Flow cytometric detection of HLA-specific antibodies as a predictor of heart allograft rejection

Background. Historically, panel reactive antibody (PRA) analysis to detect HLA,antibodies has been performed using cell-based complement-dependent cyt otoxicity (CDC) techniques. Recently, a flow cytometric procedure (FlowPRA) was introduced as an alternative approach to detect HLA antibodies, The fl ow methodology, using a solid phase matrix to which soluble HLA class I or class II antigens are attached is significantly more sensitive than CDC ass ays, How ever, the clinical relevance of antibodies detected exclusively by FlowPRAhas not been established. In this study of cardiac allograft recipi ents, FlowPRA was performed on pretransplant sera with no detectable PRA ac tivity as assessed by CDC assays. FlowPRA antibody activity was then correl ated with clinical outcome. Methods. PRA analysis by anti-human globulin enhanced (AHG) CDC and FlowPRA was performed on sera corresponding to final cross-match specimens from 21 9 cardiac allograft recipients. In addition, sera collected 3-6 months post transplant from 91 patients were evaluated. The presence or absence of anti bodies was correlated with episodes of rejection and patient survival, A re jection episode was considered to have occurred based on treatment with ant irejection medication and/or histology, Results. By CDC, 12 patients (5.5%) had pretransplant PRA >10%. In contrast , 12 patients (32.9%) had pretransplant anti-HLA antibodies detectable by F lowPRA (34 patients with only class I antibodies; 7 patients with only clas s II antibodies; 31 patients with both class I and class II antibodies). A highly significant association (P<0.001) was observed between pretransplant HLA antibodies detected by FlowPRA and episodes of rejection that occurred during the first posttransplant year. Fifteen patients died within the fir st year posttransplant. Of nine retrospective flow cytometric cross-matches that were performed, two were in recipients who had no pretransplant antib odies detectable by FlowPRA Both of these cross-matches were negative. In c ontrast, five of seven cross-matches were positive among recipients who had FlowPRA detectable pretransplant antibodies. Posttransplant serum specimen s from 91 patients were also assessed for antibodies by FlowPRA, Among this group, 58 patients had FlowPRA antibodies and there was a trend (although not statistically significant) for a biopsy documented episode of rejection to have occurred among patients with these antibodies. Conclusions. Collectively, our data suggest that pre-and posttransplant HLA antibodies detectable by FlowPRA and not AHG-CDC identify cardiac allograf t recipients at risk for rejection. Furthermore, a positive donor reactive flow cytometric cross-match is significantly associated with graft loss, Th us, we believe that detection and identification of HLA-specific antibodies can be used to stratify patients into high and low risk categories, An imp ortant observation of this study is that in the majority of donor:recipient pairs, pretransplant HLA antibodies were not directed against donor antige ns, We speculate that these non-donor-directed antibodies are surrogate mar kers that correspond to previous T cell activation. Thus, the rejection epi sodes that occur in these patients are in response to donor-derived MHC pep tides that share cryptic determinants with the HLA antigens that initially sensitized the patient.