Principle of tissue processing

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Principle of tissue processing

  1. 1. PRINCIPLE OF TISSUE PROCESSING
  2. 2. Surgical Specimen  Clinical Details  Adequate specimen  Proper Fixative
  3. 3. Gross Examination Description:  Specimen weight & measurement (approx)  Consistency  Cut section
  4. 4. Taking Samples: Edge of lesions.  Wall of cysts.  Whole specimen if small.  Direction, mark
  5. 5. Inking the Margins  To mark surgical margin.  Spread of lesion  Malignancy  Adequacy of removal  Different colors to identify margins
  6. 6. Fixation:  Specimen bits are placed in porous cassettes  Not more than 5mm thick  In 10% formalin  1mm/hour fixation  ~ 6 hour
  7. 7. Types of fixative:  Acetic acid  Formaldehyde  Ethanol  Glutaraldehyde  Methanol and Picric acid.
  8. 8. Dehydration A. 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
  9. 9. Dehydration Procedure;  1. automatic tissue processor  a. overnight  2. Baths: water, 70,95,100,100 % alcohol  3. Clearing agent: 2 baths of xylene
  10. 10. 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 .
  11. 11. 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
  12. 12. Embedding • Procedure; • 1. Place tissue cassette in melted paraffin • 2. Fill mold with paraffin • 3. Place tissue in mold • 4. Allow to cool
  13. 13. Embedding A. Orient tissue 1. cross section 2. longitudinal section B. Dissection orientation C. Avoid bubbles
  14. 14. 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.
  15. 15. 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
  16. 16. Embedding:  Paraffin block with embedded tissue  consistency to cut  Paraffin blocks taken for sectioning
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