title: Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.
abstract: We have noticed that suboptimal specimen processing and storage conditions may cause false-negative results in the detection of hepatitis C virus (HCV) RNA in plasma or serum. To establish the influence of specimen handling in a serological laboratory on the rate of detection of HCV RNA by the cDNA polymerase chain reaction (cDNA-PCR), we tested routine serum samples and fresh-frozen plasma samples from the same bleeding from confirmed anti-HCV-positive blood donors. When primers from the NS3/NS4 region were used, HCV RNA was detected in fresh-frozen plasma from 67% of the donors, whereas positive results were obtained with only 50% of the serum samples that had been subjected to routine serological procedures. Analysis of the same samples with primers from the highly conserved 5'-terminal region (5'-TR) revealed an HCV RNA detection rate of 92% for both the routine and the fresh-frozen samples. However, the yield of the amplification product in routine samples was strongly reduced compared with that in fresh-frozen plasma. Comparison of both primer sets for cDNA-PCR showed that the 5'-TR primer set was 10- to 100-fold more effective in detecting HCV RNA. We also analyzed the effect of storage of whole EDTA-blood and serum at room temperature and at 4 degrees C on the yield of the amplification product. A rapid decline in detectable HCV RNA of 3 to 4 log units was observed within 14 days when whole blood and serum were stored at room temperature. By contrast, no perceptible reduction in the cDNA-PCR signal was found in freshly prepared serum stored at 4 degrees C.
authors.affiliation: Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
authors.forename: H T
authors.initials: HT
authors.lastname: Cuypers
authors.forename: D
authors.initials: D
authors.lastname: Bresters
authors.forename: I N
authors.initials: IN
authors.lastname: Winkel
authors.forename: H W
authors.initials: HW
authors.lastname: Reesink
authors.forename: A J
authors.initials: AJ
authors.lastname: Weiner
authors.forename: M
authors.initials: M
authors.lastname: Houghton
authors.forename: C L
authors.initials: CL
authors.lastname: van der Poel
authors.forename: P N
authors.initials: PN
authors.lastname: Lelie
mesh_terms: D001483:Base Sequence; D001793:Blood Preservation; D001800:Blood Specimen Collection; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics; D005069:Evaluation Studies as Topic; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral