Evaluation of Human Leukocyte Antigen-B27 Expression in Patients with Spondylopathy/Spondylitis by Flowcytometry

Abstract

Introduction: The role of immune mediated injury in pathogenesis of Ankylosing Spondylitis (AS) is well established. HLA B27, a Major Histocompatibility Complex (MHC) class I molecule is one of the major genetic risk factors associated with the disease. Various techniques are used for testing HLA-B27 which includes Polymerase Chain Reaction (PCR) based tests, Enzyme Linked Immunosorbent Assay (ELISA) and flowcytometry. Flowcytometry has gained popularity due to ease of procedure, shortened turnaround time and cost-effectiveness. Aim: The aim of the present study was to assess the sensitivity and specificity of flowcytometry for HLA-B27 detection, taking PCR assay as the gold standard along with its association with demographic, clinicopathological and radiological parameters. Materials and Methods: This was a prospective study conducted in Department at Pathology of Shri Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, India, for a period of 18 months from January 2020 to June 2021. The study included 51 patients for which HLA-B27 typing was done cases by flowcytometry and Sequence Specific Allele (SSA) PCR/Real time PCR on peripheral blood samples. The association of HLA-B27 with clinical features {Inflammatory Back Pain (IBP), arthritis, psoriasis, uveitis, dactylitis, Inflammatory Bowel Disease (IBD), cervicitis, urethritis, diarrhoea) along with MRI findings (sacroiliitis)}, laboratory findings {C-reactive protein and Erythrocyte Sedimentation Rate (ESR)} was evaluated. The performance analysis parameters of flowcytometry were evaluated both by excluding and including the cases in grey zone taking PCR as gold standard. Statistical testing was conducted with SPSS 20.0. Chi-square test or Fisher’s-exact test were used and a p-value of less than 0.05 was taken as significant. Results: A significant association of HLA-B27 was seen only with IBP (p-value= 0.001) and sacroiliitis (p-value= 0.03). Of the 22 (43.1%) patients positive for HLA-B27 by PCR, 18(81.8%) patients were positive while the remaining 4(18.1%) were in grey zone by flowcytometry. Of the 29(56.9%) patients testing negative by PCR, 27(93.1%) patients were negative, one (3.4%) was in grey zone and one (3.4%- false positive) tested positive for HLA-B27 by flowcytometry. Sensitivity and specificity of flowcytometry for detection of HLA-B27 was found to be 100% and 96.4% respectively when grey zone cases were excluded. Conclusion: The study brings to light that flowcytometry is a fairly specific and sensitive method for HLA-B27 detection with a high Negative Predictive Value (NPV) (100%) and Positive Predictive Value (PPV) (94.7%). In the COVID-19 era, it reiterates the importance of flowcytometry for HLA-B27 especially when PCR is overburdened.

Keywords

• immune mediated
• grey zone
• polymerase chain reaction
• diagnostic technique