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
