HISTOPATHOLOGY

CLEARING & DEALCOHOLIZATION

Jenivie Morillo Jose University of Santo Tomas National Childrens Hospital

DEFINITION:
CLEARING / DEALCOHOLIZATION Process of removing alcohol from section of tissue by immersing them in ANTE MEDIUM Tissue becomes transparent as a result of ante medium raising the REFRACTIVE INDEX

MOST COMMON CLEARING AGENTS

1. BENZENE CLEARING TIME: 15-60 minutes
BEST ROUTINE CLEARING AGENT for urgent biopsies Rapid in action HIGHLY INFLAMMABLE, CARCINOGENIC

IDEAL CHARACTERISTICS:
Miscible with alcohol Miscible with paraffin wax Should not produce excessive tissue shrinkage and hardening Should not evaporate quickly in the water bath

Should make tissue transparent

MOST COMMON CLEARING AGENTS

2. CHLOROFORM CLEARING TIME: 6- 24 HOURS ONE BATH is needed Miscible with ABSOLUTE ALCOHOL Causes very little SHRINKAGE and hardening of tissues Suitable for LARGE TISSUE SPECIMENS, TOUGH TISSUES(skin, fibroid, decalcified tissues), for NERVOUS TISSUE, LYMPH NODES and EMBRYOS Slowly DISPLACES alcohol TOXIC to the LIVER

MOST COMMON CLEARING AGENTS

3. XYLENE(XYLOL)
CLEARING TIME: 15-30 minutes ONE BATH is needed Miscible with ABSOLUTE ALCOHOL COLORLESS

For URGENT BIOPSIES

Rapid in action Makes tissue TRANSPARENT

MOST COMMON CLEARING AGENTS

3. XYLENE(XYLOL) *continuation*
NOT CARCINOGENIC Cheap HIGHLY INFLAMMABLE VOLATILIZED rapidly in PARAFFIN OVEN

EXCESSIVE tissue shrinkage and

hardening MILKY if INCOMPLETELY DEHYDRATED

MOST COMMON CLEARING AGENTS

4. TOULENE
CLEARING TIME: 15-60 minutes ONE BATH is needed Miscible with ABSOLUTE ALCOHOL Rapid in action

NOT CARCINOGENIC
HIGHLY INFLAMMABLE Shrinkage of tissues

MOST COMMON CLEARING AGENTS

5. CEDAR OIL For CENTRAL NERVOUS SYSTEM, SKIN and SMOOTH MUSCLE and CYTOLOGICAL WORK CLEARING TIME: Indefinite until clear TWO BATHS Miscible with 95% alcohol BEST CLEARING AGENT Minimal shrinkage and hardening of the tissues Extremely slow Expensive

MOST COMMON CLEARING AGENTS

6. CLOVE OIL
For SKIN and SMOOTH MUSCLE CLEARING TIME: Indefinite TWO BATHS Miscible with 95% alcohol

LITTLE shrinkage
Wax impregnation is SLOW and DIFFICULT Makes tissue BRITTLE

MOST COMMON CLEARING AGENTS

7. OIL OF BERGAMOT
For SKIN and SMOOTH MUSCLE CLEARING TIME: Indefinite TWO BATHS Miscible with 95% alcohol

NOT EXTRACT aniline dyes

Used with CELLOIDIN PREPARATION Expensive

MOST COMMON CLEARING AGENTS

8. OIL OF ORIGANUM
Spanish hop oil or thyme oil or mixtures of these two, with or without turpentine For SKIN CLEARING TIME: Indefinite TWO BATHS miscible with 95% alcohol DOESNT DISSOLVE celloidin DISSOLVES ANILINE DYES Expensive

MOST COMMON CLEARING AGENTS

9. OIL OF WINTERGREEN
methyl salicylate; artificial oil For DELICATE TISSUES CLEARING TIME: Indefinite TWO BATHS

Miscible with 95% alcohol

MOST COMMON CLEARING AGENTS

9. OIL OF WINTERGREEN *continuation*
HARDENING EFFECT OF ALCOHOL can

be avoided
Mainly for CELLOIDIN SECTIONS but must be soaked by CHLOROFORM or XYLENE before mounting POOR DEHYDRANT OXIDIZES easily on STANDING

MOST COMMON CLEARING AGENTS

10. CARBON DISULFIDE For SMOOTH MUSCLE
CLEARING TIME: Indefinite TWO BATHS Minimal shrinkage of tissue VERY INFLAMMABLE, with FOUL ODOR

Wax impregnation is SLOW and DIFFICULT

MOST COMMON CLEARING AGENTS

11. CARBON TETRACHLORIDE

For ROUTINE work Similar to CHLOROFORM

MOST COMMON CLEARING AGENTS

12. CARBOL XYLENE For FRIABLE TISSUES
CLEARING TIME: up to 1 hour Made by adding ANHYDROUS CRYSTALS of PHENOL to XYLENE until no more dissolves

Good for MOIST and HUMID CLIMATES

CORROSIVE

MOST COMMON CLEARING AGENTS

13. TETRAHYDROFURAN For CELLULAR ORGANS, FRIABLE TISSUES, SKIN, ATHERMATOUS ARTERIES, HARD COLLAGENOUS SPECIMENS CLEARING TIME: 2-4 hours THREE BATHS Colorless liquid with 65C (boiling point), -108.5C (freezing point) and 0.8894 (density) Wide range solvent (including fats) Soluble in most solvents (lower alcohols, ketones, esters, chloroform, ether, acetone, benzene, zylene, dioxane, creosote)

MOST COMMON CLEARING AGENTS

13. TETRAHYDROFURAN *continuation* SUPERIOR to ORDINARY DEHYDRATING CLEARING AGENTS (can perform 2 processes at the same time) SHORTENS the TOTAL PROCESSING TIME and allowing MORE time for fixation

Causes CONJUNCTIVAL IRRITATION for about 6

MONTHS OFFENSIVE ODOR

MOST COMMON CLEARING AGENTS

14. TERPINEOL EXCELLENT for DELICATE MATERIAL

especially EYES
CLEARING TIME: 2-4 hours TWO BATHS

Miscible with 95% alcohol

Solution consists of 1 part XYLENE and 4 parts TERPINEOL Expensive

MOST COMMON CLEARING AGENTS

15. PHENOL For SMOOTH MUSCLE
CLEARING TIME: 2 hours TWO BATHS Good for PARAFFIN and CELLOIDIN WORK

DEHYDRATES and CLEARS very quickly

VOLATILE

MOST COMMON CLEARING AGENTS

16. HIGH TESTAVIATION LEAD FREE GASOLINE
EXCELLENT CLEARING AND DEALCOHOLIZATION AGENT Cedar oil, gasoline and petroleum ether cause the LEAST HARDENING of tissues

APPLICATIONS OF CLEARING IN HISTOPATHOLOGY:

1. To make tissue, embryos and parasite

transparent
2. Dealcoholization of tissue preparatory to wax impregnation 3. Dealcoholization of stained sections prior to mounting in PERMOUNT, CLARITE or CANADA BALSAM