HISTOPATH LEC - DEHYDRATION, CLEARING

DEHYDRATION

  • Removes intracellular and extracellular water from tissue

  • Follows fixation ; prior to wax infiltration

  • 70 - 95 - 100% alcohol

  • First step is a mix of FORMALIN and ALCOHOL

  • Amount: At least 10 TIMES the volume or more.

Characteristics of Ideal Dehydrating Agent:

  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

Example of Dehydrating Agents:

  1. Ethyl Alcohol

  2. Acetone

  3. Methyl Alcohol

  4. Isopropyl Alcohol

  5. Ethoxyethanol

  6. Dioxane

  7. Cellosolve

  8. Triethyl phosphate

  9. Tetrahydrofuran

  10. Polyethylene glycol

Specific Dehydrating Agents:

ETHYL ALCOHOL

  • Best dehydrating agent

  • Not very expensive

  • Not poisonous/toxic

  • Fast-acting

  • Penetrates easily

  • Rec’d for ROUTINE dehydrating

  • Boiling point: 76.3C

  • Mixes with water and inorganic solvents

  • Does not cause tissue shrinkage

  • 99.85% ethanol

METHYL ALCOHOL / METHANOL

  • Toxic dehydrating agent

  • BLOOD and TISSUE films

  • SMEAR preparations

BUTYL ALCOHOL / BUTANOL

  • PLANT and ANIMAL microtechniques

  • SLOW dehydrating agent

  • Less shrinkage and hardening

  • Rec’d for those that do not require rapid processing

  • ODOROUS

ISOPROPYL ALCOHOL

  • Substitute for ethanol

  • Not ideal for staining solutions

ACETONE

  • Highly MISCIBLE and FLAMMABLE

  • Cheap and rapid-acting

  • URGENT BIOPSIES

  • Dehydrates in ½-2 hours

  • Causes shrinkage and brittleness

  • Removes lipids

DIOXANE/DIETHYLENE DIOXIDE

  • Excellent dehydrating and clearing agent

  • Expensive and dangerous

  • CUMULATIVE TOXIC ACTION

  • Work in fume hood

  • Miscible with paraffin, alcohol and xylene

  • Less tissue shrinkage

  • Tissue can be stored for long period

CELLOSOLVE / ETHYLENE GLYCOL MONOETHYL ETHER

  • Dehydrates rapidly

  • Can be stored without distortion

  • Not harmful to tissues

  • BEST substitute for ethanol

TETRAHYDROFURAN

  • Dehydrates and clears

  • Dissolves fats

  • Non-toxic

  • Prolonged exposure → conjunctival irritation

  • Offensive odor

  • Improved results on staining procedures

  • Use in well-ventilated room

TRIETHYL PHOSPHATE

  • Soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene

  • Dehydrates sections and smears ff certain stains

  • Minimum shrinkage

  • Duration of dehydration should be kept to minimum consistent with tissues being processed

  • 1mm thick blocks = 30 minutes each alcohol

  • 5mm thick blocks = 90 minutes or longer each change

  • Tissues may be held and stored indefinitely in 70% ethanol without harm

CLEARING / DEALCOHOLIZATION

  • Removes excess alcohol from the tissue

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

  • Makes tissues translucent

  • LOW BOILING POINTS

Characteristics of a Good Clearing Agent

  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

Choice of clearing agent depends on the ff:

  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.

XYLENE

  • COMMONLY USED in routine

  • Excellent and true clearing agent

  • ½ to 1 hour

ADVANTAGES

  • Most rapid

  • URGENT BIOPSIES

  • Mounting; does not dissolve celloidin

  • Evaporates quickly

  • cheap

DISADVANTAGES

  • Highly inflammable

  • Excessive hardening and shrinkage

  • Becomes MILKY when incompletely dehydrated

TOLUENE

  • Substitute for XYLENE OR BENZENE

  • Fumes may be toxic

  • 1-2 hours

ADVANTAGES

  • Fairly rapid

  • Recommended for routine

  • Not carcinogenic

  • Doesn’t become excessively hard/brittle

DISADVANTAGES

  • Highly inflammable

  • expensive

CHLOROFORM

  • For routine clearing during embedding process

ADVANTAGES

  • For tough tissues like: skin, fibroid, decalcified tissues

  • for nervous tissue like brain, lymph nodes, embryos

  • Suitable for large tissue specimens

DISADVANTAGES

  • Relatively toxic to liver

  • Not volatile in paraffin oven

  • Doesn’t make tissue transparent

  • Evaporates quickly in water bath

BENZENE

  • Hazardous, causes aplastic anemia and cancer

  • MOST TOXIC

ADVANTAGES

  • RAPID ACTING

  • Rec’d for urgent biopsies - 15-60 mins

  • Rec’d for routine purposes

  • Volatilizes rapidly

  • Miscible with absolute alcohol

  • Does not make tissue hard/brittle

  • Minimum shrinkage

  • Makes tissue transparent

DISADVANTAGES

  • Highly inflammable

  • May cause shrinkage if left for long time

  • Excessive exposure → may become carcinogenic

CEDARWOOD OIL

  • Used to clear both paraffin and celloidin sections

  • Rec’d for clearing CNS and cytological studies of smooth muscles and skin

  • 2-3 Days

ADVANTAGES

  • Penetrates easily

  • Miscible with 95% alcohol

  • Clears celloidin in 5-6 days

  • Minimal shrinkage and hardening of tissues

  • May be left in oil indefinitely w/o damage

  • Does not dissolve out aniline dyes

  • Transparent tissues

  • Clearing often improves cutting

DISADVANTAGES

  • EXTREMELY SLOW, not for routine

  • Hard to eliminate from tissues -> slow impregnation

  • Quality is not always uniform

  • Becomes milky upon prolonged storage

  • VERY EXPENSIVE

ANILINE OIL

  • Clearing embryos, insects, delicate specimens

  • Not for routine

CARBON TETRACHLORIDE

  • Similar to chloroform

  • Cheaper

  • Considerable hardening

  • Dangerous to inhale in prolonged exposure

CLOVE OIL

  • For clearing skin and smooth muscle

  • Minimum shrinkage

  • Slow and difficult impregnation

  • Brittle tissues; removed aniline dyes; dissolved celloidin

  • Expensive

  • Not for routine

TETRAHYDROFURAN

  • Both clearing and dehydrating agent

  • Non-toxic, offensive odor

  • Well-ventilated room

METHYL BENZOATE AND METHYL SALICYLATE

  • Double embedding techniques

  • Slow acting

HISTOCLEAR

  • Recently introduced

  • Non-toxic derivative of food grade materials

Other Clearing Agents:

OIL OF BERGAMOT - for clearing skin and smooth muscle

OIL OF ORIGANUM - for clearing skin

OIL OF WINTERGREEN - artificial oil for delicate tissues

CARBON DISULFIDE - smooth muscle, foul odor (rotten egg)

CARBOL XYLENE - for friable tissues

TERPINEOL - for delicate materials like eyes

PHENOL - for smooth muscles

HIGH AVIATION LEAD FREE GASOLINE - excellent clearing agent

  • Clearing agents also remove a proportion of tissue fat (a barrier to wax impregnation)

  • Prolonged clearing time = brittleness

  • Incomplete clearing = uneven hematoxylin-eosin staining, poor nuclear chromatin patterns