Variability of RANKL and osteoprotegerin staining in synovial tissue from patients with active rheumatoid arthritis: Quantification using color video image analysis

Objective. To assess the interpatient, interbiopsy, and intrabiopsy variability of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) immunostaining within synovial tissue from rheumatoid knee joints with active synovitis, using digital image analysis. Methods. Synovial biopsy specimens were obtained from patients with rheumatoid arthritis (RA) and active synovitis. Immunohistologic analysis was performed on frozen synovial tissue biopsy specimens from 6 patients using a monoclonal antibody (Mab) to detect RANKL (626) or OPG (805 or 805 1). Patients with a minimum of 4 synovial biopsies were included in the study. Sections were evaluated by computer assisted image analysis to assess between-patient, between-biopsy, and intrabiopsy variability of OPG and RANKL protein expression. The study was designed to deliberately maximize the variability. Results. Computerized image analysis of staining with Mab to RANKL and OPG revealed variance for each antibody across the 3 components of the total variability. Conclusion. Our study shows that variability in synovial immunostaining of RANKL and OPG protein is a significant and complex problem. We discuss methods to reduce this. variability and suggest that the auspices of OMERACT may be employed to advance the study of synovium in collaborative international studies.