title: Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues.
abstract: Advantageous preservation of histology and detailed cellular morphology has rendered neutral buffered formalin (NBF) the most widely used fixative in clinical pathology. Despite excellent morphology for routine diagnostics, a major drawback of NBF fixation is its detrimental effect on DNA and RNA quality. In addition to complicating analysis of genes and transcripts in complex tissues, NBF denatures proteins and thereby hampers immunohistochemical visualization of certain antigens. In the present study, we evaluated a zinc-based fixative (ZBF) regarding its effects on tissue morphology, quality of genomic DNA, and preservation of protein immunoreactivity in a broad spectrum of tissues. Four different modes of fixation were analyzed: ZBF-paraffin embedding, NBF-paraffin embedding, ZBF-fixation prior to snap-freezing, and immediate snap-freezing. Laser-assisted microdissection, allowing retrieval of a defined number of cells for PCR, was used to study DNA quality. Genomic DNA was analyzed using primers for beta2-microglobulin and the transferrin receptor. Immunohistochemistry was performed using nine antibodies. Tissue microarray blocks were used for analysis of morphology and immunoreactivity. Only slight impairment of morphologic qualities was found after ZBF-paraffin embedding, whereas ZBF prior to freezing resulted in a more crisp morphology compared with routine cryosections. A significantly higher DNA yield was observed in samples isolated from ZBF-paraffin-embedded tissues compared with NBF-paraffin-embedded tissues. Both yield and quality of DNA was comparable in frozen tissues irrespective to prior ZBF fixation. Immunoreactivity in paraffin-embedded tissue was superior in ZBF-fixated tissue compared with NBF-fixated for a majority of tested antibodies. Furthermore, for seven out of nine antibodies, antigen retrieval pretreatment proved unnecessary in ZBF-fixated tissue. Thus, despite a slight impairment of morphology, ZBF preserves protein structures well. We conclude that ZBF is superior to NBF for analysis of DNA and protein expression. Fixation of tissues in ZBF may also be an alternative strategy to freeze storage of tissue specimens, eg, in future bio-banks.
authors.affiliation: Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden. kenneth.wester@genpat.uu.se
authors.forename: Kenneth
authors.initials: K
authors.lastname: Wester
authors.forename: Anna
authors.initials: A
authors.lastname: Asplund
authors.forename: Helena
authors.initials: H
authors.lastname: Bäckvall
authors.forename: Patrick
authors.initials: P
authors.lastname: Micke
authors.forename: Andra
authors.initials: A
authors.lastname: Derveniece
authors.forename: Ilona
authors.initials: I
authors.lastname: Hartmane
authors.forename: Per-Uno
authors.initials: PU
authors.lastname: Malmström
authors.forename: Fredrik
authors.initials: F
authors.lastname: Pontén
mesh_terms: D002295:Carcinoma, Transitional Cell / Q000473:pathology; D048429:Cell Size; D004247:DNA / Q000235:genetics*; D005404:Fixatives; D006652:Histological Techniques / Q000379:methods*; D006801:Humans; D008214:Lymphocytes / Q000473:pathology; D008297:Male; D011506:Proteins / Q000235:genetics*; D012867:Skin / Q000166:cytology / Q000473:pathology; D015032:Zinc
chemical_list: D005404:Fixatives; D011506:Proteins; D004247:DNA; D015032:Zinc