Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning

M. Hermsen, V. Volk, J. Bräsen, D. Geijs, W. Gwinner, J. Kers, J. Linmans, N. Schaadt, J. Schmitz, E. Steenbergen, Z. Swiderska-Chadaj, B. Smeets, L. Hilbrands and J. van der Laak

Laboratory Investigation 2021.

DOI

Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n=22) taken at six weeks post-transplantation. Patients were stratified for IFTA development (<10 % versus ≥10%) from six weeks to six months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi-spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at six weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development six months post-transplantation (p<0.05). CD3+CD8-/CD3+CD8+ ratios were higher in patients with <10% IFTA development (p<0.05). We observed a high correlation between CD163+ and CD4+GATA3+ cell density (R=0.74, p<0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi-spectrally imaged slides of kidney transplant biopsies.