Cut-off determination of HLA crossmatching by flow cytometry

Background: Microlymphocytotoxicity test is a gold standard for HLA crossmatching but provides low sensitivity. Emerging flow cytometry crossmatching (FCXM) provides more sensitivity, however, suitable cut-off values for FCXM must still be established for crossmatching result interpretation. Objectives: This study aimed to determine the appropriate cut-off value for FCXM in kidney transplant patients and donors. Materials and methods: A total of 30 sample pairs with known HLA typing and known antibody specificity were analyzed. Median channel fluorescence (MCF) and median channel fluorescence shift (MFS) were used to determine cut-off value using three different approaches and % accuracy was determined. Results: The result showed that the cut-off values from first approach using lowest MCF of positive control and highest MCF of negative control were 1655.99 MCF for T-cells (yielding 100% accuracy) and 43668.06 MCF for B-cells (yielding 36.67% accuracy). The cut-off values from second approach which calculated from standard deviation (SD) of negative control MCF showed SD of T-cells MCF as 203.81 (100% accuracy) and SD for B-cells MCF as 11109.30 (60% accuracy). The cut-off values from third approach derived from the lowest MFS of the expected positive results were 1753.50 for T-cells (100% accuracy) and 593.50 for B-cells (73.33% accuracy). Conclusion: The cut-off values from the third approach exhibited the highest accuracy. However, FCXM still had false positives and false negatives, underscoring the necessity for continuous refinement and adjustment of cut-off values based on the data accumulated within the laboratory to be used further for routine laboratory practice.