Anatomic Pathology / DISTINGUISHING PRIMARY BILIARY CIRRHOSIS FROM AUTOIMMUNE HEPATITIS

We studied nondiagnostic liver biopsy specimens from 20 patients with definite primary biliary cirrhosis (PBC) and 18 with definite autoimmune hepatitis (AIH) to identify distinguishing features. All patients had early-stage disease; biopsy specimens were devoid of granulomas or diagnostic features of PBC or AIH. Diagnoses were based on serologic and clinical variables. Sixteen specimens from each group were immunostained with cytokeratin 7. The density of portal tract eosinophils and number with cytokeratin 7–reactive periportal hepatocytes were quantified. Sixteen of 18 patients with AIH and 13 of 20 with PBC had no or minimal bile duct injury. Histologic activity index scores were 5.8 in AIH and 5.7 in PBC. The mean portal eosinophil score was greater in PBC than in AIH. Cytokeratin 7 identified many central bile ducts that were obscured by portal inflammation. The mean periportal cytokeratin 7–reactive hepatocyte score was greater in PBC than in AIH. Portal eosinophils and cytokeratin 7 reactivity in periportal hepatocytes are supportive of PBC rather than AIH. No morphologic features were supportive of AIH. Cytokeratin 7 reactivity in periportal hepatocytes may be an early response to PBC-induced biliary obstruction in other regions of the liver. Primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) can be difficult to distinguish.1-3 Patients with mildly active, early-stage PBC or AIH can have vague, nondescript symptoms. Serum antibody titers and liver function tests can be insufficiently elevated to be strongly supportive of either disease, and multiple serum antibody titers can be elevated, creating a nondiagnostic antibody panel. The distinction between the diseases is important. Corticosteroids, which are the mainstay of AIH therapy, usually are contraindicated in PBC. When the clinical findings and laboratory tests are inconclusive, the diagnosis can rest primarily on the morphologic features of the liver biopsy specimen. Unfortunately, liver biopsy specimens from patients with early-stage, mildly active PBC or AIH also can be nondiagnostic because they lack the characteristic morphologic features of either disease. Normal biliary epithelium is strongly cytokeratin 7 reactive.4-6 Mature, uninjured hepatocytes are cytokeratin 7 nonreactive.4,7 One author recently suggested that cytokeratin 7 immunohistochemical analysis could be helpful in the distinction of PBC and chronic hepatitis.1,8 Many of the liver biopsy specimens in this study had late-stage changes with marked bridging fibrosis or cirrhosis and characteristic morphologic features.8 The extent to which cytokeratin 7 immunohistochemical analysis could be diagnostically helpful in liver biopsy specimens from patients with early stage, mildly active PBC or AIH has not been explored fully. Three studies during the 1990s found an increased number of portal tract eosinophils in liver biopsy specimens from patients with PBC compared with patients with chronic hepatitis.9-11 The majority of studied biopsies had late-stage changes, with marked bridging fibrosis or cirrhosis, and they Anatomic Pathology / ORIGINAL ARTICLE Am J Clin Pathol 2001;116:846-853 847 © American Society of Clinical Pathologists also had characteristic morphologic features. The usefulness of portal tract eosinophils in liver biopsy specimens from earlystage, mildly active PBC and AIH also has not been studied. The goal of the present study was to evaluate whether portal tract eosinophils and cytokeratin 7 immunohistochemical analysis could be helpful in the morphologic distinction between mildly active, early-stage PBC and AIH, especially in biopsy specimens that are devoid of characteristic morphologic features. Materials and Methods The goal was to retrospectively obtain a group of nondiagnostic liver biopsy specimens from patients with earlystage, mildly active PBC or AIH who later had definite, unequivocal diagnoses of AIH or PBC. The studied liver biopsy specimens were selected from a large group of patients with PBC or AIH treated by one of us (S.C.G.) during the period January, 1, 1993, through July 1, 2000. At the time of the retrospective review, all patients with AIH in the group had “definite AIH” according to the International Autoimmune Hepatitis Scoring System (serum alanine transaminase level at least 5 times the upper limit of normal, serum IgG level at least 2 times the upper limit of normal, positive anti–smooth muscle antibodies, and a liver biopsy specimen with moderate or severe periportal or periseptal lymphoplasmacytic piecemeal necrosis), and all patients with PBC had definite PBC (positive serum antimitochondrial antibodies, serum alkaline phosphatase level >2 times the upper limit of normal, serum glutamyltranspeptidase level at least 5 times the upper limit of normal, and a liver biopsy specimen with florid duct lesion).12,13 We were not interested in characterizing these features in liver biopsy specimens with the characteristic morphologic features of PBC or AIH. Liver biopsy slides and pathology reports from the PBC and AIH patient pool were initially reviewed, and the biopsy specimens were excluded if they had extensive bridging fibrosis, cirrhosis, or characteristic morphologic features of PBC (moderate bile duct injury, bile duct–centered lymphoplasmacytic inflammation, peri–bile duct lymphoid aggregates, onion skin–type peri–bile duct fibrosis, portal or parenchymal granulomas, and isolated foreign body giant cells) or AIH (a low-magnification impression of parenchymal-centered inflammation; diffuse, moderate interface hepatitis; and moderate parenchymal inflammation, extensive hepatocyte necrosis, numerous foci of spotty inflammation with acidophilic bodies).3,12 The clinical records of patients also were reviewed, and biopsy specimens were excluded if the patient was receiving corticosteroids or ursodeoxycholic acid at the time of biopsy, had additional liver diseases including B or C viral hepatitis (at the time of liver biopsy or subsequently diagnosed), had a subsequent diagnosis other than definite PBC or AIH, or was lost to follow-up before a definitive diagnosis could be established. Included biopsy specimens were from patients who were diagnosed with definite PBC or AIH at the time of the liver biopsy or after the biopsy.12,14 None of the patients in the study had an overlap syndrome with moderate to markedly active features of both diseases.13,15-18 The final study group included nondiagnostic liver biopsy specimens from 38 patients with definite PBC or AIH. Twenty biopsy specimens were from patients with PBC, and 18 were from patients with AIH. The mean age at biopsy for patients with AIH was 42 years and for patients with PBC, 47 years. All specimens from patients with PBC were predominantly stage 1 or 2.19 Four (20%) had rare portal-portal fibrous bridges. None had extensive bridging fibrosis or cirrhosis. Thirteen patients with PBC had incorrect, nonspecific, or possible PBC diagnoses at the time of the liver biopsy; all biopsies were performed while under the care of nonauthor physicians. These patients came under the care of one of us (S.C.G.), and their disease was reclassified as probable or definite PBC 2.5 to 39 months after the liver biopsy. The other 7 patients with PBC had probable or definite PBC diagnoses made by one us (S.C.G.) at the time of liver biopsy. The majority of portal tracts in biopsy specimens from patients with AIH had portal fibrosis that was predominantly mild, and less commonly moderate. Ten biopsy specimens (56%) had rare, thin portal-portal fibrous septa. None had extensive bridging fibrosis or cirrhosis. Fourteen of 18 patients with AIH had incorrect or nonspecific hepatitis diagnoses at the time of the liver biopsy, and 3 had probable AIH. Ten patients underwent a second liver biopsy that showed characteristic AIH. A definitive AIH diagnosis was made by one of us (S.C.G.) 1 to 22 months after the initial liver biopsy. Specimen Processing and Staining All liver biopsy specimens were processed in a similar manner. Blank sections were serially cut after formalin fixation and sequentially stained with H&E, Masson trichrome, reticulin, periodic acid–Schiff without diastase, periodic acid–Schiff with diastase, Prussian blue, orcein, and H&E again. Sixteen biopsy specimens from each disease group with the greatest amounts of residual tissue in the blocks were used for cytokeratin 7 immunohistochemical staining. A 3μm-thick section from each case was placed on a charged slide. Sections were deparaffinized using sequential immersions in 2 xylene baths, 3 baths of decreasing alcohol concentrations, and 2 water baths, followed by a 1-minute Goldstein et al / DISTINGUISHING PRIMARY BILIARY CIRRHOSIS FROM AUTOIMMUNE HEPATITIS 848 Am J Clin Pathol 2001;116:846-853 © American Society of Clinical Pathologists wash in water. Slides were immersed in EDTA buffer (pH 7.0) and put into a commercial vegetable steamer at 95°C for 30 minutes. The slides were allowed to cool on the counter, remaining immersed in the heated EDTA buffer–filled containers for 5 minutes, followed by a 2-minute rinse with water while remaining in the containers. The slides were transferred into tris(hydroxymethyl)aminomethane–filled containers (pH 7.0) and allowed to undergo an additional 10 minutes of cooling on the countertop. They then were transferred to a commercial immunohistochemical autostainer (DAKO, Carpinteria, CA) and first were washed with buffer, followed by a hydrogen peroxide incubation. The latter was rinsed off, and the primary antibody was applied. Cytokeratin 7 (clone OV-TL-12/30; 1:800 dilution; DAKO) antibody was incubated over the sections for 20 minutes at room temperature. After the primary antibody was washed off, the components of the Envision-plus (DAKO) detection system were applied, including an antimouse polymer, 2 distilledwater washes, and a final diaminobenzidine incubation for 4 minutes. Sections were counterstained with hematoxylin, and cover glasses were applied. A positive control slide containing known cytokeratin-reactive tissues was stained with each batch of simultaneously stained slides. The chromogen system used was a 2-step technique that uses a horseradish peroxidase polymer conjugated with secondary antibodies. The labeled polymer does not contain avidin or biotin, and, therefore, nonspecific staining of hepatocytes from endogenous biotin is avoided.