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RACHELLE MAE F. FERRANCO, RMT

DEALCOHOLIATION / CLEARING - Due to this property of making tissues transparent

- Clearing (de-alcoholization) is the process whereby alcohol - Not all clearing agents, however, exhibit this property.
or a dehydrating agent is removed from the tissue and
CHARACTERISTICS OF A GOOD CLEARING AGENT:
replaced with a substance that will dissolve the wax with
which the tissue is to be impregnated (e.g. Paraffin) or the 1. It should be miscible with alcohol to promote rapid removal
medium on which the tissue is to be mounted (e.g. Canada of the dehydrating agent from the tissue.
balsam).
2. It should be miscible with, and easily removed by melted
paraffin wax and/or by mounting medium to facilitate
- When the dehydrating agent has been entirely replaced by impregnation and mounting of sections.
the solvent, the tissue has a translucent appearance;
hence, the use use of the term “clearing agent”. 3. It should not produce excessive shrinkage, hardening or
damage of tissue.
- Few dehydrating agents are miscible with paraffin wax
4. It should not dissolve out aniline dyes.
- When use after alcohol dehydration, the clearing agent
serves to mix with alcohol and removes it from the tissue. 5. It should not evaporate quickly in a water bath

- It should be miscible also with paraffin in order to 6. It should make tissues transparent
o facilitate the penetration of this embedding
medium. - Most clearing agents are flammable liquids that warrant
considerable caution in their use, and hstotechnologist should
- The most commonly used clearing agents for de- be aware of the large quantities used in routine processing, so
alcoholization in the embedding process are xylene, that the safest methods of use and storage can be adopted.
dioxane, chloroform and cedarwood.
- Clearing fluids with a low boiling point are generally more
- When used after the tissue section has been stained, the readily replaced by melted paraffin, although chloroform which
clearing agent will make microscopic tissue preparations has a lower boiling point than xylene in fact takes longer than
transparent due to their high index of refraction. the latter to clear.
- Aside from removing alcohol, a clearing agent must also - Viscosity also affects the speed of penetration of clearing
be miscible with Canada balsam and other resins that are agent.
used for mounting sections.
- Prolonged exposure to most clearing agents causes the tissue
- The most commonly used clearing agent for this purpose to become brittle and therefore more difficult to cut.
is xylene.
AMONG THE COMMON CLEARING AGENTS USED ARE:
- Glycerin and gum syrup are used when the tissue is to be
cleared directly from water, as in a frozen section. No de- a. Xylene (most common)
alcoholization is involved in this process. b. Toluene
c. Benzene
- The clearing agents merely improve the refractive index of d. Chloroform
the tissue. e. Cedarwood oil
f. Aniline oil
- Because of the high refractive indices of most reagents are
g. Clove oil
used for dealcoholization, tissues, particularly embryos and h. Carbon Tetrachloride
parasites become transparent so that the internal
structures become visible to the naked eye.

HISTOPATHOLOGY NOTES
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RACHELLE MAE F. FERRANCO, RMT

CLEARING AGENTS:
DISADVANTAGES:
1. XYLENE 1. It is relatively slower than xylene and benzene.
-xylene is a cololess clearing agent that is most 2. It tends to acidify in a partially filled vessel.
commonly used in histology laboratories 3. Highly concentrated solutions will emit fumes that are
-It used for clearing, both for embedding and toxic upon prolonged exposure.
mounting procedures. 4. It is more expensive.
-it is generally suitable for most routine histologic
processing schedules of less than 24 hours, and 3. BENZENE
when the tissue block size is less than 5mm. in -fast-acting
thickness. -Benzene is preferred by some as clearing agent in the
embedding process of tissues because it penetrates and
ADVANTAGES: clears tissues rapidly.
1. It is the most rapid clearing agent, suitable for
urgent biopsies which it clears within 15-30 ADVANTAGES:
minutes. 1. It is rapidly actin, hence is recommended for urgent
2. It makes tissues transparent biopsies (15-60 minutes) and routine purposes
3. It is miscible with absolute alcohol and paraffin 2. It volatilizes rapidly in paraffin oven and is therefore
4. It does not extract out aniline dyes. easily eliminated from the tissue
5. For mounting procedures, it does not dissolve 3. It is miscible with absolute alcohol
celloidin and cam, therefore, be used for celloidin 4. It does not make tissues hard and brittle
sections. 5. It causes minimum shrinkage
6. It evaporates quickly in paraffin over and can, 6. It makes tissues transparent
therefore, be readily replaced by wax during
impregnation and embedding. DISADVANTAGES
7. It is cheap. 1. It is highly inflammable
2. If a section is left in benzene for a long time,
DISADVANTAGES: considerable tissues shrinkage may be observed; hence
1. It is highly inflammable and should be tissues should be transferred to paraffin wax as soon as
appropriately stored possible
2. If used longer than 3 hours, it makes tissues 3. Excessive exposure to benzene may be extremely
excessively hard and brittle. toxic to man and may become carcinogenic or it may be
3. It causes considerable hardening and shrinkage damage the bone marrow resulting in aplastic
of tissues; hence, is not suitable for nervous anemia. If ever benzene is to be used for clearing, the
tissues and lymph nodes. laboratory should be well-ventilated.
4. Xylene becomes milky when an incompletely
dehydrated tissue is immersed in it. 4. CHLOROFORM
2. TOLUENE -can cause wax deterioration
-can be a substitute for xylene and benzene -it is not a clearer (will not make tissue transparent)
-longer cleaning time: 1-2 hours -Chloroform, when used for clearing tissues during the
-Toulene may be used as substitute for xylene or embedding process, is slower in action than xylene, but
benzene for clearing both during embedding and causes less brittleness.
mounting processes. -Thicker tissue blocks, even those up to 1 cm. in
thickness, can be processed.
ADVANTAGES: -Tissues placed in chloroform do not become
1. It is miscible with both absolute alcohol and paraffin. translucent.
2. It acts fairly rapidly and it is recommended for
routine purposes. ADVANTAGES:
3. Tissues do not become excessively hard and brittle 1. It is recommended for routine work (6-24 hours)
even left in toulene for 24 hours. 2. It is miscible with absolute alcohol
4. It is not carcinogenic. 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 tissues specimens.
5. It is not inflammable.

HISTOPATHOLOGY NOTES
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RACHELLE MAE F. FERRANCO, RMT

specimen from oil to benzene for 1/2 hour before finally placing
DISADVANTAGES: the tissue in wax
1. It is relatively toxic to the liver after 3. Quality is not always uniform and good. Tissues cleared in
prolonged inhalation; this may be prevented by cedarwood oil initially float before gradually staying to the
adequate room ventilation and proper caution when bottom as clearing proceeds. Hence, the tissue may dry-out
handling specimen. before it is completely cleared.
2. Wax impregnation after chloroform clearing is 4. Cedarwood oil becomes milky upon prolong storage
relatively low. and should be filter before use.
3. It does not make tissues transparent 5. Cedarwood oil that has been previously used to clear
4. It is not very volatile in paraffin oven; hence is acetic-alcohol fixed tissues may produce crystals with a
difficult to remove from paraffin sections. It may melting point of approximately 35 degree C and therefore
even produce considerable deterioration of the interfere with adequate clearing of of tissue. The solution must
wax. be heated to 200 degree C in order to dissolve the crystals and
5. Its vapor may attack the rubber seal used in restore the solution to its normal state
vacuum impregnating bath 6. It is very expensive.
6. Complete clearing is difficult to evaluate.
7. Tissues tend to float in Chloroform; this 6. ANILINE OIL
may be avoided by wrapping the tissues with -for embryos, insects, and other delicate specimens
absorbent cotton gauze to facilitate sinking of -has ability to clear 70% alcohol without excessive tissue
the section in solution shrinkage and hardening.
8. It evaporates quickly from a water bath
7. CLOVE OIL
5. CEDARWOOD OIL -both expensive and slow
-extremely slow clearing agent -tissues become adulterated
-becomes milky on prolonged storage -this reagent causes minimum shrinkage of tissues. -
-for CNS tissues and cytological studies however, its quality is not guaranteed due to its tendency
-for smooth muscles of the skin to become adulterated.
-Cedarwood oil is used to clear both parraffin and -wax impregnation after clearing with clove oil is slow
celloidin sections during the embedding process. and difficult.
-It requires two changes in clearing solution. -tissue become brittle, aniline dyes are removed, and
-Clearing is usually complete in 2-3 days. celloidin is dissolved.
-all of these, in addition to the expensiveness of the
ADVANTAGES: solution make it insuitable for clearing purposes.
1. It is very penetrating
2. It is miscible with 96% alcohol which removes readily. 8. CARBON TETRACHLORIDE (CCl4)
3. It clears celloidin in 5-6 days. -same action as chloroform
4. It causes minimal shrinkage and hardening of 9. TETRAHYDROFURAN
tissues 10. TERPINEOL
5. Tissues may be left in oil indefinitely without -for clearing eye specimens
considerable damage and distortion 11. OIL OF BERGAMOT
6. It does not dissolve out aniline dyes -for clearing skin and smooth muscle
7. It makes tissues transparent 12. OIL OF WINTERGREEN -for delicate (sorry, I wasn’t able to
8. Clearing with cedarwood oil often improves cutting of note this one. Listen nalang kayo kay Ma’am)
the sections 13. N-BUTYL ACETATE
-used as xylene substitute and nitrocellulose solvent
DISADVANTAGES:
1. It is an extremely slow clearing agent,hence is not
recommended for routine purposes 14. LIMONENE
2. It is hard to be eliminated from the tissues in paraffin bath, -derived from a citrus fruit
making the wax impregnation process very slow. This may be -component of various proprietary blends of
improved or hastened by transferring the transition solvents marketed as xylene substitutes
-less viscous than cedarwood oil and is similar to
the esters in clearing action and in eliminating from
wax.
15. AMYL ACETATE
Chiefly used as nitrocellulose
16. METHYL BENZOATE
17. METHYL SALICYLATE solvents in double embedding
techniques

HISTOPATHOLOGY NOTES
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RACHELLE MAE F. FERRANCO, RMT

DOUBLE EMBEDDING: use of celloidin to infiltrate and

Normal EMBEDDING: use of paraffin wax for both

N-butyl acetate and Limonene are both marketed as xylene

TAKE NOTE:
Sentences or terminologies marked with are questions
given by Dr. Liwanag during our time. I hope it could help
you CEU peeps.

HISTOPATHOLOGY NOTES