One‐tube HLA‐B27/B2708 typing by flow cytometry using two “Anti‐HLA‐B27” monoclonal antibody reagents

Background: Flow cytometry‐based methods are widely used to detect the ankylosing spondylitis‐associated HLA‐B27/B2708 antigens. However, the generally used “HLA‐B27” monoclonal antibodies (moabs) cross‐react with many HLA specificities, including the common HLA‐B7 antigen. Thus, using two “B27” moabs is highly recommended. Methods: The assay used two “HLA‐B27” reagents, FITC and PE conjugated, respectively and a PE‐Cy5 anti‐CD3 antibody. Assay verification used 51 reference subjects possessing B*2705, B*2702, and B*2708 and a range of cross‐reactive HLA antigens. A total of 1,006 consecutive patients' samples, referred for “HLA‐B27 typing”, were assayed alongside our standard flow cytometry method. A further 12 low frequency HLA‐B*27 specificities were tested. Samples reacting with one “B27” moab only were B*27 allele typed by PCR using sequence‐specific primers. Results: All patient B27/B2708 positives (28.3%) were identified by our one‐tube method which detected B*2705, B*2702, B*2708, and 8/12 other B*27 specificities. It was unaffected by HLA‐B7 and other cross‐reactive antigens but required a minor adjustment, a reduction in the volume of one of the “B27” moabs used, to avoid detecting a minority of HLA‐B57 subjects. Conclusions: Our one‐tube B27/B2708 assay is simple, robust, uses two “B27” moabs for typing precision and security, does not suffer from interference by HLA‐B7 or other cross‐reactive antigens and has the obvious advantage of using a single tube per typing. © 2009 Clinical Cytometry Society