Non-toxic acid-free glyoxal fixative for veterinary gross specimen preservation, histopathology, immunohistochemistry, and molecular analysis

Formaldehyde-based fixation is the most used chemical system for histopathological examination worldwide. However, its toxicity is well known, and preservation of gross features, proteins, and nucleic acids is not optimal. Alternative fixatives resulting in similar morphological tissue quality and costs, but with reduced toxicity and with better preservation of gross features, proteins, and nucleic acids would increase operator safety and application possibilities in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular outcomes of fixation with a newly patented, non-toxic, acid-free glyoxal (GAF) fixative with neutral-buffered formalin (NBF). Fifty-nine tissue biopsies and 21 necropsies of different animal species were analyzed. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was better preserved with GAF. Histology, histochemistry, and in situ hybridization results from GAF-fixed samples were mainly equal when compared to NBF-fixed samples, except for the loss of mast cell granules in GAF-fixed samples compared to NBF. IHC analyses showed comparable results with slight and rare protocol adjustment. DNA yields were higher and amplification of selected genes (ie, TP53 and COX1) was more efficient in GAF-fixed biopsies (P < .05). DNA and RNA yields were higher also in necropsy GAF-fixed tissues, but no difference was detected for selected gene amplification (ie, COX1, GAPDH, β-actin). Based on these data, despite not yet being economically competitive, GAF could represent a valuable alternative to NBF for standard laboratory applications, while also improving on-field sampling and teaching applications.