Skip to main content
SpringerLink
Log in

Guidelines for the Quantification of HIV and HCV in Small Volume Whole Blood Samples

  • Protocol
  • First Online:
Diagnosis of Sexually Transmitted Diseases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 903))

  • 2195 Accesses

Abstract

The quantification of Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) in whole blood provides several advantages over the quantification in plasma samples. The application of small samples of capillary blood allows for application in point-of-care diagnostic testing methods.

Here we describe two protocols of extracting viral RNA from small samples of whole blood by hybridization to biotinylated LNA-modified 2′-O-Methyl-RNA or to biotinylated DNA, indirect capturing to streptavidin-coated beads, and subsequent quantification by one-step non-nested qRT-PCR. Further, we provide some general guidelines on extraction and quantification of HIV and HCV in small volume whole blood samples.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Lavanchy D (2009) The global burden of hepatitis C. Liver Int 29(Suppl 1):74–81

    Article  PubMed  Google Scholar 

  2. Joint United Nations Programm on HIV/AIDS (UNAIDS) (2008) 2008 report on the global AIDS epidemic. World Health Organization

    Google Scholar 

  3. Mellors JW, Rinaldo CR, Gupta P, White RM, Todd JA, Kingsley LA (1996) Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272(5265):1167–1170

    Article  PubMed  CAS  Google Scholar 

  4. Ghany MG, Strader DB, Thomas DL, Seeff LB (2009) Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 49(4):1335–1374

    Article  PubMed  CAS  Google Scholar 

  5. O’Brien WA, Hartigan PM, Daar ES, Simberkoff MS, Hamilton JD (1997) Changes in plasma HIV RNA levels and CD4+ lymphocyte counts predict both response to antiretroviral therapy and therapeutic failure. Ann Intern Med 126(12):939–945

    PubMed  Google Scholar 

  6. Gross R, Bilker WB, Friedman HM, Strom BL (2001) Effect of adherence to newly initiated antiretroviral therapy on plasma viral load. AIDS 15(16):2109–2117

    Article  PubMed  CAS  Google Scholar 

  7. Bouffard P, Hayashi PH, Acevedo R, Levy N, Zeldis JB (1992) Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients. J Infect Dis 166(6):1276–1280

    Article  PubMed  CAS  Google Scholar 

  8. Moldvay J, Deny P, Pol S, Brechot C, Lamas E (1994) Detection of hepatitis C virus RNA in peripheral blood mononuclear cells of infected patients by in situ hybridization. Blood 83(1):269–273

    PubMed  CAS  Google Scholar 

  9. Manzin A, Solforosi L, Candela M, Cherubini G, Piccinini G, Brugia M, Gabrielli A, Clementi M (1996) Hepatitis C virus infection and mixed cryoglobulinaemia: assessment of HCV RNA copy numbers in supernatant, cryoprecipitate and non-liver cells. J Viral Hepat 3(6):285–292

    Article  PubMed  CAS  Google Scholar 

  10. Lotz G, Szalay F, Firneisz G, Abonyi M, Lengyel G, Telegdy L, Ibrányi E, Nemes B, Kury F, Schaff Z, Simon S (2002) Localization of hepatitis C virus RNA on human erythrocytes by RT in situ PCR technique. Scand J Gastroenterol 37(5):578–584

    Article  PubMed  CAS  Google Scholar 

  11. Hamaia S, Li C, Allain JP (2001) The dynamics of hepatitis C virus binding to platelets and 2 mononuclear cell lines. Blood 98(8):2293–2300

    Article  PubMed  CAS  Google Scholar 

  12. Sattentau QJ, Dalgleish AG, Weiss RA, Beverley PC (1986) Epitopes of the CD4 antigen and HIV infection. Science 234(4780):1120–1123

    Article  PubMed  CAS  Google Scholar 

  13. Kaiser P, Joos B, Niederöst B, Weber R, Günthard HF, Fischer M (2007) Productive human immunodeficiency virus type 1 infection in peripheral blood predominantly takes place in CD4/CD8 double-negative T lymphocytes. J Virol 81(18):9693–9706

    Article  PubMed  CAS  Google Scholar 

  14. Zhu T, Muthui D, Holte S, Nickle D, Feng F, Brodie S, Hwangbo Y, Mullins JI, Corey L (2002) Evidence for human immunodeficiency virus type 1 replication in vivo in CD14(+) monocytes and its potential role as a source of virus in patients on highly active antiretroviral therapy. J Virol 76(2):707–716

    Article  PubMed  CAS  Google Scholar 

  15. Hess C, Klimkait T, Schlapbach L, Del Zenero V, Sadallah S, Horakova E, Balestra G, Werder V, Schaefer C, Battegay M, Schifferli J (2002) Association of a pool of HIV-1 with erythrocytes in vivo: a cohort study. Lancet 359(9325):2230–2234

    Article  PubMed  Google Scholar 

  16. Bruisten S, van Gemen B, Koppelman M, Rasch M, van Strijp D, Schukkink R, Beyer R, Weigel H, Lens P, Huisman H (1993) Detection of HIV-1 distribution in different blood fractions by two nucleic acid amplification assays. AIDS Res Hum Retroviruses 9(3):259–265

    Article  PubMed  CAS  Google Scholar 

  17. Bruns T, Steinmetzer K, Ermantraut E, Stallmach A (2009) Hepatitis C virus RNA quantitation in venous and capillary small-volume whole-blood samples. J Clin Microbiol 47(10):3231–3240

    Article  PubMed  CAS  Google Scholar 

  18. Steinmetzer K, Seidel T, Stallmach A, Ermantraut E (2010) HIV load testing with small samples of whole blood. J Clin Microbiol 48(8):2786–2792

    Article  PubMed  Google Scholar 

  19. Cotten M, Oberhauser B, Brunar H, Holzner A, Issakides G, Noe CR, Schaffner G, Wagner E, Birnstiel ML (1991) 2′-O-methyl, 2′-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. Nucleic Acids Res 19(10):2629–2635

    Article  PubMed  CAS  Google Scholar 

  20. Kumar R, Singh SK, Koshkin AA, Rajwanshi VK, Meldgaard M, Wengel J (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2′-thio-LNA. Bioorg Med Chem Lett 8(16):2219–2222

    Article  PubMed  CAS  Google Scholar 

  21. Saldanha J, Lelie N, Heath A (1999) Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. WHO Collaborative Study Group. Vox Sang 76(3):149–158

    Article  PubMed  CAS  Google Scholar 

  22. Holmes H, Davis C, Heath A, Hewlett I, Lelie N (2001) An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques. J Virol Methods 92(2):141–150

    Article  PubMed  CAS  Google Scholar 

  23. Al-Soud WA, Rådström P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39(2):485–493

    Article  PubMed  CAS  Google Scholar 

  24. NCCLS (2003) Evaluation of the Linearity of quantitative measurement procedures: a statistical approach; approved guideline. NCCLS document EP6-A (ISBN 1-56238-498-8). NCCLS, Pennsylvania, USA

    Google Scholar 

Download references

Acknowledgments

We wish to thank our colleagues from Alere Technologies Peter Slickers and Ralf Ehricht for the design of the oligonucleotides used to extract, amplify, and detect HCV and HIV RNA and Kornelia Kuhn and Monique Rüttger for performing most of the experiments to test oligonucleotide sequences and optimize procedures.

Author information

Authors and Affiliations

  1. Division of Gastroenterology, Hepatology, and Infectious Diseases, Department of Internal Medicine II, Jena University Hospital, Jena, Germany

    Tony Bruns

  2. Integrated Research and Treatment Center of Sepsis Control and Care (CSCC), Jena University Hospital, Jena, Germany

    Tony Bruns

  3. Alere Technologies GmbH, Jena, Germany

    Katrin Steinmetzer

Authors
  1. Tony Bruns
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katrin Steinmetzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tony Bruns .

Editor information

Editors and Affiliations

  1. University Hospital, Institute of Medical Microbiology, Heinrich-Heine-University, Universitätssraße 1, Düsseldorf, 40225, Germany

    Colin R. MacKenzie

  2. University Hospital, Institute of Medical Microbiology, Heinrich-Heine-University, Universitätssraße 1, Düsseldorf, 40225, Germany

    Birgit Henrich

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Bruns, T., Steinmetzer, K. (2012). Guidelines for the Quantification of HIV and HCV in Small Volume Whole Blood Samples. In: MacKenzie, C., Henrich, B. (eds) Diagnosis of Sexually Transmitted Diseases. Methods in Molecular Biology, vol 903. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-937-2_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-937-2_3

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-936-5

  • Online ISBN: 978-1-61779-937-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation