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

Cristina Zanini1*, Elisa Gerbaudo2, Elisabetta Ercole1, Anna Vendramin3 and Marco Forni1

Author Affiliations

1 Research Laboratory of EuroClone S.p.A at Molecular Biotechnology Centre (MBC), University of Turin, Turin, Italy

2 Department of ImmunoHematology, A.O. Ospedale Infantile Regina Margherita (OIRM), S.Anna, Turin, Italy

3 Research Laboratory of EuroClone S.p.A, Basovizza, TS, Italy

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Environmental Health 2012, 11:59  doi:10.1186/1476-069X-11-59

Published: 4 September 2012



Formaldehyde (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.


More 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.


Differences 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.


Abolition 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.

Formalin toxicity; Alternative fixatives; Tissue micro array; RNA extraction