CHAPTER 10
CLEARING
Although the dehydrated tissue is now essentially water-free, it still cannot be
infiltrated with wax because wax and ethanol are largely immiscible. An
intermediate solvent that is fully miscible with both ethanol and paraffin wax is
needed to remove alcohol and other dehydrating solutions from tissues prior to
embedding (usually in paraffin wax), and from finished slides prior to mounting.
They are also used after sectioning to remove paraffin wax after cutting on the
microtome. 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).
Aside from removing alcohol, a clearing agent must also be miscible with
Canada balsam and other resins that are used for mounting sections. This stage
in the process is called “clearing” because many (but not all) 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. Because of the high
refractive indices of most reagents used for de-alcoholization, tissues,
particularly embryos and parasites, become transparent so that the internal
structures become visible to the naked eye. Another important role of the
clearing agent is to remove a substantial amount of fat from the tissue which
otherwise presents a barrier to wax infiltration.
The most commonly used clearing agent for this purpose is xylene. 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.
Characteristics of a Good Clearing Agent:
It should be miscible with alcohol to promote rapid removal of the
dehydrating agent from the tissue.
It should be miscible with, and easily removed by melted paraffin wax
and/or by mounting medium to facilitate impregnation and mounting of
sections.
It should not produce excessive shrinkage, hardening or damage of
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tissue.
It should not dissolve out aniline dyes.
It should not evaporate quickly in a water bath.
It make tissues transparent.
The choice of a clearing agent depends upon the following:
The type of tissues to be processed, and the type of processing to be
undertaken
The processor system to be used
Intended processing conditions such as temperature, vacuum and
pressure
Safety factors
Cost and convenience
Speedy removal of dehydrating agent
Ease of removal by molten paraffin wax
Minimal tissue damage
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. Viscosity also affects the speed
of penetration of the clearing agent. Prolonged exposure to most clearing agents
causes the tissue to become brittle and therefore more difficult to cut.
A. Xylene (Xylol)
Xylene is a colorless clearing agent that is most commonly used in histology
laboratories. Clearing time is usually 1/2 to 1 hour. It is used for clearing, both
for embedding and mounting procedures. It is generally 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. Xylene is reasonably cost effective and
works well for short-term clearing of small tissue blocks.
Xylene is one of the routinely used chemical in histology and pathology
laboratories because of its vital role in the paraffin wax tissue processing
method. It is mostly used as a clearing agent during tissue processing and as a
dewaxing agent during staining. 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. However, several toxicities
believed to be caused by intermediate products of xylene metabolism such as
methyl benzaldehyde have been reported. These include central nervous system
disorders, respiratory depression, abdominal pain, dryness and redness of skin,
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dermatitis, liver diseases, nephrotoxicity, conjunctivitis, and teratogenic and
fetotoxic effects.
Advantages:
It is the 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.
Disadvantages:
It is highly inflammable and should be appropriately stored.
If used longer than 3 hours, it makes tissues excessively hard and brittle.
It causes considerable hardening and shrinkage of tissues; hence, is not
suitable for nervous tissues and lymph nodes.
Xylene becomes milky when an incompletely dehydrated tissue is
immersed in it.
Xylene may irritate eyes, nose and respiratory tract. It can be absorbed
through the skin and cause dermatitis. At high concentrations, it is toxic
and narcotic.
Special Handling Procedures and Storage Requirements:
• Keep container tightly closed to prevent xylene from subliming and
entering the atmosphere.
• Only non-sparking tools may be used to handle xylene.
• Store in a cool and dry area away from incompatible substances (i.e.
oxidizing agents, strong acids).
• Store xylene in a flammable liquid storage cabinet.
• Wash hands thoroughly after handling xylene (even if gloves were
used).
• Remove contaminated clothing and wash before reuse.
• Keep away from heat, sparks, flames, sources of ignition (including
empty containers that will retain product residue).
• Transport chemicals in closed containers, in the smallest amounts
possible, and use aids such as carts, chemical transport carriers, etc.
• It is highly recommended that all chemicals be stored below eye level so
cracking or leaking containers are immediately visible and there is less
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potential for chemicals falling onto lab workers when pulling from
shelves.
B. Toluene
Toluene is better at preserving tissue structure and is more tolerant of small
amounts of water left behind in the tissues than xylene. However, toluene is
more expensive than xylene and more toxic, so toluene is less commonly used.
Toluene 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.
Advantages:
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 toluene
for 24 hours.
Clears overnight.
It is not carcinogenic.
Disadvantages:
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.
C. Benzene
Benzene is 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.
Advantages:
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.
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It clears overnight.
Disadvantages:
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. If ever benzene is to be used for clearing, the laboratory
should be well-ventilated.
D. Chloroform
Chloroform, when used for clearing of tissues during the embedding
process, 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 chloroform do not become translucent.
Advantages:
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.
Disadvantages:
It is relatively toxic to the liver after prolonged inhalation; this may be
prevented by adequate room ventilation.
Wax impregnation after chloroform clearing 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 in chloroform; 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.
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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. It
requires two changes in clearing solution. Clearing is usually complete in 2-3
days.
Advantages:
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.
Clearing with cedarwood oil often improves cutting of the sections.
Disadvantages:
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. Tissues cleared in cedarwood oil
initially float before gradually staying to the bottom as clearing proceeds.
Hence, the tissue may dry out before it is completely cleared. This can be
prevented by superimposing absolute alcohol on the surface of the
clearing agent. Once saturated, the specimen should then be transferred
to a fresh solution of cedarwood oil.
Cedarwood oil becomes milky upon prolonged storage and should be
filtered before use.
Cedarwood oil that has been previously used to clear acetic-alcohol fixed
tissues may produce crystals with a melting point of approximately 35°C
and therefore interfere with adequate clearing of tissue. The solution
must be heated to 200°C in order to dissolve the crystals and restore the
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solution to its normal state.
It is very expensive.
F. Aniline oil
This is not normally utilized as a routine clearing agent but it is
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, and celloidin is dissolved. All of these, in addition to the
expensiveness of the solution, make 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 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.
J. Dioxane
Dioxane is 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. The dioxane is changed 3 times within 4 hours and
the tissues are transferred directly to paraffin (3 changes are made in a total of 90
minutes). Dioxane causes greater shrinkage than xylene does. In addition, it is
dangerous. Fumes of dioxane are toxic to human especially to the liver.
Other Xylene Substitutes
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The reported toxicity and environmental pollution from unsafe disposal of
xylene led to its substitution with other less-toxic substitutes such as limonene
reagents, mineral oil mixtures, 1.7% dish washing solution, vegetable oils and
coconut oil. Though these substitutes exist, their availability in commercial
quantities in developing countries has hampered their use. All the xylene-
substitutes have to be analyzed thoroughly, before concluding which alternative
is better.
Terpenes are isoprene polymers found in essential oils originally derived
from plants, though some are now synthesized. They are the earliest transition
solvents to be used in histology and include turpentine and oils of bergamot,
cedarwood, clove, lemon, oreganum and sandalwood. In general the natural oils
are not highly pure compounds but contain several substances.
Many terpenes clear tissues and celloidin sections from 80%-95% alcohol,
render tissues transparent and have a slow gentle non-hardening action. Most are
generally regarded as safe though some have particularly strong odors which can
be overpowering, requiring good laboratory ventilation. Terpenes are moderately
effective solvents, but they too are considered toxic. Solvents in this class also
dry slowly, leave an oily residue on slides and are relatively expensive.
One of the recommended xylene substitutes from the terpene family is
Limonene, a volatile oil found in citrus peels which goes by several trade
names. It is a natural oil found in the skins of citrus fruits, such as lemons or
oranges, and in cooking is usually referred to as lemon or orange zest. Limonene
is obtained industrially by the steam distillation of orange peel which is a
byproduct of the orange juice industry. It is a clear, colorless fluid with a
distinctly citrus aroma, not unpleasant to most people, although some do not like
it.
Limonene is often sold as a xylene replacement and some technologists
substitute it for xylene in other uses, but this is not universally successful. When
used as the clearant immediately prior to cover slipping, there are some reports
that the mounting medium, usually dissolved in either toluene or xylene, does
not mix well with the limonene. In such cases, replacing the limonene with
xylene or toluene, or quickly dipping the section in either one just prior to cover
slipping should be effective. This does, of course, defeat the purpose of the
replacement to a certain degree.
Orange oil based clearing agents offer the clearing action with the lowest
hazard rating of all xylene alternatives. It is excellent for preserving fine tissue
structure, and can often be used in place of xylene with no alteration of protocol.
In using a product containing orange oils, it is important to use a product which
has been rigorously purified then stabilized. Orange oils that are neither pure nor
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stable can break down to produce compounds which will interfere with staining
procedures.
Chlorinated hydrocarbons can be effective solvents, but they are
considered toxic chemicals, posing serious health risks. Government regulations
have restricted most of the effective solvents in this class.
Coconut oil is an efficient substitute for xylene, as it is non-hazardous, less
expensive and causes less shrinkage of the tissue. It can be used as a de-
alcoholization agent in the histopathological laboratory, without losing the
quality of the histological details. The only drawback associated with coconut
oil, is its tendency to get solidified at a lower temperature. However, this can be
overcome by performing the clearing procedure in an incubator, maintaining the
required temperature.
Substitution of the conventional xylene with bleached palm oil as a
clearing agent during tissue processing and as a dewaxing agent during staining
gives good tissues, sections and histological slides. In addition, bleached palm
oil is nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to
handle, and readily available.
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