Immunohistological assessment of the synovial tissue in small joints in rheumatoid arthritis: validation of a minimally invasive ultrasound-guided synovial biopsy procedure

Abstract

The aim of the present study was to perform an immunohistological assessment of the synovial tissue from involved small joints in rheumatoid arthritis (RA) and to explore the reliability of a mini-invasive ultrasound (US)-guided technique of small joint synovial biopsy for the histopathological assessment. Synovial tissue collected during arthrotomic surgery of small joints in nine patients served as the gold standard for the validation of the histological assessment. Small hand-joint synovial biopsies from an additional nine patients with erosive RA were obtained by a mini-invasive US-guided procedure, performed percutaneously by the portal and rigid forceps technique. Using digital image analysis, the area fractions of synovial macrophages (CD68 cells), T cells (CD3 cells) and B cells (CD20 cells) were measured in all high-power fields of every sample at different cutting levels. The representative sample was defined as the minimal number of high-power fields whose mean area fraction would reflect the overall mean area fraction within a percentage mean difference of 10%. For each patient, a range of three to five large samples for surgical biopsies and a range of 8-12 samples for US-guided biopsies were collected and analysed. In arthrotomic samples, the analysis of a randomly selected tissue area of 2.5 mm2 was representative of the overall value for CD68, CD3 and CD20 cells. US-guided samples allowed histological evaluation in 100% of cases, with a mean valid area of 18.56 mm2 (range 7.29-38.28 mm2). The analysis of a cumulative area of 2.5 mm2 from eight randomly selected sections (from different samples or from different cutting levels) allowed to reduce the percentage mean difference to less than 10% for CD68, CD3 and CD20 cells. In conclusion, US-guided synovial biopsy represents a reliable tool for the assessment of the histopathological features of RA patients with a mini-invasive approach.

Figure 1

Ultrasound-guided synovial biopsy of a second metacarpophalangeal joint using the portal and forceps technique. Arrow, open forceps inside the joint; asterisk, metacarpal hand.

Figure 2

Evaluation of the minimum area required for quantitative analyses of CD68, CD3 and CD20 cells. For each of the tissue markers studied, a plot was generated to show the effects of increasing sample size (that is, the number of high-power fields examined) and the proximity of this sample mean from the overall determination of area fraction. Data represent the mean values of all cases. As the number of high-power fields examined increases, the difference between the sampling mean and the overall mean reduces. A threshold value of 10% of the overall mean (arrows) was set as providing a reasonable estimator of the true sample mean.

Figure 3

Microphotograph of an ultrasound-guided sample. H & E staining of a metacarpophalangeal sample of a rheumatoid arthritis patient (patient U-SY1), a result of multiple high-power fields (40× objective) merged into a single image (montage).

Figure 4

Number of ultrasound-guided biopsy sections required for quantitative analysis. Evaluation of the number of ultrasound-guided biopsy sections required for quantitative analyses of CD68, CD3 and CD20 cells. Reduction in the percentage mean difference can be obtained by studying 2.5 mm² from an increasing number of sections. Arrows, number of sections that allow one to achieve a percentage mean difference lower than 10%. x axis, number of sections studied; y axis, percentage mean difference.