specificities Then

19. Option 2 Matching rare phenotype cells • More caution needed • Beware ABO !! All positive Negative found • Type patient cells for relevant antigen • Match further examples if possible - to exclude other antibodies

20. Option 2 Matching rare phenotype cells All positive Caution! Underlying antibodies may be present eg anti-K, -Fya ‘FALSE’ positive result

21. ? Problem still not solved Test ‘antigen matched’ donor cells ie D- S- Fy(a-) Jk(b-) Mixture of Antibodies

22. ? Still no clues • Make eluate from ‘antigen matched’ group O cells -isolates antibody to ‘high’ - eliminates ABO problem - match ‘null’ phenotype cells (any ABO group)

23. ? Still no clues Match against enzyme treated and chemically modified cells

24. Examples of effect of enzymes and chemical modification Papain Trypsin Chymotrypsin Pronase AET 200mM Ficin DTT + + + + + + Dib/Vel - - - - - - JMH/Inb + + + - + - Sc - - - + + + LW + + - - Cromer +/- +/- - Knops +/- - + - + - - - - + - Ch/Rg + + + - - +/- Kell

25. Investigation of a broadly reacting serum • Ch and Rg are determinants on the complement receptor C4 C IAT IAT untreated papain 1 0 3 0 C4 coated C4 coat cells in vitro with Ch+Rg+ plasma 0 1 0 2 4 1 0 2 0 Anti-Ch/Rg can be inhibited by C4 in plasma 1 0 3 0 2 0 0 0 eg Ch, Rg, Kna/McCa, Yka

26. Investigation of a broadly reacting serum • Kna, McCa and Yka are antigens on the complement regulatory protein CR1 Monoclonal Antibody Immobilisation Erythrocyte Antigens C IAT IAT untreated papain 1 0 3 0 0 1 0 2 MAIEA using monoclonal anti-CR1 1 0 2 0 1 0 3 0 2 0 0 0 eg Ch, Rg, Kna/McCa, Yka

27. Assignment of antigens using MAIEA assay Peroxidase-conjugated Goat anti-human Human antibody Solubilised antigen Mouse antibody (Mab anti-CR1) Goat anti-mouse

28. D IAT IAT 180C (RT) untreated papain 4 4 1 4 4 2 2 4 3 4 0 0 4 4 2 3 0 1 2 4 0 4 4 3 3 0 2 0 0 0 eg anti-S+Jka+Fya+P1 Investigation of a mixture of antibodies Clues ! Papain resistant Papain enhanced Variable strength reactions Papain sensitive

29. Investigation of a mixture of antibodies • Observe the clues from panel • What antibodies react in this way? • Use different modes of reactivity to advantage • Phenotypes known - What can patient make ? • S+s- Jk(a-) Fy(a-) • S- Jk(a+b-) Fy(a-) • S- Jk(a-) Fy(a+b-) • S- Jk(a-) Fy(a-) Cell selection in this case

30. Investigation of a mixture of antibodies Not enough cells to choose from! • Adsorption/elutions can help to isolate the antibodies: • Phenotypes often unknown (exclusions need to be more systematic) • Several panels may be needed Adsorb onto cells positive for ONE antigen only eg S+ Jk(a-) Fy(a-) etc Test eluates for specificities

31. A Cautionary Tale

32. rr + + + + + Antibody Identification Panel Profile Cell IAT Enz 40C Rh M N S s K Fya Fyb Jka Jkb 1 R1wR1 + + 0 0 + + 0 + 3 - - 0 + + 2 R1R1 0 + + + 0 + 0 4 - - 3 R2R2 0 + 0 + 0 0 + + 0 - - - + 4 r’r + + + 0 0 0 + 0 3 - - + 5 0 + + 0 + 0 0 + 4 - - r’’r + 6 rr 0 + 0 0 0 + + + 2 - - rr 7 0 + 0 + + 0 + + 0 - - - 8 rr + + 0 + 0 + 0 0 + 2 - - rr 9 + 0 + + 0 + 0 + 0 3 - - rr + + + + - 10 + 0 0 0 0 2 - Patient 0 0 0 0

33. rr + + + + + Antibody Identification Panel Profile Cell IAT Enz 40C Rh M N S s K Fya Fyb Jka Jkb 1 R1wR1 + + 0 0 + + 0 + 3 3 - - 0 + 2 R1R1 + 0 + + 0 + 0 4 4 - - + 3 R2R2 0 + 0 + 0 0 + + 0 - - - + 4 r’r + + + + 0 0 0 + 0 3 3 - - + 5 + 0 + + 0 + 0 0 + 4 4 - - r’’r + 6 rr + 0 + 0 0 0 + + + 2 2 - - rr 7 0 + 0 + + 0 + + 0 - - - 8 rr + + + 0 + 0 + 0 0 + 2 2 - - rr 9 + + 0 + + 0 + 0 + 0 3 3 - - rr + + + + + - 10 + 0 0 0 0 2 2 - Patient 0 0 0 0 0

34. Anti-M

35. Antibody exclusion Patient phenotypes: cde/cde M-N+ S-s+ K+ Fy(a-b+) Jk(a+b-) Therefore can make: Anti-C,-D,-E, -M, -S, -Fya, Jkb

36. rr + + + + + Antibody Identification Panel Profile Cell IAT Enz 40C Rh M N S s K Fya Fyb Jka Jkb C D E Jkb 1 R1wR1 + + 0 0 + + 0 + 3 - - 0 + + 2 R1R1 0 + + 0 + 0 4 - - + 3 R2R2 0 + 0 + 0 0 + + 0 - - + 4 r’r + + + 0 0 0 + 0 3 - - + 5 0 + + 0 + 0 0 + 4 - - r’’r M Fya S ?? + 0 6 rr + 0 0 0 + + + 2 - - rr 7 0 + 0 + + 0 + + 0 - - - 8 rr + + 0 + 0 + 0 0 + 2 - - rr 9 + 0 + + 0 + 0 + 0 3 - - rr + + + + - 10 + 0 0 0 0 2 - Patient 0 0 0 0

37. 3 R2R2 0 + 0 + 0 0 + + 0 - - - rr + + + + + Antibody Identification Panel Profile Cell IAT Enz 40C Rh M N S s K Fya Fyb Jka Jkb 1 R1wR1 + + 0 0 + + 0 + 3 - - 0 + + 2 R1R1 0 + + 0 + 0 4 - - + 0 0 0 - + 4 r’r + + + 0 0 0 + 0 3 - - + 5 0 + + 0 + 0 0 + 4 - - r’’r + 6 rr 0 + 0 0 0 + + + 2 - - 7 rr 0 0 + 0 0 + + 0 0 + + 0 - - - - 8 rr + + 0 + 0 + 0 0 + 2 - - rr 9 + 0 + + 0 + 0 + 0 3 - - rr + + + + - 10 + 0 0 0 0 2 - Patient 0 0 0 0

38. Cell selection in this case • M-N+, S+s-, Fy(a-b+) • M-N+, S-s+, Fy(a+b-) • M+N- S-s+, Fy(a-b+) • M-N+, S-s+, Fy(a-b+) Homozygous expression

39. Results • Anti-S+Fya present reacting by IAT with untreated cells but not with papain treated cells • Expected characteristic for both antibodies (and anti-M if present) • Both clinically significant antibodies • Anti-M was excluded

40. Points for consideration • Anti-M usually ‘naturally occuring’ • Reacts preferentially at lower temperatures in ‘direct’ tests • Less often react by IAT • Often exhibit ‘dosage’ ie M+N-cells react stronger than M+N+ cells • Two clinically significant antibodies were mistaken for one of ? clinical significance

41. Learning points • A systematic approach to antibodyexclusion is an essential part of the antibody identification process • Use the characteristics of different antibodies to advantage • Enyzme treated cells useful in the ID process • Must consider the possibility of ALL clinically significant antibodies

42. Summary Antibody detection and identification • Fundamental to the practice of immunohaematology • Can be difficult and time consuming • Delay in patient care • Systematic approach is essential • Paucity of guidelines

43. Summary Antibody detection and identification • Optimum selection of blood for transfusion • Guide to the clinical significance of the antibody Why?

44. Summary Positive Antibody Screen • Many paths can be taken • Each laboratory should have a policy outlining their procedures

45. Hints Reaction Strength • Accurate grading of agglutination strength is vital • Clue to type of antibody and its clinical significance • If + and - in a serum, the stronger the positives more credence can be given to the negatives • Generally weak reactions- negatives might not lack the antigen but have weak expression eg Ch, Rg, Kna, Yka

46. Some general points • Knowledge of expected phases of reactivity for various antibodies will be a guide to certain specificities • Dosage effect ( M, N, S, s, Jka, Jkb) • Cells with homozygous expression for antibody exclusion • Observe haemolysis - it can mean a positive reaction

47. One last tip !

48. Red cell antigens and antibodies have characters - get to know them! ……………and don’t forget

49. Haemolysis Observe the clues !!! Enzymes Mode of reaction Dosage effect Reaction strength Temperature