HLA-B27 testing: A journey from flow cytometry to molecular subtyping.

ABSTRACT

BACKGROUND: Determination of HLA-B27 status plays an important role as adjuvant in suspected cases for diagnosis of Ankylosing Spondilytis (AS). Objectives of this study were to evaluate (i) flow cytometry method in comparison with DNA microarray for HLA-B27 typing and (ii) EUROArray HLA-B27 Direct assay for HLA-B27 allele detection along with discrimination of AS/non-AS subtypes in Indian population. METHODS: A total of 7543 patients with a presumptive clinical diagnosis of AS were referred for screening of HLA-B27. All samples were initially tested by flow cytometry, and based on its findings, 1560 samples were analyzed for the presence of HLA-B27 allele by microarray technology. A subset of samples (n = 200) were further tested by DNA sequencing for identification of HLA-B27 subtypes. RESULTS: Screening of HLA-B27 by flow cytometry reported 1551 positive (20.56%) and 5556 negative (73.65%) cases. Remaining 436 (5.78%) samples were identified within equivocal zone. Of cases (n = 1560) analyzed by microarray method, 1333 (85.44%) and 227 (14.55%) were detected microarray positive and negative, respectively. DNA sequencing identified HLA-B*2707 as the predominant subtype among cases showing ex2 positivity by microarray method. Of 200 cases, 20 cases (14 of HLA-B*07 and 6 of HLA-B*37) of HLA-B27 cross-reactive subtypes were also identified. CONCLUSION: We recommend DNA typing as a complementary tool along with flow cytometry to accomplish successful HLA-B27 phenotype determination. This is the first study among Indian population to evaluate efficacy of EUROArray to detect B27 allele and its potential to indicate the presence of nondisease-associated alleles in Indian population.