Effects of delayed freezing of liver biopsies on the detection of hepatitis C virus RNA strands.
OBJECTIVE
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 al-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.


CONCLUSIONS
Freezing of liver samples immediately after extraction is crucial to avoid false negative HCV-RNA detection results, especially for the antigenomic RNA strand.
Madejón
A
A
Department of Hepatology, Fundación Jiménez Díaz, and Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.
Manzano
M L
ML
Arocena
C
C
Castillo
I
I
Carreño
V
V
D001706:Biopsy; D005615:Freezing; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000473:pathology*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000235:genetics
D012333:RNA, Messenger; D012367:RNA, Viral; D000515:alpha 1-Antitrypsin