Clearing

True

True or False: wax and ethanol are largely immiscible.

Clearing (de-alcoholization)

is the process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the tissue is to be mounted (e.g. Canada balsam).

True

True or False: a clearing agent must also be miscible with Canada balsam and other resins that are used for mounting sections

True

True or False: clearing agents impart an optical clarity or transparency to the tissue due to their relatively high refractive index. This change in appearance is often used as an indication of the effectiveness or completeness of the clearing process.

Glycerin and gum syrup

are used when the tissue is to be cleared directly from water, as in a frozen section. No de-alcoholization is involved in this process.

True

True or False: Good Clearing Agent should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue.

True

True or False: A good clearing agent should not evaporate quickly in a water bath.

Clearing fluids with a low boiling point

are generally more readily replaced by melted paraffin, although chloroform which has a lower boiling point than xylene in fact takes longer than the latter to clear.

Xylene (Xylol)

• colorless clearing agent that is most commonly used in histology laboratories

• Clearing time is usually 1/2 to 1 hour.

• suitable for most routine histologic processing schedules of less than 24 hours, and when the tissue block size is less than 5 mm. in thickness.

• cost effective and works well for short-term clearing of small tissue blocks.

• It is also used in cover slipping, in cleaning tissue processors, as solvent to remove synthetic immersion oil from the microscope objective and in recycling of used slides

• central nervous system disorders

• respiratory depression

• abdominal pain

• dryness and redness of skin

• dermatitis

• liver diseases

• nephrotoxicity

• conjunctivitis

• teratogenic and fetotoxic effects.

several toxicities believed to be caused by intermediate products of xylene metabolism such as methyl benzaldehyde have been reported. These include:

• highly inflammable

• If used longer than 3 hours, it makes tissues excessively hard and brittle.

• It causes considerable hardening and shrinkage of tissues

• not suitable for nervous tissues and lymph nodes.

• becomes milky when an incompletely dehydrated tissue is immersed in it.

Disadvantages of Xylene

• most rapid clearing agent, suitable for urgent biopsies which it clears within 15-30 minutes

• It makes tissues transparent.

• It is miscible with absolute alcohol and paraffin.

• It does not extract out aniline dyes.

• For mounting procedures, it does not dissolve celloidin and can, therefore, be used for celloidin sections.

• It evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding.

• It is cheap.

Advantages of Xylene

Toluene

• better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene.

• more expensive than xylene and more toxic

• may be used as a substitute for xylene or benzene for clearing both during embedding and mounting processes.

• Time recommended for clearing is 1 -2 hours.

• It is miscible with both absolute alcohol and paraffin.

• It acts fairly rapidly and is recommended for routine purposes.

• Tissues do not become excessively hard and brittle even if left in for 24 hours.

• Clears overnight.

• It is not carcinogenic.

Advantages of of Toluene

• It is slower than xylene and benzene.

• It tends to acidify in a partially filled vessel.

• Highly concentrated solutions will emit fumes that are toxic upon prolonged exposure.

• It is more expensive.

Disadvantages of Toluene

Benzene

• preferred by some as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly.

• It used to be a popular routine clearing agent until recently when its highly carcinogenic properties were recognized.

• Its use for clearing purposes is therefore strongly discouraged.

• It is rapid acting, hence is recommended for urgent biopsies (15-60 minutes) and routine purposes.

• It volatilizes rapidly in paraffin oven and is therefore easily eliminated from the tissue.

• It is miscible with absolute alcohol.

• It does not make tissues hard and brittle.

• It causes minimum shrinkage.

• It makes tissues transparent.

• Clears overnight

Advantages of Benzene

• It is highly flammable.

• If a section is left in benzene for a long time, considerable tissue shrinkage may be observed. Hence, tissues should be transferred to paraffin wax as soon as possible.

• Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in aplastic anemia.

• the laboratory should be well-ventilated.

Disadvantages of Benzene

Chloroform

is slower in action than xylene, but causes less brittleness. Thicker tissue blocks, even those up to I cm. in thickness, can be processed. However, tissues placed in it do not become translucent.

• It is recommended for routine work (6-24 hours).

• It is miscible with absolute alcohol.

• It is recommended for tough tissues (e.g. skin, fibroid and decalcified tissues) for nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues.

• It is suitable for large tissue specimens.

• It is not inflammable.

Advantages of Chloroform

• It is relatively toxic to the liver after prolonged inhalation; this may be prevented by adequate room ventilation.

• Wax impregnation after it is relatively slow.

• It does not make tissues transparent.

• It is not very volatile in paraffin oven; hence, it is difficult to remove from paraffin sections. It may even produce considerable deterioration of the wax.

• Its vapor may attack the rubber seal used in vacuum impregnating bath.

• Complete clearing is difficult to evaluate.

• Tissues tend to float; this may be avoided by wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution.

• It evaporates quickly from a water bath.

Disadvantages of Chloroform

Cedarwood oil

used to clear both paraffin and celloidin sections during the embedding process. It is especially recommended for central nervous system tissues and cytological studies, particularly of smooth muscles and skin. It requires two changes in clearing solution. Clearing is usually complete in 2-3 days.

• It is very penetrating.

• It is miscible with 96% alcohol which it removes readily

• It clears celloidin in 5-6 days.

• It causes minimal shrinkage of tissues.

• Tissues may be left in oil indefinitely without considerable damage and distortion.

• It does not dissolve out aniline dyes.

• It makes tissues transparent.

• It does not harden tissues.

• It does not interfere too seriously with paraffin penetration if it is not completely removed.

• often improves cutting of the sections.

Advantages of Cedarwood oil

• It is an extremely slow clearing agent, hence, it is not recommended for routine purposes.

• It is slightly slower in penetrating than benzene.

• It is hard to eliminate from the tissues in paraffin bath, making the wax impregnation process very slow. This may be improved or hastened by transferring the specimen from oil to benzene for 1/2 hour before finally placing the tissue in wax.

• Quality is not always uniform and good.

• becomes milky upon prolonged storage and should be filtered before use.

• may produce crystals with a melting point of approximately 35°C and therefore interfere with adequate clearing of tissue.

Disadvantages of Cedarwood oil

Aniline oil

• not normally utilized

• recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.

Clove oil

• causes minimum shrinkage of tissues.

• However, its quality is not guaranteed due to its tendency to become adulterated.

• Wax impregnation after clearing oil is slow and difficult.

• Tissues become brittle, aniline dyes are removed, and celloidin is dissolved.

• All of these, in addition to the expensiveness of the solution, make it unsuitable for routine clearing purposes.

Carbon tetrachloride

• may be used in clearing tissues for embedding.

• Its properties are very similar to that of chloroform although it is relatively cheaper.

• Its disadvantage is the same as that of chloroform.

• It produces considerable tissue hardening, and is dangerous to inhale on prolonged exposure due to its highly toxic effects.

Tetrahydrofuran

• 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.

• It is non-toxic but has offensive odor and should be used in a well-ventilated room.

Dioxane

• miscible both with water and paraffin.

• It is used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation. The tissues are transferred to dioxane straight from Bouin's fluid or a formalin fixative.

• changed 3 times within 4 hours and the tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes).

• causes greater shrinkage than xylene does.

• it is dangerous.

• Fumes are toxic to human especially to the liver.