2. Introduction
The routine work associated with a surgical pathology
specimen includes gross and microscopic examination.
Importance of the Gross Aspect.
An inadequate gross dissection and sampling will invalidate
the microscopic interpretation.
Complicated specimens demand experience and knowledge
in order to be dissected, described, and sampled adequately.
3. Initial handling of specimens
The surgical pathology laboratory in close proximity to the
operating room.
Transport
Most small biopsies (needle biopsies, incisional biopsies,
endoscopic biopsies) should be placed in the chosen fixative.
Proper fixative most commonly used is formaline 10%
Proper identification and details on specimen form to be
submitted in the laboratory
4. Fixation can also be achieved with microwaves, which are
defined as electromagnetic waves with a frequency between
300 MHz and 300 GHz.
5. 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
6. 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.
7. Protocols followed in Histotechniques ;
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
8. 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
9. 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.
10. Aims of Fixation :
1. It should prevent autolysis & putrefaction of the cell.
2. It should penetrate evenly and rapidly.
3. It should harden the tissues
4. Increase the optical density
5. Should not cause shrinkage or swelling of the cells
6. Must not react with the receptor sites & thus must not interefere with the
staining procedure.
7. It must be cheap and easily available.
11. 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.
15. 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 OFACTION:
It forms cross links between amino acids of proteins thereby making them
insoluble.
It fixes 4 mm thick tissue in 8 hours .
16. ADVANTAGES :
1. Rapid penetration
2. Easy availability & cheap
3. Does not overharden the tissue
4. Fixes lipids for frozen sections
5. Ideal for mailing
DISADVANTAGES:
1. Irritant to the nose,the eyes and mucous membranes
2. Formation of precipitate of paraformaldehyde which can be prevented by adding 11-
16 % methanol.
3. Formation of black formalin pigment ,Acid formaldehyde hematin.
18. 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.
19. 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 doesn’t take place.
22. 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
23. 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
24. The duration of the procedure can be noted down as;
1. 70 % alcohol – 1 hour
2. 70 % alcohol – 1 hour
3. 95 % alcohol – 1 hour
4. 95 % alcohol – 1 hour
5. Absolute alcohol – 1 hour
6. Absolute alcohol – 1 hour
7. 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.
25. 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.
26. 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.
27. The various waxes which are used are,
1. Paraffin wax
2. Paraplast
3. Paraplast plus
4. Gelatin
5. Celloidin
29. 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.
30. Types of Moulds:
A. Leuckhart’s 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 .
33. 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
34. 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.
35. 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.
37. 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.
41. 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.
42. 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
43. Result :
The nucleus stains Blue
The cytoplasm stains pink.
44. Mounting:
Adhesives used for fixing the sections on the slides :
Albumin solution ( Mayor’s egg albumin)
Starch paste
Gelatin
48. Automation:
Automated tissue processor:
All the before mentioned procedures upto the impregnation step can be done
automatically in a single, unmanned instrument , which is theAutomatedTissue
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.
