Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues

Abstract

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher ( P < 0.05 ) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.

Standardization of Manual Method of Immunohistochemical Staining for Breast Cancer Biomarkers at Tertiary Cancer Care Center: An Audit

“…In our experience, tissue floating was observed using isopropyl alcohol; therefore, its use should be discouraged in manual IHC staining [ 9 , 10 ]. During the process, the tissue undergoes fixation and dehydration, and ethyl alcohol used in different grades (50%, 70%, 90%, 100%) is found to accurate fixative as well as dehydration solution, so no tissue floatation was seen [ 11 ].…”

Standard techniques for formalin-fixed paraffin-embedded tissue: A Pathologist’s perspective

“…Tissue fixation uses a chemical solution such as formalin can be considered a chemical reaction since tissue fixation occurs by one of these three mechanisms: fixation by covalent bond, fixation by protein coagulation, and fixation by forming precipitates formalin considered as a covalent bond fixative (29). Protein fixation by formalin done by two steps; the first one called fast step and starts by forming a covalent bonds with amine groups in amino acids (especially lysine, arginine, tyrosine, histidine, glutamine, and serine) within first 12 hours, later the slow step starts by reaction of glycol methyl groups with each other by methylene bridges [-CH2-] to connect two proteins molecule together (30), while the nucleic acid fixed by formalin through the action of methyl glycol groups that bind to free amino groups in the nucleic acid chains causing fixation of these chains, on other hand the lipoproteins fixed by formalin via the action of methyl glycol groups that produced from the hydrolysis of formalin and bind to carbon particles or Sulphur groups [-SH] recent in these compounds this will cause fixation of lipoproteins, in contrast, formalin did not react or fix the carbohydrate specially long chains from glucose because they consider as chemically inactive compounds and trapped in the other fixed cellular compounds. They fixed by methyl glycol groups (31) (Figure 3 and 4).…”

The hazardous effects of formalin and alcoholic fixative in mice: A public health perspective study

“…While the combination of this two alcohol have proven their potency as tissue fixative through their usefulness in cytology; when used alone, both can act as causal factor for shrinkage and fragility of tissue ( Adrian et al, 2000 ). An alcohol-based fixative is a safer alternate of formalin which keeps nucleic acid intact during preservation as well facilitate the microscopic and immunohistochemical investigations ( Haque et al, 2020 , Rahman et al, 2021 ).…”

“…Furthermore, alcoholic fixation has advantages over formalin fixation in terms of penetration, preservation, and a safer working environment. Furthermore, the EMA's preservation capacity has already been investigated in one of our prior experiments ( Haque et al, 2020 , Rahman et al, 2021 ). However, its health hazards due to exposure need to be explored compared to the gold standard fixative, formalin.…”