Tissue Processing

Introduction
• Histopathology – microscopic study of diseased tissue
• Tissue source
• Biopsy
• Necropsy
• The techniques for processing the tissues, whether biopsies or tissues
from necropsy so as to enable the pathologist to study them under
the microscope
Protocols
1. Receipt & Identification
2. Labeling of the specimen with numbering
3. Fixation
4. Dehydration
5. Clearing
6. Impregnation
7. Embedding
8. Section cutting
9. Staining
10. Mounting
Specimen identification
• Patient information
• History
• Laboratory code (numbers that will identify each specimen for each
patient)
Fixation
• Preserve tissues permanently in as life-like a state as possible
• The fixative should be 15 – 20 times more in volume then the
specimen
Ideal Fixative
Aims
• Should not cause shrinkage or swelling
• Prevent autolysis
of cells
• Penetrate evenly and rapidly
• Must not interfere with staining
• Harden specimen
• Cheap and easily available
• Antisepsis of the sample
• Should be good antiseptic
• Increase optical density
• Safe to use
Fixation
• The bits should of the size of approximately 2 x 2 cm & 4-6
micrometer in thickness
• These bits are then placed in metal cassettes or capsules which are
then placed in the fixative
Fixatives
• 10% Formalin
• 10 ml formalin in 90 ml distilled water
• MOA – forms cross links between amino acids of proteins thereby making
them insoluble
• Advantages – rapid penetration, available & cheap, does not over harden
tissue, ideal for mailing
• Disadvantages – irritant, forms paraformaldehyde precipitates, formation of
black formalin pigment , acid formaldehyde hematin
Fixatives
• Other fixatives
1. Glutaraldehyde
2. Osmium tetraoxide
3. Potassium dichromate
4. Mercuric chloride
5. Picric acid
6. Zenker’s fluid
7. Zenker’s Formal (Helly’s fluid)
8. Bouin’s fluid
* Why we prefer formaline over all these?S
Fixatives
• Microanatomical fixatives:
• These are used to preserve the anatomy of the tissue.
• E.g. 10% formal saline (brain), buffered formalin
• Cytological fixatives:
• These are used to fix intracellular structures.
• E.g. nuclear fixatives (Carnoy’s fluid, Clarke’s fluid), cytoplasmic fixatives
(Champy’s fluid, Regaud’s fluid)
• Histochemical fixatives:
• These are used to demonstrate the chemical constituents of the cell.
• E.g. formal saline, cold acetone, absolute alcohol
Decalcification
• Removal of the calcium salts from the specimen
• Nitric acid, formic acid, picric acid, acetic acid, citric acid, hydrochloric
acid
Dehydration
• 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
• E.g. Ethyl alcohol, Acetone, Isopropyl alcohol, Dioxane
• 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
• The procedure where in the alcohol in the tissue is replaced by a fluid
which will dissolve the wax used for impregnating the tissues
• 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.
• Paraffin wax
• Paraplast
• Paraplast plus
• Gelatin
• Celloidin
Embedding
• It is done by transferring 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.
Section cutting
• 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
Section cutting
• 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)
• Block holder
• Knife clamp screws
• Knife clamps
• Block adjustment
• Thickness gauge
• Angle of tilt adjustment
• Operating handle.
Tissue flotation bath
• It is a thermostatically controlled water bath with the inside colored
black.
• It is maintained at a temperature maintained 5 – 6 degree below the
melting point of paraffin wax.
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
• Nucleus – blue, cytoplasm – pink
Mounting
• Adhesives used for fixing the sections on the slides :
• Albumin solution (Mayor’s egg albumin)
• Starch paste
• Gelatin
• Mountants :
• DPX ( Distrene Dibutyl phthalate Xylene ).
• Canada Balsam
• Colophonium resin
• Terpene resin