*Purpose: Flow cytometric crossmatch test (FCXM) is critical to detect donor-specific antibodies (DSA) in transplant patients. Rapid, simple and sensitive FCXM procedure can improve HLA compatibility judgments and diagnosis of antibody mediated rejection. The aim of this study was to assess the optimized Halifax protocol in association with the results of virtual crossmatch (VXM) based on the class I single antigen bead (SAB) assay.

*Methods: Halifax FCXM protocol includes a 96-well tray platform, reduced wash times, increased serum to cell suspension volume ratio, shortened incubations, and higher incubation temperature. The cut-off value was determined as the median fluorescence intensity (MFI) ratio with 3 standard deviations using forty-five PRA-negative sera. Among 316 T-XMs performed, 102 cases were class I- VXM positive. Of 102 VXM (+) cases, 55 cases showed only one class I HLA antigen-positive for serum, and 47 cases showed at least two antigens-positive for serum. Normalized MFI values above 1,000 were defined as a positive-VXM.

*Results: Of 55 cases with class I VXM positive against one HLA antigen (26 HLA-A, 25 HLA-B, 4 HLA-Cw), 39 (70.9%) cases were T-FCXM positive (21 (80.8%) HLA-A and 18 (72.0%) HLA-B). T-FCXM positive cases had higher MFI values than T-FCXM negative cases (P <0.05). Class I-VXM (+)/T-FCXM (-) cases had class I MFI values ranged 1037-3440 (Table1). Overall T-FCXM using Halifax protocol revealed excellent correlation with VXM (81.4% and 98.1% sensitivity and specificity, respectively) for the identification of DSA (HLA-A/B/Cw) above 1000 MFI values.

*Conclusions: The Halifax FCXM protocol showed comparable performance to VXM based on SAB results. The application of rapid and simple new protocol in clinical laboratories can be effective in detecting sensitized patients with a shorter assay time.

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