Electron Microscopy and Other Techniques

CHAPTER 17 Electron Microscopy and Other Techniques



Introduction


This chapter will focus primarily on the role of transmission electron microscopy in the assessment of liver ultrastructure and disease. It also describes, in brief, the principles and uses of other methodologies. The special conditions required for tissue processing in each of these techniques (Table 17.1) should be carefully planned for in advance of obtaining specimens. While some of these methods are not universally available in pathology departments, other departments at one’s institution or at other centres of investigation may be consulted in cases of particular diagnostic or research interest. Procedures for fixation and processing for transmission electron microscopy are available in several of the General reading references at the end of the chapter.


Table 17.1 Liver tissue processing for various techniques




































Technique Tissue preparation
Transmission electron microscopy Glutaraldehyde fixation
Scanning electron microscopy Perfusion-fixation; critical point drying; coating with gold or platinum
Immunoelectron microscopy Glutaraldehyde/paraformaldehyde fixation
Immunoperoxidase of tissue sections Fixation in 10% neutral formalin or alternative fixative
Immunoperoxidase and immunofluorescence of frozen sections Snap freeze after embedding in OCT compound
In situ hybridisation Snap freeze after embedding in OCT compound
Flow cytometry Fresh tissue
Confocal laser scanning microscopy Snap freeze after embedding in OCT compound
Laser capture microdissection Conventional tissue sections for light microscopy
Gene array analysis Snap freeze in liquid nitrogen; store at −80oC

OCT, optimal cutting temperature.



Electron microscopy of liver biopsies


Transmission electron microscopy (TEM) continues to provide important information about the normal cellular and extracellular constituents of the liver and their alterations in disease. Recent interest in the relationships between the various sinusoidal cells of the liver has benefited from TEM studies1,2 as has investigations of hepatic progenitor cells.3 Data from standard TEM studies can be enhanced by the application of immunohistochemical stains (see Immunoelectron microscopy), digitised three-dimensional computer reconstructions46 and morphometry. TEM is sometimes limited by the lack of specificity of certain ultrastructural changes and the problem of sampling error in lesions that may not be uniformly distributed. The first of these limitations is well illustrated in cholestasis; various features of cholestasis such as loss of canalicular microvilli are easily recognised under the electron microscope, but many causes produce these changes. Sampling error can sometimes be reduced by the combination of light and electron microscopy in a single instrument.7


In diagnostic work TEM should be seriously considered under five circumstances:



1. To establish the nature of an inborn error of metabolism. In a number of storage diseases the ultrastructural changes are diagnostic or give an indication of the type of disease to be considered.8,9 Specific features are seen, for example, in type II glycogenosis, in Gaucher’s disease and in Niemann–Pick disease (Fig. 17.1). Storage diseases can and should often be diagnosed by other, usually biochemical, methods, but even then electron microscopy can reduce the period of investigation by drawing attention to a likely diagnosis. Electron microscopy may show whether a liver-cell pigment is lipofuscin or the pigment of the Dubin–Johnson syndrome (Fig. 17.2), and can therefore be helpful when this syndrome is suspected but not fully proved by light microscopy.10 In some patients with Wilson’s disease characteristic changes may be seen in liver-cell mitochondria (see below).


2. To establish the presence of viral infection. Electron microscopy of liver biopsies may prove to be important when serological test results or cultures for suspected viral infection are unavailable or incomplete. Both intranuclear and intracytoplasmic virions may be identified by the appearances of their spherical or hexagonal capsids, dense core material, surface envelopes and paracrystalline and lattice-like arrays. These features can be compared with published micrographs for identification of the candidate virion.11,12 For example, some adult patients with the unusual finding of giant-cell hepatitis on routine light microscopy have been shown to have paramyxovirus-like particles in the liver as a result of electron microscopic studies of biopsy material.13,14 Electron microscopy can also be applied to cell cultures, as shown in a recent study demonstrating 50–90 nm hepatitis C virions.15 Glutaraldehyde fixation of biopsy specimens is preferred, but viral particles can also be identified in formalin-fixed tissues which are washed and then processed for electron microscopy.


3. To establish the nature of a tumour of doubtful histogenesis. The ultrastructural features of many tumours, including neuroendocrine tumours and malignant melanomas, help in making a firm diagnosis.16 The more obvious features such as neurosecretory granules in neuroendocrine tumours survive paraffin embedding; re-embedding of paraffin material for electron microscopy should therefore be considered.


4. To establish the presence of specific drug-related changes. In liver damage due to a small number of drugs, including perhexiline maleate17 and amiodarone,1821 hepatocytes contain lysosomes filled with lamellar phospholipid material (Fig. 17.3).


5. To provide material for research. Electron microscopy offers wide potential for research into human liver disease, and it may be that future research will increase the diagnostic value of electron microscopy in this field. If liver biopsy is performed in a patient having a disease with potentially helpful or interesting ultrastructural features, small pieces of the specimen can be embedded for electron microscopy and stored indefinitely in block form. The extent to which this is done clearly depends on the resources of the particular laboratory.





Whenever electron microscopy of a liver biopsy specimen is considered, the laboratory should be contacted beforehand and arrangements made for collection and fixation of the specimen at the bedside. Proper processing of the tissue, including optimal fixation, provides the basis for accurate analysis of ultrastructural changes.



The normal liver and examples of ultrastructural changes in disease


The following description of the liver under the transmission electron microscope is a general one. It should be noted that the quality of fixation will influence the appearance of cells and organelles. The labels in the description of normal liver refer to Figs 17.4 and 17.5.




Several cell types are found in the hepatic lobules. The hepatocytes or parenchymal cells (PC) are separated from the sinusoidal endothelial cells (EC) by the space of Disse (SD), in which there are collagen fibres and stellate cells (SC), formerly known as perisinusoidal cells, Ito cells or fat-storing cells. Within the sinusoidal lumen are Kupffer cells (KC), the hepatic macrophages and large granular lymphocytes (also called pit cells) with natural killer activity.



Hepatocyte (liver cell, parenchymal cell)


Hepatocytes have similar features in different lobular regions but vary in detailed structure. For example, there are more lysosomes and mitochondria in periportal than in perivenular hepatocytes, while the converse is true for the smooth-surfaced endoplasmic reticulum. The hepatocyte is a highly polarised cell with surfaces facing the space of Disse, other hepatocytes and the bile canaliculus. The plasma membrane is specialised in these three areas. Many microvilli project into the space of Disse and into the bile canaliculus. This is a potential space formed by two or three hepatocytes in normal liver, and sometimes more in disease. The intercellular membrane of the hepatocyte is relatively smooth and forms several types of intercellular junctions.




Mitochondria


Mitochondria (M) are the sites of oxidative enzyme activity, and are involved in the metabolism of amino acids, lipids and carbohydrates. There are an average of 2200 mitochondria within the hepatocyte.22 A smooth outer limiting membrane and an inner membrane with deep infoldings, the cristae, give the mitochondria a characteristic appearance. The inner membrane surrounds the mitochondrial matrix which contains many dense granules.



Structural changes


Cristae of atypical shape, crystalline inclusions and enlarged or unusually scanty granules are found in a wide variety of conditions, and sometimes also in normal liver. Giant mitochondria are seen most often in alcoholic liver disease23 but are also found in non-alcoholic fatty liver disease24 and other conditions.25 Immunohistochemistry and immunoelectron microscopy can assist in their detection.26 They are frequently found in patients with systemic sclerosis.27 In the early stages of Wilson’s disease mitochondria show variation in shape, increased electron density, widening of the spaces between membranes, vacuolation, enlargement of matrix granules and deposition of crystalline material28,29 (Fig. 17.7). Three types of Wilsonian mitochondria are described which show intrafamilial concordance.30 Abnormal, swollen and irregular mitochondria are found in hepatocytes in Reye’s syndrome31 and other microvesicular fat syndromes.32




Endoplasmic reticulum


This is an important site of protein synthesis and transport. It also contains enzymes involved in drug and steroid metabolism. Morphologically, the endoplasmic reticulum is a cisternal membrane-bound system continuous with the nuclear envelope. It is the morphological counterpart of the microsomes. Two main types of endoplasmic reticulum can be recognised. The rough-surfaced endoplasmic reticulum (RER) is studded with ribosomes and is often arranged in a lamellar pattern. The smooth-surfaced endoplasmic reticulum (SER) lacks ribosomes and has a tubular or vesicular appearance.







Cell sap (cytosol)


The soluble portion of the cytoplasm (cell sap) contains variable amounts of glycogen, free ribosomes, microtubules, intermediate filaments and microfilaments. A few lipid droplets and scanty iron-containing granules are also seen.



Structural changes


Ferritin particles accumulate in iron storage disorders.33 Fat droplets are numerous in fatty liver, but the amount of fat varies greatly with the patient’s state of nutrition. Core particles of hepatitis B virus can be identified in the cytoplasm in many cases of chronic type B hepatitis. Cytoplasmic crystalline inclusions are seen both in normal and in diseased livers. In alcoholic hepatitis, the Mallory bodies found in ballooned hepatocytes are composed of accumulations of cytokeratin and other proteins in the form of filaments (Fig. 17.9).


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Jul 25, 2017 | Posted by in GASTROENTEROLOGY | Comments Off on Electron Microscopy and Other Techniques

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