Clearing Flashcards ionicons-v5-c

Substitution of the conventional xylene with this as a clearing agent during tissue processing and as a dewaxing agent during staining gives good tissues, sections and histological slides. It is nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle, and readily available.

Bleached palm oil

efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue. It can be used as a dealcoholization agent in the histopathological laboratory, without losing the quality of the histological details. The only drawback associated with this 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.

Coconut oil

can be effective solvents, but they areconsidered toxic chemicals, posing serious health risks. Government regulations have restricted most of the effective solvents in this class.

Chlorinated hydrocarbons

neither pure nor stable can break down to produce compounds which will interfere with staining procedures.

Orange oil based clearing agents

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 oil based clearing agents

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

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

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.

terpenes

used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation.

Dioxane

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.

Tetrahydrofuran

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.

Carbon Tetrachloride

reagent causes minimum shrinkage of tissues. However, its quality is not guaranteed due to its tendency to become adulterated. Wax impregnation after clearing with this 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.

clove oil

Aniline oil

requires two changes in clearing solution. Clearing is usually complete in 2-3 days.

Cedarwood Oil

Cedarwood Oil

Thicker tissue blocks, even those up to I cm. in thickness, can be processed

Chloroform

when used for clearing of tissues during the embedding process, is slower in action than xylene, but causes less brittleness.

Chloroform

benzene

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

benzene

better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene

toluene

better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene

toluene

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

routinely used chemical in histology and pathology laboratories because of its vital role in the paraffin wax tissue processing method

xylene

Clearing time is usually 1/2 to 1 hour. It is used for clearing, both for embedding and mounting procedures.

xylene

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

Choice of clearing agent

used when the tissue is to be cleared directly from water, as in a frozen section. No de-alcoholization is involved in this process.

Glycerin and Gum syrup

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

Clearing

Methyl benzoate or Methyl Salicylate

(2) tissue must be wrapped in cotton gauze for them to settle to the bottom of the container

Chloroform

(1) if stored for a long time, this clearing agent must be filtered before it is use

Cedarwood Oil

o The lower the boiling agent, the faster it is replaced by paraffin o Xylene has a low boiling point while chloroform has a higher boiling point (that’s why its not easily replaced by paraffin)

Viscosity

o The lower the boiling agent, the faster it is replaced by paraffin o Xylene has a low boiling point while chloroform has a higher boiling point (that’s why its not easily replaced by paraffin)

Boiling point of clearing agent

clearing agents in double embedding methods

Methyl benzoate or Methyl Salicylate

Aniline oil

o If the fixative used is acetic-alcohol, this forms crystals which are removed by heating the this at 200 degree Celsius if you still wish to use it for other tissues

Cedarwood Oil

oil becomes milky and requires filtration before use Very expensive

Cedarwood Oil

Remedy when Tissues placed in cedarwood floats before they gradually settle at the bottom

Putting absolute alcohol at the top of it

o Hard to eliminate from tissues in paraffin oven thus impregnation with parafiin would require additional time

Cedarwood Oil

Cedarwood Oil

o Clearing time: 2-3 days o Works well with celloidin and tissues are easy to cut o Minimal tissue hardening and shrinkage

Cedarwood Oil

o Can be used in tissues to be embedded using paraffin or celloidin

Cedarwood Oil

o Human symptoms of acute (short-term) inhalation and oral exposures to this include headache, weakness, lethargy, nausea, and vomiting. o Acute exposures to higher levels and chronic (long-term) inhalation or oral exposure to this produces liver in humans.

Carbon Tetrachloride

o Properties and disadvantages are similar to chloroform o Can cause minimal tissue hardening o Cheaper than chloroform

Carbon Tetrachloride

• Reproductive effects, such as decreased conception rates, decreased ability to maintain pregnancy, and an increase in the percentage of abnormal sperm were observed in animals exposed to this through inhalation. • Animal studies have noted decreased fetal weight, increased fetal resorptions, but no evidence of birth defects, in animals orally exposed to this

Chloroform

• Toxic to the central nervous system and liver after prolonged inhalation o Lower concentrations (resulting in dizziness, headache, tiredness) o At very high levels, this exposure may result in death.

Chloroform

Remedy when Tissues float in chloroform because it has a bigger density thus affecting the clearing process

Wrap tissue with cotton gauze to make it settle at the bottom of the container

• Not very soluble in paraffin and does not easily evaporate in the oven thus impregnation will take time

Chloroform

• Does not make tissue transparent

Chloroform

• Does not cause tissue hardening thus ideal for skin, fibroid tissue, decalcified tissue)

Chloroform

• Can penetrate even thick tissue sections and large tissue specimen • Does not cause tissue shrinkage that’s why its ideal for biopsies of lymph nodes and nerves

Chloroform

• Clearing time: 6-24hrs • NOT FLAMMABLE

Chloroform

(7) Can cause spontaneous abortions

toluene

(6) Not affordable

toluene

(5) Not Ideal for urgent biopsies

toluene

(4) Not carcinogenic

toluene

(3) Can cause aplastic anemia and leukemia

benzene

(2) Tissue shrinkage happens upon prolonged exposure

benzene

(1) Tissue can become brittle and excessively hard upon prolonged exposure

xylene

• Breathing high levels of this during pregnancy has been shown to result in children with birth defects and to retard mental abilities and growth. • There is evidence that exposure to this at work is associated with spontaneous abortion.

toluene

• Slower than xylene and is EXPENSIVE • Emits fume that can be toxic to humans o CNS effects (headache, dizziness, drowsiness, euphoria, hallucinations, tremor, and seizures) o Liver and kidney damage o Cardiac arrythmia

toluene

toluene

• Clearing time: 1-2hrs • Used as substitute for xylene and benzene

toluene

• Exposure to this may be harmful to the reproductive organs. • Some women workers who breathed high levels of this for many months had irregular menstrual periods. When examined, these women showed a decrease in the size of their ovaries.

benzene

• Chronic exposure to this can cause bone marrow suppression resulting to aplastic anemia • this is also a carcinogenic – acute myeloid leukemia

benzene

tissue becomes brittle and excessively hard

xylene

there is considerable tissue shrinkage

benzene

• Same as xylene, highly volatile inside the oven thus is easily replaced by paraffin wax • Miscible in alcohol and makes tissue transparent • Unlike xylene, it is preferred by some because it doesn’t cause tissue to become hard and brittle

benzene

benzene

• Repeated or prolonged skin contact with liquid this can defat the skin, causing it to crack and peel (it will dissolve the natural oils in the skin) • At very high levels of exposure, this can injure the liver and kidneys.

xylene

• Inhalation of this clearing agent vapors in small amounts can cause headache, dizziness, drowsiness, and nausea. • Effect of this on the central nervous system is attributed to the liposolubility of this in the neuronal membrane. o It has been suggested that this disturbs the action of proteins essential to normal neuronal function either by disruption of the lipid environment in which the membrane proteins function or by direct interaction with the proteins in the membranes.

xylene

• Its vapor is heavier than air and may accumulate in low-lying areas. • Children exposed to the same levels of this clearing agent vapor as adults may receive a larger dose because they have greater lung surface area:body weight ratios.

xylene

• Exposure to high levels of this clearing agent can irritate the lungs, causing chest pain and shortness of breath. • Extreme overexposure (e.g., in a confined space) can result in pulmonary edema, a potentially life-threatening condition in which the lungs fill with fluid.

xylene

• Becomes milky if there is still water in the tissue o Make sure dehydration process is done completely o Repeat dehydration using absolute alcohol

xylene

xylene

• Tissue immersed in this clearing agent for more than 3hrs become hard and brittle

xylene

• Evaporates rapidly in the oven thus can easily be replaced by the melted paraffin • Highly flammable

xylene

• Miscible with alcohol and paraffin • Makes tissue transparent • Does not affect staining

xylene

• Colorless chemical and inexpensive • Clearing time: 15-30 mins (rapid clearing agent)

xylene

• Commonly used clearing agent in routine biopsies

xylene

Tissue to Clearing Agent ratio

1:10

(1) Should be miscible with alcohol to promote rapid removal of the dehydrating agent (2) Should be miscible or is easily removed by the melted paraffin wax to facilitate impregnation of the tissue (3) Should not produce excessive tissue shrinkage, hardening and damage (4) Should not dissolve dyes (5) Should not evaporate quickly in water bath but should readily evaporate in the oven (6) Should make tissue transparent

Characteristics of a Good Clearing Agent

to facilitate the penetration of the tissue by the embedding medium which is the wax

purpose of the clearing

• The clearing of the tissue is due to the increase in the …. of the tissue caused by the clearing agents

refractive index

• After the dehydrating agent is removed, the tissue will have a translucent appearance

Clearing

• Process whereby alcohol or a dehydrating agent is removed by the tissue and replaced it with a substance that will dissolve with the wax that will be used in impregnation

Clearing