1. Interesting Case Studies from The Mayo Clinic Reference Laboratory Georgette Benidt, MT(ASCP)

2. 2 Case 1 81 year old with B-cell lymphoproliferative disorder Clinician ordered the Donath Landsteiner Test

3. 3 DONATH-LANDSTEINER (DL)

4. 4 Case 1 – objectives Significance of the test Incidence of positive tests Testing challenges

5. 5 DL Significance Paroxysymal Cold Hemoglobinuria is an ideopathic disorder occurring in <1% of hemolytic anemias IgG biphasic autoantibody (usually anti-P) Fixes complement at 4 C Activates complement at 37 C Patient needs to avoid cold exposures (MN winters, air conditioners)

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7. 7 Donath-Landsteiner Testing Challenges Need to maintain the specimens and controls at 37°C. Length of time from start to finish is minimum of 2 ½ hours Need fresh donor samples for complement and RBC’s

8. 8 Case 2 68 Y.O. male O Rh negative Myelodyplasia Syndrome Transfusion Dependent Previous Anti-K, Anti-E, Warm Autoantibody Presents now with the following results:

9. 9 DCCwEcefvKkFyaFybJkaJkbMNSsISPEG AHGC R1R1+++00+000++00++0++02+ R2R2+00++0000+0+0++0++04+ rr0000+++0++0+0+++++02+ rr0000++++0+00+0++++01+ rr0000+++00++++++0+00w+ rr0000+++00++00++0++0 W+ rr0000+++00+0++++0+00w+ rr0000+++00+0+0++0+00w+ Ror+000+++00+00+00+0+02+ Autow+ Case 2: Initial Panel

10. 10 Case 2 Do you see a pattern? Is there varying reactivity? We know that the patient has a warm autoantibody, what next? At Mayo, we absorb onto 3 different cells: R1R1, R2R2, and rr

11. 11 DCCwEcefvKkFyaFybJkaJkbMNSs ISISPEG AHGCC rr0000+++0++0+0+++++ 2+ rr0000++++0+00+0++++ 2+ R1R1 ++00 + 0+++++++0+ 02+ r"r000++++00+0+++0+0+ 4+ rr0000+++00+0++++0+0 02+ rr0000+++00+00+00+0+ 02+ rr0000+++00++0++0+0+ 02+ R1R1++000+ 0 000+0000 Absorbing Cell Case 2: R1R1 Absorbate

12. 12 DCCwEcefvKkFyaFybJkaJkbMNSs ISISPEG AHGCC r'r0+00+++00+0+++++0+ 1+ rr0000+++0++0+0+++++ 2+ rr0000++++0+00+0++++ 2+ rr00 0 0+++++++0+ 02+ rr0000+++00+0+0++0+0 02+ R2R2+00++0+00+0++++0+0 02+ rr0000+++00++0++0+0+ 02+ R2R2+00++0 0 00+00000 Absorbing Cell Case 2: R2R2 Absorbate

13. 13 DCCwEcefvKkFyaFybJkaJkbMNSs ISISPEG AHGCC r'r0+00+++00+0+++++0+ 1+ Ror+000+++00+00+0+++0 1+ r"r000++++00+0+++0+0+ 4+ rr0000++++0+00+0++++ 2+ rr0000+++00++++++0+0 3+ rr00 0 ++++++++0+ 02+ rr0000+++00+00+00+0+ 02+ rr0000+++00++00++0++ 02+ R1R1++000+000+0++++0+0 1+ rr 0000++++0+0+0+++++ 2+ rr0000++ + 00+00000 Absorbing Cell Case 2: rr Absorbate

14. 14 Case 2 What antibodies were identified: Anti-G, Anti-C, Anti-E, Anti-K, Anti- Mur, Anti-V, and Warm Auto Why do we care about underlying antibodies: Possible DHTR Difficulty of finding antigen negative blood

15. 15 Case 2 What is significant about Anti-G? Belongs to the Rh family G antigen is present on all D+ and or C+ RBCs IgG and does not fix complement Stimulus from the transfusion of C+ RBCs following trauma

16. 16 Case 2 More on anti-G For Transfusion: Provide D-, C- crossmatch compatible RBCs For OB Patients Adsorption/elution studies may be necessary to determine if anti-D is also present RhIG administration??

17. 17 Case 2 Antigen Incidence Blacks 92% Caucasians 84% Asians 100%

18. 18 Case 2: Conclusion Anti-G has been shown to be present years after the exposure of D+ or C+ RBC’s Why did we care in this case? The patient had a previous Anti-C The patient has only received Rh negative blood that we know of Do we have a rr, G+ donor?

19. 19 Case 3 20 YO female A Rh negative 38 week gestation in 2 nd pregnancy No other information available Initial panel results are:

20. 20 DCcEeKkFyaFybJkaJkbMNSsIS PEG AHGCC R1R1++00+0+0+0+0+0+0W+ R1R1++00++++++00+0+01+ R1R1++00+0++00++0+001+ R2R2+0++00++0+++00+01+ R2R2+0++0++0+0++0++01+ R2R2+0++00+0++00+0+01+ R2r+0++++0++++0+0+01+ r'r0++0+0+0+0+++++01+ r"r00+++0++W+++0++0W+ rr00+0+++0++++0+00W+ rr00+0+0+00+00+0+01+ rr00+0+0++0+00+++01+ rr00+0+0+00+00+0+0W+ AUTO 00+ Case 3: Initial Panel

21. 21 Case 3 Do you see a pattern? What should be done next? Why?

22. 22 Case 3 Possible antibody to high incidence antigen Perform phenotype Test serum against phenotypically similar cell If negative, look for multiple common antibodies If positive, consider high incidence

23. 23 Case 3 Our results Phenotypically similar cell reacted 1+ with patient serum Antibody was titered to determine if it exhibited HTLA characteristics Antibody did not have a high titer Now what?

24. 24 Case 3 DTT and papain treated cells were tested The antibody did not react with the treated cells. Antigen is assumed to be sensitive to treatments A list of high incidence antigens was compiled

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26. 26 Case 3 Based on the sensitivity of papain and DTT, a Yt(a-) cell was thawed and tested This cell was negative at AHG, and 2 more Yt(a-) cells were thawed and tested We now have our 3 negative cells to confirm the presence of an Anti-Yta The patient’s antigen status was Yta-

27. 27 Case 3 In most populations, Yta has an antigen incidence of >99.8% Yta can bind complement Yta has been shown to cause anywhere from no transfusion reactions to moderate/delayed reactions Yta has not been shown to cause HDN

28. 28 Case 4 26 Y.O. female A Rh negative Presented during pregnancy No known antibody history Patient presents now with the following results:

29. 29 DCcEeKkFyaFybJkaJkbMNSsIS PEG AHGCC RZR1++0++0++0+++0++01+ R2R2+0++00+0+0+0+++01+ r'r0++0+0+0++0+00+01+ r"r00+++0++0+++00+01+ rr00+0+0++0+0++0+01+ RZR1++0++0+0++0+00+01+ R1R1++00++++++00+0+01+ rr00+0+0+00+0+++001+ rr00+0++++00++0+001+ AUTO 00+ Case 4: Initial Panel

30. 30 Case 4 Possible Suspects Multiple allo-antibodies High-Titer-Low-Avidity High Incidence

31. 31 Case 4 Phenotype was performed Phenotypically similar cell was tested against serum and reacted 1+ AHG. Ruled out the common multiple alloantibodies. What would you do next? HTLA titers were done x2 with possible HTLA identified

32. 32 Case 4 I was not convinced of the HTLA HTLA negative cells (Ch,Rg,Kn,Mc) were run with similar results We papain and DTT treated the same panel cell to see if we could rule out antigens Papain cell still reacted DTT cell did not react, and upon repeating, reacted at micro positive.

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34. 34 Case 4 Based on the Papain and DTT results, high incidence negative cells were tested Lu(a-b-); Sc:-1,2; K null; Yt(a-); Ge:-2,- 3; Lu:-8; Lu:-6 cells were all W+ At this point, we decided to send it to New York Blood Centers to see if they could identify the antibody

35. 35 Case 4 NYBC identified an Anti-Jra We picked ourselves up, dusted off and confirmed these results with our own reagents.

36. 36 Case 4 A little about anti-Jra (Junior) Anti-Jra can bind complement Can cause transfusion reactions but no cases of HDN have been identified This antigen has an incidence of >99% in most of the population

37. 37 Case 4 What went wrong? We forgot that antibodies do not read textbooks! Jra antigen should be resistant to DTT Anti-Jra antibodies shouldn’t look like HTLAs Our patient wasn’t Japanese

38. 38 Case 4 Outcome of patient: Patient was urged to donate units while she was still pregnant in case she needed them Baby was antigen positive, but there were no complications Patient remains an allogeneic blood donor

39. 39 Conclusions HTLA’s and High Incidence antibodies can mimic each other High Incidence antibodies can titer out to HTLA levels It is important to differentiate between HTLA and High Incidence antibodies Certain patient populations will continue to form antibodies

40. 40 Conclusions It is helpful to perform phenotypes, especially on patients you expect to have multiple transfusions Tests that seem like a waste of time can sometimes surprise you! Remember to take a picture of a positive DL…you may never see another one.

41. 41 References The Blood Group Antigen Facts Book, M.E. Reid, C.L. Francis Applied Blood Group Serology, P.D. Issitt, D.J. Anstee Technical Manual, 15 th edition Mayo Clinic Transfusion Medicine SOP’s

42. 42 Thanks Craig Tauscher for helping me prepare this presentation Sheila Muenster for reviewing my presentation The MT students who had to sit through my rough draft Bob Stowers for having the DL The rest of my coworkers for their help

43. Any Questions??