Optimization and validation of a flow cytometry‐based HLA ‐ B27 assay on BD FACSLyric

The BD™ HLA-B27 GS145.2-based assay for flow cytometric HLA-B27 typing has been validated on earlier-generation cytometers but not on the increasingly adopted BD FACSLyric platform, highlighting the need for platform-specific validation. While prior transfer strategies relied on conversion of the source instrument's parameters, we performed a de novo analytical validation of the HLA-B27 Kit on the FACSLyric. Our strategy encompassed determination of the assay's gray zone and rigorous evaluation of analytical performance following CLSI H62 guidelines. A total of 125 patient samples were analyzed in parallel by real-time PCR (RT-PCR) and flow cytometry. Sixteen patients (12.8%) were HLA-B27 positive by RT-PCR. A ROC-derived mean fluorescence intensity (MFI) cut-off of 6627 achieved 100% sensitivity and 99.1% specificity. The empirically defined gray zone (6627-7073) was narrow, encompassing only two samples. To strengthen analytical robustness, bootstrapping was applied to derive a 95% confidence interval around the cut-off, yielding an expanded gray zone of 5779-7327. Using this interval, 7 samples (5.6%) were classified as equivocal, comprising four HLA-B27-positive cases and three samples carrying cross-reactive alleles (HLA-B*42, HLA-B*37, and HLA-B*44). Outside the gray zone, flow cytometry showed complete concordance with RT-PCR. MFI values above 7327 ensured unequivocal HLA-B27 positivity with 100% specificity. Precision was high (intra-assay 5.43%, between-run 1.84%, between-day 5.13%), with no carryover and reliable typing up to 24 h post-collection. These results establish a robust, reproducible, and scalable framework for accurate first-line HLA-B27 detection on the FACSLyric platform.