P033 The effect of pronase on B-cell flow cytometry crossmatch results: A case study

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Cross matching is crucial in pre-organ transplantation and FCXM is the most sensitive cell-based XM technique. Routinely in our laboratory, we pre-treat the cells with pronase to improve the sensitivity and specificity of the B-cell FCXM. Pronase treatment of B-cell is used to disrupt the Fc and CD20 to minimize nonspecific binding of immunoglobulins and interference with Rituximab respectively. Pronase-treated T cells are tested in a single tube T-cell/B-cell technique and it has been shown that pronase treatment is likely to give false-positive reactions in the T-FCXM test. T cells express fewer Fc receptors than B cells for that, pronase may expose cryptic epitopes. This phenomenon of falsely incompatible T-cell FCXM with pronase in the absence of DSA has been well documented and has been frequently seen in our laboratory. In this study, we are presenting a case of incompatible B-cell FCXM after pronase treatment. An 18-year-old Caucasian male with an end-stage renal disease secondary to Joubert syndrome was considered for renal transplantation. Prior antibody screening and single-antigen bead testing disclosed anti-HLA class I antibodies with 6% cPRA and the absence of anti-HLA class II antibodies. His mother was the potential living related donor and the recipient did not carry any DSA against his mother HLA antigen. Crossmatch was performed between the donor lymphocytes and the patient serum after incubating the isolated lymphocytes with pronase. An initial FCXM showed unexpected B-FCXM incompatibility with both current serum and a historic serum. The concurrent T-FCXM was compatible. One month later, we repeated the XM with and without pronase-treatment. Unexpectedly, pronase treatment caused incompatible B-FCXM, whereas non-treated cells were B-FCXM compatible. In all cases, the T-FCXM was compatible. The take-home message of that case that cautious should be taken in clinical labs using pronase treatment in single tube T/B FCXM, to avoid false-positive reporting of results and alternative methods for blocking Fc receptors in FCXM should be explored.