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

Details

• Alternative Title:
  J Clin Virol
• Personal Author:
  Lagheden, Camilla ; Eklund, Carina ; Kleppe, Sara Nordqvist ; Unger, Elizabeth R. ; Dillner, Joakim ; Sundström, Karin
• Description:
  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 20 minutes. 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, Viral DNA-extraction Female Formaldehyde Formalin-Fixed Paraffin-Embedded (FFPE) Genotype Genotyping Human Papillomavirus (HPV) Human Papillomavirus 16 Humans Molecular Typing Papillomavirus Infections Paraffin Embedding Sensitivity And Specificity Tissue Fixation Uterine Cervical Neoplasms Xylene Xylenes

  More +
• Source:
  J Clin Virol. 80:36-39
• Pubmed ID:
  27148635
• Pubmed Central ID:
  PMC5075962
• Document Type:
  Journal Article
• Funding:
  CC999999/Intramural CDC HHSUnited States/
• Volume:
  80
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:4e5d976e9b654bf92ca54675c045f9b8a049483cc109e0949fbaf68038e88200
• Download URL:
  https://stacks.cdc.gov/view/cdc/42220/cdc_42220_DS1.pdf
• File Type:
  [PDF - 390.47 KB ]