HISTOPATHOLOGY

CLEARING / DE-ALCOHOLIZATION

 Process of removing alcohol (dehydrating agents) from the tissue and replacing it with clearing agents that will dissolve the wax with which
the tissue is to be impregnated (paraffin) or the medium on which the tissue is to be mounted (Canada balsam)
 End-product: TRANSLUSCENT APPEARANCE OF THE TISSUE
 Process/Mechanism: Mixes with alcohol and remove it from the tissue to increase the tissue refractive index
 Most commonly used clearing agents in the embedding process are: Xylene, Dioxane, Chloroform, Cedarwood
 Should be miscible with paraffin wax and Canada balsam (Xylene)
 Glycerin and Gum syrup – clears tissue directly from water (frozen section)
 Steps requiring complete clearing of tissues: infiltration and mounting
Characteristics of Good Clearing Agents
1. Should be miscible with alcohol
2. Should be miscible with melted paraffin wax and mounting medium
3. Should not produce excessive shrinkage, hardening or damage of tissues
4. Should not dissolve out aniline dyes
5. Should not evaporate quickly in a water bath
6. Make tissues transparent

CLEARING AGENTS

AGENTS DESCRIPTION ADVANTAGE(S) DISADVANTAGE(S)

XYLENE  Suitable for MOST routine  MOST RAPID agent, clears  Highly inflammable
histological processing schedules of within 15-30mins and suitable for  Not suitable for nervous tissues
<24 hrs. and when tissue block size URGENT BIOPSIES and lymph nodes
is <5mm thickness  Does not extract out aniline dyes  Incompletely dehydrated tissue
 Clearing time: ½ to 1hr.  For CELLOIDIN PROCESS (
may cause Xylene to become
 Used in clearing, embedding, for Mounting procedure)
 Evaporates rapidly in paraffin
MILKY
mounting procedires
oven
 Cheap
TOLUENE  Substitute for xylene or benzene  Recommended for routine  Relatively slower than xylene or
 Clearing time: 1-2hrs. purposes benzene
 Not carcinogenic  Tends to acidify if container is not
 Tissue may be immersed for fully filled
24 hrs  More expensive
 Highly conc. solutions tend to emit
toxic fumes
 HISTOPATHOLOGY

BENZENE  Clearing agent in embedding  Recommended for URGENT  Highly inflammable

process BIOPSIES (15-60mins) and  Transferred to paraffin wax ASAP
routine purposes  Carcinogenic and may damage
 Evaporates rapidly in paraffin the bone marrow causing
oven, easily removes on tissues APLASTIC ANEMIA
CHLOROFORM  Thick tissue blocks can be  Recommended for routine  Relatively toxic to the LIVER
processed (1cm) work (6-24hrs)  Difficult to remove from paraffin
 Does not make tissues  For tough tissues (skin, fibroid, sections and may produce
transluscent decalcified tissues), nervous deterioration of the wax
tissues, lymph nodes and  Vapor may attach the RUBBER
embryos SEAL
 Suitable for LARGE spx.  Tissues tend to float in chloroform
(*REMEDY: wrap with absorbent
cotton gauze)
 Evaporates quickly on water bath
CEDARWOOD  Clear both paraffin and celloidin  Improves cutting of the  Extremely slow clearing agent, NOT
OIL sections sections RECOMMENDED for routine
 Recommended for CNS tissues  Very penetrating purposes
and cytological studies  Clears celloidin for 5-6 days  Hard to eliminate from the tissues in
paraffin bath
(smooth muscle and skin),  Tissues may be left in oil
 Quality is not always uniform and
dense tissues (e.g. uterus) indefinitely without good
 Clearing time: 2-3days considerable damage or  Becomes MILKY due to prolonged
distortion storage
 Does not dissolve out aniline  May produce crystals on ACETIC-
dyes ALC. FIXED TISSUES with a
melting pt. of 35°C (*REMEDY: Heat at
200°C)
ANILINE OIL  Not normally utilized as a  Recommended for clearing
routine clearing agent embryos, insects and very
 Clears 70% alc. delicate spx.

CLOVE OIL  Unsuitable for routine clearing  Minimum shrinkage of tissues  Tends to become
purposes adulterated/weaken
 Dissolves out aniline dye and
celloidin
CARBON  Same with chloroform but  Same with chloroform
TETRACHLORIDE relatively cheaper
THF
 HISTOPATHOLOGY

TERPINEOL  Clear, colorless mixture of isomers

with a FAINT PLEASANT
ODOR and very low evaporation
rate
 GOOD SUBSTITUTE for
cedarwood oil in manual processing
 For clearing eye spx
OIL OF  For skin & smooth muscle
BERGAMOT
OIL OF  For delicate tissues
WINTERGREEN
N-BUTYL  Xylene substitute and
ACETATE nitrocellulose solvent
AMYL ACETATE,  Nitrocellulose solvents in
METHYL DOUBLE EMBEDDING
BENZOATE, TECHNIQUES
METHYL
SALICYLATE
LIMONENE  From a CITRUS FRUIT
 Component of xylene substitutes
 Less viscous than cedarwood oil
and similar to the esters in
clearing action and eliminating
from wax