DECALCIFICATION

Calcification
Term applied to tissues which have been
infiltrated with calcium salts

Decalcification
Process of removing calcium ions or lime salts
from the organic cellular matrix, calcified
collagen and surrounding tissue of the bones.
Principle of Decalcification
Insoluble calcium salt are converted into
soluble calcium salts by the action of
decalcifying agent so that the tissue
become soft.

Chelating agents binds to calcium ion

present in the bone and decalcification is
carried out
Specimen Types
Amputated Limbs
Secondary to tumor, inflammation and gangrene.
Delivered without fixative.
Refrigerate if cannot be processed.
Resected specimen
Benign tumors/arthritic femoral head
Calcified tissues
Tuberculous organs, arteriosclerotic vessels
Factors Affecting Decalcification
Concentration and Volume of Decalcifying
Agent
More concentrated solutions decalcify bone more
rapidly but are more harmful to the tissue.
High concentrations and greater amount of fluid
will increase the speed of the process.
Recommended ratio = 20:1
Temperature
Heat will serve to hasten decalcification but also
increase the damaging effects on tissues.
Optimum temp = Room Temperature (18-30 C)
Agitation
Influence fluid exchange within and around the
tissue
Speeds up decalcification time (assumption)
Suspension
Specimen should be fully suspended on the
solution and makes complete contact.
Other factors
Patients age
Type of bone
Size of specimen
Ideal time required = 24-48 hours
Dense bone tissues = 14 days or longer
Methods of Decalcification
ACID METHOD
Widely used agent for decalcification
Stable, easy available, inexpensive
Carried out at room temperature
Nitric Acid
- Most common and fastest decalcifying agent
Aqueous Nitric Acid Formol Nitric Acid
Solution 10% Less tissue
Needle and small destruction
biopsies, urgent Composition:
biopsies (rapid Conc. Nitric Acid 10ml
diagnosis) Formaldehyde 5ml
Composition: d.H2O 85ml
Conc. Nitric Acid 10ml
D.H2O 100ml
Perenyis Fluid
both tissue softener and
decalcifying agent
Composition:
Nitric Acid 40 mL
Chromic Acid 30 mL
Abs. Ethyl Alcohol 30 mL
Phloroglucin-Nitric Acid
MOST RAPID
DECALCIFYING AGENT
Composition:
Conc. Nitric Acid 10 mL
Phloroglucin 1g
10% Nitric Acid 100 mL
Hydrochloric Acid
Slower and greater Von Ebners fluid
tissue distortion Teeth, small pieces of bone
Extent of decalcification
Good nuclear cannot be measured by
staining, surface chemical methods
Composition:
decalcification of
Sat. Aqueous Solution of
tissue blocks NaCl
Conc. Hydrochloric Acid
D. H2O
Formic Acid
Slow acting, less tissue distortion
Used for decalcification of research
tissue
10% Formalin-Formic acid Mixture
Formic acid
10% Formal-Saline
Formic acid-Sodium Citrate Solution
Aqueous Sodium Citrate
Formic Acid
Trichloroacetic Acid & Picric Acid
Weak and slow-acting decalcifying
agent
Doesnt require washing excess acid is
removed by 90% alcohol (improves
dehydration)
Sulfurous Acid
Weak decalcifying solution (minute bone)
Chromic Acid (Flemmings Fluid)
Fixative and decalcifying agent
Inhibits hematoxylin
Extent of decalcification cannot be
measured by chemical methods
General Procedure of Decalcification
Step Action
1 Rinse fixed section with WATER, place in DECALCIFYING
Solution
2 Completely immerse the section in a volume of
decalcifying agent equivalent to 5-10 (20) times the
volume of the specimen.
3 Check extent of decalcification.
Change the Decalcifying Solution daily. NEVER add fresh
solution to a used solution.
4 After decalcifying, wash specimen in running water
before routine tissue processing
Methods of Decalcification
CHELATING AGENT
Combine with calcium ions to form weakly
dissociated complexes and facilitate
removal of calcium salt
Ethylenediamine tetraacetic acid
(EDTA)
Use for detailed Formalin-EDTA
(Hillemann and Lee,
microscopic studies 1953)
(immunohistochemical EDTA, disodium salt
and enzyme D. H2O
staining/Electron Formaldehyde
Microscopy) EDTA, aqueous
EDTA disodium salt
D. H2O
Methods of Decalcification
ION EXCHANGE RESIN
Ammonium form of polysterene resin
Hasten decalcification by removing calcium
ions from formic-acid containing solution
Extent of decalcification cannot be
measured by chemical methods
Ion-Exchange Resin Method
Step Action
1 Spread in. thick of ion-exchange resin over
the bottom of the container
2 Place specimen on top of it.
3 Add DECALCIFYING agent (formic acid) 20-30
time volume of tissue
4 Allow tissue to stay in the solution for 1-14 days.
5 Measure extent of decalcification using physical
or X-ray method
Methods of Decalcification
ELECTROLYTIC METHOD
Process whereby calcium ions are attracted to
a negative electrode and remove from
decalcifying solution.
Electrolytic Decalcifying Solution
88% Formic acid
Hydrochloric acid
D. H2O
Electrolytic Method
Step Action
1 Suspend the bone specimen using a platinum
anode in a jar
2 Change Decalcifying solution after 8 hours.
3 Rinse specimen in alkaline water
4 Immerse specimen in lithium carbonate before
staining.
Decalcification End-point Test
Frequent monitoring if calcium is completely
removed from specimen.
Need for solution change
Acid Daily testing.
Near end-point, every 5 hours
EDTA Weekly test, unless solution changes more
frequent
Minimally calcified Tested only once
tissues
PHYSICAL/MECHANICAL TEST
Most inaccurate, damaging to tissues
Methods:
Probing
Bending the specimen
Needling, inserting pin, razor, scalpel directly into
the tissue
Pricking or slicing
Touching the specimen
Squeezing the tissue
Create artifacts (needle track), disrupt soft
tumors, cause false-positive microfractures
Radiographic Method
Ideal, sensitive, reliable
Can spot even smallest focus of calcium
Expensive
Chemical Method
Calcium released from bone into
decalcifying solution are precipitated by
chemical methods
Insoluble Calcium/Ammonium hydroxide
Calcium/Ammonium oxalate
Decalcifying fluid is tested for calcium.
When NO CALCIUM is found,
decalcification is COMPLETE.
Solution to Chemically test residual Calcium:
5% Ammonium Hydroxide Stock
28% Ammonium hydroxide + d. H2O
5% Ammonium Oxalate Stock
Ammonium oxalate + d. H2O
Working solution:
Equal parts of Ammonium Hydroxide Stock & Ammonium
Oxalate Stock
Chemical Test Procedure 1:
Step Action
1 Insert a pipette into decalcifying solution containing the
specimen.
2 Withdraw approximately 5 mL and place on test tube.
3 Add 10 mL of working solution
4 Mix well
5 Let sit overnight

Result:
Decalcification is complete when no precipitate or
turbidity is observed
If slotuion turns turbid, the solution should be changed.
 Chemical Test Procedure 2:
Step Action
1 Take 5 mL of decalcifying fluid
2 Add litmus paper/pH paper with magnetic stirrer
3 Add ammonium hydroxide, drop by drop, until indicators is
neutral (pH 7)
4 Add 5 ml ammonium oxalate. Shake well
5 Stand for 30 minutes
Result:
If white precipitate (calcium hydroxide) forms after adding NH4OH large
quantity of Ca++.
If clear, proceed with step 4. if precipitation occurs, Ca++ is present (small
amt)
If solution remains clear (step 5) decalcification is complete.
Treatment of Decalcified
Specimen
Chemical Neutralization
Immersed in lithium carbonate solution
Rinsing with water
30 mins small bones
1-4 hours large/dense bones
Treatment with alcohol (70%)
Decalcified with EDTA
Tissue Softeners
For unduly hard tissues that may damage
the microtome knife
Perenyis fluid tissue softener and
decalcifying agent
4% aqueous phenol
Molliflex
2% HCl
1% HCl in 70% alcohol