The presence of calcium salts in tissues makes them difficult. This causes knife damage, tissue cutting difficulty. Calcium may also be present in normal tissues under pathological conditions such as necrotic tissue in tuberculosis. Decalcification is the process of removing calcium salts
Why is Decalcificaition done?
- Decalcification is done to remove calcium salts from tissues and make them suitable for sectioning.
- The calcified hard tissues should first be cut into small pieces (2 to 6 mm) with a thin blade, hacksaw or sharp knife to minimize tearing of the surrounding tissues.
- This process is followed by fixation in formalin buffered or any other desired fixative.
- Tissues must be thoroughly washed after fixation and excess fixative should be removed before the specimen is subjected to decalcification.
The process of decalcification should meet the following conditions:
- Complete removal of calcium salts
- Minimal distortion of cell morphology
- No interference during staining
The process of decalcification is straightforward, but in order to be successful it requires:
- 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
What are the method of decalcification?
Before decalcification,The tissue is cut into small pieces of 3 to 5 mm in size.This helps to decalcify faster.The tissue is then suspended with waxed thread in the decalcifying medium.The thread covering of wax prevents the action of acid on thread.The volume of the decalcifying solution should be 50 to 100 times the volume of tissue.Decalcification should be checked at regular intervals.
The following are the various method of decalcification in cyto-histopathology:
- Acid decalcification
- Ion exchange resin
- Electrical ionization
- Chelating methods
- Surface decalcification
This is the method most commonly used. Various acid solutions can be used either alone or in combination with a neutralizer. The neutralizer helps prevent the cells from swelling.
The following are the usually used solutions for decalcification
- Aqueous Nitric Acid
|Nitric acid||5 ml|
|Distilled water||100 ml|
If tissue remains in the solution for a long time, the tissue may be damaged. Yellow nitric acid color should be removed with urea. But this solution gives both good nuclear staining and rapid action.
- Nitric Acid Formaldehyde
|Nitric acid||10 ml|
|Distilled water||upto 100 ml|
It has advantages such as
- Rapid action
- Good nuclear staining
- Washing with water is not required
- Formalin protects the tissues from maceration
- Formic Acid Solution
|Formic acid||5 ml|
|Distilled water||90 ml|
The decalcification is slow in this solution. If the concentration of formic acid is increased, the process is fast, but there is more tissue damage.
- Trichloroacetic Acid
This is used in small biopsies. The decalcification process is slow and therefore can not be used for dense bone or large bony pieces.
|Formal saline (10%)||95 ml|
|Trichloroacetic acid||5 gm|
Ion Exchange method
Ammonium salts of sulfonated polystyrene resin are used in this method. The salt is layered at the bottom of the container and the fluid – containing formic acid is filled. Mineral acid should not be present in the decalcifying fluid. Only complete decalcification can be determined by X – rays.The benefits of this method are :
- Faster decalcification
- Well preserved tissue structures
- Longer use of resin
Formic acid or HCl are used as an electrolyte medium. The calcium ions are moving towards the cathode. Rapid decalcification is achieved, but the heat produced can damage the cytological details.
Organic chelating agents absorb metal ions. EDTA can bind calcium to form a non – ionized soluble complex. It works best for cancer bone. This is the best way to decalcify bone marrow biopsies as it best preserves cytological details. Marrow glycogen is preserved.This method makes uses of EDTA Solution
- EDTA Solution
|Distilled water||900 ml|
The paraffin block surface layer is inverted for one hour in 5 percent HCl. About the top 30 microns is decalcified. Before cutting, it should be thoroughly washed.
Factors affecting the rate of decalcification
- Decalcifying solution concentration: The reaction is fastened by increased concentration of the decalcifying agent.
- Temperature : Decalcification rate increases with temperature rise.
- Bone density : Harder bone density takes longer to decalcify.
- Tissue thickness : Small pieces of tissue decalcify earlier.
- Agitation : Agitation increases the rate of decalcification.
Methods of determining the endpoint of decalcification
It is important to take note that Specimens should NOT be crowded together and should NOT contact the container bottom to provide complete decalcification also Over decalcified, specimen can be permanently damaged. The following procedure helps determine the correct decalcification endpoint.
The following are various methods used in determining the optimum or end point of decalcification :
- X-ray (the most accurate way)
- Chemical testing (accurate)
- Physical testing (less accurate and potentially damage of specimen)
The following solutions are needed for the chemical testing of residual calcium.
|5% Ammonium Hydroxide Stock||5% Ammonium Oxalate Stock|
|Ammonium hydroxide, 28% 5 ml||Ammonium oxalate 5 ml|
|Distilled water 95 ml||Distilled water 95 ml|
|Mix well||Mix well|
Ammonium Hydroxide/Ammonium Oxalate Working Solution:
Use equal parts of 5% ammonium hydroxide solution and 5% ammonium oxalate solution.
Chemical test procedure
- Insert a pipette into the decalcification solution containing the specimen
- Remove approximately 5 ml of the hydrochloric acid / formic acid decalcification solution from below the specimen and place it in a test tube.
- Add approximately 10 ml of the working solution for ammonium hydroxide / ammonium oxalate, mix well and let stand overnight.
- Decalcification is complete when no precipitate is observed on two consecutive test days. Repeat the test every two or three days.
Physical testing includes bending the specimen or inserting a pin, razor, or scalpel directly into the tissue.The drawback of inserting a pin, razor, or scalpel is the introduction of tears and pinhole artifacts.The slight bending of the specimen is safer and less disruptive, but it will not determine conclusively whether all calcium salts have been removed.Wash thoroughly before processing after checking for rigidity.
Note: If paraffin embedded bones are not fully decalcified, the paraffin blocks can be soaked in the same decalcification solution for a few minutes before cutting. Usually this is helpful.
Some points to remember
- After completion of decalcification, the specimen should be washed in water
- Over decalcification is more noticeable in staining of nuclei.
- Acid solutions soften bone by removing calcium salts.
- EDTA is used as chelating agent for decalcification.
- To offset the hydrolysis of nucleic acids caused by decalcification, bone marrow is often fixed in Zenker’s solution.
- During decalcification, carbon dioxide gas is released.
- Factors affecting decalcification are Size of specimen, Concentration of decalcifying solution,Time in decalcifying solution and Amount of decalcifying solution
- For proper decalcification, bone should be cut into 4-5mm thick pieces
- Chelating agents act by binding calcium ions