CLINICAL PATHOLOGY. Optimizing Diagnosis From the Medical Liver Biopsy. Value of Liver Biopsy in Diagnosis. Clinical Scenario

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5: CLINICAL PATHOLOGY Optimizing Diagnosis From the Medical Liver Biopsy ALBERT J. CZAJA* and HERSCHEL A. CARPENTER *Division of Gastroenterology and Hepatology and the Department of Pathology and Laboratory Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota The histologic assessment of acute and chronic liver disease is based on an accurate description of the pattern of injury within the tissue specimen. Interpretation of the injury pattern requires review of the associated clinical and laboratory features. The morphologic interpretation achieves relevance only if it satisfactorily explains the clinical syndrome. Clinical characterization of the liver disease as having a hepatitic or cholestatic nature is useful in focusing the histologic assessment. The principal histologic patterns have hepatitic, steatotic, biliary, granulomatous, vascular, and metabolic designations, and mixed clinical and histologic patterns are possible. The goal of this review is to optimize the yield from the liver biopsy examination by indicating the importance of a systematic analysis of the tissue specimen and the crucial need for correlating the clinical and histologic patterns of liver injury. Meaningful histologic interpretations reflect the collaborative effort of the pathologist and the clinical physician. Neoplastic changes typically are cytologic transformations rather than injury patterns, and they are not discussed here. A Clinical Scenario 38-year-old woman noticed decreased stamina over a 6-week period. She maintained a full schedule of activities as a real estate agent, but retired to bed shortly after work each day. She noted dark urine after the onset of fatigue. She also experienced a reduced appetite, mild generalized muscle ache, and scleral icterus. She had no risk factors for a viral-related, drug-induced, or toxic hepatitis. On examination, the body mass index was 21.8 kg/m 2. There were no cutaneous stigmata of chronic liver disease, hepatosplenomegaly, or shifting dullness. Laboratory studies disclosed a serum aspartate aminotransferase level of 492 U/L (normal, 31 U/L), an alanine aminotransferase level of 687 U/L (normal, 29 U/L), an alkaline phosphatase level of 185 U/L (normal, 100 U/L), and a bilirubin concentration of 4.6 mg/dl. The serum albumin level was 4.1 g/dl, and the international normalized ratio was 1.1. Serologic assays for viral infections were negative, and the serum ceruloplasmin level was normal. The serum -globulin level was 3.2 g/dl (normal, 1.6 g/dl), and the immunoglobulin G level was 4300 mg/dl (normal, 1500 mg/dl). Titers were 1:320 for antinuclear antibodies and 1:640 for smooth muscle antibodies. The liver biopsy specimen showed severe interface hepatitis (Figure 1) with dense portal plasma cell infiltration (Figure 2). The diagnosis of autoimmune hepatitis with acute onset was made, and treatment with prednisone and azathioprine was started. After 2 weeks, she felt better; the serum bilirubin level was normal; and the serum aspartate and alanine aminotransferase levels were improved. Value of Liver Biopsy in Diagnosis Unless a causative agent (virus, fungus, or bacillus) is identified within the liver specimen, the principal value of the biopsy tissue is to identify a pattern of injury from which diagnostic possibilities can be considered, assessed, validated, or discarded. 1,2 Within each broadly characterized histologic pattern, there are multiple variations, and each can have its own implication. Furthermore, the liver may be affected by more than one disease process, and the clinical and histologic patterns may be mixed or inconsistent with any one diagnosis. The differentiation of concurrent disorders and the interpretation of atypical findings require a continuous information exchange between the clinician and the pathologist. Optimal recognition of the histologic pattern requires characterization of the clinical syndrome, formulation and communication of the clinical question, procurement of an adequate tissue sample, and disciplined analytic assessment of the tissue specimen. Characterization of the Clinical Syndrome Characterization of the clinical syndrome as hepatitic, cholestatic, or mixed enhances the histologic examination. A serum aspartate or alanine aminotransferase increase that is in excess of the serum alkaline phosphatase increase reflects mainly liver cell injury, and it connotes a predominantly hepatitic process. 3 The serum alkaline phosphatase level is increased commonly in hepatitic processes, but it typically is less than 2-fold the upper limit of normal. 4,5 Conversely, a serum alkaline phosphatase increase greater than the serum aspartate and alanine aminotransferase increases reflects mainly bile duct injury, obstruction, or dysfunction, and it connotes a cholestatic process. 3 The serum bilirubin level can be increased abnormally in either a hepatitic or cholestatic disorder, and its level is not distinctive. When the serum aminotransferase levels are increased markedly and the serum alkaline phosphatase level exceeds 2-fold the upper limit of normal, then the possibility of a mixed or overlap syndrome must be raised. 5 Abbreviations used in this paper: CMV, cytomegalovirus; EBV, Epstein Barr virus by the AGA Institute /07/$32.00 doi: /j.cgh

2 August 2007 OPTIMIZING LIVER BIOPSY DIAGNOSIS 899 Figure 1. Interface hepatitis. Lymphocytic infiltrates extend from the portal tracts into acinar tissue with destruction of the limiting plate. Findings are consistent with autoimmune hepatitis, drug reaction, or viral infection. Characterization of the Optimal Liver Specimen The liver tissue specimen must be of adequate size to minimize sampling error and intraobserver variation. Cirrhosis is particularly difficult to sample reliably because of the irregular distribution of fibrosis within the liver and the frequent inability of the needle biopsy sample to show all relevant features. 6,7 A fragmented specimen, a fibrous band, collapsed stroma, or a nodular change may suggest cirrhosis, but the secure diagnosis requires the demonstration of fibrosis and a complete regenerative nodule. 8 Regenerating hepatocytes that expand against an encircling fibrous scar and produce a smooth and regular interface constitute a regenerative nodule. Multiple liver samples obtained from different sites during the same procedure will confirm cirrhosis in all specimens in only 33% of instances, 9 and intraobserver consistency in making the same diagnosis in the same tissue specimen will range from 50% to 78%, depending on the observer s adherence to predefined criteria for the diagnosis Reticulin and trichrome stains can facilitate the distinction between fibrosis and stromal collapse, and they may enhance the recognition of regenerative nodules. The ideal biopsy specimen for assessing the pattern of injury, grade of inflammation, and stage of fibrosis is 2.5 cm long and 1.4 mm wide, and it contains 6 8 portal tracts Reductions in the length of the tissue core are associated with erroneous reductions in the grade and stage of the liver disease. 13 Although it generally is believed that the bigger the biopsy specimen the better, the ideal specimen may differ among the various liver diseases and the information desired. 15 Systematic Assessment of Inflammatory Activity and Fibrosis The most effective approach to diagnosis is to first examine the tissue specimen without knowledge of the clinical and laboratory findings, followed by a second fully informed analysis. This dual approach provides an initial analysis that is free from bias. The second clinically focused examination may compensate for a less-than-optimal biopsy specimen by intensifying the histologic search, especially for a process known to have an irregular distribution within the liver or subtle morphologic features. The systematic grading and staging of the liver biopsy specimen also enhances the value of the histologic examination. 18 The severity of inflammatory activity and the degree of fibrosis in the tissue specimen are analyzed in accordance with codified criteria. These scores are also useful when compared with other scores at different times in the same patient to evaluate disease behavior. 19 The Ishak et al 20 system and the Batts and Ludwig 21 system are useful in assessing the activity and fibrosis of chronic hepatitis (Table 1). The METAVIR (name of French Cooperative Study Group) system has been applied mainly to evaluate the inflammatory activity of chronic hepatitis C 22 ; the Scheuer 23 system and the Ludwig et al 24 system have been used in assessing primary biliary cirrhosis; and the Brunt et al 25 system and its modifications 26 have been applied to nonalcoholic fatty liver disease (Table 1). Each system ensures uniformity of assessment, evaluation of all relevant aspects of the tissue specimen, and a means to compare different specimens from the same patient. Hepatitic Pattern of Liver Injury The hepatitic histologic pattern mainly reflects inflammation and injury to hepatocytes, and it commonly denotes a viral, drug, or immune reaction (Table 2) Mononuclear cell infiltration of the portal tracts and acinar tissue may be accompanied by destruction of the limiting plate of the portal tract and manifestations of interface hepatitis (Figure 1). Hepatitis viruses, hypersensitivity drug reactions, and autoimmune hepatitis are associated commonly with this pattern, 1 but it also is associated with Wilson disease, 27,28 and celiac disease. 29 Characterizing the Inflammatory Infiltrate An etiologic diagnosis can be suggested by variations in cellular reactions within the hepatitic pattern. A dense plasma Figure 2. Portal plasma cell infiltration. Plasma cells, characterized by a cytoplasmic halo around the nucleus, are present commonly in autoimmune hepatitis, but they also occur in chronic hepatitis B and C infections and drug reactions.

3 900 CZAJA AND CARPENTER CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 8 Table 1. Scoring Systems Proposed for Histologic Evaluations Chronic hepatitis Grading Periportal inflammation Portal inflammation Confluent necrosis Focal necrosis Staging fibrosis Ishak Batts and Ludwig METAVIR Lobular necrosis, 0 2 Histologic activity, 0 3 PBC Stage 1 Stage 2 Stage 3 Stage 4 Scheuer 23 Florid duct lesion Ductular proliferation Scarring Cirrhosis Ludwig 24 Portal hepatitis Periportal hepatitis Bridging necrosis or fibrosis Cirrhosis NAFLD Steatosis Ballooning Lobular inflammation Portal inflammation Fibrosis Brunt Minimal-marked CRN NOTE: Text in bold to clearly identify diseases. CRN, Collaborative Research Network; NAFLD, nonalcoholic fatty liver disease; PBC, primary biliary cirrhosis. cell infiltration suggests autoimmune hepatitis (Figure 2) or infections with the hepatitis viruses and other infectious agents. Portal lymphoid aggregates with mild acinar inflammation and minimal steatosis suggest chronic hepatitis C virus infection (Figure 3) or other viral infections when the disease is relatively inactive. The histologic manifestations associated with individual etiologic agents may vary depending on inflammatory activity, and chronic hepatitis C may be indistinguishable from other forms of an active hepatitis. Specific Infectious Agents Specific microbial agents may induce characteristic cellular changes in infected cells, such as the hepatitis B virus (ground-glass hepatocytes) (Figure 4), or specific morphologic features, such as with cytomegalovirus (Figure 5), herpes simplex or zoster virus (Figure 6), and adenovirus. Special histochemical stains can indicate the presence of a microbial agent, and techniques, such as immunohistochemical stains, in situ hybridization, and polymerase chain reaction, can identify the specific microbial agent. Serologic tests are valuable for the diagnosis of common and uncommon infections, and serum analysis by polymerase chain reaction can distinguish active from past viral infection. Distribution and Specific Types of Lesions in the Hepatic Lobule Inflammatory cells distributed in portal tracts and sinusoids in the absence of liver cell injury or liver plate alteration (Figure 7) typically reflect an immune response to systemic infection, such as the Epstein Barr virus, or a benign or malignant lymphoproliferative disorder (Table 2). Hepatocyte necrosis in an acinar zone constitutes zonal necrosis, and it can involve the periportal (acinar zone 1), midzonal (acinar zone 2), or centrilobular (acinar zone 3) areas. Acinar zone 3 necrosis is the most commonly encountered and results from ischemia, toxins, or the direct effects of drugs or their metabolites (Table 2). Zonal injury contrasts with random, multifocal, parenchymal necroses that suggest a viral infection (eg, herpes simplex, herpes zoster, or adenovirus) (Figure 8). Multinucleated hepatocytes (Figure 9) are liver cells that have fused or failed to divide after nuclear division Hepatocytes with 2 or 3 nuclei are not uncommon or pathologic. With 8 10 nuclei in giant hepatocytes, neonatal hepatitis or syncytial giant-cell hepatitis in older children and adults should be considered (Table 2). These changes in children represent the nonspecific response of the immature liver to neonatal jaundice Table 2. Hepatitic Patterns of Liver Injury Hepatitic patterns Portal and/or acinar mononuclear cell inflammation with liver cell injury Portal and sinusoidal mononuclear cell inflammation without liver cell injury or hepatic plate alterations Zonal necroses (mainly acinar zone 3) Nonzonal multifocal necroses Multinucleated hepatocytes (8 10 nuclei per cell) Viral, drug, or autoimmune hepatitis; Wilson s disease, CMV infection, and celiac disease possible Immune response to systemic infections (eg, EBV) or lymphoproliferative disorder Ischemia, toxins, drugs or their metabolites with direct cytotoxic effects Viral infections (herpes simplex, herpes zoster, and adenovirus) Neonatal hepatitis and syncytial giant cell hepatitus; viruses (paramyxoviruses, human immunodeficiency virus, EBV, and hepatitis C virus); drugs (methotrexate and 6-mercaptopurine); toxins (vinyl chloride and certain herbs); immune disorders CMV, cytomegalovirus; EBV, Epstein Barr virus.

4 August 2007 Figure 3. Portal lymphoid aggregates and minimal steatosis. The findings are consistent with a quiescent chronic hepatitis C infection. of any cause. In syncytial giant cell hepatitis, these changes result from various viruses, drugs, toxins, and disorders of the immune system. Electron microscopic examination of the liver tissue may disclose widely scattered foci of viral-like inclusions, sometimes suggestive of paramyxovirus.33 Steatotic Pattern of Liver Injury Fatty infiltration of the liver can be bland with little or no inflammatory activity, or it may be associated with a hepatitic pattern that includes hepatocyte swelling and hyalin bodies (Figure 10).25,26 Fatty changes that are largely macrovesicular are usually indicative of a metabolic or toxic disease (Table 3), including drug reactions, alcohol injury, diabetes, obesity, and Wilson disease.36 Microvesicular steatosis usually reflects mitochondrial injury (Table 3) caused by diverse genetic disorders or various drugs and mitochondrial toxins.37 Fatty infiltration of the liver is common in a general population in whom obesity is prevalent. Another co-existent but Figure 4. Ground-glass hepatocyte. Enlarged hepatocytes with homogeneous cytoplasm have prominent endoplasmic reticulum filled with hepatitis B surface antigen or reactive to certain drugs (eg, chlorpromazine, barbiturates, phenytoin, and azathioprine). OPTIMIZING LIVER BIOPSY DIAGNOSIS 901 Figure 5. Cytomegalovirus infection. Findings of an enlarged endothelial cell with swollen nucleus and intranuclear inclusion (arrow) that resembles an owl s eye typifies the viral infection. pertinent disease may be obscured by the fatty infiltration. In nonalcoholic fatty liver disease, the histologic appearance can transition from bland steatosis (no inflammation) to steatohepatitis (active inflammation with swollen, damaged, fat-filled hepatocytes). Parenchymal inflammation and hepatocyte damage may be minimal in nonalcoholic fatty liver disease, but characteristic portal inflammation, ductular proliferation, and fibrosis can be sufficient to justify the designation of steatohepatitis. Progression to cirrhosis is possible,38,39 and steatosis may decrease or disappear with advancing fibrosis. In the late stage of nonalcoholic fatty liver disease, the characteristic features may disappear and the original basis for the tissue injury cannot be surmised.38,40,41 Biliary Patterns of Liver Injury Biliary patterns of liver injury reflect large bile duct obstruction, cell-mediated bile duct destruction, or bile duct loss without inflammation (Table 4). Figure 6. Herpes simplex infection at edge of focal hemorrhagic necrosis. Eosinophilic, near-purple, nuclear inclusion with clear surrounding halo typifies an infected hepatocyte.

5 902 CZAJA AND CARPENTER CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 8 Figure 7. Macrovesicular fatty infiltration of hepatocytes with ballooning and hyalin bodies (arrows). These findings are consistent with nonalcoholic steatohepatitis or alcoholic hepatitis. Figure 9. Nonzonal foci of parenchymal necrosis. The foci of parenchymal necrosis (arrows) are typically associated with a viral infection (herpes simplex, herpes zoster, or adenovirus). Large bile duct obstruction may be complete or incomplete, acute or chronic, and is caused by a variety of conditions. The acute phase is characterized by bile duct tortuosity, portal tract edema, slight mixed portal inflammation, and ductular (cholangiolar) proliferation (Figure 11). The chronic phase is characterized by replacement of portal tract edema with fibrosis, predominance of lymphocytes within the portal tracts, and less ductular proliferation. In both the acute and chronic phases, the portal tract is expanded, and the interface with the acinar parenchyma is irregular as a result of ductular proliferation, edema, and fibrosis (known as biliary piecemeal necrosis). Hepatocytes retain bile, and they can swell, accumulate copper, and develop hyaline inclusions (cholate stasis). Other changes are periductal fibrosis (fibrous obliterative cholangitis) and bile duct loss (ductopenia). These features are nonspecific and do not implicate a particular cause. The features of large duct obstruction can be caused by mechanical obstruction, drug reactions, abdominal or systemic infection, multi-organ failure, bacterial or chemical toxins, or reactions to total parenteral nutrition (Table 4). Bile infarcts are seen only in mechanical obstruction, and they are caused by the extravasation of bile into the periportal parenchyma, causing ballooning transformation of the hepatocytes or localized necrosis. Incomplete large-duct obstruction tends to have a chronic histologic appearance. Slowly growing neoplasms, drug reactions, infections or infestations, immune reactions (primary or secondary sclerosing cholangitis), and genetic conditions, especially in children and young adults, are diagnostic considerations. In children, -1 antitrypsin deficiency, biliary atresia, cystic fibrosis, and progressive familial intrahepatic cholestasis type 3 are possibilities (Table 4). Canalicular collections of bile in the absence of parenchymal inflammation or biliary changes (pure cholestasis) typically reflects a systemic infection, drug toxicity (metabolic steroids), paraneoplastic syndrome (lympho- Figure 8. Sinusoidal inflammation with little liver cell injury or liver plate alteration. This inflammatory pattern is consistent with an immune response to a systemic infection or lymphoproliferative disorder. Figure 10. Multinucleated giant hepatocytes. This injury response is consistent with a viral infection, drug reaction, or toxic exposure. Electron microscopy may disclose widely scattered foci of viral-like inclusions.

6 August 2007 OPTIMIZING LIVER BIOPSY DIAGNOSIS 903 Table 3. Steatotic Patterns of Liver Injury Steatotic patterns Macrovesicular fatty changes predominant, little or no inflammation (steatosis) Macrovesicular fatty changes predominant, mixed portal and acinar inflammation, ductular proliferation, hepatocyte swelling and hyalin bodies (steatohepatitis) Microvesicular steatosis predominant Obesity, diabetes, drugs Obesity, diabetes, alcohol, drug reaction, Wilson s disease, diverse metabolic diseases in children Mitochondrial injury caused by genetic diseases, drugs, or toxins proliferative disease), or hereditary condition (benign recurrent intrahepatic cholestasis) (Table 4). Cell-mediated bile duct destruction is characterized by a mononuclear cell infiltration of the portal tract; cellular destructive cholangitis of a lymphocytic, pleomorphic, or granulomatous nature; and bile duct loss (Figure 12). 23,24 Initially, this is a multifocal process with inflammatory destruction of interlobular bile ducts. At later stages, the inflammatory reaction becomes more uniform because most interlobular bile ducts are lost and the process is driven by the accumulation of toxic bile acids. Primary biliary cirrhosis exemplifies this pattern, but other associations include cell-mediated immune and drug reactions, graft-versus-host disease, and cellular rejection in allografts. Bile ducts may be destroyed in sarcoidosis, but usually the bile ducts are incidental targets. Ductopenia is defined as the absence of a bile duct in proximity to each arteriole in the portal tract (Figure 13). It implies that the bile duct has been destroyed by an inflammatory process or ischemic event. Autoimmune biliary disease, drug reactions, and cholestatic sarcoidosis are considerations in adults, and Alagille syndrome, -1 antitrypsin deficiency, and defects in bile acid metabolism or transport are considerations in children (Table 4). Mechanical ischemia or ischemia related to immune reactions, such as small-duct primary sclerosing cholangitis, chronic rejection in allografts, and graft-versushost disease are important diagnostic possibilities. Fibro-obliterative cholangitis is characteristic of primary sclerosing cholangitis, but it is a nonspecific and uncommon finding (Figure 14). 42 Biliary and hepatitic patterns may co-exist and suggest variant or overlap syndromes of autoimmune hepatitis and primary biliary cirrhosis or drug reaction. Mixed patterns also may be seen in primary sclerosing cholangitis, 46 and in children with neonatal, multinucleated, giant-cell transformation who may have features of ductopenia or biliary obstruction. Granulomatous Pattern of Liver Injury Granulomas identify a nonspecific inflammatory reaction that usually is multifocal and frequently is associated with systemic disease. The differential diagnosis includes numerous infectious, toxic, inflammatory, and immune disorders (Table 5). Granulomas may have a necrotizing or nonnecrotizing component; they can be distributed in the portal tracts, hepatic parenchyma, or both regions; and they may be variably sized, ill-formed, or discrete, and associated with little or abundant inflammation. Ill-formed granulomas that destroy bile ducts and exist with heavy monoclonal infiltrates within the portal tract suggest primary biliary cirrhosis, but a drug reaction is possible, especially if many portal tracts are involved. Discrete, well-formed granulomas along prelymphatic drainage pathways in the periphery of the portal tract suggest sarcoidosis, and they typically have only a narrow cuff of lymphocytes and fibrosis (Figure 15). The granulomas of sarcoidosis can involve interlobular bile ducts, arteries, and veins in a random fashion that result in highly variable collateral histologic changes. Table 4. Biliary Patterns of Liver Injury Biliary patterns Biliary obstructive pattern with portal edema and/or fibrosis, slight mixed inflammation, ductular proliferation with/without hepatocanalicular or ductular cholestasis; fibro-obliterative cholangitis and/or bile duct loss may be present Portal and periportal mononuclear cell infiltrates with cellular destructive cholangitis and/or bile duct loss, variable fibrous expansion of portal tract; cholate stasis in advanced disease Ductopenia Canalicular cholestasis without inflammation or biliary changes Biliary obstruction (mechanical, large-duct primary or secondary sclerosing cholangitis, drug reaction, sepsis, bacterial or chemical toxins, fibrosing cholestatic hepatitis, multiple organ failure, TPN, biliary ischemia); -1 antitrypsin deficiency, biliary atresia, cystic fibrosis, and PFIC type 3 in children PBC, drug reaction, graft-versus-host disease, and cellular rejection in allografts Small-duct PSC, drug reaction, graft-versus-host disease, chronic rejection in allografts, idiopathic adulthood ductopenia, and the cholestasis of sarcoidosis; syndromic (Alagille syndrome) or nonsyndromic intrahepatic paucity of bile ducts; -1 antitrypsin deficiency, and bile acid synthetic or transport defects in children Systemic infection, drug toxicity, paraneoplastic syndrome, benign recurrent intrahepatic cholestasis PFIC, progressive familial intrahepatic cholestasis; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; TPN, total parenteral nutrition.

7 904 CZAJA AND CARPENTER Figure 11. Bile ductular proliferation, bile duct tortuosity, and cholestasis. Findings indicate biliary obstruction associated with mechanical, toxic, or drug reaction. Loose, ill-formed granulomas may be only one component of a pleomorphic inflammatory infiltrate, as in active fungal, mycobacterial, and parasitic infections or drug reactions. The presence of central necrosis indicates the likelihood of infection, and the suppurative, eosinophilic, caseous, or fibrinoid nature of the necrosis can implicate certain etiologic agents or disease processes. Granulomas that are mainly in the hepatic parenchyma suggest a hematogenous infection, whereas vague pleomorphic granulomas that are mainly in the portal tracts suggest a drug reaction. Very small, poorly formed granulomas consisting of meager collections of histocytes within the sinusoids are microgranulomas, and they are seen commonly in systemic infections. Fibrin ring granulomas also may be small, but they have a central fat vacuole, intermediate ring of fibrin, and a peripheral rim of activated macrophages. These granulomas are seen in numerous and varied clinical settings, but they are characteristic of Q fever. The recognition of granulomas within the biopsy specimen justifies the performance of special stains for fungi and acidfast bacilli. These stains frequently are negative even with Figure 12. Granulomatous destructive cholangitis with bile duct loss (florid duct lesion). This feature is characteristic of primary biliary cirrhosis, but also may occur in sarcoidosis. CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 8 Figure 13. Ductopenia. Arteriole within the portal tract lacks an adjacent bile duct. Findings are consistent with small-duct primary sclerosing cholangitis. known active infection such as tuberculosis, and they must be supplemented with appropriate cultures, skin tests, and serologic assays. Vascular Pattern of Liver Injury The vascular pattern of liver injury reflects disturbances in the hepatic blood supply or venous drainage that may be within or outside the liver and involve the arterial and venous circulations (Table 6). Localized intrahepatic arterial obstruction, as in vasculitis, thrombosis, or shunts, may cause ischemic injury to the biliary system and be evident as biliary strictures or loss of interlobular bile ducts. Venous outflow impairment can cause biliary damage and the histologic changes of bile ductular proliferation, portal inflammation, and fibrosis common to chronic incomplete biliary obstruction.47 Sinusoidal congestion and red blood cell extravasation into the space of Disse (Figure 16) indicate venous outflow obstruction caused by Figure 14. Fibro-obliterative cholangitis. The rim of fibrosis encircling an obliterated bile duct (arrow) is a finding that is characteristic of primary sclerosing cholangitis.

8 August 2007 OPTIMIZING LIVER BIOPSY DIAGNOSIS 905 Table 5. Granulomatous Patterns of Liver Injury Granulomatous patterns Diffusely distributed microgranulomas or vaguely granulomatous inflammation Necrotizing epithelioid granulomas with or without pleomorphic inflammatory component Nonnecrotizing epithelioid granulomas with little or no inflammation Fibrin ring granulomas Granulomatous bile duct destruction with dense mononuclear portal inflammation (florid duct lesion) Systemic infection or drug reaction Infectious cause, especially tuberculosis, fungal infection, or uncommon infection caused by bacteria or parasites Sarcoidosis or infection Infections (Q fever) or drug reactions (allopurinol) Primary biliary cirrhosis, drug reaction, or sarcoidosis thrombosis (Budd Chiari syndrome), caval obstruction associated with web or neoplasm, or increased right heart pressure (cardiac failure, pulmonary hypertension, or constrictive pericarditis), whereas these findings with red blood cell extravasation, subintimal edema, and concentric fibrosis of the terminal hepatic venules indicate endothelial injury and veno-occlusive disease (Figure 17). 48 Prehepatic portal vein obstruction leads to liver atrophy, hilar portal vein obstruction causes nodular transformation of the liver, and intrahepatic segmental portal vein occlusion results in focal nodular hyperplasia. 49 Nodular regenerative hyperplasia is characterized by diffuse atrophy of the perivenular hepatic plate and hypertrophy of the periportal hepatic plate in the absence of fibrosis. It may or may not have detectable venular lesions (Figure 18). Nodular regenerative hyperplasia is indicative of an intrahepatic circulatory disorder that invariably is associated with occlusion of small portal veins and occasionally with occlusion of small hepatic veins. 50,51 Noncirrhotic portal hypertension can be present, and imaging studies may suggest cirrhosis. The decreased portal blood flow deprives the distal perivenular hepatocytes of nutrients, and the compensatory arterial blood Table 6. Vascular Patterns of Liver Injury Vascular patterns Acinar zone 3 perivenular necrosis, sinusoidal congestion, extravasation of blood into space of Disse Sinusoidal congestion and endothelial intimal proliferation within terminal hepatic venules or veins Nodular hepatic parenchyma with atrophy of the perivenular hepatic plate and hypertrophy of the periportal hepatic plate in the absence of fibrosis Hepatic ischemia; venous outflow obstruction (Budd Chiari syndrome), cardiac failure, constrictive pericarditis, pulmonary hypertension Veno-occlusive disease (toxicity to pyrrolizidine alkaloids, hepatic radiation, or drug reaction, especially to chemotherapeutic agents or azathioprine) Nodular regenerative hyperplasia (obstruction of small portal veins caused by myeloproliferative disorders, hypercoagulable states, toxic or drug reactions, or autoimmune conditions); any circulatory disorder regardless of cause flow provides the proximal periportal hepatocytes with increased oxygen and a stimulus for hypertrophy. Myeloproliferative disorders, hypercoagulable states, vascular injury associated with radiation, toxins or drugs, and autoimmune disorders have been associated with this histologic finding. Tissue specimens obtained early in the disease, especially if multiple samples are taken from both lobes of the liver, are more likely to reveal the diagnosis. The features of hepatic vein thrombosis and veno-occlusive disease are distributed irregularly within the liver, and their histologic features can overlap late in the course of the disease. Metabolic Patterns of Liver Injury The common metabolic patterns of liver injury include macrovesicular and microvesicular steatosis, hepatocanalicular Figure 15. Nonnecrotizing granulomatous reaction. A discrete nonnecrotizing epithelioid granuloma is consistent with a drug reaction or sarcoidosis. Figure 16. Sinusoidal congestion and red blood cell extravasation. The vascular pattern of injury reflects venous outflow obstruction, and it may be caused by hepatic vein thrombosis, pulmonary hypertension, constrictive pericarditis, or veno-occlusive disease.

9 906 CZAJA AND CARPENTER CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 5, No. 8 Table 7. Metabolic Patterns of Liver Injury Metabolic patterns Kupffer cell hemosiderosis Hepatocellular hemosiderosis Microvesicular steatosis Hepatocanalicular cholestasis with little or no inflammation Secondary hemosiderosis, non HFE-associated genetic hemochromatosis HFE-associated genetic hemochromatosis Primary or secondary mitochondrial injury caused by metabolic disorder, toxin, or drug reaction Drug reaction, sepsis, multiple organ failure, or metabolic disorder (hyperthyroidism) Figure 17. Veno-occlusive disease with sinusoidal congestion and endothelial proliferation and obstruction of the central vein. Endothelial proliferation and obstruction of the central vein (arrow) distinguishes veno-occlusive disease from hepatic vein thrombosis. It is associated with toxicity to pyrrolizidine alkaloids (bush tea toxicity), hepatic radiation, or drug reaction (chemotherapeutic agents or azathioprine). cholestasis, Kupffer cell hemosiderosis, and hepatocellular hemosiderosis (Table 7). The genetic disorders that affect liver cell metabolism, storage, and transport are mainly in children, but milder forms are recognized in adults. These same patterns of injury also can be seen in nongenetic conditions, and the presence of hepatic steatosis is an important first clue to a metabolic syndrome of a genetic or nongenetic nature. Steatosis in childhood may simply reflect obesity, but Wilson s disease always must be excluded. Other patterns of injury seen in metabolic disorders include hepatocanalicular cholestasis (Dubin Johnson syndrome), hepatocellular hemosiderosis (HFE-associated genetic hemochromatosis), Kupffer cell hemosiderosis (usually secondary hemosiderosis but may be non HFE-associated genetic hemochromatosis), and hepatic globules positive by the periodic acid Schiff reaction ( -1 antitrypsin deficiency) (Table 7). 52,53 In patients with clinical evidence of iron overload, the presence of stainable iron mainly within the hepatocytes rather than the Kupffer cells is useful in distinguishing, respectively, HFE-associated genetic hemochromatosis from secondary hemosiderosis and non HFE-associated genetic hemochromatosis caused by ferroportin defects. 52,53 Summary The liver biopsy assessment often is critical in the evaluation of acute and chronic liver disease. The clinical classification of the disorder as hepatitic or cholestatic focuses the histologic examination. The tissue specimen may reveal the etiologic agent, but it is more likely to disclose the pattern of tissue injury from which the cause of the liver disease can be determined. The histologic patterns may have hepatitic, steatotic, biliary, granulomatous, vascular, or metabolic features, and each may have hepatitic or cholestatic clinical findings. An active dialogue between the clinician and pathologist, adequately sized tissue sample, disciplined analysis and interpretation of the histologic features, and awareness of the etiologic possibilities for each histologic pattern ensure optimal use of the biopsy specimen. Figure 18. Nodular regenerative hyperplasia. The nodular pattern (arrows) is associated with compression and atrophy of the perivenular hepatic plate and the absence of fibrosis. The finding is associated with obstruction of small portal veins as a result of myeloproliferative disorders, hypercoagulable states, toxic or drug reactions, or autoimmune conditions. References 1. Czaja AJ, Carpenter HA. Sensitivity, specificity and predictability of biopsy interpretations in chronic hepatitis. Gastroenterology 1993;105: Carpenter HA, Czaja AJ. The role of histologic evaluation in the diagnosis and management of autoimmune hepatitis and its variants. Clin Liver Dis 2002;6: Czaja AJ. Evaluation of hepatobiliary diseases. In: Stein J, ed. Internal medicine. St. Louis: Mosby, 1998: Kenny RP, Czaja AJ, Ludwig J, et al. Frequency and significance of antimitochondrial antibodies in severe chronic active hepatitis. Dig Dis Sci 1986;31: Czaja AJ. Frequency and nature of the variant syndromes of autoimmune liver disease. Hepatology 1998;28: Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis: morphologic features and the genesis of incomplete septal fibrosis. Arch Pathol Lab Med 2000;124: Wanless IR. Use of corticosteroid therapy in autoimmune hepatitis resulting in resolution of cirrhosis (editorial). J Clin Gastroenterol 2001;32:

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N Engl J Med 2004;350: Address requests for reprints to: Albert J. Czaja, MD, Mayo Clinic, 200 First Street SW, Rochester, Minnesota czaja. albert@mayo.edu; fax: (507)