An optimized method for routine HLA‐B27 screening using flow cytometry

Abstract

Flow cytometry and monoclonal antibodies are promising tools for HLA‐antigen detection. Previous approaches have been hampered by the lack of 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, calibration beads, and the anti‐HLA‐B27‐FITC/anti‐Leu4‐PE (CD3) monoclonal antibodies. The median fluorescence channel result for the HLA‐B27‐FITC signal of CD3+ T lymphocytes is compared to a decision marker. Values lower than this threshold are read as HLA‐B27 negative and those above are recommended for retesting with the classic microcytotoxicity assay on the presumption of HLA‐B27 positivity. The anti‐HLA‐B27 antibody reacts with all six HLA‐B27 subtypes and shows a weaker binding to HLA‐B7. The screening test results were compared with those from the microcytotoxicity assay for HLA‐typing in studies involving several European centers. The observed sensitivity was 100% (95% Cl: 98.6–100) and the specificity was 97.4% (95% Cl: 96.4–98.3). Other performance studies verified the reproducibility and reliability of results obtained with the screening system. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc.