EDTA Treatment of Serum Reduces T Cell and B Cell False Positivity in Flow Cytometry Crossmatching: A Multicenter...

False positive T or B cell flow cytometry crossmatching (FCXM) results present a significant challenge in transplantation decisions. In this multicentre study, we investigated the efficacy of ethylenediaminetetraacetic acid (EDTA) treatment of serum samples to enhance the quality of FCXM results by reducing false positivity at two independent laboratories. Our findings indicate that EDTA treatment of serum effectively eliminated false positivity in B cell non-pronased (NP) FCXM compared with untreated samples, possibly by lowering aggregated IgG formation from in vitro storage of patient sera. In B cell NP FCXM, EDTA-treated serum from both sensitised and non-sensitised patients exhibited comparable efficacy to B cell pronase treated (PT) FCXM, with a sensitivity of 94% and specificity of 96%. Moreover, EDTA-treated serum samples demonstrated superior performance to T cell PT FCXM, effectively reducing false positivity. In addition, EDTA-treated sera performed in par with sera pre-treated with 2-mercaptoethanol, and superior to sera treated with Dithiothreitol or with heat inactivation, in removing false B cell positivity in NP FCXM. Importantly, our analysis of 124 FCXM underscores the simplicity and effectiveness of EDTA treatment, which can be seamlessly integrated into both NP and PT FCXM. Consequently, the adoption of EDTA-treated patient serum in B cell NP FCXM reduces the need for B cell PT FCXM.