The duration of fixation influences the yield of HCV cDNA-PCR products from formalin-fixed, paraffin-embedded liver tissue.
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.
Bresters
D
D
Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Department of Virusdiagnostics, Amsterdam.
Schipper
M E
ME
Reesink
H W
HW
Boeser-Nunnink
B D
BD
Cuypers
H T
HT
D001706:Biopsy; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D005069:Evaluation Studies as Topic; D005404:Fixatives; D005557:Formaldehyde; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis; D006801:Humans; D008099:Liver / Q000821:virology*; D010232:Paraffin; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors; D014773:Virology / Q000379:methods*
D018076:DNA, Complementary; D004279:DNA, Viral; D005404:Fixatives; D012367:RNA, Viral; D005557:Formaldehyde; D010232:Paraffin