DECALCIFICATION

Awal Mir Khattak

Demonstrator MLT

B.Sc. MLT Baqai Medical University Karachi

M.Sc. Hematology Baqai Medical University Karachi
M.Phil. Medical Lab Sciences, The University of Haripur
 DEFINITION
DECALCIFICATION
Decalcification is a process of complete removal of
calcium salt from the tissues like bone, teeth, cartilage
or other hard tissues following fixation.
 AIM OF DECALCIFICATION

• To obtain satisfactory paraffin sections of bone, teeth

and cartilage.
• To remove inorganic calcium from organic collagen
matrix of hard tissues.
• Decalcification is done to assure that the specimen is
soft enough to allow cutting with the microtome
knife.
 DECALCIFYING AGENTS
• There are two types of decalcifiers

(i) Mineral acids (ii) chelating agents.

Acid decalcifier is further divided two sub types.

(i) Strong inorganic acids: Nitric acid, HCL

(ii) weak organic acid: Formic, acetic, picric acids

chelating agents: ethylenediaminetetraacetic acid

 DECALCIFYING AGENTS
(i) Strong inorganic acids: Nitric acid, HCL

• Recommended concentration: 5-10 %

• Decalcify rapidly but causing tissue swelling and

damage tissue stain ability if used for longer than 24–
48 hours.

• Strong acids are used for needle and small biopsy

specimens allowing rapid diagnosis within 24 hours.
 DECALCIFYING AGENTS
Composition

1. Aqueous nitric acid, 5–10% (Clayden, 1952).

2. Perenyi’s fluid (Perenyi, 1882).

3. Formalin-nitric acid (use inside a fume hood).

 DECALCIFYING AGENTS
(ii) Weak organic acids e.g. formic, acetic, picric

• Formic is the only weak acid used extensively and

acetic and picric acids cause tissue swelling and not
used alone decalcifiers.

• The formalin-10% formic acid mixture simultaneously

fixes and decalcifies, and is recommended for small
bone pieces or needle biopsies.
 DECALCIFYING AGENTS
• The salts, sodium formate or sodium citrate are added

to formic acid solutions making ‘acidic’ buffers.

• Buffering is used to counteract the injurious effects

of the acid.

• It is suitable for most routine surgical specimens,

particularly when immunohistochemical staining is
required.
 DECALCIFYING AGENTS
• Composition

1. Aqueous formic acid (5-10%).

2. Formic acid-formalin
• 3. Buffered formic acid
 DECALCIFYING AGENTS
• Chelating agents
• EDTA is used as chelating agents
• Although EDTA is nominally ‘acidic’, it does not act
like an inorganic or organic acid but binds metallic
ions, notably calcium and magnesium.
• EDTA will not bind to calcium below pH 3 and is faster at
pH 7–7.4. the higher pH may damage alkalisensitive
protein linkages.
 DECALCIFYING AGENTS
• EDTA does inactivate alkaline phosphatase, but
activity can be restored by the addition of magnesium
chloride.

• K2 EDTA (disodium salt) 10% or K3 EDTA (tetrasodium

salt)14% are approaching saturation and can be

simple aqueous or buffered solutions at a neutral pH

of 7–7.4, or added to formalin.

 DECALCIFYING AGENTS
• Composition
1. Formalin-EDTA
2. EDTA (aqueous), pH 7.0–7.4.
• Other methods
• Electrolytic removal of calcium ions from tissue by use
of electric current.
 Criteria for good decalcification
1. Complete removal of calcium.

2. Absence of damage to tissue cells or fibers.

3. Subsequent staining not altered.

4. Short time required for decalcification.

 FACTOR INFLUENCING RATE OF
DECALCIFICATION
1. Concentration of decalcifying agent

2. Temperature

3. Agitation

4. Suspension
 Decalcification end point test
(1) Flexibility method
• Bending or needling, if it bends easily that means
decalcification is complete . This method is Unreliable,
causes damage and distortion of tissue.
• (2) X-ray method
• Best method for determining complete decalcification
but very costly. Tissue fixed in mercuric chloride
containing fixatives cannot be tested as they will be
radio opaque.
 Decalcification end point test
(1) Chemical method
17