Purpose: Total inflammation (TI) is an important variable for the pathologic work-up of transplant kidney biopsies (TxBx) in the Banff schema. However, current standard pathologic assessment of the inflammation is imprecise and the interobserver reproducibility of the Banff grading is suboptimal. The aim of our study was to design and validate a quantitative CD45 (leukocyte common antigen) immunohistochemical (IHC) assay for precise, objective and reproducible measurement of TI in TxBx.

Methods: Transplant kidney biopsies with normal morphology (NL) (n=15), borderline change (BL) (n=17), and acute cellular rejection (ACR) (n=23) were used for the study. From each case, serial 3 [mu]m-thick sections cut in a routine histology lab were stained for CD45 on a Leica Bond RX platform using standardized IHC protocol on 2 consecutive days. Whole slide images generated on Aperio AT2 scanner were analyzed using Definiens Tissue Studio 4.1 software to measure the total number of cells and the total number of CD45 positive cells in the cortical portion of the biopsies. Linear regression analysis was used to assess the repeatability of the assay with the IHC stains performed on separate days on paired consecutive sections.

Results: Fifteen cases in which the total cell count difference in the paired sections exceeded 10 percent indicative of significant cell content mismatch were excluded from the analysis. The remaining 40 cases [NL (n=10); BL (n=16) and ACR (n=14)] showed an average number of 13234 ± SE 2072 vs. 12924 ± SE 2032 CD45 positive cells in the paired sections (p= 0.915). Linear regression analysis of day 1 vs day 2 values showed a slope of 0.9248 (95% CI) y-intercept (-776.9 to 927.7), and R2= 0.9796 in the paired sections.

Conclusions: The results demonstrate excellent repeatability for the standardized quantitative CD45 IHC assay on TxBx. The test will allow objective, precise, and reproducible quantitation of inflammation in TxBx to monitor therapeutic responses in follow-up biopsies in Clinical Trials in Organ Transplantation and also in routine biopsy setting. The test can be readily implemented in routine pathology laboratories.

CITATION INFORMATION: Walavalkar V, Bowman S, Laszik L, Ikle D, Chandran S, Vincenti F, Laszik Z. A Quantitative CD45 Immunohistochemical Assay Allows Precise and Reproducible Measurement of Inflammation in Renal Transplant Biopsies. Am J Transplant. 2016;16 (suppl 3).

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