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

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 6 weeks post-transplantation. Patients were stratified for IFTA development (<10% versus ≥10%) from 6 weeks to 6 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 6 weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development 6 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.

Graph-based description of tertiary lymphoid organs at single-cell level.

Our aim is to complement observer-dependent approaches of immune cell evaluation in microscopy images with reproducible measures for spatial composition of lymphocytic infiltrates. Analyzing such patterns of inflammation is becoming increasingly important for therapeutic decisions, for example in transplantation medicine or cancer immunology. We developed a graph-based assessment of lymphocyte clustering in full whole slide images. Based on cell coordinates detected in the full image, a Delaunay triangulation and distance criteria are used to build neighborhood graphs. The composition of nodes and edges are used for classification, e.g. using a support vector machine. We describe the variability of these infiltrates on CD3/CD20 duplex staining in renal biopsies of long-term functioning allografts, in breast cancer cases, and in lung tissue of cystic fibrosis patients. The assessment includes automated cell detection, identification of regions of interest, and classification of lymphocytic clusters according to their degree of organization. We propose a neighborhood feature which considers the occurrence of edges with a certain type in the graph to distinguish between phenotypically different immune infiltrates. Our work addresses a medical need and provides a scalable framework that can be easily adjusted to the requirements of different research questions.

Image analysis of immune cell patterns in the human mammary gland during the menstrual cycle refines lymphocytic lobulitis.

PURPOSE: To improve microscopic evaluation of immune cells relevant in breast cancer oncoimmunology, we aim at distinguishing normal infiltration patterns from lymphocytic lobulitis by advanced image analysis. We consider potential immune cell variations due to the menstrual cycle and oral contraceptives in non-neoplastic mammary gland tissue. METHODS: Lymphocyte and macrophage distributions were analyzed in the anatomical context of the resting mammary gland in immunohistochemically stained digital whole slide images obtained from 53 reduction mammoplasty specimens. Our image analysis workflow included automated regions of interest detection, immune cell recognition, and co-registration of regions of interest. RESULTS: In normal lobular epithelium, seven CD8[Formula: see text] lymphocytes per 100 epithelial cells were present on average and about 70% of this T-lymphocyte population was lined up along the basal cell layer in close proximity to the epithelium. The density of CD8[Formula: see text] T-cell was 1.6 fold higher in the luteal than in the follicular phase in spontaneous menstrual cycles and 1.4 fold increased under the influence of oral contraceptives, and not co-localized with epithelial proliferation. CD4[Formula: see text] T-cells were infrequent. Abundant CD163[Formula: see text] macrophages were widely spread, including the interstitial compartment, with minor variation during the menstrual cycle. CONCLUSIONS: Spatial patterns of different immune cell subtypes determine the range of normal, as opposed to inflammatory conditions of the breast tissue microenvironment. Advanced image analysis enables quantification of hormonal effects, refines lymphocytic lobulitis, and shows potential for comprehensive biopsy evaluation in oncoimmunology.

Task design for crowdsourced glioma cell annotation in microscopy images.

Crowdsourcing has been used in computational pathology to generate cell and cell nuclei annotations for machine learning. Herein, we broaden its scope to the previously unsolved challenging task of glioma cell detection. This requires multiplexed immunofluorescence microscopy due to diffuse invasiveness and exceptional similarity between glioma cells and reactive astrocytes. In four pilot experiments, we iteratively developed a task design enabling high-quality annotations by crowdworkers on Amazon Mechanical Turk. We applied majority or weighted vote and validated them against ground truth in the final setting. On the base of a YOLO convolutional neural network architecture, we used these consensus labels for training with different image representations regarding colors, intensities, and immmunohistochemical marker combinations. A crowd of 712 workers defined aggregated point annotations in 235 images with an average [Formula: see text] score of 0.627 for majority vote. The networks resulted in acceptable [Formula: see text] scores up to 0.69 for YOLOv8 on average and indicated first evidence for transferability to images lacking tumor markers, especially in IDH-wildtype glioblastoma. Our work confirms feasibility of crowdsourcing to generate labels suitable for training of machine learning tools in the challenging and clinically relevant use case of glioma microenvironment.

Unsupervised many-to-many stain translation for histological image augmentation to improve classification accuracy.

Background: Deep learning tasks, which require large numbers of images, are widely applied in digital pathology. This poses challenges especially for supervised tasks since manual image annotation is an expensive and laborious process. This situation deteriorates even more in the case of a large variability of images. Coping with this problem requires methods such as image augmentation and synthetic image generation. In this regard, unsupervised stain translation via GANs has gained much attention recently, but a separate network must be trained for each pair of source and target domains. This work enables unsupervised many-to-many translation of histopathological stains with a single network while seeking to maintain the shape and structure of the tissues. Methods: StarGAN-v2 is adapted for unsupervised many-to-many stain translation of histopathology images of breast tissues. An edge detector is incorporated to motivate the network to maintain the shape and structure of the tissues and to have an edge-preserving translation. Additionally, a subjective test is conducted on medical and technical experts in the field of digital pathology to evaluate the quality of generated images and to verify that they are indistinguishable from real images. As a proof of concept, breast cancer classifiers are trained with and without the generated images to quantify the effect of image augmentation using the synthetized images on classification accuracy. Results: The results show that adding an edge detector helps to improve the quality of translated images and to preserve the general structure of tissues. Quality control and subjective tests on our medical and technical experts show that the real and artificial images cannot be distinguished, thereby confirming that the synthetic images are technically plausible. Moreover, this research shows that, by augmenting the training dataset with the outputs of the proposed stain translation method, the accuracy of breast cancer classifier with ResNet-50 and VGG-16 improves by 8.0% and 9.3%, respectively. Conclusions: This research indicates that a translation from an arbitrary source stain to other stains can be performed effectively within the proposed framework. The generated images are realistic and could be employed to train deep neural networks to improve their performance and cope with the problem of insufficient numbers of annotated images.

Classifying substrate specificities of membrane transporters from Arabidopsis thaliana.

Membrane transporters catalyze the active transport of molecules across biological barriers such as lipid bilayer membranes. Currently, the experimental annotation of which proteins transport which substrates is far from complete and will likely remain so for much longer. Therefore, it is highly desirable to develop computational methods that may aid in the substrate annotation of putative membrane transport proteins. Here, we measured the similarity of membrane transporters from Arabidopsis thaliana by their amino acid composition, higher sequence order information, amino acid characteristics, or sequence conservation. We considered the substrate classes amino acids, oligopeptides, phosphates, and hexoses. Substrate classification based on the amino acid frequency yielded an accuracy of 75% or higher. Integrating additional information improved the prediction performance to 90% and higher.

Crowdsourcing of Histological Image Labeling and Object Delineation by Medical Students.

Crowdsourcing in pathology has been performed on tasks that are assumed to be manageable by nonexperts. Demand remains high for annotations of more complex elements in digital microscopic images, such as anatomical structures. Therefore, this paper investigates conditions to enable crowdsourced annotations of high-level image objects, a complex task considered to require expert knowledge. Seventy six medical students without specific domain knowledge who voluntarily participated in three experiments solved two relevant annotation tasks on histopathological images: 1) labeling of images showing tissue regions and 2) delineation of morphologically defined image objects. We focus on methods to ensure sufficient annotation quality including several tests on the required number of participants and on the correlation of participants' performance between tasks. In a set up simulating annotation of images with limited ground truth, we validated the feasibility of a confidence score using full ground truth. For this, we computed a majority vote using weighting factors based on individual assessment of contributors against scattered gold standard annotated by pathologists. In conclusion, we provide guidance for task design and quality control to enable a crowdsourced approach to obtain accurate annotations required in the era of digital pathology.

Detection of lobular structures in normal breast tissue.

BACKGROUND: Ongoing research into inflammatory conditions raises an increasing need to evaluate immune cells in histological sections in biologically relevant regions of interest (ROIs). Herein, we compare different approaches to automatically detect lobular structures in human normal breast tissue in digitized whole slide images (WSIs). This automation is required to perform objective and consistent quantitative studies on large data sets. METHODS: In normal breast tissue from nine healthy patients immunohistochemically stained for different markers, we evaluated and compared three different image analysis methods to automatically detect lobular structures in WSIs: (1) a bottom-up approach using the cell-based data for subsequent tissue level classification, (2) a top-down method starting with texture classification at tissue level analysis of cell densities in specific ROIs, and (3) a direct texture classification using deep learning technology. RESULTS: All three methods result in comparable overall quality allowing automated detection of lobular structures with minor advantage in sensitivity (approach 3), specificity (approach 2), or processing time (approach 1). Combining the outputs of the approaches further improved the precision. CONCLUSIONS: Different approaches of automated ROI detection are feasible and should be selected according to the individual needs of biomarker research. Additionally, detected ROIs could be used as a basis for quantification of immune infiltration in lobular structures.

Rule-based regulatory and metabolic model for Quorum sensing in P. aeruginosa.

BACKGROUND: In the pathogen P. aeruginosa, the formation of virulence factors is regulated via Quorum sensing signaling pathways. Due to the increasing number of strains that are resistant to antibiotics, there is a high interest to develop novel antiinfectives. In the combat of resistant bacteria, selective blockade of the bacterial cell-to-cell communication (Quorum sensing) has gained special interest as anti-virulence strategy. Here, we modeled the las, rhl, and pqs Quorum sensing systems by a multi-level logical approach to analyze how enzyme inhibitors and receptor antagonists effect the formation of autoinducers and virulence factors. RESULTS: Our rule-based simulations fulfill the behavior expected from literature considering the external level of autoinducers. In the presence of PqsBCD inhibitors, the external HHQ and PQS levels are indeed clearly reduced. The magnitude of this effect strongly depends on the inhibition level. However, it seems that the pyocyanin pathway is incomplete. CONCLUSIONS: To match experimental observations we suggest a modified network topology in which PqsE and PqsR acts as receptors and an autoinducer as ligand that up-regulate pyocyanin in a concerted manner. While the PQS biosynthesis is more appropriate as target to inhibit the HHQ and PQS formation, blocking the receptor PqsR that regulates the biosynthesis reduces the pyocyanin level stronger.