Comparison of the Fixation Effects of Four Fixatives on Frozen Sections

doi:10.3969/j.issn.1674-3245.2025.05.036

Abstract：Objective To investigate the fixation effects of four fixatives, including 10% neutral buffered formalin, methanol, 95% ethanol, and alcohol-acetate-formalin (AAF), on frozen sections. Methods A total of 34 thyroid specimens and 36 breast specimens collected from the Department of Pathology of Linfen Central Hospital between October 2023 and October 2024 were prospectively collected. After cryosectioning, the specimens were fixed with 10% neutral buffered formalin, methanol, 95% ethanol, or AAF, followed by hematoxylin-eosin (HE) staining. The histological structure and staining quality were observed under each fixative condition. Results After 1 minute of fixation, the HE staining scores for AAF and 95% ethanol were higher than those for methanol, which were higher than those for 10% neutral buffered formalin, with statistically significant differences (P < 0.05). There was no statistically significant difference between AAF and 95% ethanol (P > 0.05). After 4 minutes and 7 minutes of fixation, the HE staining scores for AAF were higher than those for 95% ethanol, which were higher than those for methanol, which were higher than those for 10% neutral buffered formalin, with statistically significant differences (P < 0.05). At 4 minutes of fixation, 10% neutral buffered formalin resulted in unclear tissue structure, poor cytoplasmic staining, nuclear shrinkage, and low nuclear-cytoplasmic contrast; methanol showed relatively clear structure, acceptable cytoplasmic staining, nuclear lysis, and good nuclear-cytoplasmic contrast; 95% ethanol showed clear structure, blurred nuclear staining, dull cytoplasmic staining, and nuclear shrinkage; AAF provided clear structure, vivid nuclear and cytoplasmic staining, and high nuclear-cytoplasmic contrast. Conclusion AAF fixative exhibited the best fixation effect on frozen sections, with bright HE staining, high nuclear-cytoplasmic contrast, clear tissue structure, accurate localization, and strong positivity, effectively improving the quality of frozen sections.