Effects of short and long-term alcohol-based fixation on Sprague-Dawley rat tissue morphology, protein and nucleic acid preservation

Abstract

Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory's trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation. Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.

Keywords: Alcohol; DNA; Fixative; Formaldehyde; Immunohistochemistry; Pathology; RNA.

Conflict of interest statement

Declaration of Competing Interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. The authors also declare that their funding sources had no direct role in the study design, data collection, analysis and interpretation of the data, in the writing of the manuscript, or in the decision to publish the work.

Copyright © 2019 Elsevier GmbH. All rights reserved.

Figures

Fig. 1.

Female Sprague-Dawley rat spleen tissues stained with: H&E, Mallory’s Trichrome, Ki67, CD3, PAX5, CD68 (10 X). Tissue samples were fixed either in 10% formalin (NBF) for 48 h (1a – 1f) or in 70% solution of Solvanol (ethyl alcohol 60%, isopropyl alcohol 40% and distilled water), for 48 h (2a – 2f), 1 week (3a – 3f), 1 month (4a – 4f), 1 year (5a – 5f), 4 years (6a – 6f), 7 years (7a – 7f).