Intro to nistopathologic techniques

• Histopathologic techniques


  Laboratory performed on a human tissue or organ sample that aims to preserve the cellular, histopathologic, histochemical, immunohistological, and cytoplasmic characteristics of cells and tissues
• Biopsy results


  Used by other physicians to correlate clinical findings with patients' history, signs and symptoms and effectiveness of therapy
• Diagnostic purpose of histopathologic techniques

  • Provide evidence of cellular changes, necrosis or apoptosis, degenerations and adaptations observable in human tissues correlated to various pathologic conditions
  • Provide evidence of the true cause of death among patients with unexplained or questionable fatalities
  • Provide evidence on successful or unsuccessful cancer treatment
• Hypertrophy


  Increase in size of cells/ enlargement of cellular cells
• Hyperplasia


  Increase in size due to multiple cells
• Atrophy


  Decrease in size of cells
• Anaplasia


  Loss of cells
• Practice purpose of histopathologic techniques

  • Allow an in-depth observation of any noticeable and significant histologic or cytologic adaptations, degenerations and injuries on human tissue or cells
  • Allow a long-term (life-long) preservation of human tissues and cells for further study and evaluation
  • Retard the progressive decomposition of human tissues and cells that may potentially hinder effective disease detection
• Overview of histopathologic techniques

  • The process involved requires a long turn-around-time (TAT)
  • Depending on availability of lab equipment and reagents, specialized histotechnologists and pathologists, TAT can range from weeks to months
  • There are multiple and sequential steps
• Steps in histopathologic techniques

  • Numbering
  • Fixation
  • Decalcification
  • Dehydration
  • Clearing/Dealcoholization
  • Impregnation/Infiltration
  • Embedding
  • Trimming
  • Sectioning
  • Staining
  • Mounting
  • Labelling
• Fresh tissue processing


  • Examined on its living tissue state
  • Protoplasmic activities are observed
  • Tissue preparation isn't permanent
• Methods of fresh tissue processing


  • Teasing or dissociation
  • Squash or crush preparation
  • Smearing preparation
  • Touch preparation (impression smear)
  • Fresh frozen section
• Numbering


  • The first and most crucial step in the histopathology section
  • Identify specimens without putting the entire name of a patient on the requisition form and on the specimen label
  • Logging the numbers and details in a receiving logbook
  • Codes vary on each institution's preferences
• Fixation


  • The process of preserving cells and tissue constituents in a life-like manner
  • Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body
  • Hardens and protects the tissue from trauma of further handling
• Mechanisms of fixation


  • Additive fixation
  • Non-additive fixation
• Main factors involved in fixation

  • pH level
  • Temperature
  • Thickness
  • Osmolality
  • Concentration
  • Duration of fixation
• Practical considerations in fixation

  • Speed
  • Rate of penetration
  • Volume
  • Duration of fixation
• Practical considerations in fixation of certain organs

  • Brain
  • Hollow organs
  • Air filled lungs
  • Eyes
  • Hard tissues
• Fixatives according to mechanism of action

  • Microanatomical fixatives
  • Cytological fixatives
• Cytological fixatives


  • Nuclear fixatives
  • Cytoplasmic fixatives
• Fixation of chemical constituents found in tissues

  • Lipids
  • Carbohydrates
  • Protein
  • Glycogen
• Mixture of fixatives

  • Electron cytochemistry
  • Acrolein
• Formaldehyde

  Widely used in 10% Formalin, made from Formaldehyde (a gas)
• Digitonin

  Cholesterol Ultrastructural demonstration
• Alcoholic Fixatives

  For Glycogen
• Loss of Glycogen can be high (60 – 80%) in aqueous solutions
• Alcoholic formaldehyde


  Better for skin compared to Neutral Buffered Formaldehyde
• Neutral Buffered Formol Saline/ Formaldehyde vapor


  For amino acid histochemistry
• Rossman's Fluid or Cold Absolute Alcohol

  For Glycogen
• Essential for patients with Glycogen Storage Diseases
• Better glycogen retention if section is coated with celloidin
• Karnovsky's


  Paraformaldehyde – Glutaraldehyde solution for Electron Cytochemistry
• Acrolein


  Can be mixed with formaldehyde or glutaraldehyde
• Formaldehyde


  Widely used in 10% Formalin, made from Formaldehyde (a gas produced from oxidation of methyl alcohol)
• Formaldehyde


  • No brittleness (Recommended for Nervous tissue preparation)
  • Colored Tissue Photography
  • Tolerant fixative, used for mailing specimens
  • Forms abundant brown pigment granules on blood – containing tissues (spleen, due to blackening of Hemoglobin)
  • Bleaching of specimen
  • Fatty Tissues stored in long time
• Cadmium or Cobalt salts


  Added to prevent dispersions, loss of natural tissue colors
• 70% alcohol


  Immerse tissues in prior to staining to restore Natural tissue color
• Glutaraldehyde


  Made up of 2 formaldehyde residues linked by 3 carbon chains
• Buffered Glutaraldehyde followed by secondary fixation with Osmium Tetroxide


  • Satisfactory for Electron Microscopy
  • Recommended for Enzyme Histochemistry and Electron Microscopy
  • Better Plasma Protein Preservation
• 10% FORMOL SALINE


  • For CNS tissues and general post – mortem tissues for histochemical examinations
  • For Silver Impregnation techniques