title: Formaldehyde substitute fixatives: effects on nucleic acid preservation.
abstract: OBJECTIVE
  In surgical pathology, formalin-fixed paraffin-embedded tissues are increasingly being used as a source of DNA and RNA for molecular assays in addition to histopathological evaluation. However, the commonly used formalin fixative is carcinogenic, and its crosslinking impairs DNA and RNA quality.
  
  
  METHODS
  The suitability of three new presumably less toxic, crosslinking (F-Solv) and non-crosslinking (FineFIX, RCL2) alcohol-based fixatives was tested for routine molecular pathology in comparison with neutral buffered formalin (NBF) as gold standard. Size ladder PCR, epidermal growth factor receptor sequence analysis, microsatellite instability (MSI), chromogenic (CISH), fluorescence in situ hybridisation (FISH) and qPCR were performed.
  
  
  RESULTS
  The alcohol-based non-crosslinking fixatives (FineFIX and RCL2) resulted in a higher DNA yield and quality compared with crosslinking fixatives (NBF and F-Solv). Size ladder PCR resulted in a shorter amplicon size (300 bp) for both crosslinking fixatives compared with the non-crosslinking fixatives (400 bp). All four fixatives were directly applicable for MSI and epidermal growth factor receptor sequence analysis. All fixatives except F-Solv showed clear signals in CISH and FISH. RNA yield and quality were superior after non-crosslinking fixation. qPCR resulted in lower Ct values for RCL2 and FineFIX.
  
  
  CONCLUSIONS
  The alcohol-based non-crosslinking fixatives performed better than crosslinking fixatives with regard to DNA and RNA yield, quality and applicability in molecular diagnostics. Given the higher yield, less starting material may be necessary, thereby increasing the applicability of biopsies for molecular studies.
authors.affiliation: Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands. cmoelans@umcutrecht.nl
authors.forename: Cathy B
authors.initials: CB
authors.lastname: Moelans
authors.forename: Daphne
authors.initials: D
authors.lastname: Oostenrijk
authors.forename: Michiel J
authors.initials: MJ
authors.lastname: Moons
authors.forename: Paul J
authors.initials: PJ
authors.lastname: van Diest
mesh_terms: D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000187:drug effects / Q000235:genetics; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D053842:Microsatellite Instability / Q000187:drug effects; D012313:RNA / Q000187:drug effects* / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D005404:Fixatives; D005557:Formaldehyde; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA