Application of alternative fixatives to formalin in diagnostic pathology

Submitted: 19 May 2011
Accepted: 10 January 2012
Published: 4 May 2012
Abstract Views: 1740
PDF: 1241
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Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.

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Supporting Agencies

The work was supported by ex-60% funds from the University of Brescia.
M. Cadei, University of Brescia
Second Department of Pathology, School of Medicine
P. Balzarini, University of Brescia
Second Department of Pathology, School of Medicine
A. Verzeletti, University of Brescia
Department of Forensic Medicine, School of Medicine
V. Cortellini, University of Brescia
Department of Forensic Medicine, School of Medicine
F. De Ferrari, University of Brescia
Department of Forensic Medicine, School of Medicine
P. Grigolato, University of Brescia
Second Department of Pathology, School of Medicine

How to Cite

Benerini Gatta, L., Cadei, M., Balzarini, P., Castriciano, S., Paroni, R., Verzeletti, A., Cortellini, V., De Ferrari, F., & Grigolato, P. (2012). Application of alternative fixatives to formalin in diagnostic pathology. European Journal of Histochemistry, 56(2), e12. https://doi.org/10.4081/ejh.2012.12

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