Cellular quantitative analysis of neuroblastoma tumor and splitting overlapping cells

© 2014 Tafavogh et al.; licensee BioMed Central Ltd. Background: Neuroblastoma Tumor (NT) is one of the most aggressive types of infant cancer. Essential to accurate diagnosis and prognosis is cellular quantitative analysis of the tumor. Counting enormous numbers of cells under an optical microscope is error-prone. There is therefore an urgent demand from pathologists for robust and automated cell counting systems. However, the main challenge in developing these systems is the inability of them to distinguish between overlapping cells and single cells, and to split the overlapping cells. We address this challenge in two stages by: 1) distinguishing overlapping cells from single cells using the morphological differences between them such as area, uniformity of diameters and cell concavity; and 2) splitting overlapping cells into single cells. We propose a novel approach by using the dominant concave regions of cells as markers to identify the overlap region. We then find the initial splitting points at the critical points of the concave regions by decomposing the concave regions into their components such as arcs, chords and edges, and the distance between the components is analyzed using the developed seed growing technique. Lastly, a shortest path determination approach is developed to determine the optimum splitting route between two candidate initial splitting points.Results: We compare the cell counting results of our system with those of a pathologist as the ground-truth. We also compare the system with three state-of-the-art methods, and the results of statistical tests show a significant improvement in the performance of our system compared to state-of-the-art methods. The F-measure obtained by our system is 88.70%. To evaluate the generalizability of our algorithm, we apply it to images of follicular lymphoma, which has similar histological regions to NT. Of the algorithms tested, our algorithm obtains the highest F-measure of 92.79%.Conclusion: We develop a novel overlapping cell splitting algorithm to enhance the cellular quantitative analysis of infant neuroblastoma. The performance of the proposed algorithm promises a reliable automated cell counting system for pathology laboratories. Moreover, the high performance obtained by our algorithm for images of follicular lymphoma demonstrates the generalization of the proposed algorithm for cancers with similar histological regions and histological structures

Plasmodium life cycle stage classification based quantification of malaria parasitaemia in thin blood smears.

Visual inspection for the quantification of malaria parasitaemiain (MP) and classification of life cycle stage are hard and time taking. Even though, automated techniques for the quantification of MP and their classification are reported in the literature. However, either reported techniques are imperfect or cannot deal with special issues such as anemia and hemoglobinopathies due to clumps of red blood cells (RBCs). The focus of the current work is to examine the thin blood smear microscopic images stained with Giemsa by digital image processing techniques, grading MP on independent factors (RBCs morphology) and classification of its life cycle stage. For the classification of the life cycle of malaria parasite the k-nearest neighbor, Naïve Bayes and multi-class support vector machine are employed for classification based on histograms of oriented gradients and local binary pattern features. The proposed methodology is based on inductive technique, segment malaria parasites through the adaptive machine learning techniques. The quantification accuracy of RBCs is enhanced; RBCs clumps are split by analysis of concavity regions for focal points. Further, classification of infected and non-infected RBCs has been made to grade MP precisely. The training and testing of the proposed approach on benchmark dataset with respect to ground truth data, yield 96.75% MP sensitivity and 94.59% specificity. Additionally, the proposed approach addresses the process with independent factors (RBCs morphology). Finally, it is an economical solution for MP grading in immense testing

Image analysis and statistical modeling for applications in cytometry and bioprocess control

Today, signal processing has a central role in many of the advancements in systems biology. Modern signal processing is required to provide efficient computational solutions to unravel complex problems that are either arduous or impossible to obtain using conventional approaches. For example, imaging-based high-throughput experiments enable cells to be examined at even subcellular level yielding huge amount of image data. Cytometry is an integral part of such experiments and involves measurement of different cell parameters which requires extraction of quantitative experimental values from cell microscopy images. In order to do that for such large number of images, fast and accurate automated image analysis methods are required. In another example, modeling of bioprocesses and their scale-up is a challenging task where different scales have different parameters and often there are more variables than the available number of observations thus requiring special methodology. In many biomedical cell microscopy studies, it is necessary to analyze the images at single cell or even subcellular level since owing to the heterogeneity of cell populations the population-averaged measurements are often inconclusive. Moreover, the emergence of imaging-based high-content screening experiments, especially for drug design, has put single cell analysis at the forefront since it is required to study the dynamics of single-cell gene expressions for tracking and quantification of cell phenotypic variations. The ability to perform single cell analysis depends on the accuracy of image segmentation in detecting individual cells from images. However, clumping of cells at both nuclei and cytoplasm level hinders accurate cell image segmentation. Part of this thesis work concentrates on developing accurate automated methods for segmentation of bright field as well as multichannel fluorescence microscopy images of cells with an emphasis on clump splitting so that cells are separated from each other as well as from background. The complexity in bioprocess development and control crave for the usage of computational modeling and data analysis approaches for process optimization and scale-up. This is also asserted by the fact that obtaining a priori knowledge needed for the development of traditional scale-up criteria may at times be difficult. Moreover, employment of efficient process modeling may provide the added advantage of automatic identification of influential control parameters. Determination of the values of the identified parameters and the ability to predict them at different scales help in process control and in achieving their scale-up. Bioprocess modeling and control can also benefit from single cell analysis where the latter could add a new dimension to the former once imaging-based in-line sensors allow for monitoring of key variables governing the processes. In this thesis we exploited signal processing techniques for statistical modeling of bioprocess and its scale-up as well as for development of fully automated methods for biomedical cell microscopy image segmentation beginning from image pre-processing and initial segmentation to clump splitting and image post-processing with the goal to facilitate the high-throughput analysis. In order to highlight the contribution of this work, we present three application case studies where we applied the developed methods to solve the problems of cell image segmentation and bioprocess modeling and scale-up

A Comprehensive Overview of Computational Nuclei Segmentation Methods in Digital Pathology

In the cancer diagnosis pipeline, digital pathology plays an instrumental role in the identification, staging, and grading of malignant areas on biopsy tissue specimens. High resolution histology images are subject to high variance in appearance, sourcing either from the acquisition devices or the H\&E staining process. Nuclei segmentation is an important task, as it detects the nuclei cells over background tissue and gives rise to the topology, size, and count of nuclei which are determinant factors for cancer detection. Yet, it is a fairly time consuming task for pathologists, with reportedly high subjectivity. Computer Aided Diagnosis (CAD) tools empowered by modern Artificial Intelligence (AI) models enable the automation of nuclei segmentation. This can reduce the subjectivity in analysis and reading time. This paper provides an extensive review, beginning from earlier works use traditional image processing techniques and reaching up to modern approaches following the Deep Learning (DL) paradigm. Our review also focuses on the weak supervision aspect of the problem, motivated by the fact that annotated data is scarce. At the end, the advantages of different models and types of supervision are thoroughly discussed. Furthermore, we try to extrapolate and envision how future research lines will potentially be, so as to minimize the need for labeled data while maintaining high performance. Future methods should emphasize efficient and explainable models with a transparent underlying process so that physicians can trust their output.Comment: 47 pages, 27 figures, 9 table

Hover-Net : simultaneous segmentation and classification of nuclei in multi-tissue histology images

Nuclear segmentation and classification within Haematoxylin & Eosin stained histology images is a fundamental prerequisite in the digital pathology work-flow. The development of automated methods for nuclear segmentation and classification enables the quantitative analysis of tens of thousands of nuclei within a whole-slide pathology image, opening up possibilities of further analysis of large-scale nuclear morphometry. However, automated nuclear segmentation and classification is faced with a major challenge in that there are several different types of nuclei, some of them exhibiting large intra-class variability such as the nuclei of tumour cells. Additionally, some of the nuclei are often clustered together. To address these challenges, we present a novel convolutional neural network for simultaneous nuclear segmentation and classification that leverages the instance-rich information encoded within the vertical and horizontal distances of nuclear pixels to their centres of mass. These distances are then utilised to separate clustered nuclei, resulting in an accurate segmentation, particularly in areas with overlapping instances. Then, for each segmented instance the network predicts the type of nucleus via a devoted up-sampling branch. We demonstrate state-of-the-art performance compared to other methods on multiple independent multi-tissue histology image datasets. As part of this work, we introduce a new dataset of Haematoxylin & Eosin stained colorectal adenocarcinoma image tiles, containing 24,319 exhaustively annotated nuclei with associated class labels

Discrete Element Modeling of the Grading- and Shape-Dependent Behavior of Granular Materials

Granular materials, such as sand, biomass particles, and pharmaceutical pills, are widespread in nature, industrial systems, and our daily life. Fundamentally, the bulk mechanical behavior of such materials is governed by the physical and morphological features of and the interactions among constituent particles at the microscopic scale. From a modeling standpoint, the particle-based discrete element method (DEM) has emerged as the most prevalent numerical tool to model and study the behavior of granular materials and the systems they form. A critical step towards an accurate and predictive DEM model is to incorporate those physical and morphological features (e.g., particle size, shape, and deformability) pertaining to the constituent particles. The main objective of this dissertation is to approach an accurate characterization and modeling of the grading- and shape-dependent behavior of granular materials by developing DEM models that incorporate realistic physical and morphological features of granular particles. Revolving around this objective, three studies are presented: image-based particle reconstruction and morphology characterization, grading and shape-dependent shearing behavior of rigid-particle systems, and granular flow of deformable irregular particles. The first study presents a machine learning and level-set based framework to re- construct granular particles and to characterize particle morphology from X-ray computed tomography (X-ray CT) imaging of realistic granular materials. Images containing detailed microstructure information of a granular material are obtained using the X-ray CT tech- nique. Approaches such as the watershed method in two dimensions (2D) and the combined machine learning and level set method in three dimensions (3D) are then utilized and implemented to segment X-ray CT images and to numerically reconstruct individual particles in the granular material. Based on the realistic particle shapes, particle morphology is characterized by descriptors including aspect ratio, roundness, circularity (2D) or sphericity (3D). The particle shapes or morphology provide important constraints to develop DEM models with particle physical and morphological features conforming to the specific granular material of interest. In the second study, DEM models incorporated with realistic particle sizes and shapes are developed and applied to study the shearing behavior of sandy soils. The particle sizes and shapes are obtained from realistic samples of JSC-1A Martian regolith simulant. Irregular-shape particles are represented by rigid clumps based on the domain overlapping filling method. The effects of particle shape irregularity on the shearing behavior of granular materials are investigated through direct shear tests, along with the comparisons from spherical particles with or without rolling resistance. The micro-mechanisms of shape irregularity contributing to the shear resistance are identified. The last study investigates the effects of particle deformability (e.g., compression, deflection or torsion), together with particle sizes and shapes, on the granular flow of flexible granular materials. A bonded-sphere DEM model is implemented with the capability of embodying various particle sizes and irregular shapes, as well as capturing particle deformability. This approach is then applied to simulate and study the behavior of flexible granular materials in cyclic compression and hopper flow tests. The effects of particle size, shape and deformability on the bulk mechanical behavior are investigated on the basis of the DEM simulation results. The importance of particle deformability to the DEM simulations of flexible granular materials is demonstrated

Nuclei & Glands Instance Segmentation in Histology Images: A Narrative Review

Instance segmentation of nuclei and glands in the histology images is an important step in computational pathology workflow for cancer diagnosis, treatment planning and survival analysis. With the advent of modern hardware, the recent availability of large-scale quality public datasets and the community organized grand challenges have seen a surge in automated methods focusing on domain specific challenges, which is pivotal for technology advancements and clinical translation. In this survey, 126 papers illustrating the AI based methods for nuclei and glands instance segmentation published in the last five years (2017-2022) are deeply analyzed, the limitations of current approaches and the open challenges are discussed. Moreover, the potential future research direction is presented and the contribution of state-of-the-art methods is summarized. Further, a generalized summary of publicly available datasets and a detailed insights on the grand challenges illustrating the top performing methods specific to each challenge is also provided. Besides, we intended to give the reader current state of existing research and pointers to the future directions in developing methods that can be used in clinical practice enabling improved diagnosis, grading, prognosis, and treatment planning of cancer. To the best of our knowledge, no previous work has reviewed the instance segmentation in histology images focusing towards this direction.Comment: 60 pages, 14 figure

INVESTIGATING INVASION IN DUCTAL CARCINOMA IN SITU WITH TOPOGRAPHICAL SINGLE CELL GENOME SEQUENCING

Synchronous Ductal Carcinoma in situ (DCIS-IDC) is an early stage breast cancer invasion in which it is possible to delineate genomic evolution during invasion because of the presence of both in situ and invasive regions within the same sample. While laser capture microdissection studies of DCIS-IDC examined the relationship between the paired in situ (DCIS) and invasive (IDC) regions, these studies were either confounded by bulk tissue or limited to a small set of genes or markers. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS), which combines laser-catapulting with single cell DNA sequencing to measure genomic copy number profiles from single tumor cells while preserving their spatial context. We applied TSCS to sequence 1,293 single cells from 10 synchronous DCIS patients. We also applied deep-exome sequencing to the in situ, invasive and normal tissues for the DCIS-IDC patients. Previous bulk tissue studies had produced several conflicting models of tumor evolution. Our data support a multiclonal invasion model, in which genome evolution occurs within the ducts and gives rise to multiple subclones that escape the ducts into the adjacent tissues to establish the invasive carcinomas. In summary, we have developed a novel method for single cell DNA sequencing, which preserves spatial context, and applied this method to understand clonal evolution during the transition between carcinoma in situ to invasive ductal carcinoma