Chapter 10: Clearing Agents in Tissue Processing

De-alcoholization

A process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which is to be impregnated or the medium on which the tissue is to be mounted.

Characteristics of a Good Clearing Agent

Makes tissues excessively hard and brittle, if used longer than 3 hrs; becomes milky when an incompletely dehydrated tissue is immersed; irritant; causes dermatitis; toxic and narcotic at high concentrations.

Xylene

A colorless clearing agent that is slower than xylene and benzene, tends to acidify in a partially filled vessel, and emits fumes in highly concentrated solutions that are toxic upon prolonged exposure.

Toluene

Preserves tissue structure, is more expensive and toxic than xylene, and serves as a substitute for xylene or benzene for clearing during embedding and mounting processes.

Benzene

Penetrates and clears tissues rapidly but is highly carcinogenic (preleukemic state).

Advantages of Clearing Agents

For clearing, embedding and mounting procedures; to remove paraffin in slides, not in actual mounting; dewaxing agent during staining; suitable for histologic processing schedules of less than 24hrs, tissue block size <5mm in thickness; works well for short-term clearing of small tissue blocks; as solvent to remove synthetic immersion oil from the microscope objective.

Urgent Biopsies

Can be processed within 15-60 minutes without making tissues hard and brittle, causing minimum shrinkage, and making tissues transparent.

Rapid Clearing Agent

Suitable for urgent biopsies, clears within 15-30 minutes, miscible with absolute alcohol and paraffin, dissolves celloidin, evaporates quickly in paraffin oven, and is cheap.

Analine Oil

May become carcinogenic and may damage the bone marrow resulting in aplastic anemia.

Chloroform

Not normally utilized as a routine clearing agent; recommended for clearing embryos, insects, and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.

Handling Procedures for Clearing Agents

Miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue; easily removed by melted paraffin wax and/or by mounting medium; should not produce excessive shrinkage, hardening or damage of tissue; should not evaporate quickly in a water bath.

Storage Requirements for Clearing Agents

Close the container tightly; store in a cool and dry area; store in a flammable liquid storage cabinet; use chemical transport carriers in closed containers and in the smallest amount; store below eye level for visibility of cracks or leaking containers.

Xylene (concentrated)

Xylene (2) is a form of xylene used in clearing processes.

Alcohol

The most common clearing agent.

Acetone

A clearing agent used in histological processes.

Dioxane

A clearing agent used in histological processes.

Cellosolve

A clearing agent used in histological processes.

Triethyl Phosphate

A clearing agent used in histological processes.

Tetrahydrofuran

A clearing agent used in histological processes.

CLOVE OIL

Slower in action than xylene, but causes less brittleness.

Thicker tissue blocks

Can be processed, even those up to 1 cm in thickness.

Advantages of CLOVE OIL

Recommended for touch tissues (skin, fibroid and decalcified tissues) for nervous tissues, lymph nodes and embryos.

Disadvantages of CLOVE OIL

Causes minimum shrinkage of tissues, but quality is not guaranteed; wax impregnation after clearing is slow and difficult; unsuitable for routine clearing purposes since tissues become brittle.

CARBON TETRACHLORIDE

May be used in clearing tissues for embedding; its properties are very similar to that of chloroform although it is relatively cheaper.

Disadvantages of CARBON TETRACHLORIDE

Relatively toxic to the liver after prolonged inhalation; produces considerable tissue hardening and is dangerous to inhale on prolonged exposure due to its highly toxic effects.

CEDARWOOD OIL

It is superior to ordinary dehydrating and clearing agents due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation.

Clearing time with DIOXANE

Clearing is usually complete in 2-3 days; clears celloidin in 5-6 days.

Advantages of DIOXANE

Very penetrating; causes minimal shrinkage of tissues; tissues may be left in oil definitely without considerable damage and distortion; does not harden tissues; improves cutting of the sections.

Disadvantages of DIOXANE

It causes greater shrinkage than xylene; it is dangerous; fumes of dioxane are toxic to humans, especially to the liver.

TERPENES

Found in essential oils originally derived from plants; many terpenes clear tissues and celloidin sections from 80-95% alcohol, render tissues transparent and have a slow gentle non-hardening action.

LIMONENE

A volatile oil found in citrus peels; clear, colorless fluid with a distinctly citrus aroma.

ORANGE OIL-BASED CLEARING AGENTS

Offer the clearing action with the lowest hazard rating of all xylene alternatives; excellent for preserving fine tissue structure and can often be used in place of xylene with no alteration of protocol.

CHLORINATED HYDROCARBONS

Can be effective solvents but they are toxic chemicals.

COCONUT OIL

Efficient substitute for xylene; non-hazardous, less expensive and causes less shrinkage of the tissue; can be used as a dealcoholization agent.

BLEACHED PALM OIL

It is a clearing agent during tissue processing and as a deqaxing agent during staining; non-toxic, non-hazardous, non-flammable, biodegradable, economic, easy to handle and readily available.