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Update and Application to Liver Biopsy Interpretation in Clinical Practice
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Case 2 -
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Nonalcoholic Steatohepatitis (NASH)
Julia C. Iezzoni
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Clinical History
The patient, a 36-year-old Caucasian female, was noted to
have mildly elevated aminotransferases during an examination performed to evaluate her as a possible
living-related-liver-donor for her mother, who suffered from end-stage liver disease due to cryptogenic
cirrhosis. Otherwise the patient was asymptomatic, denied alcohol abuse, and was not on any medications
or herbal supplements. Other laboratory values, including viral studies and autoantibodies, were
negative, and evaluation for inherited metabolic disorders detected no abnormalities. Physical
examination was remarkable for mild obesity and hepatomegaly. A liver biopsy was performed.
Diagnosis: Nonalcoholic Steatohepatitis (NASH)
Pathologic findings:
The liver biopsy shows moderate
macrovesicular steatosis, predominantly in acinar zone 3 with extension into zone 2. Scattered
hepatocytes, also within zone 3, demonstrate ballooning degeneration and Mallory's hyaline, and there is
a sparse lobular inflammatory infiltrate. On trichrome stain, zone 3 perivenular,
perisinuoidal/pericellular fibrosis is identified.
Discussion
In 1980, the term nonalcoholic steatohepatitis (NASH) was introduced by Ludwig et al. to describe a form of chronic liver injury that is morphologically
identical to alcoholic hepatitis but occurs in patients without a history of excessive alcohol
consumption. Since this initial description, NASH has been relatively underappreciated as a major cause
of chronic liver injury, likely due the lack of obvious symptoms in many cases, the perception that it
consistently follows an indolent clinical course, and the lack of a consensus regarding the histologic
criteria for the diagnosis. More recently, however, NASH has been recognized as an important and common
etiology of chronic liver disease, which in some cases, may result in cirrhosis. In addition, its
prevalence is expected to increase as the rate of obesity, a major risk factor for the development of
NASH, continues to rise within the population. Consequently, pathologists may expect to see increasing
numbers of liver biopsies performed on patients being evaluated for possible NASH. Accordingly, this
discussion will focus on the application of recent advances in the understanding of NASH to the routine
clinical evaluation of liver biopsy specimens.
Steatohepatitis is a distinctive pattern of hepatic injury that is recognized as one of
the major morphologic categories of chronic liver disease. As such, one of the initial responsibilities
of the pathologist upon review of a liver biopsy is to correctly identify a histological pattern of
chronic injury as steatohepatitis, and importantly to distinguish it from non-steatotic forms of chronic
hepatitis. The morphologic features for doing so will be discussed in detail below.
While the morphologic features of steatohepatitis are distinctive, they are etiologically
nonspecific and may be seen in association with a wide variety of clinical conditions. Most commonly,
NASH is associated with obesity, type II diabetes mellitus, insulin resistance syndromes, and/or
hyperlipidemia. In addition, however, steatohepatitis may be due to certain drugs (e.g. amiodarone,
synthetic estrogens), post-gastrointestinal bypass surgical procedures (e.g. jejunoileal bypass,
gastroplexy), and a variety of miscellaneous disorders (e.g. total parenteral nutrition-related,
abeta/hypobeta liproteinemia). Accordingly, once steatohepatitis is recognized as the pattern of liver
injury in a liver biopsy, clinical correlation is required to determine the specific etiologic
factor(s).
Classically NASH has been considered to be a disease of obese, middle-aged females often
with type II diabetes and / or hyperlipidemia. Subsequent studies have demonstrated that the
overwhelming risk factor for the of development NASH continues to be obesity, in particular, truncal
obesity. The feature is followed in relative risk by type II diabetes and hyperlipidemia. Recent
studies have documented, however, the fairly high prevalence of NASH in patients without any of these
risk factors. As such, the demographic profile of patients with NASH has expanded to include non-obese
male patients with no evidence of type II diabetes or hyperlipidemia. Furthermore, several studies have
suggested that a high percentage of cases of cryptogenic cirrhosis may be due to NASH.
Nonalcoholic steatohepatitis is not limited to adults. Studies have documented NASH in
children, and some investigators have suggested that NASH may be one of the most common causes of chronic
liver disease in school-aged children. While approximately 20% of the pediatric NASH cases occurs in
non-obese children, as in adults, a major risk factor for NASH in children is obesity. As such, in the
United States, the prevalence of NASH in children is likely to increase as a result of the rising rate of
childhood obesity. As in adults, the prognosis of NASH in children is variable, and NASH can progress to
cirrhosis in childhood.
Clinically, non-alcoholic steatohepatitis is frequently asymptomatic. Most patients who
come to medical attention for evaluation of subclinical liver disease do so because of incidental
laboratory findings. When present, the most common complaint is dull right upper quadrant pain, which
may be caused by a distended liver capsule. Less commonly, patients with NASH may describe a variety of
nonspecific constitutional symptoms, such as weakness, fatigue, and malaise. The most common laboratory
abnormality in NASH is elevated aminotransferases.
A growing body of evidence indicates that steatohepatitis, due to either alcohol-related or
non-alcohol-related etiologies, develops as the result of stress or injury to the liver in which there is
pre-existing steatosis. To this extent, hepatic steatosis is a necessary precursor lesion for the
development of steatohepatitis. According to this so-called "two hit" model for the pathogenesis of
steatohepatitis, derangements that cause steatosis are the "first hit", and subsequent injury that
results in cellular damage, inflammation and fibrosis is the "second hit". As such, an explanation of
the pathogenesis of steatohepatitis requires an understanding of why fat accumulates in hepatocytes in
the first place, as well as determination of the mechanisms that cause the unique pattern of cell damage,
inflammation and fibrosis that characterizes steatohepatitis. While these questions currently are an
active area of investigation, examination of both of these features will begin to explain the diverse
etiologies, unique morphologic features, and the variable clinical course of steatosis and
steatohepatitis.
Due to this concept of progression from steatosis to steatohepatitis to cirrhosis, the
term non-alcoholic fatty liver disease (NAFLD) has been proposed. Broader and more inclusive than the
term NASH, NAFLD designates the entire spectrum of non-alcohol induced steatotic syndromes.
Non-alcoholic fatty liver disease has been subdivided in to four types to reflect the progressive
inflammation and fibrosis after the initial development of steatosis. As such, steatosis is at one end
of the spectrum of NAFLD, with the developed fibroinflammatory counterpart, NASH, at the other.
Pathology
Morphologic features of NASH
The histologic criteria necessary for the diagnosis of NASH are not agreed upon by all
investigators. While there is general consensus that the histologic features of NASH are similar to
those of alcoholic liver disease, the specific morphologic components necessary for this diagnosis vary
among studies. Furthermore, the minimal histologic features considered sufficient for the diagnosis of
NASH have not been established. For example some studies use the sole findings of macrovesicular
steatosis and intra-acinar inflammation to categorize a case as NASH. Although these two features
literally do add up to "steatohepatitis", this minimalist definition includes cases of steatosis in
which, coincidentally, there happens to be minor degrees of parenchymal inflammation. As such, these
differences in the morphologic features considered necessary and/or sufficient for the diagnosis of NASH
may be an important source of discordant results in investigations of this disease.
Though not agreed upon universally, the following morphologic definition of NASH has been
proposed as a means to better delineate this disease as a distinct clinicopathologic entity. This
definition requires that in addition to (1) steatosis and (2) inflammation (predominantly lobular and
composed in part by neutrophils), there must be morphologic documentation of cellular damage, as
evidenced by (3) hepatocyte ballooning degeneration and/or (4) fibrosis in a perivenular,
perisinusoidal/pericelluar pattern. Early pathologic alterations are concentrated in the
perivenular/acinar zone 3 region, but as the disease progresses, the changes extend across the acinus.
Based on the severity of disease, the extent and relative proportion of these four basic features vary
from case to case. As such, NASH encompasses a wide morphologic spectrum, which ranges from minor
perivenular changes to outright cirrhosis.
As is apparent in this definition, no single light microscopic finding is considered
sufficient for the diagnosis of NASH. Instead, NASH is defined histologically by the combination of
several specific morphologic features that result in a characteristic pattern of injury. These features,
and others, frequently seen in cases of NASH but not necessary for the diagnosis, will be discussed in
detail as follows:
Steatosis
The hepatocellular steatosis that characterizes NASH is
typically macrovesicular. Microscopically, this is seen as a single, large vacuole within the hepatocyte
cytoplasm that displaces the nucleus to the periphery of the cell. The involved hepatocytes usually are
larger than the uninvolved hepatocytes. In some cases, and especially those with more marked disease
activity, the steatosis manifests as an admixture of macrovesicular steatosis and microvesicular
steatosis. In the latter form of steatosis, the hepatocyte cytoplasm is filled with many small vacuoles;
the nucleus remains centrally located, and the involved hepatocytes may or may not be enlarged.
Sometimes, the hepatocytes may contain both forms of steatosis simultaneously. In occasional cases of
NASH, microvesicular steatosis may be the principle type of fatty change observed.
The extent of steatosis varies among cases of NASH and may range from focal to diffuse
involvement. In addition, the steatosis loosely follows a zonal pattern of distribution depending on the
extent of the finding. In general, initially the steatosis is concentrated in, although not limited to,
acinar zone 3. With more extensive disease, the steatosis may be panacinar in distribution. As such,
the extent of steatosis in NASH can range from mild accumulation, usually in the perivenular region, to
near complete panacinar involvement. Exceptions to this pattern, however, have been noted. For example,
in children with NASH, the steatosis may have a diffuse or scattered distribution within the lobules from
the early stages of the disease. Furthermore, in cases of cirrhoisis due to NASH, the steatosis may no
longer be present.
By convention, the extent of steatosis is assigned a grade based on the percentage of
total hepatocytes that are steatotic, as follows: mild (<33%); moderate (34%-66%); severe (>66%).
Inflammation
Though the degree and extent of inflammtion varies with the
severity of disease activity, typically the inflammation of NASH is mild relative to other forms of
chronic hepatitis. In addition, this inflammatory infiltrate is predominantly lobular rather than
portal-based in its distribution. Indeed in adult patients, while lobular inflammation is a hallmark of
the disesase, portal inflammatory infiltrates may not be present, especially early in the course of the
disease. As the disease activity progresses, mild to moderate portal inflammatory infiltrates may become
evident. In children with NASH, this pattern of distribution of the inflammation, however, may be
reversed, with a predominance of portal inflammation over lobular infiltrates. In either age group,
however, intense portal inflammation with interface activity raises concerns about other etiologies of
chronic hepatitis, such as viral or autoimmune disease, rather than steatohepatitis.
The lobular infiltrates in NASH are characterized by an admixture of neutrophils and
chronic inflammatory cells (mainly lymphocytes with occasional plasma cells and rare eosinophils).
Although the presence of neutrophils is a classic feature of steatohepatitis, in some cases, chronic
inflammatory cells may be the predominant cell type within the lobular infiltrates; this especially is
seen in cases with mild disease activity. When present, the portal infiltrates are composed mainly of
chronic inflammatory cells.
The neutrophils, in addition to being scattered throughout the lobules, may be clustered
around hepatocytes with intracytoplasmic aggregates of Mallory's hyaline. This phenomenon, known as
satellitosis, is attributed to the chemotactic properties of Mallory's hyaline, which result in
accumulation of neutrophils around the involved hepatocytes.
Hepatocyte ballooning degeneration
Ballooning degeneration is a prominent
morphologic manifestation of hepatocyte damage in NASH. It is thought to result from impairment of the
secretory capacity of the injured hepatocytes, which in turn causes intracellular fluid accumulation. As
a result, the ballooned hepatoctyes are swollen and enlarged (two times normal size), with "diluted"
cytoplasm, which appears finely granular or reticulated. The nuclei of the affected hepatocytes may be
enlarged and often display accentuated nuclear membranes and prominent nucleoli. In early cases, groups
of ballooned hepatocytes typically are noted in zone 3 near steatotic hepatocytes. With advancing
disease, they are generally found adjacent to fibrous septa. Although a potentially reversible form of
injury, ballooning degeneration can result in hepatocyte necrosis.
Fibrosis
The pattern of fibrosis in NASH is distinctive. Initially, the
fibrosis occurs in in the perivenular area of acinar zone 3, usually in association with hepatocytes that
demonstrate other lesions of steatohepatitis, such as steatosis or ballooning degeneration. The collagen
is deposited along the sinusoids and characteristically appears to invest single or small groups of
hepatocytes in a pattern referred to as "pericellular" or "chicken-wire" fibrosis. This pattern is seen
to best advantage with connective tissue stains, such as a trichrome stain. Fibrosis in the portal and
periportal regions, in contrast to the perivenular zones, is generally a minor feature, at least during
the earlier stages of the disease. This pattern of fibrosis distinguishes steatohepatitis from other
forms of chronic hepatitis in which fibrosis is initially portal based. Progressive injury in NASH,
however, results in portal and periportal fibrosis, with eventual central-portal and portal-portal septum
formation (bridging) and ultimately cirrhosis.
Other morphologic features
Mallory's hyaline
Mallory's hyaline is a common finding in NASH. When
present, Mallory's hyaline is located in hepatocytes that have undergone ballooning degeneration. On
routine hematoxylin-and-eosin stained tissue sections, it is recognizable as intracytoplasmic,
perinuclear eosinophilic material that ranges in shape from short chunky clumps to elongated rope-like
cords. Of note, Mallory's hyaline often is absent in pediatric cases of NASH.
Since Mallory's hyaline is a manifestation of cellular injury, some studies have required
the its presence for a case to be diagnosed as NASH. The identification of Mallory's hyaline in liver
biopsies, however, is prone to significant inter-observer variation, and as a marker of cellular injury,
it only may be identifiable later in the disease course than ballooning change. As such, Mallory's
hyaline may not be as dependable a marker of cellular injury as ballooning degeneration. In our opinion,
therefore, in the background of steatosis, ballooning degeneration, and lobular inflammation, the
presence of Mallory's hyaline is additional evidence in support of the diagnosis of NASH, but its
presence in not necessary for the histologic diagnosis.
Recently, immunohistochemical staining for ubiquitin has been proposed as a means to
improve the sensitivity and reliability of detection of Mallory's hyaline in cases of suspected NASH.
Mallory's hyaline is composed of complex aggregates of cytokeratins 7, 18, and 19 admixed with ubiquitin
and other proteins. Ubiquitin itself is present normally in a variety of cell types and is an
intracellular protein that binds to other proteins to target them for proteolysis. As such, ubiquitin
may serve as a marker of cellular injury. Whether the presence of immunoreactive ubiquitin reliably
distinguishes NASH from simple steatosis, however, awaits further studies.
Apototic bodies
In addition to ballooning degeneration, the
hepatocellular injury in NASH may manifest as apoptotic (acidophil) bodies. These result from apoptotic
cell death and are noted as small, eosinophilic globular fragments of cytoplasm that are usually located
in the sinusoids.
Lipogranulomas
Though neither specific for nor diagnostic of
steatohepatitis, lipogranulomas occur frequently in NASH. They result from the rupture of a lipid-laden
hepatocyte, which in turn, leads to the development of a foreign-body type granulomatous reactive
response. Morphologically they are composed of the steatotic hepatocyte and a surrounding accompaniment
of mononuclear cells, Kupffer cells, and occasionally, an eosinophil. Frequently they are quite small,
composed of just a few cells. Lipogranulomas may be associated with focal fibrosis. This type of
fibrosis, however, should not be confused with the perisinusoidal fibrosis of steatohepatitis, and there
is no evidence to suggest that this localized change contributes to progressive fibrosis.
Megamitochondria
Enlarged mitochondria, known as giant or
megamitochondria, may be seen in NASH, but they also can occur in a range of other conditions. By light
microscopy, they appear as hepatocellular, cytoplasmic, discrete eosinophilic bodies, 3 to 10 um
diameter, that have a globoid, or less often, needle-like shape. They are bright red on trichrome stain,
and their negativity upon PAS staining helps to distinguish them from alpha-1-antitrypsin globules.
Ultrastructurally they contain abnormal cristae and paracrystalline inclusions, which may be a
manifestation of injury or an adaptive change.
Glycogenated hepatocyte nuclei
Glycogenated hepatocellular nuclei are a
common finding in NASH, but this change is seen in a wide variety of other livers lesions, including
Wilson's disease. As such, glycogenated hepatocellular nuclei is a common though etiologically
nonspecific feature in NASH.
Morphologic Grading and Staging of Nonalcoholic Steatohepatitis
The standard practice in the evaluation of liver biopses from patients with chronic
hepatitis is to analyze and report the degree of necroinflammatory activity and extent of fibrosis by
following a semiquantitative scoring system. The morphologic features of NASH, however, are sufficiently
different from those of other forms of chronic hepatitis so as to preclude the use of currently available
necroinflammatory grading and fibrosis staging strategies. Accordingly, a semiquantitative scoring
system for analyzing and reporting the severity of steatohepatitis in liver specimens that takes into
account the morphologic features unique to this entity has been proposed by Brunt
et al. Analagous to the Histologic Activity Index (HAI), which is used in reporting chronic
hepatitis, this newly proposed system assigns cases of NASH a grade based on the degree of
necroinflammatory activity and a stage based on the extent of fibrosis.
As described above, no single morphologic feature defines the necroinflammatory activity
of NASH. Instead, the necroinflammatory activity of NASH is characterized by the combination of
steatosis, hepatocyte ballooning, lobular inflammation, and to a lesser degree, portal inflammation.
Accordingly, the proposed necroinflammatory grading scheme assesses this constellation of morphologic
features (Table 1). Based on the extent of these multiple features, a global grade of necroinflammatory
activity is assigned; it may range from mild (Grade 1) to moderate (Grade 2) to severe (Grade 3).
Table 1: Grading the degree of necroinflammatory activity in NASH
| Grade 1 (Mild) | Steatosis: predominantly macrovesicular, involves up to 66% of the hepatocytes. |
| Ballooning occasionally observed; zone 3 hepatocytes. |
| Lobular inflammation mild and scattered neutrophils and occasional chronic inflammatory cells. |
| Portal inflammation none or mild. |
| Grade 2 (Moderate) | Steatosis: any degree; usually mixed macrovesicular and microvesicular. |
| Ballooning present; zone 3 hepatocytes. |
| Lobular inflammation neutrophils may be noted associated with ballooned hepatocytes and/or pericellular fibrosis; mild chronic inflammation may be present. |
| Portal inflammation mild to moderate. |
| Grade 3 (Severe) | Steatosis: typically >66% (zone 3 or panacinar); commonly mixed macrovesicular and microvesicular. |
| Ballooning marked; predominantly zone 3. |
| Lobular inflammation scattered neutrophils and chronic inflammatory cells; neutrophils may appear concentrated in zone 3 areas of ballooning and perisinusoidal fibrosis. |
| Portal inflammation mild or moderate; not predominant or marked. |
The fibrosis staging system of this proposed scheme takes into account the unique zone 3
perivenular perisinusoidal/pericellular fibrosis that characterizes steatohepatitis (Table 2). As such,
the fibrosis stage reflects both the location and extent of fibrosis, and it may range from zone 3
perivenular perisinusoidal/pericellular fibrosis (Stage 1) to outright cirrhosis (Stage 4).
Table 2 Staging the extent of fibrosis
| Stage 1 | Zone 3 perivenular, perisinusoidal/pericelluar fibrosis, focal or extensive. |
| Stage 2 | As above plus focal or extensive periportal fibrosis. |
| Stage 3 | Bridging fibrosis, focal or extensive. |
| Stage 4 | Cirrhosis, with or without foci of residual perisinusoidal fibrosis. |
Based on additional studies, modifications may be made to these criteria for grading and
staging steatohepatitis. This proposed scheme, however, currently provides a template for standardizing
the analysis and reporting the morphologic features of NASH.
Differential diagnosis
1) Steatosis with nonspecific lobular inflammation
In cases with marked
disease activity, the morphologic pattern of injury in NASH is sufficiently characteristic such that the
pathologic diagnosis generally is straightforward. At the other end of the disease spectrum, however,
very mild or early steatohepatitis may be difficult to distinguish from a steatotic liver that
demonstrates nonspecific lobular inflammation. In these latter cases, the lobular inflammatory
infiltrate typically consists of sparse, mainly chronic inflammatory cells; lobular neutrophils, a
characteristic feature of NASH, are few or absent. While this paucity of neutrophils may help in the
distinction between these two possibilities, chronic inflammatory cells may be the predominant lobular
inflammatory cell type in cases of NASH with mild disease activity. As such, there are cases where it
may be impossible on morphologic grounds alone to distinguish between steatosis with nonspecific lobular
inflammation and very early or mild NASH.
2) Wilson's disease
In early Wilson's disease the liver biopsy typically
shows only microvesicular or macrovesicular steatosis and increased numbers of glycogenated nuclei. With
progression, a predominantly lymphocytic portal inflammatory infiltrate, with or without piecemeal
necrosis, becomes apparent. Although Mallory's hyaline may be present in Wilson's disease, it is usually
seen in cases of acute fulminant hepatitis or cirrhosis rather than early mild disease. When present in
cases of Wilson's disease, Mallory's hyaline is in a periportal location as opposed to the zone 3
distribution characteristic of NASH. Copper deposition, if present, favors Wilson's disease. Overall
pattern of inflammation and fibrosis in Wilson's disease is that of a chronic active hepatitis, not
steatohepatitis.
3) Chronic hepatitis C
Mild macrovesicular steatosis is a common finding
in chronic hepatitis C. This feature, in conjunction with the lobular inflammation typically present in
chronic hepatitis C, may suggest the diagnosis of NASH. However, the macrovesicular steatosis of chronic
hepatitis C typically is mild and is scattered thoughout the lobule; it is not as extensive as can be
seen in NASH and does not demonstrate the zone 3 accentuation that characterizes steatohepatitis. In
addition, in hepatitis C, the portal inflammation is greater than lobular inflammation, a pattern
opposite to that seen in NASH. Neutrophils, Mallory's hyaline and ballooned hepatocytes also are not
features of hepatitis C. Furthermore, in hepatitis C, if fibrosis is present, it demonstrates a
portal/periportal predominance over zone 3 perivenular fibrosis, and lacks the pericellular fibrosis that
typifies NASH.
4) Co-morbid conditions
Different disease processes may occur
simultaneously in the liver. The pattern of injury seen in the liver biopsy may reflect the features of
both diseases. For example, a patient may have chronic hepatitis C, as documented by serologic testing,
as well as be at high risk for NASH, due to marked obesity and type II diabetes. In such circumstances,
the pathologist sometimes is asked to speculate on which disease process has the greater contribution to
the overall liver injury. Fortunately, some of the histologic features of NASH (for example the
pericellular fibrosis) are sufficiently distinctive to aid the pathologist in this somewhat daunting
assignment.
References
- Bacon BR, Farahvash MJ, Jannery CG, Neuschwander-Tetri BA. Nonalcoholic steatohepatitis: An expnaded clinical entitity. Gastroenterol 1993;104:1103-9.
- Brunt EM. Nonalcoholic steatohepatitis: Definition and pathology. Sem Liver Disease 2001;21:3-16.
- Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: A proprosal for grading and staging the histologic lesions. Am J Gastroenterol 1999;94:2467-74.
- Caldwell SH, Oelsner DH, Iezzoni JC, Hespenheide EE, Battle EH, Driscoll CJ. Cryptogenic cirrhosis: Clincal characterization and risk factors for underlying disease. Hepatol 1999;29:664-9.
- Day CP, James OFW. Steatohepatitis: A tale of two "hits"? Gastroenterol 1998;114:842-5.
- Day CP, James OFW. Hepatic Steatosis: Innocent bystander or guilty party? Hepatol 1998;27:1463-5.
- Falck-Ytter Y, Younossi Z, Marchesini G, McCullough AJ. Sem Liver Disease 2001;21:17-26.
- Lee RG. Nonalcoholic steatohepatitis: Tightening the morphologic screws on a hepatic rambler. Hepatol 995;21:1742-3.
- Ludwig J, Viggiano TR, McDill DB, Ott BJ. Non-alcoholic steatohepatitis. Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980;55:434-8.
- Rashid M, Roberts EA. Nonalcoholic steatohepatitis in children. J Ped Gastroenterol Nutr 30:48-53, 2000.
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