Frequent False-Positive Reactions in Pronase-Treated T-Cell Flow Cytometric Cross-match Tests

Abstract Objective Pretransplantation cross-match (XM) is essential in organ transplantation. The flow cytometric XM (FCXM) is the most sensitive cell-based XM technique. Pronase treatment is used to improve the sensitivity and specificity of the B-cell FCXM. Thus, pronase-treated (PT) T cells are tested in a single tube T-cell/B-cell technique. Observing discrepancies between PT and pronase–nontreated (PN) T- FCXM results, we investigated their incidence, clinical significance, and possible causes. Methods We tested 226 serum samples from 167 kidney transplantation candidates or posttransplantation follow-up patients using PT and PN T-FCXM in parallel using 3-color and 2-color immunofluorescence staining, respectively. We reviewed panel-reactive antibody (PRA) and donor-specific antibody (DSA) status as well as HLA data and clinical outcomes. Results The T-FCXM positive rate was significantly higher among PT versus PN tests (24.3% vs 11.1%; P < .001). Less than half of the PT-positive cases were positive in the PN test (45.5%; 25/55). Discrepancies were observed in 30 cases (13.3%), all of which gave PT(+)/PN(−) results. Our findings suggested that PT(+)/PN(−) results might arise from non-HLA antibodies. Class I DSA-positive rate (6.3% vs 2.2%; P = .45) and antibody-mediated rejection rate (0% vs 16.3%; P = .32) were not different between PT(+)/PN(−) and PT(−)/PN(−) groups. Moreover, 2 cases of PT(+)/PN(−) were observed among HLA-A, B, DR–identical donor-recipient pairs. Conclusion Pronase treatment is prone to give false-positive reactions in T-FCXM test probably due to the participation of non-HLA antibodies including autoantibodies. Patients might be inappropriately excluded from receiving organs. In laboratories using PT single tube T/B FCXM, caution is needed to avoid false-positive reporting of results.