About two billion people worldwide have been infected with the hepatitis B virus and about 350 million live with chronic infection. Besides, an estimated 600 000 persons die each year due to the acute or chronic consequences of hepatitis B. The course of HBV infection is a dynamic process and is influenced by many factors including viral, host, and exogenous factors. Clinical suspicion of acute viral hepatitis usually does not necessitate biopsy; however, persistence of clinical symptoms or biochemical evidence of hepatotrophic viral infection for more than six months duration necessitates a liver biopsy - in several instances to primarily establish the histopathological diagnosis, to grade and stage the hepatic changes (determine management and prognosis), to document the severity and extent of the hepatic inflammation, as a guide to therapy or to monitor the changes of liver histology while on treatment. Moreover, improvement in liver histology can be used as an endpoint in clinical trials for new forms of therapy. Additionally, in some cases there is considerable clinical overlap between the states of exacerbation of chronic hepatitis and acute hepatitis. Biopsy is helpful in these cases too. Nevertheless, it must be mentioned that with the current trends and various guidelines, the indications for liver biopsy in chronic hepatitis B are somewhat reduced. It is hoped that with non-invasive markers the number of liver biopsies will reduce further. This article provides an overview of the histopathology of chronic hepatitis B virus infection.
Keywords: Chronic hepatitis B, fibrosis, hepatitis B virus infection, histopathology
How to cite this article: Das P, Ahuja A, Gupta SD. Overview of the histopathology of chronic hepatitis B infection. Hep B Annual 2012;9:49-85
The term Chronic Viral Hepatitis [CVH] encompasses three components: Chronicity [time period clinically or concomitant healing and repair pathogenetically], viral etiology [current knowledge identifies Hepatitis B with or without Hepatitis D and Hepatitis C as the causative agents], and hepatitis [indicating necrosis and inflammation of the liver]. This is reflected as a pattern or triad of clinical, biochemical, and histological alterations in association with either hepatitis B (HBV), C (HCV) or D (HDV) virus infection. The clinical definition of CVH necessitates persistent inflammation of the liver, in a setting of an identifiable hepatotrophic viral infection of ≥ 6 months duration, evidenced by clinical symptomatology and / or biochemical abnormalities. Although on histology, the presence of inter-phase or piecemeal necrosis is considered as the morphological hallmark of chronic viral hepatitis, it must be emphasized that interphase hepatitis is not a mandatory diagnostic feature of chronic hepatitis, as in some cases inflammation is restricted to the portal triads without discernible interphase hepatitis.
Hepatitis B continues to remain an important cause of hepatitis, especially in certain regions of the world. Unfortunately many of these regions are relatively densely populated and even low rates of infection get translated to enormous numbers of individuals. About two billion people worldwide have been infected with the virus and about 350 million live with chronic infection. An estimated 600 000 persons die each year due to the acute or chronic consequences of hepatitis B.
The course of HBV infection is a dynamic process and influenced by many factors including viral, host, and exogenous factors. Clinical suspicion of acute viral hepatitis usually does not necessitate biopsy; however, persistence of clinical symptoms or biochemical evidence of hepatotrophic viral infection for more than six months duration necessitates a liver biopsy: in several instances to primarily establish the histopathological diagnosis; to grade and stage the hepatic changes (determine management and prognosis), to document the severity and extent of the hepatic inflammation; as a guide to therapy or to monitor the changes of liver histology while on treatment. Moreover, improvement in liver histology can be used as an endpoint in clinical trials for new forms of therapy., Additionally, in some cases there is considerable clinical overlap between the states of exacerbation of chronic hepatitis and acute hepatitis. Biopsy is helpful in these cases. Nevertheless, it must be mentioned that with the current trends and various guidelines, the indications for liver biopsy in chronic hepatitis B are somewhat reduced. It is hoped that with non-invasive markers the number of liver biopsies will reduce further [hopefully the tribe of liver pathologists would still not be included in the list of endangered species!].
It has to be understood that HBV does not cause direct cytopathic injury, but initiates an immune response that results in liver damage. The histological changes in the liver of patients with HBV infection is a reflection of the interaction between viral replication and host immune response that attempts to eradicate the virus. The necrosis and inflammation can be 'graded' and the end result of the healing and repair by way of fibrosis or the chronicity of these changes ('stage') can be readily assessed in a liver biopsy. The stage of any disease is a measure of how far it has progressed in its natural history, characterized by the varying extent of portal or porto-portal fibrosis in the early stages and cirrhosis to the end stage. It also means that as the histological changes are a sum total of the effects of necrosis, inflammation, and fibrosis, it is expected that changes in the transaminases can at best serve as a marker of some of these components, and will not correlate with the histological severity of the disease.
Adequacy of Liver Biopsies
Assessment of the possible hepatic changes primarily depends on the availability of an adequate size liver biopsy, preferably obtained with a 2.5 cm or longer, 16-gauge or wider needle. It has been estimated that a liver biopsy, 1 cm in length, obtained by a 24G external Menghini biopsy needle, contains six full portal triads or 0.8 ± 0.5 portal triads per square millimeter. However, for routine histopathological examination, the presence of two full portal triads and a medium-size hepatic artery will suffice for an adequate liver biopsy. However, if any pathology can be ascertained even in a smaller liver core; it must be reported., It must also be kept in mind that when sampling liver tissue using core needles, if only the sub-capsular parenchyma is sampled; a cautious approach is required while staging, as this area normally shows prominent fibrosis. It must be appreciated that even in an adequate biopsy; the interpretation of the biopsy often carries an intra-observer and inter-observer variation of 10-20%, even among experienced pathologists. Thus close cooperation between the attending gastroenterologist and histopathologist is essential. Both need to understand each other and such institutional dialects are of immense practical importance [as important as the language of the published literature] in managing patients.
Fixation of Liver Biopsies
It needs to be emphasized that the risk in liver biopsy, as far as the patient's health is concerned, is perhaps far greater when an improperly fixed or processed sample is interpreted by the histopathologist than the procedure itself.
Liver biopsies should be fixed in neutral buffered 10% Formalin immediately. The volume of formalin should be ideally 10-15 times in volume, in relation to the biopsy. This is easily achieved given the size of routine liver biopsies and the available containers. It is not necessary to mount the specimen on a board [like a mucosal biopsy] to keep the specimen straight. In case a frozen sample is to be preserved for molecular biology studies, one has to check with the laboratory. After routine processing of formalin fixation, the biopsies are embedded in paraffin and around five-micron-thick sections are evaluated, after relevant staining. For all routine evaluations such as grading, staging, and immunohistochemistry such sections are sufficient. It is advisable to study the serial sections, as in chronic hepatitis, the changes may be patchy.
A properly fixed and properly processed sample is one of the most important requisites for a meaningful interpretation of biopsies.
Role of Special Stains
In general a Hematoxylin and Eosin [H and E]-stained section provides all the information required for the overall evaluation and grading of chronic hepatitis B. However, special stains aid in staging of the disease. Immunohistochemistry provides invaluable information in certain instances.
A comprehensive histopathological evaluation of liver biopsy in chronic hepatitis B infection utilizes a standard panel of special stains as well as selective immunohistochemistry. These methods are especially useful in grading and staging of chronic hepatitis and establishing a diagnosis of cirrhosis. In most of the institutions the standard initial set of special stains includes reticulin [Gordon and Sweets silver impregnation or an equivalent], Masson trichrome, and diastase-pre-treated periodic acid Schiff (PAS + D). Periodic acid-Schiff (PAS), Shikata's orcein [or equivalents], and iron stain are used only rarely. At our institute we routinely perform PAS with diastase, reticulin, and Masson's trichrome stains, along with H and E, for interpretation of liver biopsies. [Table 1] provides a list of special stains and their use. The details are beyond the scope of this chapter.
Table 1: Special stains used in evaluation of liver biopsies in chronic viral hepatitis
Macroscopic changes in chronic hepatitis B infection are not of importance in routine practice. However, with excellent imaging techniques the gastroenterologist [and colleagues in radio-diagnosis] is often privy to changes that histopathologists rarely get to see. With transplantation programs in several institutions, explants have added to the gross pathology of the liver, earlier available only at autopsies! The macroscopic changes in chronic hepatitis B vary from a normal gross appearance to scarred multinodular hepatic cirrhosis. In chronic hepatitis without significant fibrosis, the changes are generally unremarkable. As the stage progresses to cirrhosis widespread fibrous scarring and diffuse macro-nodular or mixed macro- and micronodular cirrhosis can be identified. Although a mixed micro- and macro-nodular or macronodular cirrhosis is most common in HBV infection; micronodular cirrhosis can sometimes be seen and is often associated with concomitant alcoholic liver disease. According to the constituents of the cell cytoplasm, the hepatic nodules can be beefy-red or yellowish-green, secondary to cholestasis. Necrosis can be seen secondary to outgrown blood supply or thrombosed portal veins. In some cases large nodules should arouse the suspicion of an adenomatous / dysplastic nodule or even hepatocellular carcinoma. It must be emphasized that a bile-stained liver tumor, for all practical purposes, is hepatocellular carcinoma.
Common histological changes in chronic hepatitis B include:
Hepatocellular injury – characterized by apoptotic bodies and liver cell dropout
Inflammation – predominantly lymphocytic: in the portal tracts (Portal triaditis) at the interface between the parenchyma and portal connective tissue (Interface hepatitis / Piece-meal necrosis) in the parenchyma, at the site of the liver cell dropout (Lobular hepatitis)
Repair of the damage – activation of Kupffer cells and hepatocellular regeneration
Fibrosis – scarring with a fibrous expansion of portal areas that can extend to link adjacent vascular structures (bridging fibrosis). As the disease progresses, the scarring can completely surround groups of liver cells, and, along with hepatocellular regeneration, produce the nodules of cirrhosis.
Spilling over of the inflammatory cells beyond the limiting plate of the portal tract [piece-meal or interface hepatitis] has been one of the foremost, although not the mandatory histopathological criteria for identifying chronic hepatitis. According to the International Working Group classification of viral hepatitis, this feature, when present, is described as 'active hepatitis'; in contrast hepatitis which do not show this spill-over was termed as 'inactive hepatitis'. However, terminology such as 'active' and 'inactive' can also be used to denote clinical progression of the disease. As the clinical and histological connotations were often mistakenly used interchangeably, the term 'chronic active hepatitis' is no longer used currently present.
Other than the interphase hepatitis these inflammatory cells can also infiltrate and destroy the hepatocytes in zones 2 or 3. These lesions indicate 'lobular hepatitis'. The old terminology of 'chronic lobular hepatitis,' to denote a predominant or exclusive lobular rather than portal inflammation is also no longer in use, although current reports carry descriptions of inflammation within the lobule.
As inflammation in the lobule is invariably associated with necrosis, foci of inflammatory cells are an indication of necrosis of single or groups of hepatocytes ['spotty' or 'confluent' necrosis, respectively]. In these lesions the mononuclear inflammatory cells from the portal tract, infiltrate the hepatocytes and cause their premature apoptosis, resulting in nuclear shrinkage, hyperchromasia, fragmentation, eosinophilia, and pulling away of the cytoplasm from the adjacent hepatocytes, giving them a round contour (acidophil or Councilman bodies). Such acidophil bodies are generally scattered and few. A large number of such bodies are seen in acute hepatitis, indicating a more extensive necrosis. An area of confluent necrosis between a central vein and a central vein or a central vein and a portal tract, is called bridging hepatic necrosis. The term pan-lobular or pan-acinar hepatic necrosis is used for necrosis affecting the entire lobule or acinus. Such extensive forms of necrosis are generally uncommon in chronic hepatitis. It must be mentioned that as interface hepatitis is necroinflammation at the interface, between the parenchyma and the mesenchyma, bridging necrosis is a form of severe interface hepatitis.
Another feature of hepatocellular injury is a hydropic degeneration of hepatocytes [ballooned hepatocytes]. Ballooned hepatocytes, indicative of cell injury, are secondary to the dilatation of endoplasmic reticulum. These cells are swollen, round, and show clear abundant cytoplasm with nuclear enlargement, prominent nucleoli, and frequent multinucleation. These swollen hepatocytes may exert pressure over the adjacent hepatocytes, resulting in their shrinkage, angulations, rounding up or disintegration. Compression of bile canaliculi may result in intrahepatic cholestasis.
Hepatocyte damage around the hepatic venules (zone 3), as described in dogs or elsewhere in the lobule, can be picked up by periodic acid Schiff staining after diastase digestion [to remove staining by glycogen normally present in hepatocytes], as in these areas, macrophages with intracytoplasmic periodic acid Schiff (PAS)-positive diastase-resistant phagocytosed material pile up along with numerous inflammatory cells. In confluent necrosis, the reticulin framework of the liver cell plate gets disorganized and may collapse or come together, giving an illusion of thick bands of fibrosis [pseudo-fibrous bands]. When there is difficulty in differentiating extensive collapse from fibrosis by stains such as reticulin, it is advisable to resort to a elastic tissue stain [Orcein, Victoria Blue]. Normally an elastic tissue is confined to the portal tract or around larger vessels and additionally in the region of the fibrosis, but not in necrotic areas.
Hepatocytes are capable of regeneration following injury. The process of hepatic regeneration occurs simultaneously with hepatocyte damage. The features of regeneration are characterized by binucleation, nuclear enlargement, and / or nuclear prominence. The hepatocytes are often arranged in cords of two cells [double cord pattern] or into small cylinders, called hepatocyte rosettes. However, if the rosettes are extensive, it is advisable to rule out autoimmune hepatitis.
Additional changes include varying degrees of cholestasis [which may stain, similar to ceroid pigment-laden macrophages or Kupffer cells] and varying degrees of bile ductular proliferation in the peri-portal area or zone 1 [Figure 1] and [Figure 2].
Figure 1: (a) Photomicrograph showing interphase hepatitis (black arrow indicates piecemeal necrosis) [H and E × 40], (b) Ballooned hepatocytes and spotty necrosis (black arrow) is seen [H and E × 200], (c) Ground glass hepatocytes show voluminous, abundant, granular cytoplasm, with peripheral cytoplasmic clearing and central nuclei [HandE × 400], (d) Core biopsy of liver showing bridging hepatic necrosis. Inset is a reticulin stain of the same biopsy showing a collapsed reticulin framework with formation of ‘passive septae’ (black arrow) [H and E × 40; Inset- Reticulin stain × 40]
In a patient with chronic hepatitis B an increase in the severity of parenchymal damage should indicate the possibilities of: (a) Super-infection with other viruses such as delta, A, E, and C; (b) reactivation of hepatitis B; (c) superimposed drug-induced hepatitis; (d) discontinuation or withdrawal of interferon therapy.
The characteristic inflammatory cells in CH are usually mononuclear CD4+ T helper lymphocytes. Plasma cells can also be noted. These infiltrates are usually denser than those seen in acute HBV infection. The portal tract may be of normal size, expanded, and rounded-up, with varying degrees of edema and fibrosis. Macrophages with PAS-positive diastase–resistant cytoplasmic material can be seen. The PAS-positive material is generally lipofuschin pigment released from the necrotic hepatocytes. Occasionally the inflammatory cells encroach the portal blood vessels, associated with venous thrombosis. When present these changes pose a diagnostic dilemma, especially in a post-liver-transplant setting, as on morphology it is impossible to differentiate them from chronic liver graft rejection. When interphase hepatitis prevails, the nature of the inflammatory cells alters, as most of these cells are now CD8+ / cytotoxic T cells. These cells then infiltrate or percolate in-between the adjacent hepatocytes. Often lymphocytes can be identified in the hepatocyte cytoplasm, morphologically resembling emperipolesis.
Lymphocytes in the liver in chronic hepatitis
It is well-recognized that the nature of injury in CHB [and for that matter in most chronic viral hepatitis] is immunological. Thus, lymphocyte infiltration plays a major role in determining the histological changes. In general, the majority are T cells with around 50-60% of T helper lymphocytes and approximately 25% T-cytotoxic lymphocytes. B lymphocytes comprise of 15% of the inflammatory infiltrate, and the other cells, including NK, 10%. Expression of HLA antigens paralleled inflammatory activity. The most number of cells, especially in portal tracts, were found to be CD45RO+ lymphocytes (activated T cells, memory cells of both CD4 and CD8 subpopulations). The CD4 / CD8 ratio was > 1.5. The T cells mainly localized in the portal tracts with formation of interface hepatitis are of the alpha–beta type (95.3%alphabeta T cells; 4.7% gammadelta T cells).
There appears to be an active regulation of T-cell activity within the liver. Thus, Raziorrouh et al., have found that immunoregulatory CD244 and PD-1 are highly co-expressed on virus-specific CD8+ T-cells in chronic HBV infection. To add to these findings, Miyaaki et al., have identified that the ratio of Foxp3(+) Tregs in CD3(+) T cells increases more in patients with severe activity than those with mild activity.(P = 0.04). Serum alanine aminotransferase level (R = 0.402, P = 0.025) and the ratio of the CD8(+) T cell plus CD56(+) NK cell against the CD4(+) T cell (R = 0.53, P = 0.005) correlated with the ratio of Foxp3(+) Tregs in CD3(+) T cells.
Natural Killer Cells (CD 56+) are found in areas of lobular and interface necrosis, but the levels are lower than in the normal ones. The number of NK cells is reported to be more in HBV than HCV. Although the number of these cells remains the same irrespective of the necroinflammatory score, the numbers tend to decrease relatively as the fibrosis increases.
The scarring in chronic hepatitis,, is usually an extension of the portal stroma; in addition peri-venular and peri-cellular fibrosis may also develop. However, in bridging the hepatic necrosis between the central vein and portal tract, the resultant fibrous scar will have a matching distribution. With aging, the scar acquires deposition of the collagen, with an increased amount of elastic fibers. The fibrous septa, in addition, carry mononuclear inflammatory cells. These scars can be highlighted by the reticulin stain along with the Masson's trichrome stain for type I and type III collagen, sirius red stain for type 1 and type 3 collagen, and Victoria blue or orcein stains for elastic fibers. Under polarized light, sirius red further helps to differentiate the collagen types; as the fine type I collagen appears green and thicker type III collagen appears yellow. Immunohistochemical stains for alpha smooth muscle actin (SMA) highlight the stellate cells responsible for fibrosis; and CD34 stains the scar. It is believed that hepatic stellate cells are activated by oxidant stress or by various cytokines, for example, TGF-β, IL-1, and IL-4. As the scarring progresses the hepatic stem cells become activated and form bile ductules at the scar parenchyma interphase. The ductules produce type V collagen, and thus, the septa become voluminous and join each other to form cirrhotic nodules. With the Hematoxylin and Eosin stain the elastic fibers appear as faintly eosinophilic hyaline material, while the collagen appears as a glassy, thick, dark eosinophilic material, and thus it is not very difficult to differentiate the fiber types in a scar.
Various studies had been performed using various formulae to estimate hepatic fibrosis by non-invasive techniques, especially using parameters such as serum AST level or platelet count. However, most of these techniques were restricted by their reproducibility.
Transient elastography used for measuring liver stiffness appears promising, but currently liver biopsy remains the gold standard for the assessment of fibrosis.
Ground glass hepatocytes
Ground glass hepatocytes,, when present, are a relatively specific finding. These are round enlarged hepatocytes with a eosinophilic granular / glassy cytoplasm and thickened cytoplasmic borders. The peripheral-most part of the cytoplasm shows shrinkage and appears as a clear space, just beneath the cytoplasmic margin [Figure 1]c. The nucleus is usually small and situated eccentrically,
It must be emphasized that ground glass hepatocytes are not seen in acute hepatitis except in severely immunosuppressed individuals. Hence, their presence generally indicates chronic hepatitis B. This pointer is helpful in differentiating acute hepatitis from an exacerbation of chronic hepatitis B on biopsy.
Ground glass hepatocytes may be stained by the Orcein or Victoria Blue stains. Immunofluorescence and immunohistocehemistry are other methods for detection of such cells. Currently immunohistochemistry is the accepted standard method, in view of its sensitivity. The staining patterns of the three methods, however, are similar. It must be emphasized that the sensitive methods pick up cells that contain HBsAg and in some cases the cells may not necessarily appear to be ground-glass.
The section on immunohistochemistry gives an account of the relevance of this technique in the identification of various HBV antigens in the liver.
Sanded nuclei: Hepatitis core antigen within the nuclei appear as eosinophlic inclusions described as sanded nuclei and these inclusions stain with chromotrope-aniline blue.
Role of immunohistochemistry
The major use of immunohistochemistry in chronic viral hepatitis is to identify Hepatitis B and Hepatitis C viral antigens., The pattern of staining helps to confirm the presence of HBV infection and can also provide information regarding viral replication. It must be emphasized that Hepatitis B surface antigen is not demonstrable in the hepatocytes in acute hepatitis, by the current staining methods, except in highly immunosuppressed individuals [e.g., HIV infection, immunosuppressive therapy, fibrosing cholestatic hepatitis, etc.]. Thus, demonstration of HBsAg in a liver biopsy invariably indicates chronic hepatitis B.
Hepatitis B virus is a unique DNA virus that replicates through reverse transcription of its mRNA. It behaves more like a retrovirus than a DNA virus. HBV consists of a 28 nm central core containing the genome (a single molecule of partially double-stranded DNA) and a specific DNA polymerase with a surrounding core protein shell. The core is commonly found in the nuclei of infected hepatocytes, with the outer HBsAg coat being acquired from the cytoplasm of the hepatocyte. The core antigen is antigenically distinct from the HBsAg. This allows for a separate detection of the core antibody (anti-HBc) and surface antibody (anti-HBs). A further viral antigen, termed HBeAg, can be detected in the serum, together with DNA polymerase. HBeAg is a subunit of HBcAg. HBeAg positivity implies viral replication and is an indicator of high infectivity. Mutants that do not produce HBeAg exist; these 'pre-core mutants' can cause severe hepatitis.
Hepatitis B surface antigen: The topographical distribution of HBsAg in a liver biopsy can be seen as discrete (staining of scattered individual cells), clusters and sheets of positive-staining cells, and the staining pattern can be diffuse cytoplasmic, sub-membranous or membranous. Cytoplasmic positivity, in the presence of ground glass cells, confirms the diagnosis of chronic HBV. Diffuse membranous staining for the surface antigen, associated with core antigen staining, indicates a high replicative state. Chu and Liaw have concluded that membranous staining of HBsAg on the hepatocyte correlates excellently with the serum HBV-DNA level and thus can be recognized as a sensitive and specific marker of active hepatitis B virus replication. However, intracytoplasmic staining for surface antigen indicates a relatively low level of viral replication (Bianchi L, 1994).
Hepatitis B Core Antigen: Staining of Hepatitis B Core Antigen Is Predominantly Nuclear and Less Commonly Cytoplasmic or Membranous., the Presence of Nuclear Staining Correlates Well With Active Viral Replication, as Indicated by Hbv Dna, Dna Polymerase, and Hbe Ag in the Liver and Serum., However, Cytoplasmic Staining for the Core Antigen Correlates Best With the Hepatocyte Regenerative Activity., a Further Study by Chu and Liaw Reveals That the Degree of Expression of Hbcag in the Hepatocyte Nucleus, but not in the Cytoplasm, Can Accurately Reflect the Level of Viral Replication in Patients With Chronic Hepatitis B. the Cytoplasmic and Membranous Expression of Hbcag Correlates to a High Degree With Liver Inflammatory Activity. Ramakrishna Et Al., Have Shown That in Hbeag-Positive Individuals, Nuclear Core Antigen Positivity on Immunohistochemistry Correlates With the Dna Titer and Cytoplasmic Core Antigen Localization Correlates With the Histological Activity; Whereas, in Hbeag-Negative Individuals, Nuclear Localization Correlates With the Dna Titer, Histological Activity, and Fibrosis, and Cytoplasmic Localization Correlates With the Dna Titer and Fibrosis, but not With Histological Activity [Table 2].[Figure 3]
Table 2: Interpretation of immunohistochemical staining [Figure 3]
Figure 3: Immunohistochemical stain for HbsAg showing strong, diffuse, membranous, and cytoplasmic [Fig. 3a × 100], membranous [Fig 3b × 100], and discrete staining of the hepatocyte [Fig 3c × 40]. Stain for HBcAg showing a discrete nuclear [Fig 3d × 40]; both nuclear and cytoplasmic positivity [Fig 3e and f × 100]
Hepatitis B envelope antigen: HbeAg staining generally follows that for a core antigen in chronic hepatitis B infection. Staining for an 'e' antigen, along with the core antigen is indicative of active viral replication. It could be either nuclear or cytoplasmic., The viral elimination phase is associated with sero-conversion from HBeAg to HBe antibody and on immunohistochemistry by nuclear and cytoplasmic HBeAg staining.
Hepatitis B X antigen: HBX Ag staining is more widely present in hepatocytes of patients with chronic hepatitis B, and therefore, it is more sensitive than either the surface or core antigen, to demonstrate HBV infection by IHC. It represents integration of the segment of the viral genome into the host genome; therefore, it is expressed independent of the other viral antigens.
For practical purposes only, HBsAg and HBcAg immunostaining are relevant, easily feasible, and important. On the other hand HBe Ag and HBX Ag staining are not performed routinely. The serological tests for HBeAg are more easily available and their relevance and use need no elaboration.
In addition to immunohistochemistry, the indirect immunofluorescent technique is also applied for the detection of various antigens in the liver tissue. Immunofluorescence provides a distinctive distribution pattern of HBsAg, in various types of acute and chronic hepatitis. However, the ease and ability to perform staining on paraffin sections that are relatively permanent and can be archived makes immunohistochemistry the current method of choice for the detection of various hepatitis B virus antigens in tissues routinely.
An understanding of the role of identifying various antigens in liver biopsies in CHB depends on the appreciation of the natural history of HBV infection. Three clinicopathological phases of chronic hepatitis B virus (HBV) infection are identified classically. However, as will be described later, currently based on the HBeAg status, five such phases or stages are described. It is not necessary that these stages are sequential.
Immune tolerance: Immune tolerance of HBV may result when maternally derived hepatitis B e antigen (HBeAg) crossing the placenta may result in the elimination of T helper cells responsive to HBeAg / HBcAg. This phase can last for periods ranging from a few weeks to 10 or more years, until immune tolerance is lost. The immune tolerance phase is associated with high levels of viraemia, normal alanine aminotransferase (ALT) levels, and minimal histological lesions. More than 30-40% of hepatocytes have predominantly nuclear hepatitis B core antigen (HBcAg). The rate of loss of HbeAg is low.
Immune clearance: Immune clearance of HBV is the second phase. This is associated with intermediate levels of viraemia, fluctuating ALT levels, and active and ongoing hepatitis. Approximately 20-30% of the hepatocytes have HBcAg, predominantly in their cytoplasm. Expression of pre-core defective HBV mutants during chronic HBV infection may lead to a reduction in the secretion of HBeAg and may trigger the beginning of the immunoelimination phase. The mechanism of the intrahepatic shift of HBcAg from the nucleus to the cytoplasm and the decreased levels of viraemia in this phase may be, at least in part, secondary to liver damage and regeneration.
Latent infection with residual integrated HBV: In this final phase, undetectable viraemia is associated with normal ALT levels and no virus-induced liver damage. With regard to hepatocyte expression of HBsAg in chronic HBV infection, membrane staining of HBsAg on hepatocytes has been shown to correlate well with the presence of viraemia. The degree of cytoplasmic hepatitis B surface antigen (HBsAg) expression inversely correlates with the level of viraemia. Therefore, HBsAg carriers with high levels of viraemia have low levels of cytoplasmic hepatitis B surface antigen (HBsAg) expression, while those with low levels of viraemia have high levels of cytoplasmic HBsAg expression. However, several exceptions have been identified. High levels of viraemia associated with high levels of cytoplasmic HBsAg expression have been recognized in patients with fibrosing cholestatic hepatitis. In contrast, low levels of viraemia associated with low levels of cytoplasmic HBsAg expression are recognized in patients with hepatitis C virus, but not hepatitis D virus superinfection.
Overall inflammatory activity does not seem to correlate with the expression of HBsAg and HCVAg. HBcAg expression, however, accompanies chronic hepatitis B of moderate and severe activity.
Fatty change has been reported to a varying extent in chronic hepatitis B. Fatty change associated with CHB is generally less common in children than in adults.
In one study from India fatty change was found in 66.6% of chronic hepatitis B in comparison to 70% in chronic hepatitis C patients. The extent of steatosis was not necessarily uniform. In one study the steatosis involved < 33% of hepatocytes in 90% of the cases.
Steatosis has been found to be more common in men than women. Most studies indicate that steatosis is independently associated with body mass index, serum triglyceride, apolipoprotein B, uric acid, and fasting blood glucose, and thus the metabolic syndrome. Correlation with HbeAg, fibrosis, viral load, and other factors have not been consistent.,,,
The reason for steatosis in HBV has thus been a matter of debate. Although, the currently available data indicates that the association between steatosis and HCV is specific, this may not be the case in HBV-infected patients. Steatosis in CHB could be a co-factor related to the associated metabolic syndrome rather than the result of HBV infection.
Histological Classification Schemes and Scoring Systems Used in Chronic Hepatitis Reporting
The current value of liver biopsies in CHB not only lies in providing a confirmation of the diagnosis and a description of the changes described; but also in the semi-quantitative assessment of histological changes. Such an assessment provides a guideline for therapy and also for comparing sequential changes in a given patient, especially as a follow-up to treatment.
The initiation of the current histological classification first commenced with the publication by an International group in 1968,39 which was revised in 1977. These seminal articles provided codified terminologies such as chronic persistent, chronic lobular, and chronic active hepatitis.
This old system is no longer in use because it does not include the disease stage and etiology, and cannot be correlated with the newer semi-quantitative scoring systems.
The first most widely used semi-quantitative system for reporting chronic hepatitis was the Histological Activity Index (HAI) in 1981.
For determining the HAI, points or numbers are awarded to histological changes and these are added to a score or the index. The HAI is thus a sum total of three major components of histological changes [Table 3]:
Even as necrosis and inflammation are the direct result of liver injury, fibrosis is a consequence of necrosis and inflammation. Hence necro-inflammation and fibrosis are assessed or scored separately.
The first category includes two important types of necrosis: piecemeal necrosis and bridging necrosis. Although subgroups B, C, and D score piecemeal necrosis, subgroups E and F refer to a combination of piecemeal necrosis and bridging necrosis. Thus, bridging necrosis cannot be scored separately.
One of the major criticisms of HAI was that the categories were not uniform and had scores ranging from 0-10 for I, 0-4 for II to IV, and several intervening numbers were omitted. In category I, there were seven subgroups (A-G), but the scores 2, 7, 8, and 9 were missing. Similarly categories II to IV had four subgroups (A-D) with the score 2 missing in all of them. More importantly it was felt that pathogenetically fibrosis was the end-result of necrosis and inflammation, and the subsequent healing and repair. Hence, necro-inflammation and fibrosis were two different processes that should be separated and not added up to provide a composite score.
This resulted in the concept of grading and staging of histological activity in chronic hepatitis. Grading is used to describe the intensity of the necro-inflammatory activity, while Staging is an indication of architectural alteration. These signify progression of the disease toward cirrhosis or end-stage liver disease. This histological activity is important for the patient and the clinician because it provides a measure of severity of the hepatitis at the time of biopsy, and this is not always matched by abnormal liver function tests. Histological changes in CHB are a sum total of the necrosis, immunological and inflammatory reactions, as well as the subsequent result, such as, fibrosis. Serum enzymes alone do not reflect all these aspects, and hence, cannot be expected to correlate with the histological changes.
Various classification systems have been proposed to grade and stage CHB
Recommended approach of classifying of chronic hepatitis grading and scoring
There is no universal acceptance of a single system for evaluating chronic hepatitis.
However the International System is more often used for Chronic Hepatitis B, while the Metavir System is more popular, while assessing Chronic HCV. Whatever the system used, it is essential that both the clinician and pathologist not only understand the system thoroughly, but are equally comfortable with the system adopted. In general there is good correlation between the two systems of scoring.
A histopathological report should be descriptive and should communicate sufficient and appropriate information about the disease state to the physician. The final impression should ideally include: (a) presence / absence of chronic hepatitis, (b) the grade of inflammation, (c) stage of injury and chronicity, wherever possible, and (d) the etiology (either based on the demonstration of viral antigens in the biopsy or due to compatible histology). Finally, the accumulated scoring for the histological activity grading and staging should be mentioned and the scoring system used should be indicated, with the maximum possible scores. In using these scores as endpoints in early clinical trials, it was decided that a two-point or greater decrease in the grading or one-point in the staging was considered to be a clinically significant 'histological improvement or deterioration,' and trials were evaluated by comparing the proportion of patients in each treatment cohort showing improvement.
It is essential to remember that the final score of grading for a single patient may not be comparable between the two patients. Thus a grade of 8 could be arrived at by both, two points each for interface hepatitis, confluent hepatitis, spotty necrosis, and portal inflammation or by three points each for interface hepatitis and spotty necrosis, and two points for portal inflammation! The combination is endless. Thus the real value of this scoring lies in assessing the progress in an individual patient.
It must be mentioned that several studies, by different observers, have evaluated variations in scoring biopsies. Most studies have been conducted with regard to Chronic Hepatitis C, but are applicable to Chronic Hepatitis B. In general within specialists the agreement is acceptable.,, The assessment of fibrosis or staging, which has a bearing on the progression of disease, has less variation than grading.
Although there is always a concern regarding who is reporting the biopsy it must be emphasized that other factors, which actually have a bearing on reporting, such as the size of the biopsy, is often neglected. These seemingly trivial matters unfortunately become the focus of 'extracting' reports, by stretching the existing knowledge to the endless horizons of unnecessary bravado, resulting in avoidable errors, embarrassment, blame-games, and above all a bad relationship among colleagues [beside the unfortunate patient whose score of overall suffering rises by several points!]. Obviously such methods of practice are more risky and more expensive than not getting a report or even a repeat liver biopsy.
Severity of histological changes in different phases of chronic hepatitis B
At this stage it is best to review changes in relation to the current understanding of the natural history of Hepatitis B virus infection based on the HBeAg status. Five stages are recognized instead of the classical three indicated earlier in the section, on immunostaining.
Immune tolerance phase: Generally seen in perinatal infection characterized by high HBeAg postivity, high HBV replication, mild or low necroinflammation, and no or slow progression to fibrosis. The liver biopsy thus shows little or no changes, but HBs Ag and HBcAg are detectable.
Immune reactive phase: This is seen several years after the immune tolerance phase or much sooner following infection in adulthood. HBeAg and HBV replication is relatively lower than in the previous phase. There is moderate-to-severe necroinflammation and more rapid progression to fibrosis than in the previous phase. HbsAg and sometimes HbcAg [depending on the extent of replication] are detectable on immunohistochemistry.
Inactive HBV carrier state: This phase may follow seroconversion from HBeAg to anti-HBe antibodies. It is characterized by very low or undetectable serum HBV DNA levels and normal aminotransferases. As a result of immunological control of the infection, this state confers a favorable long-term outcome, with a very low risk of cirrhosis or HCC, in a majority of patients. HBsAg loss occurs spontaneously. Thus, histological changes are few and often HbsAg-positive hepatocytes are the only finding.
HbeAg-negative CHB: Regarded as a sub-population of CHB, HbeAg-negative and / or anti-HBe-positive CHB is defined as a hepatitis B surface antigen (HBsAg)-positive, active virus replication and liver disease. In the clinical setting HBeAg negative CHB is reserved for those with positive HBsAg and negative HBeAg for more than six months, with an elevated ALT level and detectable DNA in the serum, by hybridization techniques. Although originally believed to be restricted to Mediterranean countries, a worldwide prevalence has been noted. There is periodic reactivation with a pattern of fluctuating levels of HBV DNA and aminotransferases and active hepatitis. These patients are HBeAg-negative, and harbor HBV variants with nucleotide substitutions in the precore and / or the basal core promoter regions, unable to express or expressing low levels of HBeAg. HBeAg-negative CHB is associated with low rates of prolonged spontaneous disease remission. It is important and sometimes difficult to distinguish the true inactive HBV carriers from patients with active HBeAg negative CHB, in whom phases of spontaneous remission may occur. The former patients have a good prognosis with a very low risk of complications, while the latter patients have active liver disease with a high risk of progression to advanced hepatic fibrosis, cirrhosis, and subsequent complications, such as, decompensated cirrhosis and HCC. In a study by Jie et al. on 743 Chinese patients, 35.9% of the patients were HBeAg negative. This group of patients was younger (27.1 vs. 32.7 years) than those of CHB with HBeAg positivity, had lower HBV DNA levels, higher ALT levels, and higher HAI necro-inflammatory scores in association with increasing DNA levels; in contrast to the decreasing HBV DNA levels, in association with higher ALT levels and higher HAI necro-inflammatory scores in HbeAg-positive patients. Although serum HBV DNA levels correlated positively with HAI-NI and HAI-F scores in both HBeAg-positive and HBeAg-negative patients, the total liver HBV DNA levels correlated with HAI-NI and HAI-F scores in anti-HBe-positive patients only. Hence, the quantitative measurement of intrahepatic HBV DNA was a valuable marker of histological status for the liver in anti-HBe-positive patients with CHB only, and it could give an insight into the prognosis and the ideal time for the cessation of antiviral treatment. In another study by Shao et al., (2007), on 213 patients, the authors did not find any correlation between the serum viral DNA levels in either HbeAg-positive or negative patients. The authors also noted an older age of prevalence in HbeAg-negative CHB patients.
HBsAg negative phase: In the 'HBsAg-negative phase,' after HBsAg loss, low-level HBV replication may persist with detectable HBV DNA in the liver. Generally, HBV DNA is not detectable in the serum, while anti-HBc antibodies with or without anti-HBs are detectable. HBsAg loss is associated with improvement of the outcome, with reduced risk of cirrhosis, decompensation, and HCC. However, immunosuppression may result in reactivation of hepatitis with histological changes, sometimes resembling acute hepatitis. In these cases HBsAg positive cells on immunohistochemistry provide a clue to diagnosis.
Histology and Treatment Decision
The aim of management, with or without biopsy, is to achieve a sustained suppression of HBV replication and remission of liver disease, as well as, to prevent progression to cirrhosis and HCC. As HBsAg seroconversion is rarely achieved, current treatment concepts are aimed at (i) sustained suppression of viral replication, (ii) normalization of aminotransferase levels, (iii) histological improvement in the case of hepatitis B e-antigen (HBeAg) positive disease, and (iv) HBeAg loss or seroconversion to anti-HBe. As a standardized definition of sustained response is not available, treatment endpoints vary greatly between different trials.
Assessment of disease activity over time is thus of great importance in the management of CHB patients. As biopsies are invasive procedures there have been attempts to reduce their requirement to a reasonable minimum, and rightly so. Hence, virological and serological response rates are gaining more acceptability than histological response to therapy. However, here are certain instances where biopsies are considered essential in the diagnosis and management of CHB.
Current treatment guidelines suggest that patients with a wild-type virus (HBe antigen-positive), serum HBV DNA >105 copies / mL, and alanine amino transferase (ALT) levels greater than two times the upper limit of normal (ULN) [ALT level of > 2 x ULN] are obvious candidates for antiviral therapy. A previous study has demonstrated that even in the presence of normal ALT levels, 20% of the patients with CHB infection had piecemeal necrosis and 10% had severe hepatic fibrosis or cirrhosis., It should also be kept in mind that, as patients develop advanced fibrosis; the serum ALT levels tend to decrease. Thus, modified treatment guidelines also suggest that a liver biopsy should be considered in patients ≥ 40 years of age, with elevated HBV DNA, and borderline normal or slightly elevated ALT levels; because some patients with persistently normal ALT levels have significant hepatic pathology upon biopsy examination, which have been infected with HBV, genotype C. In addition to the importance of serological variance, an increased rate of significant histology in CHB patients with high viral load has been linked to increased age. Some studies have reported age ≥ 45 years as an independent predictor of significant histology. Whatever be the HBe serological status and treatment, the efficacy of antiviral treatment is assessed by repeated measurements of viral load and serum ALT levels, in principle, every 12-24 months. In patients who are HBe antigen-positive, the efficacy of antiviral treatment is shown by the loss of HBe antigen, followed by the appearance of anti-HBe antibodies (HBe seroconversion), a reduction in viral load below 2 x 104IU / mL, and normalization of serum aminotransferase. In patients who are HBe antigen-negative or HBe antigen-positive, but do not seroconvert after short-term treatment, and who receive nucleoside or nucleotide analogs, the treatment objective is for the HBV DNA to become undetectable by real-time PCR.,
The role of various parameters in predicting the response to therapy for CHB has been reviewed in detail by Kau, Vermehren, and Sarrazin. The histological parameters that are of importance in predicting a response in HbeAg-positive CHB include a high necroinflammatory activity score and fibrosis, when treated with PEG IFN alpha 2b. With regard to nucleosides / nucleotides, the reports are conflicting, with one report of this predicting response and another not showing any such correlation. Staging or fibrosis does not appear to be an important parameter related to the response to necleuoside / nucleotide therapy. Few studies have looked at baseline grading scores as predictive variables of treatment response in HBeAg-negative disease. No correlation has been found between the HAI score and combined biochemical and virological response in PEG IFN alfa-2a and / or LAM and ADF treatment.
The impact of steatosis on response to antiviral treatment has been poorly studied. One study reports no association between steatosis and response to antiviral treatment, with either PEG IFN alfa-2a or a combination of PEG IFN alfa-2a plus LAM.
Based on these observations various guidelines have recommended liver biopsy in different groups of CHB patients. Unfortunately there does not appear to be a single consensus, with the European Association for the Study of the Liver (EASL) guideline being more generous than others on the requirement for liver biopsy. In general the following are the recommendations of some guidelines.
The AASLD Guidelines 2009 suggest that liver biopsy should be done, taking into consideration the age [> 40 years], the new suggested upper limits of normal for ALT [30U / L for men and 19U / L for women], HBeAg status, HBV DNA levels, and other clinical features suggestive of chronic liver disease or portal hypertension.
Liver biopsy should be considered if diagnosis of Chronic Hepatitis B is made by other criteria for the purpose of grading and staging, prior to therapy. Patients with moderate / severe activity and significant fibrosis require treatment.
In case of HBeAg +, persistently high HBV DNA [> 20,000 IU / mL]
Biopsy is recommended if there is three-to-six months of raised ALT
According to the EASL guidelines, patients should be considered for treatment when HBV DNA levels are above 2000 IU / ml (i.e., approximately 10,000 copies / ml) and / or the serum ALT levels are above the upper limit of normal (ULN) for the laboratory, and liver biopsy (or non-invasive markers when validated in HBV-infected patients) shows moderate-to-severe active necroinflammation and / or fibrosis using a standardized scoring system (for example at least grade A2 or stage F2 by METAVIR scoring) (A1). Thus biopsies are considered more frequently than the AASL guidelines. The situation where biopsies are not considered to be necessary include: most patients under 30 years of age with persistently normal ALT levels and a high HBV DNA level (usually above 107 IU / ml), without any suspicion of liver disease, and without a family history of HCC or cirrhosis. These patients do not require immediate liver biopsy or therapy. Follow-up is mandatory (B1) in this group of patients.
Several other guidelines are available and these have been reviewed by Sethy and Goenka and interested readers may refer the article for details.
Influence of Chronic Co-Infection of Hepatitis B With Hepatitis D Virus, Hepatitis C Virus, and Human Immunodeficiency Virus on Liver Histology
Coinfection with Hepatitis D virus (HDV, delta agent) alters the course of acute hepatitis B, favors chronic evolution, and enhances the severity of disease., There are no specific features of chronic HBV and HDV infection, except that HDV can be stained in the nuclei of hepatocytes.
Several studies have elucidated differences between the histological features of hepatitis B and C: ground glass hepatocytes in CHB and lymphoid follicles, more fatty change, and bile duct damage in CHC.,,
It must be emphasized that both HBV and hepatitis C virus (HCV) co-infection do occur. In one study it was found that in comparison to chronic HBV infection alone, the dual infection serum HBV DNA (37% vs. 71.9%, P < 0.005) and ground-glass hepatocytes (37% vs. 66.7%, P < 0.01) were less frequently identified. In the mixed infection group, the HCV-RNA was also less frequently detectable than the control group (60% vs. 92.3%, P < 0.001) and had a significantly higher fibrosis score. It was difficult to make a diagnosis of a dual infection histologically.
Human immunodeficiency virus (HIV) co-infection increases the risk of HBV-associated, end-stage liver disease. The presence of HIV infection increases the risk of chronicity after exposure to HBV. HIV reduces the rate of spontaneous HBsAg and HBeAg seroconversion. Hence, the prevalence of HBeAg-negative chronic hepatitis B as well as the HBV-inactive carrier state tend to be lower in HBV / HIV-co-infected individuals. Despite higher serum HBV-DNA levels, hepatic necroinflammation tends to be milder in HBV / HIV-co-infected individuals. Nevertheless, the enhanced replication levels of HBV in HIV-co-infected patients may result paradoxically in a progression to more severe liver fibrosis.
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Hepatic Histology in Treatment-naïve Children With Chronic Hepatitis B Infection Living in the United States and Canada
Norberto Rodriguez-Baez, Karen F. Murray, David E. Kleiner, Simon C. Ling, Philip Rosenthal, Kristen Carlin, Kara Cooper, Kathleen B. Schwarz, Sarah J. Schwarzenberg, Jeffrey H. Teckman, Marc G. Ghany, Ahmad Samer Alawad
Journal of Pediatric Gastroenterology & Nutrition. 2020; 71(1): 99