Fixation on Histology

Xylene Substitutes


Xylene SubstitutesXylene is a chemical commonly used in the histology lab as a clearing agent. Clearing agents are used to make the slides easier to read, by making the tissue transparent, or clear. Clearing is a step that occurs during tissue processing, after water has been removed from a tissue. Xylene is used because wax is not soluble in water. Alcohol replaces the water in the tissue. Xylene is miscible with alcohol and will replace the alcohol. Wax can now infiltrate the tissue, and the tissue can move on to embedding.


The problem with xylene… well there are a lot of problems with xylene. In the 70’s studies began to confirm that xylene was dangerous. Not only is it flammable, but it is also not great when breathed in for extended periods of time. The vapor is absorbed through the lungs, while the liquid can be absorbed through the skin, then once in the body it is metabolized by the liver. Prolonged exposure in high concentrations became associated with respiratory tract symptoms, anemia, and other symptoms generally associated with central nervous system issues. Xylene is still widely used, but there are now more safety precautions to help prevent these types of risks. Clinical labs are required to do yearly xylene exposure testing to maintain CAP accreditation under ANP .08216 Formaldehyde/Xylene Safety. If the limits are exceeded, they must develop an action plan for addressing the overexposure and do a repeated test. Xylene substitutes became popular as another way of combating these threats. Some of the substitutes however, come with their own set of risks.


The trick to xylene substitutes is that they have to be miscible with alcohol and paraffin wax as the whole point of their usage was to do the job that water could not. There are different types of xylene substitutes, one of which is terpenes. Terpenes are a type of hydrocarbon compound found in plants, specifically citrus plants. Terpenes are known for their strong citrus odor, which a lot of people object to, and they also run the risk of allergic reactions. Another popular type of xylene substitute is aliphatic hydrocarbons. These don’t have the odor problem and are typically what is used in the substitutes you will see offered by vendors.


You will likely have to make certain other adjustments to your procedures if you are going to use a xylene substitute. For example, many xylene substitutes will require additional changes during processing for the xylene substitute to fully replace the alcohol. Some xylene substitutes are a little less effective at penetration or wax removal. A lab tech may need to re-assess their processing times, as well as their staining and dewax times.


In addition, some xylene substitutes will not absorb water, whereas xylene can absorb small amounts of water, which is useful if a tissue was not fully dehydrated, so this is something else to be aware of, though water tolerance can differ by brand of substitute.

Ultimately, neither xylene substitutes nor xylene is perfect. It is up to your lab to decide what makes sense for you and be aware of the risks, and procedural changes needed to use the chemical safely.



Jenifer M. Langman (1994) Xylene: Its toxicity, measurement of exposure levels, absorption, metabolism and clearance, Pathology, 26:3, 301-309, DOI: 10.1080/00313029400169711