3 HISTOPATHOLOGY

• Histopathologic Techniques


  Involves different procedures that have been adopted for the preparation of materials and tissue for microscopic examination
• 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. Labeling
• There is a 13th step which is decalcification (done or placed in between fixation and dehydration; for bones including teeth)
• All in all, it will take 24-48 hours to do
• Receptionist

  • Validates if the specimen is adequate or good for tissue processing
  • 1st person that will receive the specimen and put it into the container
• Container

  • Contain fixatives
  • Proportional to the size of the specimen (larger than the organ; the organ or specimen should be fully submerged)
  • Clear - to see the organ
  • Unbreakable - no leaking
  • Wide mouthed bottle - easy access

  
• The receptionist must not accept specimens that are only placed in plastic
• Medical Technologists assist pathologists in autopsies
• Portion of the specimen will be placed in tissue cassettes and the rest of the organ will be contained
• Specimen Source

  • Bilateral organs (Extremities, kidneys, lungs and ovaries)
  • Miscellaneous (age, sex, ward)
• Numbering


  1. Date and time

  2. Name of the patient (must be indicated in the log book)

  3. Specimen Number (C- Cytology, A- Anatomical, S- Surgical)
• 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
• Effects of Fixatives


  • Inhibit bacterial growth and reduce the risk of infections
  • Act as mordant or accentuator- accelerating the staining process
• Types of Fixatives According to Actions

  • Microanatomic Fixative (10% Formol Saline, 10% Neutral Buffered Formalin)
  • Cytological Fixative (Nuclear fixatives: Flemming's Fluid, Bouin's Fluid, Heidehain's Susa; Cytoplasmic Fixatives: Kelly's Fluid, Orth's Fluid)
  • Histochemical Fixative (10% Formol Saline, Absolute Ethyl Alcohol, Acetone)
• Types of Fixatives According to Composition

  • Simple Fixative (Aldehydes: Formaldehyde, Glutaraldehyde; Metallic Fixative: Mercuric Chloride, Chromate Fixatives, Lead Fixatives)
  • Compound Fixative (Use of two or more chemicals for fixation)
• Dehydration


  Removing of intracellular and extracellular water and fixatives in the tissue
• Dehydrating Agents


  • Alcohol (particularly ethanol)
  • Acetone
  • Dioxane
  • Tetrahydrofuran
  • Cellosolve (Ethylene glycol monoethyl ether)
• Clearing


  Removing of dehydrating agents and will be replaced by the clearing agent
• Impregnation


  • Also known as Infiltration or Wax 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 Wax Impregnation


  • Paraffin: the simplest, most common and best embedding medium used for routine tissue processing
  • Surface area of the tissue is larger; clearing agent is removed; specimen is further hardened
• 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
• Sectioning


  • Also known as Cutting or Microtomy
  • The process by which processed tissue is cut into uniformly thin slices to facilitate studies under microscope

  
• Kinds of Microtome


  • Rocking Microtome
  • Rotary Microtome
  • Sliding Microtome
  • Freezing Microtome
  • Rotary Microtome (Cryostat or Cold Microtome)
  • Ultrathin Microtome
• Staining


  Tissue constituent are demonstrated in sections by direct interaction with dye or staining solution producing coloration of the active tissue component
• Haematoxylin and Eosin Staining (H&E)

  • Utilizes micro-anatomical studies of tissue
  • It is a regressive staining method
  • Haematoxylin is used to stain nuclear components
  • Eosin is used for cytoplasmic components
• Mounting


  • Mounting medium: the solution in which the specimen is embedded, generally under a cover glass
  • Adhesive agent to protect the specimen
• Labeling


  1. Date and time

  2. Name of the patient

  3. Specimen Number (C- Cytology, A- Anatomical, S- Surgical)
• Specimens for Examination


  • Gynecological Specimen
  • Non-Gynecological Specimen
  • Urine
• Gynecological Specimen


  Performed regularly even in pregnant women without undue risk (Example: Vaginal smear)
• Non-Gynecological Specimen


  • Respiratory Tract specimens: Sputum, Bronchoalveolar lavage (BAL)
• Urine


  Determine the presence of urethral cancer (Pattern called "ferning")