Formaldehyde fixation may be the gold standard for preserving cells and tissues but alcohol-based fixation can deliver improved results in biomolecular techniques - with the benefit of safer, greener chemistry. Advances in tissue interrogation methods and the sustainability of manufacturing are setting the stage for a non-toxic fixative future.
formaldehyde advantages come at the expense of high toxicity
Formaldehyde is a gas produced industrially by the catalytic oxidation of methanol. Paraformaldehyde is polymerized formaldehyde in a powder form. Both are used in fixation solutions in life science labs. Biologists fixing cells or tissues for staining are most often familiar with paraformaldehyde solutions. Histology and pathology clinical labs more commonly use formalin, a saturated formaldehyde solution.
Formaldehyde fixation was discovered accidentally by Ferdinand Blum in the 1890s while investigating its antimicrobial properties. Blum noticed that the skin on his fingers had hardened from contact with the compound. He was intrigued since at that time alcohol fixation had been the norm for preserving tissues. Further tests showed that formaldehyde fixation worked well with common histology stains. Formalin-fixed tissue samples had less shrinkage than alcohol-fixed tissues. Formaldehyde-based fixation became and remains the most popular method in histology labs.
Modern methods still show that paraformaldehyde is the most effective at preserving the overall cell content and distribution of cellular components. Today histology labs standardly use formalin-fixed paraffin-embedded (FFPE) blocks of tissue. This fixation method faithfully preserves cellular and tissue morphology for pathology lab diagnostics. Its popularity is also based on the excellent stability of stored FFPE samples.
From a sustainability perspective, the potential environmental impacts of formaldehyde can be thwarted. Used formalin can be effectively neutralized with Neutralex® or FormaGO® for disposal. Alternatively, formalin solutions can be recycled by labs to reduce both lab running and environmental costs.
The main problem lies in its toxicity to users. A brief inhalation of formaldehyde gives you pause and a burning sensation around your nose and eyes. Lifetime exposure to the lungs and nose is worrisome. The EPA lists formaldehyde as a probable human carcinogen. Fume hoods can make formaldehyde-based fixation safer for microscopic techniques. Health risks from inhalation are less avoidable in macroscopic, medical settings.
The sustainability of Ethanol and methanol
The path to lowering carbon footprints from life science work can be paved in part by how lab materials are manufactured. Ethanol is well established due to investment in biofuels. Methanol is an emerging opportunity.
Ethanol is a low environmental risk solvent already used in different grades by life science labs with exciting, recent advances in sustainable manufacturing. Today only about 5% of the ethanol produced in the world is a synthetic ethanol petroleum product, as compared to ~57% in 1952. (A good start!) The rest is produced by fermentation using renewable plant feedstock. This is considered bio-based or bio-renewable ethanol. In terms of its use as a biofuel, ethanol can be net zero carbon because CO2 from burning ethanol is offset by the carbon capture of the feedstock crop.
Bio-based ethanol has had a bumpy path to sustainable production not without scientific controversy. However, the most exciting breakthroughs are based on research using cellulosic feedstocks that are scaled up in industrial plants. While it is a longer production process, cellulosic (non-food crop) feedstocks do not impair food security. Brazil leads the world in production plants using waste from sugarcane crops.
On the other hand, nearly all methanol is produced with fossil fuels. The technology exists for renewable methanol but the main barrier is cost. It’s a shame because according to the Methanol Institute, compared to conventional fuels, renewable methanol cuts carbon dioxide emissions by up to 95%, reduces nitrogen oxide emissions by up to 80%, and completely eliminates sulfur oxide and particulate matter emissions. With an increasing demand for bio-based options in the life science industry, companies are expanding their offerings.
The Life Science business of Merck KGaA, Darmstadt, Germany, which operates as Millipore Sigma in the U.S. and Canada, has the broadest portfolio of bio-based solvents for the life science industry and an outstanding reputation for supporting sustainability and Green Chemistry in particular. I wanted to know their outlook on this space. Jeffrey Whitford, Head of Sustainability & Social Business Innovation, kindly shared, “We're on a continual journey to evolve our approach. We learn, take steps forward and learn more and adjust. It's progressive learning in practice. Ultimately, where we want to land is using waste as the feedstock. While biobased ethanol will be more expensive for the time being due to the scale of availability, over time as the bio-based industry expands, the price should come down. The market and demand signals are important so that there is further investment in the expansion of the industry.” To me, this is the right outlook to enable life science to practice environmental stewardship. It’s a journey.
The end-of-life impacts of these alcohols are in the hands of scientists. After ethanol or methanol is used scientific facilities have a choice to burn this material with a hazardous waste service or recycle it on site with distillation,(i.e. Rotovapor equipment). The University of Colorado Boulder Green Labs program provides a cost-saving program to recycle ethanol. Over several years 120 gallons of ethanol have been diverted from hazardous waste for reuse by Biochemistry Cell Culture labs with a purchasing savings of $1500 per year. If you are considering this type of program it’s worth noting that technically mixed alcohols can be separated by adding fractional distillation to customized solvent recycling equipment. In any used solvent or fixation solution scenario, environmental health and safety can help guide scientists on meeting local regulations and the expected environmental life-cycle impact of disposal methods.
Improved technical results
Given the safety profile of formalin and paraformaldehyde and molecular analyses in use today for research and diagnostics, there is growing interest in alcohol-based tissue fixation. In the recent paper, Md. Asabur Rahman et al. Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues, the authors generated supportive data and referenced the body of evidence for substituting formalin fixation with safer and cheaper alcohol-based alternatives.
Ethanol, methanol and acetic acid (EMA) fixation has several advantages over formalin. EMA penetrates tissues faster. EMA is relatively non-toxic whereas Formalin is noxious and highly toxic. Formalin is more expensive than EMA fixative. Alcohol-fixed tissue sections have increased immunogenicity so depending on the epitope, lower antibody concentrations may be used. The quantity and purity of nucleic acid recovered from EMA-treated tissues is higher. DNA and RNA recovered from alcohol fixed paraffin embedded (AFPE) tissue samples have improved integrity which is better retained over extended storage times compared to FFPE tissue samples.
Conclusion
The fixation method will always impact sample yield and quality. To be fair, there is no universally perfect fixation for nucleic acid detection, protein staining or immunochemistry. Experimental conditions must be optimized for targets and interrogative biomolecular techniques.
To those scientists and clinical histologists using alcohol-based fixation methods, I say, that’s the spirit!
