Measurement of cytokine and adhesion molecule expression in synovial tissue by digital image analysis

Objective-Digital image analysis (DIA) offers the opportunity to quantify t he stained area and staining intensity when synovial tissue (ST) is investi gated by immunohistochemical analysis. This study aimed at determining the sensitivity of DIA compared with semiquantitative analysis (SQA). Methods-Paired ST samples were obtained from the knee joint of 10 patients with rheumatoid arthritis (RA) with active disease and after follow up when complete clinical remission was achieved. ST samples of 10 subjects with n oninflammatory knee pain served as controls. Immunohistochemistry with anti bodies against interleukin 1 beta (IL1 beta) and vascular cell adhesion mol ecule 1 (VCAM-1) was applied using two staining proto cols with 3-amino-9-e thylcarbazole (AEC) or p-diethylaminobenzaldehyde (DAB) as dye. All section s were analysed semiquantitatively (0-4) and DIA of up to a maximum of 60 h igh power fields (HPF). The average integrated optical density was calculat ed as the product of the stained area (corrected for total tissue area) and the optical density. Results-Both SQA and DIA enabled the assessment of differences in IL1 beta and VCAM-1 expression between ST from active PA, RA in remission, and contr ols. SQA and DIA showed excellent correlations (IL1 beta r(s)=0.867; p<0.00 01: VCAM-1 r(s)=0.828; p<0.0001). A limited analysis of one region with six HPF still allowed adequate discrimination compared with an extended analys is of three regions with a total of 60 HPF. In general, the red dye (AEC) r esulted in better discrimination than the brown (DAB) staining. Conclusion-DIA offers a reliable, reproducible, and sensitive analysis of S T sections stained for cytokines and adhesion molecules.