P001: DSA SOLID-PHASE CROSSMATCHING DEMONSTRATES THAT PRONASE-TREATED B CELLS SOMETIMES FAIL TO BIND ANTI-CLASS I IgG

Background The observation that MHC Class I expression on B cells is greater than MHC Class I expression on T cells has led to the interpretation that Tpos/Bneg flow crossmatches are not detecting MHC Class I antibodies. While this proves true much of the time, we have previously reported that Tpos/Bneg flow crossmatches can be mediated by Bw4/Bw6 and HLA C antibodies. We now extend this observation to include other commonly encountered MHC Class I antibodies. Methods Ficoll-purified donor PBMC were mildly treated with pronase (0.02 mg/ml at 37 C for 15 min) to reduce B cell MdX for IgG binding with the negative control serum. This treatment does not remove all FcR, nor does it reduce MHC Class I and Class II expression. In our program any positive flow crossmatch is confirmed by solid phase donor crossmatches (DSA) using the manufacturers protocol. Observations We present 5 cases where a Tpos/Bneg result was caused by Class I DSA detectable by single antigen bead testing. Donor lysate-derived Class I crossmatches confirmed IgG binding for HLA-A1, HLA-A2, and HLA-B60. Conclusions These results caution against a blanket interpretation that Tpos/Bneg crossmatches indicate “safe” non-Class I antibody binding. Follow-up analysis is warranted for such flow crossmatch results. We recommend using donor-derived antigen in addition to commercially available single antigen testing to confirm or rule out the presence of DSA.