title: Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, F56 Huddinge, 14188 Stockholm, Sweden.
authors.forename: Camilla
authors.initials: C
authors.lastname: Lagheden
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, F56 Huddinge, 14188 Stockholm, Sweden.
authors.forename: Carina
authors.initials: C
authors.lastname: Eklund
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, F56 Huddinge, 14188 Stockholm, Sweden.
authors.forename: Sara Nordqvist
authors.initials: SN
authors.lastname: Kleppe
authors.affiliation: Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Unger
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, F56 Huddinge, 14188 Stockholm, Sweden; Medical Epidemiology & Biostatistics, Karolinska Institutet, SE-17177 Stockholm, Sweden. Electronic address: Joakim.Dillner@ki.se.
authors.forename: Joakim
authors.initials: J
authors.lastname: Dillner
authors.affiliation: Department of Laboratory Medicine, Karolinska Institutet, F56 Huddinge, 14188 Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Sundström
mesh_terms: D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D005557:Formaldehyde; D005838:Genotype; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D058889:Molecular Typing / Q000379:methods*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016612:Paraffin Embedding; D012680:Sensitivity and Specificity; D016707:Tissue Fixation; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D014992:Xylenes
chemical_list: D004279:DNA, Viral; D014992:Xylenes; D005557:Formaldehyde
keywords: DNA-extraction; Formalin-Fixed paraffin-Embedded (FFPE); Genotyping; Human papillomavirus (HPV); Xylene