Tissue processing

Aak.dhiman@gmail.c
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TISSUE PROCESSING

CONTENTS
 Introduction
 Factors influencing Processing
 Stages of processing
-Fixation
-Dehydration
-Clearing
-Impregnation
-Embedding
-Sectioning
-Staining
-Mounting
• Automated Tissue Processor
• Bibliography

INTRODUCTION
 Proper handling of tissue specimens is critical to
ensure that an accurate diagnosis is obtained from
patient tissue samples.
 Regardless of the methodology, tissue samples
requiring processing need to be placed in fixative as
soon as possible after excision from the patient.
 This is essential to prevent autolysis, which could
destroy diagnostic elements, and to prepare the
tissue for the rigors of the reagents used in
subsequent processing steps.

Definition:
“Tissue processing” describes the steps required to
take animal or human tissue from fixation to the
state where it is completely infiltrated with a suitable
histological wax and can be embedded ready for
section cutting on the microtome.
Aim:
The aim of tissue processing 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 , and yet soft enough not to damage the
knife or tissue.

FACTORS INFLUENCING
PROCESSING
1.Viscosity: soultions with low viscosity
smaller sized molecules faster penetration
rate and vice-versa.
 Melted paraffin wax has a low viscosity which
enhances the impregnation rate.
2. Agitation: increases the flow of solutions around
the tissue.
 The mechanism for agitation in automated
processors: either vertical/rotary oscillation or
pressurized removal and replacement of fluids at

3. Heat: increase in temperature improves the fluid
exchange and penetration rate.
 Excessive exposure to heat can cause shrinkage
and hardening of the tissue which negatively
affects subsequent staining and
immunohistochemistry.
4. Vacuum & Pressure: increase fluid mobility,
thus increasing the infiltration rate and decreasing
the time necessary to complete each processing
step.
 Vacuum aids the removal of air pockets in porous
tissue, e.g. lung.

5. Processing solvent contamination: The number of
blocks on each run, tissue type, size, frequency of the
runs, use of sponges and cross-contamination of
processor solvents will influence how often solutions
should be rotated between stations or changed to
maintain processing quality.
6. Size: Tissue slice below 4mm are satisfactory.

Receipt &
Identification
Labeling of the
specimen with
numbering
Dehydration Clearing Impregnation
Staining
Sectioning
Embedding
PROTOCOL
Fixation
Mounting

FIXATION
 The first and most critical step in
specimen handling.
 Fixation denatures proteins rendering
the cell and its components resistant to
further autolysis.
 Complete fixation also allows the tissue
to withstand the negative effects of
subsequent processing reagents.
 Although samples are typically
received in fixative, it is best to begin
processing with a fixative station as the
natural diffusion of water from the
tissue will have diluted the fixative in

 The size and type of
specimen in the tissue
cassette determines the
time needed for complete
fixation and processing to
occur.
 The tissue should be
dissected to 2–4 mm in
thickness and trimmed to a
size that allows complete
flow of reagents around the
tissue in the cassette.
 Ideally tissue should be
separated according to size
and/or type, and processed
using different schedules.
 The most commonly used
reagent for the fixation of

Post-fixation treatment
 Specimens fixed in alcoholic fixatives should be followed
with alcohol to prevent re-introduction of water to the tissue
specimen.
 Picric acid fixatives will color the tissue bright yellow. A rinse
of the tissue in 50-70% alcohol for 4-6 hours will remove
excess fixative. This can also be accomplished by treating
the cut tissue section on the glass slide with a dilute
carbonate solution.
 A few drops of 1% eosin can be added to the specimen
container 30 minutes prior to processing to assist in
visualizing small tissue fragments during embedding.
 The pink color of the tissue remains during processing, but
washes out during subsequent staining. Incomplete removal
of the eosin can interfere with fluorescent procedures.

DEHYDRATION
 Dehydration displaces the residual fixative as well as
cellular water.
 Correct dehydration schedules during tissue processing
should only remove the free water, leaving the bound
water intact.
 Graded alcohols are used in dehydration to remove
free water and keep the bound water in place.
 The removal of the bound water will produce
overprocessing artifacts such as shrinkage, ‘parched
earth’ effect and abnormal staining, as well as dry,
brittle tissues during microtomy.

 Incomplete dehydration will impair the penetration of
the clearing reagents into the tissue, leaving the
specimen soft and non-receptive to paraffin wax
infiltration.
 Ethanol, reagent alcohol and isopropanol work well in
tissue processing.
 Glycol ether dehydrants are an effective alternative to
alcohols in tissue processing. Due to their gentle
nature they are used undiluted.

DEHYDRATING AGENTS
1) Alcohol- expensive; dehydrates quickly.
2) Ethyl Alcohol- very expensive
3) Isopropyl Alcohol- used in routine, easily available
and cheap.
4) Methyl Alcohol- unpleasant odour; poisonous.
5) Acetone- causes excess shrinkage and hardening;
can be used for small biopsies; dehydrates quickly.
6) Dioxane- dehydrating as well as clearing agent, toxic
and carcinogenic.

1) 70% alcohol
For 90 minutes
4.) Absolute
alcohol-I
For 90 minutes
2) 80% alcohol
For 90 minutes
6.) Absolute alcohol-III
For 1 hour
5) Absolute alcohol-II
For 1 hour
3) 95% alcohol
For 90 minutes

CLEARING/DEALCOHOLIZATIO
N
 Clearing agents must be miscible with both the
preceding anhydrous alcohols/dehydrants to
effectively remove them, and with the ensuing
paraffin wax to allow complete infiltration.
 A good clearant will also dissolve lipids which can
impede the wax penetration.
 This is an intermediate stage between dehydration
and Infiltration.

Clearing should have following properties:
1. Rapid penetration of tissues
2. Rapid removal of dehydrating agent
3. Ease of removal by melted paraffn wax
4. Minimal tissue damage
5. Low flammability
6. Low toxicity
7. Low cost
Clearing agents suitable for routine use:
1. Xylene :
 Highly inflammable, rarely used.
 Over exposure causes hardness of tissue.
 Quick in action, satisfactory.

2. Toluene :
Similar to xylene, less damaging than xylene in case
of prolonged exposure but it is more inflammable and
volatile.
Suitable for automated processing.
3. Chloroform :
Slower in action, can be used for blocks thicker than
1 mm.
It produces highly toxic gases.
It is most commonly used in CNS specimens.
Requires longer time (overnight) for action.
4. Benzene:
 Similar to xylene, not used as it is carcinogenic.

5. Petrol:
 Not used because of various additives.
6. Cedar wood oil:
 Good clearing agent but slow in action.
Expensive; used for research purposes.
7. Clove oil:
Same as cedar oil.

How is it done?
Toluene-I (60
minutes)
Toluene-II (90
minutes)
Toluene-III (90
minutes)

WAX INFILTRATION/
IMPREGNATION
The various waxes which are used
are:
1. Paraffin wax
2. Resin
3. Agar
4. Gelatin
5. Celloidin
6. Low viscosity nitrocellulose(LVN)

BLOCK
FORMATION/EMBEDDING
 It is done by transferring the tissue which has been
cleared off the alcohol to a mould filled with molten
wax & is allowed to cool and solidify.
 After solidification, a wax block is obtained which is
then sectioned to obtain ribbons.
Types of Moulds:
A. Leuckhart’s Moulds
B. Glass or Metal petri-dishes
C. Watch glass
D. Paper boats .

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.
Paraffin wax
 It is a polycrystalline mixture of solid hydrocarbons
produced during the refining of coal and mineral oils.
 It is about two thirds the density and slightly more
elastic than dried protein.
 Paraffin wax is traditionally marketed by its melting
points which range from 39°C to 68°C.
 The properties of paraffin wax are improved for
histological purposes by the inclusion of substances
added alone or in combination to the wax: blended
with ceremic, bees wax, rubber etc.
 This improves ribbonning, increases hardness,
decrease melting point, improve adhesion between

OTHER WAXES:
 Ester wax: M.P- 46-48 C; used in Base sledge microtome.
 Resins: for electron microscopy.
 Agar: can be used in double embedding with paraffin wax
for friable tissue.
 Gelatin: used when frozen section of friable tissue is
required; also useful in uterine curettings.
 Celloidin: not used in routine; useful in neurological
specimens; supplied in the form of wool dampened with
alcohol; working strength- 2 to 4%
 LVN: used in preference to celloidin since it forms harder
blocks; thinner sections can be made; highly explosive –
exposure to direct sunlight should be avoided.
Note: Nail, dense fibrous tissue, keratin masses, EMC need
treatment with 4% phenol in 70% alcohol.

General Embedding Procedure
1. Open the tissue cassette, check against worksheet entry to
ensure the correct number of tissue pieces are present.
2. Select the mould, there should be sufficient room for the
tissue with allowance for at least a 2 mm surrounding
margin of wax.
3. Fill the mould with paraffin wax.
4. Using warm forceps select the tissue, taking care that it
does not cool in the air; at the same time.
5. Chill the mould on the cold plate, orienting the tissue and
firming it into the wax with warmed forceps. This ensures
that the correct orientation is maintained and the tissue
surface to be sectioned is kept flat.

6. Insert the identifying label or place the labeled embedding
ring or cassette base onto the mould.
7. Cool the block on the cold plate, or carefully submerge it
under water when a thin skin has formed over the wax surface.
8. Remove the block from the mould.
9. Cross check block, label and worksheet.

ORIENTATION OF TISSUE IN THE
BLOCK
 Correct orientation of tissue in a mould is the most important
step in embedding.
 Incorrect placement of tissues may result in diagnostically
important tissue elements being missed or damaged during
microtomy.
 Elongated tissues are placed diagonally across the block.
 Tubular and walled specimens such as vas deferens, cysts
and gastrointestinal tissues are embedded so as to provide
transverse sections showing all tissue layers.
 Tissues with an epithelial surface such as skin, are
embedded to provide sections in a plane at right angles to
the surface (hairy or keratinised epithelia are oriented to face
the knife diagonally).
 Multiple tissue pieces are aligned across the long axis of the

SECTIONING/CUTTING
 It is the procedure in which the blocks which have been
prepared are cut or sectioned and thin strips of varying
thickness are prepared.
 The instrument by which this is done is called as a
Microtome. Most microtomes use a steel blade and are used
to prepare sections of animal or plant tissues for histology.
TYPES OF MICROTOMES:
1) Sliding
2) Rotary
3) Rocking
4) Freezing
5) Base sledge

ROTARY MICROTOME
 It is the most commonly used.
 Also known as Minnot’s Rotary microtome.
 In this the Block holder moves up and down while the
knife remains fixed.
 It is suitable for cutting of small tissues & serial
sections can be taken on it.

Parts of a Microtome ( Rotary ):
A. Block holder
B. Knife clamp screws
C. Knife clamps
D. Block adjustment
E. Thickness gauge
F. Angle of tilt adjustment
G. Operating handle.

STEPS IN SECTIONING:
1. Rough cutting of the block at 20 micron to expose the
tissue.
2. Side cutting of the block to get trim section and serial
section.
3. Put block in a bowl with ice cubes.
4. Keep in referigerator (2 to 8 degree celsius) for 1-2 hours.
5. Cut the sections at 4 microns.
6. Transfer the section on a water bath to remove wrinkles.
7. Take section on albuminized slide.
8. Before Staining, put the slides on hot plate to loosen the
wax and fixation of issue on a slide.

STAINING
 Staining of the section is done
to bring out the particular
details in the tissue under study
.
 The most commonly used stain
in routine practice is
Haematoxylin & Eosin (H&E)
stain.
 H & E is a charge-based,
general purpose stain.
 Hematoxylin stains acidic
molecules in shades of
blue(nucleus)

HAEMATOXYLIN & EOSIN(AUTO
SLIDE STAINER) PROCEDURE
 Bring section to water.
 Stain with Haematoxylin for 8-10 minutes.
 Wash with water for 2-3 minutes.
 Differentiate in 1% acid alcohol for 15-18 seconds.
 Wash with water for 1 minute.
 Bluing in scott’s tap water for 3-5 minutes.
 Wash with water for 2-3 minutes.
 Stain with eosin for 45 seconds-3 minutes.
 Wash with water for 1 miute.
 Dehydration with alcohol for 1 minute- 3 changes.
 Clearing in Xylene for 3-5 minutes.
 Mounting Labelling Submission

MOUNTING
Adhesives used for fixing the sections on the slides :
 Albumin solution ( Mayor’s egg albumin)
 Starch paste
 Gelatin
Mountants :
 DPX
(Dibutylphthalate Polystyrene Xylene)
 Canada Balsam
 Colophonium resin
 Terpene resin

TISSUE
PROCESSING(MANUAL)
PERCENTAGE AGENT USED DURATION
10% FORMAL SALINE OVERNIGHT
70% ALCOHOL 2 HOURS
90% ALCOHOL 1.5 HOURS
ABSOLUTE ALCOHOL-I 1.5 HOURS
ABSOLUTE ALCOHOL-II 1 HOUR
ABSOLUTE ALCOHOL-III 1 HOUR
- CHLOROFORM OVERNIGHT
- TOLUENE-I 1.5 HOURS
- TOLUENE-II 2 HOURS
- TOLUENE-III 1.5 HOURS
- PARAFFIN WAX-I 2.5 HOURS
- PARAFFINWAX-II 4 HOURS

AUTOMATED TISSUE
PROCESSOR
 All the before mentioned procedures upto the
impregnation step can be done automatically in a
single, unmanned instrument , which is the Automated
Tissue processor.
Advantages :
 It provides constant agitation during every step which
ensures better fixation & processing.
 It reduces the work load & in turns improves the overall
output of the laboratory.

RESTORATION OF
SPECIMENS
 Keep the tissue in 70% alcohol (70ml), glycerol
(80ml), and dithionite (1 gram).
 Leave the tissue in the solution in a sealed
container, overnight.
 Perform the processing starting from the
dehydration step.

 Bancroft’s Theory and Practice of Histological
Techniques (8th edition)
 www.slideshare.net
 Image courtesy: GMSH pathology Department

Tissue processing

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