Assessment and Differential Diagnosis of Pathological Features

Naked-eye examination and description of biopsy specimens

Although naked-eye examination and description are of limited diagnostic value, they reduce the possibility of specimen identification error. The pathologist should make sure that the whole specimen has been adequately sectioned by comparing the size of the sectioned and stained tissue with the measurement recorded on macroscopic examination. Naked-eye examination also helps in the selection of suitable areas for electron microscopy. The contour and colour of needle biopsy specimens in the fixative container, in the paraffin block or on the glass slide itself may provide some preliminary diagnostic impressions, barring any technical artefacts imposed by unusual specimen handling or staining. Normal liver gives rise to cylinders of even colour and thickness, which do not fragment easily. By this standard, needle biopsies can usually be categorised as one of the following three types, based on their contours ( Fig. 4.1 ): (1) normal contour (suggesting relatively intact architecture without advanced fibrosis, although significant pathology such as hepatitis, cholestasis or other findings may nonetheless be present); (2) irregular contour (suggesting the presence of chronic disease, with focal regions of narrowing due to substantial fibrosis or cirrhosis); and (3) fragmented biopsy (consistent with cirrhosis, primary hepatocellular carcinoma or metastatic tumour). Such impressions obviously require further confirmation on microscopy. Cholestasis imparts a green colour, whereas fatty liver is pale brown or yellow and may float in the fixative. In cholesterol ester storage disease and Wolman’s disease the specimen is bright orange; this should warn the pathologist of the need to keep some tissue for frozen sectioning and electron microscopy. A black or very dark brown colour is characteristic of the Dubin–Johnson syndrome. Metastatic tumour, like fibrous tissue, is often white. Congested liver is deep red in colour.

Routine microscopy

Routine microscopy of liver biopsies should include systematic assessment of overall structure, portal tracts and their contents, terminal hepatic venules, hepatocytes and sinusoidal cells. Some pathologists use a pro forma or checklist in order to avoid omitting relevant data.

It is often helpful to make certain that several relatively common lesions have not been missed, such as cholestasis, steatosis, hemosiderosis, ground-glass hepatocellular inclusions, periportal alpha-1-antitrypsin globules and bile duct loss. The pathologist should bear in mind that some liver biopsies show very few changes that are specifically diagnostic and may result in a diagnosis of ‘non-specific changes” or ‘near-normal liver’. The diagnosis in some of these cases only later comes to light and is a recognized condition such as autoimmune hepatitis, drug-induced liver injury or a fatty liver-related lesion.

The following sections are intended to help in the evaluation of pathological changes. Most of the information is also found in other parts of the book, under individual diseases. There is inevitably some repetition, because many of the listed features are found in combination. The final part of the chapter contains guidance on the differential diagnosis of a number of specific pathological findings.

Structural changes, collapse and fibrosis

Minor structural changes are difficult to assess in sections stained with haematoxylin and eosin (H&E), and may indeed be missed altogether. Examination of a connective tissue preparation is therefore often important. Normal liver tissue shows a hierarchy of ramifying portal tracts of varied sizes which are present in needle and wedge biopsy samples ( Fig. 4.2 ). The subdivisions of these portal tracts parallel the hierarchy of hepatic artery and portal vein branches and bile ducts as they distribute throughout the liver and can thereby be roughly subdivided into segmental, area, conducting (septal) and terminal portal tract units ( see Fig. 5.1 ). For detection of the most minor abnormalities an uncounterstained silver impregnation for reticulin is generally best, although pericellular fibrosis is most easily detected in sections stained for collagen.

Using these methods, an impression may be gained that, although portal tracts and terminal venules are normally related to each other, the portal tracts are enlarged and perhaps even linked by fibrous septa. This is consistent with mild chronic viral hepatitis or with one of the conditions in which portal changes typically predominate; these include biliary tract disease, haemochromatosis, congenital hepatic fibrosis and schistosomiasis. If, by contrast, the reticulin framework of the parenchyma is distorted, lesions characterised by lobular damage should be considered. These include acute and chronic hepatitis as well as forms of biliary disease in which there is also hepatocellular damage, notably primary biliary cirrhosis. Venous congestion leads to regular condensation of perivenular reticulin.

Recent collapse and fibrosis are sometimes difficult to distinguish, even with the help of good collagen stains. A stain for elastic tissue can help to resolve this problem because the presence of elastic fibres outside the portal tracts is an indication of long-standing disease. Collagen stains are helpful for the recognition of blocked veins, for example, in necrotic areas, alcoholic liver disease, venous outflow obstruction and epithelioid haemangioendothelioma. Collagen staining is important for the detection of pericellular fibrosis, as already indicated, and should therefore be used whenever there is substantial steatosis or a suspicion of steatohepatitis.

The histological diagnosis of cirrhosis is fully discussed in Chapter 10 . Once cirrhosis has developed, the pattern of fibrosis is one of the features that may help to determine its cause. In primary or secondary biliary cirrhosis, for example, fibrosis expanding and linking the portal tracts is a more important early factor in pathogenesis than hepatocellular regeneration; this is reflected in the morphological picture of broad perilobular septa surrounding irregularly shaped islands of parenchyma ( see Fig. 5.11 ). In hereditary haemochromatosis and chronic venous outflow obstruction the impression is also of fibrosis rather than regeneration as the principal pathogenetic factor. In these diseases with a long precirrhotic phase of fibrosis, transected parenchymal peninsulas may be mistaken for true regenerative nodules. This is particularly common just deep to the liver capsule. Isolated subcapsular nodules in an otherwise not nodular biopsy should therefore be interpreted with caution.

Hepatocellular damage

There is a broad histological spectrum of possible hepatocellular damage, ranging from subtle changes affecting the appearance of the cytoplasm or specific organelles to obvious hepatocyte ballooning, apoptosis or necrosis. Normal hepatocytes are polygonal in shape, with abundant pale-staining granular cytoplasm rich in glycogen. An occasional apoptotic body (acidophil body) may be seen in normal liver. When present, apoptotic bodies are typically seen within sinusoidal spaces following extrusion from liver-cell plates ( Fig. 4.3 ). These ovoid bodies are highly eosinophilic and sometimes require through-focusing on microscopic examination because their thickness is not confocal with the surrounding tissue. In cholestasis from any cause, and in donor livers shortly after transplantation, there is often an increase in the number of apoptotic bodies as well as in mitotic figures in hepatocytes. Abundant apoptotic bodies are found in acute hepatitis from any cause. They were first described by Councilman in yellow fever, so that the term ‘Councilman body’ should, strictly speaking, be confined to that disease ( see Ch. 6 ).

Hepatocytes may demonstrate cytoplasmic ‘ground-glass’ change in a variety of conditions ( Table 4.1 ). The affected liver cells have a pale pink homogeneous appearance resembling frosted glass ( Fig. 4.4 ). The change may involve all or a portion of the hepatocyte cytoplasm or may be in the form of a rounded or crescentic inclusion, sometimes with a surrounding artefactual empty white space. A common example is the hepatitis B surface antigen-containing ground-glass inclusion seen in individuals with chronic hepatitis B ( Fig. 4.4A , and see Fig. 9.13 ). Such inclusions are scattered randomly through the lobular parenchyma but sometimes are numerous. Use of certain medications (e.g. barbiturates) and occasionally hepatocellular cholestasis can result in similar appearances, but confined to perivenular hepatocytes ( Figs 4.4B,D , and see Fig. 8.1 ), where it is referred to as ‘pseudo-ground-glass’ change. Recipients of transplants (liver, cardiac, bone marrow) may also show ground-glass-like inclusions containing an abnormal type of glycogen ( Fig. 4.4C ). These have a predilection for periportal hepatocytes, as do the ground-glass inclusions of Lafora’s disease (myoclonus epilepsy). Assessment of the clinical setting together with the staining methods shown in Table 4.1 usually clarifies the cause of the ground-glass change.

Moderate hepatocyte swelling is sometimes due to adaptive hyperplasia of smooth endoplasmic reticulum in response to drugs or, occasionally, is due to cholestasis. Perivenular hepatocyte swelling may be seen in allograft biopsies soon after liver transplantation due to preservation injury of the donor liver ( see Fig. 16.2 ). More severe swelling with rounding of the cell outlines is a feature of cell damage ( Fig. 4.5A ). It may accompany canalicular cholestasis ( see Figs 5.2 and 16.9 ), but is most characteristically found in various forms of hepatitis ( Fig. 4.5A and see Fig. 6.2 ) where it is recognised by disruption of the liver-cell plates and by accompanying inflammatory cell infiltration. The liver-cell swelling seen in viral, drug and autoimmune hepatitis differs from that seen in hepatocellular ballooning of steatohepatitis where the liver cells have a clarified appearance and wisp-like strands of rarefied cytoplasm, sometimes with Mallory–Denk bodies ( Fig. 4.5B and see Fig. 7.8 ). The term hepatocyte ballooning therefore has taken on a special significance when examining liver biopsies for evidence of steatohepatitis, as is further discussed in Chapter 7 . In microvesicular steatosis, the cytoplasm of hepatocytes is expanded by minute fat droplets which are sometimes too small to resolve by routine microscopy. The frequent presence of larger fat vacuoles and the clinical context should help to make the diagnosis. Another type of hepatocyte swelling is seen in feathery degeneration ( Fig. 4.5C ), where intracellular cholestasis with retention of bile and bile salts results in mild hepatocyte enlargement and pale, rarefied and reticular, often vacuolated cytoplasm. Feathery degeneration is most often seen in association with large bile-duct obstruction.

Death of individual hepatocytes or small groups of these cells is loosely called focal necrosis ( Fig. 4.6 ), although the mechanism may in fact be apoptosis, or even a combination of both ( necroapoptosis ). The distinction cannot always be made easily by routine microscopy unless apoptotic bodies are seen. Focal necrosis is associated with accumulation of inflammatory cells of various types, including macrophages. Spotty necrosis ( Fig. 4.6 ) is a term used for the same lesion in the context of acute hepatitis. Focal necrosis is a common finding which does not in itself indicate primary disease of the liver because it is often part of a non-specific reaction to disease elsewhere in the body. While degenerating hepatocytes or cell fragments are sometimes seen within the focal inflammatory infiltrate, the inflammatory reaction is usually more obvious than the necrosis, and the latter is assumed to have taken place because of a gap in a liver-cell plate (liver-cell ‘dropout’).

Hepatocyte death by coagulative necrosis ( Fig. 4.3 ) is usually clear from its perivenular location and involvement of a contiguous group of hepatocytes in the zone of diminished perfusion. Necrotic hepatocytes show distinctive cytoplasmic eosinophilia, abnormal sizes and contours, and nuclear pyknosis and karyorrhexis. Perivenular (centrilobular, acinar zones 3) coagulative necrosis is usually seen following hypotensive or septic shock, or after hypoperfusion due to left ventricular failure or hepatic artery thrombosis. If several days have elapsed since the episode(s) of liver hypoperfusion, there sometimes is a reactive sinusoidal neutrophil infiltrate adjacent to the necrotic hepatocytes, particularly if the patient has been maintained on pressor agents.

Confluent necrosis

Confluent necrosis ( see Fig. 8.4 ) refers to substantial areas of liver-cell death. The most common cause of this type of necrosis in biopsy material is hepatitis, whether viral, drug-related or autoimmune, in which case the necrosis is accompanied by an inflammatory reaction. Confluent necrosis with little or no inflammation is seen in hypoperfusion of the hepatic parenchyma ( Fig. 4.6 ), as in shock or left ventricular failure, and in heatstroke ( see Fig. 12.2 ). Paracetamol (acetaminophen) poisoning produces a similar lesion ( see Fig. 8.4 ). In all the aforementioned examples the necrosis is typically perivenular. A predilection for mid-zonal (acinar zones 2) necrosis is seen with yellow fever ( see Fig. 6.3 ) and dengue virus infections. Some poisons, including ferrous sulphate and phosphorus, typically cause periportal (zone 1) necrosis. Haphazardly distributed areas of necrosis are found in disseminated herpesvirus infections (e.g. herpes simplex, varicella; see Fig. 15.4 ) and in mycobacterial diseases. Tumour necrosis may be so extensive that no recognisable tumour tissue is present in the section; in such cases the reticulin pattern may help to establish a diagnosis.

If severe and extensive, confluent necrosis may form bridges linking vascular structures and is referred to as bridging necrosis . Linking of portal tracts to each other is common in conditions in which portal tracts are widened, for example, by chronic hepatitis or biliary tract disease. Linking of perivenular areas to each other is found in some examples of parenchymal hypoperfusion and venous outflow obstruction ( Fig. 4.6 ).

Bridging hepatic necrosis linking terminal hepatic venules (centrilobular veins) to portal tracts ( Figs 4.7 and 4.8 ) deserves specific notation by the pathologist because of its potential association with more severe disease. Central-to-portal bridging necrosis is a fairly common feature of acute hepatitis of viral type; in such cases the bridges show inflammation, loss of hepatocytes and reticulin condensation, without significant fibrosis or elastic fibres. It is also seen in exacerbations of chronic hepatitis. Old bridges contain elastic fibres as well as collagen fibres. Such bridging fibrosis is an important component of the more severe examples of both chronic viral and autoimmune hepatitis. Contraction of collagen-rich bridges may produce rapid and severe distortion of the normal hepatic microstructure, with correspondingly rapid progression to cirrhosis.

Panlobular (panacinar) and multilobular (multiacinar) necrosis ( see Fig. 6.11 ) are terms used to describe confluent necrosis involving entire single lobules or several adjacent lobules, respectively. They are further discussed in Chapter 6 . Massive hepatic necrosis describes loss of virtually all hepatic parenchyma and is characteristically seen in acute liver failure (ALF) of viral, drug, autoimmune or unknown causation. Histologically there is widespread hepatocyte loss with collapse of reticulin accompanied by outgrowth of periportal bile ductular structures (neocholangioles) derived from activated hepatic progenitor cells. Such livers grossly are reduced in size and show capsular wrinkling due to loss of subcapsular parenchyma. The term submassive necrosis is used in certain cases which present clinically as ALF to describe severe loss of liver parenchyma (as in massive hepatic necrosis) but accompanied by foci of regenerative hyperplasia and nodules that are visible on both gross and histological examination ( Fig. 4.9 ). The presence of regenerative nodules and, depending on the individual case, evidence of early fibrosis are consistent with a more protracted time course, possibly of several months, during which the hepatitis may have been subclinical. Submassive necrosis and a clinical chronology consistent with ALF should be distinguished from acute-on-chronic liver failure (ACLF) , a condition seen in individuals who already have underlying cirrhosis or well-established chronic liver disease. ACLF is characterised by rapid progression of liver injury and one or multiorgan failure triggered by one or more factors (e.g. gastrointestinal tract haemorrhage, alcohol misuse) with a high risk of mortality within 3 months ( see Ch. 10 ). In such cases the pathologist’s role, if liver biopsy has been obtained, is to verify the presence (or absence) of cirrhosis or chronic disease with advanced fibrosis. This determination has direct impact on subsequent clinical management decisions for the patient.