histo

• 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
• 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 the 12 steps in Histopathologic Techniques
• 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 (e.g. Extremities, kidneys, lungs and ovaries)
  • Miscellaneous (e.g. age, sex, ward)
• Numbering

  Basic information needed: Date and time, Name of the patient, 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

  • Microanatomic Fixative (e.g. 10% Formol Saline, 10% Neutral Buffered Formalin)
  • Cytological Fixative (e.g. Nuclear fixatives: Flemming's Fluid, Bouin's Fluid, Heidehain's Susa; Cytoplasmic Fixatives: Kelly's Fluid, Orth's Fluid)
  • Histochemical Fixative (e.g. 10% Fomol Saline, Absolute Ethyl Alcohol, Acetone)
• Fixatives by Composition

  • Simple Fixative (e.g. 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
• 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 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
• Types of Tissue Impregnation

  • Parrafin wax
  • Celloidin
  • Gelatin
  • Plastic
• 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
• 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
• Microtome


  • Machine or instrument used for cutting sections of tissue
  • Blade and holder
  • Thin slices; cannot do manually
• Kinds of Microtome


  • Rocking Microtome
  • Rotary Microtome
  • Sliding Microtome
  • Freezing Microtome
  • Ultrathin Microtome
• Cryostat Microtome

  • For cutting serial sections of tissue specimen
  • Also known as COLD MICROTOME
  • Permits rapid penetration of tissue biopsies for surgical pathology
• 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

  • 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
  • Can be used as a pair
• Mounting


  • MOUNTING MEDIUM: the solution in which the specimen is embedded, generally under a cover glass
  • Adhesive agent to protect the specimen
  • It may be liquid, gum or resinous, soluble in water, alcohol or other solvents and be sealed from the external atmosphere by non-soluble ringing media
• Labelling


  • Date and time
  • Name of the patient
  • Specimen Number (C- Cytology, A- Anatomical, S- Surgical)
• Specimens for Examination


  • Gynecological Specimen (e.g. Vaginal smear)
  • Non-Gynecological Specimen (e.g. Sputum, Bronchoalveolar lavage (BAL))
  • Urine (to determine the presence of urethral cancer - pattern called "ferning")