Steatosis, Steatohepatitis and Related Conditions

Macrovesicular steatosis

Macrovesicular steatosis is common. It is frequently apparent by non-invasive imaging and may be accompanied by moderate abnormalities of serum aminotransferases, alkaline phosphatase and γ-glutamyl transpeptidase. Liver tests may be normal. There are many causes of macrovesicular steatosis, of which the most common are listed in Box 7.1 . It is usually not possible to determine the cause of uncomplicated large-droplet steatosis from histological examination alone.

The lipid in macrovesicular steatosis accumulates in hepatocytes because of increased triglyceride synthesis or decreased excretion. Increased synthesis results from availability of excess free fatty acids and fatty acid precursors and from reduced fatty acid oxidation. Reduced excretion is a result of diminished apoprotein production, seen for example in protein malnutrition and alcohol abuse. Current investigations of fatty liver disease indicate that this view is overly simplistic. The pathogenesis of hepatic steatosis is far more complex and is affected by phenotypic variations in enzymes involved in lipid metabolism, in inflammation and in fibrosis, as well as zonal representation of specific enzymes and lipid moieties involved in lipid metabolism (lipid zonation) within the lobule/acinus. For example, fatty acid oxidation is at a higher rate in periportal regions than elsewhere, in part at least because of higher content of the oxidative enzyme carnitine palmitoyltransferase-1 in periportal hepatocytes. The spectrum of possible phenotypic variations among individuals is obviously considerable and influences the distribution and type of steatosis and its evolution to steatohepatitis and cirrhosis. For the pathologist, the impact of such data is to serve as a practical reminder that all fatty livers are not the same and that each warrants careful examination for the lobular distribution of steatosis, the type of steatosis (i.e. macro- vs microvesicular) and any unusual associated histological features ( Figs 7.3 and 7.4 ).

Macrovesicular steatosis provides the background on which the important lesions of alcoholic and non-alcoholic steatohepatitis (NASH) develop. Moreover, macrovesicular steatosis is increasingly being recognised as a significant risk factor for hepatocellular carcinoma, even without preceding fibrosis or cirrhosis, particularly when widely prevalent risk factors for metabolic syndrome such as obesity and diabetes are present. Most steatosis is perivenular (centrilobular regions/acinar zones 3). Alcohol use, adult obesity, diabetes and corticosteroid therapy typically show this location. Increasing amounts of steatosis extend to progressively involve mid-zonal and periportal regions (acinar zones 2 and 1, respectively). With increasing amounts of macrovesicular fat there sometimes are interspersed clusters or patches of hepatocytes with microvesicular steatosis, probably reflecting the evolution of large lipid vacuoles from progressive coalescence of small lipid droplets. By contrast, periportal steatosis is more common in children with non-alcoholic fatty liver disease (NAFLD; discussed later), in patients on parenteral nutrition, in kwashiorkor and protein-calorie malnutrition, and it is sometimes seen in acquired immunodeficiency syndrome (AIDS; Fig. 7.4 ). In focal fatty change more or less rounded foci of steatosis are seen in an otherwise normal liver and may be mistaken for neoplasms on imaging ( Fig. 7.5 ). In many cases the cause is unknown, but exposure of the capsular surface of the liver to insulin (e.g. in diabetics receiving peritoneal dialysis and intraperitoneal insulin) or in liver tissue adjacent to a metastatic insulinoma the lesion may become apparent radiologically.

The histological grade of steatosis should be reported based on the percentage of hepatocytes which contain lipid vacuoles. One commonly used scoring system includes grades of minimal (<5%), mild (5%–30%), moderate (30%–60%) and marked (>60%). Provision of a numerical assessment to the nearest percentile is also recommended (e.g. ‘marked macrovesicular steatosis is present involving approximately 90% of the parenchyma’). Periodic acid–Schiff and trichrome stains can be helpful in the assessment, providing contrast of the large lipid vacuoles against the more darkly stained background cytoplasm of hepatocytes. Digitised computer image analysis is an alternative method of grading but from a practical standpoint is better suited to research settings.

Occasionally lipid-laden hepatocytes rupture and the fat is then taken up by macrophages. The resulting lesion is a lipogranuloma ( Fig. 7.6 ). Lipogranulomas are situated within the lobules, often near terminal venules. Serial sectioning may be needed to identify the fat in the centre of the lesion. Lipogranulomas may undergo fibrosis, but this does not appear to contribute to progressive liver disease and must be distinguished from the more important pericellular fibrosis characteristic of steatohepatitis (discussed later). Globules within portal tracts are usually the result of uptake of ingested or injected mineral oils by macrophages, rather than uptake of lipids ( Fig. 7.7 ). Lipopeliosis —the formation of large intrasinusoidal fat cysts following release of lipid from hepatocytes after transplantation—is described in Chapter 16 .

The differential diagnosis of macrovesicular steatosis includes microvesicular steatosis. The presence of several fat vacuoles in one hepatocyte has to be distinguished from true microvesicular steatosis (see the following section) in which vacuoles are generally less than 1 μm in diameter and may even be invisible in paraffin sections by light microscopy. The distinction is clinically important. The location of the nucleus helps to differentiate the two conditions. A second differential diagnosis is from stellate-cell hyperplasia ( Fig. 7.8 ), in which the vacuoles are not in hepatocytes but in perisinusoidally located stellate cells. Their nuclei are compressed into a crescentic shape by the vitamin A-rich globules. Stellate-cell hyperplasia may be unexplained, but should lead to investigation of possible overuse of vitamin A or other retinoids.

Microvesicular steatosis

In this serious and sometimes fatal condition, finely divided fat accumulates in hepatocyte cytoplasm as a result of mitochondrial damage leading to impaired β-oxidation. Causes include acute fatty liver of pregnancy ( Ch. 15 ), hepatotoxic drugs such as valproate and nucleoside analogues ( Ch. 8 ), mitochondrial DNA depletion and deletion syndromes, foamy degeneration in the alcoholic (discussed later) and total parenteral nutrition ( Box 7.2 ). Another cause, Reye’s syndrome, has declined sharply in incidence in recent years. In neonates and children, mitochondrial hepatopathies may need consideration. Viral infections occasionally give rise to similar changes.

Histologically, the cytoplasmic lipid is seen to be very finely divided and is not always obvious in paraffin sections. It can be stained with oil red O in frozen sections. The affected hepatocytes are often swollen. Their nuclei remain central ( Fig. 7.2 ).

The differential diagnosis is from macrovesicular steatosis and from conditions in which hepatocytes are swollen for other reasons, such as hepatitis. As discussed in Chapter 13 , phospholipids and sphingolipids accumulate in various metabolic disorders. Cholesterol esters accumulate in hepatocytes in Wolman’s disease and cholesterol ester storage disease, and glycogen accumulates in glycogen storage disease and diabetics with glycogenic hepatopathy (discussed later).

It bears noting that the terms ‘macrovesicular steatosis’ and ‘microvesicular steatosis’ are preferable for use (in lieu of the colloquial ‘macrosteatosis’ and ‘microsteatosis’).

Systematic histological approach to macrovesicular fatty liver disease

Histological evaluation in AFLD and NAFLD should take into account not only the presence of large-droplet steatosis, but also evidence of hepatocellular damage, inflammation, fibrosis and siderosis which may also be present. The diagnosis of steatohepatitis should be rendered based on specific histological criteria (described in detail later). In AFLD and NAFLD there may be relatively inconspicuous apoptotic bodies. On the one hand, increased necroinflammatory activity in NASH may be accompanied by numerous apoptotic bodies. Focal lobular inflammation (usually clusters of lymphocytes and activated Kupffer cells) may be seen ( Fig. 7.9 ) but does not constitute steatohepatitis. Hepatocyte ballooning, on the other hand, is a major feature of both early and of well-developed steatohepatitis ( Figs 7.10 and 7. 11 ), for which careful inspection is warranted. Ballooned hepatocytes are often identifiable even at low magnification ( Fig. 7.10 ). They show watery and oedematous, wispy and rarefied cytoplasm ( Fig. 7.11 ). A variety of factors cause this type of ballooning, including perturbed metabolic pathways, cytoskeletal damage (particularly of keratins 8 and 18) and endoplasmic reticulum stress. These ballooned hepatocytes appear to be moribund but yet ‘undead’, still capable of producing a variety of factors such as Sonic hedgehog which exerts both paracrine and autocrine effects. Combination immunostaining for cytokeratins 8 and 18 (CK8/18) helps to demonstrate affected cells which show either absent or decreased cytoplasmic staining ( Fig. 7.11 , inset ). The most fully developed histopathological picture of steatohepatitis ( Fig. 7.12 ) includes macrovesicular steatosis, hepatocyte ballooning, inflammation, intracellular Mallory–Denk bodies and perivenular fibrosis (discussed in detail later).

Uncomplicated steatosis in the majority of cases is not associated with significant portal tract inflammation. However, focal minimal or mild portal lymphocytic infiltrates are sometimes present in either steatosis or steatohepatitis. More active cases of NASH with advanced fibrosis sometimes present diagnostic difficulties because of substantial chronic portal inflammation (including lymphoid aggregates), which may raise the possibility of chronic hepatitis as an alternative diagnosis. Any histological doubt on this issue should be resolved by discussion with the clinician and investigations to exclude causes of chronic hepatitis when necessary. Some adult and paediatric patients with NAFLD show positive serum anti-nuclear and/or anti-smooth-muscle antibodies , raising a clinical suspicion of autoimmune hepatitis (AIH). However, these autoantibodies are usually low-titre and are considered non-specific. The characteristic portal lymphoplasmacytic inflammation, interface hepatitis and regenerative rosettes of AIH are typically absent in the majority of such cases. Rarely, anti-mitochondrial antibody may be positive. The pathologist should therefore be alert to this common diagnostic problem of NAFLD with autoantibodies, and recognise that changes of AIH are not usually present.

A connective tissue stain is important to evaluate the extent of any fibrosis and its distribution in the steatotic liver. When there is fibrosis in AFLD and NAFLD, it is usually present as a feature of steatohepatitis and is seen in centrilobular regions (acinar zone 3), as will be described later. Alternatively (and less frequently), there may be portal and periportal fibrosis accompanied by chronic inflammation ( Fig. 7.13 ). This distribution is more common in paediatric NAFLD and in morbidly obese individuals.

An iron stain should also be reviewed for siderosis . Mild iron overload in Kupffer cells and/or hepatocytes may be seen in alcoholic patients because of altered intestinal iron absorption and in up to one-third of individuals with NAFLD due to dysmetabolic iron overload syndrome (DIOS) ( Fig. 7.14 ). Iron overload in this setting increases oxidative stress and hepatocyte apoptosis. Significant hepatocellular siderosis should always prompt consideration of possible primary (genetic) iron overload.

Several systems exist to assess steatosis, inflammation and hepatocellular damage in NAFLD. Matteoni and colleagues characterised the spectrum of NAFLD according to four subtypes: NAFLD subtype 1 (simple steatosis), NAFLD subtype 2 (steatosis with inflammation), NAFLD subtype 3 (steatosis with hepatocellular ballooning degeneration; Fig. 7.10 ) and NAFLD subtype 4 (NASH; Fig. 7.12 ) . (Subtypes 3 and 4 are now both considered NASH.) Increased morbidity and mortality were associated with types 3 and 4. The NAFLD Activity Score (NAS) is used to determine the presence of NASH and is discussed later (see the ‘Non-alcoholic steatohepatitis’ section). Similar scoring systems have not been available for AFLD, although a recently proposed system utilises histological features (degree of fibrosis, degree of neutrophil infiltrates, type of cholestasis and presence of megamitochondria) to predict 90-day mortality. Ultimately, the choice and use of a scoring system vary among pathologists and institutions and the system(s) used may be selected for specific clinical and research needs. At minimum, though, the pathologist needs to be able to determine when steatohepatitis is present and what degree of fibrosis, if any, has developed, because these features have impact on therapy and prognosis.