SECTIONING

• Sectioning

  Also termed as "Microtomy". Process by which tissue block is trimmed and cut into uniformed thin slices known as "sections" to facilitate microscopy.

  
• Types of Tissue Sections

  • Paraffin Sections = 4 – 6 μm
  • Celloidin Sections = 10 – 15 μm
  • Frozen Sections (Cryostat) = 4 μm
• Microtome


  Cutting engine in histopathology. Cuts sections at a predetermined thickness by sliding the block into a cutting tool attached to the machine.

  
• Parts of Microtome

  • Block Holder
  • Knife Carrier and Knife
  • Pawl, Ratchet Feed Wheel, & Adjustment Screws
• Principle of Microtome

  Spring-balanced teeth (pawl) is brought into contact and turns a ratchet feed wheel (connected to a micrometer screw), which is rotated, moving the tissue block at a predetermined distance towards the knife for cutting sections.
• Rocking Microtome (Cambridge)


  • Most SIMPLE type of microtome
  • Invented by Paldwell Trefall (1881)
  • Thickness of sections = 10 – 12 μm
  • Used to cut small and large blocks of paraffin tissue
  • NOT recommended for serial sections
  • Tissues are cut in slightly curved planes
  • Restrictions in sizes of the tissue block that can be cut
  • Difficulty of reorienting the block

  
• Rotary Microtome


  • Most COMMON type of microtome
  • Invented by Minot (1885 – 1886)
  • Purpose: Cutting of paraffin embedded tissues
  • Thickness of sections = 4 – 6 μm
  • Knife is fixed in horizontal position
  • Knife is placed in a blade-up position (relatively dangerous)
  • Heavier knife is used, so there is less vibration
  • Cutting angle of knife is adjustable hence, it can cut harder tissues
  • It can cut celloidin embedded sections by using special holder to set the knife obliquely
  • Heavier and more stable and complex than Rocking Microtome

  
• Sliding Microtome

  • Most DANGEROUS type of microtome
  • Invented by Adams (1789)
  • Purpose: Cutting of celloidin embedded tissues
  • Thickness of sections = 7 – 9 μm
  • Base Sledge: Less dangerous than Standard Sliding, block holder can be moved back and forth, knife is stationary but angle is adjustable, knife is long (24 cm) hence requires less honing, favored in laboratories where hard tissue and large blocks are sectioned, suited for sectioning specimens embedded in all forms of media
  • Standard Sliding: Most dangerous type, knife can be moved back and forth, block holder is stationary, it is difficult to attach knife guards
• Ultrathin Microtome


  • Purpose: Cutting of sections for Electron Microscopy
  • Thickness of sections = 0.5 – 1 μm
  • Equipped with a glass knife or gem grade diamond knife
  • Used to cut very thin sections of tissue embedded in epoxy resin
  • Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a TEM
  • Tissues are usually embedded in plastic

  
• Types of Sections

  • Paraffin Section
  • Celloidin Section
  • Frozen Section
• Frozen Section


  Used for rapid diagnosis (patient is still in the O.R.), enzyme histochemistry, demonstration of soluble substances, immunofluorescent and immunocytochemical staining, specialized silver stains
• Methods of Preparing Frozen Section

  1. Cold Knife Procedure: Freezing microtome is required

  2. Cold Microtome Procedure (Cryostat Method): Cryostat is required
• Freezing Microtome

  • Invented by Queckette (1848)
  • Purpose: Cutting of frozen sections, demonstration of fats and neurologcal structures, cut tissues with heat-sensitive structures, cut undehydrated sections for rapid diagnosis
  • Thickness of sections = 10 – 15 μm
  • Freezing Agent: Rapid, intermittent burst of CO2
  • Second cooling device for lowering temperature of knife is also incorporated in most machines
  • Dew Line: Point in which section may be cut at 10 μm
• Cold Microtome (Cryostat)


  • Cryostat is a refrigerated apparatus, usually Rotary Microtome
  • Temperature of the cold chamber immediately hardens tissues to facilitate immediate cutting
  • Capable of freezing fresh tissues within 2 – 3 mins
  • Purpose: Used in fresh tissue microtomy, for preparing thin sections of fresh frozen tissues for fluorescent antibody staining, or histochemical enzyme studies, most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis
  • Temperature = – 5 to – 30°C, average = – 20°C
  • Thickness of sections = 4 μm
  • Cryostat is often housed in the frozen section room, close to the operating room to allow direct consultation between surgeon and pathologist
• Both Freezing Microtome and Cryostat immediately hardens tissues to facilitate immediate cutting
• Microtome Comparison

  • Rocking: 10 – 12 μm, Serial sections of large blocks
  • Rotary: 4 – 6 μm, Paraffin blocks
  • Base – Sledge: 7 – 9 μm, Celloidin blocks
  • Standard Sliding
  • Ultrathin: 0.5 – 1 μm, Sections for EM
  • Freezing: 10 – 15 μm, Frozen sections, Fats and neurological structures, Heat-sensitive structures, Undehydrated structures
  • Cryostat: 4 μm, Frozen sections, Fluorescent antibody staining, Histochemical enzyme studies, Urgent biopsies for intraoperative diagnosis
• Care of the Microtome

  • Microtome should be placed on a stable bench, away from air drafts, doorways, and passing staff
  • After sectioning, all accumulated paraffin and small pieces of tissues must be brushed away with a soft brush
  • Microtome MUST ALWAYS be covered when not in use
  • After drying the machine and knife holder, the parts should be wiped with xylol (xylene)
  • Prolonged and continuous application of the painted parts with xylene should be AVOIDED
  • Movable parts should be oiled thoroughly to prevent rusting
  • ALWAYS REMOVE the knife or blade before cleaning
  • NO FLUID MUST ENTER the inside of the instrument when cleaning
  • When cleaning the blade, avoid dragging anything along the cutting edge

  
• Safety Measures

  • Staff should not be distracted when using microtome due to risks of injury from extremely sharp blades
  • Non-slip flooring in the vicinity of the microtomes is preferable
  • Use forceps or brush NOT FINGERS in picking up sections or wax fragments from blade or block face
  • Use hand wheel lock when changing blocks
  • Knife or blade should be removed from the microtome when the instrument is left unattended or when cleaning the instrument
  • Used blades should be disposed in "sharps" or "used blades"
  • NEVER place a knife or blade on the bench or in a box with cutting edge FACING UP
• Microtome Knives

  • Plane-concave: 1 side flat, 1 side concave, cuts celloidin embedded tissues on flat side, cuts paraffinembedded tissues on concave side (25mm)
  • Biconcave: Both sides concave, recommended for cutting paraffin embedded tissues on rotary microtome (120mm)
  • Plane-wedge: Both sides flat (straight), recommended for frozen sections and extremely hard and tough specimen embedded in paraffin blocks (100 mm)

  
• Cutting Facet (Bevel)


  Found on the tapered edge of all knives, sides are more acutely inclined towards each other, forming the actual cutting edge of all knives
• Bevel Angle


  Angle formed between the cutting edges, normal angle 27 – 32°, maintained by means of slide-on back, a spring-loaded semi-circular metal sheet slipped onto the knife
• Clearance Angle


  Angle formed between the surface of the block and the cutting edge of the knife
• Wedge Angle


  Angle formed by the sides of the wedge knife
• Good Cutting Edges


  • Should be made of good quality steel
  • Must be able to cut good sections from a paraffin wax block about 2 – 3 μ thick (or 4 μ) without causing any serration noted on examination
• Bevel angle


  Angle formed between the cutting edges
• Cutting facet (bevel)


  • Found on the tapered edge of all knives
  • Sides are more acutely inclined towards each other, forming the actual cutting edge of all knives
• Normal bevel angle

  27 - 32°
• Maintaining bevel angle

  1. Slide-on back, a spring-loaded semi-circular metal sheet slipped onto the knife

  2. Holds the cutting edge at a constant, correct angle
• Each knife should have its own corresponding back which SHOULD NOT be interchanged with another, to keep the bevel angle
• Good cutting edges

  • Made of good quality steel
  • Must be able to cut good sections from a paraffin wax block about 2 - 3 μ thick (or 4 μ) without causing any serration
  • Cutting edge must be thinner than the section being cut
  • Too soft cutting edges are likely to become dull easily
  • Too hard cutting edges are likely to produce nicks or jagged edges and irregularities on the knife thereby producing tears or striation on the tissue sections during cutting
• Safety razor blades


  • May be used for partially calcified materials, paraffin, and frozen sections
  • Unsatisfactory for sections less than 10 μ
• Perfect and optimum cutting angle

  • Sides of the wedge knife are inclined at an angle of 15°
  • Maximum penetration of the tissues
  • Minimizes distortion
• Knife inclination

  • 5 - 10° clearance angle from the cutting plane
  • To prevent uneven sections, or alternate thin and thick sections
  • So that cutting facet (bevel) will not compress the block during the process of cutting
• Badly nicked knives with blunted ends have to undergo sharpening
• Ensures optimum sectioning of tissue blocks

  
• Prevents gross irregularities on tissue sections
• Jagged ends, if not corrected, will produce tears or striations in tissue sections
• Honing

  • Hard sharpening
  • Involves coarse honing to remove gross nicks and honing proper to grind the cutting edge of the knife on a stone to acquire an even edge
• Hone


  A natural sharpening stone or hard grinding surface that serves to remove nicks and irregularities on the knife edges
• Types of hones (sharpeners)

  • Belgium yellow
  • Arkansas
  • Fine carborundum