Flow cytometric detection of complement-activating HLA antibodies

Background: Antibodies to HLA are implicated in allograft rejection; as a consequence, tests to identify them are indicated in organ transplantation. Flow cytometry (FC) may improve the outcome of organ transplantation. Although increasingly used, most of the available FC techniques do not discriminate between complement and noncomplement-activating antibodies. However, the clinical relevance of noncomplement-activating antibodies is controversial. Therefore, many laboratories still use the conventional complement-dependent microlymphocytotoxicity crossmatch (CDC-XM) alone or in combination with FC. Methods: We describe a novel flow cytometric complement-dependent cytotoxicity crossmatch assay (FC-CDC-XM) that clearly identifies complement-binding HLA antibodies. With the aid of a three-color FACScan flow cytometer with a standard argon ion laser, we used fluorescence-conjugated anti-CD3 for the identification of T lymphocytes, anti-CD 19 antibodies for the identification of B lymphocytes, and propidium iodide (PI) for the identification of damaged cells. Fluorescence-conjugated anti-CD3 and anti-CD19 antibodies were detected in the green (FL1) and orange (FL2) ranges of the spectrum. PI fluorescence was measured with the red (FL3) fluorescence channel. Gating of lymphocytes was performed using side scatter versus FL1 or FL2 dot plots for the identification of T and B lymphocytes, respectively. Results: FC-CDC-XM was able to correctly identify all antibodies tested as specific to HLA class I (n = 18) or II (n = 4). Conclusions: The new FC-CDC-XM is more sensitive and more objective than the standard CDC-XM and can be used for pre- and posttransplant diagnoses and for the detection of other antibody-mediated cytotoxic reactions. (C) 2004 Wiley-Liss, Inc.