A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue is the predominant preparation for diagnostic histopathological evaluation and increasingly the biospecimen on which molecular diagnostics are performed. However, formalin is carcinogenic and results in cross-linking of proteins and nicking and alterations of nucleic acids. Alternative fixatives, including 70% ethanol, improved biomolecular integrity; however, they have yet to replace neutral-buffered formalin (NBF). Herein, we describe the phosphate-buffered ethanol 70% (BE70) fixative. The histomorphology of BE70-fixed tissue is very similar to that of NBF; however, it is a non-cross-linking fixative and lacks the carcinogenic profile of formaldehyde-based fixatives. RNA isolated from tissue fixed in BE70 was of substantially higher quality and quantity than that was recovered from formalin-fixed tissue. Furthermore, the BE70 fixative showed excellent RNA and DNA integrity compared with that of NBF fixative based on real-time polymerase chain reaction analysis results. Immunohistochemical staining was similar for the antigen tested. In conclusion, BE70 is a non-cross-linking fixative that is superior to NBF and 70% ethanol with reference to biomolecule recovery and quality from paraffin-embedded tissue. Additional studies to compare the histomorphologic and immunohistochemical performance and utility in a clinical setting are required.

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

“…This is not the case for the "gold standard" NBF, as emphasized in the work of Chung et al, (2018) in which scientists reported a progressive decrease of H&E staining intensity following 1 week to 6 months fixation. Finally, we observed some artifactual changes that have been previously described in alcohol-based fixed tissues, in particular shrinkage and hardening effects independently by fixation times (Perry et al, 2016). However, the degree of these alterations did not significantly influence the establishment of a proper diagnosis in our samples.…”

The Paraffin-Embedded RNA Metric (PERM) for RNA Isolated from Formalin-Fixed, Paraffin-Embedded Tissue

“…An alternative fixative showed the highest PERM values (mean = 121.23), while neutral buffered formalin showed the lowest PERM values (mean = 32.02) among tested fixatives (11). RNA isolated from tissue fixed in the alternative fixative resulted in a mean C q value of 34.46 for HPRT , whereas the C q value was higher in samples generated from formalin-fixed tissues (mean C q = 41.27).…”

“…In a retrospective study, PERM values were also correlated with RT-qPCR data (11). An alternative fixative showed the highest PERM values (mean = 121.23), while neutral buffered formalin showed the lowest PERM values (mean = 32.02) among tested fixatives (11).…”

“…We have additionally evaluated a series of alcohol-based alternative fixatives (11). The effect of fixatives on RNA integrity was evaluated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) amplification of the hypoxanthine-guanine phosphoribosyl transferase ( HPRT ) gene.…”

A Comparative Study of Fixatives for Axolotl Blastema Tissue

“…The key step of histological analysis of a tissue needs a proper fixation method and consequently availability of appropriate fixatives for that purpose. A proper fixative fluid should fulfill the following criteria: 1) prevention of autolysis, 2) preservation of structures between and 3) within the cells and inhibition of contamination by bacterial or molds growth (Perry et al, 2016). Therefore, comparison of various fixative solutions for blastema tissue in order to choose the proper one for the desired purpose would offer valuable information to utilize in histological evaluation in regeneration studies.…”