principle of tissue processing
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PRINCIPLE OF TISSUE PROCESSING
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Surgical Specimen
Clinical Details Adequate specimen Proper Fixative
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Gross Examination
Description: Specimen weight &
measurement (approx)
Consistency Cut section
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Taking Samples:
Edge of lesions. Wall of cysts. Whole specimen if
small. Direction, mark
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Inking the Margins
To mark surgical margin.
Spread of lesion Malignancy Adequacy of
removal Different colors to
identify margins
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Fixation:
Specimen bits are placed in porous cassettes
Not more than 5mm thick
In 10% formalin 1mm/hour fixation ~ 6 hour
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Types of fixative: Acetic acid Formaldehyde Ethanol Glutaraldehyde Methanol and Picric acid.
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DehydrationA. Definition: removal of water
B. Rationale: for paraffin embedding/sectioning
C. Steps
1. wash out fixative
2. graded series of alcohol 70%, 95%, 100%, 100%
3. replace water by diffusion
4. not too long, not too short
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DehydrationProcedure; 1. automatic tissue
processor a. overnight 2. Baths: water,
70,95,100,100 % alcohol
3. Clearing agent: 2 baths of xylene
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Clearing: • replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium.Choice of a clearing agent depends upon the following - Intended processing conditions such as temperature, vacuum and pressure. - Safety factors. - Cost and convenience. - Speedy removal of dehydrating agent . - Ease of removal by molten paraffin wax . - Minimal tissue damage .
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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.
Embedding
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Embedding• Procedure;• 1. Place tissue cassette
in melted paraffin• 2. Fill mold with
paraffin• 3. Place tissue in
mold• 4. Allow to cool
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EmbeddingA. Orient tissue
1. cross section
2. longitudinal section
B. Dissection orientation
C. Avoid bubbles
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Precaution while embedding in wax
The wax is clear of clearing agent. No dust particles must be present. Immediately after tissue embedding, the wax must be rapidly
cooled to reduce the wax crystal size.
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There are four main mould systems and associated embedding protocols presently in use :• 1- traditional methods using paper boats• 2- Leuckart or Dimmock embedding irons or metal containers• 3- the Peel-a-way system using disposable plastic moulds and• 4- systems using embedding rings or cassette-bases
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Embedding:
Paraffin block with embedded tissue
consistency to cut Paraffin blocks taken
for sectioning
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