HLA‐B27 screening by flow cytometry

Abstract

A flow cytometric assay for lymphocyte HLA‐B27 expression using a two‐color direct immunofluorescent assay was compared to traditional microlymphocytotoxicity testing on 209 clinical samples. For the flow cytometric assay, whole blood was mixed with a monoclonal anti‐B27 conjugated to fluorescein‐isothiocyanate (FITC) and anti‐CD3 conjugated to phycoerythrin (PE). The samples were analyzed with flow cytometry by gating on CD3 positive events and anti‐B27 staining intensity was evaluated as median channel fluorescence of the histogram peak. The median channel fluorescence was least with B27 negative and B7 negative samples (84 ± 17), intermediate with samples that were B27 negative but B7 positive (118 ± 13), and greatest with samples that were B27 positive (155 ± 13). In addition to cross‐reactivity with the B7 antigen (n = 38), the monoclonal anti‐B27 cross‐reacted with HLA‐B37 positive samples (n = 3) and HLA‐B39 positive samples (n = 3). Using a median channel fluorescence cutoff of 136, 39 of the 40 B27 positive samples gave positive results in the flow cytometric assay for a sensitivity of 97.6%. The specificity was 95.9% with 7 false positives of 169 B27 negative samples. The flow cytometric HLA‐B27 assay is a convenient, useful screening test. For greatest specificity, samples positive by flow cytometry should be confirmed by conventional microlymphocytotoxicity or by use of other monoclonal antibodies directed against B27. © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc.