Spatial resolution requirements for acquisition of the virtual screening slide for digital whole-specimen breast histopathology

We examined the effect of lateral spatial resolution and reader specialty on the accuracy of detection of breast cancer. The motivation for this pilot study was the need to acquire and display very large data sets in whole-specimen 3D digital breast histopathology imaging. The ultimate goal is to determine the minimum resolution adequate for detection of malignancy. Twenty-three histologic slides were selected from breast pathology cases and digitized at 2 sampling distances (3.2 and 1.9 microm pixels). Images were viewed by 14 pathologists, of whom 5 had breast pathology as their primary specialty. The readers assessed the likelihood of malignancy on a 5-point Likert scale, and provided a provisional diagnosis. For the detection task, sensitivity, specificity, overall accuracy of detection, and area under the receiver-operator curve were calculated. An overall diagnostic score, and scores grouped by malignancy type, were also computed. Outcome measures were examined for significant resolution and specialty effects. Increasing the lateral resolution significantly improved accuracy in diagnosis (P=.004) but no effect was found for detection. Breast specialists achieved significantly higher scores for all outcome measures except specificity. Differences in performance between the 2 groups of readers tended to be greater for the diagnostic task compared to detection, especially at the higher resolution. However, specimen coverage may also be a significant factor. Factors related to the readers may have also affected performance in this study. Based on these results, a more comprehensive study should examine pixel sizes between 0.7 and 1.9 microm.     

Whole-specimen histopathology: a method to produce whole-mount breast serial sections for 3-D digital histopathology imaging

AIMS: To develop a method for preparing diagnostic-quality, whole-mount serial sections of breast specimens while preserving 3-D conformation. This required supporting the fresh specimen prior to breadloafing and refining the conventional tissue processing method. The overall goal is to use digital images of whole-specimen histopathology to improve the estimation of extent of disease. METHODS AND RESULTS: To maintain a 3-D conformation, the specimen is suspended in 3.5% agar at 55 degrees C. The block is sliced at 5-mm intervals. Sectioning is performed after extended fixation in 4% formaldehyde from paraformaldehyde in 0.1 m Millonig's buffer, followed by paraffin processing using a non-routine schedule and extended paraffin infiltration. Whole-mount serial breast sections are produced with features of equal or superior quality to that which can be achieved using conventional methods. The method is compatible with some immunohistochemical stains but requires further optimization for others. CONCLUSIONS: The technique is currently suitable for research applications. With the reduction in processing time achievable with microwave-assisted processing, there is the potential for its use as a routine clinical method. This tool may improve the accuracy of margin estimates and identification of multifocality in breast cancer; further evaluation is necessary.     

虚拟显微镜技术在病理的应用

 虚拟显微镜技术应用于形态学教学能显著提高教学和学习的效率,与显微数码互动系统联合应用优势互补;临床主要应用于病理远程会诊、切片存档,是一项重要的辅助技术。随着虚拟显微镜技术的发展与完善,虚拟显微镜技术将普及应用,推动病理技术和病理诊断的不断发展。     

A three-dimensional digital atlas of the starling brain

Because of their sophisticated vocal behaviour, their social nature, their high plasticity and their robustness, starlings have become an important model species that is widely used in studies of neuroethology of song production and perception. Since magnetic resonance imaging (MRI) represents an increasingly relevant tool for comparative neuroscience, a 3D MRI-based atlas of the starling brain becomes essential. Using multiple imaging protocols we delineated several sensory systems as well as the song control system. This starling brain atlas can easily be used to determine the stereotactic location of identified neural structures at any angle of the head. Additionally, the atlas is useful to find the optimal angle of sectioning for slice experiments, stereotactic injections and electrophysiological recordings. The starling brain atlas is freely available for the scientific community.     

A digital three-dimensional atlas of structure/function relationships

The techniques and issues relevant to the development of a digital three-dimensional atlas of brain structure and function are discussed. The goals were to develop the appropriate methods for an interactive and quantitative stereotactic atlas to describe both anatomy and physiology. The work presented here used serial sections from unstained, thionein-stained and 2-deoxyglucose autoradiographic data sets.     

Design and characterization of a digital image acquisition system for whole-specimen breast histopathology

We have developed a digital histopathology imaging system capable of producing a three-dimensional (3D) representation of histopathology from an entire lumpectomy specimen. The system has the potential to improve the accuracy of surgical margin assessment in the treatment of breast cancer by providing finer sampling and 3D visualization. A scanning light microscope was modified to allow digital photomicrography of a stack of large (up to 120x170 mm2) histology slides cut serially through the entire specimen. The images are registered and displayed in 2D and 3D. The design of the system, which reduces or eliminates the appearance of 'tiling' and 'seam' artefacts inherent in the scanning method, is described and its resolution, contrast/noise and coverage properties are characterized through measurements of the modulation transfer function (MTF), depth of field (DOF) and signal difference to noise ratio (SDNR). The imaging task requires a lateral resolution of 5 microm, an SDNR of 5 between relevant features, 'tiling artefact' at a level below the detectability threshold of the eye, and 'seam artefact' of less than 5-10 microm. The tests demonstrate that the system is largely adequate for the imaging task, although further optimizations are required to reduce the degradation of coverage incurred by seam artefact.     

A three-dimensional digital atlas database of the adult C57BL/6J mouse brain by magnetic resonance microscopy

A comprehensive three-dimensional digital atlas database of the C57BL/6J mouse brain was developed based on magnetic resonance microscopy images acquired on a 17.6-T superconducting magnet. By using both manual tracing and an atlas-based semi-automatic segmentation approach, T2-weighted magnetic resonance microscopy images of 10 adult male formalin-fixed, excised C57BL/6J mouse brains were segmented into 20 anatomical structures. These structures included the neocortex, hippocampus, amygdala, olfactory bulbs, basal forebrain and septum, caudate-putamen, globus pallidus, thalamus, hypothalamus, central gray, superior colliculi, inferior colliculi, the rest of midbrain, cerebellum, brainstem, corpus callosum/external capsule, internal capsule, anterior commissure, fimbria, and ventricles. The segmentation data were formatted and stored into a database containing three different atlas types: 10 single-specimen brain atlases, an average brain atlas and a probabilistic atlas. Additionally, quantitative group information, such as variations in structural volume, surface area, magnetic resonance microscopy image intensity and local geometry, were computed and stored as an integral part of the database. The database augments ongoing efforts with other high priority strains as defined by the Mouse Phenome Database focused on providing a quantitative framework for accurate mapping of functional, genetic and protein expression patterns acquired by a myriad of technologies and imaging modalities.     

Going glass to digital: virtual microscopy as a simulation-based revolution in pathology and laboratory science

The recent technological advance of digital high resolution imaging has allowed the field of pathology and medical laboratory science to undergo a dramatic transformation with the incorporation of virtual microscopy as a simulation-based educational and diagnostic tool. This transformation has correlated with an overall increase in the use of simulation in medicine in an effort to address dwindling clinical resource availability and patient safety issues currently facing the modern healthcare system. Virtual microscopy represents one such simulation-based technology that has the potential to enhance student learning and readiness to practice while revolutionising the ability to clinically diagnose pathology collaboratively across the world. While understanding that a substantial amount of literature already exists on virtual microscopy, much more research is still required to elucidate the full capabilities of this technology. This review explores the use of virtual microscopy in medical education and disease diagnosis with a unique focus on key requirements needed to take this technology to the next level in its use in medical education and clinical practice.     

ImmunoMembrane: a publicly available web application for digital image analysis of HER2 immunohistochemistry

Tuominen V J, Tolonen T T & Isola J
(2012) Histopathology  60, 758–767
ImmunoMembrane: a publicly available web application for digital image analysis of HER2 immunohistochemistry Aims: Assessment of the human epidermal growth factor receptor 2 (HER2) with immunohistochemistry (IHC) is routine practice in clinical pathology laboratories. Visual classification of the staining reaction (usually into 0/1+, 2+ or 3+) is subjective and prone to significant inter‐ and intra‐observer variation. In this study, we describe ImmunoMembrane, an easy‐to‐use HER2 IHC analysis software, which is freely available as a web application, requiring no download or installation. Methods and results: ImmunoMembrane uses colour deconvolution for stain separation and a customized algorithm for cell membrane segmentation. A quantitative score (IM‐score, 0–20 points) is generated according to the membrane staining intensity and completeness. Specimens are classified into 0/1+, 2+ or 3+ based on IM‐score cut‐offs defined using a training set. The classification and membrane segmentation are presented as a pseudo‐coloured overlay image. With a validation set (144 HercepTest^®‐stained whole tissue sections), ImmunoMembrane matched well with the pathologist’s visual classification (weighted kappa κ_w = 0.80), as well as fluorescence in‐situ hybridization (FISH) (IHC disagreement 3.5%, n = 144) and chromogenic in‐situ hybridization (CISH) (IHC disagreement 2.8%, n = 144). Conclusions: We anticipate that publicly available web applications, such as ImmunoMembrane, will accelerate the adoption of automated image analysis in clinical diagnostics of HER2 IHC. ImmunoMembrane is freely accessible at: http://jvsmicroscope.uta.fi/immunomembrane/ .     

Auto-analysis of immunohistochemical findings for breast cancer using specified software and virtual microscopy

Currently, the therapeutic strategy for a breast cancer patient is designed according to their histopathological, immunohistochemical and molecular findings. These findings are obtained through the collected efforts of many individual pathologists or medical technologists (MTs) and are, thus, limited by intra-observer error and potentially subjective decision making. Twenty five breast cancer specimens collected between November 2009 and February 2010 were examined for immunohistochemical expressions of estrogen receptor (ER), progesterone receptor (PgR), HER2, Ki-67, Topoisomerase II alpha (Topo IIalpha). Fifty one cancer specimens collected November 2009 and June 2010 were examined for human epidermal growth factor 2 (HER2). Immunohistochemical staining was performed using auto-stainers (Ventana) and the results were stored digitally after examination by virtual microscopy (Hamamatsu Photonics). Data analysis was performed with the Genie/Aperio software package on a desk-top computer. For all the antibodies used expect for HER2, concordant results were obtained in 100% of 24 ER positive cases. Ki-67 index (r=0.96) and Topo IIalpha index (r=0.95) also showed a significant correlation (p<0.001). For HER2, all four specimens with Hercep-score 2 by ocular observation but auto-analysis score 1 revealed no HER2 gene amplification. Well-organized auto-analysis is more likely to result in an objective observation and to provide a means by which to standardize the methods for immunohistochemical detection of breast cancer.     

A computer based handbook and atlas of pathology

The Diagnostic Encyclopaedia Workstation (DEW) is a computerized handbook of pathology intended for use in diagnostic practice. It consists of a combination of a personal computer (PC), a video disc player (VDP), for which a specially developed disc is used, two monitors, a mouse and software. The hard disc of the computer contains textual information on diagnoses in categories such as macroscopy, common histology, immunopathology, clinical observations and prognosis and case histories. This information is frequently illustrated by pictures on a video disc which is automatically addressed by the computer software. All pictures, at present some 3000, pertain to case histories which are included in the system. Also integrated are classification aids in two categories: diagnostic criteria and differential diagnosis. Advantages of DEW over the use of conventional manuals are 1) the extensive volume of text, 2) the large number of high quality illustrations, 3) the immediate access to cross references and 4) the potential for continuous revision.     

Three-dimensional localisation based on projectional and tomographic image correlation: an application for digital tomosynthesis.

Accurate three-dimensional tumor localisation in Radiotherapy, is critical to the treatment outcome, particularly when high dose gradients are present. A number of techniques have been proposed for the localisation of anatomical structures or markers. The present study proposes an approach to a concurrent maximisation of localisation accuracy and efficiency by correlation of tomographic and projectional images. The method introduces an element of direct verification and interactive optimisation of the process. Tomographic images are used for the identification of a point of interest. Its position is computed within the treatment co-ordinate system and verification of this position is achieved by obtaining the beam's eye view of the identified point on two projection radiographs. The key element of the approach is that all images used should be part of one single image data set. The implementation of this localisation method, as part of the functionality of a Digital Tomosynthesis prototype, has provided an integrated facility for localisation, of optimised accuracy and precision, while easy and efficient to use. The considerations are general and apply in principle to any imaging system that can augment tomographic images with projections.     

A breast density index for digital mammograms based on radiologists’ randing

The purpose of this study was to develop and evaluate a computerized method of calculating a breast density index (BDI) from digitized mammograms that was designed specifically to model radiologists’ perception of breast density. A set of 153 pairs of digitized mammograms (cranio-caudal, CC, and mediolateral oblique, MLO, views) were acquired and preprocessed to reduce detector biases. The sets of mammograms were ordered on an ordinal scale (a scale based only on relative rank-ordering) by two radiologists, and a cardinal (an absolute numerical score) BDI value was calculated from the oridinal ranks. The images were also assigned cardinal BDI values by the radiologists in a subsequent session. Six. mathematical features (including fractal dimension and others) were calculated from the digital mammograms, and were used in conjunction with single value decomposition and multiple linear regression to calculate a computerized BDI. The linear correlation coefficient between different ordinal ranking sessions were as follows: intraradiologist intraprojection (CC/CC):r=0.978; intraradiologist interprojection (CC/MLO):r=0.960; and interradiologist intraprojection (CC/CC):r=0.968. A separate breast density index was derived from three separate ordinal rankings by one radiologist (two with CC views, one with the MLO view). The computer derivedBDI had a correlation coefficient (r) of 0.907 with the radiologists’ ordinalBDI. A comparison between radiologists using a cardinal scoring system (which is closest to how radiologists actually evaluate breast density) showedr=0.914. A breast density index calculated by a computer but modeled after radiologist perception of breast density may be valuable in objectively measuring breast density. Such a metric may prove valuable in numerous areas, including breast cancer risk assessment and in evaluating screening techniques specifically designed to improve imaging of the dense breast.     

Characterization and classification of psittacine atherosclerotic lesions by histopathology,digital image analysis,transmission and scanning electron microscopy

Atherosclerosis is a degenerative and inflammatory vascular disease characterized in mammals and birds by the accumulation of inflammatory cells, lipids, calcium, and formation of large fibrofatty lesions within the intima of arteries resulting in the disorganization of the arterial wall and stenosis of the lumen. Despite the high incidence of atherosclerosis in parrots and the high number of case reports, there are few pathologic investigations and the ultrastructural study of the lesions has not been documented. Sixty-three major arteries were collected from 24 psittacine birds of 11 species during routine post-mortem examinations. Samples from the major arteries were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde, and processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additional samples were fixed in 10% formalin and embedded in paraffin for histological examination. Additional histochemical stains for calcium, elastic fibres, and lipid were performed. Toluidine blue-stained 0.5 µm-thick resin sections were also obtained. Digital image analysis was performed to provide objective quantitative information on the different lesions. The histopathology and ultrastructure of psittacine atherosclerosis were found to be similar to other avian and mammalian species. Seven lesion types could be described, which were similar to the human classification system. Digital image analysis, TEM, and SEM helped to further describe the lesions and refine the classification system. TEM findings were similar to other avian and mammalian species with the notable presence of macrophage-derived and smooth muscle cell-derived foam cells and extracellular lipid. SEM revealed various stages of endothelial surface defects and, occasionally, adherent blood cells.     

数字化颅颌面骨三维解剖图谱的建立

目的:研究和建立数字化颅颌面骨三维解剖图谱.方法:使用自主研发的医学图像处理和三维建模系统生成颅颌面诸骨的精确三维模型,然后采用 Visual Studio 2008开发三维解剖图谱系统,设计强大而灵活的界面布局和操作控制,充分发挥数字化图谱的虚拟可视化功能.结果:成功研制了数字化颅颌面骨三维解剖图谱系统 Dacas,人机交互式界面方便灵活,可以任意选取感兴趣区放大、旋转观察,对各个组成部分的名称、解剖标志、及其上、下级层次结构深入了解,具有逼真的三维显像和方便的教学学习功能.结论:数字化颅颌面骨组织三维解剖图谱是一种强有力的新型解剖工具,在口腔、颌面等多个学科均有很好的推广应用前景.