44-P : ANTIBODIES AGAINST DENATURED C-LOCUS ANTIGENS IN TWO PATIENTS WITH VENTRICULAR ASSIST DEVICES (VAD)

Aim To investigate discrepancies between different solid phase HLA antibody detection platforms. Methods Sera from two patients implanted with a VAD were tested for Class I and II antibodies using FlowPRA screening beads, HLA phenotype beads (flow cytometry), single antigen beads (SAB; by Luminex) and a cell based crossmatch assay (flow cytometric). Antibody profiles were analyzed for each patient on all platforms. Results No HLA Class I antibodies were detected by the FlowPRA assay while antibodies to HLA-C∗01, C∗12 and C∗15 were identified by SAB testing. To resolve these discrepencies, sera were tested with HLA phenotype beads and a flow cytometric crossmatch using cell lines expressing individual HLA-C locus antigens. These assays were negative for antibodies to HLA-C, supporting the results of the FlowPRA screen. Hence, the SAB assay was the outlier. Importantly, the HLA-C locus expressing cell lines that were crossmatch negative were the same cell lines from which solubilized HLA-C locus antigens were isolated and conjugated to SABs. Conclusions There should be agreement among the solid phase platforms currently in use to detect and identify HLA antibodies. As reported here, some antibodies detected on the SAB platform (using recombinant HLA antigen targets) are undetectable by other solid phase assays and cell based assays (where the HLA antigens are in native configuration). The adherence of purified recombinant HLA-C antigens to a plastic matrix likely results in conformational changes and/or exposure of cryptic epitopes not seen with native antigens. We speculate that the HLA-C locus antibodies to denatured HLA-C antigens developed as a consequence of VAD implantation. Since antibodies to denatured antigens do not appear to be clinically relevant, at least in the short term, it is important to determine whether antibodies detected in the SAB assay are to native or denatured antigens. Failure to do so can negatively impact a patient’s access to an allograft.