Decalcification

(1)

DECALCIFICATION

(2)

Calcification



Term applied to tissues which have been

infiltrated with calcium salts

Decalcification



Process of removing calcium ions or li

me salts

from the organic cellular matrix, calcified

collagen and surrounding tissue of the bones.

(3)

Calcification



Term applied to tissues which have been

infiltrated with calcium salts

Decalcification



Process of removing calcium ions or li

me salts

from the organic cellular matrix, calcified

collagen and surrounding tissue of the bones.

(4)

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

(5)

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

(6)

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.

(7)

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

(8)

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

(9)

Methods of Decalcification

ACID METHOD



Widely used agent for decalcification



Stable, easy available, inexpensive



Carried out at room temperature

(10)

Nitric Acid

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

(11)

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 1 g  10% Nitric Acid 100 mL

(12)

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

(13)

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

(14)

Trichloroacetic Acid & Picric Acid

Weak and slow-acting decalcifying

agent

Doesn’t require washing – excess acid is

removed by 90% alcohol (improves

(15)

Sulfurous Acid



Weak decalcifying solution (minute bone)

Chromic Acid (Flemming’s Fluid)



Fixative and decalcifying agent



Inhibits hematoxylin



Extent of decalcification cannot be

(16)

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

(17)

Methods of Decalcification

CHELATING AGENT



Combine with calcium ions to form weakly

dissociated complexes and facilitate

removal of calcium salt

(18)

Ethylenediamine tetraacetic acid

(EDTA)

Use for detailed

microscopic studies

(immunohistochemical

and enzyme

staining/Electron

Microscopy)

 Formalin-EDTA

(Hillemann and Lee, 1953)

 EDTA, disodium salt  D. H2O

 Formaldehyde

 EDTA, aqueous

 EDTA disodium salt  D. H2O

(19)

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

(20)

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

(21)

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

(22)

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

(23)

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

(24)

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

(25)

Radiographic Method

Ideal, sensitive, reliable

 Can spot even smallest focus of calcium

(26)

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,

(27)

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

(28)

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

(29)

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