Histopathology - Finals Q1

• Microtomy - process by which processed tissue, most commonly a paraffin embedded tissue, is trimmed and cut into uniformly thin slices or "sections" to facilitate studies under the microscope
• Paraffin Sections - This is used for paraffin embedded tissue blocks which may be cut by rocking and rotary microtome.
• Celloidin Sections - For celloidin embedded tissues cut by sliding microtome (10-15um)
• Frozen Sections or Cryostat - cut from tissues that have been fixed and frozen with CO2 (4um)
• Block holder/ CHUCK


  Where the tissue block is held in position, a part that holds the block during cutting
• Knife carrier & Knife (Old)

  Disposable blades, used for actual cutting of tissue
• Knife carrier & Blade (New)


  Blade and blade carrier used for cutting tissue nowadays
• Rotating wheel


  The part that manipulates to start the process, moves mechanically to start cutting
• Parts that manipulate to line up the tissue block with the knife/cutting edge

  • Pawl
  • Ratchet feed wheel
  • Adjustment screw
• Cutting edge


  Sharp part of the blade
• Rocking (Cambridge) Microtome (10 – 12 um)

  • Purpose: for cutting serial sections of large blocks of paraffin embedded tissues.

  • Invented by Paldwell Trefall; most simple type.

  • Knife and the block holder are brought together by upward and vertical motions.

  • Produces ribbons that is continuous.

  • Disadvantages:

  o Restrictions in size of tissue block that can be cut. o Difficulty of reorienting the block.
• Rotary (Minot) Microtome (3-5 um in Gregorios; or 4-6 um)

  • Purpose: for cutting paraffin embedded sections, and uses in researchInvented by Minot; most common type.

  • The device operates with a staged rotary action such that the actual cutting is part of the rotary motion. o Knife is fixed in a horizontal

  position.

  • It is heavier and more stable than

  the rocking microtome, is more complex in design and construction, and is therefore more expensive.
• Sliding Microtome


  • Purpose: for cutting celloidin embedded sections
  • Invented by Adams
  • Most dangerous
• Types of Sliding Microtome

  • Base-Sledge Microtome
  • Standard Sliding microtome
• Base-Sledge Microtome


  • Less dangerous compared with standard sliding because the movable part is the block holder and the one remain stationary is the knife
  • Purpose: for cutting tough tissue blocks which may offer great resistance to the knife
  • Also used to cut tissues embedded in all forms of media and for cutting resin embedded decalcified tissues
• Standard Sliding microtome


  • Most dangerous because the movable part is the knife and the one that remains fixed or stationary is the block holder
  • Knife is move backward and forward during sectioning
• Freezing Microtome / Cold Knife

  For cutting unembedded frozen sections, 10-15 um
• Used to cut

  • Undehydrated thin to semi-thin sections of fresh, frozen tissues
• For demonstration of

  • Fats and other neurological structures
• Also used to cut

  • Tissues with heat sensitive structure
• Cutting process


  The knife is moved whilst the tissue block remains static, same as sliding microtome
• Freezing agents

  • Liquefied nitrogen
  • Isopentane cooled by liquid nitrogen
  • Freon 2.2
  • Carbon dioxide
  • Dry ice
  • Aerosol sprays (cryokwik)
• Liquefied nitrogen


  Generally used in histochemistry, Intra-operative diagnosis, and most rapid. Main disadvantage is soft tissues is liable to crack due to rapid expansion (Ice crystallization inside the tissue)
• Isopentane cooled by liquid nitrogen


  Liquid at room temperature (RT). Tissue is frozen, affixed on a cork disc, aluminum foil or cryostat chuck
• Freon 2.2


  High thermal conductivity
• Carbon dioxide

  At -50 Celsius
• Aerosol sprays (cryokwik)


  For small tissues except the muscle. Fluorinated Hydrocarbons (Cryokwik) – rapid freezing of any tissues
• Cryostat / Cold Microtome (3-5 um; or 4 um in Gregorios)

  • maintained at temp between -5 to -30C
• Ultrathin Microtome (0.5 – 1 um)

  • Purpose: for cutting sections for Electron Microscopy.

  • Equipped with a glass or gem grade diamond knife is used to cut

  very thin sections (typically 60 to 100 nanometer) of tissue

  embedded in epoxy resin.

  • Also used with its glass knife or an industrial grade diamond knife

  to cut semi-thin sections prior to thin sectioning.

  • Thin sectioning for the TEM is often done with a gem quality diamond knife.
• Cold Knife Procedure

  1. Requires freezing microtome

  2. Tissue block thickness is 3-5 mm

  3. Immediately harden tissues to facilitate immediate cutting

  4. Dew Line – point where the tissues may be cut at 10 um
• Cold Microtome Procedure/Cryostat method

  Requires a cryostat
• Plane-Concave Knife


  • One side of the knife is flat while the other is concave
  • Less concave sides are recommended for cutting celloidin-embedded tissue blocks on a sliding microtome
  • More concave sides are used to cut paraffin sections on base-sledge, rotary or rocking microtome
• Biconcave Knife


  With both sides concave, recommended for cutting paraffin-embedded sections on a rotary microtome
• Plane-Wedge Knife


  Have both sides straight, recommended for frozen sections or for cutting extremely hard and tough specimens embedded in paraffin blocks, using a base sledge type or sliding microtome
• Bevel Angle


  The angle formed between the cutting edges, normally about 27° to 32°
• Good cutting edge


  • Made of good quality steel
  • Able to cut good sections from a paraffin wax block about 2-3 microns thick, without any serration noted on examination
  • Too soft cutting edges are likely to become dull easily
  • Too hard edges are likely to produce nicks or jagged edges and irregularities on the knife edge, thereby producing tears or striation on the tissue sections during cutting
• Clearance angle/Tilt Angle/ Inclination Angle


  • Angle formed between the surface of the block and the cutting edge of the knife
  • The perfect and optimum cutting angle is 15°, causing maximum penetration of the tissues and minimizing distortion
  • To prevent uneven sections, or alternate thin and thick sections, the knife should be inclined with a 5-10° clearance angle from the cutting plane
• Wedge angle


  Angle formed by the sides of the wedge knife set at 14-15°
• In board exams, 5-10° is the actual angulation used
• The knife or blade should be removed from the microtome when the instrument is left unattended or when cleaning the instrument