Segmentation of nuclei regions from histological images is an important task for automated computer-aided analysis of histological images, particularly in the presence of impermissible color variation in the color appearance of stained tissue images. While color normalization enables better nuclei segmentation, accurate segmentation of nuclei structures makes color normalization rather trivial. In this respect, the paper proposes a novel deep generative model for simultaneously segmenting nuclei structures and normalizing color appearance of stained histological images.This model judiciously integrates the merits of truncated normal distribution and spatial attention. The model assumes that the latent color appearance information, corresponding to a particular histological image, is independent of respective nuclei segmentation map as well as embedding map information. The disentangled representation makes the model generalizable and adaptable as the modification or loss in color appearance information cannot be able to affect the nuclei segmentation map as well as embedding information. Also, for dealing with the stain overlap of associated histochemical reagents, the prior for latent color appearance code is assumed to be a mixture of truncated normal distributions. The proposed model incorporates the concept of spatial attention for segmentation of nuclei regions from histological images. The performance of the proposed approach, along with a comparative analysis with related state-of-the-art algorithms, has been demonstrated on publicly available standard histological image data sets.
Numerous deep learning-based solutions have been proposed for histopathological image analysis over the past years. While they usually demonstrate exceptionally high accuracy, one key question is whether their precision might be affected by low-level image properties not related to histopathology but caused by microscopy image handling and pre-processing. In this paper, we analyze a popular NCT-CRC-HE-100K colorectal cancer dataset used in numerous prior works and show that both this dataset and the obtained results may be affected by data-specific biases. The most prominent revealed dataset issues are inappropriate color normalization, severe JPEG artifacts inconsistent between different classes, and completely corrupted tissue samples resulting from incorrect image dynamic range handling. We show that even the simplest model using only 3 features per image (red, green and blue color intensities) can demonstrate over 50% accuracy on this 9-class dataset, while using color histogram not explicitly capturing cell morphology features yields over 82% accuracy. Moreover, we show that a basic EfficientNet-B0 ImageNet pretrained model can achieve over 97.7% accuracy on this dataset, outperforming all previously proposed solutions developed for this task, including dedicated foundation histopathological models and large cell morphology-aware neural networks. The NCT-CRC-HE dataset is publicly available and can be freely used to replicate the presented results. The codes and pre-trained models used in this paper are available at https://github.com/gmalivenko/NCT-CRC-HE-experiments
In recent years, deep neural networks (DNNs) have demonstrated remarkable performance in pathology applications, potentially even outperforming expert pathologists due to their ability to learn subtle features from large datasets. One complication in preparing digital pathology datasets for DNN tasks is variation in tinctorial qualities. A common way to address this is to perform stain normalization on the images. In this study, we show that a well-trained DNN model trained on one batch of histological slides failed to generalize to another batch prepared at a different time from the same tissue blocks, even when stain normalization methods were applied. This study used sample data from a previously reported DNN that was able to identify patients with early stage non-small cell lung cancer (NSCLC) whose tumors did and did not metastasize, with high accuracy, based on training and then testing of digital images from H&E stained primary tumor tissue sections processed at the same time. In this study we obtained a new series of histologic slides from the adjacent recuts of same tissue blocks processed in the same lab but at a different time. We found that the DNN trained on the either batch of slides/images was unable to generalize and failed to predict progression in the other batch of slides/images (AUC_cross-batch = 0.52 - 0.53 compared to AUC_same-batch = 0.74 - 0.81). The failure to generalize did not improve even when the tinctorial difference correction were made through either traditional color-tuning or stain normalization with the help of a Cycle Generative Adversarial Network (CycleGAN) process. This highlights the need to develop an entirely new way to process and collect consistent microscopy images from histologic slides that can be used to both train and allow for the general application of predictive DNN algorithms.
Despite notable advancements, the integration of deep learning (DL) techniques into impactful clinical applications, particularly in the realm of digital histopathology, has been hindered by challenges associated with achieving robust generalization across diverse imaging domains and characteristics. Traditional mitigation strategies in this field such as data augmentation and stain color normalization have proven insufficient in addressing this limitation, necessitating the exploration of alternative methodologies. To this end, we propose a novel generative method for domain generalization in histopathology images. Our method employs a generative, self-supervised Vision Transformer to dynamically extract characteristics of image patches and seamlessly infuse them into the original images, thereby creating novel, synthetic images with diverse attributes. By enriching the dataset with such synthesized images, we aim to enhance its holistic nature, facilitating improved generalization of DL models to unseen domains. Extensive experiments conducted on two distinct histopathology datasets demonstrate the effectiveness of our proposed approach, outperforming the state of the art substantially, on the Camelyon17-wilds challenge dataset (+2%) and on a second epithelium-stroma dataset (+26%). Furthermore, we emphasize our method's ability to readily scale with increasingly available unlabeled data samples and more complex, higher parametric architectures. Source code is available at https://github.com/sdoerrich97/vits-are-generative-models .
The integrity of the reconstructed human epidermis generated in vitro could be assessed using histological analyses combined with immunohistochemical staining of keratinocyte differentiation markers. Computer-based analysis of scanned tissue saves the expert time and may improve the accuracy of quantification by eliminating interrater reliability issues. However, technical differences during the preparation and capture of stained images and the presence of multiple artifacts may influence the outcome of computational methods. Using a dataset with 598 unannotated images showing cross-sections of in vitro reconstructed human epidermis stained with DAB-based immunohistochemistry reaction to visualize 4 different keratinocyte differentiation marker proteins (filaggrin, keratin 10, Ki67, HSPA2) and counterstained with hematoxylin, we developed an unsupervised method for the detection and quantification of immunohistochemical staining. The proposed pipeline includes the following steps: (i) color normalization to reduce the variability of pixel intensity values in different samples; (ii) color deconvolution to acquire color channels of the stains used; (iii) morphological operations to find the background area of the image; (iv) automatic image rotation; and (v) finding markers of human epidermal differentiation with clustering. Also, we created a method to exclude images without DAB-stained areas. The most effective combination of methods includes: (i) Reinhard's normalization; (ii) Ruifrok and Johnston color deconvolution method; (iii) proposed image rotation method based on boundary distribution of image intensity; (iv) k-means clustering using DAB stain intensity. These results should enhance the performance of quantitative analysis of protein markers in reconstructed human epidermis samples and enable comparison of their spatial distribution between different experimental conditions.
Histological tissue examination has been a longstanding practice for cancer diagnosis where pathologists identify the presence of tumors on glass slides. Slides acquired from laboratory routine may contain unintentional artifacts due to complications in surgical resection. Blood and damaged tissue artifacts are two common problems associated with transurethral resection of the bladder tumor. Differences in histotechnical procedures among laboratories may also result in color variations and minor inconsistencies in outcome. A digitized version of a glass slide known as a whole slide image (WSI) holds enormous potential for automated diagnostics. The presence of irrelevant areas in a WSI undermines diagnostic value for pathologists as well as computational pathology (CPATH) systems. Therefore, automatic detection and exclusion of diagnostically irrelevant areas may lead to more reliable predictions. In this paper, we are detecting blood and damaged tissue against diagnostically relevant tissue. We gauge the effectiveness of transfer learning against training from scratch. Best models give 0.99 and 0.89 F1 scores for blood and damaged tissue detection. Since blood and damaged tissue have subtle color differences, we assess the impact of color processing methods on the binary classification performance of five well-known architectures. Finally, we remove the color to understand its importance against morphology on classification performance.
Breast cancer early detection is crucial for improving patient outcomes. The Institut Catal\`a de la Salut (ICS) has launched the DigiPatICS project to develop and implement artificial intelligence algorithms to assist with the diagnosis of cancer. In this paper, we propose a new approach for facing the color normalization problem in HER2-stained histopathological images of breast cancer tissue, posed as an style transfer problem. We combine the Color Deconvolution technique with the Pix2Pix GAN network to present a novel approach to correct the color variations between different HER2 stain brands. Our approach focuses on maintaining the HER2 score of the cells in the transformed images, which is crucial for the HER2 analysis. Results demonstrate that our final model outperforms the state-of-the-art image style transfer methods in maintaining the cell classes in the transformed images and is as effective as them in generating realistic images.
In this paper, we present a Neural Preset technique to address the limitations of existing color style transfer methods, including visual artifacts, vast memory requirement, and slow style switching speed. Our method is based on two core designs. First, we propose Deterministic Neural Color Mapping (DNCM) to consistently operate on each pixel via an image-adaptive color mapping matrix, avoiding artifacts and supporting high-resolution inputs with a small memory footprint. Second, we develop a two-stage pipeline by dividing the task into color normalization and stylization, which allows efficient style switching by extracting color styles as presets and reusing them on normalized input images. Due to the unavailability of pairwise datasets, we describe how to train Neural Preset via a self-supervised strategy. Various advantages of Neural Preset over existing methods are demonstrated through comprehensive evaluations. Notably, Neural Preset enables stable 4K color style transfer in real-time without artifacts. Besides, we show that our trained model can naturally support multiple applications without fine-tuning, including low-light image enhancement, underwater image correction, image dehazing, and image harmonization. Project page with demos: https://zhkkke.github.io/NeuralPreset .
Visual microscopic study of diseased tissue by pathologists has been the cornerstone for cancer diagnosis and prognostication for more than a century. Recently, deep learning methods have made significant advances in the analysis and classification of tissue images. However, there has been limited work on the utility of such models in generating histopathology images. These synthetic images have several applications in pathology including utilities in education, proficiency testing, privacy, and data sharing. Recently, diffusion probabilistic models were introduced to generate high quality images. Here, for the first time, we investigate the potential use of such models along with prioritized morphology weighting and color normalization to synthesize high quality histopathology images of brain cancer. Our detailed results show that diffusion probabilistic models are capable of synthesizing a wide range of histopathology images and have superior performance compared to generative adversarial networks.
Stain color variation in histological images, caused by a variety of factors, is a challenge not only for the visual diagnosis of pathologists but also for cell segmentation algorithms. To eliminate the color variation, many stain normalization approaches have been proposed. However, most were designed for hematoxylin and eosin staining images and performed poorly on immunohistochemical staining images. Current cell segmentation methods systematically apply stain normalization as a preprocessing step, but the impact brought by color variation has not been quantitatively investigated yet. In this paper, we produced five groups of NeuN staining images with different colors. We applied a deep learning image-recoloring method to perform color transfer between histological image groups. Finally, we altered the color of a segmentation set and quantified the impact of color variation on cell segmentation. The results demonstrated the necessity of color normalization prior to subsequent analysis.
Computer assisted diagnosis in digital pathology is becoming ubiquitous as it can provide more efficient and objective healthcare diagnostics. Recent advances have shown that the convolutional Neural Network (CNN) architectures, a well-established deep learning paradigm, can be used to design a Computer Aided Diagnostic (CAD) System for breast cancer detection. However, the challenges due to stain variability and the effect of stain normalization with such deep learning frameworks are yet to be well explored. Moreover, performance analysis with arguably more efficient network models, which may be important for high throughput screening, is also not well explored.To address this challenge, we consider some contemporary CNN models for binary classification of breast histopathology images that involves (1) the data preprocessing with stain normalized images using an adaptive colour deconvolution (ACD) based color normalization algorithm to handle the stain variabilities; and (2) applying transfer learning based training of some arguably more efficient CNN models, namely Visual Geometry Group Network (VGG16), MobileNet and EfficientNet. We have validated the trained CNN networks on a publicly available BreaKHis dataset, for 200x and 400x magnified histopathology images. The experimental analysis shows that pretrained networks in most cases yield better quality results on data augmented breast histopathology images with stain normalization, than the case without stain normalization. Further, we evaluated the performance and efficiency of popular lightweight networks using stain normalized images and found that EfficientNet outperforms VGG16 and MobileNet in terms of test accuracy and F1 Score. We observed that efficiency in terms of test time is better in EfficientNet than other networks; VGG Net, MobileNet, without much drop in the classification accuracy.
In digital pathology, different staining procedures and scanners cause substantial color variations in whole-slide images (WSIs), especially across different laboratories. These color shifts result in a poor generalization of deep learning-based methods from the training domain to external pathology data. To increase test performance, stain normalization techniques are used to reduce the variance between training and test domain. Alternatively, color augmentation can be applied during training leading to a more robust model without the extra step of color normalization at test time. We propose a novel color augmentation technique, HistAuGAN, that can simulate a wide variety of realistic histology stain colors, thus making neural networks stain-invariant when applied during training. Based on a generative adversarial network (GAN) for image-to-image translation, our model disentangles the content of the image, i.e., the morphological tissue structure, from the stain color attributes. It can be trained on multiple domains and, therefore, learns to cover different stain colors as well as other domain-specific variations introduced in the slide preparation and imaging process. We demonstrate that HistAuGAN outperforms conventional color augmentation techniques on a classification task on the publicly available dataset Camelyon17 and show that it is able to mitigate present batch effects.
Automatic cell segmentation is an essential step in the pipeline of computer-aided diagnosis (CAD), such as the detection and grading of breast cancer. Accurate segmentation of cells can not only assist the pathologists to make a more precise diagnosis, but also save much time and labor. However, this task suffers from stain variation, cell inhomogeneous intensities, background clutters and cells from different tissues. To address these issues, we propose an Attention Enforced Network (AENet), which is built on spatial attention module and channel attention module, to integrate local features with global dependencies and weight effective channels adaptively. Besides, we introduce a feature fusion branch to bridge high-level and low-level features. Finally, the marker controlled watershed algorithm is applied to post-process the predicted segmentation maps for reducing the fragmented regions. In the test stage, we present an individual color normalization method to deal with the stain variation problem. We evaluate this model on the MoNuSeg dataset. The quantitative comparisons against several prior methods demonstrate the superiority of our approach.
Performance of deep learning algorithms decreases drastically if the data distributions of the training and testing sets are different. Due to variations in staining protocols, reagent brands, and habits of technicians, color variation in digital histopathology images is quite common. Color variation causes problems for the deployment of deep learning-based solutions for automatic diagnosis system in histopathology. Previously proposed color normalization methods consider a small patch as a reference for normalization, which creates artifacts on out-of-distribution source images. These methods are also slow as most of the computation is performed on CPUs instead of the GPUs. We propose a color normalization technique, which is fast during its self-supervised training as well as inference. Our method is based on a lightweight fully-convolutional neural network and can be easily attached to a deep learning-based pipeline as a pre-processing block. For classification and segmentation tasks on CAMELYON17 and MoNuSeg datasets respectively, the proposed method is faster and gives a greater increase in accuracy than the state of the art methods.
Although generative adversarial network (GAN) based style transfer is state of the art in histopathology color-stain normalization, they do not explicitly integrate structural information of tissues. We propose a self-supervised approach to incorporate semantic guidance into a GAN based stain normalization framework and preserve detailed structural information. Our method does not require manual segmentation maps which is a significant advantage over existing methods. We integrate semantic information at different layers between a pre-trained semantic network and the stain color normalization network. The proposed scheme outperforms other color normalization methods leading to better classification and segmentation performance.
This paper addresses the problem of liver cancer segmentation in Whole Slide Image (WSI). We propose a multi-scale image processing method based on automatic end-to-end deep neural network algorithm for segmentation of cancer area. A seven-levels gaussian pyramid representation of the histopathological image was built to provide the texture information in different scales. In this work, several neural architectures were compared using the original image level for the training procedure. The proposed method is based on U-Net applied to seven levels of various resolutions (pyramidal subsumpling). The predictions in different levels are combined through a voting mechanism. The final segmentation result is generated at the original image level. Partial color normalization and weighted overlapping method were applied in preprocessing and prediction separately. The results show the effectiveness of the proposed multi-scales approach achieving better scores compared to the state-of-the-art.
Stain variation is a phenomenon observed when distinct pathology laboratories stain tissue slides that exhibit similar but not identical color appearance. Due to this color shift between laboratories, convolutional neural networks (CNNs) trained with images from one lab often underperform on unseen images from the other lab. Several techniques have been proposed to reduce the generalization error, mainly grouped into two categories: stain color augmentation and stain color normalization. The former simulates a wide variety of realistic stain variations during training, producing stain-invariant CNNs. The latter aims to match training and test color distributions in order to reduce stain variation. For the first time, we compared some of these techniques and quantified their effect on CNN classification performance using a heterogeneous dataset of hematoxylin and eosin histopathology images from 4 organs and 9 pathology laboratories. Additionally, we propose a novel unsupervised method to perform stain color normalization using a neural network. Based on our experimental results, we provide practical guidelines on how to use stain color augmentation and stain color normalization in future computational pathology applications.
Normalizing unwanted color variations due to differences in staining processes and scanner responses has been shown to aid machine learning in computational pathology. Of the several popular techniques for color normalization, structure preserving color normalization (SPCN) is well-motivated, convincingly tested, and published with its code base. However, SPCN makes occasional errors in color basis estimation leading to artifacts such as swapping the color basis vectors between stains or giving a colored tinge to the background with no tissue. We made several algorithmic improvements to remove these artifacts. Additionally, the original SPCN code is not readily usable on gigapixel whole slide images (WSIs) due to long run times, use of proprietary software platform and libraries, and its inability to automatically handle WSIs. We completely rewrote the software such that it can automatically handle images of any size in popular WSI formats. Our software utilizes GPU-acceleration and open-source libraries that are becoming ubiquitous with the advent of deep learning. We also made several other small improvements and achieved a multifold overall speedup on gigapixel images. Our algorithm and software is usable right out-of-the-box by the computational pathology community.
This article introduces a generalization of the discrete optimal transport, with applications to color image manipulations. This new formulation includes a relaxation of the mass conservation constraint and a regularization term. These two features are crucial for image processing tasks, which necessitate to take into account families of multimodal histograms, with large mass variation across modes. The corresponding relaxed and regularized transportation problem is the solution of a convex optimization problem. Depending on the regularization used, this minimization can be solved using standard linear programming methods or first order proximal splitting schemes. The resulting transportation plan can be used as a color transfer map, which is robust to mass variation across images color palettes. Furthermore, the regularization of the transport plan helps to remove colorization artifacts due to noise amplification. We also extend this framework to the computation of barycenters of distributions. The barycenter is the solution of an optimization problem, which is separately convex with respect to the barycenter and the transportation plans, but not jointly convex. A block coordinate descent scheme converges to a stationary point of the energy. We show that the resulting algorithm can be used for color normalization across several images. The relaxed and regularized barycenter defines a common color palette for those images. Applying color transfer toward this average palette performs a color normalization of the input images.
The recognition of color texture under varying lighting conditions is still an open issue. Several features have been proposed for this purpose, ranging from traditional statistical descriptors to features extracted with neural networks. Still, it is not completely clear under what circumstances a feature performs better than the others. In this paper we report an extensive comparison of old and new texture features, with and without a color normalization step, with a particular focus on how they are affected by small and large variation in the lighting conditions. The evaluation is performed on a new texture database including 68 samples of raw food acquired under 46 conditions that present single and combined variations of light color, direction and intensity. The database allows to systematically investigate the robustness of texture descriptors across a large range of variations of imaging conditions.