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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1992 Dec;30(12):3220–3224. doi: 10.1128/jcm.30.12.3220-3224.1992

Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

H T Cuypers 1, D Bresters 1, I N Winkel 1, H W Reesink 1, A J Weiner 1, M Houghton 1, C L van der Poel 1, P N Lelie 1
PMCID: PMC270636  PMID: 1333489

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.

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Selected References

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  1. Baggiolini M., Dewald B. The neutrophil. Int Arch Allergy Appl Immunol. 1985;76 (Suppl 1):13–20. doi: 10.1159/000233730. [DOI] [PubMed] [Google Scholar]
  2. Bresters D., Mauser-Bunschoten E. P., Cuypers H. T., Lelie P. N., Han J. H., Jansen P. L., Houghton M., Reesink H. W. Disappearance of hepatitis C virus RNA in plasma during interferon alpha-2B treatment in hemophilia patients. Scand J Gastroenterol. 1992;27(3):166–168. doi: 10.3109/00365529208999943. [DOI] [PubMed] [Google Scholar]
  3. Busch M. P., Wilber J. C., Johnson P., Tobler L., Evans C. S. Impact of specimen handling and storage on detection of hepatitis C virus RNA. Transfusion. 1992 Jun;32(5):420–425. doi: 10.1046/j.1537-2995.1992.32592327714.x. [DOI] [PubMed] [Google Scholar]
  4. Chayama K., Saitoh S., Arase Y., Ikeda K., Matsumoto T., Sakai Y., Kobayashi M., Unakami M., Morinaga T., Kumada H. Effect of interferon administration on serum hepatitis C virus RNA in patients with chronic hepatitis C. Hepatology. 1991 Jun;13(6):1040–1043. [PubMed] [Google Scholar]
  5. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2451–2455. doi: 10.1073/pnas.88.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choo Q. L., Weiner A. J., Overby L. R., Kuo G., Houghton M., Bradley D. W. Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis. Br Med Bull. 1990 Apr;46(2):423–441. doi: 10.1093/oxfordjournals.bmb.a072408. [DOI] [PubMed] [Google Scholar]
  7. Cuypers H. T., Winkel I. N., van der Poel C. L., Reesink H. W., Lelie P. N., Houghton M., Weiner A. Analysis of genomic variability of hepatitis C virus. J Hepatol. 1991;13 (Suppl 4):S15–S19. doi: 10.1016/0168-8278(91)90016-5. [DOI] [PubMed] [Google Scholar]
  8. Farci P., Alter H. J., Wong D., Miller R. H., Shih J. W., Jett B., Purcell R. H. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med. 1991 Jul 11;325(2):98–104. doi: 10.1056/NEJM199107113250205. [DOI] [PubMed] [Google Scholar]
  9. Follett E. A., Dow B. C., McOmish F., Yap P. L., Hughes W., Mitchell R., Simmonds P. HCV confirmatory testing of blood donors. Lancet. 1991 Oct 19;338(8773):1024–1024. doi: 10.1016/0140-6736(91)91891-w. [DOI] [PubMed] [Google Scholar]
  10. Fong T. L., Shindo M., Feinstone S. M., Hoofnagle J. H., Di Bisceglie A. M. Detection of replicative intermediates of hepatitis C viral RNA in liver and serum of patients with chronic hepatitis C. J Clin Invest. 1991 Sep;88(3):1058–1060. doi: 10.1172/JCI115368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Garson J. A., Tuke P. W., Makris M., Briggs M., Machin S. J., Preston F. E., Tedder R. S. Demonstration of viraemia patterns in haemophiliacs treated with hepatitis-C-virus-contaminated factor VIII concentrates. Lancet. 1990 Oct 27;336(8722):1022–1025. doi: 10.1016/0140-6736(90)92487-3. [DOI] [PubMed] [Google Scholar]
  12. Houghton M., Weiner A., Han J., Kuo G., Choo Q. L. Molecular biology of the hepatitis C viruses: implications for diagnosis, development and control of viral disease. Hepatology. 1991 Aug;14(2):381–388. [PubMed] [Google Scholar]
  13. Lelie P. N., Cuypers H. T., Reesink H. W., van der Poel C. L., Winkel I., Bakker E., van Exel-Oehlers P. J., Vallari D., Allain J. P., Mimms L. Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays. J Med Virol. 1992 Jul;37(3):203–209. doi: 10.1002/jmv.1890370310. [DOI] [PubMed] [Google Scholar]
  14. Ogata N., Alter H. J., Miller R. H., Purcell R. H. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3392–3396. doi: 10.1073/pnas.88.8.3392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schlauder G. G., Leverenz G. J., Amann C. W., Lesniewski R. R., Peterson D. A. Detection of the hepatitis C virus genome in acute and chronic experimental infection in chimpanzees. J Clin Microbiol. 1991 Oct;29(10):2175–2179. doi: 10.1128/jcm.29.10.2175-2179.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Thaler M. M., Park C. K., Landers D. V., Wara D. W., Houghton M., Veereman-Wauters G., Sweet R. L., Han J. H. Vertical transmission of hepatitis C virus. Lancet. 1991 Jul 6;338(8758):17–18. doi: 10.1016/0140-6736(91)90006-b. [DOI] [PubMed] [Google Scholar]
  17. Tsukiyama-Kohara K., Iizuka N., Kohara M., Nomoto A. Internal ribosome entry site within hepatitis C virus RNA. J Virol. 1992 Mar;66(3):1476–1483. doi: 10.1128/jvi.66.3.1476-1483.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Van der Poel C. L., Cuypers H. T., Reesink H. W., Weiner A. J., Quan S., Di Nello R., Van Boven J. J., Winkel I., Mulder-Folkerts D., Exel-Oehlers P. J. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet. 1991 Feb 9;337(8737):317–319. doi: 10.1016/0140-6736(91)90942-i. [DOI] [PubMed] [Google Scholar]
  19. Wang J. T., Wang T. H., Sheu J. C., Lin S. M., Lin J. T., Chen D. S. Effects of anticoagulants and storage of blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus. J Clin Microbiol. 1992 Mar;30(3):750–753. doi: 10.1128/jcm.30.3.750-753.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weiner A. J., Kuo G., Bradley D. W., Bonino F., Saracco G., Lee C., Rosenblatt J., Choo Q. L., Houghton M. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet. 1990 Jan 6;335(8680):1–3. doi: 10.1016/0140-6736(90)90134-q. [DOI] [PubMed] [Google Scholar]
  21. van der Poel C. L., Bresters D., Reesink H. W., Plaisier A. A., Schaasberg W., Leentvaar-Kuypers A., Choo Q. L., Quan S., Polito A., Houghton M. Early antihepatitis C virus response with second-generation C200/C22 ELISA. Vox Sang. 1992;62(4):208–212. doi: 10.1111/j.1423-0410.1992.tb01200.x. [DOI] [PubMed] [Google Scholar]
  22. van der Poel C., Cuypers H., Reesink H., Choo Q. L., Kuo G., Han J., Quan S., Polito A., Verstraten J., van de Wouw J. Risk factors in hepatitis C virus-infected blood donors. Transfusion. 1991 Oct;31(8):777–779. doi: 10.1046/j.1537-2995.1991.31892023508.x. [DOI] [PubMed] [Google Scholar]

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