clearing part 1

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

• is one of the routinely used chemical in histology and pathology laboratories

5

Xylene- tissue block size is less than ___ mm

methyl benzaldehyde

several toxicities believed to be caused by intermediate products of xylene metabolism such as ____________ have been reported. These include central nervous system disorders, respiratory depression, abdominal pain, dryness and redness of skin, dermatitis, liver diseases, nephrotoxicity, conjunctivitis, and teratogenic and fetotoxic effects.

Xylene

• 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 is cheap

Xylene

• 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

Xylene

does not extract out aniline dyes

Xylene

• 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 node

Xylene

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

• 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

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, so _____ 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

Toluene

• It is miscible with both absolute alcohol and paraffin.

• It acts fairly rapidly and is recommended for routine purposes

Toluene

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

• Clears overnight.

• It is not carcinogenic

Toluene

• It is slower than xylene and benzene.

• It tends to acidify in a partially filled vessel

Toluene

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

• It is more expensive

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

Benzene

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

• It is miscible with absolute alcohol.

• It does not make tissues hard and brittle.

• It causes minimum shrinkage.

• It makes tissues transparent.

• It clears overnight

Benzene

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

Benzene

• It is highly flammable.

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

Benzene

Excessive exposure to _______ 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

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

Chloroform

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

• It is miscible with absolute alcohol.

• It is not inflammable

Chloroform

• 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

Chloroform

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

• Wax impregnation after ______ clearing is relatively slow

• It does not make tissues transparent

• Complete clearing is difficult to evaluate

Chloroform

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

• 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

Chloroform

• 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

• It evaporates quickly from a water bath

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

Cedarwood Oil

• 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

Cedarwood Oil

• 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 _________ often improves cutting of the sections

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

Cedarwood Oil

• Quality is not always uniform and good. Tissues cleared in _______ 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

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 solution to its normal state.

• It is very expensive.