Clearing

Clearing or de–alcoholization is the process whereby Alcohol is removed from the tissue and replaced with substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or the medium on which the tissue is to be mounted (e.g. Canada Balsam)

When used after Alcohol dehydration, the clearing agent serves to mix with Alcohol and remove it from the tissue. It should be miscible also with paraffin in order to facilitate the penetration of this embedding medium. The most commonly used clearing agents for de–alcoholization in the embedding process are Xylene, Dioxane, Chloroform and cedarwood.

When used after the tissue section has been stained, the clearing agent will make microscopic tissue preparations transparent due to high index of refraction. Aside from removing Alcohol, a clearing agent must also be miscible with Canada Balsam and other resins that are used for mounting sections. The most commonly used clearing agent for this purpose is Xylene.

Glycerine and gum syrup are used when the tissue is to be cleared directly from water, as in Frozen Section. No de–alcoholization is involved in this process. The clearing agents merely improved the refractive index of the tissue.

Characteristics of a good clearing agent

1. It should be miscible with Alcohol to promote rapid removal of the dehydrating agent from the tissue.

2. It should be miscible with paraffin and / or mounting medium to facilitated impregnation and mounting of sections.

3. It should not produce excessive tissue shrinkage and hardening.

4. It should not evaporate quickly with waterbath.

5. It should not make the tissue transparent.

Commonly used clearing agents

A. Xylene

Xylene is a colorless clearing agent most commonly used today. Clearing time is usually ½ to 1 hour. It is used for clearing, both for embedding and mounting procedures.

Advantages:

1. It is the most rapid clearing agent, suitable for urgent biopsies, which it clears within 15 – 30 minutes.

2. It makes tissue transparent.

3. It is miscible with absolute alcohol and paraffin.

4. It does not extract out aniline dyes.

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

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

7. It is cheap.

Disadvantages

1. It is highly flammable.

2. If used longer than 3 hours, it makes tissue excessively hard and brittle.

3. It causes considerable hardening and shrinkage of tissues; hence, is not suitable for nervous tissues and lymph nodes.

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

B. Toluene

Toluene may be used as a substitute for Xylene or Benzene for clearing both during embedding and mounting process. Time recommended for clearing is 1 – 2 hours.

Advantages:

1. It is miscible with both absolute alcohol and paraffin.

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

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

4. It is not carcinogenic.

Disadvantages:

1. It is relatively slower than Xylene and Benzene.

2. It tends to acidify in a partially filled vessel.

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

4. It is more expensive.

C. Benzene

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

Advantages:

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

2. It volatizes rapidly in paraffin oven and is therefore easily eliminated from the tissue.

3. It is miscible with absolute alcohol.

4. It does not make tissues hard and brittle.

5. It causes minimum shrinkage.

6. It makes tissues transparent.

Disadvantages:

1. It is highly flammable.

2. 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.

3. 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. If ever benzene is to be used for clearing, the laboratory should be well–ventilated.

D. Chloroform

Chloroform is used for routine clearing of tissues during the embedding process.

Advantages:

1. It is recommended for routine work (6 – 24 hours)

2. It is miscible with absolute alcohol

3. 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.

4. It is suitable for large tissue specimens.

5. It is not inflammable.

Disadvantages:

1. It is relatively toxic to the liver after prolonged inhalation; adequate room ventilation and proper caution may prevent this when handling the specimen.

2. Wax impregnation after Chloroform clearing is relatively slow.

3. It does not make tissue transparent.

4. Its vapor may attack the rubber seal used in vacuum impregnation bath.

5. Complete clearing is difficult to evaluate.

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

7.     It evaporates quickly from a waterbath.

E. Cedarwood oil

Cedarwood oil is 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, and requires two changes in clearing solutions; clearing is usually complete in 2 – 3 days.

Advantages:

1. It is very penetrating.

2. It is miscible with 96% alcohol which it removes readily.

3. It clears celloidin in 5 – 6 days.

4. It causes minimal shrinkage and hardening of tissues.

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

6. It does not dissolve out aniline dyes.

7. It makes tissue transparent.

8. Clearing with cedarwood oil often improves cutting of the sections.

Disadvantages:

1. It is extremely slow clearing agent, hence, is not recommended for routine purposes.

2. It is hard to be eliminated 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 ½ hours before finally placing the tissue in wax.

3. Quality is not always uniform and good. Tissues cleared in cedarwood oil initially float before gradually sinking to the bottom as clearing proceeds. Hence, the tissue may dry out before it is completely cleared. Superimposing absolute alcohol on the surface of the clearing agent can prevent this. Once saturated, the specimen should then be transferred to a fresh solution of cedarwood oil.

4. Cedarwood oil become milky upon prolonged storage and should be filtered before use.

5. Cedarwood oil that has been previously used to clear acetic – alcohol fixed tissues may produce crystals with a melting point of approximately 35^oC and therefore interfere with adequate clearing of tissue. The solution must be heated to 200^oC in order to dissolve the crystals and restore the solution to its normal state.

6. It is very expensive.

F. Aniline oil

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

G. Clove oil

This reagent causes minimum shrinkage of tissues. However, its quality is not guaranteed due to its tendency to become adulterated. Wax impregnation after clearing with Clove oil is slow and difficult. Tissues become brittle, aniline dyes are removed, celloidin is dissolved. All of these, plus the expensiveness of the solution, makes it unsuitable for routine clearing purposes.

H. Carbon Tetrachloride

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.

I. Tetrahydrofuran

Tetrahydrofuran is superior to ordinary dehydrating and clearing agent 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 an offensive odor and should be used in a well–ventilated room.