Validation and correlation of flow cytometric analysis of human leucocyte antigen-B27 in spondyloarthritis

Abstract

Spondyloarthritis (SpA) refers to a group of diseases primarily affecting the axial skeleton and the peripheral joints, characterised by common clinical manifestations and a genetic predisposition to human leucocyte antigen-B27 (HLA-B27). While the significance of HLA-B27 in SpA is well-recognised, its exact pathogenic role remains unclear. In fact, there are three hypotheses of significance which attempt to explain this role: the arthritogenic peptide hypothesis, HLA-B27 misfolding and unfolded protein response hypothesis, and the HLA-B27 homodimer hypothesis. Apart from understanding the underlying mechanisms by which HLA-B27 contributes to the development of SpA, detection of this genetic marker plays a crucial role in the diagnosis of this disease. However, the lack of local testing necessitates the referral of samples to a reference laboratory overseas, resulting in delays to diagnosis and management. The study aimed to validate the use of flow cytometry for the local HLA-B27 detection using the BD FACSCantoTM II flow cytometer, with results compared against the polymerase chain reaction (PCR) method performed at a reference laboratory. The two methods demonstrated complete concordance, with flow cytometry offering significantly reduced turnaround times. During the course of the study, the flow cytometric assay was adapted to a newly introduced flow cytometer platform, BD FACSLyricTM which required the establishment of a new cut-off value. A single discordant result was identified when comparing the results of the two flow cytometers, underscoring the need for further validation. Correlative analyses revealed no statistically significant associations between HLA-B27 expression and gender, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Additionally, clinical findings indicated a higher prevalence of peripheral manifestations among female participants. Finally, magnetic resonance imaging (MRI) assessments revealed no apparent association between HLA-B27 expression and the severity of sacroiliitis.