Evaluation of two commercial and three home-made fixatives for the substitution of formalin: a formaldehyde–free laboratory is possible

Abstract

BackgroundFormaldehyde (HCHO) is a gas (available as a 37% concentrated solution, stabilized with methanol). The 10% dilution (approximately 4% formaldehyde) has been used as a fixative since the end of the 19th century. Alternative fixatives are also commercially available or may be prepared in-house in laboratories. Statements by the IARC, along with other USA agencies (CalEPA, RoC/NTP) on the carcinogenicity of formaldehyde for humans renders its substitution in Pathology Departments necessary since the annual use of formalin may exceed 3,500 liters for a medium-large laboratory.To achieve a “formalin-free laboratory” we tested straightforward-to-make fixatives along with registered reagents offered as formalin substitutes.MethodsMore than two hundreds specimens were fixed in parallel with in-laboratory made fixatives PAGA (Polyethylenglycol, ethyl Alcohol, Glycerol, Acetic acid), two zinc-based fixatives (ZBF, Z7), and commercially-available alternatives (RCL2 and CellBlock). Tissue micro arrays were used for morphological and immunohistochemical comparison. Extraction of RNA was carried out to evaluate preservation of nucleic acids.ResultsDifferences compared to formalin fixation were evident in alcohol-based fixatives, mainly restricted to higher stain affinity and considerable tissue shrinkage. Conversely, nuclear detail was superior with these alcohol-based formulas compared to formalin or glyoxale-based recipes. RNA extraction was superior for Z7, PAGA and RCL2 with regard to concentration but relatively comparable regarding quality.ConclusionsAbolition of the human carcinogen formaldehyde from pathology laboratories is possible even in contexts whereby commercial alternatives to formalin are unavailable or are too expensive for routine use, and aspiration devices are lacking or not adequately serviced. The use of known formulations, possibly with simple and not-noxious (“alimentary grade”) constituents, comparable with registered proprietary products, may expand the search for the ideal fixative combining satisfactory morphology with improved preservation of nucleic acids and proteins as well as being easy and safe to dispose of.

Queen Triggerfish Reproductive Biology in U.S. Caribbean Waters

“…), or polyethylene glycol–ethyl alcohol–glycerol–acetic acid (PAGA) fixative (Zanini et al. ) for up to 2 weeks and then transferred to 70% isopropanol. Gonad samples were processed using standard histological procedures for triggerfish species (Lang and Fitzhugh ; Kelly‐Stormer et al.…”

Medullospheres from DAOY, UW228 and ONS-76 Cells: Increased Stem Cell Population and Proteomic Modifications

“…Cell-blocks were subsequently fixed overnight in a formaldehyde-free fixative as previous described [33]. After routine processing and inclusion in paraffin, pilot sections were cut and stained with Hematoxylin and Eosin.…”

Tannic Acid Solution: A Better Fixative Solution Than Formalin for Elastin and Collagen—Toxic and Morphological Assessment

“…These compounds must prevent postmortem tissue decay during long‐term storage, preserving cells and tissues in a “life‐like” state. However, they should also enable appropriate staining methods to be applied, such as those involving antigen–antibody reactions and those depending on proper preservation of DNA and RNA molecules (van Essen et al, ; Moelans et al, ; Zanini et al, ; Kennel et al, ).…”

“…Consequently, the quest for formaldehyde substitutes has been mainly motivated by its hazardous nature and the fact that it does not ensure complete DNA and RNA recovery, which is essential for a number of molecular biology tests (Masuda et al, ; Buesa, ; van Essen et al, ; Moelans et al, ; Zanini et al, ).…”

“…Numerous alternatives are commercially available (Zanini et al, ). These include mainly alcoholic and nonalcoholic solutions; solutions with or without acetic acid (Moelans et al, ), zinc‐based solution (Lykidis et al, ) and solutions containing less than 10% of formalin (Buesa, ; van Essen et al, ).…”