Electron Microscopy

Electron Microscopy

Ashima Agarwal

Frances V. White

  • I. INTRODUCTION. Transmission electron microscopy (EM) has been used by surgical pathologists over the past 50 years for the diagnosis of a wide range of diseases in various organ systems (Table 53.1). The method allows for the visualization of subcellular morphology, with appreciation of disease processes and structural abnormalities that cannot be resolved by light microscopy. EM is an essential part of the work-up of medical renal diseases, peripheral nerve diseases, muscle diseases, and primary ciliary dyskinesia. It is also useful in evaluating metabolic and inherited diseases, providing an initial differential diagnosis, or ruling in or out a specific disease process. The role of EM in the diagnosis of neoplasms has decreased since the advent of immunohistochemical and molecular techniques, but it is still an ancillary tool for the evaluation of atypical tumors or when other techniques yield indeterminate results. Although not usually needed, EM is occasionally used to demonstrate infectious agents or evidence of drug toxicity.

    In recent years, EM has been combined with immunohistochemical and in situ hybridization methods, allowing antigen detection and localization at the subcellular level. These combined methods require special fixation and processing protocols. Although immunoelectron microscopy is currently used primarily in research laboratories, the method is now considered to be a promising diagnostic technique in oncologic surgical pathology, in particular, for the identification and localization of targets for gene therapy.

  • II. METHODOLOGY. Tissue for EM must be immediately fixed. A thin slice of tissue should be immersed in a cold fixative such as buffered glutaraldehyde 2% to 4%, or buffered glutaraldehyde plus paraformaldehyde, and then diced into 1-mm cubes using a sharp, clean scalpel blade. Specimens are usually postfixed in osmium tetroxide, dehydrated in ethanol, and then embedded in an epoxy resin or plastic. Semithin (1-μm-thick) sections are cut from the blocks and stained with toluidine blue or methylene blue, and light microscopic examination of the semithin sections is used to select the blocks from which thin sections are cut and placed on grids. The thin sections are usually stained with uranyl acetate and lead citrate; other stains, however, may be selected to enhance electron contrast of specific particles or structures depending on the tissue type and diagnostic question. Tissue processing typically takes a couple of days, but with microwave techniques, grids can be ready within 5 hours postfixation.

    For some disease processes, if glutaraldehyde-fixed tissue is not available, EM can be performed on formalin-fixed wet tissue or paraffin-embedded tissue. Wet tissue is preferable to paraffin-embedded tissue, but previous prompt fixation in formalin is essential. Autopsy material, whether fixed in glutaraldehyde or formalin, is often unsatisfactory due to the prolonged postmortem interval prior to fixation.

    A focused differential diagnosis based on integration of the clinical history and light microscopic findings is essential for the correct interpretation of ultrastructural findings. Except for the most routine specimens, the pathologist should personally review the semithin sections by light microscopy and select the blocks for further processing. In addition, the pathologist should communicate his differential diagnosis to the electron microscopist and specify the cell type and subcellular structures of interest. Obviously, sampling error is minimized and the most
    information is obtained when the pathologist is directly involved in scanning the tissue grids.

  • III. KIDNEY. EM, in conjunction with routine histology and immunofluorescence, is an essential part of the work-up of medical renal biopsies to diagnose glomerular disease. It is also performed on renal allograft biopsies when recurrent or de novo glomerular disease is suspected. The protocol for triaging renal biopsies
    is presented in detail elsewhere in this manual. Because EM makes it possible to visualize the individual components of the glomerular capillary wall, including endothelium, glomerular basement membrane, and visceral epithelial cells, it is used for identification and localization of discrete electron-dense deposits in glomeruli, either of immunoglobulins or amyloid and amyloid-like proteins. The glomerular basement membrane can also be evaluated for abnormal thickening, thinning, and/or splitting and for the presence of electron lucent, granular, or other deposits. Tubular basement membranes, arterioles, and the interstitium can also be evaluated by EM for pathogenic changes, for example, nonimmune deposits such as amyloid, light chain dense deposits, and cryoglobulins. Chapter 19 describes in detail the ultrastructural findings in specific renal diseases.

    TABLE 53.1 Examples of Subcellular Features Used for Classification of Disease by EM

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    Oct 20, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Electron Microscopy

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