Sansonno D, Iacobelli A R, Cornacchiulo V, Distasi M, Dammacco F
Department of Biomedical Sciences and Human Oncology, University of Bari Medical School, Italy.
Clin Exp Rheumatol. 1995 Nov-Dec;13 Suppl 13:S29-32.
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.
丙型肝炎病毒（HCV）相关抗原（Ags）在肝组织中难以检测且定义不清，其解读也存在不确定性。HCV感染时免疫组化失败可能归因于特异性抗血清的亲和力、感染组织中抗原的水平、抗原决定簇表达的不稳定以及可能改变或破坏病毒表位的固定剂的使用。因此，针对急性和慢性肝炎患者以及肝细胞癌（HCC）患者的肝活检，已设计出优化组织标本处理各阶段的策略。
采用两阶段间接免疫染色程序，对7例急性和23例慢性HCV感染患者以及4例HCV相关HCC患者未固定的低温冻存肝切片进行HCV-Ags检测。使用针对HCV相关结构和非结构蛋白的单克隆抗体混合物作为一抗试剂。
所有7例急性肝炎患者中，50%-70%的肝细胞发现有HCV-Ags，而23例慢性肝炎患者中有10例（43.5%）肝细胞中HCV-Ags比例≤20%（P<0.05）。免疫反应信号仅表现为细胞质中弥漫性或粗大颗粒状沉积物。细胞核不着色。虽在细胞质不同区域观察到细颗粒状、膜下积聚，但未发现清晰的膜状模式。在急性肝炎中，HCV-Ag阳性肝细胞分布于小叶内，与坏死区域和炎性细胞积聚区域直接相关，而在慢性肝炎中，免疫反应细胞与坏死灶无明显关联。所有HCC患者均检测到HCV-Ag免疫沉积物。肿瘤细胞中的免疫反应信号主要位于细胞质，很少位于细胞核。与非肿瘤区域相比，肿瘤区域的特异性信号显著更高。
免疫组织学是鉴定肝组织中HCV相关蛋白的有力工具。使用新鲜冷冻组织（可能保留其HCV抗原结构）以及针对HCV相关蛋白的单克隆抗体混合物可显著提高敏感性，这可能是由于每个探针可分别作用于不同靶蛋白。HCV感染在肝细胞质中的显示表明这是HCV复制的主要部位，而其在恶性细胞中的存在提示该病毒可能在HCC发病机制中起重要作用。
