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Background: Identification of bladder layers is a necessary prerequisite to bladder cancer diagnosis and prognosis. We present a method of multi-class image segmentation, which recognizes urothelium, lamina propria, muscularis propria, and muscularis mucosa layers as well as regions of red blood cells, cauterized tissue, and inflamed tissue from images of hematoxylin and eosin stained slides of bladder biopsies., Methods: Segmentation is carried out using a U-Net architecture. The number of layers was either, eight, ten, or twelve and combined with a weight initializers of He uniform, He normal, Glorot uniform, and Glorot normal. The most optimal of these parameters was found by through a seven-fold training, validation, and testing of a dataset of 39 whole slide images of T1 bladder biopsies., Results: The most optimal model was a twelve layer U-net using He normal initializer. Initial visual evaluation by an experienced pathologist on an independent set of 15 slides segmented by our method yielded an average score of 8.93 ± 0.6 out of 10 for segmentation accuracy. It took only 23 min for the pathologist to review 15 slides (1.53 min/slide) with the computer annotations. To assess the generalizability of the proposed model, we acquired an additional independent set of 53 whole slide images and segmented them using our method. Visual examination by a different experienced pathologist yielded an average score of 8.87 ± 0.63 out of 10 for segmentation accuracy., Conclusions: Our preliminary findings suggest that predictions of our model can minimize the time needed by pathologists to annotate slides. Moreover, the method has the potential to identify the bladder layers accurately. Further development can assist the pathologist with the diagnosis of T1 bladder cancer.

Acute infections of the middle ear are the most commonly treated childhood diseases. Because complications affect children's language learning and cognitive processes, it is essential to diagnose these diseases in a timely and accurate manner. The prevailing literature suggests that it is difficult to accurately diagnose these infections, even for experienced ear, nose, and throat (ENT) physicians. Advanced care practitioners (e.g., nurse practitioners, physician assistants) serve as first-line providers in many primary care settings and may benefit from additional guidance to appropriately determine the diagnosis and treatment of ear diseases. For this purpose, we designed a content-based image retrieval (CBIR) system (called OtoMatch) for normal, middle ear effusion, and tympanostomy tube conditions, operating on eardrum images captured with a digital otoscope. We present a method that enables the conversion of any convolutional neural network (trained for classification) into an image retrieval model. As a proof of concept, we converted a pre-trained deep learning model into an image retrieval system. We accomplished this by changing the fully connected layers into lookup tables. A database of 454 labeled eardrum images (179 normal, 179 effusion, and 96 tube cases) was used to train and test the system. On a 10-fold cross validation, the proposed method resulted in an average accuracy of 80.58% (SD 5.37%), and maximum F1 score of 0.90 while retrieving the most similar image from the database. These are promising results for the first study to demonstrate the feasibility of developing a CBIR system for eardrum images using the newly proposed methodology., Competing Interests: Moberly and Gurcan are directors of Otologic, Inc. The other authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Automatic identification of tissue structures in the analysis of digital tissue biopsies remains an ongoing problem in digital pathology. Common barriers include lack of reliable ground truth due to inter- and intra- reader variability, class imbalances, and inflexibility of discriminative models. To overcome these barriers, we are developing a framework that benefits from a reliable immunohistochemistry ground truth during labeling, overcomes class imbalances through single task learning, and accommodates any number of classes through a minimally supervised, modular model-per-class paradigm. This study explores an initial application of this framework, based on conditional generative adversarial networks, to automatically identify tumor from non-tumor regions in colorectal H&E slides. The average precision, sensitivity, and F1 score during validation was 95.13 ± 4.44%, 93.05 ± 3.46%, and 94.02 ± 3.23% and for an external test dataset was 98.75 ± 2.43%, 88.53 ± 5.39%, and 93.31 ± 3.07%, respectively. With accurate identification of tumor regions, we plan to further develop our framework to establish a tumor front, from which tumor buds can be detected in a restricted region. This model will be integrated into a larger system which will quantitatively determine the prognostic significance of tumor budding.

Automatic and accurate detection of positive and negative nuclei from images of immunostained tissue biopsies is critical to the success of digital pathology. The evaluation of most nuclei detection algorithms relies on manually generated ground truth prepared by pathologists, which is unfortunately time-consuming and suffers from inter-pathologist variability. In this work, we developed a digital immunohistochemistry (IHC) phantom that can be used for evaluating computer algorithms for enumeration of IHC positive cells. Our phantom development consists of two main steps, 1) extraction of the individual as well as nuclei clumps of both positive and negative nuclei from real WSI images, and 2) systematic placement of the extracted nuclei clumps on an image canvas. The resulting images are visually similar to the original tissue images. We created a set of 42 images with different concentrations of positive and negative nuclei. These images were evaluated by four board certified pathologists in the task of estimating the ratio of positive to total number of nuclei. The resulting concordance correlation coefficients (CCC) between the pathologist and the true ratio range from 0.86 to 0.95 (point estimates). The same ratio was also computed by an automated computer algorithm, which yielded a CCC value of 0.99. Reading the phantom data with known ground truth, the human readers show substantial variability and lower average performance than the computer algorithm in terms of CCC. This shows the limitation of using a human reader panel to establish a reference standard for the evaluation of computer algorithms, thereby highlighting the usefulness of the phantom developed in this work. Using our phantom images, we further developed a function that can approximate the true ratio from the area of the positive and negative nuclei, hence avoiding the need to detect individual nuclei. The predicted ratios of 10 held-out images using the function (trained on 32 images) are within ±2.68% of the true ratio. Moreover, we also report the evaluation of a computerized image analysis method on the synthetic tissue dataset.

In pathology, Immunohistochemical staining (IHC) of tissue sections is regularly used to diagnose and grade malignant tumors. Typically, IHC stain interpretation is rendered by a trained pathologist using a manual method, which consists of counting each positively- and negatively-stained cell under a microscope. The manual enumeration suffers from poor reproducibility even in the hands of expert pathologists. To facilitate this process, we propose a novel method to create artificial datasets with the known ground truth which allows us to analyze the recall, precision, accuracy, and intra- and inter-observer variability in a systematic manner, enabling us to compare different computer analysis approaches. Our method employs a conditional Generative Adversarial Network that uses a database of Ki67 stained tissues of breast cancer patients to generate synthetic digital slides. Our experiments show that synthetic images are indistinguishable from real images. Six readers (three pathologists and three image analysts) tried to differentiate 15 real from 15 synthetic images and the probability that the average reader would be able to correctly classify an image as synthetic or real more than 50% of the time was only 44.7%.

The World Health Organization (WHO) has clear guidelines regarding the use of Ki67 index in defining the proliferative rate and assigning grade for pancreatic neuroendocrine tumor (NET). WHO mandates the quantification of Ki67 index by counting at least 500 positive tumor cells in a hotspot. Unfortunately, Ki67 antibody may stain both tumor and non-tumor cells as positive depending on the phase of the cell cycle. Likewise, the counter stain labels both tumor and non-tumor as negative. This non-specific nature of Ki67 stain and counter stain therefore hinders the exact quantification of Ki67 index. To address this problem, we present a deep learning method to automatically differentiate between NET and non-tumor regions based on images of Ki67 stained biopsies. Transfer learning was employed to recognize and apply relevant knowledge from previous learning experiences to differentiate between tumor and non-tumor regions. Transfer learning exploits a rich set of features previously used to successfully categorize non-pathology data into 1,000 classes. The method was trained and validated on a set of whole-slide images including 33 NETs subject to Ki67 immunohistochemical staining using a leave-one-out cross-validation. When applied to 30 high power fields (HPF) and assessed against a gold standard (evaluation by two expert pathologists), the method resulted in a high sensitivity of 97.8% and specificity of 88.8%. The deep learning method developed has the potential to reduce pathologists' workload by directly identifying tumor boundaries on images of Ki67 stained slides. Moreover, it has the potential to replace sophisticated and expensive imaging methods which are recently developed for identification of tumor boundaries in images of Ki67-stained NETs.

Pathology Image Informatics Platform (PIIP) is an NCI/NIH sponsored project intended for managing, annotating, sharing, and quantitatively analyzing digital pathology imaging data. It expands on an existing, freely available pathology image viewer, Sedeen. The goal of this project is to develop and embed some commonly used image analysis applications into the Sedeen viewer to create a freely available resource for the digital pathology and cancer research communities. Thus far, new plugins have been developed and incorporated into the platform for out of focus detection, region of interest transformation, and IHC slide analysis. Our biomarker quantification and nuclear segmentation algorithms, written in MATLAB, have also been integrated into the viewer. This article describes the viewing software and the mechanism to extend functionality by plugins, brief descriptions of which are provided as examples, to guide users who want to use this platform. PIIP project materials, including a video describing its usage and applications, and links for the Sedeen Viewer, plug-ins, and user manuals are freely available through the project web page: http://pathiip.org Cancer Res; 77(21); e83-86. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The liver vessels, which have low signal and run next to brighter bile ducts, are difficult to segment from MR images. This study presents a fully automated and adaptive method to segment portal and hepatic veins on magnetic resonance images. In the proposed approach, segmentation of these vessels is achieved in four stages: (i) initial segmentation, (ii) refinement, (iii) reconstruction, and (iv) post-processing. In the initial segmentation stage, k-means clustering is used, the results of which are refined iteratively with linear contrast stretching algorithm in the next stage, generating a mask image. In the reconstruction stage, vessel regions are reconstructed with the marker image from the first stage and the mask image from the second stage. Experimental data sets include slices that show fat tissues, which have the same gray level values with vessels, outside the margin of the liver. These structures are removed in the last stage. Results show that the proposed approach is more efficient than other thresholding-based methods. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Fat accumulation in the liver causes metabolic diseases such as obesity, hypertension, diabetes or dyslipidemia by affecting insulin resistance, and increasing the risk of cardiac complications and cardiovascular disease mortality. Fatty liver diseases are often reversible in their early stage; therefore, there is a recognized need to detect their presence and to assess its severity to recognize fat-related functional abnormalities in the liver. This is crucial in evaluating living liver donors prior to transplantation because fat content in the liver can change liver regeneration in the recipient and donor. There are several methods to diagnose fatty liver, measure the amount of fat, and to classify and stage liver diseases (e.g. hepatic steatosis, steatohepatitis, fibrosis and cirrhosis): biopsy (the gold-standard procedure), clinical (medical physics based) and image analysis (semi or fully automated approaches). Liver biopsy has many drawbacks: it is invasive, inappropriate for monitoring (i.e., repeated evaluation), and assessment of steatosis is somewhat subjective. Qualitative biomarkers are mostly insufficient for accurate detection since fat has to be quantified by a varying threshold to measure disease severity. Therefore, a quantitative biomarker is required for detection of steatosis, accurate measurement of severity of diseases, clinical decision-making, prognosis and longitudinal monitoring of therapy. This study presents a comprehensive review of both clinical and automated image analysis based approaches to quantify liver fat and evaluate fatty liver diseases from different medical imaging modalities., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Follicular lymphoma (FL) is one of the most common lymphoid malignancies in the western world. FL cases are stratified into three histological grades based on the average centroblast count per high power field (HPF). The centroblast count is performed manually by the pathologist using an optical microscope and hematoxylin and eosin (H&E) stained tissue section. Although this is the current clinical practice, it suffers from high inter- and intra-observer variability and is vulnerable to sampling bias., Methods: In this paper, we present a system, called Follicular Lymphoma Grading System (FLAGS), to assist the pathologist in grading FL cases. We also assess the effect of FLAGS on accuracy of expert and inexperienced readers. FLAGS automatically identifies possible HPFs for examination by analyzing H&E and CD20 stains, before classifying them into low or high risk categories. The pathologist is first asked to review the slides according to the current routine clinical practice, before being presented with FLAGS classification via color-coded map. The accuracy of the readers with and without FLAGS assistance is measured., Results: FLAGS was used by four experts (board-certified hematopathologists) and seven pathology residents on 20 FL slides. Access to FLAGS improved overall reader accuracy with the biggest improvement seen among residents. An average AUC value of 0.75 was observed which generally indicates "acceptable" diagnostic performance., Conclusions: The results of this study show that FLAGS can be useful in increasing the pathologists' accuracy in grading the tissue. To the best of our knowledge, this study measure, for the first time, the effect of computerized image analysis on pathologists' grading of follicular lymphoma. When fully developed, such systems have the potential to reduce sampling bias by examining an increased proportion of HPFs within follicle regions, as well as to reduce inter- and intra-reader variability.

An estimated 6.5 million patients in the United States are affected by chronic wounds, with more than US$25 billion and countless hours spent annually for all aspects of chronic wound care. There is a need for an intelligent software tool to analyze wound images, characterize wound tissue composition, measure wound size, and monitor changes in wound in between visits. Performed manually, this process is very time-consuming and subject to intra- and inter-reader variability. In this work, our objective is to develop methods to segment, measure and characterize clinically presented chronic wounds from photographic images. The first step of our method is to generate a Red-Yellow-Black-White (RYKW) probability map, which then guides the segmentation process using either optimal thresholding or region growing. The red, yellow and black probability maps are designed to handle the granulation, slough and eschar tissues, respectively; while the white probability map is to detect the white label card for measurement calibration purposes. The innovative aspects of this work include defining a four-dimensional probability map specific to wound characteristics, a computationally efficient method to segment wound images utilizing the probability map, and auto-calibration of wound measurements using the content of the image. These methods were applied to 80 wound images, captured in a clinical setting at the Ohio State University Comprehensive Wound Center, with the ground truth independently generated by the consensus of at least two clinicians. While the mean inter-reader agreement between the readers varied between 67.4% and 84.3%, the computer achieved an average accuracy of 75.1%., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In this paper, we describe the design and development of a multitiered content-based image retrieval (CBIR) system for microscopic images utilizing a reference database that contains images of more than one disease. The proposed CBIR system uses a multitiered approach to classify and retrieve microscopic images involving their specific subtypes, which are mostly difficult to discriminate and classify. This system enables both multi-image query and slide-level image retrieval in order to protect the semantic consistency among the retrieved images. New weighting terms, inspired from information retrieval theory, are defined for multiple-image query and retrieval. The performance of the system was tested on a dataset including 1666 imaged high power fields extracted from 57 follicular lymphoma (FL) tissue slides with three subtypes and 44 neuroblastoma (NB) tissue slides with four subtypes. Each slide is semantically annotated according to their subtypes by expert pathologists. By using leave-one-slide out testing scheme, the multi-image query algorithm with the proposed weighting strategy achieves about 93% and 86% of average classification accuracy at the first rank retrieval, outperforming the image-level retrieval accuracy by about 38 and 26 percentage points, for FL and NB diseases, respectively.

Follicular lymphoma (FL) is one of the most common types of nonHodgkin lymphoma in the U.S. Diagnosis of FL is based on tissue biopsy that shows characteristic morphologic and immunohistochemical (IHC) findings. Our group's work focuses on the development of computer-aided image-analysis techniques to improve the FL grading. Since centroblast enumeration needs to be performed in malignant follicles, the development of an automated system to accurately identify follicles on digital images of lymphoid tissue is an important step. In this letter, we describe an automated system to identify follicles in IHC-stained tissue sections. A unique feature of the system described here is the use of texture and color information to mimic the process that a human expert might use to identify follicle regions. Comparison of system-generated results with expert-generated ground truth has shown promising results with a mean similarity score of 87.11%.

Follicular lymphoma (FL) is one of the most common lymphoid malignancies in the western world. FL has a variable clinical course, and important clinical treatment decisions for FL patients are based on histological grading, which is done by manually counting the large malignant cells called centroblasts (CB) in ten standard microscopic high-power fields from H&E-stained tissue sections. This method is tedious and subjective; as a result, suffers from considerable inter and intrareader variability even when used by expert pathologists. In this paper, we present a computer-aided detection system for automated identification of CB cells from H&E-stained FL tissue samples. The proposed system uses a unitone conversion to obtain a single-channel image that has the highest contrast. From the resulting image, which has a bimodal distribution due to the H&E stain, a cell-likelihood image is generated. Finally, a two-step CB detection procedure is applied. In the first step, we identify evident nonCB cells based on size and shape. In the second step, the CB detection is further refined by learning and utilizing the texture distribution of nonCB cells. We evaluated the proposed approach on 100 region-of-interest images extracted from ten distinct tissue samples and obtained a promising 80.7% detection accuracy.

Cystic Fibrosis (CF) is the most common lethal genetic disorder in the Caucasian population, affecting about 30,000 people in the United States. It results in inflammation, hence thickening of airway (AW) walls. It has been demonstrated that AW inflammation begins early in life producing structural AW damage. Because this damage can be present in patients who are relatively asymptomatic, lung disease can progress insidiously. High-resolution computed tomographic imaging has also shown that the AWs of infants and young children with CF have thicker walls and are more dilated than those of normal children. The purpose of this study was to develop computerized methods which allow rapid, efficient and accurate assessment of computed tomographic AW and vessel (V) dimensions from axial CT lung images. For this purpose, a full-width-half-max based automatic AW and V size measurement method was developed. The only user input required is approximate center marking of AW and V by an expert. The method was evaluated on a patient population of 4 infants and 4 children with different stages of mild CF related lung disease. This new automated method for assessing early AW disease in infants and children with CF represents a potentially useful outcome measure for future intervention trials.

We present a pathological image analysis system for the computer-aided prognosis of neuroblastoma, a childhood cancer. The image analysis system automatically classifies Schwannian stromal development of pathological tissues and determines the grade of differentiation. Due to the demanding computational cost of processing large digitized slides, the system was implemented on a cluster of computers with automated load balancing within a multi-resolution framework. In our experiments, the overall accuracies for stromal classification and the grade of differentiation were 96.6% and 95.3%, respectively. Additionally, the multi-resolution framework reduced the run time of the single resolution approach by 53% and 34% on average for stromal classification and grade of differentiation, respectively. For these two cases, parallelization on a 16-node cluster reduced the sequential run time by 92% and 88% on average. Accuracy and efficiency of these techniques are promising for the development a computer-assisted neuroblastoma prognosis system.

Neuroblastoma is a childhood cancer of the nervous system. Current prognostic classification of this disease partly relies on morphological characteristics of the cells from H&E-stained images. In this work, an automated cell nuclei segmentation method is developed. This method employs morphological top-hat by reconstruction algorithm coupled with hysteresis thresholding to both detect and segment the cell nuclei. Accuracy of the automated cell nuclei segmentation algorithm is measured by comparing its outputs to manual segmentation. The average segmentation accuracy is 90.24+/-5.14%