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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.