Clearing

32問 • 2年前

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it 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).

De-alcoholization

They impart an optical clarity or transparency to the tissue due to their relatively high refractive index.

Clearing agents

Commonly used clearing agent

Xylene

No de-alcoholization involved this section

Frozen section

Characteristics of Clearing agents -Miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue - Miscible with, and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections. - Not produce excessive shrinkage, hardening or damage of tissue. - Not dissolve out aniline dyes - Not evaporate quickly in a water bath - Make tissue transparent

Choice of clearing agents depends on the following - Type of tissue to be processed - Type of processing to be undertaken - Processor system to be used -Intended processing conditions such as temperature vacuum and pressure

It also affects speed of penetration of the clearing agent

Viscosity

Advantages -Most rapid clearing agent, suitable for urgent biopsies, clears withing 15-30 minutes -Make tissues transparent -Miscible with absolute alcohol and paraffin -Does not extract out aniline dyes -Mounting procedures, does not dissolve celloidin and can be used for celloiding sections -Evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnatin and embedding -Cheap

Xylene

- Colorless clearing agent - Most common in histology lab - Clearing time : 1/2 - 1 hr - Used for clearing, both embedding and mounting procedures -Cost effective and works well for short-term clearing of small tissue blocks - Routinely used due to its vital role in paraffin wax tissue processing method. - Clearing agent during tissue processing, and dewaxing agent during staining. - Used in cover slipping as solvent to remove synthetic slides.

Xylene

Clearing time of Xylene

30 mins to 1 hour

When metabolized, it can caused toxicity • Nervous system disorders • Respiratory depression • Abdominal pain • Dryness and redness of skin • Dermatitis • Liver diseases • Nephrotoxicity • Conjunctivitis • Teratogenic and ferotoxic effects

Xylene(xylol)

- Better at preserving tissue structure - Most tolerant of small amounts of water left behind in the tissue - More expensive and more toxic and less used -Used as a substitute for xylene or benzene for clearing, embedding and mounting process

Toluene

What is the clearing time of toluene?

1-2 hours

Advantages • Miscible with both absolute alcohol and paraffin •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 • Not carcinogenic

Toluene

- Penetrates and clears tissues rapidly -Popular routine clearing agent but strongly discouraged due to high carcinogenic properties

Benzene

Advantages •Rapid acting, recommended for urgent biopsies (15-60 minutes) and routine purposes. • Volatilizes rapidly in paraffin oven and therefore easily eliminated from the tissue • Miscible with absolute alcohol • Does not make tissues hard and brittle • Minimum shrinkage • Makes tissues transparent • Clears overnight

Benzene

Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in _________. When used, laboratory should be well-ventilated

Aplastic anemia

-Slower action than xylene, but causes less brittleness. -Thicker tissue blocks, even those up to 1 cm in thickness, can be processed -Tissues do not become translucent

Chloroform

Advantage • Recommended for routine work (6-24 hours). • Miscible with absolute alcohol • Recommended for tough tissues (skin fibroid and decalcified tissues) for nervous tissue, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues • Suitable for large tissue specimens • Not inflammable

Chloroform

-Clear both paraffin and celloidin sections during embedding process -Recommended for CAN tissues and cytological studies(smooth muscles and skin) -Requires two changes -Usually complete in 2-3 days

Cedarwood oil

Advantages • Very penetrating • Miscible with 96% alcohol which it removes readily. • Clears celloidin in 5-6 days • Causes minimal shrinkage of tissues • Tissues may be left in oil indefinitely without considerable damage and distortion • Does not dissolve out aniline dyes • Makes tissues transparent •Does not harden tissues •Does not interfere too seriously with paraffin penetration if it is not completely removed • Clearing with cedarwood oil often improves cutting of the sections

Cedarwood oil

An extremely slow clearing agent, not for routine purposes

Cedarwood oil

-Not normally utilized as a routine clearing agent but recommended for clearing embryos, insects, and very delicate specimens. - Can 70% alcohol without shrinkage and hardening

Aniline oil

• Causes minimum shrinkage • Not guaranteed due to tendency to become adulterated. • Wax impregnation after clearing is slow and difficult •Tissues become brittle, aniline dyes are removed, celloidin is dissolved. • Expensive and not suitable for routine clearing purposes.

Clove oil

- Similar to chloroform but cheaper -Disadvantage are similar to chloroform, producing considerable tissue hardening and dangerous when inhaled on prolongned exposure

Carbon tetrachloride

- Miscible both water and paraffin - If important, due to tissues may be embedded with paraffin within 4 hours after fixation - Transferred straight from Bouin's fluid or a formalin fixative -Changed 3 times within 4 hours and tissues are directly transferred to paraffin - Causes greater shrinkage, dangerous due to toxic fumes that can cause liver damage

Dioxane

- Isoprere polymers, Found in essential oils - Earliest transition solvents -Many terpenes clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have slow gentle non-hardening action. - Strong odors, requires good ventilation. - Moderately effective solvents but toxic - Slow drying, leave oil residue in slides - Expensive

Terpenes

-Volatile oil found in citrus peels -When used immediately prior to cover slipping, the mounting medium usually dissolved in either toluene or xylene, does not mix well with limonene.

Limonene

-Lowest hazard rating -Excellent for preserving fine tissue structure -When using a product contain orange oils, rigorous purification is required to stabilized -Neither stable nor pure can break down to produce compounds which will interfere with staining procedures

Orange oil based clearing agents

- Effective but toxic and a serious health hazard. - Restricted

Chlorinated hydrocarbons

- Non-hazardous, less expensive, - Less shrinkage -Useful as de-alcoholization agent in histopathology laboratory, without losing the quality of the histological details. - Tendency to solidify at lower temperature. - Require temperature to be maintained

Coconut oil

-Clearing agent during tissue processing and dewaxing agent during staining. - Nontoxic, nonhazardous, nonflammable, biodegradable, economic, easy to handle, readily available.

Bleached palm oil

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MAHATHIR ALPHA · 55問 · 2年前

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