Decalcification

DECALCIFICATION

Calcification  

Term applied to tissues which have been infiltrated with calcium salts

Decalcification  

Process of removing calcium ions or lime li me salts from the organic cellular matrix, calcified collagen and surrounding tissue of the bones.

Calcification  

Term applied to tissues which have been infiltrated with calcium salts

Decalcification  

Process of removing calcium ions or lime li me 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 





Patient’s 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 Solution 10% 



Needle and small biopsies, urgent biopsies (rapid diagnosis) Composition:  

Conc. Nitric Acid 10ml D.H2O 100ml

Formol Nitric Acid 



Less tissue destruction Composition:   

Conc. Nitric Acid 10ml Formaldehyde 5ml d.H2O 85ml

Perenyi’s 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 tissue distortion Good nuclear staining, surface decalcification of tissue blocks

 Von Ebner’s fluid  



Teeth, small pieces of bone Extent of decalcification cannot be measured by chemical methods Composition: 

 

Sat. Aqueous Solution of 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 Doesn’t require washing – excess acid is removed by 90% alcohol (improves dehydration )

Sulfurous Acid 

Weak decalcifying solution (minute bone)

Chromic Acid (Flemming’s 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 microscopic studies (immunohistochemical and enzyme staining/Electron Microscopy)



Formalin-EDTA (Hillemann and Lee, 1953)   

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EDTA, disodium salt D. H2O Formaldehyde

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 ½ 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 tissues

Tested only once

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  



5% Ammonium Oxalate Stock  



28% Ammonium hydroxide + d. H2O  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)  •

•

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