Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis

Abstract

Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA) of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry was used to detect CD3+ T cells, CD38+ plasma cells and CD68+ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments, and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland-Altman plots showed no systemic differences between measurements. The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes (R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials.

Figure 1

Acquisition, analysis and output for a digital image analysis system. Acquisition and analysis of immunohistochemical staining of CD3⁺ T lymphocytes in synovial tissue using a digital image analysis system [10]. Three different areas from each biopsy sample, which are representative of the whole tissue section are selected. During analysis, staining thresholds are set for primary staining (i.e. CD3⁺ T lymphocytes), nuclear staining and background staining. The output is generated in a spreadsheet as the total number of positive cells per square millimetre of synovial tissue.

Figure 2

Mean change in number of positive cells versus the difference in change in positive cells. Shown are scatter plots of the mean change in the number of positive cells versus the difference in change of positive cells between two measurements by observer 1 for (a) CD3⁺ lymphocytes, (b) CD38⁺ plasma cells and (c) CD68⁺ macrophages. The dotted line represents the mean ± 2 × standard deviation.