HISTOPATH LEC - DEHYDRATION, CLEARING

Dehydration

The process of removing intracellular and extracellular water from tissue.

Fixation

The step that precedes dehydration in tissue processing.

1. Dehydrates RAPIDLY without producing shrinkage or distortion

2. Should NOT EVAPORATE VERY FAST.

3. Should BE ABLE TO DEHYDRATE EVEN FAT TISSUES

4. Should NOT HARDEN TISSUES EXCESSIVELY

5. Should NOT BE TOXIC TO THE BODY

6. Should NOT REMOVE STAINS

7. Should NOT BE A FIRE HAZARD

Ideal Dehydrating Agent

Ethyl Alcohol

The best dehydrating agent, fast-acting, and not toxic.
Recommended for Routine
76.3 C Boiling Point
99.85%

Methyl Alcohol

A toxic dehydrating agent used for blood and tissue films.
Smear preparation.

Butyl Alcohol / Butanol

Plant and animal microtechniques.
Slow.
Recommended for those that doesn’t require rapid processing.
Odorous.

Isopropyl Alcohol

A substitute for ethanol, not ideal for staining solutions.

Acetone

A, cheap, rapid-acting, miscible and flammable dehydrating agent that causes shrinkage and removes lipids.

Dehydrates in ½ to 2 hours

Urgent biopsies

Dioxane / Diethylene dioxide

An excellent dehydrating and clearing agent, but expensive and dangerous.

Cumulative toxic action. Work in fumehood.

Tissue can be stored for long period.

Cellosolve / ETHYLENE GLYCOL MONOETHYL ETHER

A rapid dehydrating agent that is not harmful to tissues.

Tetrahydrofuran

A non-toxic dehydrating agent that dissolves fats and has an offensive odor.

Prolonged exposure → conjunctival irritation

Use in well-ventilated room

Triethyl Phosphate

A dehydrating agent with minimum shrinkage, soluble in various solvents.

Dehydrates sections and smears ff certain stains

30 minutes each alcohol

1mm thick blocks

90 minutes or longer each change

5mm thick blocks

70% ethanol

Tissues may be held and stored indefinitely in (BLANK) without harm

Clearing

The process of removing excess alcohol from tissue and making it translucent.

Replaces substance that dissolves the wax where the tissue is to be impregnated.

Low boiling points

1. Should be miscible with alcohol

2. Should be miscible with paraffin wax and/or mounting medium

3. Should not produce excessive tissue shrinkage and hardening

4. Should not dissolve aniline dyes

5. Should not evaporate quickly

6. Should not make tissue transparent

Good Clearing Agent Characteristics

1. Type of tissue, type of processing

2. Processor system used

3. Processing conditions: temperature, vacuum, pressure

4. Safety factors

5. Cost and convenience

6. Speedy removal of dehydrating agent

7. Ease of removal by molten paraffin wax

8. Minimal tissue damage.

Choice of clearing agent depends on the following:

Xylene

½ to 1 hour

A commonly used clearing agent that is rapid but highly flammable.

Xylene

Clearing Agent :

Advantages:
Most rapid
URGENT BIOPSIES
Mounting; does not dissolve celloidin
Evaporates quickly
cheap

Disadvantages:
Excessive hardening and shrinkage
Milky when incompletely dehydrated

Toluene

1-2 hours

A substitute for xylene or benzene,

fairly rapid but also highly flammable.

Chloroform

Routine clearing during embedding process

Used for tough tissues (skin, fibroid, decalcified tissues) nervous tissue (brain, lymph nodes, embryos), large tissue specimens, but relatively toxic to the liver.

Not volatile and doesn’t make tissue transparent, evaporates quickly.

Benzene

MOST TOXIC. causes aplastic anemia and cancer.

A rapid-acting , recommended for urgent biopsies at 15-60 minutes. Recommended for routine purposes. Rapid volatilization.

Cedarwood Oil

2-3 days

Clears both paraffin and celloidin.

Clearing CNS and cytological studies of smooth muscle and skin.

A slow-clearing agent that penetrates easily and minimizes tissue shrinkage.

VERY EXPENSIVE. EXTREMELY SLOW.

Aniline Oil

Used for clearing delicate specimens like embryos and insects.

Carbon Tetrachloride

Similar to chloroform but cheaper, with considerable hardening effects. Dangerous to inhale in prolonged exposure.

Clove Oil

A clearing agent for skin and smooth muscle, but slow and expensive.
Brittle tissues, removed aniline dyes, dissolved celloidin.

Tetrahydrofuran

Both clearing and dehydrating agent

Non-toxic, offensive odor

Well-ventilated room

METHYL BENZOATE AND METHYL SALICYLATE

Double embedding techniques
Slow acting

Histoclear

A non-toxic clearing agent derived from food-grade materials.

Prolonged Clearing Time

Can lead to brittleness in tissues.

Incomplete Clearing

Results in uneven hematoxylin-eosin staining and poor nuclear chromatin patterns.

oil of bergamot

clearing skin and smooth muscle

oil of origanum

clearing skin

oil of wintergreen

aritificial oil for delicate tissues

carbon disulfide

smooth muscle, foul odor like rotten egg

carbol xylene

for friable tissues

terpineol

delicate materials like eyes

phenol

smooth muscles

high aviation lead free gasoline

excellent clearing agent