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TISSUE PROCESSING

The document discusses tissue processing, which is essential for preparing tissue specimens for microscopic examination. It outlines the stages of processing, including dehydration, clearing, impregnation with paraffin wax, and embedding, as well as factors influencing these processes. Additionally, it covers labeling methods, automated and manual processing techniques, and alternative embedding media.

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Waheed ahmad
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8 views25 pages

TISSUE PROCESSING

The document discusses tissue processing, which is essential for preparing tissue specimens for microscopic examination. It outlines the stages of processing, including dehydration, clearing, impregnation with paraffin wax, and embedding, as well as factors influencing these processes. Additionally, it covers labeling methods, automated and manual processing techniques, and alternative embedding media.

Uploaded by

Waheed ahmad
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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TISSUE PROCESSING

DR. NEELAKSHI
SINGH
MDS –First Year
POINTS TO BE DISCUSSED
• Labeling of tissues
• What is tissue processing
• Stages of tissues processing
• Factors influencing the rate of
processing
• Dehydration & Dehydrating fluids
• Clearing & Clearing Agents
• Impregnation with paraffin wax
• Embedding
Orientation of tissues
• Automated tissue processing
• Manual tissue processing
• Embedding centers.
• Alternative embedding media
• Restoration of tissue dried in
processing
INTRODUCTION
The pathologist responsible for tissue
diagnosis is heavily dependent on the
competence of his histotechnologist.
The ability to consistently turn out slides of
high quality, well sectioned and well stained
on a wide variety of tissue is indeed an art.
Tissues and cells examined outside the
body with an imaging system are an artifact
of the original. It is essential therefore, to
ensure that the artifact produced at any
stage during tissue handling and processing
is constant.
For microscopic examination tissue specimen
must be thin enough to permit the passage
of transmitted light. To achieve this, tissue
slices need to be approximately 4-6µm in
thickness, although for highly cellular tissues
(such as lymph node) 2-3µm & when
examining larger cells & processes, such as
those of nervous system, 10-20µm. This is
facilitated by infiltrating & embedding the
tissue in a support medium. Although there is
no perfect medium for these purposes,
paraffin wax has proved the most universally
popular.
LABELLING OF TISSUES

1. THIN WHITE CARD

2. TISSUE TEK SYSTEM

3. AUTOMATIC EMBOSSING MACHINES

4. BAR CODES
What is Tissue
Processing?
Paraffin wax is not miscible with water
and since many fixatives are water
based there must be some treatment
of tissues to allow paraffin wax
impregnation, which is called tissue
processing.
Aim is to embed the tissue in a solid
medium firm enough to support the
tissue and give it sufficient rigidity to
enable thin sections to be cut.
STAGES OF TISSUE
PROCESSING
1. DEHYDRATION: To remove fixative &
water from the tissue & replace them
with dehydrating fluid.

2. CLEARING: To replace the dehydrating


fluid with a fluid that is totally miscible
with both the dehydrating fluid & the
embedding medium.

3. IMPREGNATION: To replace the clearing


agent with the embedding medium.

4. EMBEDDING / BLOCKING
DEHYDRATION
The first stage of processing is the removal of
aqueous fixative fluids from the tissues by
various compounds. Some are hydrophilic &
attract water from the tissues, whereas others
effect dehydration by repeated dilution of the
aqueous tissue fluid.
DEHYDRATING FLUIDS
Ethanol
IndustrialMethylated
Spirit(denatured alcohol)
Methanol
Propan-2-ol,Isopropyl Alcohol
Acetone
Additives to dehydrating agents-
phenol,anhydrous copper
sulphate
CLEARING
When the dehydrating agent has been
entirely replaced by the solvents
(Hydrocarbon solvents, refractive index
similar to protein) which are miscible
with both dehydrating agents & paraffin
wax, the tissue has a translucent
appearance, hence the term clearing.

The criteria for choosing a suitable


clearing agent are:
Speedy removal of dehydrating agent
Ease of removal by molten paraffin wax
Minimal tissue damage
Flammability
Toxicity
-Clearing fluids with low boiling point:
readily replaced.
-Prolonged exposure clearing agents:
tissue become brittle & difficult to
section.
-Enclosed tissue processors have
charcoal filters: reduces the amount of
harmful fumes.

CLEARING AGENTS:
1.XYLENE & TOLUENE
2.CHLOROFORM
3.CNP 30 &INHIBISOL
4.CITRUS FRUIT OIL
5.CEDAR WOOD OIL
Impregnation with Paraffin wax
Paraffin wax continues to be the most
popular embedding medium for histology,
because of following reasons:
-it is cheap
-easily handled
-section production provides few
difficulties
-wide range of melting points
PROPERTIES
-it is a hydrocarbon produced in cracking of
mineral oil.
-its melting point range between 40-70°C
Orientation of Tissues
For most tissue blocks, sections are cut
from the largest area of tissues but
there are many important exceptions.
-tubular structures are cut in cross
section e.g. arteries.
-skin and other epithelial biopsies are cut
in a plane right angle to the surface and
oriented so that the surface is cut first.
-muscle biopsies are sectioned in both
transverse and longitudinal planes.
-a particular tissue feature may be
present on one aspect only.
EMBEDDING / BLOCKING
Tissue is finally transferred from the last
wax bath to a mould filled with molten
paraffin wax, inverting the tissue to free
all surface of air bubbles & orienting the
intending cutting surface so that it faces
the base of the mould.

LEUCKHART’S L PIECES: They consist of


two L shaped pieces of metal, usually
brass which are laid on a metal or glass
plate to form an oblong. By adjusting the L
pieces the relative shape & size of the
mould can be modified according to the
size & number of tissues.
 Ice trays, paper boats, embedding
cassettes can also be used.
Technique
 Metal moulds should be sprayed or
smeared lightly with mould release fluid.
 The mould is filled with appropriate
paraffin wax.
 The tissue is transferred rapidly to the
wax containing based mould with a pair
of warm forceps. Care should be taken
that the forceps are not too hot & that
excessive pressure is not used.
Electrically heated forceps are ideal for
this purpose.
 Ensure that no air bubbles are trapped &
place the tissue, with the surface to be
sectioned down, against the base of the
mould. Allow the wax in the bottom of the
mould to be solidify sufficiently to hold
the tissue in place.
 The block should be immediately
transferred to a refrigerated surface, a
tray of ice or, once a skin has formed
on the surface, immersed in a trough
of cold water. This is done to promote
the formation of smaller paraffin wax
crystals.
 Once solidification is complete the
block may be removed from the
mould. Mould release spray should
ensure that this happens easily, but if
it does stick, a sharp tap should
release it.
Automated Tissue Processing
CAROUSEL TYPE:
ENCLOSED TISSUE PROCESSOR
Manual Tissue Processing
CONTAINER FLUID TIME(HRS)
1 70%ALCOHOL 0900 TO 1000
2 95%ALCOHOL 1000 TO 1100
3 95%ALCOHOL 1100 TO 1300
4 100%ALCOHOL 1300 TO 1430
5 100%ALCOHOL 1430 TO 1600
6 100%ALCOHOL 1600 TO 1730
7 100%ALCOHOL OVERNIGHT
8 XYLENE 0900 TO 1000
9 XYLENE 1000 TO 1130
10 WAX WITH VACCUM 1130 TO 1230
11 WAX WITH VACCUM 1230 TO 1400
12 WAX WITH VACCUM 1400 TO 1600
EMBEDDING CENTRES
1. An area for holding cassettes
containing processed tissues awaiting
embedding. This is a warm area where
the wax infiltrating and surrounding the
tissues remains molten.

2. An embedding area, usually well


illuminated, with a wax dispenser. A
warm surface is provided to maintain
paraffin wax in a molten state in the
embedding molds. A cold spot may be
included so that a layer of solid wax
may be induced in the base of the
mould to hold the tissues in position.
Heated forcep wells are also included.

3. A refrigerator surface to assist in the


solidification of the blocks.
Alternative Embedding
Media
Other waxes-water soluble e.g.-
polyethylene glycol.
-ester wax
-polyester wax
-microcrystalline wax
Resins-for electron microscopy, for
thin sectioning, for high resolution and
for undecalcified bone.eg-
acrylic,epoxy,urea-formaldehyde
Agar-main use is that of a cohesive
agent for small friable pieces of
tissues after fixation.
These fragments are embedded in
molten agar and when solidified and
trimmed, processed to paraffin wax.
Gelatin –use in the production of
sections of whole organ and in frozen
sectioning.
Celloidin ,double embedding
RESTORATION OF TISSUE DRIED IN
PROCESSING
Sometimes due to overfilling of the
cassette basket or under filling the
reagent bath results in tissue drying
before wax impregnation and such
tissues cannot be regarded as normal.
This tissue can be treated with –
70% Ethanol- 70ml
Glycerol - 30ml In a
sealed vessel
Dithionite - 1g
For several hours, followed by
processing commenced at the
dehydrating stage in the usual manner
which will allow some interpretation of
the stained sections.

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