Xylene, also known as xylol or dimethylbenzene, is an aromatic hydrocarbon known for its wide usage in tissue processing, staining and cover slipping in the histology laboratory. Xylene is a colourless, sweet-smelling liquid or gas occurring naturally in petroleum, coal and wood tar, and is so named because it is found in crude wood spirit. Xylene can easily leak into soil, surface water or groundwater after spills or as a result of a leak during storage or burial at a waste site.

For further information:

Public Health England has information here.

Xylene in the path lab

In histological processing, xylene’s high solvency factor allows maximum displacement of alcohol and renders the tissue transparent, enhancing paraffin infiltration. In staining procedures, its excellent dewaxing and clearing capabilities contribute to well-stained slides.

Associated health risks

Acute (short-term) inhalation of xylene vapours in humans may cause irritation of the eyes, nose, and throat, gastrointestinal effects, eye irritation, and neurological effects.

Chronic (long-term) inhalation exposure of humans to mixed xylenes results primarily in central nervous system (CNS) effects, such as headache, dizziness, fatigue, tremors, and incoordination; respiratory, cardiovascular, and kidney effects have also been reported.

Further information regarding the health aspects of xylene:

IARC –  Summaries and Evaluations – Xylenes  available here.

US Environmental Protection Agency – Xylenes available here.

T Rajan S, Malathi N. Health hazards of xylene: a literature review. J Clin Diagn Res. 2014 Feb;8(2):271-4. doi: 10.7860/JCDR/2014/7544.4079. Epub 2014 Feb 3. PMID: 24701554; PMCID: PMC3972585.

Kandyala R, Raghavendra SP, Rajasekharan ST. Xylene: An overview of its health hazards and preventive measures. J Oral Maxillofac Pathol. 2010 Jan;14(1):1-5. doi: 10.4103/0973-029X.64299. PMID: 21180450; PMCID: PMC2996004.

Environmental impacts

Xylene evaporates quickly, meaning that most xylene that gets into soil or water is released into the air and broken down by sunlight and less harmful chemicals within a couple of days. However, in underground water, it may take several months for xylene to degrade.

Safe disposal

An overview of the details on a company’s responsibility for hazardous waste disposal in the UK can be accessed here and there are companies which will collect and dispose of xylene safely.

Alternatives to xylene

A blog post published by the National Society for Histotechnology gives an overview of xylene alternatives and can be accessed here.

Natural and chemical alternatives

There are a number of publications comparing natural and chemical xylene substitutes in histology and xylene alternatives are available commercially.

Swamy SR, Nandan SR, Kulkarni PG, Rao TM, Palakurthy P. Bio-Friendly Alternatives for Xylene – Carrot oil, Olive oil, Pine oil, Rose oil. J Clin Diagn Res. 2015 Nov;9(11):ZC16-8. doi: 10.7860/JCDR/2015/16384.6731. Epub 2015 Nov 1. PMID: 26673687; PMCID: PMC4668515.

Alwahaibi N, Aljaradi S, Alazri H. Alternative to xylene as a clearing agent in histopathology. J Lab Physicians. 2018 Apr-Jun;10(2):189-193. doi: 10.4103/JLP.JLP_111_17. PMID: 29692586; PMCID: PMC5896187.

Negi A, Puri A, Gupta R, Chauhan I, Nangia R, Sachdeva A. Biosafe alternative to xylene: A comparative study. J Oral Maxillofac Pathol. 2013 Sep;17(3):363-6. doi: 10.4103/0973-029X.125199. PMID: 24574653; PMCID: PMC3927336.

Case Study: Elimination of xylene from a commercial laboratory

Matti Kiupel has kindly allowed us to share  his experiences of xylene free processing on our sustainability pages. Matti deliberately does not provide the names of the different systems as he does not want to advertise for a specific vendor, if you have specific questions about a system, please contact him directly.

We have gone completely xylene free for a number of years. We started with the back end by switching to an automated xylene free HE stainer and coverslipper and replaced this after 5+ years of use a while ago with the newest model of the series. The system comes with a couple of advantages. It is self-contained and doesn’t use alcohol or xylene and doesn’t expose anybody to fumes. It individually labels slides avoiding floaters like the typical “dunk and dip” systems and it guarantees consistency of the staining for each individual slide making QA/QC much easier. It is fully automated from staining slides to coverslipping them and allows you to modify your HE stains based on tissue type or difference in prior processing, etc., e.g., decalcified versus non-decalcified slides, necropsy versus biopsy cases or skin versus brain tissues. Cost and maintenance are the major factors to be considered when making the switch.

It took us much longer to become completely xylene free as I was very skeptical that the removal of paraffin and processing of tissue could be done with the same quality as our xylene based processing system. We used a 13 hour protocol that delivered excellent processing results. We switched three years ago to a xylene-free tissue processor. While I was very quickly convinced that the tissue quality was not affected based on a number of trial runs, I was concerned about the molecular aspects. There had been a number of studies by different hospitals in human medicine that have shown equal or better quality of IHC etc. We ran a series of antibodies and in-situ probes on parallel processed tissues and could see no differences and the amount and quality of extracted nucleic acid was also excellent. For the last three years we had no issues with molecular testing using this system and the results are excellent. In addition, the isopropyl alcohol processing can be done much faster and we routinely run an 8 hour protocol that equals the quality of our previously processed tissues after 13 hours when comparing the final HE slides. This time saving can have a major impact on your work flow. We have tested the shorter protocols and found an acceptable quality with a 2 hour protocol that we offer for stat cases. Lastly, you can run two different protocols simultaneously at the same time.

We are not using xylene substitutes, they are expensive, don’t work as well, smell like citrus etc. We don’t use the typical alcohol deparaffinization at all. We are using mainly isopropyl alcohol and the amount of waste generated is much smaller to begin with. This allows you to deparaffinize in much shorter time with the same quality of deparaffinization and better preservation of antigenicity and nucleic acid. You need to immerse tissues at higher temperature (85 C)  into wax, so you need a special type of wax that works in these temperatures and special processors that have seals that work in these temperatures. There are numerous companies that sell these new processors and numerous large hospital systems have also switched to this system and have done in house validation of this method.xylene protocol. 1.5 hour processing is still ok for stat cases, but you see reduction in quality and I would want that routinely for things like derm cases etc.

I deliberately did not provide the names of the different systems as I don’t want to advertise for a specific vendor, if you have specific questions about a system, please just contact me directly.

Matti Kiupel

Dr. med. vet. Dr. habil. Matti Kiupel, BS, MS, PhD, DACVP

Fachtierarzt für Veterinär Pathologie

Professor, Department of Pathobiology and Diagnostic Investigation

Michigan State University Veterinary Diagnostic Laboratory

College of Veterinary Medicine, 4125 Beaumont Road 152A, Lansing, MI 48910, USA