Aim Rituximab (Rtx) is a humanized monoclonal antibody (Ab) to CD20 receptor expressed on B cells. Sera from patients who have received Rtx will have positive B-cell crossmatches (XMs) challenging the detection of clinically relevant Abs. There is a commercially available Ab advertised as blocking the binding of Rtx to CD20. We wanted to test if the addition of this anti-Rituximab (anti-Rtx) Ab to our XMs will prevent false positive (FP) results, allowing the detection of potential donor specific Abs (DSA). Methods Negative control (NCS) and patient sera were spiked or not with Rit. Samples were incubated or not with anti-Rtx Ab (MB2A4, GeneTex) for 20 min. at known concentrations. These mixtures were used for XMs (flow cytometric (FC) and complement dependent cytotoxic (CDC)). Our FCXM uses a 256 channel scale. B-cell FCXM MCS above 9 is weak positive and above 20 is positive. Results Addition of anti-Rtx blocked Rtx binding to B-cells: NCS spiked with Rtx causes positive B-cell XMs, which were negative in the presence of anti-Rtx (Table 1.A). Anti-Rtx prevented FP B-cell XMs in two samples from patients known to have received Rtx (Table 1.B). Anti-Rtx blocks Rtx in a dose-dependent manner. Anti-Rtx allowed the detection of DSA: A serum with anti-DQ2 Abs (4000 MFI) was spiked with Rtx and incubated with anti-Rtx before crossmatching with cells expressing DQ2 antigen or not. The addition of anti-Rtx converted the XM with DQ2-negative cells to negative, but the XM with DQ2-positive cells was weakly positive, as was the XM performed with untreated serum (Table 2). Conclusions Treatment of the sera of patients under Rtx immunotherapy with anti-Rtx before FC and CDC XMs prevents FP results and allow the detection of clinical relevant Abs. Download : Download high-res image (183KB) Download : Download full-size image Download : Download high-res image (97KB) Download : Download full-size image
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