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OBJECTIVES
HISTOLOGY TECHNIQUES
Define dehydration, Clearing, Infiltration/
Impregnation and Embedding process.
Explain the tissue processing functions.
State various dehydration and clearing
solutions.
Elaborating tissue processing at level
dehydration, Clearing, Infiltration/
Impregnation and Embedding including action,
preparation and indication.
SMS2143
LECTURE 3
INTRODUCTION
Cont..
Once fixed or decalcified, the tissue must be
treated to allow the cutting of the thin sections
required for viewing under the microscope.
The procedures designed to prepare the tissue for
embedding by using paraffin wax are collectively
known as Tissue Processing.
Processing.
Then the tissue will be immersed in the liquid
paraffin wax solution before the tissue undergoes
embedding process.
First, the sample is dehydrated by immersion in a
series of aqueous alcohol solutions gradually
moving to pure alcohol.
Finally the tissue is embedded in paraffin wax,
which enables the cutting of sections of between 4 10 microns thickness.
The movement through the series of baths in Tissue
Processing occurs either by hand or by means of an
automated processor.
The tissue is then soaked in an appropriate solvent
to remove the alcohol (clearing).
DEHYDRATION
TISSUE PROCESSING
The
Purpose
: preparation for the tissue for
embedding by using paraffin wax
Divided into 4 steps which are ::
Dehydration
Clearing
Infiltration & Impregnation
Embedding
purpose of the dehydration process is:
To remove water and fixative from tissue
To replace the space with solution that can be mixed
with paraffin
This
is because wet fixed tissue CANNOT be
directly infiltrated with paraffin wax.
This process is done by using ALCOHOL solution
solution..
The technique that is used to perform this process
is SERIAL DILUTION.
DILUTION.
The tissue will be soak in a series dilution of
ascending alcohols such as (70% 95%
100%)
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List of commonly used DEHYDRANTS
Cont
DEHYDRANT
Tissue
can be brittle if it is left too long in high
conc. of alcohol
Most essential step
Tissue transferred directly from dH2O to high
[alcohol] would cause distortion of tissue elements
because of mixing dH2O + alcohol
More delicate object to be processed, more
numerous steps required
Failure to do this properly will prevent the tissue
from becoming impregnated successfully later with
paraffin wax or celloidin
Other dehydrants agent used in histopath lab
Agents
Advantages
Disadvantages
Ethyl alcohol
Fast action
Shrink tissue
Isopropyl alcohol
Non toxic, good
penetration
Tissue harden
Methyl alcohol
Fast action
Toxic
Butyl alcohol
Less shrinkage & has
hardening effect for
long term
Slow dehydration
capability
Spirit
Fast action
Slow dehydration
capability
2-propanone
Fast action
Less expensive
Shrink tissue
Tetrahydrofuran
Less expensive
Volatile &
flammable
Method
Tissue block are placed
in perforated containers
or cassette
To allow thorough
penetration
Identification number
place in the cassette
Lid securely closed
Tiring fragments of
tissue should be
wrapped in a single layer
of lens paper
Ethanol
Methanol
Acetone
DESCRIPTION
Flammable
Expensive
Mixture of water and organic material
(hydrophilic)
Flammable
Rarely use
Colorless
Flammable
Used for urgent biopsy
Rarely used because it can cause tissue
damage and low in penetration
Other dehydrants can be used, but have major disadvantages.
Dioxane can be used without clearing, but has toxic fumes.
The process can be carried out by:
transferring tissue direct from fixative into methylated spirit
(for 1 6 hrs) or 70%, 80% & 90% alc.
absolute alcohol 1 6 hrs
absolute alcohol 1 12 hrs
Delicate tissue (brain, spinal cord, embryo) better carried
in stages using varying grades of alcohol
Other reagents acetone, cellosolve, butanol and
isopropanol
When dehydrating tissue, a large bulk of fluid must always
be used - 1:10
Dehydrant most common - ethyl alcohol, isopropyl alcohol
(99%) is the best substitute for ethyl alcohol
Cont..
During
dehydration, tissues are passed through a
series of progressively increasing conc. of alcohol
With one change in each conc.
Duration of 1 hr in each
Keep covered to avoid evaporation
Starting [alcohol] is usually 80%
For soft tissue 30%, 50%, 70%
Important that the final bath of alcohol is pure and
change 3X
Free from water
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Cont..
CLEARING
The step following dehydration is called "clearing"
It is also referred as dealcoholization
The purpose of the clearing process ::-
As a result, when the tissue is completely infiltrated with
the clearing agent, it becomes translucent.
This change in appearance is often used as an indication of
the effectiveness or completeness of the clearing process.
Important characteristic in choosing clearing agent
alcohol removal from tissue to make it transparent
Clearing agents will replace the alcohol and enable paraffin to
penetrate into tissue
Can be mix with dehydrant
Tissue will seen shining and transparent when soak in
cleaning agent
It consists the replacement of dehydrant with a substance
that will be miscible with the embedding medium (paraffin).
The term "clearing" comes from the fact that the clearing
agents often have the same refractive index as proteins.
Can be mix with paraffin wax
DESCRIPTION
Advantages
Disadvantages
Xylene
Tissue cant be
soak more than
6 hours
Carcinogenic
Flammable
Fast action
Non-toxic
Less expensive
Tissue become
transparent
Shrink tissue
Harden tissue
Flammable
Toluene
More suitable
but dangerous
Fast action
Less expensive
Flammable
Slightly toxic
Benzene
Highly in toxicity Fast
Carcinogenic
Less expensive
Minimum tissue
shrinkage
Flammable
Toxic
Carcinogenic
Expensive
Suitable for
CNS tissue
Chloroform Low in reaction
Carcinogenic
Poisonous
Non flammable
Minimum tissue
hardening
Tissue not brittle
Slow clearing
capacity
Toxic
Cont..
The most common clearing agent is Xylene.
Xylene is reasonably cost effective and works well for
short--term clearing of small tissue blocks.
short
Long-term immersion of tissue in Xylene results in tissue
Longdistortions.
INFILTRATION & IMPREGNATION
CLEARING AGENT
Agents
After clearing, the tissue specimen are transferred to a bath of molten
(liquid) paraffin wax for infiltration and impregnation process
During this combined process :: Xylene is eliminated from tissue by diffusion (infiltration)
wax diffuses into the tissue pores to replace the clearing agent
(impregnation)
Temperature 50 560C
Time 2 3 hrs
Vacuum impregnation oven reduces the time to 15 min
The purpose of the infiltration & impregnation process :: Empty space (pores) in the tissue after removal of watery solution will
be filled with paraffin wax
Hardens of the tissue which helps in section cutting
Most tissue processing is done using automated machines that carry
out the steps automatically.
AUTOMATED TISSUE PROCESSOR
Paraffin wax is the most common embedding
media
there should be at least 1 change of wax
to completely remove the clearing agent
Types of wax depends :
nature of material to be embedded thickness
temperature of working area
change 3X 1 hrs if needed
Inadequate impregnation leads to drying and shrinking of
tissue
Cracks and crumble develop
High temperature will over harden the tissues
Manual and automated tissue processing
Good technician can produce equally good result with
manual procedure
Problem with automated processing
electricity supply
lack of service support
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Cont..
Automated tissue processing :
arranged in a circle
with a timing device
containers hold reagent and paraffin wax
transfer arm changes position of the casket thru the
reagents
the final dip is in the final paraffin wax
the cassettes are opened and the tissues are embedded
Changing of solution :
More than 100% alcohol, xylene and paraffin wax are kept
in a series
after using more than 2 -3 days the last solution in a series
are replaced
previously used ones are moved forward
while the first one are discarded
EMBEDDING PROCESS
definition of embedding process is to infiltrate,
support & enclose specimens, which in turn
produce tissue block that subsequently cut into
thin sections.
The capability of the embedding medium are
The
substances capable of being converted from liquid to solid
form
has the ability to infiltrate or penetration
The histological cassettes were transfer through the
following solvent ::Solution / Reagents
1.70% alcohol
Duration /
Temperature
Description
1 hr
2.90% alcohol
1 hr
3.100% alcohol
1 hr
4.100% alcohol
1 hr
5.100% alcohol
1 hr
6.100% alcohol
1 hr
7.Histo-Clear (or Xylene)
1 hr
8.Histo-Clear (or Xylene)
1 hr
9.Histo-Clear (or Xylene)
1 hr
10.Molten wax
2 hr, 6065oC
11.Molten wax
2 hr, 6065oC
Steps 1-6 give gentle, but complete
dehydration to remove aqueous fixative
and any tissue water content
Steps 7 - 9 are the 'clearing' fluid which
is totally miscible with the dehydrating
alcohol and wax embedding
agent. Other clearing fluids are Xylene
and chloroform but Histo-Clear is safer
to use routinely.
Steps 10 -11 infiltration & impregnate the
tissue with molten wax for the final
embedding stage which sets specimens
in blocks of paraffin wax from which
sections may be cut.
Cont
Embedding, the final stage of the tissue processing
The embedding process is carried out on a Embedding centre
(machine) using stainless steel cassette moulds of several routine sizes
(suitable for large numbers of small and moderate dimensions).
The plastic labeled cassettes are incorporated into the finished block
The tissue is finally transformed into a tissue block of wax
The tissue block gives the tissue support and shape for the next step,
which is sectioning (cutting).
The
common embedding media that are used
currently are
paraffin
carbowax
celloidin
plastic
The tissues, trimmed prior to processing to fit inside
cassettes (25 x 20 x 4 mm maximum), may have
shrunk considerably after the process and are
orientated in the wax mould to present required
features/planes on the final cutting surface.
1. Paraffin embedding
solid at normal room temperature
heat renders paraffin fluid permeate the tissue
hardness of paraffin matched the hardness of the tissue
soft parafins advantages: finer grained & less brittle.
(melting point: 45 C to 50 C only used in cool temp. 59 F
Hard parafins advantages; thinner section & better
support.
(melting point: 56 C to 62 C suitable for lab temp 72 F
Routine work paraffin with high melting point 5656-58 C
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Paraffin embedding techniques
Cont
Cakes of paraffin placed in clean metal melted down in
paraffin oven
After tissue completely dehydrated & cleared, they are
immersed in melted paraffin for 22-4 hours
Usually changes of paraffin needed to eliminate traces of
solvent (clearing agent) which prevent the paraffin from
hardening properly
Rapid chilling of the melted paraffin is recommended
because slow cooling of a liquid form crystallized block
(large crystals)
Rapid cooling fine crystalline structure capable of fitting,
closely to the individual cells give adequate support
The surface of the section to be cut should be placed
parallel to the bottom of the mould
After dehydration, the tissues is embedded in paraffin
(melting temperature 5656- 58 C)
The blocks are transferred to pure paraffin in the oven for 1
hour & second pot of melted paraffin for 22-3hours block is
completely infiltrated with melted paraffin
Always use bluntblunt-nose warm forceps for making the transfer
of tissues to the mould
Then pour the melted paraffin over the tissue
Warm the forceps again & orient the tissue
Pathologist may notch the opposite side to be cut
Make sure that there is no air bubble
2. Wax embedding
Cont..
Identification label must accompany the specimen
through all types of tissue processing
Label
Place the label against the side of the mould containing
the molten wax
Adjacent to the tissue
Label must not be in the way of the knife blade
The encased blocks are removed by tapping on the
bench
The hard wax is ready to be cut
Involves
the use of alcohols, inflammable
solvents and waxes on automatic tissue
processors
Bunsens: represents a fire risk
Electrical plugs and sockets should be made
sparkles to lessen the chances of vapor igniting
Wax baths of tissue processors should have
safety cutcut-outs to prevent over
over--heating
3. Plastic embedding
Resin plastic embedding mainly used for electron
microscope and the resins must be handled with care as
many are toxic can cause dermatitis and must worn a
gloves
Eg. butyl or methyl methacrylate produce harmful fumes
and best handled in a fume cupboard. Methyl
methacrylate is very hard & good for embedding
undercalcified bone.
The soft plastics sectioned with a standard steel
microtome blade do not require glass or diamond
knives
COMPARISON OF TISSUE PROCESSING METHOD
BETWEEN
LIGHT MICROSCOPE & ELECTRON MICROSCOPE
Description
Sample Size
Fixative
Light
Electron
1 cm3
1 mm3
Formaldehyde
Glutaradehyde
Post-Fixation
None
Osmium Tetroxide
Dehydration
Graded Alcohol
Alcohol or Acetone
Clearing Agent
Xylene / Toluene
Propylene Oxide
Embedding Material
Microtome Knife
Section Thickness
Stains
Paraffin Wax
Various Plastics
Standard Steel Blade
Glass or Diamond
5 - 10m
60 - 90 nm
Colored dyes
Heavy Metals
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BRING SECTION TO WATER
Bring section to water is the term used to rehydrates
the tissue specimen again.
These procedures are in reverse order of tissue processing
method or against the Histotechnique procedure in
histopathology laboratory.
Normally in Histotechnique the tissues specimen are
processed in order to removes the water substances from
the tissue
However the Bring section to water method are done
to introduce back the water substance into the tissue again
This methods are done normally for staining or grossing
procedure
Cont
Bring section to water procedure are
done in the following steps
The
Xylene
3 minutes
Xylene
3 minutes
Xylene
3 minutes
Alcohol
3 minutes
Alcohol
3 minutes
90% alcohol 3 minutes
70% alcohol 3 minutes
Tap water / Distill Water
Staining / Grossing
