Interesting Case Studies from The Mayo Clinic Reference Laboratory

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) Biphasic- going from warm to cold Anti-P is part of the globoside blood group system. It consists of P, Pk, and LKE. P negative RBC�s are very rare in adults. History of the test: PCH was one of the first types of antibody induced hemolytic anemia. It was first described by Dressler in 1854. In 1880, Rosenbach demonstrated hemolysis by having people place their hands and feet in ice water. The DL test was named after Donath and Landsteiner who first described the hemolysis in 1904. PCH has a very dramatic presentation. The autoantibody binds to the patient�s red cells in the extremities when the patient is exposed to cold. Once the patient has warmed back up to 37 degrees, intravascular hemolysis occurs. The symptoms of acute attacks include: sudden onset of fever, shaking chills, abdominal cramps, back pain, and intravascular hemolysis. This disease used to be seen in patient�s with advanced forms of syphilis. Because syphilis is treated successfully at early stages, the disease isn�t seen as often. Secondary PCH can be seen in children recovering from measles, mumps, chicken-pox, and infectious mono. Most of the time it appeared to be transient hemolysis after infections. The antibody most often implicated in PCH is auto-anti-P. The P blood group was implicated and described by Philip Levine in 1963. If transfusion is necessary, P- RBC�s would survive better, but any RBC�s would survive the same as the patient�s own cells. Biphasic- going from warm to cold Anti-P is part of the globoside blood group system. It consists of P, Pk, and LKE. P negative RBC�s are very rare in adults. History of the test: PCH was one of the first types of antibody induced hemolytic anemia. It was first described by Dressler in 1854. In 1880, Rosenbach demonstrated hemolysis by having people place their hands and feet in ice water. The DL test was named after Donath and Landsteiner who first described the hemolysis in 1904. PCH has a very dramatic presentation. The autoantibody binds to the patient�s red cells in the extremities when the patient is exposed to cold. Once the patient has warmed back up to 37 degrees, intravascular hemolysis occurs. The symptoms of acute attacks include: sudden onset of fever, shaking chills, abdominal cramps, back pain, and intravascular hemolysis. This disease used to be seen in patient�s with advanced forms of syphilis. Because syphilis is treated successfully at early stages, the disease isn�t seen as often. Secondary PCH can be seen in children recovering from measles, mumps, chicken-pox, and infectious mono. Most of the time it appeared to be transient hemolysis after infections. The antibody most often implicated in PCH is auto-anti-P. The P blood group was implicated and described by Philip Levine in 1963. If transfusion is necessary, P- RBC�s would survive better, but any RBC�s would survive the same as the patient�s own cells.

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7. 7 Donath-LandsteinerTesting Challenges Need to maintain the specimens and controls at 37�C. Length of time from start to finish is minimum of 2 � hours Need fresh donor samples for complement and RBC�s The reason to keep the specimens at body temp is so that the test doesn�t start itself before we are ready.The reason to keep the specimens at body temp is so that the test doesn�t start itself before we are ready.

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: Explain what I mean by transfusion dependent. IE 2 units every 2 weeks.Explain what I mean by transfusion dependent. IE 2 units every 2 weeks.

9. 9 Point out the variable reactivity and positive auto controlPoint out the variable reactivity and positive auto control

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 Explain difference between stroma and intact rbc�s. Explain why we don�t autoabsorb on recently tx�d patientsExplain difference between stroma and intact rbc�s. Explain why we don�t autoabsorb on recently tx�d patients

11. 11 Explain why the anti-K and anti-E remain Use pointer to show different reactions Point out the extra reactionsExplain why the anti-K and anti-E remain Use pointer to show different reactions Point out the extra reactions

12. 12 Explain why the anti-K remains, but the anti-E does not Again, point out the different reactions. Point out the extra reactionsExplain why the anti-K remains, but the anti-E does not Again, point out the different reactions. Point out the extra reactions

13. 13 Again, point out why the anti-E remains, but the anti-K does not Point out the reactions. Point out the extra reactions Note the new 3+ reaction vs the other 1+ 3+ low incidence = anti-Mur 1+ anti-V See if anyone has ideas for the extra reactions D C V Mur Again, point out why the anti-E remains, but the anti-K does not Point out the reactions. Point out the extra reactions Note the new 3+ reaction vs the other 1+ 3+ low incidence = anti-Mur 1+ anti-V See if anyone has ideas for the extra reactions D C V Mur

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 Would this patient be one that you would want to run low incidence antigens on? Do you think that this patient has made everything he can? Explain that Mur is a significant antibody.Would this patient be one that you would want to run low incidence antigens on? Do you think that this patient has made everything he can? Explain that Mur is a significant antibody.

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? In this case, we have tested all of our donors that he has received units from and not found the rrG+ donor. This supports the evidence that he had received C+ RBC�s in the past.In this case, we have tested all of our donors that he has received units from and not found the rrG+ donor. This supports the evidence that he had received C+ RBC�s in the past.

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

20. 20 Looking at this panel, what do you suspect for antibodies? Point out negative control.Looking at this panel, what do you suspect for antibodies? Point out negative control.

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? Explain what HTLA�s are.Explain what HTLA�s are.

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 The cells that were treated were phenotype matched. By destroying the antigen, it means that the cell was negative.The cells that were treated were phenotype matched. By destroying the antigen, it means that the cell was negative.

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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- Insert Reid�s page about YtaInsert Reid�s page about 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 Once again, point out the autocontrol.Once again, point out the autocontrol.

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. Once again, point out it was phenotypically similar.Once again, point out it was phenotypically similar.

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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. Insert Reid�s page on JraInsert Reid�s page on Jra

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 Explain that there isn�t any rationale between why some people form antibodies and others do not. There are HLA experiments ongoing that are trying to find a link between antibody formers.Explain that there isn�t any rationale between why some people form antibodies and others do not. There are HLA experiments ongoing that are trying to find a link between antibody formers.

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, 15th 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??