Tissue Processing: Department of Oral Pathology and Microbiology
Tissue Processing: Department of Oral Pathology and Microbiology
Tissue Processing: Department of Oral Pathology and Microbiology
TISSUE PROCESSING
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CONTENTS:
Introduction
Principles of Tissue Processing
Factors effecting rate of processing
Dehydration
Clearing
Embedding
Vacuum impregnation
Automatic tissue processing
Manual tissue processing
Alternative embedding media
Problems during Tissue Processing
Conclusion
References
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Introduction:
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Transportation: Tissue processing begins with the clinician
surgically removing a tissue specimen from the patient.
The tissue is placed in a container (often containing a
fixative) and transported to the lab. At this stage, the
tissue is called gross tissue.
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Labelling of tissues :
The labels should also indicate number of pieces being put into
one cassette,ordinary ink should not be used as this may be
dissolved in the reagents used during processing.
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Tissue and its labell is placed in tissue processing baskets. These
are perforated small metal containers , primarily designed for use
with automatic tissue containers , but may be used in a manual
system.
Small porcelain pots which are perforated have also been used for
this purpose .Printed ,graphite pencilled, typewritten, stencilled
or indian ink written labels are satisfactory .
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More reliable way is use of tissue tek system in which tissue
identification is written over cassette .
Better way is use of bar codes,within bar codes are all the
relevant patient details.
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Fixatives :
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Fixative should be ten times the volume of tissue
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Formic Acid Decontamination of Fixed Blocks
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Principle of tissue processing
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Tissue processing is concerned with the diffusion of various
substances into and out of stabilize porous tissues.
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Factors influencing the rate of processing :
Agitation :
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Heat :
It increases the rate of penetration. Temperatures limited to 45
degrees can be used effectively.
Higher temperatures adversely affect staining and
immunochemistry.
Viscosity :
Most of fluids used during standard dehydration and clearing
have similar properties.
Vacuum :
Reduce the pressure are of use during impregnation by paraffin
wax.
With dense and fatty tissues ,reduces the impregnation time
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Water is present in tissues in free and bound (molecular) forms.
Tissues are processed to the embedding medium by removing
some or all of the free water. During this procedure various
cellular components are dissolved by dehydrating fluids.
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•Dehydration is necessary in all infiltration methods, except where
tissues are simply externally supported by an aqueous embedding
medium.
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•While well fixed tissues can be transferred directly to 95%
ethanol, incompletely fixed tissues may exhibit artifacts if placed
directly in higher alcohols.
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To minimize tissue distortion from diffusion currents, delicate
specimens are dehydrated in a graded ethanol series from water
through 10%-20%-50%-95%-100% ethanol.
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Ethyl alcohol has been the dehydrating reagent of choice. The
period of immersion of the tissue specimen in various alcohols
and their various strengths will affect the hardness of the tissue
specimen.
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Wet fixed tissues (in aqueous solutions) cannot be directly
infiltrated with paraffin. First, the water from the tissues must
be removed by dehydration. This is usually done with a series of
alcohols, say 70% to 95% to 100%.
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Other dehydrants can be used, but have major disadvantages.
Acetone is very fast, but a fire hazard, so is safe only for
small, hand-processed sets of tissues. Dioxane can be used
without clearing, but has toxic fumes.
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Dehydrating fluids
ETHANOL INDUSTRIAL METHANOL Propan-2-ol , DIOXANE (DIETHYL
METHYLATED isopropyl DIOXIDE)
SPIRITED ( alcohol
DENATURED CH3CHOHCH3
ALCOHOL)
AgtX and ethyl alcohol are both good lipid extractors and are
miscible with all clearing reagents used in tissue processing.
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Ethyl alcohol is a controlled substance requiring record keeping
by the end user. AgtX is not a controlled substance and requires
no record keeping.
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1. Absolute methyl alcohol cannot be uses in most closed
processors.
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Additives to dehydrating agents
Phenol:
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Artifacts during Dehydration
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A problem that leads to very poor staining of the nucleus. In
the United States this problem is referred to as smudginess or
blue halo effect, and in the United Kingdom as “nuclear
meltdown.”
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Removal of the dehydrant with a substance that will be miscible
with the embedding medium (paraffin).
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Boiling point of the clearing agent gives an indication of its
speed of replacement by molten paraffin wax.Fluids with low
boiling point are more readily replaced .
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Enclosed tissue processors have charcoal filters,thereby
reducing the amount of harmful fumes from clearing
agents entering the environment.
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‘Agitation’ option on processors is useful as it improves
penetration of reagents and speed of processing. Several
baths of each reagent are required to ensure complete
processing
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Reagents must be changed in accordance with an appropriate
schedule (to account for the number of cycles or number of
cassettes or length of time) as each will become contaminated
with the previous one and contamination can cause incomplete
processing of the block.
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Clearing is the transition step between dehydration and
infiltration with the embedding medium.
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The term clearing arises because some solvents have high
refractive indices (approaching that of dehydrated fixed tissue
protein) and, on immersion, anhydrous tissues are rendered
transparent or clear.
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Criteria for choosing a suitable clearing agent are :
safety factors
Flammability,Toxicity.
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Technique for clearing
Similar to dehydration
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Those in xylene, benzene and toulene should be given one change
after 30-60 minutes
Cedarwood oil
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Artifacts during Clearing
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slower than xylene renders tissue less non inflammable but
CHLOROFROM brittle than benzene, highly toxic
toulene and xylene
CITRUS FRUIT OILS Non-toxic and Miscible with water so small mineral deposits
strong odour can be discharged in tissue, such as
through ordinary copper
waste pipes.
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XYLENE, BENZENE Fairly rapid in action Tissue becomes
AND TOLUENE clearer as alcohol is
replaced
CEDARWOOD OIL Best reagent for Tissue can be left in Can be used for tissue
research and this reagent for long like skin and dense
treatment of delicate periods without fibrous tissue
tissue as it has least damage
hardening effect.
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Ideally an infiltrating and embedding medium should be:
• soluble in processing fluids
• suitable for sectioning and ribboning
• molten between 30°C and 60°C
• translucent or transparent; colorless
• stable
• homogeneous
• capable of flattening after ribboning
• non-toxic
• odorless
• easy to handle
• inexpensive
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Infiltration (Interpenetration)
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Double embedding is the process by which tissues are first
embedded or fully infiltrated with a supporting medium such as
agar or nitrocellulose, then infiltrated a second time with wax in
which they are also embedded.
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Process Fixation Dehydration Clearing Infiltration
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IMPREGNATION WITH PARAFFIN WAX
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Tissue-wax adhesion depends upon crystal morphology of
the embedding medium. Small, uniform sized crystals
provide better physical support for specimens through close
packing.
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Paraffin wax has advantage for the different climatic
regions of the world.
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Low melting point (i.e., softer) paraffin wax is the most suitable
for soft, friable tissue & a higher melting point paraffin wax
(i.e., harder) for tough tissue.
Additives
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Increase the hardness of the wax & thus the support for
tougher tissues; to increase the plasticity of the wax & thus
improve the ribboning qualities & presumably change the
sectioning from point to point cleavage to a continuous flow
shearing; & to produce a more uniform crystalline structure
to the wax.
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Many of additives have a higher melting points than paraffin
wax and consequently made the tissue more brittle .
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Technique for impregnation
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TIME FOR IMPREGNATION
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2 Clearing agent employed
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3 Use of vacuum embedding oven
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Vacuum impregnation :
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Tissues which benefit from vacuum impregnation are lung,
muscle,spleen, decalcified bone,skin and tissue from the
central nervous system.
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The apparatus consists of an air tight embedding oven
attached to an exhaust pump, the degree of vacuum achieved
being controlled by an attached mercury manometer or
vacuum gauge.
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To avoid contamination of the wax in the bath by water or oil used
in the vacuum pump, the pump is connected to a thick walled
vessel (flask or bottle) which act as a trap; this in turn is connected
to the vacuum chamber and to a mercury manometer
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Inadequate infiltration with paraffin
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Embedding is the process by which tissues are surrounded by a
medium such as agar, gelatin, or wax which when solidified
will provide sufficient external support during sectioning.
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•Araldite is about the same as methacrylate, but requires a
more complex embedding process. Epon is routinely used for
electron microscopy where very thin sections are required.
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Embedding tissue to produce wax block
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•Select an embedding base mould (rubber, metal or plastic, or
use L pieces) large enough to give a clear rim of wax all around
the tissue.
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•Place the surface to be sectioned face down in the embedding
mould. Gently press flat into solidifying wax onto a cool surface
to ensure a flat and even orientation for sectioning.
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•With Tissue tek system ,paraffin wax is dispensed
automatically from a nozzle into a suitably sized mold which is
then placed on a small cool area to allow the wax at the base
of the mold to semi-congeal.
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•When this has been done the base of cassette is placed on top
and together they are placed on the cold plate so the paraffin
wax can cool quickly, thus ensuring a small crystalline
structure.
•After the paraffin wax has solidifies the mold is removed and
the block is then ready for sectioning and afterwards can be
immediately filed .
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Different moulds that can be used -
Leuckhart’s L Pieces
Paper boats
Cheap and convenient. Tissue can be stored still in their paper
boats . One or more tissue can be blocked in one boat.
Long life
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Watch glasses
For small pieces of tissue
Glycerin should be used
Test tubes
For small fragments which have been processed in
them throughout
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Artefacts resulting from the embedding process
This allows the tissue to fall out or vibrate during the cutting
procedure. Vibration of the tissue specimen results in an
artefact that resembles a venetian blind, with compressed
zones of tissue separated by open spaces .
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These wrinkles usually extend the length of the section
and stain more intensely since the stain has access to
both surfaces of the wrinkle.
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•An unclean water bath may also contain remnants of previous
tissues, which may become incorporated into the present tissue
section
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liver tissue present in the lung
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Orientation of tissues :
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Tubular structures ,eg arteries and vas deferens are cut in
cross section.
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If the tissue is fairly homogeneous (like a piece of liver) and
roughly equal in vertical and horizontal dimensions, it doesn't
really matter how you orient it in the block.
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For example, in a cross section of skin, the knife should cut
through the subdermal fat first, then the collagenous dermis,
then the epidermis and finally the keratinous layer.
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Automated Tissue Processing :
these have reduced the time
taken to process routine tissue
by manual methods at least
24 hrs.
are of 2 types-
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On the older carousel type machine , minimum time at any
stage was 30 minutes. The newer carousel machines can be
programmed electronically and , like the enclosed processor
type, minimum of 15 minutes per station can be achieved.
: Usually have a twelve stage cycle with the last two usually
reserved for paraffin wax infiltration. Static beakers are
provided to hold the processing fluids & tissues held in
individual containers & cassettes in a suspended basket are
mechanically moved from one to another. Agitation is
provided either by vertical or rotating movements of the
basket.
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ENCLOSED PUMP FLUID TYPE TISSUE PROCESSOR
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Processing machine maintainance :
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Important points:
Fluid and wax containers must be filled to appropriate level
and correctly located in machine.
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container fluid Time(hrs)
1. 10% FORMALIN 0
2. 70% ALCOHOL ½
3. 95% ALCOHOL ½
4. 100% ALCOHOL ½
5. 100 % ALCOHOL 1
6. 100% ALCOHOL 1
7. 100% ALCOHOL 1
8. 100% ALCOHOL / XYLENE ½
9. XYLENE 1
10. XYLENE 2
11. WAX 2
12. WAX 4
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2. Short processing schedules
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CONTAINER FLUID VACUUM HEAT TIME, MINUTES
1. 10% FORMALIN Y 45 20
2 95% ALCOHOL Y 45 5
3 95% ALCOHOL Y 45 5
4 100% ALCOHOL Y 45 5
5 100% ALCOHOL Y 45 5
6 100% ALCOHOL Y 45 5
7 100% ALCOHOL Y 45 5
8 ABSOLUTE ALCOHOL Y 45 5
9 XYLENE Y 45 5
10 XYLENE Y 45 5
11 WAX Y 45 5
12 WAX Y 45 5 94
Manual Tissue Processing :
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Alternative embedding media :
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4.RESINS-
-Used for the production of ultra thin sections of electron
microscopy.
-Tissue is dehydrated and infiltrated with resin in monomeric
form which is subsequently polymerized, either chemically or
physically to give a hard, glass clear block.
-Extra hardness provided by resins permits sections of upto
0.5 -2.0µm.
6. AGAR-
-It mainly act as a cohesive agent for small friable pieces of
tissue after fixation. These fragments are embedded in molten
Agar and when solidified and trimmed , processed to paraffin
wax 97
Restoration of tissue dried in processing
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ARTIFACTS
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(A) Bone marrow in the brain appears to be a focus of
inflammation
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(B) A piece of small
intestine adjacent to the
brain.
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During macro-sectioning of the fixed gross specimens
prior to processing, care should be taken to keep the
surface of the cutting block free of tissue debris.
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CONCLUSION:
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REFERENCES
•http://www.nationaldiagnostics.com/article_info.php/articles_id/
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•Http://in.Answers.Yahoo.Com/question/index?Qid=201104030
41752aaamfzq
•Http://www.Alneurosciencecenter.Uab.Edu/word%20docs/core%
20c/histology-problems%20with%20staining.Pdf
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THANK YOU
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