Emerging spatially resolved molecular imaging techniques, such as co-detection by indexing (CODEX), have enabled researchers to uncover distinct cellular structures in histological kidney sections. Spatial proteomics can provide users with the intensity level of proteins synthesized in the tissue in the same histology tissue section. However, the mapping of cell type proportions and molecular signatures can be challenging which might have contributed to the limited use of these technologies in clinical practice. Developing a computational model that handles such highdimensional whole-slide imaging (WSI) data from CODEX requires applying advanced machine learning techniques to address common challenges such as interpretability, efficiency, and usability. In this study, we propose a computational pipeline for CODEX mapping on biopsy images that features an automated registration module that utilizes nuclei segmentation in both modalities. Our pipeline provides an explainable prediction and mapping of cell type clusters on histology and analyzes the heterogeneity of molecular features in the predicted clusters. For mapping, we used an unsupervised clustering analysis of uniform manifold approximation and projection (UMAP)- reduced features to enable visualizing the predicted clusters onto the histological tissue image. To test our proposed pipeline, we used a high-dimensional CODEX panel that comprises 44 markers and visualized the intensities and the predicted clusters on whole slide images (WSI) in a set of renal histology samples collected atIndiana University. Our results delineated 14 distinct cell clusters which demonstrated high fidelity between labeled objects and specific markers. Notably, 88% of cells in the “podocytes” dominant UMAP cluster were found to have a high level of podocalyxin, although it is adjacent to two other clusters dominated by renal vasculature cells. Out of 626 features examined, 44 were central to the “podocyte” cluster, accounting for approximately 50% of its variance (p < 0.05). This study can improve the understanding of the cell type proportions and kidney functions of tissue structures, which can contribute to the human biomolecular kidney atlas; a step towards substantial advancements in the field of kidney cell biology research.
Artificial intelligence (AI) has extensive applications in a wide range of disciplines including healthcare and clinical practice. Advances in high-resolution whole-slide brightfield microscopy allow for the digitization of histologically stained tissue sections, producing gigapixel-scale whole-slide images (WSI). The significant improvement in computing and revolution of deep neural network (DNN)-based AI technologies over the last decade allow us to integrate massively parallelized computational power, cutting-edge AI algorithms, and big data storage, management, and processing. Applied to WSIs, AI has created opportunities for improved disease diagnostics and prognostics with the ultimate goal of enhancing precision medicine and resulting patient care. The National Institutes of Health (NIH) has recognized the importance of developing standardized principles for data management and discovery for the advancement of science and proposed the Findable, Accessible, Interoperable, Reusable, (FAIR) Data Principles with the goal of building a modernized biomedical data resource ecosystem to establish collaborative research communities. In line with this mission and to democratize AI-based image analysis in digital pathology, we propose ComPRePS: an end-to-end automated Computational Renal Pathology Suite which combines massive scalability, on-demand cloud computing, and an easy-to-use web-based user interface for data upload, storage, management, slide-level visualization, and domain expert interaction. Moreover, our platform is equipped with both in-house and collaborator developed sophisticated AI algorithms in the back-end server for image analysis to identify clinically relevant micro-anatomic functional tissue units (FTU) and to extract image features.
Collagen and elastin are prominent components in both normal and abnormal tissues, and their presence and distribution have great significance for fibrosis- and cancer-related processes. Collagen and elastin quantification in the context of fibrosis, often associated with irreparable organ injury, can predict the disease severity and patient prognosis. In the context of cancer, specific spatial collagen signatures are known to influence tumor microenvironments while identification of elastin is important in the context of treatment of metastatic cancers. Traditional methods to quantify collagen and elastin vary in accuracy, cost, and ease of use. Using DUET microscopy on H&E slides, high-resolution collagen and elastin mapping is possible without added staining steps or expensive optical instrumentation. We demonstrate this approach in chronic kidney disease (CKD), coronary artery disease (CAD), and for identifying vascular elastin in colon cancers.
Accurate quantification of renal fibrosis has profound importance in the assessment of chronic kidney disease (CKD). Visual analysis of a biopsy stained with trichrome under the microscope by a pathologist is the gold standard for evaluation of fibrosis. Trichrome helps to highlight collagen and ultimately interstitial fibrosis. However, trichrome stains are not always reproducible, can underestimate collagen content and are not sensitive to subtle fibrotic patterns. Using the Dual-mode emission and transmission (DUET) microscopy approach, it is possible to capture both brightfield and fluorescence images from the same area of a tissue stained with hematoxylin and eosin (H&E) enabling reproducible extraction of collagen with high sensitivity and specificity. Manual extraction of spectrally overlapping collagen signals from tubular epithelial cells and red blood cells is still an intensive task. We employed a UNet++ architecture for pixel-level segmentation and quantification of collagen using 760 whole slide image (WSI) patches from six cases of varying stages of fibrosis. Our trained model (Deep-DUET) used the supervised extracted collagen mask as ground truth and was able to predict the extent of collagen signal with a MSE of 0.05 in a holdout testing set while achieving an average AUC of 0.94 for predicting regions of collagen deposits. Expanding this work to the level of the WSI can greatly improve the ability of pathologists and machine learning (ML) tools to quantify the extent of renal fibrosis reproducibly and reliably.
The incorporation of automated computational tools has a great amount of potential to positively influence the field of pathology. However, pathologists and regulatory agencies are reluctant to trust the output of complex models such as Convolutional Neural Networks (CNNs) due to their usual implementation as black-box tools. Increasing the interpretability of quantitative analyses is a critical line of research in order to increase the adoption of modern Machine Learning (ML) pipelines in clinical environments. Towards that goal, we present HistoLens, a Graphical User Interface (GUI) designed to facilitate quantitative assessments of datasets of annotated histological compartments. Additionally, we introduce the use of hand-engineered feature visualizations to highlight regions within each structure that contribute to particular feature values. These feature visualizations can then be paired with feature hierarchy determinations in order to view which regions within an image are significant to a particular sub-group within the dataset. As a use case, we analyzed a dataset of old and young mouse kidney sections with glomeruli annotated. We highlight some of the functional components within HistoLens that allow non-computational experts to efficiently navigate a new dataset as well as allowing for easier transition to downstream computational analyses.
One of the strongest prognostic predictors of chronic kidney disease is interstitial fibrosis and tubular atrophy (IFTA). The ultimate goal of IFTA calculation is an estimation of the functional nephritic area. However, the clinical gold standard of estimation by pathologist is imprecise, primarily due to the overwhelming number of tubules sampled in a standard kidney biopsy. Artificial intelligence algorithms could provide significant benefit in this aspect as their high-throughput could identify and quantitatively measure thousands of tubules in mere minutes. Towards this goal, we use a custom panoptic convolutional network similar to Panoptic-DeepLab to detect tubules from 87 WSIs of biopsies from native diabetic kidneys and transplant kidneys. We measure 206 features on each tubule, including commonly understood features like tubular basement membrane thickness and tubular diameter. Finally, we have developed a tool which allows a user to select a range of tubule morphometric features to be highlighted in corresponding WSIs. The tool can also highlight tubules in WSI leveraging multiple morphometric features through selection of regions-of-interest in a uniform manifold approximation and projection plot.
Diabetic Nephropathy (DN) progression is stratified into several stages with different levels of proteinuria, albuminuria, and physical characteristics as observed by pathologists. These physical changes are primarily visible within a patient’s glomeruli which function as filtration units for blood returning for oxygenation. As DN stage increases, it is possible to observe the thickening of the glomerular basement membrane, expansion of the mesangium, and development of nodular sclerosis. Classification of different stages of DN by pathologists is based on semiqualitative assessments of these characteristics on an individual glomerulus basis. Being able to probabilistically infer stage membership of individual glomeruli based on a combination of easily observable and hidden image features would be an invaluable tool for furthering our understanding of the drivers of DN progression. Markov Particle filters, included in the bnlearn package in R, were used to query a Bayesian Network (BN) constructed using the structural Hill-Climbing algorithm on a set of glomerular features. These features included both traditional characteristics such as glomerular area and number of mesangial nuclei as well as more abstract features derived from Minimum Spanning Trees (MST) to quantify spatial distribution of mesangial nuclei. Our results using images from multiple institutions suggest that these abstract features exercise a variable influence on DN stage membership over the course of disease progression. Further research incorporating clinical data will give nephrologists a “white box” visual of quantitative factors present in DN patients.
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