CLEARING
Review:
• DEHYDRATION
- WATER MOLECULE IS REMOVED FROM THE
TISSUE
• CLEARING
- DEHYDRATING AGENT IS REPLACED BY
CLEARING AGENT
• IMPREGNATION
- TISSUE IS INFILTRATED WITH A
SUPPORTING MEDIUM
CLEARING (DE-ALCOHOLIZATION)
• process whereby alcohol or a dehydrating agent
is removed from the tissue and replaced with a
substance that will dissolve the wax with which
the tissue is to be impregnated (e.g. paraffin)
• A step after dehydration and before
impregnation
WHY IS CLEARING REQUIRED DURING PROCESSING?
• to remove alcohol from tissues and is replaced
by fluid which is miscible with wax with which
tissue must be impregnated
• necessary as the dehydrating agents are not
miscible with paraffin
- Clearing agent is miscible with both
dehydrating agent as well as paraffin wax
Why is the process of removing alcohol called as
clearing?
• because many (but not all) clearing agents
impart an optical clarity or transparency to the
tissue due to their relatively high refractive
index
HISTOPATHOLOGY LABORATORY
CLEARING
• remove a substantial amount of fat from the
tissue which otherwise presents a barrier to
wax infiltration
CHARACTERISTICS OF A GOOD CLEARING AGENT
• should be miscible with alcohol
• should be miscible with paraffin wax
• should not produce excessive shrinkage,
hardening or damage of tissue
• should not dissolve out aniline dyes
• should not evaporate quickly in a water bath
• Should make tissues transparent
How much volume of clearing agent is required?
• 10 times the volume of the tissue
• Ratio: 1:10
CLEARING AGENTS
Aromatic Hydrocarbons (Xylene, Toluene, and Benzene)
XYLENE (XYLOL)
• most commonly used in histology laboratories
• cost effective
• colorless clearing agent
• used for clearing both for embedding and
mounting procedures
• 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
• evaporates quickly in paraffin oven
• Disadvantages:
- highly flammable
- makes tissues excessively hard and brittle, if
the tissue is left for more than 3 hours in
xylene
- causes shrinkage of tissues thus, not
suitable for nervous tissues and lymph
nodes
LECTURE 3 (MIDTERMS) 1
 CLEARING
- may irritate eyes, nose and respiratory tract
- causes dermatitis
- becomes milky when an incompletely
dehydrated tissue is immersed in it
TOLUENE
• better at preserving tissue structure
• more tolerant of small amounts of water left
behind in the tissues than xylene
• Clearing time: 1-2 hours
• causes less hardening of tissues
• not carcinogenic
• Disadvantages:
- slower than xylene and benzene
- toxic upon prolonged exposure
- more expensive
BENZENE
• penetrates and clears tissues rapidly
• Clearing time: 15-60 minutes
• does not make tissues hard and brittle
• evaporates rapidly in paraffin oven and is
therefore, easily eliminated from the tissue
• Disadvantages:
- highly flammable
- causes considerable tissue shrinkage if left
in benzene for a long time
- extremely toxic to man in excessive
exposure
- carcinogenic –acute myeloid leukemia
- damage the bone marrow resulting in
aplastic anemia
HISTOPATHOLOGY LABORATORY
CHLOROFORM
• Clearing time: 6-24 Hours
• not flammable
• recommended for tough tissues, nervous
tissues, lymph nodes and embryos
• causes minimum shrinkage and hardening of
tissues
• recommended for tough tissues
• can penetrate even thick sections and large
tissue specimens
• Disadvantages:
- does not make tissues transparent
- evaporates quickly from a water bath
- tissues tend to float
o may be avoided by wrapping the
tissues with absorbent cotton gauze
to facilitate sinking of the section in
solution
- attacks rubber and plastics so it cannot be
used on most modern automatic tissue
processors
- toxic to the liver and central nervous system
after prolonged inhalation-hepatitis,
depression and irritability
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

paraffin bath, making the wax impregnation
process very slow
- Tissues cleared in cedarwood oil initially
float before gradually staying to the bottom
- becomes milky upon prolonged storage
- very expensive

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

• XYLENE 1 = 20 MINS
• XYELENE 2 = 20 MINS
• XYLENE 3 = 45 MINS
- At the end of the clearing, tissue must look
transparent

LECTURE 3 (MIDTERMS) 3