P010 Heterogeneous flow cytometry crossmatch reactions in the presence of patient sera with pan-dr antibodies

Abstract

Listen

Translate article

Aim During routine clinical evaluations of antibody profiles using single antigen bead (SAB) assays, several instances of widespread reactivity exclusively to DR antigens (‘pan-DR’ antibodies) were observed. Additional assays were performed to investigate reactivity of these pan-DR positive sera against different antigenic conformations present on bead products and donor cells. Methods Four serum samples taken two months apart from two hematopoietic cell transplant candidates were evaluated. Mixed bead and C1q assays (One Lambda, Inc) were performed to verify that the antibodies being detected were indeed against DR antigens and not cryptic epitopes that are present on SAB. Flow cytometry crossmatch (FCXM) was performed against two different donor cells. ELISA was performed to assess the involvement of AT1R antibodies in positive FCXM reactions based on reports of the capacity of anti- AT1R to cause positive FCXM. Results All sera were negative for Class II antibodies using mixed beads and C1q screening assays, contradicting the Pan-DR reactions with SAB. From patient#2, FCXM showed that serum #3 was T+/B + crossmatched against cell #1 but became T-/B- crossmatched against cell #2. From patient #1, serum #1 was borderline T+/B + when crossmatched against cell #1 but became T+/B- when crossmatched against cell #2. FCXM with all serum samples from both patients against cell#2 were negative. Additionally, auto-crossmatches for both patients were negative despite the presence of auto-antibodies. There was no association between AT1R antibody level and pan-DR observation. Conclusions The presence of antibodies against both naive and denatured antigens was indicated in pan-DR positive SAB/negative mixed beads, and in the positive T-cell FCXM and/or B-cell FCXM reactions. It is plausible that pan-DR antibodies described by the SAB data are not HLA antibodies at all but rather non-HLA antibodies with cross reactivity between cryptic HLA-DR antigens and yet unidentified non-HLA epitopes on lymphocytes.