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Liver tissue examination

      1. Introduction

      Examination of surgical and needle liver biopsies comprises mainly histopathological study, and may serve diagnostic and/or research purposes. Diagnostic investigations require close collaboration with the clinician to assure optimal reliability and information. Of paramount importance are adequate biopsies and impeccable histopathological and histochemical techniques [
      • Desmet V.
      • Fevery J.
      Liver biopsy.
      ].

      2. Histopathology

      Histopathology of the liver biopsy allows diagnosis of acute hepatitis, its degree of severity, and in most cases differentiation from chronic hepatitis.
      Since histopathologically acute viral hepatitis B appears essentially similar to other forms of acute hepatitis, an aetiological diagnosis is less reliable. Acute viral hepatitis B may show different patterns as described below [
      • Ferrell L.D.
      • Theise N.D.
      • Scheuer P.J.
      Acute and chronic viral hepatitis.
      ,
      ].
      Acute hepatitis with spotty necrosis is the classical picture of self-limiting acute hepatitis. Cell damage tends to predominate in the centrolobular areas [
      • Kobayashi K.
      • Hashimoto E.
      • Ludwig J.
      • Hisamitsu T.
      • Obata H.
      Liver biopsy features of acute hepatitis C compared with hepatitis A, B and non-A, non-B, non-C.
      ]. Distinction can be made between an early, fully developed, late, and residual stage [
      • Bianchi L.
      • De Groote J.
      • Desmet V.J.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • et al.
      Acute and chronic hepatitis revisited.
      ]. Acute hepatitis with bridging necrosis represents more severe hepatitis with confluent areas of necrosis of the lytic type. The necrosis may link afferent and efferent vascular landmarks (portal-central bridging necrosis) [
      • Boyer J.L.
      • Klatskin G.
      Pattern of necrosis in acute viral hepatitis. Prognostic value of bridging (subacute hepatic necrosis).
      ]. Extensive confluent necrosis is often followed by collapse of the denuded reticulin framework, resulting in scarring fibrous septa. Older scars can be identified by their elastin content [
      • Scheuer P.J.
      • Maggi G.
      Hepatic fibrosis and collapse: histological distinction by orcein staining.
      ]. Acute hepatitis with panlobular or multilobular necrosis is more severe, seen in fulminant hepatitis. Acute hepatitis with periportal necrosis features periportal interface hepatitis (piecemeal necrosis). The occurrence of this lesion in acute hepatitis B was considered an indicator of possible transition to chronicity [
      • Bianchi L.
      • De Groote J.
      • Desmet V.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • et al.
      Morphological criteria in viral hepatitis.
      ]; as was also bridging hepatic necrosis [
      • Boyer J.L.
      • Klatskin G.
      Pattern of necrosis in acute viral hepatitis. Prognostic value of bridging (subacute hepatic necrosis).
      ]. It appears that in the later stage of acute hepatitis (after 2 months) both lesions are unfavorable prognostic signs [
      • Desmet V.J.
      Acute viral hepatitis: hepatitis B.
      ]. The most reliable predictor of chronicity is the demonstrable presence of hepatitis B virus (HBV) antigens in scattered hepatocytes [
      • Houthoff H.J.
      • Niermeijer P.
      • Gips C.H.
      • Arends A.
      • Hofstee N.
      Hepatic morphologic findings and viral antigens in acute hepatitis B. A longitudinal study.
      ].
      The histological differentiation of acute hepatitis B from viral-like drug-induced and auto-immune hepatitis may sometimes be difficult. Chronic hepatitis is recognizable by predominance of portal and periportal changes, possible presence of elastin-containing septa, and detectable viral antigens (hepatitis B surface antigen, HBsAg; hepatitis B c antigen, HBcAg) on immunostaining. Differentiation from bile duct obstruction and acute alcoholic hepatitis is feasible.
      The histopathology of chronic viral hepatitis B comprises the common lesions of any form of chronic hepatitis and some specific features [
      • Ishak K.G.
      Pathologic features of chronic hepatitis: a review and update.
      ].

      2.1 Common features of chronic hepatitis

      Portal infiltration. Most portal tracts are infiltrated by inflammatory cells, predominantly lymphocytes [
      • Baptista A.
      • Bianchi L.
      • De Groote J.
      • Desmet V.J.
      • Ishak K.G.
      • Korb G.
      • et al.
      The diagnostic significance of periportal hepatic necrosis and inflammation.
      ]. Interface hepatitis (previously termed piecemeal necrosis) is typical of more active disease. It corresponds to lymphocytic infiltration at the interface between connective tissue (portal tracts and septa) and parenchyma, associated with apoptotic death of local hepatocytes [
      • Ishak K.G.
      Pathologic features of chronic hepatitis: a review and update.
      ,
      • Lau J.Y.
      • Xie X.
      • Lai M.M.
      • Wu P.C.
      Apoptosis and viral hepatitis.
      ]. The lesion may be minimal, mild or severe. True interface hepatitis must be differentiated from periportal necro-inflammation in hepatitis A, from ‘biliary piecemeal necrosis’ in chronic biliary diseases, and from spill-over of inflammatory cells unassociated with liver cell damage [
      • Baptista A.
      • Bianchi L.
      • De Groote J.
      • Desmet V.J.
      • Ishak K.G.
      • Korb G.
      • et al.
      The diagnostic significance of periportal hepatic necrosis and inflammation.
      ]. Intralobular focal necro-inflammation (spotty necrosis) varies in extent with the severity of disease. Confluent lytic necrosis (bridging, panlobular, multilobular) characterizes severe disease, and clinical exacerbations of chronic disease [
      • Villari D.
      • Raimondo G.
      • Brancatelli S.
      • Longo G.
      • Rodino G.
      • Smedile V.
      Histological features in liver biopsy specimens of patients with acute reactivation of chronic type B hepatitis.
      ]. Hepatitic rosettes correspond to clusters of surviving hepatocytes in areas of extensive necro-inflammation [
      ]. Most authors consider bridging necrosis an ominous prognostic finding [
      • Cooksley W.G.E.
      • Bradbear R.A.
      • Robinson W.
      • Harrison M.
      • Halliday J.W.
      • Powell L.W.
      • et al.
      The prognosis of chronic active hepatitis without cirrhosis in relation to bridging necrosis.
      ] although the lesion may also heal [
      • Chen T.
      • Liaw Y.F.
      The prognostic significance of bridging hepatic necrosis in chronic type B hepatitis: a histopathologic study.
      ]. Apparently bridging necrosis carries a sinister long-term prognosis when associated with interface hepatitis, whereas the cirrhogenic evolution of chronic disease marked by interface hepatitis (‘chronic active hepatitis’) is accelerated by bridging hepatic necrosis [
      • Bianchi L.
      • De Groote J.
      • Desmet V.J.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • et al.
      Acute and chronic hepatitis revisited.
      ].
      Hepatic fibrosis in chronic hepatitis occurs mostly in a septal pattern, comprising so-called active and passive septa [
      • Bianchi L.
      • De Groote J.
      • Desmet V.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • et al.
      Morphological criteria in viral hepatitis.
      ]. Active septa are rich in cells; they represent extensive interface hepatitis eventually leading to portal-portal septal fibrosis [
      • Bianchi L.
      • De Groote J.
      • Desmet V.J.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • et al.
      Acute and chronic hepatitis revisited.
      ]. Passive septa carry few or no inflammatory cells, are sharply delineated, and derive from postnecrotic collapse after confluent necrosis. The mesenchymal cells responsible for fibrosis comprise portal/septal myofibroblasts, interface myofibroblasts and activated intralobular hepatic stellate cells [
      • Cassiman D.
      • Libbrecht L.
      • Desmet V.
      • Aertsen P.
      • Denef C.
      • Roskams T.
      Hepatic stellate cell/myofibroblast subpopulations in fibrotic human and rat livers.
      ]. Parenchymal regeneration is evidenced by thickening of liver cell plates (two to three cells wide), and increased numbers of bi- and tri-nucleated hepatocytes [
      • Ishak K.G.
      Pathologic features of chronic hepatitis: a review and update.
      ]. Activation of liver progenitor cells also participates in regeneration in chronic hepatitis [
      • Libbrecht L.
      • Desmet V.
      • Van Damme B.
      • Roskams T.
      Deep intralobular extension of human hepatic ‘progenitor cells’ correlates with parenchymal inflammation in chronic viral hepatitis: can ‘progenitor cells’ migrate?.
      ]. Parenchymal regeneration in between a restructuring fibrous scaffold, leads to progressive disturbance of the lobular architecture and results in cirrhosis, usually of the macronodular type [
      • Desmet V.
      Liver lesions in hepatitis B viral infection.
      ]. Necro-inflammation may continue in the cirrhotic stage (active cirrhosis) or burn out (inactive cirrhosis) [
      • Desmet V.
      Liver lesions in hepatitis B viral infection.
      ]. The common elementary lesions of chronic hepatitis constitute the base for grading and staging in chronic liver disease (see below).

      2.2 Specific features of chronic hepatitis B

      The most distinctive histological feature for identifying HBV aetiology is the ‘ground-glass hepatocyte’ [
      • Hadziyannis S.
      • Gerber M.A.
      • Vissoulis C.
      • Popper H.
      Cytoplasmic hepatitis B antigen in ‘ground-glass’ hepatocytes of carriers.
      ]. It has a finely granular, and faintly eosinophilic cytoplasm due to proliferation of the smooth endoplasmic reticulum containing accumulated hepatitis B surface antigen [
      • Yamada G.
      • Nakane P.K.
      • Hepatitis B.
      core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labelled antibody method.
      ]. Ground-glass cells are highlighted by special stains, including Shikata's orcein or aldehyde fuchsin [
      • Shikata T.
      • Uzawa T.
      • Yoshiwara N.
      • Akatsuka T.
      • Yamazaki S.
      Staining methods of Australia antigen in paraffin section - detection of cytoplasmic inclusion bodies.
      ] and Victoria blue [
      • Tanaka K.
      • Mori W.
      • Suwa K.
      Victoria blue-nuclear fast red stain for HBs antigen detection in paraffin sections.
      ]. Ground-glass hepatocytes can be specifically stained by more sensitive immunohistochemistry (see below).
      Ground-glass appearance must be differentiated from a similar outlook due to drug-induction, to cyanamide toxicity, to Lafora's disease, and to fibrinogen storage disease [
      • Callea F.
      • De Vos R.
      • Togni R.
      • Tardanico R.
      • Vanstapel M.J.
      • Desmet V.J.
      Fibrinogen inclusions in liver cells: a new type of ground-glass hepatocyte. Immune light and electron microscopic characterization.
      ]. Ground-glass inclusions from several causes may co-exist in the same patient and in the same hepatocyte [
      • Alonso-Marti C.
      • Moreno A.
      • Barat A.
      • Solera J.C.
      • Oliva H.
      Co-existence of hepatocyte ground-glass inclusions from several causes.
      ].
      A study on the frequency of characteristic features for chronic hepatitis B, C, autoimmune and cryptogenic hepatitis concluded that the respective histological patterns have low sensitivity, but high specificity and predictability [
      • Czaja A.J.
      • Carpenter H.A.
      Sensitivity, specificity, and predictability of biopsy interpretations in chronic hepatitis.
      ].

      3. Immunohistochemistry, in situ hybridization, electron and immuno-electron microscopy

      Immunohistochemical staining with specific antibodies for HBV antigens allows to specify the HBV aetiology of chronic hepatitis and the viral phase in the disease [
      • Ferrell L.D.
      • Theise N.D.
      • Scheuer P.J.
      Acute and chronic viral hepatitis.
      ,
      • Gerber M.A.
      • Thung S.N.
      The diagnostic value of immunohistochemical demonstration of hepatitis viral antigens in liver.
      ]. In situ hybridization (ISH) of viral DNA is helpful in detecting HBV infection and its topography in the infected cells. Various direct and indirect immunofluorescence and immunoperoxidase procedures can be applied on fresh frozen and paraffin embedded tissue. The sensitivity of immunoperoxidase methods can be enhanced by techniques of antigen retrieval and signal amplification like the Immunomax [
      • Merz H.
      • Malisius R.
      • Mannweiler S.
      • Zhou R.
      • Hartmann W.
      • Orscheschek K.
      • et al.
      ImmunoMax: a maximized immunohistochemical method for retrieval and enhancement of hidden antigens.
      ] and the Envision™+ system [
      • Sabattini E.
      • Bisgaard K.
      • Ascani S.
      • Poggi S.
      • Piccioli M.
      • Cessarelli C.
      • et al.
      The EnVision™+ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate™, SCA, LABL and SABC techniques.
      ].
      In acute hepatitis B, very little or no viral antigens are demonstrable [
      • Bianchi L.
      • Gudat F.
      Immunopathology of hepatitis B.
      ], except in the very early phase [
      • Edgington T.S.
      • Chisari F.V.
      Immunological aspects of hepatitis B virus infection.
      ] and according to one study in patients infected with a mutant HBV (‘silent HBV’) [
      • Uchida T.
      • Shimojima S.
      • Gotoh K.
      • Shikata T.
      • Mima S.
      Pathology of livers infected with ‘silent’ hepatitis B virus mutant.
      ].
      In chronic hepatitis B, antigen localization patterns vary. The course of chronic hepatitis B comprises three successive phases: virus tolerance (viral replication) phase, virus clearance and residual integration phase [
      • Chu C.M.
      • Karayiannis P.
      • Fowler M.J.F.
      • Monjardino J.
      • Liaw Y.F.
      • Thomas H.C.
      Natural history of chronic hepatitis B virus infection in Taiwan. Studies of hepatitis B virus DNA in serum.
      ,
      • Chen D.S.
      Natural history of chronic hepatitis B virus infection: new light on an old story.
      ,
      • Lok A.S.F.
      • McMahon B.J.
      Chronic hepatitis B.
      ].
      During the immunotolerant, viral replicative phase there is only mild hepatocellular damage and inflammation. Immunostaining reveals predominant nuclear localization of HBcAg [
      • Naoumov N.V.
      • Portmann B.C.
      • Tedder R.S.
      • Ferns B.
      • Eddleston A.L.W.F.
      • Williams R.
      Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection.
      ]. HBcAg and hepatitis B e antigen (HBeAg) generally have a coincident cellular expression [
      • Lindh M.
      • Savage K.
      • Rees J.
      • Garwood L.
      • Horal P.
      • Norkrans G.
      • et al.
      HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B.
      ], but the ratio of HBcAg to HBeAg may differ in subcellular locations. Strong cytoplasmic HBeAg is a marker of high viral replication [
      • Naoumov N.V.
      • Portmann B.C.
      • Tedder R.S.
      • Ferns B.
      • Eddleston A.L.W.F.
      • Williams R.
      Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection.
      ,
      • Lindh M.
      • Savage K.
      • Rees J.
      • Garwood L.
      • Horal P.
      • Norkrans G.
      • et al.
      HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B.
      ]. HBsAg is found in the cytoplasm of some hepatocytes and in the membrane of numerous cells in a honeycomb-like pattern [
      • Chu C.M.
      • Liaw Y.F.
      Membrane staining for hepatitis B surface antigen in hepatocytes: a sensitive and specific marker of active viral replication in hepatitis B.
      ].
      The viral clearance phase is characterized by immune elimination of virus-infected hepatocytes, seroconversion from HBeAg to anti-HBe, and reduction of viral replication [
      • Loriot M.A.
      • Marcellin P.
      • Bismuth E.
      • Martinot-Peignoux M.
      • Boyer N.
      • Degott C.
      • et al.
      Demonstration of hepatitis B virus DNA by polymerase chain reaction in the serum and the liver after spontaneous or therapeutically induced HBeAg to anti-HBe or HBsAg to anti-HBs seroconversion in patients with chronic hepatitis B.
      ]. Liver histopathology consists of more severe lesions, including confluent necrosis of variable extent [
      • Liaw Y.F.
      • Yang S.S.
      • Chen T.J.
      • Chu C.M.
      Acute exacerbation in hepatitis B e antigen – positive chronic type B hepatitis – a clinico-pathological study.
      ]. Immunostaining reveals nuclear, cytoplasmic and membranous HBcAg. Cytoplasmic HBcAg correlates predominantly with liver damage [
      • Naoumov N.V.
      • Portmann B.C.
      • Tedder R.S.
      • Ferns B.
      • Eddleston A.L.W.F.
      • Williams R.
      Detection of hepatitis B virus antigens in liver tissue. A relation to viral replication and histology in chronic hepatitis B infection.
      ,
      • Chu C.M.
      • Liaw Y.F.
      Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis.
      ] and proliferation [
      • Chu C.M.
      • Yeh C.T.
      • Sheen I.S.
      • Liaw Y.F.
      Subcellular localization of Hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B.
      ]. HBsAg shows weak cytoplasmic positivity in some hepatocytes and a membranous staining pattern [
      • Chen D.S.
      Natural history of chronic hepatitis B virus infection: new light on an old story.
      ]. HBeAg has been found localized in nucleus and cytoplasm of hepatocytes [
      • Lindh M.
      • Savage K.
      • Rees J.
      • Garwood L.
      • Horal P.
      • Norkrans G.
      • et al.
      HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B.
      ,
      • Villari D.
      • Pollicino T.
      • Spinella S.
      • Russo F.
      • Campo S.
      • Rodino G.
      • et al.
      Hepatitis Be antigen detection in formalin fixed liver biopsy specimens. A tool to investigate wild-type and e-minus variant HBV infection.
      ], and in liver cell membranes [
      • Yamada G.
      • Takaguchi K.
      • Matsueda K.
      • Nishimoto H.
      • Takahashi M.
      • Fujiki S.
      • et al.
      Immunoelectron microscopic observation of intrahepatic HBeAg in patients with chronic hepatitis B.
      ].
      Some virus-infected hepatocytes apparently escape immune elimination, resulting in persistence of viral infection and integration of viral DNA into the host genome (viral integration phase) [
      • Hadziyannis S.J.
      • Lieberman H.M.
      • Karvountzis G.G.
      • Shafritz D.A.
      Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus.
      ]. If damage during the virus clearance phase was not extensive, the liver may recover to only minimal abnormalities [
      • Su I.J.
      • Lai M.Y.
      • Hsu H.C.
      • Chen D.S.
      • Yang P.M.
      • Chuang S.M.
      • et al.
      Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection.
      ]. In case several exacerbations occurred [
      • Davis G.L.
      • Hoofnagle J.H.
      Reactivation of chronic type B hepatitis presenting as acute viral hepatitis.
      ] the liver may have developed cirrhosis [
      • Chen D.S.
      Natural history of chronic hepatitis B virus infection: new light on an old story.
      ]. Active replication of HBV has ceased, but HBsAg is produced by hepatocytes that contain integrated HBV-DNA [
      • Shafritz D.A.
      • Hadziyannis S.J.
      Molecular pathobiology of persistent hepatitis B virus infection in relation to chronic liver disease and primary hepatocellular carcinoma.
      ]. HBsAg accumulates in clusters of hepatocytes. HBcAg is usually undetectable [
      • Martinot-Peignoux M.
      • Boyer N.
      • Colombat M.
      • Akremi R.
      • Pham B.N.
      • Ollivier S.
      • et al.
      Serum hepatitis B virus DNA levels and liver histology in inactive HBsAg carriers.
      ]. Mild inflammation may persist for some time, but substantial necro-inflammation should alert for a possible superinfection with another virus [
      • Perillo R.P.
      • Brunt E.M.
      Hepatic histologic and immunohistochemical changes in chronic hepatitis B after prolonged clearance of HBeAg and hepatitis B surface antigen.
      ].
      Some patients show ongoing active disease after HBeAg seroconversion, due to persistence of a precore stop mutant HBV with deficient HBeAg synthesis [
      • Naoumov N.V.
      • Schneider R.
      • Grötzinger T.
      • et al.
      Precore mutant hepatitis B virus infection and liver disease.
      ] or other mutations [
      • Naoumov N.V.
      • Eddleston A.L.W.F.
      Host immune response and variations in the virus genome: pathogenesis of liver damage caused by hepatitis B virus.
      ]. Immunostaining in such cases reveals cytoplasmic HBcAg [
      • Naoumov N.V.
      • Schneider R.
      • Grötzinger T.
      • et al.
      Precore mutant hepatitis B virus infection and liver disease.
      ,
      • Park Y.N.
      • Han K.H.
      • Kim K.S.
      • Chung J.P.
      • Kim S.
      • Park C.
      Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants.
      ,
      • Bonino F.
      • Rosina F.
      • Rizzetto M.
      • Rizzi R.
      • Chiaberge E.
      • Tardanico R.
      • et al.
      Chronic hepatitis in HBsAg carriers with serum HBV-DNA and anti-HBe.
      ] and precore peptide [
      • Dienes H.P.
      • Gerken G.
      • Goergen B.
      • Heermann K.
      • Gerlich W.
      • Meyer zum Büschenfelde K.H.
      Analysis of the precore DNA sequence and detection of precore antigen in liver specimens from patients with anti-hepatitis-B-e positive chronic hepatitis.
      ].
      HBxAg has been visualized in the nucleus and cytoplasm of hepatocytes, more widely present than HBsAg or HBcAg, perhaps representing a more sensitive immunohistochemical test for HBV infection [
      • Wang W.L.
      • London W.T.
      • Lega L.
      • Feitelson M.A.
      HB×Ag in the liver from carrier patients with chronic hepatitis and cirrhosis.
      ]. Its presence in hepatocellular carcinoma cells in HBV+ patients supports its involvement in hepatocarcinogenesis [
      • Wang W.L.
      • London W.T.
      • Feitelson M.A.
      Hepatitits B×antigen in hepatitis B virus carrier patients with liver cancer.
      ].
      ISH for detection of HBV sequences has been performed with radioactively and with chemically labeled probes, the former yielding higher sensitivity, the latter better resolution. ISH identified the hepatocyte cytoplasm as the site of HBV replication [
      • Negro F.
      • Pacchioni D.
      • Monardini A.
      • Bussolati G.
      • Bonino F.
      In situ hybridization in viral hepatitis.
      ,
      • Naoumov N.V.
      • Daniels H.M.
      • Davison F.
      • Eddleston A.L.W.F.
      • Alexander G.J.M.
      • Williams R.
      Identification of hepatitis B virus-DNA in the liver by in situ hybridization using a biotinylated probe. Relation to HBcAg expression and histology.
      ]. Combination of ISH with immunostains for HBV antigens revealed that HBV DNA is present in numerous antigen negative cells, but co-localizes mainly with cytoplasmic and less with nuclear HBcAg [
      • Naoumov N.V.
      • Daniels H.M.
      • Davison F.
      • Eddleston A.L.W.F.
      • Alexander G.J.M.
      • Williams R.
      Identification of hepatitis B virus-DNA in the liver by in situ hybridization using a biotinylated probe. Relation to HBcAg expression and histology.
      ,
      • Lau J.Y.N.
      • Naoumov N.V.
      • Alexander G.J.M.
      • Williams R.
      Rapid detection of hepatitis B virus DNA in liver tissue by in situ hybridization and its combination with immunohistochemistry for simultaneous detection of HBV antigens.
      ]. Hepatocytes with cytoplasmic HBsAg accumulation are not virus replicating [
      • Bianchi L.
      • Gudat F.
      Chronic hepatitis.
      ].
      In electron microscopy, HBc particles appear as spherical structures, 24–27 nm in diameter, in the nucleoplasm, and in the hyaloplasm between hepatocellular organelles [
      • Bianchi L.
      • Gudat F.
      Chronic hepatitis.
      ].
      HBsAg appears as filaments within the cisternae of the endoplasmic reticulum [
      • Bianchi L.
      • Gudat F.
      Chronic hepatitis.
      ]. Ground-glass hepatocytes exhibit a marked hyperplasia of the endoplasmic reticulum which dislocates the cytoplasmic organelles to the cell periphery. Within the cisternae, there are typical filaments giving a positive reaction for HBsAg and pre-S by immuno-electron microscopy [
      • Yamada G.
      • Nakane P.K.
      • Hepatitis B.
      core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labelled antibody method.
      ,
      • Bianchi L.
      • Gudat F.
      Chronic hepatitis.
      ].

      4. Grading and staging of chronic viral hepatitis B: semiquantitative scoring

      The old classification of chronic hepatitis [
      • De Groote J.
      • Desmet V.J.
      • Gedigk P.
      • Korb G.
      • Popper H.
      • Poulsen H.
      • et al.
      A classification of chronic hepatitis.
      ] was essentially a rough grading system, distinguishing milder from more severe variants of disease. Progress in aetiological insight and treatment options necessitated a revised classification.
      The new classification of chronic hepatitis is based on aetiology (viral, autoimmune, drug-induced and cryptogenic), also taking into account the histological grade of disease activity and the stage of evolution in terms of fibrosis and architectural derangement (cirrhosis) [
      • Desmet V.J.
      • Gerber M.
      • Hoofnagle J.H.
      • Manns M.
      • Scheuer P.J.
      Classification of chronic hepatitis: diagnosis, grading and staging.
      ,
      International Working Party
      Terminology of chronic hepatitis, hepatic allograft rejection, and nodular lesions of the liver: summary of recommendations developed by an international working party, supported by the World Congresses of Gastroenterology, Los Angeles, 1994.
      ]. Grading and staging is done in (preferably standardized) verbal descriptions, for instance: mild, moderate and severe chronic hepatitis as grades; and mild, moderate, severe fibrosis and cirrhosis as stages [
      • Desmet V.J.
      • Gerber M.
      • Hoofnagle J.H.
      • Manns M.
      • Scheuer P.J.
      Classification of chronic hepatitis: diagnosis, grading and staging.
      ].
      Several semiquantitative scoring systems have been proposed, in which numerical scores are assigned to different grades and stages of chronic hepatitis. These methods are primarily indicated in the context of therapeutic trials or research projects; they are not intended to replace verbal reports in routine diagnostic practice [
      • Desmet V.J.
      • Gerber M.
      • Hoofnagle J.H.
      • Manns M.
      • Scheuer P.J.
      Classification of chronic hepatitis: diagnosis, grading and staging.
      ,
      • Hübscher S.G.
      Histological grading and staging in chronic hepatitis: clinical applications and problems.
      ].
      The first successful scoring system specifically designed for chronic hepatitis is widely known as the Knodell histological activity index (HAI) [
      • Knodell R.G.
      • Ishak K.G.
      • Black W.C.
      • Chen T.S.
      • Craig R.
      • Kaplowitz N.
      • et al.
      Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis.
      ]. Scores for the individual lesion categories are added to obtain an overall ‘histological activity index’. This broadly used system is subject to a number of criticisms, the most important being that scores for necro-inflammation (grading) are added to those for fibrosis (staging) [
      • Desmet V.J.
      • Gerber M.
      • Hoofnagle J.H.
      • Manns M.
      • Scheuer P.J.
      Classification of chronic hepatitis: diagnosis, grading and staging.
      ,
      French METAVIR Cooperative Study Group
      Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C.
      ].
      Subsequent scoring systems have taken these deficiencies into account and comprise more simple and more complex prescriptions. Several simple and easily applicable systems are available [
      • Scheuer P.J.
      Classification of chronic viral hepatitis: a need for reassessment.
      ,
      • Batts K.P.
      • Ludwig J.
      Chronic hepatitis. An update on terminology and reporting.
      ]. The French METAVIR group devised a simple algorithm for standardized grading [

      Bedossa P, Poynard T, METAVIR Cooperative Study Group. An algorithm for the grading of activity in chronic hepatitis C. Hepatology 1996;24:289–293.

      ], and an assessment of fibrosis (staging) in five categories [
      • Poynard T.
      • Bedossa P.
      • Opolon P.
      Natural history of liver fibrosis progression in patients with chronic hepatitis C.
      ]. Simpler scoring systems provide less information but tend to be more reproducible than complex ones [
      • Goldin R.D.
      • Goldin J.G.
      • Burt A.D.
      • Dhillon P.A.
      • Hubscher S.
      • Wyatt J.
      Intra-observer and inter-observer variation in the histopathological assessment of chronic viral hepatitis.
      ].
      An updated version of the Knodell HAI [
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • Denk H.
      • et al.
      Histological grading and staging of chronic hepatitis.
      ] separates grading from staging and is more detailed to provide more information. It assures adequate interobserver reliability [
      • Westin J.
      • Lagging L.M.
      • Westjal R.
      • Norkrans G.
      • Dhillon P.
      Interobserver study of liver histopathology using the Ishak score in patients with chronic hepatitis C virus infection.
      ].
      Several problems are inherent to all scoring systems available: use of arbitrary scores that are not mathematically valid, observer variation, sampling variability and aetiological diversity [
      • Hübscher S.G.
      Histological grading and staging in chronic hepatitis: clinical applications and problems.
      ]. Accuracy and consistency of scoring procedures can be improved by previous planning and agreement between clinician(s) and pathologist(s), by excluding biopsy specimens of inadequate size and quality, by agreement on definition of histological criteria at the start of the study, by preferable use of dual observers, by avoiding long time intervals between scoring of biopsies, and by checking intra- and interobserver variation [
      • Ishak K.
      • Baptista A.
      • Bianchi L.
      • Callea F.
      • De Groote J.
      • Gudat F.
      • Denk H.
      • et al.
      Histological grading and staging of chronic hepatitis.
      ,
      • Scheuer P.J.
      • Standish R.A.
      • Dhillon A.P.
      Scoring of chronic hepatitis.
      ]. In general, staging of fibrosis and architectural changes has proved to be more reproducible than grading of necro-inflammatory lesions [
      French METAVIR Cooperative Study Group
      Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C.
      ]. Semiquantitative scoring of liver biopsies has been extensively applied in clinical trials for new treatments of chronic hepatitis B, C and D [
      • Dusheiko G.M.
      New treatments for chronic viral hepatitis B and C.
      ].
      Morphometry has been used as well for quantitation, mostly for assessment of fibrosis [
      • Chevallier M.
      • Guerret S.
      • Chossegros P.
      • Gerard F.
      • Grimaud J.A.
      A histological semiquantitative scoring system for evaluation of hepatic fibrosis in needle liver biopsy specimens: comparison with morphometric studies.
      ,
      • Pilette C.
      • Rousselet M.C.
      • Bedossa P.
      • Chappard D.
      • Oberti F.
      • Rifflet H.
      • et al.
      Histopathological evaluation of liver fibrosis: quantitative image analysis versus semi-quantitative scores. Comparison with serum markers.
      ]. Image analysis based automated quantification of liver fibrosis was claimed to be a sensitive, precise, objective and reproducible method for quantification, thus supplementing scoring systems that are more based on distribution patterns of fibrosis [
      • Masseroli M.
      • Caballero T.
      • O'Valle F.
      • Del Moral R.M.G.
      • Perez-Milena A.
      • Del Moral R.G.
      Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semiquantitative indexes of fibrosis.
      ]. The potential usefulness of a mathematical scoring system based on fractal geometry for quantifying irregular patterns of fibrosis as observed in chronic hepatitis was recently addressed [
      • Dioguardi N.
      • Grizzi F.
      • Bossi P.
      • Roncalli M.
      Fractal and spectral dimension analysis of liver fibrosis in needle biopsy specimens.
      ,
      • Moal F.
      • Chappard D.
      • Wang J.
      • Vuillemin E.
      • Michalak-Provost S.
      • Rousselet M.C.
      • et al.
      Fractal dimension can distinguish models and pharmacologic changes in liver fibrosis in rats.
      ].

      5. Precancerous lesions

      Both HBV and HCV infections may lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) [
      • Okuda K.
      Hepatocellular carcinoma.
      ,
      • Chisari F.V.
      Viruses, immunity and cancer: lessons from hepatitis B.
      ]. Some cellular changes and nodular lesions are considered premalignant or precursors of HCC. Recognition and reporting of such lesions is important for adequate patient surveillance. Liver cell dysplasia (LCD), which may be of the large cell type [
      • Anthony P.P.
      • Vogel C.L.
      • Barker L.F.
      Liver cell dysplasia: a premalignant condition.
      ] or of the small cell type [
      • Watanabe S.
      • Okita K.
      • Harada T.
      • Kodama T.
      • Numa Y.
      • Takemoto T.
      • et al.
      Morphologic studies of the liver cell dysplasia.
      ] belongs to this type of change.
      Large cell LCD in biopsies of patients with chronic hepatitis B (or C) is an independent risk factor for the development of HCC [
      • Libbrecht L.
      • Craninx M.
      • Nevens F.
      • Desmet V.
      • Roskams T.
      Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with non-cirrhotic and cirrhotic chronic viral hepatitis.
      ]. Small cell LCD might originate from hepatic progenitor cells [
      • Libbrecht L.
      • Desmet V.
      • Van Damme B.
      • Roskams T.
      The immunohistochemical phenotype of dysplastic foci in human liver: correlation with putative progenitor cells.
      ] and may represent an early step in carcinogenesis [
      • Libbrecht L.
      • Craninx M.
      • Nevens F.
      • Desmet V.
      • Roskams T.
      Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with non-cirrhotic and cirrhotic chronic viral hepatitis.
      ]. Clusters of LCD measuring less than 1 mm in diameter are termed ‘dysplastic foci’. Lesions measuring more than 1mm in diameter are designated as nodules [
      International Working Party
      Terminology of nodular hepatocellular lesions.
      ].
      A macroregenerative nodule (measuring 0.8 cm or more) is particularly common in macronodular cirrhosis. Histologically it shows hyperplastic liver parenchyma but no cellular atypia nor disorder in muralia arrangement. Dysplastic nodules do display atypical features, but not severe enough to qualify for frank HCC. Dysplastic nodules are subclassified as low or high grade, and considered distinct stages in the multistep process of hepatocarcinogenesis [
      • Kojiro M.
      Premalignant lesions of hepatocellular carcinoma: pathologic viewpoint.
      ]. Foci and nodules should be carefully identified and reported [
      ,
      • Le Bail B.
      • Bernard P.H.
      • Carles J.
      • Balabaud C.
      • Bioulac-Sage P.
      Prevalence of liver cell dysplasia and association with HCC in a series of 100 cirrhotic liver explants.
      ].

      6. Special cases of chronic viral hepatitis B

      Short reference is made to several situations in which liver tissue examination may be contributory.

      6.1 Coinfection with hepatitis D virus

      Coinfection with hepatitis D virus (HDV, delta agent) alters the course of acute hepatitis B, favours chronic evolution and enhances severity of disease [
      • Verme G.
      • Amoroso P.
      • Lettieri G.
      • Pierri P.
      • David E.
      • Sessa F.
      • et al.
      A histologic study of hepatitis delta virus liver disease.
      ]. Microvesicular steatosis of hepatocytes (morula cells) was a notable feature in an outbreak of HDV infection in Venezuelan Indians [
      • Buitrago B.
      • Popper H.
      • Hadler S.C.
      • Thung S.N.
      • Gerber M.A.
      • Purcell R.H.
      Specific histologic features of Santa Marta hepatitis. A severe form of hepatitis delta-virus infection in northern South America.
      ,
      • Lefkowitch J.H.
      • Goldstein H.
      • Yatto R.
      • Gerber M.A.
      Cytopathic liver injury in acute delta virus hepatitis.
      ]. Specific immunostaining allows to confirm HDV aetiology. HDAg is detected in hepatocyte nuclei [
      • Di Bisceglie A.M.
      • Negro F.
      Diagnosis of hepatitis delta virus infection.
      ] and in the cytoplasm and plasmalemma of hepatocytes [
      • Kojima T.
      • Callea F.
      • Desmyter J.
      • Sakurai I.
      • Desmet V.J.
      Immuno-light and electron microscopic features of chronic hepatitis D.
      ]. Double immunostaining reveals separate expression of HDAg versus HBsAg and HBcAg; co-expression in the same cell is found though rarely with HBcAg [
      • Kojima T.
      • Callea F.
      • Desmyter J.
      • Sakurai I.
      • Desmet V.J.
      Immuno-light and electron microscopic features of chronic hepatitis D.
      ,
      • Ryley N.G.
      • Heryet A.R.
      • Goldin R.
      • Monjardino J.
      • Saldanha J.
      • Fleming K.A.
      Co-expression of markers for hepatitis delta and hepatitis B viruses in human liver.
      ].
      ISH for HDV RNA may be more sensitive than immunostaining for HDAg [
      • Negro F.
      • Pacchioni D.
      • Monardini A.
      • Bussolati G.
      • Bonino F.
      In situ hybridization in viral hepatitis.
      ,
      • Negro F.
      • Bonino F.
      • Di Bisceglie A.
      • Hoofnagle J.H.
      • Gerin J.L.
      Intrahepatic markers of hepatitis delta virus infection. A study by in situ hybridization.
      ,
      • Pacchioni D.
      • Negro F.
      • Chiaberge E.
      • Rizzetto M.
      • Bonino F.
      • Bussolati G.
      Detection of hepatitis Delta virus RNA by a nonradioactive in situ hybridization procedure.
      ].

      6.2 Recurrent HBV infection post transplantation

      Recurrent HBV infection post transplantation may either show the typical features observed in non-transplanted liver, or, more rarely, atypical patterns of liver damage. Atypical patterns include hepatocyte ballooning, fatty change and a distinctive cholestatic syndrome, which also may occur in combination [
      • Davies S.E.
      • Portmann B.C.
      • O’Grady J.G.
      • Aldis P.M.
      • Chaggar K.
      • Alexander G.J.M.
      • et al.
      Hepatic histological findings after transplantation for chronic hepatitis B virus infection, including a unique pattern of fibrosing cholestatic hepatitis.
      ]. Hepatocyte ballooning typically occurs without significant inflammation, but shows high level viral replication reflected in diffuse nuclear and cytoplasmic immunostaining for HBcAg and HBeAg [
      • Harrison R.F.
      • Davies M.H.
      • Goldin R.D.
      • Hübscher S.G.
      Recurrent hepatitis B in allografts: a distinctive form of rapidly developing cirrhosis.
      ]. Ballooning associated with steatosis may explain the term ‘steatoviral hepatitis’ [
      • Phillips M.J.
      • Cameron R.
      • Flowers M.A.
      • Blendis L.M.
      • Greig P.D.
      • Wanless I.
      Post transplant recurrent hepatitis B viral liver disease.
      ].
      The terms ‘fibrosing cholestatic hepatitis’ (fibrosing cytolytic hepatitis, fibroviral hepatitis) refer to a distinctive pattern of injury associated with rapidly progressive graft failure [
      • Davies S.E.
      • Portmann B.C.
      • O’Grady J.G.
      • Aldis P.M.
      • Chaggar K.
      • Alexander G.J.M.
      • et al.
      Hepatic histological findings after transplantation for chronic hepatitis B virus infection, including a unique pattern of fibrosing cholestatic hepatitis.
      ,
      • Harrison R.F.
      • Davies M.H.
      • Goldin R.D.
      • Hübscher S.G.
      Recurrent hepatitis B in allografts: a distinctive form of rapidly developing cirrhosis.
      ,
      • Phillips M.J.
      • Cameron R.
      • Flowers M.A.
      • Blendis L.M.
      • Greig P.D.
      • Wanless I.
      Post transplant recurrent hepatitis B viral liver disease.
      ,
      • Hübscher S.G.
      • Portmann B.C.
      Transplantation pathology.
      ]. The histological pattern comprises periportal fibrosis surrounding ductular structures, prominent hepatocyte ballooning, bilirubinostasis, and mild or absent inflammation. Immunostaining reveals markedly positive cytoplasmic HBcAg. This stage may be followed by extensive postnecrotic collapse, fibrosis and ductular reaction.
      This lesional pattern was also reported in other immunodeficiency states, including HIV infection [
      • Fang J.
      • Wright T.
      • Lau J.
      Fibrosing cholestatic hepatitis in a patient with HIV and hepatitis B.
      ] and following renal transplantation [
      • Booth J.C.L.
      • Goldin R.D.
      • Brown J.L.
      • Karayiannis P.
      • Thomas H.C.
      Fibrosing cholestatic hepatitis in a renal transplant recipient associated with the hepatitis B virus precore mutant.
      ].

      6.3 Association of HBV with other viral infection

      Dual and triple infections with HBV, HCV and HDV occur. Histopathologically, there are no specific findings to suggest the possibility of multiple infections [
      • Colombari R.
      • Dhillon A.P.
      • Piazzola E.
      • Tomezzoli A.A.
      • Angelini G.P.
      • Capra F.
      • et al.
      Chronic hepatitis in multiple virus infection: histopathological evaluation.
      ]. Immunohistochemical analysis may reveal suppression of HBsAg and HBcAg by simultaneous HCV infection [
      • Guido M.
      • Thung S.N.
      • Fattovich G.
      • Cusinato R.
      • Leandro G.
      • Cecchetto A.
      • et al.
      Intrahepatic expression of hepatitis B virus antigens: effect of hepatitis C virus infection.
      ]. HCV infection is suspected on the basis of characteristic histological features [
      • Goodman Z.D.
      • Ishak K.G.
      Histopathology of hepatitis C virus infection.
      ] and positive immunostaining with specific antibody [
      • Verslype C.
      • Nevens F.
      • Sinelli N.
      • Clarysse C.
      • Pirenne J.
      • Depla E.
      • et al.
      Hepatic immunohistochemical staining with a monoclonal antibody against HCV-E2 to evaluate antiviral therapy and reinfection of liver grafts in hepatitis C viral infection.
      ]. Co-infection of HBV with cytomegalovirus (CMV) infection can be identified by typical nuclear inclusions, abnormal basophilic granular cytoplasm, microabcesses, and immunohistochemical staining for CMV antigen [
      ]. Co-infection of HBV with HIV usually results in diminished histological activity [
      • Goldin R.D.
      • Fish D.E.
      • Hay A.
      • Waters J.A.
      • McGarvey M.J.
      • Main J.
      Histological and immunohistochemical study of hepatitis B virus in human immunodeficiency virus infection.
      ], although more severe activity was also reported [
      • Housset C.
      • Pol S.
      • Carnot F.
      • Dubois F.
      • Nalpas B.
      • Housset B.
      • et al.
      Interactions between human immunodeficiency virus-1, hepatitis delta virus and hepatitis B virus infections in 260 chronic carriers of hepatitis B virus.
      ]. Immunostaining revealed more diffuse staining for HBV and HDV antigens in tissue specimens of HIV coinfected patients [
      • Goldin R.D.
      • Fish D.E.
      • Hay A.
      • Waters J.A.
      • McGarvey M.J.
      • Main J.
      Histological and immunohistochemical study of hepatitis B virus in human immunodeficiency virus infection.
      ].

      6.4 Association of HBV with concomitant liver disease

      Histopathological study often reveals clinically unsuspected concomitant liver disease, such as alpha-1-antitrypsin deficiency, alcoholic liver disease, primary sclerosing cholangitis, haemochromatosis, amongst others [
      • Desmet V.
      • Fevery J.
      Liver biopsy.
      ].

      7. Special analyzes

      Inventive investigations in different ways of tissue examination are applied to broaden the insight in pathophysiology. Examples include: semiquantitative evaluation of activated hepatic stellate cells [
      • Kweon Y.O.
      • Goodman Z.D.
      • Dienstag J.L.
      • Schiff E.R.
      • Brown R.A.
      • Burkhardt E.
      • et al.
      Decreasing fibrogenesis: an immunohistochemical study of paired liver biopsies following lamivudine therapy for chronic hepatitis B.
      ]; immunohistochemical studies on lymphocyte subsets, antigen presenting cells, adhesion molecules and apoptosis markers [
      • Huang S.N.
      • C C.T.
      • Tsai S.L.
      • Liaw Y.F.
      Histopathology and pathobiology of hepatotropic virus-induced liver injury.
      ,
      • Moreno-Otero R.
      • Gracia-Buey L.
      • Mateos F.
      • Garcia-Monzon C.
      Pathogenesis of chronic viral hepatitis: lessons from immunohistochemistry.
      ]; study of cytokines [
      • Maher J.H.
      Cytokines: overview.
      ]; elution of liver infiltrating lymphocytes [
      • Koziel M.J.
      Cytokines in viral hepatitis.
      ]; microdissection of sublobular regions of high and low degree of liver damage and their relationship to HBV variants [
      • Kojima N.
      • Horiike N.
      • Michitaka K.
      • Onji M.
      In situ detection of mutated hepatitis B virus in microdissected, formalin-fixed liver tissue from patients with chronic hepatitis B.
      ]; and cDNA microarray based analysis of differences in gene expression between chronic hepatitis B and C [
      • Honda M.
      • Kaneko S.
      • Kawai H.
      • Shirota Y.
      • Kobayashi K.
      Differential gene expression between chronic hepatitis B and C hepatic lesion.
      ].
      To meet future requirements, pathology evaluates appropriate techniques of tissue preparation for high throughput molecular analysis [
      • Gillespie J.W.
      • Best C.J.
      • Bichsel V.E.
      • Cole K.A.
      • Greenhut S.F.
      • Hewitt S.M.
      • et al.
      Technical advance. Evaluation of non-formalin tissue fixation for molecular profiling studies.
      ].

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