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

Disclosures I have no relevant financial relationships to disclose

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

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

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

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

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

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

Case #1: Ficin Panel

Case #1: LISS-IgG Panel

Case #1: DTT-Treated RBCs

Case #1: Patient's Phenotype

Case #1: Selected RBCs - Phenotype Similar

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

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

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

Case #1: Selected RBCs

Case #1

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

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

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

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 071611

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 071611

Case #2: LISS & Ficin Panels

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

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

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

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?

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

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

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

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

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 071611

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

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

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

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

Case #3: IRL Initial Testing Group O, R1R2 DAT negative NoCal SatSunriseSem 071611

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

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

Case #3: Selected RBCs

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

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

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

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

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)