MICROTOMYPARAFFIN & FROZEN SECTIONS

Prepared by: Ms. BR Tsauses

Lecturer: Anatomical

Pathology 2A (ANP611S)

9 March 2020

OBJECTIVE

Basic principles of microtomy are applicable to both paraffin and frozen sections.

Describe the techniques necessary to provide quality microscope slides for clinical and research histology.

MICROTOMY

Microtomy – tissue sectioning and attached it to a surface for microscopic examination

Microtomy is performed on paraffin wax embedded tissue blocks.

The Instrument used in Microtomy is called a microtome, derived from Greek word “mikros” meaning small.

The instrument uses an advancing mechanism which moves the paraffin block for a predetermined distance until it comes into contact with the cutting blade (knife).

The specimen moves vertically past the cutting surface and a tissue is produced.

Good technique is achieved through on-going practice.

TYPES OF MICROTOME

Rotary microtome

Base sledge microtome

Rotary rocking microtome

Sliding microtome

Ultra microtome

ROCKING MICROTOME

Name derived from rocking action of the cross arm.

Oldest in design, cheap, simple to use.

ROTARY MICROTOME

Types: manual, automated, fully automated

MANUAL ROTARY MICROTOME

SEMI-AUTOMATED ROTARY MICROTOME

FULLY AUTOMATED ROTARY MICROTOME

PARTS OF ROTARY MICROTOME

KNIFE HOLDER BASE

Anchors knife holder to microtome stage. Must be stationary and locked during microtomy.

COARSE HANDWHEEL

Moves block holder either towards knife or away from knife.

MICRON ADJUSTMENT

Micron settings for section thickness from 1 – 60 microns on most microtomes.

ADVANCEMENT WHEEL

Turns in one direction and advances the block towards the knife at specified microns.

SAFETY LOCK

Safety locks – prevent wheel from releasing and having the block holder come down towards the blade while a block is inserted or removed.

BASE SLEDGE MICROTOME

Commonly used in neuropathology or ophthalmic pathology.

Originally designed to cut sections of very hard tissue or large tissues e.g. whole brain.

SLIDING MICROTOME

Designed mainly for cutting celloidin embedded blocks of tissue.

Celloidin: A semisolid solution of pyroxylin in ether and alcohol. Used to embed specimens for microscopy before they are sectioned and placed on slides. A specimen embedded in celloidin.

ULTRA MICROTOME

Used exclusively for electron microscopy.

Prepares ultra thin sections.

CRYOSTAT MICROTOME (CRYOTOME)

Cutting sections of frozen tissue

MICROTOME KNIVES

Disposable steel blades:

Disposable blades have a sharp cutting edge that can produce 2-4 microns sections.

Glass and diamond knives:

Used in EM for resin embedded blocks

Blades are incorporated into the microtome

Blades are sometimes coated with polytetrafluoroethylene (PTFE) which allows ribbons to be sectioned easily.

DISPOSABLE BLADES

Manufactured from high quality stainless steel, has different grades according to thickness.

SETTING UP THE MICROTOME

The blade should be adjusted to optimize the clearance angle, to eliminate that occur when ribboning of the tissue

Clearance angle – the distance between the lower facet angle and the surface of the block face.

Do not over-tightening the blade in the clamping device

PARAFFIN SECTION CUTTING –MATERIALS REQUIRED

1. Flotation (water) bath, thermostatically controlled

2. Slides, 76x25 mm (1.0-1.2mm thick)

3. Slide drying oven

4. Fine pointed/curved forceps

5. Camel haired brush

6. Slide rack

7. Ice tray

8. Chemical resistant pencil or pen

SECTION ADHESIVES

Sections tend to detach from the slides during staining, adhesives are employed to keep the section attached to the slides.

Adhesives:

Poly-L-lysine ,

3-aminopropyltriethoxysilane (APES),

Charged/ plus slides:

Have permanent positive charge, which is resistant to cell and tissue loss during staining.

SECTIONING

First step, trimming the tissue blocks

Second step, cutting sections

Third step, float out sections

Fourth step, dry sections

FROZEN SECTIONS

Frozen sections are required for some urgent and specialized histopathological test.

Stained sections produced within few minutes by the simple act of freezing

Frozen section can be reported within 10 minutes

Results are communicated to the surgeon within a short period

USE OF FROZEN SECTIONS

Rapid production of sections for intra-operative diagnosis

Diagnosis and research enzyme histochemistry

Direct immunofluorescence requires fresh tissue to diagnose blistering diseases of the skin and in renal pathology

Diagnostic and research non-enzyme Histochemistry e.g lipids and carbohydrates

Silver demonstration in neuropathology

Fresh tissue for microbiology culture and sensitivity

Cytogenetic test –analysing the morphology of chromosomes

FREEZING OF FRESH (UNFIXED) TISSUE

Tissue for freezing should be fresh.

Specimen need to be frozen as rapidly as possible without creating freezing artefacts

Chemicals used;

liquid nitrogen (-190 °C),

Dry ice (-70 °C),

carbon dioxide gas (-70 °C)

isopentane cooled by liquid nitrogen (-150 °C)

The method of choice is isopentane and liquid nitrogen

FROZEN TISSUE SECTIONING

Specialized equipment called a cryostat is used to section frozen sections

Sections are cut temperatures of around -20°C

When tissue is frozen, the interstitial water in the tissue turns to ice, ice act as the embedding medium

Non-fatty section unfixed tissues section well at -25°C

Coated or charged slides should be used

CRYOSTAT

The cryostat: a refrigerated cabinet in which a special microtome is contained

Electronic temperature control

Automatic defrost mechanism

Cryoembedding medium

Requires decontamination and sterilization

RAPID BIOPSY FOR INTRA-OPERATIVE DIAGNOSIS

Frozen sections provide a valuable tool in rapid diagnosis of tissues during surgery.

Pathologist selects a piece of tissue, the tissue is frozen using one of several techniques (discussed).

Frozen cut tissue sections are immediately submerged in cold acetone or 95% alcohol

Sections are stained immediately by rapid haematoxylin and eosin (H&E) stain or methylene blue

FREEZE DRYING VS FREEZE SUBSTITUTION

Freeze drying and freeze substitution are dehydrating techniques which the water is gently removed from a frozen tissue.

Freeze drying-Rapid-freezing of fresh tissue at -160 °C, and subsequent removal of water molecules by sublimation in a vacuum at a higher temperature (-40 °C)

Freeze substitution dissolves the ice in the frozen tissue specimen by using an organic solvent and the technique is conducted at low temperature

TROUBLESHOOTING

TROUBLESHOOTING

TROUBLESHOOTING

TROUBLESHOOTING

TROUBLESHOOTING

TROUBLESHOOTING

REFERENCES

Suvarna, S. K., Layton, C., Bancroft, J.D. 2013. Bancroft’s Theory and practice of Histological techniques, Expert consult . 7th Edition. Churchill Livingstone, Elsevier.

Leica Microsystems. Instruction Manual Leica RM2235 V1.3 Nussloch: Leica Microsystems, 2008.

Rolls GO, Farmer NJ, Hall JB. Artifacts in Histological and Cytological Preparations. Melbourne: Leica Microsystems, 2008;106.

Rolls G. 101 Steps to Better Histology. Melbourne: Leica Microsystems, 2008.

Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. London: Butterworths, 1985.

Santoianni RA, Hammami A. Nuclear Bubbling an Overlooked Artifact. The Journal of Histotechnology, 1990;13;135-136.