CLEARING HISTOPATHOLOGY LABORATORY

Review: CLEARING
• DEHYDRATION • remove a substantial amount of fat from the
- WATER MOLECULE IS REMOVED FROM THE tissue which otherwise presents a barrier to
TISSUE wax infiltration
• CLEARING
CHARACTERISTICS OF A GOOD CLEARING AGENT
- DEHYDRATING AGENT IS REPLACED BY
• should be miscible with alcohol
CLEARING AGENT
• should be miscible with paraffin wax
• IMPREGNATION
• should not produce excessive shrinkage,
- TISSUE IS INFILTRATED WITH A
hardening or damage of tissue
SUPPORTING MEDIUM
• should not dissolve out aniline dyes
CLEARING (DE-ALCOHOLIZATION) • should not evaporate quickly in a water bath
• process whereby alcohol or a dehydrating agent • Should make tissues transparent
is removed from the tissue and replaced with a
substance that will dissolve the wax with which How much volume of clearing agent is required?
the tissue is to be impregnated (e.g. paraffin) • 10 times the volume of the tissue
• A step after dehydration and before • Ratio: 1:10
impregnation CLEARING AGENTS
WHY IS CLEARING REQUIRED DURING PROCESSING? Aromatic Hydrocarbons (Xylene, Toluene, and Benzene)
• to remove alcohol from tissues and is replaced XYLENE (XYLOL)
by fluid which is miscible with wax with which • most commonly used in histology laboratories
tissue must be impregnated • cost effective
• necessary as the dehydrating agents are not • colorless clearing agent
miscible with paraffin • used for clearing both for embedding and
- Clearing agent is miscible with both mounting procedures
dehydrating agent as well as paraffin wax • is generally suitable for most routine histologic
processing schedules of less than 24 hours and
when the tissue block size is less than 5mm in
thickness
• Clearing time: 15-30 mins, 1 hour at the most
(most rapid clearing agent)
• Suitable for urgent biopsies
• miscible with alcohol and paraffin
• does not extract out aniline dyes
Why is the process of removing alcohol called as • evaporates quickly in paraffin oven
clearing? • Disadvantages:
• because many (but not all) clearing agents - highly flammable
impart an optical clarity or transparency to the - makes tissues excessively hard and brittle, if
tissue due to their relatively high refractive the tissue is left for more than 3 hours in
index xylene
- causes shrinkage of tissues thus, not
suitable for nervous tissues and lymph
nodes

LECTURE 3 (MIDTERMS) 1
 CLEARING HISTOPATHOLOGY LABORATORY

- may irritate eyes, nose and respiratory tract

TOLUENE
• better at preserving tissue structure
• more tolerant of small amounts of water left CHLOROFORM
behind in the tissues than xylene • Clearing time: 6-24 Hours
• Clearing time: 1-2 hours • not flammable
• causes less hardening of tissues • recommended for tough tissues, nervous
• not carcinogenic tissues, lymph nodes and embryos
• Disadvantages: • causes minimum shrinkage and hardening of
- slower than xylene and benzene tissues
- toxic upon prolonged exposure • recommended for tough tissues
- more expensive • can penetrate even thick sections and large
tissue specimens
BENZENE
• Disadvantages:
• penetrates and clears tissues rapidly
- does not make tissues transparent
• Clearing time: 15-60 minutes
- evaporates quickly from a water bath
• does not make tissues hard and brittle
- tissues tend to float
• evaporates rapidly in paraffin oven and is
o may be avoided by wrapping the
therefore, easily eliminated from the tissue
tissues with absorbent cotton gauze
• Disadvantages: to facilitate sinking of the section in
- highly flammable solution
- causes considerable tissue shrinkage if left - attacks rubber and plastics so it cannot be
in benzene for a long time used on most modern automatic tissue
- extremely toxic to man in excessive processors
exposure - toxic to the liver and central nervous system
- carcinogenic –acute myeloid leukemia after prolonged inhalation-hepatitis,
- damage the bone marrow resulting in depression and irritability
aplastic anemia
CEDARWOOD OIL
• used to clear both paraffin and celloidin
sections during the embedding process
• Clearing time: 2-3 days
• causes minimal shrinkage of tissues
• does not harden tissues
• recommended for central nervous system
tissues and cytological studies, particularly
smooth muscle and skin
• Disadvantages:
- extremely slow clearing agent

LECTURE 3 (MIDTERMS) 2
 CLEARING HISTOPATHOLOGY LABORATORY

- hard to eliminate from the tissues in

ANILINE OIL
• recommended for clearing embryos and insects,
and very delicate specimens
• does not cause excessive tissue shrinkage and
hardening

CARBON TETRACHLORIDE
• properties are very similar to that of chloroform
• produces considerable tissue hardening
• dangerous to inhale on prolonged exposure due
to its highly toxic effects

TYPICAL CLEARING SEQUENCE

LECTURE 3 (MIDTERMS) 3