Histology DECALCIFICATION

DECALCIFICATION

 3. DECALCIFICATION

Bone and teeth normally contain inorganic salts which comprises 65% of bone composition. 

Of this amount 85% is calcium phosphate, 10% calcium carbonate and remaining 5%

include chlorides and fluorides of calcium and magnesium. The organic part which makes

35% of bone composition comprises collagen fibers and different proteoglycans. Thus

rigidity of bones is due to the high proportion of deposited minerals. This deposition of

minerals in the matrix occurs during bone formation by a process name as calcification.

³Decalcification is opposite to calcification. It is a process by which

calcium salts are removed from a calcified biopsy by means of decalcifying

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Certain tissues are pathologically calcified, e.g. thyroid, larger blood vessels in old people,

cancerous biopsies and different abscesses. When we receive a bony biopsy or

pathologically calcified biopsy, it is very difficult to section it due to the presence of

calcium salts and so decalcification of such biopsies is necessary. 

By decalcification, the tissue biopsy is softened and its sectioning is facilitated.

Decalcification is preceded by fixation and followed by tissue processing. It is very

necessary to fix a tissue biopsy before taking it to decalcifying agents. This is because the

agents are acids so have a detrimental effect on unfixed biopsies, e.g. organic matter

(collagen) is first destroyed followed by nucleic acids destruction by acids.

3.1 Decalcifying Agents

The chemicals used for decalcification process are termed as decalcifying agents. An acid is

the essential part of a decalcifying agent and a second substance is usually used to prevent

distortion of tissue. Buffer solutions of pH 4.4 ± 4.5 and chelating agents, e.g. EDTA can

also be used.

The four acids most commonly used for removing calcium salts from tissues are formic,

nitric, trichloroacetic and hydrochloric acids. The speed at which the calcium salts are

broken up out of the tissue is subject to the strength, temperature and volume of the

decalcifying agent.

Box 3

An increase in either the concentration of acid or the temperature at which decalcification takes

place, can decrease the time required for decalcification but this results in poor staining and partial

digestion of tissue. These effects do not apply to EDTA which may be used at 40 ± 60o C

successfully. 

3.1.1 Formic Acid

Formic acid [HCOOH] is recommended for post-mortem and research tissue. It take 2 ± 7

days for decalcification so not suitable for urgent tissue biopsies. 

Merits:

i. By using this solution excellent staining results are obtained which make it excellent

for research work.

Demerits:

i. At 5% strength, it is slow in action, so unsuitable for urgent work; however the

process can be speeded by increasing the formic acid content upto 25ml in the given

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ii. But by increasing the concentration in excess of 8%, disturbs the opacity of solution.

Formula

Formic Acid (specific gravity 120 gravity) 5ml

Distilled water 90ml

Formalin 40% 5ml

3.1.2 Nitric Acid

Nitric Acid is recommended for urgent tissue biopsies (3 ± 4mm thickness). The time

required for decalcification is from 1 ± 3 days.

Merits:

i. This is a rapidly acting decalcifying agent and permits good nuclear staining.

Demerits:

i. Good nuclear staining is obtained but it is not as good as that obtained by formic

acid.

ii. It is very reactive so biopsy may be impaired if kept for longer periods than required.

iii. In addition to this, it develops a yellow colour due to formation of nitrous acid. This

enhances decalcification but impairs staining. To avoid this 0.1% urea is added

which does not affect the efficiency of the acid.

Formula

i. Nitric Acid Formaldehyde

Nitric acid (specific gravity 1.41) 10ml

Formalin 40% 5 ± 10 ml

Distilled water upto 100ml

ii. Aqueous Nitric Acid

Nitric Acid (specific gravity 1.41) 5 ± 10 ml

Distilled water upto 100ml

3.1.3 E.D.T.A. 

This is a very slow decalcifying agent recommended for detailed microscopical studies

where time is not an important factor. The time required for decalcification is 3 weeks,

during which the solution must be changed at intervals of 3 days and 1 day in final stage.

Merits:

i. Histological artifacts are minimized by the solution.

ii. The staining results are also excellent.

Demerits:

i. It is slow in action and chelating agent so tends to harden the tissue.

Formula

EDTA disodium salt 5.5g

10% neutral formalin 100ml

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This solution was introduced as a fixative for ova, but it has gained popularity in recent

years as a good decalcifying agent. It is mostly used for softening of tissues in case of dense

fibrous tissues.

Formula

Nitric Acid 10% 40ml

Absolute Ethanol 30ml

Chromic Acid 0.5% 30ml

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The use of this fluid is recommended for teeth and staining results are good but staining

results of nucleus are comparatively poorer. Various formulae have been given for this, but

following gives good results:

Formula

Saturated NaCl 36% 50ml

Distilled water 50ml

HCl 08ml

3.1.6 Ion Exchange Resins

The use of an ion exchange resin into the decalcifying solution has been claimed to speed

up the process of decalcification, and to improve staining. The principle is that the calcium

ions are removed from the solution by the resin, thereby increasing the rate of solubility of

the calcium from the tissue.

3.2 Confirmation of Decalcification

The process of decalcification can be assessed by the following ways:

3.2.1 X-rays:

X-ray examination is the most satisfactory method depending upon the facilities available.

These are used because bone is radio-opaque due to presence of calcium salts. When the

calcium salts are removed, tissue becomes transparent for such rays. When mercuric

chloride has been used as a fixative, x-rays cannot be applied because this fixatives renders

tissue radio-opaque. 

3.2.2 Chemical Test:

A chemical test is a simple and reliable method when x-rays are not available. It is a two

stage test which relies upon the discovery of broken up calcium in the decalcifying liquid. A

positive result at either stage indicates that further decalcification of tissue in fresh fluid is

required and the test should be repeated after a suitable interval.

3.2.2.1 Method:

ƒ Decant 5ml of the used decalcifying fluid into a clean test tube and add a small piece of

litmus paper.

ƒ Add strong ammonia of specific gravity 0.88 drop by drop while agitating the tube until

the litmus paper just turnss blue, showing alkalinity.ƒ If the solution becomes turbid at this stage, calcium is present in considerable amounts

and the tissue should be transferred to fresh decalcifying fluid.

ƒ If the solution remains clear proceed with the second stage of the test. Add 0.5ml

saturated aqueous ammonium oxalate, mix and allow to stand for 30 minutes. Any

turbidity creating during this period shows the presence of calcium and re

drenching of the tissue in crisp decalcifying liquid is vital.