Application of alternative fixatives to formalin in diagnostic pathology

  • L. Benerini Gatta | l.benerini@virgilio.it University of Brescia, Italy.
  • M. Cadei University of Brescia, Italy.
  • P. Balzarini University of Brescia, Italy.
  • S. Castriciano Copan Italia, Italy.
  • R. Paroni Copan Italia, Italy.
  • A. Verzeletti University of Brescia, Italy.
  • V. Cortellini University of Brescia, Italy.
  • F. De Ferrari University of Brescia, Italy.
  • P. Grigolato University of Brescia, Italy.

Abstract

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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Author Biographies

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
Published
2012-05-04
Section
Original Papers
Supporting Agencies
The work was supported by ex-60% funds from the University of Brescia.
Keywords:
fixation, histochemistry, immunohistochemistry, molecular biology
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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