Why is dehydrating & clearing tissue specimens important?
it is necessary to prepare the tissue for infiltration and embedding in a non-aqueous medium (usually paraffin)
* Dehydration allows paraffin infiltration & prepares tissues for sectioning by removing water from tissues
* Clearing makes tissues receptive to infiltration by removing dehydrating agents
* also improves tissue transparency & enhances light penetration
What are properties of a Closed (fluid transfer) Tissue Processor?
- hold tissues stationary & pumps reagents in and out
- heat & vacuum are available if needed
- less choice of fixatives
- cannot use mercury or dichromate containing fixatives
- has less health hazards as volatile reagents aren’t open to the air
What chemicals are used for clearing tissue specimens?
Aromatic hydrocarbons
* xylene (most common), toluene (for EM), benzene, chloroform, aniline oil
Universal solvents
* dioxane, tertiary butanol, tetrahydrofuran
Xylene substitutes
* Limonene reagents, Aliphatic hydrocarbons (alkanes)
Acetone
Others
* petroleum ether, methyl salicylate (wintergreen oil), cedar wood oil, carbon tetrachloride, clove oil
What are properties of a Open (tissue transfer) Tissue Processor?
- tissues are transferred from 1 solution to the next
- volatile reagents are hard to keep at appropriate levels due to offgassing
- poses a healthough & fire risk as the reagents are open to the air
- allows for a wider choice of fixatives
- heat & vacuum are available
What are Epoxy Resins used for?
- as an alternatice to paraffin
- Epoxy resins are the required embedding media for electron microscopy & ultrastructural examination
- allows for very thin sections to be obtained (60-90nm)
- sections are cut with a diamond knife
What is Celloidin?
Where is it used?
What is a disadvantage?
- An alternative to paraffin
- generic term for nitrocellulose
- Used in research & neuropath labs
- Very time consuming process & dangerous
- andyrous ether & nitrocellulose are explosives
What is the purpose of decalcification?
to remove calcium from bones & bony tissue in preparation for infiltration & sectioning
What are some differences in standard vs short processing schedules?
- short processing time (1-4hrs)
- routine processing time (8-18+ hrs, usually overnight)
- short schedules
- usues shorter steps & stronger/faster reagents
- is used for urgent speciments or biopsies
What are properties of paraffin based on its melting point?
High melting point
* paraffin contains more polymers & becomes harder
* provides better support for tissues
* thinner sections are easier to obtain but ribboning becomes harder
Low melting point
* wax is softer & provides less support
* thin sections are difficult to obtain & ribboning becomes easier
What is Glycol methacylate (GMA)?
What is it used for?
What are disadvantages of using it?
- an alternative to paraffin
- acrylic resin, miscible in water
- provides excellent support for hard tissues
- bone marrow, undecalcified bone, kidney & lymph node biopsies
- allows very thin sections (1-2 microns) to be cut
- glass knives must be used to cut sections
- sections do not adhere well to glass slides & staining is difficult
- GMA chemicals are hazardours & fume hood must be used
What are the uses of frozen sections?
- done prior to fixation
- performed when rapid diagnosis is needed
- used when staining techniques will not work for routinely processes tissue
- ex. demonstration of fat
- used for many enzymes & IHC techniques
- fixation & the heat involves in processing inactivates most enzymes & some antigens
- IHC is unsatisfactory if tissue has been fixed & processed routinely
What is Carbowax?
How is it used?
What is a disadvantage of using it?
- an alternative to paraffin
- infiltrates tissue directly after aqueous fixation
- dehydration & clearing are not required
- fat can be demonstrated on section in small amounts
- due to not being dissolved through dehydration
- requires long impregnation time for CNS tissues
- embedded tissue sections tend to dissolve when placed in waterbath
What is infiltration?
- the permeation of tissue with a supporting medium
- holds celss & intercellular structures in their proper relationship for sectioning
When is paraffin unsuitable to use as an infiltration medium?
When
* processing reagents remove or destroy tissue components that are the object of investigation
* sections are required to be thinner than normal
* the use of heat may adversely affect the tissue
* the infiltration medium (paraffin) may not be sufficiently hard to support the tissue
What are the ways to test the completion of decalcification?
1. Physical test
* bending, probing, or trimmin of the specimen. Can cause damage or artifacts
2. Chemical (calcium oxalate) test
* discarded decalcification fluid is neutralized and ammonium oxalate is added and allowed to stand for 30 min.
* turbidity= presence of calcium, clear= complete decalcification
3. Radiography
* xray taken of the specimen to get visual confirmation of decalcification
Why should low grade alcohols be used after formalin?
- Prevents osmotic shock which can disrupt or damage delicate tissue structures & cause substantial tissue shrinkage
- Prevents precipitation of phosphate salts into tissues from phosphate buffered fixatives which can cause difficulty with microtomy
What are the additives for paraffin and their properties?
1. beeswax
* reduces crystal size, increases stickiness & adhesion
2. rubber
* reduces brittleness, increases stickiness & makes the formation of ribbons easier
3. other waxes
* produces smooth texture & smaller crystal size
4. plastics/polymers
* increases hardness & support
What chemicals are used for dehydration?
Alcohols
1. ethyl (best)
2. methyl
3. isopropyl
4. butyl
Acetone (for fatty tissue)
Universal Solvents
1. dioxane
2. tertiary butanol
3. tetrahydrofuran
Cellosolve (ethylene glycol or poly ethylene glycol monoethyl ether
[PEG] )
What is tissue clearing?
removal of the alcohol used for dehydration
makes tissues receptive to the infiltration medium
What is tissue dehydration?
the removal of aqueous fluids (water & fixatives) from the tissues
What is a universal solvent?
reagents which perform bothe the dehydration & clearing steps
Types of decalcification
Acid decalcification
* most widely used
* includes: nitric acid, HCl, Formic acid
Chelating agents
* substances that combine with Ca+ ions & other salts to form weakly dissociated complexes & facilitate the removal of calcium salt
* includes: EDTA
Ion Exchange Resin
Electrophoresis
* Ca+ ions move thru decalcification solution to cathode
Microwave
* tissues are places in decalcifying agent & placed in microwave
What are the effects of water being present in clearing agents?
- leads to incomplete clearing
- can leave tissues soft & non-receptive to infiltration
