AbstractBackgroundSome 50 clinical laboratories in the Benelux perform flow cytometric HLA‐B27 screening and participate in the Benelux external quality assessment scheme operational since 1995. Results from this scheme indicate that cross‐reactivity of HLA‐B27 monoclonal antibodies (mAbs) is a major problem.MethodsWe analyzed cross‐reactivity patterns of commercially available mAbs for HLA‐B27 screening. Three clones of HLA‐B27 mAb (ABC‐m3, n = 3; FD705; and GS145.2) from five manufacturers were evaluated. Test cells were selected as to express HLA‐B antigens with known serologic cross‐reactions (HLA‐B7, B12, B13, B16, B17, B22, B37, B40, B41, B42, B47, and B48). Cells without B27 cross‐reactive antigens (B5, B8, B14, B15, B21, and B35) and cells positive for B27 were included as controls. All tests were performed and interpreted as recommended by the manufacturers. Cross‐reactivity was defined as increased fluorescence intensity in comparison with the baseline reactivity observed with the corresponding immunoglobulin G isotype control mAb.Results and ConclusionsAll mAbs tested showed cross‐reactivity, ranging from weak (±) to strong (+), with different antigens and different degrees of intensity—ABC‐m3: (±) B12, B16, B17, B41, B47, and B48 and (+) B7, B13, B22, B37, B40, and B42; GS145.2: (±) B13, B17, B22, B40, and B47 and (+) B7, B16, B37, B42, and B48; FD705: (±) B12, B13, B16, and B48 and (+) B17, B37, and B47. If one mAb had been used for HLA‐B27 screening, ABC‐m3 would have yielded nine false‐positive B27 assignments, FD705 would have yielded seven, and GS145.2 would have yielded two. This problem largely canbe avoided by the combined use of two different mAb clones. The combination of FD705 and GS145.2 yielded the best results, with one false‐positive HLA‐B27 assignment among the 99 HLA‐B27− samples of this highly selected panel. Cytometry Part B (Clin. Cytometry) 54B:28–38, 2003. © 2003 Wiley‐Liss, Inc.