Potpourri of Reference Lab Cases 2017 CBBS Annual Meeting

1. Potpourri of Reference Lab Cases2017 CBBS Annual Meeting Gina Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ) American Red Cross Southern California Region regina.leger@redcross.org

2. Disclosures I have no relevant financial relationships to disclose

3. Objective • Review the findings and resolution of at least two complex or unusual cases worked up by the Immunohematology Reference Laboratory (IRL)

4. Patient's Information • Age, gender • Race/ethnicity • Diagnosis • Pregnancy history • Transfusion history • Transplantation history • Antibody history • Medications

5. Routine IRL Workup • ABO and Rh (phenotype 1st case) • DAT • Panels: • 5' RT, then polyethylene glycol (PEG) added, 15' 37C → anti-IgG • Ficin 37C & AGT • Additional testing: LISS; Dithiothreitol (DTT)-tr'd RBCs in LISS

6. Ficin/ Papain DTT Possible Specificity neg pos M, N, S, s*; Ge2, Ge4; Xga; Fya, Fyb; Ch/Rg negneg Indian; JMH pos weak Cromer; Knops; Lutheran; AnWj; MER2 variable negYta pos neg Kell; LW; Dombrock pospos A, B; H; P1; Rh; Lewis; Kidd; Fy3; Diego; Scianna; Co; Ge3; Oka; I, i; P, LKE; Ata; Csa; Era; Jra; Lan; Vel*; Sda Usefulness of Enzymes & DTT *variable Modified from Reid et al. The Blood Group Antigen FactsBook, 3rded, 2012

7. Case #1 • 76 year old Japanese female • Surgical patient  in surgery, but doing well • Dx: osteoarthritis • Hb = 13.8 g/dL, Hct= 39.7% • No transfusion record • Group AB, D+ • DAT negative • Antibody detection: 1+ gel & LISS • Autocontrol negative

8. Case #1: IRL initial serology Group AB, R2R2, DAT neg(IgG & C3)

9. Case #1: Ficin Panel

10. Case #1: LISS-IgG Panel

11. Case #1: DTT-Treated RBCs

12. Case #1: Patient's Phenotype

13. Case #1: Selected RBCs - Phenotype Similar

14. Case #1 Impressions: • Reactivity in PEG-IgG similar to LISS-IgG (no enhancement) • Reactivity enhanced by enzyme pretreatment • Reactivity not destroyed by DTT pretreatment • Phenotype similar RBCs reacted the same as random RBCs  probable antibody to high prevalence antigen

15. Case #1 Next steps: • Test selected RBCs negative for high prevalence antigens: • Not denatured by ficin or DTT • Patient was noted to be Japanese,  concentrate on phenotypes more common in Japanese

16. Ficin/ Possible Papain DTT High Prevalence Antibody neg pos Ch/Rg; Ge2, Ge4 negneg Indian; JMH pos weak Cromer; Knops; Lutheran; AnWj; MER2 variable negYta pos neg Kell; LW; Dombrock pos posRh; Kidd; Fy3; Diego; Scianna; Co; Ge3; Oka; I, i; P, LKE; Ata; Csa; Era; Jra; Lan; Vel*; Sda Use of Enzyme- & DTT-Treated RBCs *variable Modified from Reid et al. The Blood Group Antigen FactsBook, 3rded, 2012

17. Case #1: Selected RBCs

18. Case #1

19. Case #1

20. Case #1

21. Case #1 Conclusion: anti-Jra Surgery concluded. No units needed for transfusion.

22. Jra • High prevalence antigen, recently raised to blood group system status  JR Blood Group System • Named after antibody producer, Rose Jacobs, not for "Junior", in 1970 • Jr(a–) phenotype is found more commonly in Japanese, Asians • Antigen fully developed at birth • Resistant to enzyme, DTT and glycine-acid EDTA treatment

23. Anti-Jra • Anti-Jra usually IgG • Rare antibody, clinical significance not well established • 2fatal case HDFN reported (1 mother had previous massive transfusion) • Incompatible transfusions sometimes result in HTR • Difficult to obtain Jr(a–) units! • Encourage blood donation • Recruit siblings

24. Case #2 • 82 y/o female • Diagnosis: anemia • Hb/Hct not available (done in Dr. office) • Transfused 2 years ago • Group AB, D+ • Previous anti-M (RT) + warm auto • 2 units requested for today

25. Case #2: Initial Testing • Previous history at American Red Cross IRL • Current sample: Group AB, D+ confirmed DAT: anti-IgG 4+, anti-C3 1+, ct 0

26. Case #2: LISS & Ficin Panels

27. Case #2: Allogeneic Adsorption with ZZAP-Treated RBCs • Plasma adsorbed 4X with ZZAP-treated RBCs still reacting strongly with untreated RBCs; either • very high titer autoantibody or • antibody directed to antigen destroyed by ZZAP

28. Case #2: Testing ZZAP-Adsorbed Plasma vs ZZAP-Treated RBCs

29. Case 2 Impressions: • Reactivity in LISS-IgG and ficin-AGT consistent with previously reported warm autoantibody • Reactivity of ZZAP-adsorbed plasma not consistent with previous workup • Antibody reacted with ficin-treated RBCs, but not with ZZAP-treated RBCs,  antibody must not react with DTT-treated RBCs

30. Case #2 Next steps: • Try adsorption with untreated RBCs to exclude presence of common alloantibodies • Identify reactivity in ZZAP-adsorbed plasma? • Test plasma with DTT-treated RBCs • Test eluate?

31. Case #2: Adsorption of Autoantibody With Untreated RBCs in Presence of PEG No common alloantibodies underneath the autoantibody

32. Case #2: Testing ZZAP-Adsorbed Plasma for Antibodies to DTT-Sensitive Antigens

33. Case #2: Additional Testing With LW(a−b−) RBCs Anti-LW

34. Case #2: Test Plasma With DTT-Treated RBCs Autoantibody in plasma reacts with DTT-treated RBCs  not anti-LW, but has broad specificity

35. Case #2: Eluate Autoantibody in eluate not anti-LW

36. Case #2: Conclusions • Warm autoantibody of broad specificity reactive by LISS, ficin and saline AGT and with DTT-treated RBCs • Anti-LW (presumably autoantibody) reactive by LISS AGT detected in adsorbed plasma • Previous anti-M not demonstrable • No common alloantibodies detected in PEG-adsorbed plasma • 2 group AB, D− RBCs screened with adsorbed plasma for transfusion

37. LW Antigen • Named for Landsteiner and Wiener in 1963 who in 1940 injected rabbits with rhesus monkey RBCs  "anti-Rh" later called anti-LW • LW antigens (LWa, LWb and Lwab) are well expressed on cord D+ and D− RBCs; D− RBCs in adults have less LW than D+ RBCs • DTT destroys LW antigens, but ficin/papain and sialidase have no effect • Acquired loss of LW antigen is temporary, detected by presence of anti-LW • Very few individuals with genetically LW(a−b−) RBCs are known • The only true LW− RBCs are Rhnull phenotype

38. Anti-LW • Typically IgG, may be IgM • Optimally reactive at 37C • No data supporting HTR • Mild HDFN due to autoanti-LWab reported • Only four examples of alloanti-LWabhave been described in LW(a−b−) individual • When LW antigens are suppressed, the anti-LWabmay mimic alloantibody & is more common than autoanti-LWa • Transfusion of D− RBCs has been successful in many cases

39. Case #3 • 35 year old female • Adnexal mass • Hb/Hct not in chart • Race/ethnicity not in chart • No transfusion record • Group O, D+ • Solid phase 3+ on 1 of 3 RBCs, ? on 2 of 3 RBCs • No specificity on several panels

40. Case #3: IRL Initial Testing • Group O, R1R2 • DAT negative

41. Case #3: LISS Panel Anti-Jka reacting weakly in LISS-IgG only with double-dose Jk(a+) RBCs.

42. Case #3: Patient's Phenotype Patient's RBCs are Jk(a−b−), Jk:−3 !

43. Case #3: Selected RBCs

44. Case #3: Jk(a−b−)Selected RBCs Anti-Jk3 in PEG-IgG

45. Case #3: Conclusion • Group O, R1R2; Jk(a−b−) • Plasma: anti-Jka weakly reactive by LISS-IgG and anti-Jk3 detected by PEG-IgG • Transfusion recommendation: Group O, D+, Jk:−3

46. Jk3 • High prevalence antigen identified in 1959 • Jk(a−b−) RBCs are Jk:−3, rare null phenotype found most commonly in Polynesians • Kidd antigens well developed at birth • Resistant to treatment with ficin, DTT and glycine acid EDTA • Jk:−3 RBCs are resistant to 2M urea lysis

47. Anti-Jk3 • Produced by immunized Jk(a−b−) • Sometimes present with separable anti-Jka or -Jkb • Reacts equally well with Jk(a+b−), Jk(a+b+) and Jk(a−b+) RBCs • Usually IgG • Reactivity enhanced with protease-treated RBCs • Immediate & delayed HTR • Mild HDFN

48. Review of IRL Cases: • Anti-Jra (antibody to high prevalence antigen, found predominantly in Japanese) • Autoanti-LW under broadly reactive warm autoantibody Anti-Jk3 (antibody to high prevalence antigen, found predominantly in Pacific Islanders and Asians)