DECALCIFICATION Flashcards
is the removal of calcium ions from the bone through histological process thereby making the bone flexible and easy for pathological investigation.
Decalcification
The removal of_____ deposits is essential for good embedding procedure as well as to obtain soft sections of the bone using the microtome.
calcium
is usually cared out between the fixation and dehydration. Bone must obviously be processed in this way, but other tissues may also contain calcified areas.
Decalcification
Strong mineral acids such as_______ and ______ are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. However, these strong acids also damage cellular morphology.
nitric and hydrochloric acids
Mineral acid decalcifiers are not recommended for delicate tissues such as______ because they are not as aggressive, organic acids such as_______ are better suited to bone marrow and other soft tissues.
bone marrow
acetic and formic acid
act more slowly than mineral acids, and will require extended treatments to decalcify cortical bone.
Organic acids
Formic acid in a ______ concentration is the best all-around decalcifier.
Some commercial solutions combine formic acid with formalin to fix and decalcify tissues at the same time.
10%
Chelating agents such as______ can remove calcium and is not as harsh as mineral or organic acids.
ethylenediaminetetracetic acid (EDTA)
The use of EDTA is limited by the fact that it penetrates tissue and works slowly so it is not suitable for urgent specimens.
It is used at a concentration of approximately_____ as a neutralized solution.
14%
The rate at which EDTA will decalcify is_____ dependent.
It is generally used at_____. It works more rapidly at____ but some tissue elements can be damaged at alkaline _____.
pH
pH7.0
pH10
pH10
Techniques For Increasing The Efficiency Of Decalcification
Microwave treatment
lon-exchange resins
Electrolytic decalcification or electrolysis
has been used with hydrochloric acid decalcifiers but the raised temperature may damage morphology and cause staining artefacts.
Microwave treatment
have been incorporated into some decalcification protocols.
They are added to the container holding the decalcifier and take up the ionized calcium maintaining the effectiveness of the acid.
If acid decalcifiers are used in adequate volumes and replaced regularly the use of such resins is probably unnecessary.
lon-exchange resins
is done by placing the bone in acid decalcifier and attached to an electrode through which current is applied.
Experimental situations showed calcium was removed with the least tissue damage.
However, it has not found wide acceptance because of the potential to cause heat damage to the specimen.
It is a slow process and not suited for routine daily use.
Electrolytic decalcification or electrolysis
Factors Influencing The Rate Of Decalcification
• Concentration and Volume
• Temperature.
• Agitation.
• Tissue Size and Consistency.
The_____ of active agent will affect the rate at which calcium is removed and so does the____ of the decalcifying agent.
concentration
volume
It must be remembered that the concentration of active agent and volume of the decalcifying agent will be depleted as it combines with calcium and so it is wise to use a large volume of decalcifying agent and renew it several times during the decalcification process.
The recommended ratio of fluid to tissue is____
20:1
Increased temperature will______ the decalcification rate but will also increase the rate of_____ so must be employed with great care.
speed up
tissue damage
Gentle______ may increase the rate slightly by slowly dislodging debri or precipitates from the tissue thereby influencing fluid exchange from the tissue into the fluid and vice versa.
agitation
Ideal decalcification time is about______
Larger tissues need longer decalcification periods.
Dense tissues usually require_____ or longer to decalcify.
24-48 hours.
2 weeks
Measuring Extent Of Decalcification
X-ray
Chemical test
Physical test
Incomplete decalcification can lead to
Tissue distortions
Over decalcification causes problems with
Staining
Best method in determining wether decalcification is complete
XRAY
Will reveal tiny residual calcium deposits and allow further treatment if required
Xray
: can be applied when some acid decalcifiers are used (particularly formic acid).
______is added to a sample of the final change of decalcified that has been neutralized with ammonium hydroxide.
If calcium is present a precipitate of _____will form indicating that decalcification is probably incomplete and a longer time in decalcifying agent is required.
Ammonium oxalate solution
calcium oxalate
: require manipulation, bending probing or trimming of the specimen to “feel” for remaining calcified areas.
While this method may be successful in experienced hands it is generally considered to be unreliable.
PHYSICAL TEST
- Place the pieces of calcified tissues into a____ beaker filled with______
- Label the beaker with your group number and section. Present the container to the laboratory stockroom for storage until the process is completed.
- Allow the tissues to decalcify for_____
500 ml; 10% nitric acid.
12-24 hours.
Testing Extent of Decalcification:
- Remove_____ of used decalcification fluid from the tissue processing vessel
- Add__________ drop by drop until the pH of the solution is neutral to pH paper.
- Add____ of saturated_______ and shake well
- Allow to stand at room temperature for_____
5 ml
ammonium hydroxide
5ml; ammonium oxalate
30 minutes.
Interpretation of Results
Formation of a precipitate after the addition of_____ indicates the presence of substantial amounts of calcium in the spent fluid. The fluid should be changed and the tissue further decalcified.
Formation of a precipitate after ________addition shows less calcium. If the solution remains clear for 30 minutes after addition of the ____, the tissue is essentially calcium free.
ammonium hydroxide
ammonium oxalate
