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Updated: May 23, 2026
Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation
Published on: December 25, 2017
How much formalin is enough to fix tissues?
René J Buesa1, Maxim V Peshkov
1rjbuesa@yahoo.com
Insights
Optimal tissue fixation for histopathology requires 48 hours using neutral buffered formalin (NBF) at a 2:1 volume ratio. This ensures proper tissue fixation and infiltration for diagnostic purposes.
Area of Science:
- Histopathology
- Tissue processing
- Diagnostic pathology
Background:
- Standardized tissue fixation is crucial for accurate histopathological diagnosis.
- Optimizing fixation parameters can improve tissue processing and diagnostic quality.
Purpose of the Study:
- To determine the optimal fixation period and volume ratio of neutral buffered formalin (NBF) for various human tissues.
- To evaluate the impact of fixation parameters on microtomy quality, pH, and specific staining.
Main Methods:
- Sixty human tissue samples (breast, uterus, liver, skin, fat) were fixed with NBF at varying times (8, 24, 48 hours) and volume ratios (1:1, 2:1, 5:1, 10:1) at room temperature.
- Tissues were processed using isopropyl alcohol and mineral oil mixtures.
- Pathologists evaluated slide suitability for diagnosis, and microtomy quality was assessed.
- pH, immunostaining (vimentin), and histochemical reactions (periodic acid, reticular fibers) were analyzed.
Main Results:
- A 48-hour fixation period was found to be optimal, with skin and fat being the most challenging tissues for infiltration.
- Fixation volume ratios did not significantly impact microtomy quality.
- Fixation time and volume ratio combinations did not affect NBF pH or specific staining results.
- A NBF volume to tissue volume ratio of 2:1 for 48 hours at 20-22°C ensured proper fixation and infiltration for tested tissues.
Conclusions:
- 48 hours of fixation with NBF at a 2:1 volume ratio is sufficient for proper fixation and infiltration of various human tissues.
- Accelerated fixation (around 10 hours) may be achievable at 45°C with pressure and agitation.
- The findings suggest a standardized approach for optimal tissue preparation in histopathology.
Abstract:
A total of sixty samples from human breast, uterus, liver, skin and abdominal fat were fixed for 8; 24 and 48 hours at a room temperature of 20 to 22°C with neutral buffered formalin (NBF) with volume to tissue ratios of 1:1; 2:1; 5:1 and 10:1 and manually processed with isopropyl alcohol and mineral oil mixtures. All the slides prepared were evaluated as suitable for diagnostic purposes by nine pathologists from three different Russian histopathology institutions. The microtomy quality differences between the samples was not statistically significant for the different fixation volume ratios tested, but the differences between fixation periods and tissues types were, with 48 hours being the optimum fixation period, with skin and fat the most difficult to infiltrate. Neither the time and volume ratio combinations affected the pH of NBF or the immunostaining for vimentin in uterus or the histochemical periodic acid reaction or reticular demonstration fibers in liver. Fixing tissues with a ratio of NBF volume to tissue volume of 2:1 for 48 hours at 20-22°C was enough to assure a proper fixation and infiltration of the tested tissues and there is no objective reason to expect that other tissues will not behave similarly. It is suggested that in order to obtain good fixation and paraffin wax infiltration in around 10 hours, the fixation with NBF at 2:1 should be at 45°C with pressure and agitation.
