The presence of calcium salts in the tissue makes them hard. This causes damage to the knife and difficulty cutting tissue. Calcium is usually found in bones and teeth. Calcium may also be present in normal tissues in pathological conditions, such as necrotic tissue in tuberculosis.Therefore,the aim of tissue decalcification is to remove calcium salts from the tissues and make them amenable for sectioning or easy to section.In order to carry out the process of decalcification,the tissues has to be prepared.
Preparation of tissues for decalcification
First, the calcified hard tissue should be cut into small pieces( 2 to 6 mm) with a thin blade, hacksaw or sharp knife to minimize the tearing of the surrounding tissue.This is followed by fixation of the tissues in buffered formalin or any other desired fixative. The tissue is then thoroughly washed in order to remove excess fixative before being subjected to decalcification.
Before getting to the method of decalcification,it is important to note a decalcification method should satisfy the following conditions:
- Complete removal of calcium salts
- Minimal distortion of cell morphology
- No interference during staining
Therefore in order to attain such precisions and satisfy the above condition for a successful process of decalcification,the following is required.
- A careful preliminary assessment of the specimen
- Thorough fixation
- Preparation of slices of reasonable thickness for fixation and processing
- The choice of a suitable decalcifier with adequate volume, changed regularly
- A careful determination of the endpoint
- Thorough processing using a suitable schedule
The following are the five most used methods of decalcification
- Acid decalcification
- Ion exchange resin
- Electrical ionization
- Chelating methods
- Surface decalcification
1. Acid Decalcification
This is the most common method used. Various acid solutions can be used alone or in combination with a neutraliser. The neutralizer helps to prevent cell swelling.
In this method,the following are the commonly used decalcification solutions
Nitric Acid Formaldehyde
|Nitric acid||10 ml|
|Distilled water upto||100 ml|
It has the following advantages
- Rapid action
- Good nuclear staining
- Washing with water is not required
- Formalin protects the tissues from maceration
Aqueous Nitric Acid
|Nitric acid||5 ml|
|Distilled water||100 ml|
With aqueous nitric acid,If the tissue remains in the solution for a longer period of time,this may damage the tissues.The yellow color of nitric acid should be removed with urea. However, this solution leads to a good nuclear staining and also to a fast effect.
Formic Acid Solution
|Formic acid||5 ml|
|Distilled water||90 ml|
With this solution the decalcification is slow. When the formic acid concentration is increased, the process is fast, but the tissue damage is greater.
|Formal saline (10%)||95 ml|
|Trichloroacetic acid||5 gm|
This is used for small biopsies. The decalcification process is slow and therefore can not be used for dense bones or large pieces of bone.
2. Ion Exchange method
This process uses ammonium salts of sulfonated polystyrene resin. The salt is layered on the bottom of the container and filled with a liquid containing formic acid. The decalcifying liquid should not contain mineral acid. X- rays is the only method used to determine the complete decalcification or to determine the end point of decalcification.
The advantages of this method are
- Faster decalcification
- Well preserved tissue structures
- Longer use of resin
3. Electrolytic Method (electrolysis process)
Here formic acid or HCl are used as electrolytic medium. The calcium ions move in the direction of the cathode. Rapid decalcification is achieved, but the heat generated can damage the cytological details of the tissue.
4. Chelating Agents
Organic chelating agents absorb metal ions. EDTA (calcium edta) can bind calcium and form a non- ionised soluble complex. It works best on cancerous bones. This is the best method to decalcify bone marrow biopsies because the cytological details are best preserved. Bone marrow glycogen is preserved.EDTA Solution is an example of a chelating agent decalcifying fluid as is composed of the following
|Distilled water||900 ml|
5. Surface Decalcification
The surface layer of the paraffin blocks is converted into 5% HCl for one hour. About 30 micrometers have been decalcified. It should be thoroughly washed before cutting.
How to Decalcify tissues
- The tissue is cut into small pieces from 3 to 5 mm in size.This helps to decalcify more quickly.
- The tissue is then hung or suspended in decalcified medium with waxed thread.Coating of wax on the thread helps in preventing the action of acid on the thread.
- The volume of the decalcification solution should be 50 to 100 times the tissue volume. The decalcification should be checked on a regular basis.
What are the factors affecting rate of Decalcification
- Concentration of the decalcification solution: A higher concentration of the decalcification agent fastens the response or the faster the process of decalcification.
- Temperature : The decalcification rate increases with the increase in temperature.
- Bone density : harder bone density takes longer to decalcify.
- Tissue Thickness : Small pieces of tissue decalcify earlier
- Agitation : Agitation improves the decalcification rate.
How to Determine the end point of decalcification
There are three method commonly used to determine the optimum or end point of decalcification which are X-ray (the most accurate way) ,Chemical testing (accurate) and the Physical testing (less accurate and potentially damage of specimen).These methods helps to determine or to detect whether Calcium salts has been totally removed from the tissue in order to render the tissue soft.
1. Chemical test method
The following solutions are needed to test residual calcium chemically.
5% Ammonium Hydroxide Stock(Mix well the following ingredients)
|Ammonium hydroxide, 28%||5 ml|
|Distilled water||95 ml|
5% Ammonium Oxalate Stock (Mix well the following ingredients)
|Ammonium oxalate||5 ml|
|Distilled water||95 ml|
Use equal parts of the 5 percent ammonium hydroxide solution and 5 percent ammonium oxalate solution.
Chemical test method Procedure
- Insert a pipette into the decalcifying solution that contains the sample.
- Under the sample approx. 5 ml of the hydrochloric acid / formic acid decalcifying solution are taken and put into a test tube.
- Add about 10 ml of the ammonium hydroxide / ammonium oxalate working solution, mix well and leave overnight.
- Decalcification is complete when no precipitation is observed for two consecutive test days.
- Repeat the test every two or three days.
Physical Test Method
The physical tests include bending the specimen or directly inserting a pin, razor or scalpel into the tissue.The disadvantage of inserting a pen, razor, or scalpel is the insertion of cracks and needle puncture artifacts.A slight bending of the specimen is safer and less annoying, but can not clearly determine whether all calcium salts have been removed. After checking the stiffness, wash thoroughly before use.
If the bones embedded in paraffin are not completely decalcified, the paraffin blocks can be soaked in the same decalcifying solution for a few minutes before cutting. This is usually helpful.
X-ray Test Method
This makes used of X-ray to determined the end point of decalcification