Cytotoxic flow cytometric crossmatch in renal transplantation: a single assay to simultaneously detect antibody binding and cytotoxicity.

The complement-dependent lymphocytotoxicity crossmatch (CDC-XM) detects cytotoxic parameters of preformed antibodies. The flow cytometric crossmatch (FCXM) is used to detect the binding of recipient antibodies to donor cells. Because these two assays provide different information, both methods are often performed to assess the compatibility of donor-recipient pairs. The aim of this study was to develop a single assay that can simultaneously detect antibody binding and cytotoxicity. A procedure called cytotoxic flow cytometric crossmatch (cFCXM) that determines cell death and antibody binding simultaneously was developed. The assay was validated in parallel with extended incubation CDC-XM. Receiver operating characteristic analysis was used to determine the cut-off level. Furthermore, pretransplantation sera from seven recipients with pretransplantation donor-specific antibodies (DSA) and negative CDC-XM were retrospectively tested for cFCXM (4 without antibody-mediated rejection (AMR) and three with AMR). The optimal method for the simultaneous detection of antibody binding and cytotoxicity in a single assay has been determined. Four of four patients (100%) with pretransplantation DSA and without AMR had negative cFCXM in both parameters. Of three patients with pretransplantation DSA who developed AMR, two patients (66.7%) had positive B-cell cFCXM in both parameters, and 1 patient (33.3%) had positive T-cell cFCXM in a binding parameter only. The first patient had anti-DR9, DR53, DQ9, the second patient had anti-A11, DR12 and the last one had an anti-B46 in their pretransplantation sera. These 3 cases experienced biopsy-proven AMR after living-donor kidney transplantation. The newly developed assay, cFCXM, can simultaneously determine cytoxicity and antibody binding using a single platform. Furthermore, this assay can detect clinically significant HLA alloantibodies undetectable by conventional crossmatches. The cFCXM could serve as a new tool for the detection of a recipient's alloantibodies.

Thammanichanond D ，Athimang W ，Paisooksantivatana K ，Mongkolsuk T ，Ingsathit A ，Worawichawong S ，Kitpoka P ，Jirasiritham S ，Kantachuvesiri S ... -  《-》

Preformed complement-activating low-level donor-specific antibody predicts early antibody-mediated rejection in renal allografts.

Lawrence C ，Willicombe M ，Brookes PA ，Santos-Nunez E ，Bajaj R ，Cook T ，Roufosse C ，Taube D ，Warrens AN ... -  《-》

Reappraisal of HLA antibody analysis and crossmatching in kidney transplantation.

Lee PC ，Ozawa M 《-》

Cytotoxicity and antibody binding by flow cytometry: a single assay to simultaneously assess two parameters.

Saw CL ，Bray RA ，Gebel HM 《-》

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

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. 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. 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). 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.

Won DI ，Jeong HD ，Kim YL ，Suh JS ... -  《CYTOMETRY PART B-CLINICAL CYTOMETRY》