Simultaneous detection of antibody binding and cytotoxicity in flow cytometry crossmatch for renal transplantation

Background: The anti-HLA antibody can be detected using either a complement-dependent lymphocytotoxicity (CDC) assay or a flow cytometry crossmatch (FCXM) in renal transplantation. Discordant results are often obtained because the two methods detect different reaction phases between the donor lymphocytes and the recipient sera. This study was intended to confirm that antibody binding and cytotoxicity to the lymphocytes can be detected simultaneously in a single FCXM assay, cytotoxic FCXM. Methods: In the cytotoxic FCXM, the antibody binding to the lymphocytes was measured using anti-IgG-FITC, and the cytotoxicity using 7-aminoactinomycin D (7-AAD) after adding complement. For an evaluation of two test parameters, the cytotoxicity test moiety (dead-cell percentage) was compared with the anti-human globulin (AHG)-CDC, and the antibody-binding test moiety (sample/control fluorescence ratio) with the conventional FCXM in 77 positive and 30 negative crossmatches. Results: In the cytotoxic FCXM, both antibody binding and cytotoxicity could be assessed in a single anti-IgG-FITC/7-AAD plot. Regarding the correlation between the presence of HLA antibodies and the test result, the cytotoxicity parameter (r = 0.55) appeared to be more suitable than that of the AHG-CDC (r = 0.50) but the antibody-binding parameter (r = 0.83) was worse than that of the conventional FCXM (r = 0.93). The sensitivity of both parameters of the cytotoxic FCXM was not significantly different from each conventional counterpart (P = 0.33 and P = 0.22, respectively). Conclusions: The simultaneous detection of Ab binding and cytotoxicity was possible by the cytotoxic FCXM with the test efficiencies similar to the conventional counterparts. If this new assay is improved through the further studies to optimize the critical assay variables, this may be used as an alternative to the conventional assays to acquire more information on the characteristics of the recipient's HLA alloantibodies.