Effects of delayed freezing of liver biopsies on the detection of hepatitis C virus RNA strands

Background/Aims: There are no data about the influence of handling conditions of liver biopsies on the integrity of viral RNAs. We studied the influence of the time delay between obtaining and freezing the liver biopsy on the stability of intrahepatic positive and negative hepatitis C virus RNA (HCV-RNA) strands. Methods: Liver samples from 30 anti-HCV patients were included. For each case, one portion of the liver biopsy (first sample) was immediately frozen (20–28 s), while the other section (second sample) was kept at room temperature (1–30 min) before freezing. Each experimental time point was performed in triplicate using liver samples from three different patients. Semi-quantitative analysis of the positive and negative HCV-RNA strands and of the α1-antitrypsin mRNA was performed by a Tth-based reverse-transcription polymerase chain reaction. Results: A significant time-related decrease in both positive (r=−0.8412, p=0.001) and negative (r= −0.8539, p=0.001) HCV-RNA strand titres was found in the second liver fractions. There were no appreciable changes in RNA titres in those samples frozen after less than 3 min. The RNA titres decreased in all but two samples incubated for 4–30 min. Thus, 3/15 (20%) and 7/11 (64%) of these samples lost positive and negative HCV-RNA strands, respectively. Alpha-1-antitrypsin mRNA titres decreased significantly (r=−0.8935, p=0.01) in those samples kept at room temperature for more than 4 min. Conclusion: Freezing of liver samples immediately after extraction is crucial to avoid false negative HCV-RNA detection results, especially for the anti-genomic RNA strand.