Histopathology LAB

• Histotechnique


  Process of removing tissue from the body and producing microscopic slides for the demonstration of minute tissue structures
• Histology


  Study of normal tissues
• Histopathology


  Changes that occur in the tissue as a result of disease
• Histopathologic Technique


  Application of histotechniques for the diagnosis of diseases
• Histopathologic Technique


  1. Specimen Collection

  2. Gross Examination

  3. Fixation

  4. Dehydration

  5. Clearing

  6. Impregnation/Infiltration

  7. Embedding

  8. Trimming

  9. Sectioning.

  Staining 10. Mounting

  11. Labelling

  12. Microscopic Examination
• Biopsy


  Tissue samples coming from a living person
• Autopsy


  Tissue samples coming from a dead person
• Gross Examination


  Initial step; describing the macroscopic appearance of the sample
• Fixation


  To preserve the microscopic anatomy of the tissue using a chemical agent (10% Formalin)
• Fixative


  Chemical preservative where samples are immediately placed prior to further examination
• Specimen must be placed in a proper fixative 10-20x its volume
• Regions of interest: suspected tumor, inflammation, other lesions
• Narrative Description


  • SCO femoral head weighing 120 grams and measuring 7.0cm in length and up to 5.0 cm in diameter. CSS tan-white bony and cartilaginous tissue, with no obvious gross abnormality. Examination of cut surface of the specimen show areas of soft brown-red, hemorrhagic and bony tissue.
• Commonly used terms to describe specimen


  • Shape: fist size, wedge shape, triangular, squarish, rectangular, linear, round
  • Color: yellow, green, red, white, black, brown, combinations*
  • Consistency: soft, firm, hard, waxy, greasy, caseous/not round
• Dehydration


  Removing both intracellular and extracellular water from the tissue, replaced by wax to give the sample a firm consistency
• Dehydration


  6 changes of alcohol: (1-3) 70%, 80%, 95%; (4-6) 100%
• Clearing


  Replacing alcohol with a (xylol/xylene) that is miscible with wax with which the tissue will be impregnated
• Clearing


  3x submersion to Xylol
• Impregnation or Infiltration


  A liquid medium replaces clearing agent to fill all the cavities and solidify to provide a firm consistency
• Impregnation or Infiltration


  3x impregnation to wax
• Paraffin wax is the most commonly used medium for impregnation
• Dehydration and impregnation is a 12 hour process
• Embedding


  The tissue is transferred to a mold where it is submerged in the medium that is allowed to solidify
• Trimming


  Small paraffin blocks are trimmed into appropriate sizes to facilitate sectioning
• Prior to sectioning, place the tissue blocks in the freezer/refrigerator/ice-cold water for 5-10 minutes to harden/solidify it faster
• Sectioning


  Cut into very thin sections (Microtomy) with the use of a special cutting tool; Microtome(rotary)
• Mayer's egg albumin is used as an adhesive; smear thin layer on the slide surface where the tissue will be placed
• Initial thickness setting is 15-20 um, routine procedures use 3-5 um
• Floating Out


  Usage of water to straighten the ribbon
• Water bath is maintained at 45-50°C
• Sections are fished using the labelled slide with adhesive
• Drying of sections


  Placing the slide in a drying oven to melt out the paraffin, drying oven melting point is 55-60°C, slides must be cooled down to 37°C before staining
• Paraffin should not be stained
• Staining


  Tissues are placed into slides; staining dyes are applied into tissue sections to allow the visualization and differentiation of microscopic structures
• Hematoxylin


  Basic/nucleus/blue stain
• Eosin


  Acidic/cytoplasm/pink,orange stain
• Staining Procedure


  1. 10-15 dips Xylol x3=deparaffinization

  2. 5 dips Absolute alcohol x2

  3. 10 dips 95% alcohol x2

  4. Wash with tap water

  5. 20mins Hematoxylin=Nuclear staining

  6. Wash in tap water

  7. 1 quick dip acid alcohol=decolorization

  8. 5dips tap water

  9. 30dips ammonia water=blueing

  10. 10dips tap water and 95% alcohol

  11. 5mins Eosin=cytoplasmic stain

  12. 10-15dips x2 95% alcohol

  13. 10-15dips x3 absolute alcohol=dehydration

  14. 10-15dips x2 Xylol=clearing

  15. Start and end with Xylol
• Mounting


  Placing a thin piece of glass over the tissue section; provides protection from scratches, improved optical qualities for viewing and for storage
• Labelling


  Use the frosted end of the slide, pencil or diamond pen
• Microscopic Examination


  Performed by Pathologist