HISTOPATH

• Methods of fresh tissue examination


  • Teasing or Dissociation
  • Squash Preparation or Crushing
  • Smear Preparation
  • Frozen section
• Freezing Agents


  • Liquid nitrogen
  • Isopentane
  • Carbon dioxide gas
  • Aerosol spray
• Processing of tissues


  1. Fixation

  2. Dehydration

  3. Clearing

  4. Impregnation

  5. Embedding

  6. Trimming

  7. Sectioning

  8. Staining

  9. Mounting

  10. Labeling
• Fixation


  First and most critical step, preserves the morphological and chemical integrity of the cell, hardens and protects the tissue for easier cutting, prevents degeneration, decomposition, putrefaction, distortion
• Main factors involved in fixation

  • Hydrogen ion concentration
  • Temperature
  • Thickness of section
  • Osmolality
  • Concentration
  • Duration of fixation
• Types of fixative according to composition

  • Simple fixative
  • Compound fixative
• Types of fixative according to action

  • Microanatomical
  • Cytological
  • Histochemical
• Cytological fixatives


  • Nuclear
  • Cytoplasmic
  • Histochemical
• Aldehyde fixatives


  • Formaldehyde
  • 10% Formol-saline
  • 10% Neutral buffered formalin
  • Formol-corrosive
  • Alcoholic formalin
  • Glutaraldehyde
• Methods of removing pigments left by formalin

  1. Kardesewitch's method

  2. Lillies method

  3. Picric acid method
• Metallic fixatives


  • Mercuric chloride
  • Chromate fixative
  • Lead fixative
• Mercuric chloride fixatives


  • Zenker's fluid
  • Zenker-formol (Helly's)
  • Heidenhain's Susa
• Chromate fixatives


  • Chromic acid
  • Potassium dichromate
  • Regard's (Muller's)
  • Orth's fluid
• Picric acid fixatives


  • Bouin's solution
  • Brasil's alcoholic picroformol
• Alcohol fixatives


  • Methyl alcohol
  • Isopropyl alcohol
  • Ethyl alcohol
  • Carnoy's fluid
  • Newcomer's fluid
• Osmium tetroxide fixatives


  • Flemming's solution
  • Flemming's without acetic acid
• Secondary fixation


  Placing an already fixed tissue in a second fixative
• Post-chromatization


  A form of secondary fixation which utilizes 2.5-3% potassium dichromate (acts as a mordant)
• Washing-out


  Removing excess fixative using tap water, 50-70% alcohol, alcoholic iodine
• Factors that affect fixation time

  • Size and thickness of tissue
  • Presence of mucus
  • Presence of fats
  • Presence of blood
  • Temperature
  • Agitation
• Methods to avoid chemical fixation

  • Freeze-drying
  • Freeze substitution
  • Fresh frozen tissue sectioning
• Decalcification methods


  • Acids
  • Chelating agents
  • Ion exchange resin
  • Electrophoresis
• Acid decalcifying agents


  • Nitric acid
  • Hydrochloric acid
  • Formic acid
  • Trichloroacetic acid
  • Sulfurous acid
  • Chromic acid
• EDTA (versene)


  Chelating agent that combines with calcium ions to form weakly dissociated complex to facilitate removal, inactivates alkaline phosphatase activity so magnesium chloride is added
• Electrophoresis


  Positively charged ions are attracted to negatively electrode, satisfactory for small bone fragments
• Factors influencing rate of decalcification

  • Concentration and volume of decalcifying agent
  • Temperature
  • Mechanical agitation
  • Size of the tissue
• Tests to measure completeness of decalcification


  • Physical or mechanical
  • X-ray
  • Chemical method (calcium oxalate test)
• Dehydration


  Removal of intercellular and extracellular water from the tissue, increasing concentration of alcohol
• Dehydrating agents


  • Alcohol (ethyl, methyl, butyl)
  • Acetone
  • Diethylene dioxide
  • Cellosolve
  • Triethyl phosphate
  • Tetrahydrofuran
• Clearing


  Removal of alcohol (de-alcoholization) and replacement with a clearing agent to make the tissue transparent
• Factors affecting decalcification


  • Concentration and volume of decalcifying agent
  • Temperature
  • Mechanical Agitation
  • Size of the tissue
• Tests to measure completeness of decalcification

  • Physical or Mechanical
  • X-ray
  • Chemical Method (Calcium Oxalate Test)
• Dehydration


  Removal of intercellular and extracellular water from the tissue
• Increasing concentration of alcohol


  • Routine - starts with 70% usually ethyl alcohol
  • Embryonic tissues - starts with 30% of ethyl alcohol
  • 10:1 ratio of dehydrating agent and tissue
• Dehydrating agents


  • Ethyl alcohol
  • Methyl alcohol
  • Butyl alcohol
  • Acetone
  • Diethylene dioxide
  • Cellosolve (Ethylene glycol monoethyl ether)
  • Triethyl phosphate
  • Tetrahydrofuran
• Ethyl alcohol


  Most common, best dehydrating agent
• Methyl alcohol


  Toxic, employed for blood and tissue films
• Butyl alcohol


  Utilized for plants and micro-techniques
• Acetone


  Utilized for urgent biopsies
• Diethylene dioxide


  Dehydrating and clearing agent