LBP05: B CELL POSITIVE FLOWCYTOMETRY CROSS MATCH DUE TO DONOR SPECIFIC ANTIBODIES OF IgM ISOTYPE

Abstract

Aim Analysis of HLA specific antibodies (Ab) is an important component of monitoring of transplant recipients (TR). It includes Ab concentration, epitope specificity, isotype and ability to bind complement. In this study, we have reported positive B lymphocyte flow cytometry cross match (FC CM) owing to donor specific Ab (DSA) of IgM isotype directed to HLA-DQ7. Methods Analysis of anti-HLA Ab was performed in DTT treated serum using single antigen bead (SAB) Luminex technology. Complement binding activity of DSA was investigated using C1q solid phase assay (One Lambda, Inc). PCR-SSOP method was used for HLA typing (One Lambda, Inc). CM of TR serum with donor lymphocytes was performed in the following modifications: T cells CDC-AHG (±DTT), B cells CDC x4 washes (± DTT). B cell CM was performed at 4 °C and room temperatures. FC CM was performed as tri-color assay using secondary FITC-conjugated Ab to human IgG or IgM. In order to block falsely positive reactivity due to surface IgM on the B cells, blocking titration assay using unlabeled anti-human IgM was performed. Results Second kidney TR was typed: A2, 2; B44, 60; DR4, 13; DQ8, 6; DR53, 52. First living donor (LD) was typed A2, 25; B18, 60; DR4, 13; DQ7, 6; DR53,52, and current LD was typed A2,68; B65,60; DR13,13; DQ7,6; DR52,52. At the time of workup broad specificity of anti-Class I Ab was detected, whereas anti-class II reactivity was represented by anti-DQ7 Ab only. The strongest blocking activity of unlabeled anti-human IgM Ab was observed at 40.0 μg/ml concentration. The results of CM assays and Ab analysis are presented in the Table 1 below. a – MFI, mean fluorescence intensity, b – ASHI score, c – MCS, median channel shift, d – tested by Luminex screening IgM assay. Conclusions These findings account for B cell IgG negative CDC CM results despite the high level of DSA. We conclude that under certain circumstances detection of DSA of IgM isotype may provide new insights regarding transplantation in patients presenting with negative B-cell CDC and positive FC CM.