Gianni Bussolati et al., Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples. Plos One

Gianni Bussolati1, Laura Annaratone1, Enrico Berrino2, Umberto Miglio2, Mara Panero2, Marco Cupo1, Patrizia Gugliotta1, Tiziana Venesio2, Anna Sapino1,2, Caterina Marchiò1,3

1Department of Medical Sciences, University of Turin, Turin, Italy
2Candiolo Cancer Institute – Fondazione del Piemonte per l’Oncologia (FPO), IRCCS, Candiolo, Italy
3Pathology Division, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy


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.