HISPATHOLOGY

• Histopathologic Techniques - involves different procedures that have been adopted for the preparation of materials and tissue for microscopic examination
• 12 steps in Histopathologic Techniques:

  • Numbering
  • Fixation
  • Dehydration
  • Clearing
  • Wax impregnation
  • Embedding
  • Blocking
  • Trimming
  • Sectioning
  • Staining
  • Mounting
  • Labelling
• Decalcification - done or placed in between fixation and dehydration; for bones including teeth
• RECEPTIONIST - Validates if the specimen is adequate or good for

  tissue processing
• Bilateral organs

  • Extremities
  • kidneys
  • lungs
  • ovaries
• Miscellaneous

  • age
  • sex
  • ward
• NUMBERING - Basic information needed
• FIXATION - Most critical step in histopathological techniques
• Primary aim - preserve the morphology and chemical constituents of the tissue
• Secondary aim - protect and harden the specimen for further handling
• Fixatives - Act as mordant or accentuator for staining
• Microanatomic Fixative

  • Involves small tissue or organ
  • 10% Formol Saline
  • 10% Neutral Buffered Formalin
• Cytological Fixative - Involves body fluid or secretion
• Nuclear fixatives - Flemming’s Fluid, Bouin’s Fluid, Heidehain’s Susa
• Cytoplasmic Fixatives - Kelly’s Fluid, Orth’s Fluid
• Histochemical Fixative

  • Involves tissue containing labile substances
  • 10% Formol Saline
  • Absolute Ethyl
  • Alcohol
  • Acetone
• -TYPES OF FIXATIVES

  ACCORDING TO ACTIONS

  • Microanatomic Fixative
  • Cytological Fixative
  • Histochemical Fixative
• -TYPES OF FIXATIVES

  ACCORDING TO COMPOSITION

  • Simple Fixative
  • Compound Fixative
• Simple Fixative

  • Uses only on chemical for fixation
  • Aldehydes: Formaldehyde, Glutaraldehyde
  • Metallic Fixative: Mercuric Chloride, Chromate Fixatives, Lead Fixatives
• Compound Fixative - The use of two or more chemicals for fixation
• DEHYDRATION

  • Utilizing chemical known as dehydrating agents

  • Removing of intracellular and extracellular water and fixatives in the tissue
• CLEARNING - Removing of dehydrating agents
• Xylene – most commonly used in clearing
• Alcohol - most commonly used in dehydration
• IMPREGNATION

  • Also known as INFILTRATION OR WAX IMPREGNATION

  • The process whereby the clearing agent is completely removed from the tissue and replace by a medium that will completely fill all the tissue cavities.
• PARAFFIN - the simplest, most common and best embedding medium used for routine tissue processing
• EMBEDDING

  • Also known as CASTING OR BLOCKING

  • 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
• FOUR TYPES OF TISSUE IMPREGNATION:

  • Parrafin wax
  • Celloidin
  • Gelatin
  • Plastic
• BLOCKING - Allows the medium to solidify to produce tissue block
• TRIMMING

  • Process of removing excess wax after embedding

  • Can use knife/blade or heated spatula
• SECTIONING

  • Also known as CUTTING OR MICROTOMY

  • The process by which processed tissue is cut into uniformly thin slices to facilitate studies under microscope
• MICROTOME - machine or instrument used for cutting sections of tissue
• KINDS OF MICROTOME

  • Rocking Microtome
  • Rotary Microtome
  • Sliding Microtome
  • Freezing Microtome
  • Cryostat Microtome
  • Ultrathin Microtome
• Rocking Microtome - Simplest and oldest type of microtome
• Rotary Microtome - for cutting serial sections of tissue specimen
• Sliding Microtome - Most dangerous type of microtome
• Freezing Microtome - For urgent surgical biopsies specimen
• Cryostat Microtome

  • Permits rapid penetration of tissue biopsies for surgical pathology.
  • Also known as COLD MICROTOME
• Ultrathin Microtome - Specimen for electron microscope
• STAINING - Tissue constituent are demonstrated in sections by direct interaction with dye or staining solution producing coloration of the active tissue component