Establishment of a flow cytometric platelet crossmatching assay and its clinical application in platelet refractoriness

Platelet refractoriness is caused by antibodies against human leukocyte antigens or human platelet antigens. However, readily applicable assays that assist in selecting immunologically compatible platelet units remain limited. We established a flow cytometric platelet crossmatching assay and assessed its clinical utility by interpreting the results in conjunction with post‐transfusion corrected count increments (CCI). Platelets were incubated with serum that may contain anti‐platelet antibodies. Using flow cytometry, CD41‐expressing platelets were gated, and the median fluorescence intensity (MFI) of fluorescein isothiocyanate (FITC)‐conjugated anti‐human IgG was measured. The MFI ratio was calculated as test sample MFI divided by baseline negative control MFI. The cutoff value and limit of detection (LoD) were estimated. Platelet crossmatching was performed using residual segments of transfused platelet units and patient serum, and the results were retrospectively interpreted in conjunction with CCIs and clinical findings. The MFI ratios were clearly distinguishable among the three groups: high‐positive controls, low‐positive controls, and known negative samples ( p < 0.001). The cutoff value was calculated to be 1.35, and the LoD was 1.53. In total, eight platelet transfusion events in five patients were analyzed. Four cases were interpreted as non‐immune refractoriness, and the remaining four showed adequate post‐transfusion platelet increments. All corresponding platelet crossmatching results were negative, which was considered appropriate given that the assay is designed to reflect immune refractoriness. The flow cytometric platelet crossmatching assay was established and demonstrated to be applicable. The assay can help predict transfusion outcomes in alloimmunized patients and contribute to the selection of compatible blood units.