AN OPTIMIZED METHOD FOR ROUTINE HLA-B27 SCREENING USING FLOW-CYTOMETRY

Flow cytometry and monoclonal antibodies are promising tools for HLA-a ntigen detection. Previous approaches have been hampered by the lack o f a carefully standardized system for calibration and sample analysis. A new system for HLA-B27 screening was developed using a FACScan flow cytometer, software for automated calibration and analysis, calibrati on beads, and the anti-HLA-B27-FITC/anti-Leu4-PE (CD3) monoclonal anti bodies. The median fluorescence channel result for the HLA-B27-FITC si gnal of CD3+ T lymphocytes is compared to a decision marker. Values lo wer than this threshold are read as HLA-B27 negative and those above a re recommended for retesting with the classic microcytotoxicity assay on the presumption of HLA-B27 positivity. The anti-HLA-B27 antibody re acts with all six HLA-B27 subtypes and shows a weaker binding to HLA-B 7. The screening test results were compared with those from the microc ytotoxicity assay for HLA-typing in studies involving several European centers. The observed sensitivity was 100% (95% CI: 98.6-100) and the specificity was 97.4% (95% CI: 96.4-98.3). Other performance studies verified the reproducibility and reliability of results obtained with the screening system. (C) 1994 Wiley-Liss, Inc.