1. Potpourri of Reference Lab Cases 2017 CBBS Annual Meeting
Gina Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
American Red Cross Southern California Region

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. Usefulness of Enzymes & DTT
Ficin/ Papain DTT Possible Specificity
neg pos M, N, S, s*; Ge2, Ge4; Xga; Fya, Fyb; Ch/Rg
neg neg Indian; JMH
pos weak Cromer; Knops; Lutheran; AnWj; MER2
variable neg Yta
pos neg Kell; LW; Dombrock
pos pos A, B; H; P1; Rh; Lewis; Kidd; Fy3; Diego; Scianna; Co; Ge3; Oka; I, i; P, LKE; Ata; Csa; Era; Jra; Lan; Vel*; Sda
*variable
Modified from Reid et al. The Blood Group Antigen FactsBook, 3rd ed, 2012
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. Use of Enzyme- & DTT-Treated RBCs
Ficin/ Possible Papain DTT High Prevalence Antibody
neg pos Ch/Rg; Ge2, Ge4
neg neg Indian; JMH
pos weak Cromer; Knops; Lutheran; AnWj; MER2
variable neg Yta
pos neg Kell; LW; Dombrock
pos pos Rh; Kidd; Fy3; Diego; Scianna; Co; Ge3; Oka; I, i; P, LKE; Ata; Csa; Era; Jra; Lan; Vel*; Sda
*variable
Modified from Reid et al. The Blood Group Antigen FactsBook, 3rd ed, 2012
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
2 fatal 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
NoCal SatSunriseSem

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
NoCal SatSunriseSem

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. Untreated RBCs LISS-IgG DTT-treated RBCs LISS-IgG
Case #2: Eluate
Untreated RBCs LISS-IgG
DTT-treated RBCs LISS-IgG
RBCs
Eluate
Last Wash #1 4+ 0 #2 #3 #4 #5 #6
Autoantibody in eluate not anti-LW
NoCal SatSunriseSem

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-LWab have been described in LW(a−b−) individual
When LW antigens are suppressed, the anti-LWab may 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
NoCal SatSunriseSem

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)