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

J Clin Virol. 2016 Jul;80:36-9. doi: 10.1016/j.jcv.2016.04.016. Epub 2016 Apr 27.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Viral / isolation & purification*
  • Female
  • Formaldehyde
  • Genotype
  • Human papillomavirus 16 / genetics*
  • Humans
  • Molecular Typing / methods*
  • Papillomavirus Infections / diagnosis*
  • Paraffin Embedding
  • Sensitivity and Specificity
  • Tissue Fixation
  • Uterine Cervical Neoplasms / virology*
  • Xylenes

Substances

  • DNA, Viral
  • Xylenes
  • Formaldehyde