[Morphology and molecular pathology: detection of hepatitis C virus RNA sequences in stained sections by microscopy-directed selective extraction].
We have developed a method for extracting and detecting HCV (hepatitis C virus) RNA from histologically identified lesions in post-stained paraffin sections of human liver tissues. After confirming the lesion by light microscopy, this method enabled us to extract HCV RNA selectively from hepatic nodules and surrounding cirrhosis respectively. We examined 6 HCV positive patients with 7 hepatic nodules [5 hepatocellular carcinomas (HCC) and 2 adenomatous hyperplasias (AH)] and surrounding cirrhotic tissue. After staining with hematoxylin and confirming the histologic lesions under the microscopy, the same amount of tumor and cirrhotic tissues were collected from the slide sections. RNA was extracted by GITC method (Chomczynsky's method). Positive and negative strand RNAs of HCV-NT region were amplified by reverse-transcription and double PCR methods (RT/double PCR) followed by the Southern blotting and the hybridization with HCV-NT probe. Southern blotting disclosed positive strand HCV-RNA in all tumors except one case as well as surrounding cirrhotic livers. This one case had no detectable band in any samples. Negative strand HCV-RNAs could be detected in 4 cases with 5 tumors (4 HCCs and 1 AH) and surrounding cirrhotic livers, suggesting that HCV replication (proliferation) persisted in these tissues. We have confirmed that HCV-RNA sequences are present in liver tissues in several stages of hepatocarcinogenesis (cirrhosis, AH and HCC). These findings suggest that HCV is involved in hepatocarcinogenesis in chronic hepatitis C and cirrhosis. Our method is the first report of RNA extraction from the post-stained paraffin sections allowing precise histopathologic identification of the lesions. This method will be useful for the molecular studies of archival tissues as well as for the morphological examination, because it has the characteristics of both studies.
Saito
K
K
Division of Pathology and Laboratory Medicine, Toyama City Hospital.
D000368:Aged; D015139:Blotting, Southern; D005260:Female; D016174:Hepacivirus / Q000235:genetics; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006651:Histocytochemistry; D006801:Humans; D008297:Male; D008853:Microscopy; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification*
D012367:RNA, Viral