Decalcification: Unveiling Structures Beneath the Mineral Veil What is it? Decalcification removes calcium salts from tissues like bone and teeth, making them soft and sliceable for microscopic analysis. Why do it? Hardened tissues can't be sectioned effectively and interfere with staining. Decalcification allows clear visualization of cellular and structural details. How is it done? Different methods exist, like using weak acids or chelating agents, each with its pros and cons. The choice depends on tissue type, processing time, and desired preservation level. Knowing when to stop: Monitoring techniques like X-rays or physical assessment help determine the optimal endpoint to avoid over-decalcification and tissue damage. Beyond bone: Decalcification finds applications in diverse fields like paleontology, pathology, and cancer research.

1. DECALCIFICATION OF
TISSUE IN
LABORATORY
TOUFEEQ MALLICK

2. CONTENT
 Introduction
 Factors controlling rate of decalcification
 Methods of decalcification
 End point determination of decalcification

3. INTRODUCTION
The presence of calcium salt in the tissues make them very
firm to hard and this may damage the knife.
So, it is often necessary to remove calcium salt from the
tissue and to make it soft for cutting in a microtome.
The process of removal calcium salt from the
tissue is known as decalcification.

4. FACTORS CONTROLLING THE RATE OF DECALCIFICATION
 Concentration:- Directly proportional to the rate of
decalcification.
 Temperature:- Directly proportional to the rate of
decalcification.
 Density of bone:- Hard bone takes longer time to be
decalcification.
 Thickness of tissue:- Directly in proportional to the rate of
decalcification.

5. THE METHOD OF DECALCIFICATION
Acid decalcification
Chelating agent solution
Ion exchange resin
Electrical ionization
Surface decalcification

6. ACID DECALCIFICATION
This is the commonest method of decalcification. Acid makes the soluble
calcium salt, and thereby calcium is removed form the tissue .
Strong acid:-
The strong acids are used in 5-10% concentration .They are rapid in action .
However, careful attention is needed to prevent tissue damage.
Example:- HCL, HNO3
Weak acid:-
Somewhat gentle and take more time to complete the decalcification than
strong acids..
Example:- C2HCl3O2, HCOOH.

7. Chemicals Concentration Advantages Disadvantages
Aqueous nitric acid 5% 1. Rapid action
2. Good nuclear stain
Gives yellow colour to
the tissue
Nitric acid formaldehyde 10% 1. Rapid action
2. Less chance to tissue
damage.
3. Long time washing by
water is not needed.
Not a very good nuclear
stain
HCL acid 8% 1. Rapid action
2. Ideal decalcification
agent for the tooth
Nuclear staining is not
very good
Trichloroacetic acid 5gm in 95ml formal
saline
1. Good for small
biopsies
2. Good nuclear stain
1. Not good for hard
bony tissue
2. Slower in action and
takes 4-5 days
Formic acid 5% Decalcifying agent of
choice in routine
laboratory process.
Slow acting and takes
many days for
decalcification

8. CHELATING AGENT SOLUTION
Chelating agents are organic substances that
adsorb metals. EDTA is the most commonly
chelating agents in the routine laboratory
decalcifications. It binds with calcium of the
hydroxyapatite crystals and forms a non-
ionized soluble complex.
EDTA 5.5gm
Formalin 100ml
Distilled water 900ml

9. Advantages:-
• Morphological preservation of tissue
• Suitable to do various other laboratory Tests
• Very good for bone marrow trephine biopsy
Disadvantages:-
• Very slow process.
• Maintenance of pH around 7 is necessary.
• Thin tissue is needed.

10. ION-EXCHANGE RESIN METHOD & ELECTROLYSIS
METHOD
 Ion-exchange resin method:-
In this technique an ion-exchange resin is used along with an
organic acid as decalcifying liquids. It also produces faster
decalcification with preservation of morphological details of the
tissue.
 Electrolysis method:
In this process electrolysis of the tissue is done in a solution of HCL
acid and formic acid. Calcium from the tissue moves to the cathode
plate. This is a very rapid method of decalcification and takes only a
few hours to decalcify the bone. There is a risk of tissue damage in
this technique.

11. SURFACE DECALCIFICATION
The surface layer of paraffin blocks is
inverted in 1% HCL acid for 1hr. The
exposed top 30 μm tissue of the paraffin
block is decalcified. The block should be
washed thoroughly before cutting. Only
the first few paraffin sections are
expected to be free from calcium.

12. END POINT DETERMINATION OF DECALCIFICATION
1. Radiographic examination
2. Physical test
3. Chemical test

13. RADIOGRAPHIC EXAMINATION & PHYSICAL TEST
 Radiographic Examination:-
X-ray examination of the tissue is the most accurate technique to detect the end
point of decalcification. However, this is a costly procedure, and the pre
decalcification radiograph is also needed to assess the extent of decalcification.
 Physical test: -
This is a crude test and it does not accurately detect the end point of
decalcification . The tissue is bent or a pin is introduced within the tissue. In case
of adequate decalcification, it is expected that the tissue will be soft and could
be bent easily. Pin also should penetrate easily within the tissue. However, the
tissue damage may occur by making a hole or by bending it.

14. CHEMICAL TEST
This test is done to assess the presence of calcium in the decalcifying solution.
 Method:-
1. 5 ml of the decalcifying agent from the container containing the tissue is
withdrawn.
2. Mix the decalcifying agent with 5 ml of ammonium hydroxide and
ammonium oxalate mixture solution.
3. The mixture is kept overnight.
4. Any precipitation is noted.
5. The presence of precipitation indicates that the decalcifying agent contains
calcium and decalcification is not completed.

15. CONCLUSION
Decalcification is a crucial step in preparing calcified tissues for
microscopic examination.
Understanding different methods and their considerations
empowers researchers and pathologists to choose the optimal
approach for their specific needs.
This technique unlocks the hidden world within calcified tissues,
advancing our knowledge in various fields and contributing to
accurate diagnoses and effective treatments.