title: HLA-B27 detection - comparison of genetic sequence-based method and flow cytometry assay.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Skalska
authors.affiliation: Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kozakiewicz
authors.affiliation: Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland.
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Maśliński
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Rheumatology, Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Jurkowska
keywords: DNA sequencing; HLA-B27; allelic variants; flow cytometry