Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples

PLOS ONE, Dec 2019

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.

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Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples

August Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples Gianni Bussolati 0 1 Laura Annaratone 0 1 Enrico Berrino 1 Umberto Miglio 1 Mara Panero 1 Marco Cupo 0 1 Patrizia Gugliotta 0 1 Tiziana Venesio 1 Anna Sapino 0 1 Caterina Marchiò 0 1 0 Department of Medical Sciences, University of Turin , Turin , Italy , 2 Candiolo Cancer Institute - Fondazione del Piemonte per l'Oncologia (FPO), IRCCS , Candiolo , Italy , 3 Pathology Division, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino , Turin , Italy 1 Editor: Cesario Bianchi, Universidade de Mogi das Cruzes , BRAZIL 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. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Italian Ministry of Education, University and Research (MIUR ex-60%-2016-17 to CM and PRIN 2015HAJH8E), by AIRC, Associazione Italiana per la Ricerca sul Cancro (MFAG13310 to CM), and by 5xMille Ministero Salute 2011 - FPRC onlus AIRC 5xMille Molecular Clinical Oncology Extension program - Ref 9970 (to AS). The funders had no Introduction 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]. Over time, 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. On the other hand, 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 role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. (category 1B/2) and mutagen. This regulation may exert a heavy impact on diagnostic pathology. Reaction to this status of affairs is presently limited to adoption of protective procedures, designed to prevent excessive exposure to formaldehyde vapors. To be accepted by the scientific community, an ªalternative fixativeº should likely be an aldehyde, thus acting in a chemical reaction similar to that of formalin and affecting proteins and nucleic acids in a comparable way. This would avoid dramatic effects on IHC and molecular procedures and permit their use with minor adjustments, so that the bulk of the acquired and internationally accepted diagnostic parameters would not be lost. In addition, a possible alternative fixative should be relatively cheap, ideally in line with formalin, so as not to increase the final cost of histopathologic examinations. Moreover, it should not be toxic to avoid further restrictions. Finally, it should be rather fast, without affecting present turn around times. Glyoxal (aka ethanedial, oxalaldehyde) was proposed in 1943 [ 4 ] as a fixative alternative to formalin since it is a simple di-aldehyde. As reported by Harke & HoÈffler [ 5 ] glyoxal does not appear to evaporate from solution. Indeed, the reported Henry law constant of 3.38 × 10± 4Pa m3/mol [ 6 ] indicates that glyoxal is essentially non-volatile with regard to the aqueous phase. Glyoxal is not classifiable as a human carcinogen [ 7 ], nevertheless is irritating to skin and eyes [ 7 ]. Tumor-promoting activity of glyoxal h (...truncated)


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Gianni Bussolati, Laura Annaratone, Enrico Berrino, Umberto Miglio, Mara Panero, Marco Cupo, Patrizia Gugliotta, Tiziana Venesio, Anna Sapino, Caterina Marchiò. Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples, PLOS ONE, 2017, Volume 12, Issue 8, DOI: 10.1371/journal.pone.0182965