Immunohistochemical detection of hepatitis C virus-related proteins in liver tissue

Hepatitis C virus (HCV)-associated antigens (Ags) are hard to detect and poorly defined in liver tissue, and are of uncertain interpretation. The failure of immunohistochemistry in HCV infection may be due to the affinity of specific antisera, the levels of Ags in infected tissues, the labile and unstable expression of antigenic determinants, and the use of fixatives that may alter or destroy viral epitopes. Strategies to optimize all stages of tissue specimen processing have therefore been devised in the liver biopsies of patients with acute and chronic hepatitis, and those with hepatocellular carcinoma (HCC).

HCV-Ags were detected with a two-stage indirect immunostaining procedure on unfixed cryostat liver sections from 7 acute and 23 chronic HCV-infected patients, and from 4 patients with HCV-associated HCC. A mixture of monoclonal antibodies directed to structural and non-structural HCV-related proteins were used as the primary reagents.

HCV-Ags in 50-70% of the hepatocytes were found in all seven acute hepatitis patients compared with < or = 20% hepatocytes (P < 0.05) in 10 out of 23 patients (43.5%) with chronic hepatitis. Immunoreactive signals appeared as diffuse or coarse granular deposits in the cytoplasm only. The nuclei were unstainable. No clear membranous pattern was found, although fine granular, submembranous accumulation in distinct areas of the cytoplasm was observed. In acute hepatitis, HCV-Ag positive hepatocytes were distributed in the lobules in direct relation to the areas of necrosis and inflammatory cell accumulation, whereas in chronic hepatitis the immunoreactive cells were not clearly related to the necrotic foci. HCV-Ag immunodeposits were demonstrated in all patients with HCC. The immunoreactive signal in neoplastic cells was primarily located in the cytoplasm and rarely in the nuclei. As compared with the non-neoplastic zones, neoplasia demonstrated a significantly higher specific signal.

Immunohistology is a powerful tool for the identification of HCV-related proteins in liver tissue. Sensitivity was significantly enhanced by the use of fresh-frozen tissues, which presumably preserve their HCV antigen structure, and by a mixture of monoclonal antibodies directed against HCV-related proteins, possibly on account of the separate access of each probe to different target proteins. The demonstration of HCV infection in hepatocyte cytoplasm indicates that this is the primary site of HCV replication, while its presence in malignant cells suggests that the virus could be substantially involved in the pathogenesis of HCC.