Practical Updates in Liver Pathology: Grading, Staging, and Nomenclature
Section 1 -
Neil Theise, M.D.
Romil Saxena, M.D.
34 year old woman, hepatitis C positive; biopsy for staging and grading.
Diagnosis: Chronic hepatitis, mildly active with transition to cirrhosis
(modified ishak stage 3/4), compatible with hepatitis C.
47 year old man, hepatitis C positive; ultrasound guided biopsy of left lobe, for staging and
Diagnosis: Chronic hepatitis, mildly active, ? with portal fibrosis
(subcapsular samples), compatible with hepatitis C.
General pathology of chronic viral hepatitis
Microscopic Appearances: Patterns of Necrosis, Inflammation and Fibrosis
Hepatocyte injury and inflammation in chronic hepatitis is now generally referred to as
"activity," which is "graded" (see below). Distribution of inflammatory cells may vary from case to case
or even in sequential biopsies from the same patient. However, all cases of chronic viral hepatitis are
distinguished by a relatively dense monocytic infiltration of the portal tracts. If nearly all the
inflammation is confined within the portal tracts and if hepatocyte injury is scant or absent, the
pattern corresponds to what was originally classified as "chronic persistent hepatitis" by the
International Working Group. This phrase, however, is no longer in use. When there is more obvious
inflammation and apoptosis of hepatocytes involving the limiting plate and lobular hepatocytes, the
hepatitis may be termed active, though the phrase 'chronic active hepatitis' also should avoided.
Mononuclear infiltration of portal tracts is the defining lesion of chronic hepatitis of
any cause. Some or all portal tracts may be involved and the portal infiltrates are usually much more
dense than those seen in acute viral hepatitis. The portal tracts may be of normal size or appear
widened by the influx of mononuclear cells. The infiltrate includes predominantly CD4+helper/inducer
T-lymphocytes with an admixture of plasma cells. Some portal macrophages may also be seen to
contain periodic acid-Schiff (PAS)-positive, diastase-resistant material and iron pigment, representing
the removal of necrotic hepatocyte debris.
Portal inflammation will often fill and expand the portal fibrous stroma, pushing
structures aside without obvious injury. Lymphoid aggregates or fully formed follicles may be seen;
while most common in hepatitis C, they are also seen in other forms of hepatitis. However, inflammation
with damage or even destruction of bile ducts may be seen, particularly in hepatitis C. Inflammation may
also encroach on the portal blood vessels, in particular the portal veins, endophlebitis may be present,
and there may be associated fresh or organizing venous thrombosis; such lesions may be particularly
evident with trichrome stained sections.
The region of liver tissue where the hepatic parenchyma comes into contact with the
mesenchymal stroma of the intact or scarred portal tract may be referred to as an interface region.
Thus, hepatocyte apoptosis and inflammation of this area are generally referred to as "interface
hepatitis", this being the currently favored term. Previously, this classical histologic feature of
active chronic hepatitis, was called "piecemeal necrosis", referring to the way in which the limiting
plate of hepatocytes was eroded in a piecemeal i.e. focal, fashion.
In regions of interface hepatitis, there is a predominantly mononuclear infiltration, though
in these regions, FAS-ligand positive CD8+ suppressor/cytotoxic T-cells predominate. Close contact of
hepatocytes with these lymphocytes, and with macrophages and plasma cells, is seen. Emperipolesis, the
invagination of lymphocytes into hepatocytes, has been described. While the inflammation interweaving
amongst hepatocytes at the interface is sufficient by itself for describing the lesion as interface
hepatitis, dead or dying hepatocytes may also be seen in these areas. These hepatocytes will show the
typical features of cells undergoing apoptosis, including nuclear hyperchromasia and disintegration,
cytoplasmic eosinophilia and fragmentation, and a pulling away from adjacent cells with rounding of the
Lobular hepatitis and confluent necrosis
Another form of activity in chronic viral hepatitis is that found within the hepatic lobule away from
the portal areas or septal scars. Such lesions may be referred to as "lobular hepatitis" or "spotty
necrosis." Cases in which such lesions predominate have historically been referred to as "chronic
lobular hepatitis," though, like the terms chronic persistent and chronic active hepatitis, this
classification is no longer in favour. The inflammatory infiltrates in these areas of activity are the
same as those seen in interface hepatitis, but its relative importance to the development of scarring and
progression in chronic viral hepatitis remains uncertain.
Some foci of lobular hepatitis are relatively devoid of mononuclear cells. In these areas
there may be rare or numerous acidophil bodies, macrophages containing PAS-positive, diastase-resistant
material indicating prior cell death, or simply cellular debris and loosely aggregated collagen and
reticulin fibres. Such lesions are similar to the lobular damage found in acute viral hepatitis and tend
to focus on the hepatic venules. If large areas are involved, one may refer to the lesion as "confluent
necrosis" and, if such areas span from central vein to central vein or from central vein to portal tract,
then the term "bridging necrosis" is used. Such bridging necrosis is considered the most ominous finding
in terms of progression toward scarring and cirrhosis. In some cases, when one or more entire lobule has
been destroyed, it may be referred to as panlobular or multilobular collapse. In these areas, portal
tracts will be in abnormally close proximity, separated only by regions filled with necrotic debris,
macrophages, loose or more mature collagen, and elastic fibres. A ductular reaction, which is now
recognized as activation of an hepatic stem cell compartment is also present.
Fibrosis and hepatocyte regeneration
While some patients with chronic viral hepatitis do not show fibrous scarring, most will
have some. Increasing fibrosis is now assessed as advancing "stages" of disease. The scarring will
usually develop as an extension of the portal stroma, though perivenular and pericellular fibrosis may
also be seen. The perivenular fibrosis usually develops following collapse and condensation of the
reticulin meshwork in an area of confluent or bridging necrosis. The resulting scars are bland and
generally acellular, and often lack an extensive accompanying interface hepatitis. These septa will
either link central veins to neighbouring portal tracts or to other hepatic veins and, in these cases,
probably represent an area of healed bridging necrosis. If these scars persist, mature forms of collagen
will deposit as will elastin fibres demonstrated by Orcein or Victoria blue stains. Thus, deposition of
elastic fibres suggests increasing age of the scar.
Fibrous expansion of the portal tract probably results from a more active process of injury
and repair. There is as yet little agreement on the use of the terms periportal fibrosis and portal
fibrosis. Whichever term is used, it refers to fibrous stroma extending from the portal tract beyond its
usual boundaries. This fibrosis is usually conceptualized as following Rappaport's acinus zone 1 and,
thus, eventually leads to linking of one portal tract to another. These septa usually contain
mononuclear inflammatory cells as described above and may be associated with interface hepatitis. The
fibrosis is usually mature, darkly staining with trichrome stains, and containing abundant type I
collagen in addition to type III collagen and reticulin fibres. Again, elastin fibres are
easily demonstrated, indicating that the scars are of at least several months duration.
Immunohistochemical staining for a- smooth muscle actin highlights numerous
activated stellate cells in these regions and these cells are thought to be largely responsible for most
of the scarring.
Regeneration of hepatocytes becomes increasingly evident in parallel with the formation of
fibrous septa and advancing stages of disease. The thickening of liver cell plates to two or three cells
thick evidences this regeneration. Haematoxylin and eosin (H&E) or silver stains for reticulin can
demonstrate these changes. An incomplete periportal nodular transformation may thus be noted around
portal tracts that are becoming fibrotic. However, in the later stages of developing cirrhosis,
hepatocyte regeneration markedly diminishes in parallel with an increasingly proliferative ductular
reaction at the mesenchymal/parenchymal interface. The implication is that as hepatocytes reach
replicative senescence there is activation of the intrahepatic facultative stem cell compartment.
Regression of Fibrosis and Cirrhosis
Development of scarring in a chronically diseased liver is actually the result of a
balance in favor of matrix deposition in a liver dynamically producing and degrading matrix at all time
points. That fibrosis can regress with elimination of viral activity and infection has now been
demonstrated in all forms of chronic viral heaptitis. Cirrhosis itself, however, has generally been
thought to be an "endstage" of chronic liver disease and therefore not reversible. However, exceptions
to this concept have been noted in many liver diseases over the years.
In 2000, Ian Wanless and colleagues examined sequential biopsies from a patient with chronic
hepatitis B infection who, with successful anti-viral treatment and elimination of viral infection,
apparently reversed the scarring and re-established some degree of functional, non-cirrhotic parenchyma.
These changes were documented on biopsy. Wanless' suggestion that cirrhosis was reversible when the
impetus to hepatic injury ceases was exceedingly controversial at the time. The formal paper was finally
in a particularly interesting fashion: all in the same volume of the journal there appeared his paper,
responses/critiques written by each of the reviewers, and then his response to their comments. Despite
the controversial nature of the proposal, however, the concept rapidly entered the realm of general
knowledge as clinical papers exploring the impact of successful eradication of HCV infection on stage of
disease in patients with paired pre- and post-biopsy specimens demonstrated the possibility of regression
of fibrosis and, in some cases, regression of cirrhosis.
Wanless and colleagues described eight histologic features of regression as the "hepatic
repair complex" which can be grouped into features evident of three regenerative phenomena:
fragmentation and regression of scar (e.g. delicate, perforated septa, isolated thick collagen fibers,
delicate, periportal fibrous spikes, hepatocytes within, or splitting septa), evidence of prior, now
resolving vascular derangements (e.g. portal tract remnants, hepatic vein remnants with prolapsed
hepatocytes abberant parenchymal veins) and parenchymal regeneration in the form of hepatocyte "buds."
These hepatocyte buds are small clusters of hepatocytes, usually emerging from within portal/septal
stroma; the interpretation that these represented a regenerative phenomenon rather than entrapment of
parenchyma by active scarring was supported by Falkowski et al.
Multiple Chronic Viral Infections
Co-infection by multiple hepatotropic viruses
Since hepatitis B, C and D viruses are all parenterally spread, it is not surprising that
dual or triple infection of these viruses occurs. Viral interference appears to be most common in
co-infections. Experimentally, in chimpanzees, non-A non-B hepatitis (presumable HCV) interfered with
both acute hepatitis A and chronic hepatitis B. Interference has also been demonstrated in clinical
settings, usually HCV interfering with HBV replication, though the reverse has also been reported when
HBV infection followed HCV infection. The clinical course, however, does not seem to be consistently
altered by such co-infections.
Histopathologically, there are no specific findings to suggest the possibility of multiple infections
and the diagnosis usually depend on serological investigations. However, immunohistochemical
investigations have revealed some important features. In particular, it seems that there is suppression
of HBV core antigen by simultaneous HCV infection. It may be reasonable to suggest that, in a patient
serologically positive for hepatitis B surface antigen (HBsAg), the absence of demonstrable tissue
staining for HBV antigens indicates that clinical suspicion of co-infection not only with HDV, but also
with HCV, should be raised.
Co-infection by hepatotropic viruses and HIV
Since HBV, HCV, HDV and the human immunodeficiency virus (HIV) are all parenterally
acquired, and HBV and HIV are also frequently transmitted sexually, coinfection of these hepatitis
viruses and HIV is often seen clinically. Additionally, co-infection between HIV and HAV has been
identified, though this does not seem to lead to a difference in HAV clinical course. HIV
co-infection with HEV has been reported and transmission to the fetus and maternal mortality may be
increased in this setting.
Though acute hepatitis B is less commonly icteric in the setting of HIV infection, viral
persistence and development of chronic infection is more common. Most studies indicate a diminished
histological activity of chronic hepatitis B, though greater activity has also been reported.
Reactivation of hepatitis B has also been reported with HIV co-infection. Rarely, the
fibrosing cholestatic variant of hepatitis B is seen, as it is in hepatitis C. As far as HDV
is concerned, co-infection with HIV seems to worsen liver damage as evidenced by increased serum
aminotransferase levels and worsened histological severity. 140 Replication of both HBV by
itself and HDV-together is increased and may be demonstrated by more diffuse staining for hepatitis B and
delta antigens in tissue specimens.
Co-infection of HCV and HIV is also common. As HIV-infected individuals are
surviving longer with new antiviral therapies, the importance of HCV infection in these patients
increases. Acute hepatitis C seems to be more often symptomatic and much more likely to result in liver
failure when HIV is simultaneously acquired. Many studies indicate that untreated HIV with significant
immunosuppression increases the severity and accelerates the course of chronic hepatitis C, though
successful anti-HIV treatment accompanied by immune reconstitution seems to lead, generally, to a more
typical HCV course. However, it has been reported that even with minimal or absent immunosuppression in
such treated patients, fibrosis is still more prevalent and more rapid, though the mechanism is
uncertain. Conversely, it has also been suggested that HCV is a poor prognostic indicator for
HIV disease and patients with co-infection, even with successful immune reconstitution have increased
mortality and morbidity due to viral interactions directly or to interactions between the large battery
of medications required to treat both infections.
Differential diagnosis of chronic hepatitis
Many disease states can mimic chronic viral hepatitis and other, non-viral conditions can
cause the same chronic hepatitic patterns of injury. To fully evaluate this in a biopsy or resection
specimen, clinical history must be considered. In all cases, if a diagnosis of chronic viral hepatitis
is contemplated, seroligical tests for hepatitis B, C and D must be obtained. In the absence of positive
viral serology, the following diseases must be considered.
Other causes of chronic hepatitis
Infecting viruses are not the only aetiology of the typical histological features of
chronic hepatitis. Autoimmune hepatitis is characterized by autoantibodies such as anti-nuclear
antibodies or anti-liver kidney microsome (LKM) antibodies. Histologically, abundant plasma cells in the
inflammatory infiltrate and widespread parenchymal collapse are hints that an autoimmune process rather
than a viral infection is present. Serological studies demonstrating autoantibodies, in the absence of
those confirming viral infection, should establish the diagnosis. However, one caveat is that in chronic
hepatitis C and less commonly in chronic hepatitis B, serological studies may indicate the presence of
circulating autoantibodies. In the case of hepatitis C, false positive results of the early antibody
tests in patients with hypergamma globulinemia and antinuclear antibodies accounted for some of these
cases. It is also clear, however, that in chronic infection with HBV, HCV or HDV there may be breakdown
of self tolerance and induction of autoantibodies. For example, chronic hepatitis B infection
may stimulate the formation of circulating immune complexes of viral antigens and reactive antibody.
These complexes may fix complement and induce tissue damage such as vasculitis, arthritis and
glomerulonephritis. Chronic hepatitis C is also associated with autoimmune phenomena. HCV
and less frequently HBV are associated with mixed cryoglobulinemia. Autoantibodies including
antinuclear antibodies may be seen in chronic hepatitis C but are generally of low titer.
Metabolic diseases may also have chronic hepatitic patterns of injury. a1
-antitrypsin deficiency can have the same necroinflammatory lesions and pattern of scarring, but
histologic demonstration of a1-antitrypsin globules by H&E,
PAS staining after diastase digestion, or immunohistochemistry will confirm the diagnosis. Typically,
liver disease is seen in individuals with ZZ phenotype. Phenotypes such as SS or a heterozygous pattern
are not predictably related to chronic liver disease. Care must be taken, however, to exclude
concomitant viral infection serologically, as it appears that most chronic hepatitides in the setting of
metabolic diseases are due to a superimposed viral infection.
Similarly, Wilson disease can cause the same pattern of injury, but histological
confirmation, while suggested by abundant Mallory bodies, fatty change, or even some copper accumulation
in periportal hepatocytes, can be achieved only in combination with clinical and biochemical
findings. The diagnostic tests are complex but attempt to establish abnormalities of copper
metabolism by documenting low levels of serum ceruloplasmin or by finding evidence of increased tissue
stores of copper. Provocative testing with urine copper measurements before and after penicillamine
administration has been most extensively validated in children. However, none of the test
results is pathognomonic for Wilson disease and may be abnormal in patients with other causes of abnormal
copper retention as is seen in patients with primary biliary cirrhosis or primary sclerosing cholangitis.
Wilson disease should be remembered as a confounding imposter and considered in the differential
diagnosis of non-viral chronic hepatitis irrespective of age.
Hereditary hemochromatosis (HH) is a common inherited disorder that also results in chronic
liver disease, but not typically in individuals under the age of 40 years. HH typically does
not have any significant chronic inflammatory response, so in a clinical setting suspicious for HH, but
with chronic hepatitis-like changes seen on biopsy, one should investigate other etiologies.
Some drugs can also cause chronic hepatitis, including: a -methyldopa, isoniazid,
oxyphenasatin, nitrofurantoin and diclofenac. Some of these drugs may actually induce autoantibodies,
suggesting induction of an autoimmune hepatitis. Again, the absence of serological markers of
viral infection will help diagnostic accuracy and careful history taking will often reveal the offending
Diseases that mimic chronic viral hepatitis
Any disease leading to dense lymphoplasmacytic infiltrate in the portal tracts may mimic
chronic viral hepatitis. For example, primary biliary cirrhosis (PBC), not only often has a dense portal
infiltrate, but may also have extensive interface and lobular hepatitis. A diagnostic biopsy for this
disease demonstrating granulomatous destruction of bile ducts is unlikely to cause this confusion. A
later-stage lesion, with ductular proliferation and features of chronic cholestasis adjacent to fibrotic
septa, is also unlikely to cause confusion. However, non-diagnostic early lesions of PBC may be
difficult to distinguish from chronic viral hepatitis, particularly type C, as the latter may have marked
bile-duct damage and even loss. In these instances, clinical correlation is important, as PBC
predominantly affects women, is characterized by elevation of alkaline phosphatase and gamma-glutamyl
transpeptidase associated with elevated IgM and the presence of mitochondrial antibody. Aminotransferase
values are generally less than 5 times the upper limit of normal but may fluctuate to higher levels.
Similar confusion may be encountered, although less frequently, with a non-diagnostic biopsy specimen for
primary sclerosing cholangitis. Again, clinical correlation for possible chronic inflammatory bowel
disease (usually ulcerative colitis) and characteristic bile duct abnormalities on imaging studies are
Lymphoma or leukemic infiltrates may also mimic the inflammatory infiltrate of chronic viral
hepatitis, particularly when the infiltrate is most prominently located in the portal tracts with
overflow into neighboring sinusoids. In such cases, careful inspection of the lesion will reveal an
absence of true interface hepatitis at the margins of portal tracts where the malignant cells extend past
hepatocytes that are atrophic, but not truly apoptotic. Similarly, parenchymal hepatocytes may appear
atrophic, but acidophil bodies are generally absent. The typical scarring of progressive chronic
hepatitis is also not usually present in such specimens. Most important, of course, is that the
infiltrating cells will have features suggesting a lymphoproliferative disorder, including monomorphism
or marked atypia.
Semi-Quantitative Scoring in Chronic Hepatitis
Adequacy of biopsy sampling for grading and staging
Though one might argue that this section should follow those following, which describe
the history, use, and varied schema for grading and staging of chronic viral hepatitis, the importance of
the topic and the lack of consistent application of well-documented principles warrants it being
highlighted at the start. As summarized by Guido and Rugge, there are two basic issues which
bear on adequacy of tissue acquisition for grading and staging: 1) can a small random sample adequately
reflect the overall state of a diffuse liver disease, such as chronic hepatitis? 2) Can sample size
affect accuracy of histologic assessments?
The answer to both questions is a clear "yes." If sufficient
tissue is obtained, then the little needle biopsy core of tissue, estimated
to be 1/50,000 of the total liver parenchyma can reflect the status of disease in the organ as a whole.
However, it would seem that, indeed, there is little attention paid to what is "sufficient" in routine
hepatology and hepatopathology practice. Recent studies suggest a simple and clear answer: a total of
2.0 cm of liver tissue, containing 11 to 15 portal tracts is necessary to avoid underscoring of grade and
stage of disease. Less than that and there are likely to be significant inaccuracies in assessment.
An additional sampling problem concerns small biopsy specimens obtained from the subcapsular
region. For perhaps as much as 1 cm below the capsule, increased stroma, including septum formation and
perhaps focal nodularity, may be within the spectrum of normal. Thus, such biopsy specimens may
overestimate stage of disease.
It cannot be overemphasized, therefore, that the pathologist must assess samples of tissue
provided either by hepatologists or radiologists for adequacy before
supplying an assessment of grade (necroinflammatory activity) and stage (scarring) to the clinician and
the patient. It behooves the pathologist to educate their clinical colleagues to supply adequate
samples, either by obtaining one long needle core or through repeat passes. It should particularly be
made clear to radiologists that they should eschew the easier approach, into the left lobe, for the more
traditional right lobe sampling, thus making it more likely to avoid sampling the subcapsular region
Development and Application of Scoring Systems
The first scoring system specifically designed for the study of chronic hepatitis was
that of Knodell and colleagues. The purpose of their histogical activity index (HAI) was to
follow the course of chronic hepatitis in asymptomatic patients, in whom conventionally used clinical
events such as jaundice, ascites and encephalopathy could not be evaluated. The HAI was based on four
components: periportal and bridging necrosis; intralobular degeneration and focal necrosis; portal
inflammation; and fibrosis. The scores allotted to these components were respectively 0-10, 0-4, 0-4 and
0-4, giving a maximum possible total of 22. The system and its components were used extensively after
its publication, for the evaluation of therapy and for correlation of histological changes with other
factors such as viral load, viral genotype and liver function tests, principally in chronic hepatitis B
Several other scoring systems followed. Of these, the most notable are those of Batts and
Ludwig and the METAVIR group (a co-operative group of French investigators). An important
feature of these systems recognized that grading of necroinflammatory activity should be separated from
staging (i.e. the extent of fibrosis, structural distortion, development of cirrhosis). In recognition
of this, the "Knodell scoring system" was modified by Ishak and colleagues in 1995.
An alternative approach to the problem of assessing the extent of pathological change, in
particular of fibrosis, has been the use of morphometry. Additionally, there is currently an active hunt
for the "holy grail" of non-invasive approaches to grading and, particularly, staging. However, none of
these non-invasive approaches, using serologic assessments or radiologic/ultrasonagraphic measurements
have yet reached appropriate levels of sensitivity and specificity to make them clinically reliable.
Intra- and interobserver variation
Histological evaluation of liver biopsies includes a subjective element and is therefore
liable to both inter- and intra-observer variation; this must be taken into account when interpreting
numbers generated by different pathologists or by the same pathologist at different times.
Staging appears to be less subject to variation than grading. Moreover a simple
scoring system proved more reliable than a complex one when intra- and inter-observer variation were
investigated. Variation was reduced in a study by the METAVIR group when biopsies were
evaluated simultaneously by two observers.
Handling data from semi-quantitative analysis
When semi-quantitative scoring data are evaluated, it is important to bear in mind the nature
of the generated numbers. The numbers represent categories rather than measurements, and cannot
therefore be used as real numbers in statistical analyses. Even adding the numbers
representing different grading components (e.g.interface hepatitis, confluent necrosis, lobular activity
and portal inflammation) together to create a total grading score can lead to inaccuracies, because the
scales for these components are linear, and differ from each other. At best, a total grading score
serves only to give an approximate idea of the severity of a chronic hepatitis; it usually gives no
information on the types of liver damage and inflammation involved. It follows that statistical
evaluation must be appropriate for categories rather than measurements, and that correlations should be
made with individual grading components rather than with grading totals.
Uses and choice of scoring methods
Simple scoring methods not requiring much time may be used routinely, but like all
methods involving subjective assessment of categories they are prone to intra- and interobserver
variation. Scoring should not be undertaken unless pathologist and clinician agree that it is likely to
be helpful for patient management. The METAVIR group's algorithm is an example of a simple, carefully
evaluated method easily applicable to routine use. In some centres, scores form part of the
evaluation of patients for possible antiviral therapy. There is then merit in using the same system for
pre- and post-treatment evaluation. In general, the more complex schemes are likely to enable smaller
therapeutic effects to be recognized. Whichever system is chosen for a particular purpose, the user
should ensure that the features evaluated and the numbers generated are appropriate for the particular
purpose. If necessary, an existing system can be modified for an individual project. Features not
included in the scoring system, such as bile-duct damage and immunohistochemical findings can be
separately recorded. In interpreting the results of any scoring system, it is relevant to note that
grading scores greater than zero can sometimes be generated in biopsies form patients without liver
In addition to the role of scoring in routine reporting and in the evaluation of therapies,
it plays an important part in clinical research. Among the many examples using a variety of scoring
methods are the observations that patients infected with different HCV serotypes show relatively little
difference in histological activity, that cirrhosis commonly develops during long-term
follow-up in chronic hepatitis C, and that progression of fibrosis in this diseases is related to age at
infection, male sex and alcohol intake. Indeed, biopsy based evaluation of outcomes are
central for determining efficacy of anti-viral clinical interventions and require detailed quantitative
grading and staging of disease activity.
Individual Types of Chronic Viral Hepatitis
There are more histological similarities than differences between the various viral
hepatitides. All share the same basic features of liver-cell damage and inflammation, and the principal
histological patterns of acute hepatitis may all be found in association with any of the viruses, with
the possible exception of panlobular necrosis, which is rarely, if ever, due to HCV infection. Likewise,
the histological features of chronic hepatitis overlap for hepatitis B and C; thus, it is not currently
possible to differentiate reliably between the viruses on histological grounds alone. However, there are
different, sometimes characteristic patterns associated with some of the agents, and these will now be
described. They represent tendencies rather than definitive differential diagnostic criteria.
Histologically, acute hepatitis B infection appears essentially similar to other forms of
acute hepatitis. There are varying numbers of apoptotic bodies scattered through the lobule, perivenular
confluent necrosis and, in the most severe cases, bridging necrosis and parenchymal collapse. Similarly,
chronic hepatitis B can have all the typical hallmarks of chronic hepatitis from any cause: interface
hepatitis, lobular activity and lymphoplasmacytic infiltrates in portal tracts and focally in the
lobules. One other major clinicopathologic variation of hepatitis B occurring following orthotopic liver
transplant and, more rarely, in other settings of immune compromise is called "fibrosing cholestatic
hepatitis." This variant is rapidly
progressive, has a poor prognosis and a distinctive histopathology: fibrous expansion of portal tracts,
extensive ductular reaction often with ductular cholestasis, and marked bilirubinostasis in
The most distinctive histological feature that readily distinguishes chronic HBV infection is
the so-called ground-glass hepatocyte. These cells have a finely granular cytoplasmic inclusion which
consists of proliferated endoplasmic reticulum containing abundant HBsAg that pushes the cell contents,
including the nucleus, to the side, usually leaving a visible halo separating the inclusion from the cell
membrane. Ground-glass cells can be highlighted by histochemical stains such as Shikata's orcein and
Victoria blue stains as well as by immunohistochemical staining. Such hepatocytes are usually
scattered singly and often non-uniformly throughout the liver. In cirrhosis, some nodules may contain
abundant ground-glass cells, while other nodules are totally devoid of them. The differential diagnosis
suggested by these cells includes oncocytic hepatocytes, cyanamide toxicity, Lafora's disease, and
fibrinogen storage disease. Nuclear inclusion may also be seen on routine H&E stained
biopsy specimens of chronic hepatitis B. These pale pink, very finely granular inclusions are referred to
as 'sanded nuclei' and contain abundant core material. Confirmation can be obtained with
immunohistochemical staining for HBcAg.
Detection of HBV in tissue sections
Immunohistochemical staining of hepatitis B surface and core antigens is straightforward
and widely applied. The patterns of staining in a given specimen can confirm the presence of HBV
infection and can also provide information regarding viral replication.
HBcAg. Immunohistochemical staining for core
antigen shows a predominant localization in hepatocyte nuclei and, less prominently, in the cytoplasm or
associated with the cell membrane. The presence of nuclear staining correlates well with
active viral replication as indicated by HBV DNA, DNA polymerase and HBeAg in the liver and serum.
On the other hand, cytoplasmic staining for core antigen correlates best with hepatocyte
Immunostaining for HbcAg is an important clinical adjunct to histochemical
assessment of biopsy specimens from HBV infected patients for particular reasons. First, while absence
of staining for core antigen in a biopsy specimen may simply be due to sampling error, as expression can
be variable in different parenchymal regions, co-infection with other hepatotropic viruses may suppress
HBV replication and, thus, core antigen expression. Therefore, in the absence of core immunostaining,
clinical investigation of possible co-infection is important. On the other hand, diffuse
staining of nuclei throughout a specimen generally suggests unbridled viral replication in the setting of
immune compromise, such as following organ transplantation or in HIV infection. Likewise, in this
setting, the pattern of core antigen detection warrants additional clinical investigation, to rule out
perhaps unsuspected causes of immunosuppression.
HBsAg. While immunohistochemical demonstration HBV surface in the
presence of ground-glass cells can readily confirm the diagnosis of chronic HBV, such staining can also
give information about the replicative state of the virus. On the one hand, there may be extensive
membranous staining for surface antigen, which is usually associated with core antigen staining; such a
pattern would indicate a high replicative state for the virus. Alternatively, there may be
intracytoplasmic staining for surface antigen, not only in ground-glass cells, but also in many more
cells without apparent inclusions. Such staining may range from a faint perinuclear blush to a dense
signal throughout the cytoplasm corresponding to the ground-glass cells. This pattern of staining
indicates an inability of the hepatocytes to secrete fully-formed viruses and, if these cells are
numerous, one may expect a relatively low level of viral replication.
The hepatitis C virus (HCV) was first identified in 1989 by molecular cloning and was
found to be the agent responsible for a majority of sporadic and post-transfusion hepatitides, previously
referred to as non-A non-B hepatitis. Since its identification, it has become evident that HCV is a
globally widespread infectious agent which has been present for more than five decades. It has been
calculated that there are more than 170 million chronic carriers worldwide, i.e. approximately 3% of the
world's population. With its high rates of chronic infection, progreesion to cirrhosis and end stage
liver disease, and development of hepatocellular carcinoma, it represents a formidable public health
HCV is a small (30-38nm) single-stranded RNA virus with a lipoid envelope. It is
classified as a subgenus of the Flaviviridae family. Consisting of a large open reading frame, the
genome contains approximately 9400 nucleotides. At the 5' end is a highly conserved region
with significant homology between different subtypes of HCV and is thus useful for amplification in
polymerase chain reaction assays. The polyprotein produced from translation of this open reading frame
yields ten mature proteins which have been identified.
Variations in genomic sequences in viral isolates from around the world have allowed for
phylogenetic classification of six viral genotypes which, in turn, contain many subtypes. There are
clear differences in genotype prevalences in different geographic regions, as well as differences between
genotypes and their subtypes in terms of treatment responses, but confirmation of any association between
different genotypes and severity or progression of disease is still lacking. Whether this is due to
cohort study design or a true lack of difference remains unclear.
Histopathology of chronic hepatitis C
The histopathology of chronic infection is variably active and progressive, with all the
features of any chronic viral hepatitis as described above. The severity of changes in the biopsy does
not correlate well with symptoms or with the serum liver enzymes at the time of biopsy and therefore the
biopsy may provide information independent of serum tests. In fact, asymptomatic patients, even those
without serum aminotransferase elevations, may have significantly abnormal histology.
While not specific, some histological features may be considered characteristic of HCV
infection, when seen in the otherwise typical chronic hepatitis biopsy. These changes include prominent
lymphoid aggregates or even fully developed lymphoid follicles with germinal centres, prominent bile duct
damage, and steatosis. The lymphoid aggregates, with or without germinal centres, are not restricted to
chronic hepatitis C, as they may also be seen in autoimmune hepatitis and chronic hepatitis B.
However, they are far more common with HCV infection and their identification should raise the
possibility of HCV infection. When present, they contain activated B-cells, surrounded by follicular
dendritic cells and a B-cell mantle zone, and are surrounded by an outer T-cell zone suggesting that they
are fully functioning lymphoid follicles. The lymphocytic infiltration of the liver is
otherwise similar to that seen with other causes of chronic hepatitis, though a striking bead-like
appearance of intrasinusoidal lymphocytes reminiscent of infectious monocleosis can sometimes be
The fatty change seen in hepatitis C is usually a mixed micro-and macrovesicular steatosis of
mild to moderate degree. Such steatosis may relate to increased body mass index (particularly in
genotype 1), though direct viral effects also seem to be involved (genotype 3). More severe
degrees of steatosis, particularly if associated with Mallory bodies, neutrophilic infiltration and other
histological features of fatty liver disease, suggest concomitant injury by any of the aetiologies of
such disease, including alcohol, obesity, diabetes mellitus, and hyperlipidemia syndromes.
The bile-duct injury seen in hepatitis C may be relatively mild with focal reactive changes in
epithelium, with vacuolation, stratification and crowding. More severe injury, however, with pyknosis of
cholangiocytes and lymphocytic infiltration is frequently identified. Bile-duct loss may also occur.
Confusion with early stages of primary biliary cirrhosis must therefore be guarded against, though
clinical information should establish the diagnosis.
Hepatic iron stores are frequently increased in chronic hepatitis C. Reticulo-endothelial
iron deposition is likely to be secondary to necroinflammatory activity and is associated with a poorer
response to interferon treatment. When hepatocytic iron is also seen, particularly in advance
of cirrhosis, it may be associated with concomitant genetic hemochromatosis, porphyria cutanea tarda,
increased oral uptake, previous treatment with Ribavirin for the HCV infection, and alcohol use.
In cirrhosis, parenchymal iron is often related to the cirrhosis itself.
Hepatitis C and alcoholic liver injury
It is well recognized that in many populations, alcoholic liver disease and chronic hepatitis C often
co-exist. Alcoholics with alcohol-related liver disease are at significantly increased risk for
developing chronic HCV infection, while those without alcohol-related liver disease are not. In fact,
several studies report an increase in the frequency of HCV markers as the histological severity of
alcoholic liver disease increases. These findings suggest that HCV infection enhances the injury induced
by alcohol or, conversely, that alcoholic liver disease increases the risk of HCV infection or
progression of HCV infection to chronicity.
From the diagnostic point of view, if biopsy specimens taken for documentation of alcoholic
liver disease contain features more typical of chronic hepatitis, a significant proportion will be
hepatitis C positive. When histological features of both diseases are identified, the speed
of progression is greater. Survival for individuals with both diseases is significantly lower than for
those with only one disease or the other. The precise mechanisms for these effects are not
known, but are hypothesized to include enhancement of immune-mediated antiviral damage to alcohol-altered
hepatocyte membranes; alcohol inhibition of liver regeneration; enhancement by alcohol of viral
replication and concomitant immune suppression.
As noted above, mild to moderate steatosis is often identified in association with hepatitis C
and, by itself, neither suggests nor excludes the possibility of concomitant alcoholic liver disease.
However, more severe fatty change, involving entire lobules, should raise the possibility of concomitant
alcoholic liver injury, particularly if associated with steatohepatitis (in the form of ballooning
degeneration of hepatocytes or neutrophilic infiltration) or perivenular/pericellular fibrosis. In fact,
scarring with this distribution in the absence of fatty change may suggest alcohol injury in the past and
should be noted as such in chronic hepatitis C biopsy specimens.
Type D (delta) Hepatitis
Hepatitis D virus (HDV) was first discovered by Rizzetto and colleagues as a nuclear antigen in the
hepatocytes of HBV-infected patients. HDV was originally thought to be an antigenic variant
of HBV, and referred to as the delta antigen. It was subsequently recognized as a separate infectious
viral agent, but a highly unusual one in that is defective in terms of replicative ability and requires
co-infection with HBV for propagation
Histopathology of hepatitis D
Histologically, acute and chronic hepatitis D infections look like any other acute or chronic
hepatitis, with the same range of necroinflammatory lesions and patterns of scarring. Activity in
chronic hepatitis D is usually fairly high with widespread interface and lobular hepatitis. The single
distinguishing feature is the sanded nucleus, similar to that seen occasionally in hepatitis B. In
hepatitis D this feature is due to HDAg accumulating in the nucleus of infected cells.
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