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 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.


CONCLUSIONS
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.
Crotti
Tania N
TN
Rheumatology Unit, Repatriation General Hospital, Daw Park, Adelaide, South Australia, Australia.
Ahern
Michael J
MJ
Lange
Kylie
K
Weedon
Helen
H
Coleman
Mark
M
Roberts-Thomson
Peter J
PJ
Haynes
David R
DR
Smith
Malcolm D
MD
D000368:Aged; D000911:Antibodies, Monoclonal; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003116:Color; D005260:Female; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008875:Middle Aged; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D013194:Staining and Labeling; D013583:Synovial Membrane / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human