It's About Everything: Validation and Optimization of 96-Well Tray Flow Cytometry Crossmatch

Flow cytometric crossmatch (FCXM) is widely used in many centers as part of pretransplant risk assessment to detect donor-specific anti-HLA antibodies. The limited number of crossmatches that can be performed during on-call work-up for deceased donors within reasonable time remains the main obstacle to accommodating the majority of highly sensitized listed patients to be tested by the standard tube FCXM method. This limitation often directs the organs to nonsensitized patients and deprives highly sensitized patients who could be compatible if their sera were included in the crossmatch test. The goal of this study is to optimize a 96-well tray FCXM protocol that allows more sera to be crossmatched without prolonging the overall procedural time while maintaining quality and sensitivity of the assay. The new method was validated against use of the standard tube method and included total of 63 crossmatches performed simultaneously by both methods using 20 donors' cells with patients' sera, pooled positive controls tested on different dilutions, and commercial negative control. In the new protocol we modify various assay parameters including tube platform, incubation time, amount of reagent antibody cocktail, and cell volumes. An overall concordance of 98% was achieved with the protocols with slight improvement in sensitivity (2 negative B-cell reactions converted to positive in presence of weak donor-specific antibodies and mild T-cell reactivity could be picked up at 1:80 diluted positive control by the tray method only). The median channel fluorescence values of the 2 methods were essentially equivalent for both T and B crossmatches (r² of 0.98 and 0.97, respectively). In conclusion, 96-well tray assay has the potential to increase the probability of highly sensitized patients receiving transplants by allowing increased number of crossmatches to be performed with significant reduction in turnaround time and assay cost. Furthermore, the enhanced sensitivity of the assay will provide more accurate information about sensitization status and strength of donor-specific antibodies to treating physicians, allowing them to choose the best therapeutic option and to provide better patient care.