Fixation on Histology

Alternatives to Formalin

Fixation is an essential step in the histology process that preserves the tissue and makes the tissue receptive to dyes during staining. Click here to check out a recent post on the types of fixation. Most stains are designed for formalin fixed paraffin embedded tissue, so much so that histologists recognize FFPE as meaning just that. Despite its common usage, formalin does have its critics because of the safety risks. Formaldehyde is classified by OSHA as a human carcinogen, associated with respiratory difficulty, and skin and nasal irritation. Exposure in the laboratory is limited, and measures are taken to keep histotechs safe, but there have still been many studies focused on exploring alternatives to formalin in the histology lab.

Alternatives to FormalinOne such study examines the usability of honey as a formalin alternative. This study, published in the Journal of Histotechnology in 2006, used rat liver and kidney tissues, fixed in concentrations of honey ranging from 10%-100% diluted with distilled water. Though higher concentrations were found unsuccessful, their results concluded that honey concentrations of 10%-20% at room temperature gave similar results to that of formalin fixed tissue. Additional studies have since found similar results supporting honey’s use as an alternative to formalin. For example, a 2012 article in Biotechnic & Histochemistry noticed minor histomorphological differences, but confirmed that correct diagnosis was not impeded. A similar Journal of Histotechnology study from 2014 confirms again the use of honey as an acceptable fixative, particularly for nuclei. It does however caution against its use for connective tissue because of homogenization of collagen fibers in their results.


Studies have also been conducted testing the use of khandsari as a fixative alternative to formalin. Khandsari is a type of unprocessed sugar made from sugar cane syrup. More popular in India, it is sometimes seen as a healthier alternative to sugar because it contains minerals and other nutrients and isn’t chemically treated like processed sugars. This study on khandsari and jaggery syrup (another similar unrefined sugar cane substance), concluded similar results to the honey studies, including the limitations around connective tissue. It was also tested whether jaggery syrup would hold up over time, comparing the jaggery samples to formalin over a 6-month duration using special stains. This study confirmed that there was no significant differences between the jaggery syrup and formalin.


Another common alternative that has been studied on multiple occasions is the use of ethanol-based fixatives. One such Journal of Histotechnology study from 2000 proposes that ethanol based fixatives produced superior preservation of tissue morphology. One downside of the ethanol-based fixative is that it was not believed to kill viruses and bacteria in the tissue. Though universal precautions should always be taken, this may add additional risk, and be unsettling for the employees of the lab, particularly during the current COVID pandemic.


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