Abstract
Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.
Highlights
Fixation of histological specimens in formalin is in practice since over a century [1, 2] and still represents the procedure of choice for tissue preservation [3]
Additional and ancillary techniques such as immunohistochemistry (IHC) and molecular analyses have been optimized in formalin-fixed paraffin embedded (FFPE) tissues, so that a sudden change of fixative is presently considered as impractical being potentially detrimental to the quality of diagnostic pathology
Morphology of the 30 tissues included in the cohort fixed in phosphate buffered formalin (PBF) or in glyoxal acid-free (GAF) was considered by analyzing nuclear features such as nuclear shape and distribution of chromatin, staining characteristics, shrinkage around glandular structures or cellular aggregates
Summary
Fixation of histological specimens in formalin is in practice since over a century [1, 2] and still represents the procedure of choice for tissue preservation [3]. Additional and ancillary techniques such as immunohistochemistry (IHC) and molecular analyses have been optimized in formalin-fixed paraffin embedded (FFPE) tissues, so that a sudden change of fixative is presently considered as impractical being potentially detrimental to the quality of diagnostic pathology. Environmental authorities are increasingly concerned for the objective toxicity of this volatile reagent, so that a banning of formalin from 2016 has been proposed in the European Community. This has been stated by the EC Regulation n.605/2014 of 05.06.2014 that modifies the EC Regulation n.1272/2008 defining formalin as a carcinogen. Acid-free glyoxal as a potential novel fixative role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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