Section 1 -

Chronic Hepatitis Neil Theise, M.D. Romil Saxena, M.D.

Case 1A 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. Case 1B 47 year old man, hepatitis C positive; ultrasound guided biopsy of left lobe, for staging and grading. 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. Portal inflammation 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. Interface hepatitis 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 cell surface. 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 toxin. 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 very helpful. 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 alone. 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 and C. 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 disease. 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. Hepatitis B 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 hepatocytes. 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 regenerative activity. 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. Hepatitis C 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 challenge world-wide. 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 seen. 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. Suggested Reading Colombari R, Dhillon AP, Piazzola E, Tomezzoli AA, Angelini GP, Capra F, Tomba A, Scheuer PJ. Chronic hepatitis in multiple virus infection: histopathological evaluation. Histopathology 1993;22:319-25. 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