Purpose: The interobserver reproducibility of the current standard semiquantitative (Banff) grading of inflammation in renal transplant biopsies (TxBx) is suboptimal. We have recently developed a highly reproducible quantitative CD45 (leukocyte common antigen) immunoperoxidase assay to measure total inflammation (TI) in TxBx. The goal of the current study is to validate a quantitative CD45 assay on an immunofluorescent (IF) platform with the future aim of developing multiplexing technologies for tissue-based phenotypical and genotypic analysis.

Methods: Thirty two serially cut 2 [mu]m-thick consecutive sections of formalin-fixed paraffin-embedded (FFPE) tissues from 2 TxBx were stained for CD45 on a Leica Bond RX platform using a standardized indirect IF protocol on 2 consecutive days (sections 1-2, 5-6, etc. stained on day 1, and sections 3-4, 7-8, etc. stained on day 2). Whole slide scans of the IF sections generated on a Zeiss Axio Imager Z2 scope were analyzed using the ImageJ software. The CD45 positive cells were quantitated in each image and linear regression analysis was used to assess the reproducibility and the repeatability of the assay performed on the same day and on separate days on consecutive sections.

Results: The quality of 3 sections from 3 different pairs was suboptimal and therefore these 3 pairs were excluded from the analysis. Linear regression analysis showed the following parameter estimates and their 95% confidence intervals for the two analyses (i: same day; ii: separate day): (i) slope = 0.9761 (0.7474, 1.205), y intercept = 0.0565 (-0.7164, 0.8293), and R2 = 0.8892; (ii) slope = 0.9343 (0.5226 to 1.346), y-intercept = 0.1845 (-1.261 to 1.630), and R2 = 0.7739.

Conclusions: The results demonstrate excellent reproducibility and repeatability for the standardized quantitative CD45 IF assay on FFPE TxBx. The test will allow precise quantitation of inflammation in TxBx to monitor therapeutic responses in follow-up biopsies in Clinical Trials in Organ Transplantation and in routine biopsy setting. The assay lays the foundation for future development and implementation of tissue-based IF multiplexing tests in transplantation.

CITATION INFORMATION: Laszik Z, Dobi D, Laszik L, Ikle D, Chandran S, Vincenti F. A Novel Quantitative CD45 Immunofluorescence Assay Can Measure Inflammatory Load in Renal Transplant Biopsies with Excellent Reproducibility and Repeatability. Am J Transplant. 2016;16 (suppl 3).

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