DECALCIFICATION, WHICH DEALS WITH THE COMPLETE REMOVAL OF CALCIUM SALT FROM A BONY AND DECALCIFIED TISSUE.

1. DECALCIFICATION AND TESTING FOR END POINT OF DECALCIFICATION
BY
DR. A. B. AJILEYE

2. Introduction
• Decalcification is a process whereby calcium ion or salts are completely removed from a bony or
calcified tissue. The deposition of calcium salts {calcium phosphate, calcium carbonate and
calcium fluorites}, usually makes the cutting of fine sections by the usual methods very difficult.
The most suitable method of decalcification depends on the strength, temperature and volume
of the decalcifying solution as well as on the size, consistency of the tissue and the type of
investigation to be carried out.
• Calcium salts occur normally in bones and in teeth, or in some pathological conditions. There are
two types of pathological calcification:
• The first form is referred to as dystrophic calcification in which the calcium salts are deposited in
the cells or surrounding tissues which have been damaged or injured by disease, e.g
Tuberculosis or Cancerous changes.
• The second form of calcification is seen in other lesions and may occur as a result of the
pathological processes; it is referred to as metastatic deposition. For example, in
hyperthyroidism, there is a change in calcium metabolism causing an increase in blood calcium
level.

3. • A good decalcifying agent should remove all calcium without any adverse effect on the cells or
tissue fibres and with no impairment of subsequent staining or impregnation.
• Conditions Which Increase Rate of Decalcification
Concentration of acid: the higher the concentration of acid, the faster the rate of decalcification
of tissue but with a concomitant damage to cell structures.
Heat: high temperatures sometimes up to 60°C in an oven tremendously increase the rate at
which decalcification of tissues in an acid solution takes place.
• General Methods of carrying out Decalcification
Ion-Exchange Resins
Electrolytic Decalcification
Use of mineral Acid Decalcification
Surface Decalcification

4. • Ion-Exchange Resins: This involves, the incorporation of an ion exchange resin {an
ammonium form of polysterene resin} into the decalcifying solution has been claimed to
speed up the process of decalcification and to improve staining. The principle is that the
resin removes the calcium from the decalcifying fluid thereby increasing the rate at which
calcium salt is dissolved from the bony tissue into the resin. The dismerit of this method is
that you cannot test for end point of decalcification with chemical method. The end point of
decalcification for this method is best achieved using X-ray method
• Electrolytic Decalcification: This is a process whereby hydrochloric acid and formic acid are
used as electrolytic medium in an electrolytic bath for the decalcification of bony tissue. The
principle of this technique is based on the continuous passage of a 6 volt direct current
through the decalcifying fluid. The decalcifying fluid used is equal volumes of 8% HCL and
10% formic acid. The cathode is a brass or carbon plate while the anode is a platinum wire.
Calcium ions being positively charged are attracted towards the cathode. This keeps the
decalcifying fluid free of calcium ions, hence an increase in the rate of decalcification. Heat,
up to 45°C is generated during this process and contributes a great deal to the speed of
decalcification much more than the electrolysis itself.

5. • Surface Decalcificattion: sometimes during initial trimming of the block, unsuspected areas
of calcification become apparent through tears in the sections and resistance against the
knife. The block should be removed from the microtome and immersed for an hour or more
in either 10% formic acid or 5% nitric acid. Progressive decalcification will occur, with little
or no adverse effect on subsequent staining. The block should be washed briefly in water
dried and replaced in the microtome for cutting.
• Use of mineral Acid: this is carried out by suspending the tissue with the help of a waxed
thread in a beaker containing the calcifying fluid solution which is 10% nitric acid. The
thread used to suspend the tissue is waxed so as to avoid the effect (cutting) of the acid on
the thread. Suspension is necessary to enable the decalcifying fluid penetrate the bony
tissue easily from different angles. The end point of the decalcification can be tested at
interval, usually daily bases.

6. • CRITERIA OF A GOOD DECALCIFYING AGENT
• It should remove calcium ions completely
• It should not damage cells and tissues
• It should not impair subsequent histochemistry
• It should be reasonably fast
DECALCIFYING AGENTS
• NITRIC ACID: this is recommended for urgent biopsies, it decalcifies quickly but causes damage
to tissue and inhibit nuclear staining if its application is prolong. Because of this, it is used for
rapid decalcification of small piece of bone. It is used in a concentration of 5% to 10% in distilled
water. The time required for decalcification is 1 to 3 days
• FORMIC ACID: this is recommended for post-mortem and research tissue. The time required for
decalcification is 2 to 7 days. This decalcifying fluid permit excellent staining results and it is
regarded as being the best decalcifying solution for routine purposes for routine use, 8% formic
acid in distilled water is satisfactory.
• TRICHLORO ACETIC ACID: this is recommended for small pieces of delicate tissue which require
decalcification. The time necessary for decalcification is 4 to 5 days

7. • EBNER’S FLUID: the use of this fluid is particularly useful for decalcifying teeth and the time for
decalcification is 3 to 5 days
• ETHYLENE DIAMINE TETRA ACETIC ACID {E.D.T.A}: These are organic compounds which bind
metals. EDTA also called sequestrine or versene combines with calcium ions toform soluble
non-ionized compounds without destroying tissue structure. Prolong exposure to EDTA is
without detriment to the subsequent staining of tissues. Modified chemical techniques either
Allen and Rosen or Seilly can be used to determine the end point of decalcification.
• Other decalcifying fluids include;
Gooding and stewart;s fluid
Jenking’s fluid
Perenyi’s fluid
Smith’s fluid
Calex,
RDC.

8. • DETERMINATION OF END POINT OF DECALCIFICATION
• It is very important that end point of decalcification be determined regularly so that tissues do
not overstay in the decalcifying fluid. Excessive exposure of tissue to decalcifying fluid causes
disruption and loss of tissue constituents. Staining reactions are also impaired if tissue overstays
in the decalcifying fluid.
• There are 3 {three} major ways of determining the endpoint of decalcification of a tissue:
• X-ray method
• Needling and Palpation method
• Chemical method
• X-RAY: Tissues are put in compartment designed like the ice cube tray, where this is not
available; tissues are put on a simple hard paper and exposed to x-ray. The x-ray film is
developed and areas which appear clear and transparent indicate areas of calcium salts. This is
the best and most reliable method of determining the end point of decalcification. A major
disadvantage of x-ray method is that it cannot be used for tissues which have been fixed in radio
opaque solutions such as those which contain mercuric chloride.

9. • Other disadvantages of the x-ray method are
• The machine is expensive and has to be operated by a specialist
• Excessive exposure to x-ray causes leukemia
• NEEDLING AND PALPATION: Tissue is palpated and a long needle is passed through it as several
areas. If needle passes freely, calcium is assumed to be absent. During needling and palpation,
artefacts are introduced into the tissue and small spicules of bone are often missed out. It is
not a good method for determination of end point of decalcification and the method has since
been abandoned.
• CHEMICAL METHOD.
• Is a simple and reliable method, it is a two stage test which depends on the detection of
dissolved calcium in the decalcifying fluid. A positive result at either stage indicates that further
decalcification of the tissue in fresh fluid is required and the test should be repeated after a
suitable interval.
• The principle of the test is based on the addition of strong ammonia to a solution, which
contains calcium. The solution becomes cloudy due to the precipitation of calcium hydroxide at
an alkaline pH. Cloudy solution of calcium oxalate can also be formed if saturated aqueous
solution of ammonium oxalate is added to the decalcifying fluid.

10. DETERMINATION OF END POINT OF DECALCIFICATION BY THE CHEMICAL METHOD
Decant 5 ml of used decalcifying fluid into a clean test tube.
Add a small piece of red litmus paper to the fluid.
Add strong ammonia drop wise while shaking until the red litmus paper turns blue
Check for turbidity at this stage, if no turbidity, proceed to step 5.
Add 0.5ml of saturated aqueous ammonium oxalate, mix and observe for 30 minutes.
• NEUTRALISATION OF ACID
• After decalcifying a tissue in an acid solution, it is necessary to remove the acid. If acid is not
removed, it will interfere with subsequent histochemistry of tissue. Neutralisation is
accomplished by treating tissue with 5% lithium sulphate or sodium sulphate overnight.
Tissue can also be washed in running water overnight to remove acid. If tissue has been
decalcified in an alcoholic decalcifying fluid such as Perenyi’s fluid, the tissue should be
transferred directly to 70% alcohol for 12 to 18 hours