Variability of RANKL and Osteoprotegerin Staining in Synovial Tissue from Patients with Active Rheumatoid Arthritis: Quantification Using Color Video Image Analysis

TANIA N. CROTTI, MICHAEL J. AHERN, KYLIE LANGE, HELEN WEEDON, MARK COLEMAN, PETER J. ROBERTS-THOMSON, DAVID R HAYNES, and MALCOLM D. SMITH

ABSTRACT.

Objective. To assess the interpatient, interbiopsy, and intrabiopsy variability of receptor activator of nuclear factor kB 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 8051). 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 intra-biopsy 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. (J Rheumatol 2003;30:2319-24)

Key Indexing Terms:

BONE
RHEUMATOID ARTHRITIS
OSTEOPROTEGERIN
RECEPTOR ACTIVATOR OF NUCLEAR FACTOR kB LIGAND
QUANTITATIVE ANALYSIS
IMMUNOHISTOCHEMISTRY

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From the Rheumatology Unit and Department of Pathology, Repatriation General Hospital, Daw Park, and Department of Pathology, The University of Adelaide, Adelaide, Australia.

Supported by grants from the National Health and Medical Research Council, Department of Veteran Affairs, Arthritis Foundation of Australia, the Clive and Vera Ramaciotti Foundation, the J.H. and J.D. Gunn Foundation, and the Rebecca L. Cooper Medical Research Foundation.

T.N. Crotti, B Health Sc, Hons, Research Fellow, University of Adelaide; M.J. Ahern, MD, FRACP, Associate Professor in Medicine;

K. Lange, BSc (Ma and Comp Sciences), Hons, Statistician, Flinders University of South Australia, Adelaide; H. Weedon, Research Assistant; M.D. Smith, PhD, FRACP, Professor in Medicine, Rheumatology Research Unit, Repatriation General Hospital; M. Coleman, MB, BS, FRCPA, Deputy Director, Department of Pathology; P.J. Roberts-Thomson, DPhil (Oxon) FRACP, Head of Immunology, Department of Immunology, Allergy and Arthritis, Flinders Medical Centre; D.R. Haynes, Bch Sc, PhD, Senior Lecturer, University of Adelaide, Adelaide, South Australia

Address reprint requests to Prof. M.D. Smith, Department of Medicine, Flinders Medical Centre and Repatriation General Hospital, Adelaide, South Australia. E-mail: malcolm.smith@rgh.sa.gov.au

Submitted August 8, 2002; revision accepted April 14, 2003.

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