ABSTRACT
The extent of hepatitis C virus (HCV) RNA loss during increasing periods of fixation of liver tissue in formalin was examined. For this purpose human liver tissue, known to be HCV RNA positive and stored at -70 degrees C until use, was cut into small slices (n = 9), which were fixed in phosphate-buffered formalin for increasing periods of time before embedding in paraffin. Nucleic acids were extracted from each slice of formalin-fixed, paraffin-embedded liver tissue and HCV RNA loss during fixation was semi-quantified by testing 10-fold dilutions of each extract in an HCV cDNA-PCR assay. The endpoint dilution for HCV RNA detection by cDNA-PCR in liver slices fixed in buffered formalin for 8-24 h was comparable to the endpoint dilutions found for 'fresh', non-fixed liver slices. After fixation for 2-3 days the endpoint dilution for HCV RNA detection was 10(2) to 10(3)-fold less. After 2-4 weeks of formalin-fixation, HCV RNA was detectable from undiluted nucleic acid extracts only. It is concluded that formalin-fixed, paraffin-embedded liver biopsies can be used for HCV RNA detection by cDNA-PCR, on condition that the liver tissue has been embedded in paraffin within 24 h of formalin-fixation.
Subject(s)
DNA, Viral/genetics , Hepacivirus/genetics , Hepacivirus/isolation & purification , Liver/virology , Virology/methods , Biopsy , DNA, Complementary/genetics , Evaluation Studies as Topic , Fixatives , Formaldehyde , Hepatitis C/diagnosis , Humans , Paraffin , Polymerase Chain Reaction , RNA, Viral/genetics , RNA, Viral/isolation & purification , Time FactorsABSTRACT
In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV) cDNA-PCR. From 16 of the same non-A, non-B hepatitis patients and from 5 of the same control patients formalin-fixed, paraffin-embedded liver tissue from the same biopsy was available also for HCV cDNA-PCR. In 13 of 22 non-A, non-B hepatitis patients HCV-RNA could be detected in plasma as well as in liver tissue. In the other 9 non-A, non-B hepatitis patients and in 6 control patients, no HCV-RNA was detectable in either plasma or liver tissue. The comparison between HCV cDNA-PCR results in fresh frozen versus formalin-fixed, paraffin-embedded liver biopsies showed that although detection of HCV-RNA in both correlated 100% the quantity of HCV-RNA was lower in the formalin-fixed, paraffin-embedded liver biopsies of 5 of 8 patients for whom end-point dilution titration of liver RNA was performed. We conclude that using the procedures described HCV-RNA can be reliably detected in both fresh-frozen and formalin-fixed, paraffin-embedded liver biopsies and that HCV cDNA-PCR in liver tissue may become an important assay, especially for monitoring anti-viral therapy.