2. LEARNING OBJECTIVES
At the end of this course students should be able to
explain in detail the following:
Sample reception and Grossing
Fixation
Tissue processing
Embedding
Microtomy
Staining
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3. INTRODUCTION
Histopathology- the branch of biology which deals
with the study of diseased tissue under the
microscope
Histology- the study of normal tissue
The purpose of its study is to compare normal
tissue from abnormal tissue
Pathologist/histotechnologist understands the
different between normal and abnormal tissue
based on their:
Size, shape and nucleus to cytoplasmic ratio 3
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4. INTRODUCTION-CONT
Also use for the purpose of the diagnosing
Cancer stage
Precancerous stage
Other inflammatory disease
COMMON TERMINOLOGIES
Biopsy
Autopy
Autolysis
Putrefaction
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5. Tissue specimens received in the pathology laboratory have
a request form that lists the patient information and
history along with a description of the site of origin
How to Prepare Tissue for Light Microscope
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7. SPECIMEN ACCESSIONING
Tissue specimens 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 specimens are accessioned by giving them a
number that will identify each specimen for each
patient.
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8. GROSS EXAMINATION
During grossing, tissues removed from the body
for diagnosis are examined by a pathologist,
pathology assistant, or pathology resident.
Gross examination consists of describing the
specimen and placing all or parts of it into a
small plastic cassette which holds the tissue
while it is being processed to a paraffin block.
Initially, the cassettes are placed into a fixative.
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9. GROSS EXAMINATION-CONT
Only soft tissue can be cut into small blocks and
processed directly
Bony specimens need to be decalcified before
processing
Stones and teeth require special treatment
After grossing specimen should be kept according
to accession number
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10. GROSS EXAMINATION-CONT
Well preserved specimens with representative
lesion are kept for
Teaching
Research
Museum
For future reference, they kept from 6 months to 1
year
The specimens which are not required or not useful
for any of the above purpose should be discarded.
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11. Decalcification
Bone specimens as well as calcified tissues are the
most type here.
The calcium must be removed before embedding to
allow sectioning.
A variety of reagents have been used to decalcify
tissue such as mineral acids, organic acids, EDTA,
and electrolysis.
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12. Strong mineral acids such as nitric and
hydrochloric acids can be used
Strong acids will remove large quantities of
calcium at a rapid rate, but they will cause
damage of cellular morphology
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13. Organic acids such as acetic and formic acid
However, they act more slowly on dense cortical
bone.
EDTA can remove calcium safely, it works slowly, it
penetrates tissue poorly, but it is expensive in large
amounts.
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14. HOW IS MICROSCOPIC EXAMINATION OF TISSUES
DONE???
To observe the tissue under the microscope the
following steps are followed:
• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding
• Sectioning
• Staining
• Mounting
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15. FIXATION
The purpose of fixation is to preserve tissues permanently in
as life-like state as possible
Fixation should be carried out as soon as possible after
removal of the tissues to prevent autolysis
Formaldehyde is the most comely used fixative
Therefore, a variety of fixatives are available for use,
depending on the type of tissue present and features to be
demonstrated.
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16. • The technique of getting fixed tissue into
paraffin is called tissue processing. The main
steps in this process are dehydration and
clearing.
Tissue Processing
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18. 1. Dehydration: Gradual removal of water from
the tissue using ascending grades of ethanol to
prevent tissue shrinking
2. Clearing: Replacement of alcohol in tissue by
clearing fluid like xylene, benzene
3. Infiltration and Embedding:
- Tissues are impregnated in paraffin
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19. 4. Cutting:
- Paraffin block are cut by microtome using
metal knife, into thin sections ~ 3-5µ
6. Mounting/picking of sections:
- Sections are mounted on glass slides, allow to
drain,placed on a hot plate/ hot oven until ready for
staining
7. Staining:
- Haematoxylin and Eosin (H&E) stain
- Variable stains are used for specific tissues. 19
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21. • Once the tissues have been embedded, they must be
cut into very thin sections (3 to 5 microns) that can
be placed on a slide.
• This is done with a microtome. The important
thing for proper sectioning is to use a very sharp
knife/blade
Sectioning
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23. Frozen Sections
Frozen sections are performed
with an instrument called a
cryostat.
The cryostat is just a refrigerated
box containing a microtome.
The temperature inside the
cryostat is about -20 to -30 C.
The tissue sections are cut and
picked up on a glass slide.
The sections are dried and then
stained.
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24. Staining
The embedding process must be reversed in order
to get the paraffin wax out of the tissue and allow
water soluble dyes to penetrate the sections
Therefore, before any staining can be done, the
slides are "deparaffinized" by running them
through xylene then, to alcohols and lastly to water
There are no stains that can be done on tissues
containing paraffin
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25. Automated stainer
Frozen sections are stained by
hand, because this is faster for
one or a few individual
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26. Coverslipping
The stained section on the
slide must be covered with
a thin piece of glass to
protect the tissue from
being scratched, and to
preserve the tissue section
and for long storage
without any harm
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