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

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Protocol publication

[…] All high-power (× 40) microscopic fields (HPFs) were examined on an Olympus microscope (BX51; Olympus, Tokyo, Japan), captured using a digital camera (Olympus) and transferred to a computer platform. The resultant colour images were of dimension 2,048 × 1,536 pixels, RGB format, with a 24-bit-per-pixel resolution. For each acquisition session, the microscope, camera and computer were calibrated according to a standardized procedure. The images obtained were stored in an uncompressed TIFF format and were examined using the image-analysis system ImageJ 1.35 s (National Institutes of Health, Bethesda, MD, USA). Image segmentation was performed by RGB colour discrimination using threshold ranges such that a binary overlay was created covering only the positively stained areas. This threshold was determined by two distinct observers and was kept constant for all measurements for the same marker. A separate binary mask was created that identified the total tissue area in each image, so the final parameter of analysis was the area fraction. To speed up the image analysis process, all procedures were performed using an ad hoc macro program for each marker. [...] The area fraction of immunoreactivity for each marker was measured on multiple HPFs for each patient. The interfield variability was determined as the percentage difference between the mean area fraction when all HPFs from each patient were considered and the mean area fraction calculated from randomly selected individual HPFs. For surgical biopsies, this analysis was performed with multiple sets of data (10 sets) of an increasing number of randomly chosen HPFs from all available specimens of each patient.For US-guided biopsies the same analysis was performed with an increasing number of samples at different cutting levels. To increase the sampling efficiency, the number of HPFs required to reduce the percentage mean difference to less than 10% of the total sample mean was used as the variability threshold for the assay, as previously reported [].The variance of the measurements was reduced into its contributing factors (patient, sample and cutting level) and was analysed by analysis of variance using a general linear model and nested design. All samples at three different cutting levels were analysed for the computations. Both samples and cutting levels were nested in the patient variable. The F values for each analysis were provided. Differences were considered significant at P < 0.05. This variance component analysis was carried out using the STATISTICA data analysis software system (version 7.1, 2005; StatSoft, Inc., Tulsa, OK, USA). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Arthritis, Rheumatoid, Joint Diseases