Rheumatology (May 2015)
Original paper HLA-B27 detection – comparison of genetic sequence-based method and flow cytometry assay
Abstract
Objectives : The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection – a genetic sequence-based method and a flow cytometry assay. Material and methods : Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by flow cytometry assay using GS145.2 monoclonal antibody specific for HLA-B27. DNA was isolated from the whole blood. Genes coding for HLA-B27, -B40 and -B47:01 were detected by polymerase chain reaction using the MW02/MW09 primer pair. Then, positive samples were sequenced in order to discriminate allelic variations of the HLA-B27 gene. Results of sequencing were analysed using Chromas LITE 2.1.1 software, BLAST software and the IMGT/HLA database. Ambiguous samples were additionally analysed by polymerase chain reaction using E91 and E136 primers amplifying a 135-bp fragment of the human HLA-B27 gene. Results : Among 300 samples, 76 were HLA-B27-positive on the basis of flow cytometry analysis. Genetic sequence analysis confirmed positivity of 73 from among 76 samples. Two hundred twenty six samples were HLA-B27-negative, whereas the result of one sample analysis was ambiguous. Fifty-three samples were identified as allelic variation 27:05, 19 samples as allelic variation 27:02, and one sample as allelic variation 27:07. Conclusions : This study shows that the genetic sequence-based method and the flow cytometry assay give consistent results in 99% of cases. The performed genetic analysis proves that the majority of HLA-B27-positive samples belong to the 27:05 allelic variation, which is strongly associated with high risk of ankylosing spondylitis.
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