In Situ hybridization and its diagnostic applications in pathology

In situ hybridization (ISH) is a technique by which specific nucleotide sequences are identified in cells or tissue sections. These may be endogenous, bacterial or viral, DNA or RNA. On the basis of research applications, the technique is now being translated into diagnostic practice, mainly in the areas of gene expression, infection and interphase cytogenetics. Diagnostic applications are most often based on short nucleotide sequences (oligomers) labelled with non-isotopic reporter molecules, and sites of binding may be localized by histochemical or immunohistochemical methods. The technique can be applied to routinely fixed and processed tissues; with some targets, it is even possible to obtain hybridization in autopsy material. ISH has been used to detect messenger RNA (mRNA) as a marker of gene expression, where levels of protein storage are low; for example, to confirm an endocrine tumour as the source of excess hormone production. Its application in infectious diseases has to date been mainly in viral infections, such as the typing of human papillomavirus (HPV) or the detection of Epstein–Barr virus by the presence of small nuclear RNAs (EBERs). The expression of mRNAs for histone proteins has been used to detect cells in S phase, and related methods may be applied to detect apoptotic cells. Using probes to chromosome-specific sequences, it is possible to detect aneuploidy, and to document changes in specific chromosomes, which may have prognostic significance in some tumours, such as B-cell chronic lymphatic leukaemia. Using sequence-specific probes, translocations can be identified, such as the t(11;12) of Ewing's sarcoma. This review presents an outline of the technique of in situ hybridization and discusses areas of current and potential diagnostic application. © 1997 John Wiley & Sons, Ltd.     

Electron microscopy in diagnostic renal pathology

The high magnification of the electron microscope enables observations not possible by light microscopy, and electron microscopy is considered to be an essential component of human diagnostic renal pathology. Ultrastructural features may enable a diagnosis to be made where the light microscopy is apparently normal, for example minimal change, thin membrane disease, hereditary nephropathy, and fibrillary and immunotactoid glomerulonephritis. In addition, it can provide information to confirm or elucidate a diagnosis, as in immune complex glomerulonephritis, renal amyloidosis, dense deposit disease, and diabetes.When samples for electron microscopy are inadequate, valuable diagnostic information can be obtained from ultrastructural investigations on reprocessed paraffin- embedded or frozen material.Histopathology departments without electron microscopes should prepare resin-embedded or appropriately fixed tissue which can be sent away for electron microscopy. In addition, semithin resin sections may provide useful information at the level of the light microscope.     

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。     

Low magnification transmission electron microscopy in diagnostic pathology

Transmission electron microscopy examination of selected diagnostic specimens, that is, renal and nerve biopsies, can be greatly facilitated by the implementation of slot grid preparations that are inherently devoid of any grid bars. Such preparations are conducive to screening and photography at both low and intermediate modes of magnification without any apparent loss of resolution. The use of this technique in diagnostic electron microscopy is simplified by sequential en bloc staining with both uranyl and lead salts.     

Utility of whole slide imaging and virtual microscopy in prostate pathology

Whole slide imaging (WSI) has been used in conjunction with virtual microscopy (VM) for training or proficiency testing purposes, multicentre research, remote frozen section diagnosis and to seek specialist second opinion in a number of organ systems. The feasibility of using WSI/VM for routine surgical pathology reporting has also been explored. In this review, we discuss the utility and limitations of WSI/VM technology in the histological assessment of specimens from the prostate. Features of WSI/VM that are particularly well suited to assessment of prostate pathology include the ability to examine images at different magnifications as well as to view histology and immunohistochemistry side-by-side on the screen. Use of WSI/VM would also solve the difficulty in obtaining multiple identical copies of small lesions in prostate biopsies for teaching and proficiency testing. It would also permit annotation of the virtual slides, and has been used in a study of inter-observer variation of Gleason grading to facilitate precise identification of the foci on which grading decisions had been based. However, the large number of sections examined from each set of prostate biopsies would greatly increase time required for scanning as well as the size of the digital file, and would also be an issue if digital archiving of prostate biopsies is contemplated. Z-scanning of glass slides, a process that increases scanning time and file size would be required to permit focusing a virtual slide up and down to assess subtle nuclear features such as nucleolar prominence. The common use of large blocks to process prostatectomy specimens would also be an issue, as few currently available scanners can scan such blocks. A major component of proficiency testing of prostate biopsy assessment involves screening of the cores to detect small atypical foci. However, screening virtual slides of wavy fragmented prostate cores using a computer mouse aided by an overview image is very different from screening glass slides using a microscope stage. Hence, it may be more appropriate in this setting to mark the lesional area and focus only on the interpretation component of competency testing. Other issues limiting the use of digital pathology in prostate pathology include the cost of high quality slide scanners for WSI and high resolution monitors for VM as well as the requirement for fast Internet connection as even a subtle delay in presentation of images on the screen may be very disturbing for a pathologist used to the rapid viewing of glass slides under a microscope. However, these problems are likely to be overcome by technological advances in the future.     

Applications of electron microscopy to diagnostic pulmonary pathology

Viruses and other possible causative agents should be sought light and electron microscopically in all cases of ill-defined diseases including "sarcoid." Ideally, tissue should be prepared for electron microscopic examination as soon as a specimen is obtained; however, when this has not been done, tissue preserved in formalin solution can be used. Viruses, some bacteria, and other agents suspected on the basis of light microscopic findings can be verified electron microscopically by reprocessing paraffin-embedded tissue from areas that show smudge cells, focal necrosis with atypical cellular proliferation, and nuclear inclusions. Electron microscopically, all dying cells show swelling and rupture of cellular organelles and membranes; reactive changes include proliferation of branching tubules and paracrystalline and other types of proteinaceous precipitates (inclusions) in both the nucleus and cytoplasm. Qualitative and quantitative changes of cellular organelles, fibrils, microvilli, and intercellular junctions reflect hyperplasia, metaplasia, or dysplasia of the cell and may enable identification of the diseases, e.g., desquamative interstitial pneumonia. In various conditions, basal laminae become irregular, disruptive, or reduplicated following epithelial necrosis and regeneration. Electron microscopic evidence of immunologic damage to basal lamina and cells and immuno-electron-microscopic features of the lung in general require further studies. Electron microscopic features of transbronchial biopsy specimens may be diagnostic in cases of alveolar proteinosis, histiocytosis X, and amyloidosis. Ultrastructural abnormalities of cilia are common; primary ciliary defects are rare. Finally, light microscopic, scanning electron microscopic, and x-ray energy-dispersive spectrometric examinations of paraffin-embedded sections appear most practical for the pathologic evaluation of cases of pneumoconiosis.     

Use of virtual microscopy for didactic live-audience presentation in anatomic pathology

Didactic presentations on the topic of anatomic pathology in front of a live audience have been largely dependent on the use of standard 2 x 2 inch projection slides (Kodachromes) of selected still images from the topic at hand. Because of the highly visual nature of the specialty of anatomic pathology, this method has had some serious limitations. With the advent of digital imaging techniques and the availability of new electronic software for the projection of images, new possibilities have become available for didactic presentations in anatomic pathology in front of a large, live audience. We describe a method whereby large digital images or "virtual slides" were produced from digitally scanned whole-mount sections of histologic glass slides and projected using a combination of PowerPoint (Microsoft Corp, Redmond, WA) and virtual microscopy in front of a live audience. To provide a seamless transition between the two presentation formats, the personal computer-based PowerPoint slides were hyperlinked to a browser-based virtual microscope viewer. The presenter, with the use of a mouse, was able to "move" the image of the scanned slide on the screen, to transition seamlessly among various magnifications, and to rapidly select from the whole-mount scanned slide among any areas of interest pertinent to the topic. Thus, the visual experience obtained by the audience simulated that of viewing a glass slide at a multi-headed microscope during a glass slide tutorial. Because this most closely approximates the experience of reviewing glass slides under the microscope for practicing pathologists, the educational experience of the presentation is greatly enhanced by the use of this technique. Also, this method permits making this type of presentation available to a much larger group of individuals in a live audience.     

Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology

Until recently, the investigation of protein phosphorylation was limited to biochemical studies of enzyme activities in homogenized tissues. The availability of hundreds of phosphorylation state-specific antibodies (PSSAs) now makes possible the study of protein phosphorylation in situ, and is opening many exciting opportunities in investigative and diagnostic pathology. This review illustrates the power of PSSAs, especially in immunohistochemical applications to human disease and animal models. Technical considerations, including antibody specificity and lability of phosphoepitopes, are covered, along with potential pitfalls, illustrated by a case study. In the arena of oncology, PSSAs may prove especially valuable in directly demonstrating the efficacy of chemotherapies targeted at protein kinase cascades. Novel applications of PSSAs are also beginning to reveal molecular mechanisms of inflammatory, degenerative, and toxin-induced diseases.     

Ultrastructural postembedding immunogold labeling: applications to diagnostic pathology

Ultrastructural postembedding immunogold labeling combines the advantages of routine transmission electron microscopy and detection of antigenic epitopes. As a result, morphologic findings can be accurately interpreted. Localization of morphologic correlates for the various antigenic determinants allows a good definition of the applications and limits of immunocytochemistry in diagnostic surgical pathology. In some cases, immunofluorescence and immunocytochemistry results at light- microscopic levels can be either validated or determined to be nonspecific. Application of ultrastructural immunolabeling to surgical pathology is no longer a distant possibility but rather a reality. The technique provides reliable and reproducible results. Image analysis and microanalytical techniques applied to ultrastructural immunolabeling serve objectively to analyze and clearly to portray results. This paper describes experience with ultrastructural immunolabeling of various renal diseases and neoplasms by means of an easy-to-use methodology that can be applied to the evaluation of specimens in a diagnostic surgical pathology laboratory.     

Virtual 3D microscopy using multiplane whole slide images in diagnostic pathology

To reproduce focusing in virtual microscopy, it is necessary to construct 3-dimensional (3D) virtual slides composed of whole slide images with different focuses. As focusing is frequently used for the assessment of Helicobacter pylori colonization in diagnostic pathology, we prepared virtual 3D slides with up to 9 focus planes from 144 gastric biopsy specimens with or without H pylori gastritis. The biopsy specimens were diagnosed in a blinded manner by 3 pathologists according to the updated Sydney classification using conventional microscopy, virtual microscopy with a single focus plane, and virtual 3D microscopy with 5 and 9 focus planes enabling virtual focusing. Regarding the classification of H pylori, we found a positive correlation between the number of focus planes used in virtual microscopy and the number of correct diagnoses as determined by conventional microscopy. Concerning H pylori positivity, the specificity and sensitivity of virtual 3D microscopy using virtual slides with 9 focus planes achieved a minimum of 0.95 each, which was approximately the same as in conventional microscopy. We consider virtual 3D microscopy appropriate for primary diagnosis of H pylori gastritis and equivalent to conventional microscopy.     

Quicktime virtual reality technology in light microscopy to support medical education in pathology.

The new computer-based interactive technologies in medicine, such as virtual reality (VR), have revolutionized education. The use of virtual microscopic images would be invaluable in the training of cyto-histopathologists. However, due to the vast amount of digital information on a scanned, conventional cyto-histological slide, which is enormous by current data storage standards, these systems are expensive and not widely used in pathological medicine. The authors propose an inexpensive system based on quicktime virtual reality (QTVR) technology (by Apple Computers Inc.), which accommodates a wide area of a slide at high magnification, generating a 'virtual slide' which makes it possible to navigate by conventional input devices. Commercial softwares that stitch consecutive, adjacent images of cyto-histological preparations onto a QTVR panorama were used. QTVR files have the ability to stand on their own as self-contained, multimedia applications and also have the ability to generate multinode scenes by means of 'hot spots'. QTVR 'movies' can be played on Macintosh or Windows platforms, and on major web browsers. Virtual slides by QTVR is an inexpensive system of high educational value, which allows the creation of multimedia databases of cyto-histological preparations that can exist on an internet server or can be distributed on removable media.