Histopathology

• Histopathology

  • Microscopic study of the tissue affected by disease.
• Pathologists are specialized doctors who examine the diseased tissues.
• Histopathological Specimens

  • Histo-Surgical Pathology Specimen
  • Cytopathology Specimen
  • Autopsy/Post-mortem Specimen
• Tissue Processing

  • Solid structures and tissues must be preserved and carefully processed
• Tissue Processing (SEQUENCE)

  1. Fixation
  2. Decalcification*
  3. Dehydration
  4. Clearing
  5. Infiltration/ impregnation
  6. Embedding
  7. Trimming
  8. Section- Cutting
  9. Staining
  10. Mounting
  11. Ringing (omitted)
  12. Labeling
• Fixation

  • Preservation of the tissue by physical or chemical means
• Goals of Fixation:

  • Primary Aim: Preserve the morphologic and chemical integrity of the cell in as life like manner.
  • Secondary Aim: Harden and protect the tissue from damage of further handling
• Decalcification

  • Special processing technique done to remove calcium salts from calcified tissues
• Methods of decalcification:

  • Use of acid decalcifying agents
  • Use Chelating agents
  • Use of Ion exchange resins
  • Via Electrophoresis
• Dehydration

  • Removal of water from tissue in preparation for tissue impregnation
• Clearing

  • Intermediate process wherein the dehydrating agent in the tissue is removed and replaced with the clearing agent.
• Clearing

  • aka. De-alcoholization
  • Primary application is to make the tissues transparent
• Infiltration/Impregnation

  • Process whereby clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities.
• Types of Infiltrating / Embedding Media:

  1. Paraffin Wax
  2. Celloidin
  3. Gelatin
  4. Resins
• Embedding (Casting / Blocking / Molding)

  • Process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.
• Trimming

  • Done prior to cutting to expose the tissue in the paraffin block
• Sectioning (Cutting /Section-Cutting)

  • Done to produce thin slices of tissues using the microtome
• Staining

  • Process of giving color to the tissue
• Most commonly used Staining technique:

  • Hematoxylin and Eosin Technique
• Mounting

  • Process of putting a coverslip to the prepared sample
• Ringing

  • process of sealing the margins of the cover slip
• Labeling

  • process of indicating the year and specimen number on one end of the prepared slide for proper identification
• Quality Assurance and Documentation

  • Histopath Reports
  • Signatories
• Histopath Reports

  • Surgical Pathology
  • Cytopathology report
  • Autopsy report
• Signatories

  • Request Forms
  • Result Forms
• Quality Assurance and Documentation

  Reports and their Retention

  1. Autopsy Forensic Reports ----- Permanently
  2. Surgical Pathology Reports --- 10 years
  3. Cytogenetics Reports -------- 20 years
  4. Requisition Forms ----------- 2 years
• Histopathological Equipment

  • Microtome
• Types of Microtome

  • Rocking Microtome
  • Rotary Microtome
  • Sliding Microtome
  • Freezing Microtome
  • Ultra thin Microtome
• Care of the Microtome

  • After sectioning, all accumulated paraffin and small pieces of tissues must be brushed away with a soft brush and not allowed to stay in microtome, since this may later interfere with cutting of tissue blocks.
• Care of the Microtome

  • Parts should be wiped with xylol/xylene.
• Care of the Microtome

  • Movable portions should be oiled thoroughly to prevent rusting.
• Histopathological Equipment

  The following equipment are also required during the process of sectioning:

  • Microscope
  • Water bath
  • Drying oven
  • Forceps/Sable hairbush
  • Frosted end clean glass slides
  • Coplin jars/Slotted staining dishes
• Histopathological Equipment

  Water Bath

  • Temperature of the water should be about 6 - 10 °C below the melting point of paraffin wax.
• Histopathological Equipment

  Drying oven

  • its temperature setting is at the melting point of the wax to allow the sections to dry.
• Histopathological Equipment

  Forceps / Sable hairbrush

  • needed for handling sections during cutting, and for removing folds and creases on the sections during “Floating out” in water bath.
• Histopathological Equipment

  Frosted end clean glass slides

  • 76 x 25 mm slides that are 1.0 - 1.2 mm thick are usually preferred because they do not break easily.