André Huisman

Digital pathology: current status and future perspectives

Al‐Janabi S, Huisman A & Van Diest P J 
(2012) Histopathology 61, 1–9

Automatic nuclei segmentation in H&E stained breast cancer histopathology images.

The introduction of fast digital slide scanners that provide whole slide images has led to a revival of interest in image analysis applications in pathology. Segmentation of cells and nuclei is an important first step towards automatic analysis of digitized microscopy images. We therefore developed an automated nuclei segmentation method that works with hematoxylin and eosin (H&E) stained breast cancer histopathology images, which represent regions of whole digital slides. The procedure can be divided into four main steps: 1) pre-processing with color unmixing and morphological operators, 2) marker-controlled watershed segmentation at multiple scales and with different markers, 3) post-processing for rejection of false regions and 4) merging of the results from multiple scales. The procedure was developed on a set of 21 breast cancer cases (subset A) and tested on a separate validation set of 18 cases (subset B). The evaluation was done in terms of both detection accuracy (sensitivity and positive predictive value) and segmentation accuracy (Dice coefficient). The mean estimated sensitivity for subset A was 0.875 (±0.092) and for subset B 0.853 (±0.077). The mean estimated positive predictive value was 0.904 (±0.075) and 0.886 (±0.069) for subsets A and B, respectively. For both subsets, the distribution of the Dice coefficients had a high peak around 0.9, with the vast majority of segmentations having values larger than 0.8.

Creation of a fully digital pathology slide archive by high-volume tissue slide scanning

Digital slide scanners for scanning glass slides are becoming increasingly popular because current scanners are fast enough and produce good enough images for diagnostic purposes, education, and research. Also, the price for storing vast amounts of data has decreased over the last years, and this trend is expected to continue. Where most laboratories use their scanners mainly for education and research with limited financial and technical implications, we decided to face the huge challenges of prospectively setting up a fully digital pathology slide archive, primarily aiming to optimize the preparation and running of clinicopathological conferences. In this article, we describe the setup of our digital archiving solution and discuss the technical challenges we had to overcome. To give insight in the performance of our digital archive, we provide some statistics as well. We also present our thoughts on future developments in the area of digital slide scanning.

Whole slide images for primary diagnostics in dermatopathology: a feasibility study

Background During the last decade, whole slide images (WSI) have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation and quality assurance testing. However, WSI have not regularly been used for routine diagnosis, because of the lack of validation studies. Aim To test the validity of using WSI for primary diagnosis of skin diseases. Materials and methods 100 skin biopsies and resections which had been diagnosed light microscopically one year previously were scanned at 20× magnification, and rediagnosed by six pathologists (every pathologist assessed his own cases), having the original clinical information available, but blinded to the original diagnoses. The WSI diagnoses were compared to the initial light microscopy diagnosis and classified as concordant, slightly discordant (without clinical consequences) or discordant. Results The light microscopy and the WSI based diagnosis were concordant in 94% of the cases. The light microscopy and WSI diagnosis were slightly discordant in 6% of the cases. For one of the slightly discrepant cases the WSI diagnosis was considered better, while the original diagnosis was preferred for the other five cases. There were no discordant cases with clinical or prognostic implications. Conclusion Primary histopathological diagnosis of skin biopsies and resections can be done digitally using WSI.

Whole slide images for primary diagnostics of gastrointestinal tract pathology: a feasibility study

During the last decade, whole slide images have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation, and quality assurance testing. However, whole slide images have as yet not much been used for up-front diagnostics because of the lack of validation studies. The aim of this study was, therefore, to test the feasibility of whole slide images for diagnosis of gastrointestinal tract specimens, one of the largest areas of diagnostic pathology. One hundred gastrointestinal tract biopsies and resections that had been diagnosed using light microscopy 1 year before were rediagnosed on whole slide images scanned at ×20 magnification by 5 pathologists (all reassessing their own cases), having the original clinical information available but blinded to their original light microscopy diagnoses. The original light microscopy and whole slide image-based diagnoses were compared and classified as concordant, slightly discordant (without clinical consequences), and discordant. The diagnoses based on light microscopy and the whole slide image-based rediagnoses were concordant in 95% of the cases. Light microscopy and whole slide image diagnosis in the remaining 5% of cases were slightly discordant, none of these were with clinical or prognostic implications. Up-front histopathologic diagnosis of gastrointestinal biopsies and resections can be done on whole slide images.

Development of 3D chromatin texture analysis using confocal laser scanning microscopy

Introduction: Analysis of nuclear texture features as a measure of nuclear chromatin changes has been proven to be useful when measured on thin (5–6 μm) tissue sections using conventional 2D bright field microscopy. The drawback of this approach is that most nuclei are not intact because of those thin sections. Confocal laser scanning microscopy (CLSM) allows measurements of texture in 3D reconstructed nuclei. The aim of this study was to develop 3D texture features that quantitatively describe changes in chromatin architecture associated with malignancy using CLSM images. Methods: Thirty-five features thoughtfully chosen from 4 categories of 3D texture features (discrete texture features, Markovian features, fractal features, grey value distribution features) were selected and tested for invariance properties (rotation and scaling) using artificial images with a known grey value distribution. The discriminative power of the 3D texture features was tested on artificially constructed benign and malignant 3D nuclei with increasing nucleolar size and advancing chromatin margination towards the periphery of the nucleus. As a clinical proof of principle, the discriminative power of the texture features was assessed on 10 benign and 10 malignant human prostate nuclei, evaluating also whether there was more texture information in 3D whole nuclei compared to a single 2D plane from the middle of the nucleus. Results: All texture features showed the expected invariance properties. Almost all features were sensitive to variations in the nucleolar size and to the degree of margination of chromatin. Fourteen texture features from different categories had high discriminative power for separating the benign and malignant nuclei. The discrete texture features performed less than expected. There was more information on nuclear texture in 3D than in 2D. Conclusion: A set of 35 3D nuclear texture features was used successfully to assess nuclear chromatin patterns in 3D images obtained by confocal laser scanning microscopy, and as a proof of principle we showed that these features may be clinically useful for analysis of prostate neoplasia.

Digital slide images for primary diagnostics in breast pathology: a feasibility study

Digital slide images have been used in many areas of pathology such as teaching, research, digital archiving, teleconsultation, and quality assurance testing. However, they have not much been used as yet for upfront diagnostics. The aim of this study was therefore to test the feasibility of digital slide image-based diagnosis of breast specimens. Sections of 100 breast specimens previously diagnosed conventionally were scanned and rediagnosed on digital slide images by the same pathologists who performed the initial light microscopy-based diagnosis. The digital slide image diagnoses were compared with the light microscopy diagnoses and classified as concordant, slightly discrepant (without clinical or prognostic consequences), or discrepant. The original light microscopy- and digital slide image-based diagnoses were concordant in 93% and slightly discrepant in 6% of cases. There was only 1 discrepant case with clinical or prognostic implication to the patient. However, for this case, no final agreement could be achieved. For 4 of the 6 slightly discrepant cases, digital slide image diagnosis was considered the better one, whereas the original diagnosis was preferred in only 1 case. In addition, for 1 case categorized as slightly discrepant, both the digital slide image and conventional diagnosis were imperfect according to 2 reviewing breast pathologists. This study demonstrates that upfront histopathologic diagnosis of breast biopsies and resections can reliably be done on digital slide image.

Whole slide images as a platform for initial diagnostics in histopathology in a medium-sized routine laboratory

Introduction Whole slide imaging is the process of digitizing glass slides and the creation of Whole Slide Images (WSI), which enable the examination of pathology samples on a computer screen in a manner comparable to light microscopy. WSI have been used for different applications in pathology but their use for primary diagnostics is still limited. Implementing WSI for primary diagnostics would be a turning point necessitating extensive validation to unravel pitfalls and difficulties that could be encountered within the routine workflow. This article is aimed to describe the gradual integration of WSI into routine pathology diagnostics in a medium-sized routine pathology laboratory. Methods This project was started with optimizing the digital work environment including the setting up of validation studies, scanning preferences, storing WSI and the implemented adjustments to the workflow for the laboratory and the pathologist. Afterwards scanning glass slides was initiated in the department of pathology at the Atrium Medical Center, Heerlen, The Netherlands, for performing primary diagnostics of breast biopsies. Later this was extended to other specimen types including resections. Results The validation studies yielded a high concordance rate between WSI and conventional diagnoses. Routine primary WSI based diagnosis was possible in 82.1% of cases. Failure of digital diagnosis was mainly related to poor image quality and logistic problems. Conclusion The quality of the currently produced WSI is sufficient for primary diagnostics in 82.1% of the cases. Improving image quality, adequate retrieval and controlling scanning error will definitely encourage the wide adaptation in routine diagnostics.

Prognostic value of automatically extracted nuclear morphometric features in whole slide images of male breast cancer

Numerous studies have shown the prognostic significance of nuclear morphometry in breast cancer patients. Wide acceptance of morphometric methods has, however, been hampered by the tedious and time consuming nature of the manual segmentation of nuclei and the lack of equipment for high throughput digitization of slides. Recently, whole slide imaging became more affordable and widely available, making fully digital pathology archives feasible. In this study, we employ an automatic nuclei segmentation algorithm to extract nuclear morphometry features related to size and we analyze their prognostic value in male breast cancer. The study population comprised 101 male breast cancer patients for whom survival data was available (median follow-up of 5.7 years). Automatic segmentation was performed on digitized tissue microarray slides, and for each patient, the mean nuclear area and the standard deviation of the nuclear area were calculated. In univariate survival analysis, a significant difference was found between patients with low and high mean nuclear area (P=0.022), while nuclear atypia score did not provide prognostic value. In Cox regression, mean nuclear area had independent additional prognostic value (P=0.032) to tumor size and tubule formation. In conclusion, we present an automatic method for nuclear morphometry and its application in male breast cancer prognosis. The automatically extracted mean nuclear area proved to be a significant prognostic indicator. With the increasing availability of slide scanning equipment in pathology labs, these kinds of quantitative approaches can be easily integrated in the workflow of routine pathology practice.

Myocardial fibrosis and pro-fibrotic markers in end-stage heart failure patients during continuous-flow left ventricular assist device support

OBJECTIVES During support with a left ventricular assist device (LVAD), partial reverse remodelling takes place in which fibrosis plays an important role. In this study, we analysed the histological changes and expression of fibrotic markers in patients with advanced heart failure (HF) during continuous-flow LVAD (cf-LVAD) support. METHODS In 25 patients, myocardial tissue at the time of LVAD implantation (pre-LVAD) was compared with tissue from the explanted left ventricle (post-LVAD). Interstitial fibrosis and cardiomyocyte size were analysed pre- and post-LVAD. Plasma was obtained from all patients before and during LVAD support. Plasma levels, cardiac mRNA and protein expression of brain natriuretic peptide (BNP), galectin-3 (Gal-3), connective tissue growth factor (CTGF), osteopontin (OPN) and transforming growth factor β-1 were determined. RESULTS Fibrosis increased during cf-LVAD unloading (P < 0.05). Cardiomyocytes elongated (P < 0.05), whereas cross-sectional area did not change. BNP, Gal-3, CTGF and OPN were significantly elevated pre-LVAD in comparison with controls. BNP decreased significantly after 1 month of cf-LVAD support (P < 0.001) to near-normal levels. Pro-fibrotic markers remained elevated in comparison with controls. CONCLUSIONS cf-LVAD support is associated with lengthening of cardiomyocytes, without alterations in diameter size. Remarkably, myocardial fibrosis increased as well as circulating pro-fibrotic markers. Whether the morphological changes are a direct effect of reduced pulsatility during cf-LVAD support or due to HF progression requires further investigation.