HISTOPATH SECTIONING

Microtomy / Sectioning

The technique of cutting thin sections of tissues for microscopic examination.

Paraffin Sections

Tissue sections that are 4-6 micrometers thick.

Celloidin Sections

Tissue sections that are 10-15 micrometers thick.

Frozen Sections

• Tissue sections that are 4 micrometers thick, prepared using a cryostat.

• For Rapid Diagnosis (in less than 15 mins)

• For enzyme histochemistry and demonstration of soluble substance.

• Immunofluorescent & immunocytochemical staining ; Specialized silver stains

Method to obtain frozen sections

Cold knife procedure (freezing microtome)

Tissue block – 3-5 mm

Dew Line – point in which section may be cut at 10 um

Cryostat (Cold microtome method)

Liquid Nitrogen

Generally used in histochemistry and the most rapid.

Isopentane

Freezing agent that is liquid at RT. (Cooled by liquid nitrogen)

Aerosol spray

also known as cryokwik

Freon 2.2

freezing agent with high thermal conductivity

Honing

The process of sharpening the cutting edge of a microtome knife.

To remove gross NICKS.

Hone or Oil Stones

Honing is carried out using what?

Belgium yellow

Gives the best result of honing.

Arkansas

More polishing effect than Belgium yellow.

Fine Carborundum

Recommended for badly nicked knives.

Glass plate honing

With finely powdered aluminum oxide made into paste with water = abrasive

Diamantine

To be used for final polishing.

Heel-to-Toe Movement where the edge is the first

Honing is carried out at what motion?

20-30 strokes

The Minimum Required number of strokes in Honing.

Depends on the condition of Knife*

Stropping

The process of polishing and sharpening the cutting edge of a microtome knife.

To remove BURRS.

Horse Leather or Leather strop

Carried out using paddle strop made of ?

• Treated with oil prior to use.

• DO NOT USE MINERAL OIL!!

Toe-to-Heel with the edge is the Last.

Stropping is carried out at what motion?

40-120 double strokes

The Minimum Required number of strokes in Stropping.

Considerations in Honing and Stropping

• Stropping may be carried out without prior honing.

• Stropping must be done after the process of honing.

• Disposable blades will not require honing and stropping.
  

Deparaffinization

The process of removing excess paraffin wax from tissues on a slide.

Methods of Deparaffination

• Passing the slide over a flame using alcohol lamp

• Immersion of slides in Xylene

• Placing the slides inside the oven (55-60 degC)

Fishing out

The removal of tissue ribbons from a float out bath during microtomy.

Orientation

Process of placing ribbon in precise position.

Floatation water bath

• It is a thermostatically controlled bath used to remove wrinkles and folds; to flatten the ribbons.

• Temperature when in use is 6-10 degC lower than the wax melting point; between 45-50 degC

Drying of slides

• Leaving slides in a 37°C incubator overnight

• Placing an oven 50-60°C for 2 hours

• Drying using a hot plate at 45–55 °C for 30–45 minutes

Adhesives

Promote attachment of (ribbons) tissues to the slide, and prevent detachment.

Mayer’s egg albumin

Routine Tissue Adhesive; a combination of equal amounts of egg white and glycerine + thymol crystals (to prevent growth of molds)

Poly-L-Lysine

Adhesive recommended for Immunohistochemistry

APES (3-aminopropyltrhiethoxysilane)

Adhesive very useful for Cytology

Sodium silicate

commercial syrup 1:10 and dilution with strong adhesive property.

• Dried Albumin

• 1% Gelatin

• Gelatin- Formaldehyde mixture

• Starch Paste

• Plasma

Other Adhesives used

Block Holder/ CHUCK

where the Tissue block is held in position.

Rotating wheel

mechanically used / moved / manipulate to start the cutting process.

Pawl, ratchet feed wheel & adjustment crew

used to line up the block in the proper position with a knife.

Rocking Microtome / Cambridge

• Most simple Microtome

• Preparing serial sections of large paraffin blocks.

Trefall

Inventor of Rocking Microtome

10-12 um

Thickness of sections in Rocking Microtome

• Difficulty in re-orienting (repositioning) the block,

• Restriction in the size of block that can be cut (standard size of tissue holder, little tissue)

Disadvantage of Rocking Microtome

Rotary Microtome

• most common type

• Heavier, therefore more stable

• To cut paraffin embedded tissues.

4-6 um

Thickness of sections in Rotary Microtome

Minot

Inventor of Rotary Microtome

Sliding Microtome

• Most dangerous (Exposed knife)

• To cut celloidin-embedded tissues

• To cut extreme hard/tough tissues

7-9 um

Thickness of section in sliding Microtome

Adams

Inventor of Sliding Microtome

Base Sledge

Less dangerous because the movable part is block holder, the one that remains stationary is the knife.

for tough and large sections.

Standard Sliding

Most dangerous because the movable part is the knife, the one that remains stationary is the block holder.

For Celloidin-embedded tissues.

Freezing Microtome

• To cut tissues with heat sensitive structure

• For demonstration of fats and other neurological structures.

• Uses an intermittent burst of CO2 to freeze the block holder and tissue, along with a second cooling device for lowering the temperature of the knife (which will contribute to the immediate hardening of tissues).

CO2

Used as a freezing agent to immediately harden fresh tissues in order to facilitate immediate cutting.

Often used while using freezing microtome

Queckette

Inventor of Freezing Microtome

10-15 um

Thickness of sections in the freezing microtome.

Cryostat or cold Microtome

• A refrigerated apparatus (microtome enclosed in cold chamber)

• For fresh microtomy

• For PREPARING THIN SECTIONS OF FRESH FROZEN TISSUES (no Fixation, dehydration, clearing) for fluorescent antibody staining or histochemical enzyme studies

-5 to -30C (averagely at 20C)

fresh tissue microtomy is refrigerated at _______?

Ultrathin Microtome

• For cutting tissue for Electron Microscopy

• Usually embedded in plastic.

0.5 um

Thickness of section in the ultrathin microtome.

Plane Concave

• A 25-mm-long double-purpose knife.

• one side flat (celloidin-embedded tissue)

• one side concave (Paraffin-embedded tissues)

Biconcave

• 120mm long, longest knife.

• Both sides are concave for paraffin-embedded tissue.

Plane wedge

• Usually 100mm long

• both sides are flat for frozen sections and extremely hard and tough tissues.

Bevel angle (27-32 deg)

Angle formed between the cutting edges.

Clearance angle (5-10 deg)

Angle formed between the surface of the block and the cutting edge of the knife.

Wedge angle (15 deg)

Angle formed by the sides of the wedge knife.

Disposable blades

• Cheaper and conventional knife

• 2-4 um

Glass knives

• trimming and semi-thin sectioning for EM

• can be used for Infiltrating Eye Specimen.

Diamond knives

• For resin block for EM

• more expensive but durable.