Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2016 Jul:80:36-9.
doi: 10.1016/j.jcv.2016.04.016. Epub 2016 Apr 27.

Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples

Camilla Lagheden  1 , Carina Eklund  1 , Sara Nordqvist Kleppe  1 , Elizabeth R Unger  2 , Joakim Dillner  3 , Karin Sundström  1
Affiliations
Comparative Study

Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples

Camilla Lagheden et al. J Clin Virol. 2016 Jul.

Abstract

Background: Formalin-fixed paraffin-embedded (FFPE) samples can be DNA-extracted and used for human papillomavirus (HPV) genotyping. The xylene-based gold standard for extracting FFPE samples is laborious, suboptimal and involves health hazards for the personnel involved.

Objectives: To compare extraction with the standard xylene method to a xylene-free method used in an HPV LabNet Global Reference Laboratory at the Centers for Disease Control (CDC); based on a commercial method with an extra heating step.

Study design: Fifty FFPE samples were randomly selected from a national audit of all cervical cancer cases diagnosed in Sweden during 10 years. For each case-block, a blank-block was sectioned, as a control for contamination. For xylene extraction, the standard WHO Laboratory Manual protocol was used. For the CDC method, the manufacturers' protocol was followed except for an extra heating step, 120°C for 20min. Samples were extracted and tested in parallel with β-globin real-time PCR, HPV16 real-time PCR and HPV typing using modified general primers (MGP)-PCR and Luminex assays.

Results: For a valid result the blank-block had to be betaglobin-negative in all tests and the case-block positive for beta-globin. Overall, detection was improved with the heating method and the amount of HPV-positive samples increased from 70% to 86% (p=0.039). For all samples where HPV type concordance could be evaluated, there was 100% type concordance.

Conclusions: A xylene-free and robust extraction method for HPV-DNA typing in FFPE material is currently in great demand. Our proposed standardized protocol appears to be generally useful.

Keywords: DNA-extraction; Formalin-Fixed paraffin-Embedded (FFPE); Genotyping; Human papillomavirus (HPV); Xylene.

PubMed Disclaimer

Conflict of interest statement

Competing interest None.

Figures

Figure 1
Figure 1
Box plot for betaglobin count in log10 (copies/microliter). Inadequate samples/pairs removed (n=9).
Figure 2
Figure 2
Box plot for HPV16 copy number in log10(copies/microliter). Inadequate samples/pairs removed (n=5).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. International Agency for Research on Cancer. Monographs. Lyon, France: International Agency for Research on Cancer; 2012. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. - PMC - PubMed
    1. Bzhalava D, Eklund C, Dillner J. International standardization and classification of human papillomavirus types. Virology. 2015;476:341–4. Epub 2015/01/13. - PubMed
    1. Soderlund-Strand A, Uhnoo I, Dillner J. Change in population prevalences of human papillomavirus after initiation of vaccination: the high-throughput HPV monitoring study. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2014;23(12):2757–64. Epub 2014/11/09. - PubMed
    1. Alvarez-Aldana A, Martinez JW, Sepulveda-Arias JC. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus. Pathology, research and practice. 2015;211(2):150–5. Epub 2014/12/03. - PubMed
    1. Steinau M, Patel SS, Unger ER. Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues. The Journal of molecular diagnostics: JMD. 2011;13(4):377–81. Epub 2011/06/28. - PMC - PubMed

Publication types