HISTOPATHOLOGIC TECHNIQUES

• 12 STEPS IN HISTOPATHOLOGIC TECHNIQUES

  1. Numbering
  2. Fixation
  3. Dehydration
  4. Clearing
  5. Wax Impregnation
  6. Embedding
  7. Blocking
  8. Trimming
  9. Sectioning
  10. Staining
  11. Mounting
  12. Labelling
• Fixation


  Most critical step in histopathologic techniques
• Primary aim in Fixation

  preseve the morphology and chemical constituents of the tissue
• Secondary aim of fixation

  protect and harden the specimenfor further handling
• TYPES OF FIXATIVES ACCORDING TO ACTIONS

  1. Microanatomic fixative
  2. Cytological fixative
  3. Histochemical fixative
• TYPES OF FIXATIVE ACCORDING TO COMPOSITION

  1. Simple fixative
  2. Compound fixative
• Microanatomic Fixative


  involves small tissue or organ
• Cytological fixative


  involves body fluid or secretion
• Two more fixatives under cytological fixative

  1. Nuclear fixative
  2. Cytoplasmic fixative
• Nuclear Fixative

  1. Flemming's fluid - osmium tetroxide
  2. Bouin's fluid - picric acid
  3. Heidehain's susa - mercuric chloride
• Cytoplasmic Fixatives

  1. Kelly's fluid - made up of 40% concentration of mercuric chloride and strong aldehyde
  2. Orth's fluid - Potassium dichromate, sodium sulfate, plus strong aldehyde
• Histochemical Fixative


  Involves tissue containing labile substances
• Simple fixative


  only one chemical for fixation
• Compound fixative


  two or more chemicals for fixation
• Dehyration


  Removing of intracellular and extracellular water and fixatives in the tissue
• Most commonly use dehydrating agent

  Acohol. Particularly, ethanol
• Clearing


  Removing of dehydrating agents and will be replaced by the clearing agent.
• Most commonly used clearing agent

  Xylene
• Impregnation


  The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities.
• Paraffin


  simplest, most common and best embedding medium used for routine tissue processing
• Embedding


  The process by which the impregnated tissue is placed into a precisely arrange position in a mold containing medium which is then allowed to solidify
• Blocking


  Allows the medium to solidify to produce tissue block
• Trimming


  Process of removing excess wax after embedding
• Sectioning


  The process by which processed tissue is cut into uniformly thin slices to facilitate studies under microscope o Making tissueribbons
• Microtome


  machine or instrument used for cutting sections of tissue
• Staining


  Tissue constituent are demonstrated in sections by direct interaction with dye or staining solution producing coloration ofthe active tissue component
• Haematoxylin


  used to stain nuclear components
• Eosin


  used for cytoplasmic components
• Mounting


  Use of a mounting medium - the solution in which the specimen is embedded, generally under a cover glass.
• Labelling


  Date and timeName of the patientSpecimen Number