Measurement of Cytokine and Adhesion Molecule Expression in Synovial Tissue by Digital Image Analysis

Objective: Digital image analysis (DIA) offers the opportunity to quantify the stained area and staining intensity when synovial tissue (ST) is investigated 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 non-inflammatory knee pain served as controls. Immunohistochemistry with antibodies against interleukin 1beta (IL1beta) and vascular cell adhesion molecule 1 (VCAM-1) was applied using two staining protocols with 3-amino-9-ethylcarbazole (AEC) or p-diethylaminobenzaldehyde (DAB) as dye. All sections were analysed semiquantitatively (0-4) and DIA of up to a maximum of 60 high power fields (HPF). The average integrated optical density was calculated 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 IL1beta and VCAM-1 expression between ST from active RA, RA in remission, and controls. SQA and DIA showed excellent correlations (IL1beta rs=0.867; p<0.0001: VCAM-1 rs=0.828; p<0.0001). A limited analysis of one region with six HPF still allowed adequate discrimination compared with an extended analysis of three regions with a total of 60 HPF. In general, the red dye (AEC) resulted in better discrimination than the brown (DAB) staining.

Conclusion: DIA offers a reliable, reproducible, and sensitive analysis of ST sections stained for cytokines and adhesion molecules.