Tissue Processing: Dr. Saket Kumar 21 August 2012

Tissue Processing
Dr. Saket Kumar

21st August
2012
HISTOLOGY :
It is the branch of science which deals with the gross &
microscopic study of normal tissue .
HISTOPATHOLOGY :
It is the branch of science which deals with the gross &
microscopic study of tissue affected by disease.
Tissue for study can be obtained from:
Biopsies
Autopsies
HISTOTECHNIQUES:
The techniques for processing the tissues, whether biopsies,
larger specimen removed at surgery, or tissues from autopsy so
as to enable the pathologist to study them under the microscope.
Protocols followed in Histotechniques ;

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Receipt & Identification

Labeling of the specimen with numbering
Fixation
Dehydration
Clearing
Impregnation
Embedding
Section cutting
Staining
Mounting
Specimen identification and

labeling:
Tissue specimen received in the

surgical pathology laboratory
have a request form that lists
the patient information and
history along with a description
of the site of origin.
The specimen are accessioned

by giving them a number that
will identify each specimen for
each patient
Fixation
It is a process in which a specimen is treated by

exposing it to a fixative for a particular period of
time in order to facilitate the succeeding steps.
The purpose of fixation is to preserve tissues

permanently in as life-like a state as possible.
The fixative should be 15 20 times more in volume

then the specimen.
Aims of Fixation :
1.
It should prevent autolysis & putrefaction of the cell.

It should penetrate evenly and rapidly.
It should harden the tissues
Increase the optical density
Should not cause shrinkage or swelling of the cells
Must not react with the receptor sites & thus must
not interefere with the staining procedure.
It must be cheap and easily available.
2.
3.
4.
5.
6.
7.
The bits should of the size of approximately 2 x 2 cm

& 4- 6 micrometer in thickness for optimum fixation
to take place.
These bits are then placed in metal cassettes or

capsules which are then placed in the fixative.
Tiny biopsies or small specimen can be wrapped in a

filter paper and then put in a cassette & fixed.
Simple Fixatives
Formalin
The most commonly used fixative is Formalin .
It is prepared by mixing 40 % Formaldehyde gas in 100

w/v of distilled water.
The resultant mixture is 100 % Formalin.
Routinely, 10 % formalin is used which is prepared by

mixing 10 ml of 100 % formalin in 90 ml of distilled
water.
MECHANISM OF ACTION:
It forms cross links between amino acids of proteins
thereby making them insoluble.
It fixes 4 mm thick tissue in 8 hours .
ADVANTAGES :
1.
5.
Rapid penetration
Easy availability & cheap
Does not overharden the tissue
Fixes lipids for frozen sections
Ideal for mailing
DISADVANTAGES:
1.
Irritant to the nose,the eyes and mucous membranes

Formation of precipitate of paraformaldehyde which can
be prevented by adding 11- 16 % methanol.
Formation of black formalin pigment , Acid formaldehyde
hematin.
2.
3.
4.
2.
3.
Other Simple Fixatives

Glutaraldehyde
Osmium
Tetraoxide
Pottasium
Mercuric
Dichromate
Chloride
Other Simple Fixatives

(contd.)
Picric
acid
Zenker's
fluid
Zenkers
Formal (Hellys Fluid)
Bouins
Fluid
Compound Fixatives
Microanatomical fixatives:
These are used to preserve the anatomy of the
tissue.
Cytological fixatives:
These are used to fix intracellular structures.
Histochemical fixatives :
These are used to demonstrate the chemical
constituents of the cell.
Microanatomical
.
Fixatives
10 % Formal saline :
It is a microanatomical fixative.
Ideal for fixation of brain.
Buffered formalin:
Due to the presence of buffer, the pH of the solution

remains at neutral or near neutral.
As a result, Formalin pigment formation doesnt take
place.
Cytological
Fixatives
Nuclear fixatives :
Carnoys Fluid
Clarkes Fluid
Newcomers Fluid
Flemmings Fluid
Cytoplasmic Fixatives :
Champys Fluid
Regauds Fluid
Histochemical
Formal saline
Cold acetone
Absolute alcohol
Fixatives:
Decalcification:
.
.
.
.
.
.
It is the process of removal of the calcium salts from

the specimen.
The various agents used for decalcifying are;
Nitric acid
Hydrochloric acid
Formic acid
Picric acid
Acetic acid
Citric acid
Dehydration:
It is the process in which the water content in the tissue

to be processed is completely reduced by passing the
tissue through increasing concentrations of
dehydrating agents.
The various dehydrating agents used are ;
Ethyl alcohol
Acetone
Isopropyl alcohol
Dioxane
The duration of the procedure can be noted down as;

1.
2.
3.
4.
5.
6.
7.
.
70 % alcohol 1 hour
70 % alcohol 1 hour
95 % alcohol 1 hour
95 % alcohol 1 hour
Absolute alcohol 1 hour
Dehydration is done so that the wax i.e Paraffin
wax, which is used for impregnation, can be easily
miscible as it is immiscible with water.
CLEARING ( DEALCOHOLIZATION):
It is the procedure where in the alcohol in the tissue is
replaced by a fluid which will dissolve the wax used for
impregnating the tissues .
The various clearing agents used are ;
Cedar wood oil : The best agent but is expensive.
Benzene : It is carcinogenic.
Xylene : It is most commonly used.
Chloroform: Toxic and expensive.
Impregnation:
In this the tissue is kept in a wax bath containing

molten paraffin wax for 6 8 hours .
The wax is infiltrated in the interices of the tissue

which increases the optical differentiation & hardens
the tissue & helps in easy sectioning of the tissue.
The various waxes which are used are,
1.
Paraffin wax
2.
Paraplast
3.
Paraplast plus
4.
Gelatin
5.
Celloidin
Jar containing molten paraffin wax:
Embedding :
It is done by transfering the tissue which has been

cleared of the alcohol to a mould filled with molten
wax & is allowed to cool & solidify.
After solidification, a wax block is obtained which is

then sectioned to obtain ribbons.
Types of Moulds:
A.
Leuckharts Moulds:
L- shaped brass pieces which is placed in opposing
positions & can be manipulated to increase or
decrease the size of the block to be prepared.
B.
Glass or Metal petri dishes :
C.
Watch glass
D.
Paper boats .
Leuckharts moulds :
Paraffin block
Section 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.
.
.
.
.
.
TYPES OF MICROTOMES:
Sliding
Rotary
Rocking
Freezing
Base sledge
Rotary Microtome:
It is the most commonly used.
Also known as Minnots 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.
Tissue floatation bath:

It is a thermostatically
controlled water bath with
the inside coloured black.
It is maintained at a
temperature maintained 5
6 degree below the melting
point of paraffin wax.
Electronic tissue floatation bath:
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 stain.
Procedure :
1.
Deparaffinization with xylene.
2.
Hydration
3.
Wash under water
4.
Stain with Haematoxylin for 15 min
5.
Wash with water
6.
Differentiate with 1 % acid alcohol
7.
Wash with water for 10 min
8.
Stain with 1% Eosin for 2 min
9.
Wash with water.
10. Dehydration
11. Clearing with xylene
12. Dry
13. Mount
Result :
The nucleus stains Blue
The cytoplasm stains pink.
Mounting:
Adhesives used for fixing the sections on the slides :
Albumin solution ( Mayors egg albumin)
Starch paste
Gelatin
Mountants :
DPX ( Distrene Dibutyl phthalate Xylene ).
Canada Balsam
Colophonium resin
Terpene resin
Automation:
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.
Automatic stainer:
Automatic stainer
References:
Textbook
of Medical Laboratory
Technology. Ramnik Sood.
Histotechnology and
Cytotechnology- Manipal
Academy of Higher Education.
www.en.wikipedia.org
http://en.wikipedia.org/wiki/File:Em
physema_H_and_E.jpg
Thank
You !

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Tissue Processing

Dr. Saket Kumar

Tissue for study can be obtained from:

Protocols followed in Histotechniques ;

Receipt & Identification

Specimen identification and

Tissue specimen received in the

The specimen are accessioned

It is a process in which a specimen is treated by

The purpose of fixation is to preserve tissues

The fixative should be 15 20 times more in volume

It should prevent autolysis & putrefaction of the cell.

The bits should of the size of approximately 2 x 2 cm

These bits are then placed in metal cassettes or

Tiny biopsies or small specimen can be wrapped in a

The most commonly used fixative is Formalin .

It is prepared by mixing 40 % Formaldehyde gas in 100

The resultant mixture is 100 % Formalin.

Routinely, 10 % formalin is used which is prepared by

Irritant to the nose,the eyes and mucous membranes

Other Simple Fixatives

Other Simple Fixatives

Formal (Hellys Fluid)

Due to the presence of buffer, the pH of the solution

It is the process of removal of the calcium salts from

It is the process in which the water content in the tissue

The duration of the procedure can be noted down as;

Cedar wood oil : The best agent but is expensive.

Xylene : It is most commonly used.

Chloroform: Toxic and expensive.

In this the tissue is kept in a wax bath containing

The wax is infiltrated in the interices of the tissue

The various waxes which are used are,

Jar containing molten paraffin wax:

It is done by transfering the tissue which has been

After solidification, a wax block is obtained which is

Glass or Metal petri dishes :

It is the procedure in which the blocks which have

The instrument by which this is done is called as a

Also known as Minnots Rotary microtome.

In this the Block holder moves up and down while the

It is suitable for cutting of small tissues & serial

Parts of a Microtome ( Rotary ) :

Knife clamp screws

Angle of tilt adjustment

Tissue floatation bath:

Electronic tissue floatation bath:

Adhesives used for fixing the sections on the slides :

Albumin solution ( Mayors egg albumin)

Automated tissue processor:

It provides constant agitation during every step

It reduces the work load & in turns improves the

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