1. Serological Cases: What keeps us up at Night Stephanie Goe, MT (AAB-P) June 6, 2013

2. Solving Problem Workups Unusual and rare antibodies present complex and challenging serological workups for the blood banker. This can be due to lack of experience, exposure and appropriate reagents. 2

3. Dilemma.. “How do you know what you don’t know” How does one identify an antibody they have never seen ??? Some antibodies react similarly. However, they can be completely benign, capable of producing a mild transfusion reaction or death. 3

4. Goals The objectives of this case study are: 1.Presentation of a workup involving a clinically significant high incidence antibody  Patient Information  Serological techniques used for identification  Challenges of finding compatible units 2.Compare and contrast a clinically significant and nonsignificant antibody 3.Discuss and show that “Antibodies don’t react by the book” 4

5. Case Study Patient Information –54 year old Caucasian female –Diagnosis: acute kidney injury, rhabdomylosis, multiple traumas –Pregnancy and transfusion history unknown –Referring hospital transfused 6 rbcs as follows: * 2 rbcs on 2/26/13- DAT negative; no antibodies identified * 2 rbcs on 2/28/13 * 2 rbs on 03/15/2013- DAT negative; anti-c,-E, cold autoantibody 5

6. What now? Specimen sent to IRL We rec’d both pre and post transfusion samples 10 days after patient initially presented to referring hospital All samples had substantial hemolysis ranging from 600 to 3000 mg % free hemoglobin 6

7. Hmmm… How to proceed? Pretransfusion sample: * ABO/Rh: O Positive * DAT: Negative with polyspecific AHG * Used to phenotype for common antigens *Patient phenotype: C+,E-,c-,e+,M+,N+,S-,s+,K+,k+,Fy(a- b+),Jk(a+b+) 7

8. Initial Testing Post Transfusion Specimen: * ABO/Rh: O Positive * DAT: w+ with polyspecific AHG & complement * Specimen grossly hemolyzed 8

9. Initial Testing Initial selected cell panel run in PEG which included a phenotypically similar cell.  IS results all 3+ except for the autocontrol  Reactions at IgG ranged from w+ to 1+  Panel tested in LISS yielded similar results 9

10. Initial Panels LISS PEG Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFyaFybJkaJkbMNSs 1++00+++0++0++++ 3+2+1+3+ 2++00+0++00+0++0 2+1+3+ 3++00+0+0++0+00+ 2+1+3+ 4++00+++0++00+0+P.S.3+2+1+3+ AC 0000✓0✓

11. Now what? Similar strong IS reactivity with IgG carryover in panel cells Phenotypically similar cell reactive Hx of cold autoantibody; however, negative autocontrol! Cold screen performed 11

12. Cold Screen Results 4C37C SC14+3+ SC24+3+ AC00 I Neg4+3+ I Neg4+3+

13. Information Gained A cold reacting antibody present. However, not a cold autoantibody. Autocontrol is negative.

14. Panel with 0.01M DTT Treated Serum LISS Test Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFyaFybJkaJkbMNSs 1++00+++0++0++++ 001+ 2++00+0++00+0++0 00 3++00+0+0++0+00+ 00 4++00+++0++00+0+P.S.001+ AC 000✓0✓

15. Panel with Control Serum LISS Test Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFyaFybJkaJkbMNSs 1++00+++0++0++++ 3+2+ 2++00+0++00+0++0 3+2+ 3++00+0+0++0+00+ 3+2+ 4++00+++0++00+0+P.S.3+2+ AC 000✓0✓

16. Panel with 0.2M DTT Treated Cells LISS Test Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFyaFybJkaJkbMNSs 1++00+++0++0++++ 1+ 2++00+0++00+0++0 w+ 3++00+0+0++0+00+ 1+ 4++00+++0++00+0+P.S.1+ AC 000✓0✓

17. Recap IgM reactivity abolished with 0.01M DTT treated serum (IgG component still present) Diminished reactivity with 0.2M DTT treated cells Reactive phenotypically similar cell Negative autocontrol ………Possible high??

18. What are Some Candidates? Patient is Caucasian IS reactions Diminished reactivity with 0.2M DTT treated cells Possible PP1Pk, I, H, Vel ? 18

19. Panel Reflecting R 2 R 2 2x PEG Adsorbed Sera Results Rh systemKellDuffyKiddMNSs R2R2 DCcEeKkFyaFybJkaJkbMNSs 1++00+++0++0++++ 0✓0✓ 2++00+0++00+0++0 0✓0✓ 3++00+0+0++0+00+ 0✓0✓ 4++00+++0++00+0+P.S.0✓0✓ Differential Cells R 1 P 1 +; M+N-S+s-;K-; Fy(a+b-); Jk(a-b+) R 2 P 1 +; M-N+S-s+;K-; Fy(a-b+); Jk(a+b+) rr P 1 -; M+N+S-s+;K-; Fy(a-b+); Jk(a+b-)

20. Additional Phenotyping Patient typed with the following antisera: Anti-H-P1-I-Vel Pos Neg

21. Antigen Negative Cells Tested LISS PEG Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFy a Fy b JkaJkbMNSsVel 1++00++++++0++++00000✓0✓ 2++00+0++00+0++000000✓0✓

22. Prewarm Panel Prewarm Rh systemKellDuffyKiddMNSs IgG DCcEeKkFyaFybJkaJkbMNSs 1++00++++++0++++ 2+ 2++00+0++00+0++0 3++00+0+0++0+00+ 4++00+++0++00+0+P.S.2+ AC 0✓0✓

23. RESt TM Adsorbed Sera 1X LISS Rh systemKellDuffyKiddMNSs IS 37 o C IgG DCcEeKkFyaFybJkaJkbMNSs 1++00++++++0++++ 3+ 2++00+0++00+0++0 3++00+0+0++0+00+ 4++00+++0++00+0+P.S.3+

24. Anti-Vel Antibody to the high incidence antigen that is present in > 99 % of the population Can be both IgM and IgG in nature IgM complement binding anti-Vel can sometimes present as hemolysins Anti-Vel can cause severe hemolytic transfusion reactions Anecdotal reports of successful Vel+ transfusions. Not recommended. 24

25. A Tale of Two Antibodies….. “It was the very best of times. It was the very worst of times” - Charles Dickens Let’s compare a similarly reacting nonsignificant antibody to our anti-Vel

26. Ex. Nonsignifcant anti-I and cold autoantibody PEG KellDuffyKiddMNSs IS IgG DCcEeKkFyaFybJkaJkbMNSs 3+1+ 100+0+0+0++0++0+ 3+1+ 200+0+0+000+0+0+ 3+1+ 3+0+0+0+00+00+0+ 3+1+ 400+0+0+00+00+0+I- 3+0✓0✓ AC 1+0✓0✓ 26

27. Comparisons Anti-Vel IS reactivity IgM carryover Negative autocontrol Anti-I and cold autoantibody IS Reactivity IgM carryover Negative autocontrol at IgG

28. Contrast Anti-Vel Cord cells reactivity not diminished Reactivity not diminished by prewarm technique Anti-I and Cold autoantibody Cord cell reactivity diminished Reactions eliminated by prewarm technique

29. Cautionary Tale Exercise caution using the prewarm technique as significant alloantibodies can be eliminated. If you have IS reactions and a negative autocontrol, proceed with caution 29

30. …But it’s Not What the Textbook Said Not all antibodies behave in textbook fashion. Our anti-Vel behaved differently in several ways: (1) Cord cells did not react weaker (2) RESt TM did not adsorb out the anti-Vel 30

31. ..and Our Patient? We believe our patient exhibited an anamnestic response to the initial transfusion and was undergoing a transfusion reaction. We initiated an ARDP search and imported 2 rbcs. 31

32. Works Cited Lomas-Francis C, Reid M, The Blood Antigens Fact Book 2 nd ed. New York City, New York: New York Blood Center; 2004 Anstee J, Issitt Peter, Applied Blood Group Serology 4 th ed. Durham, North Carolina: Montgomery Scientific Publications; 1998 32