Decalcification is the process of removal of calcium from decalcified tissue and making suitable for section cutting.

In presence of calcium salts makes the tissue hard and brittle, which will cause difficulty in section cutting and damage to the microtome knife.

DECALCIFICATION

Selection of tissue Fixation Decalcification Detection of end point Neutralization Washing

STEPS

Thin sizes of bone and other calcified hard tissues are obtained by using fine toothed forceps or hack saw .Have to take thickness of tissue at 4-5 mm.

SELECTION OF TISSUE

Adequate fixation should be needed before decalcification otherwise tissue will be damaged in acid decalcification.

Bony tissue is fixed in 10% buffered neutral formalin for 2-4 days,

For bone marrow zenkers formalin is used.

FIXATION

Different steps of decalcification are By using dilute mineral acid By using ion exchange resins By chelating agent Electrophoretic decalcification

DECALCIFICATION

Complete removal of calcium Minimal tissue damage Shouldn't interfere the staining reaction Speed of decalcification The factors which influence the speed of

decalcification are Heat Strength of acid Agitation

CRITERIA OF GOOD DECALCIFYING AGENT

The acid which is used for decalcification should be simple solution or mixed with other reagents especially with fixative or buffered solution.

Different type of decalcifying fluid Gooding and stewarts fluid Formic acid(90%)-100ml Formalin-50ml Distilled water-850ml By using formic acid gives a good routine decalcifying

fluid it will give reasonable speed and minimum tissue damage.

Formaldehyde gives protection to the tissue from acids. Decalcification by using this solution with in 2-4 days

depends on the thickness and degree of decalcification

BY USING DILUTE MINEREAL ACID

Conc. HCL-15ml Nacl-175gm Distilled water-up to 1lt 0.5 % HCl should be added daily till

decalcification is complete.This is a moderately rapid decalcification.

VON-EBNER’S FLUID

Absolute alcohol-73ml Chloroform-10ml Acetic acid-3ml HCl-4ml Distilled water-10ml More amount of fluid is needed that is 40-50

times the volume of tissue After decalcification the tissue is directly

transfer to several changes of absolute alcohol till the acid is removed from the tissue.

JENKER’S FLUID

PH-4.5 7% citric acid monohydrate-5ml 7.54% anhydrous ammonium citrate-95ml 1% zinc sulphate-2ml Chloroform-2 drops calcium ions are soluble at PH 4.5 This is slower in action but there is no

damage to the tissue.

CITRATE-CITRIC ACID BUFFER

Nitric acid recause the formation of yellow dis coloration to the tissue and it will interfere with subsequent staining reaction

The formation of yellow dis coloration can be prevented by adding 1% urea to pure nitric acid. But it is having only a temporary effect

FLUID CONTAINING NITRIC ACID

Concentrated HNO3-5-10ml Distilled water-up to 100ml It is a good protein decalcifying fluid Rapid in action but it will cause damage to

the tissue It will give brilliant staining reaction

AQUEOUS NITRIC ACID

Formalin-5ml Conc. HNO3-7.5ml Distilled water-up to 100ml Formalin prevents the softening effect of

nitric acid on the cell

FORMALIN NITRIC ACID

Conc. HNO3-10 ml Phloroglucin-1gm When bubbling stops at 100 ml of 10%

nitric acid to this solution Phloroglucin protect the tissue from

softening and gives brilliant staining effect

PHLOROGLUCIN NITRIC ACID

10% HNO3-40mlAbsolute alcohol-30ml0.5% chromic acid-30ml It’s very slow in action for bones. But

excellent for small deposits of calciumIt will cause little hard to the tissueThe end point detection is by x-ray

PERENYS FLUID

It is use to remove calcium ions from the fluid that will make more rapid rate of solubility of calcium from the tissue and time taken for decalcification can be reduced

The resins commonly used as ammonium forms of suphonated poly styrene resins

It’s layered on the bottom of the container to a depth of a rod 1cm and the specimen is allowed to rest on it

The volume of fluid will be 20-30 times the bulk of the specimen

Formic acid containing decalcifying fluid will be better results

After use with resins the tissue must be washed twice in diluted HCL and followed by washing in running tap water for 3 times

BY USING ION EXCHANGE RESINS

These are organic compounds having capacity to bind with calcium metals

Tissue decalcified by this method showing minimum of artifacts and good staining results

CHELATING AGENTS

The fixative used is 10% neutral formal saline

After fixation the tissue is transffered to 50 times its bulk of 55% sequestrine buffer of ph 7.4 is prepared in phosphate buffer

The fluid is changed daily for determination of end point

After decalcification , the tissue is transffered to 70% alcohol for dehydration

PROCEDURE

HILLEMANN’S AND LEE FLUID EDTA disodium salts – 5.5 gm Distilled water – 90ml Formalin – 10ml NEUTRAL EDTA It is a cloudy solution it can be neutralised

by adding 2.5 gm of NaOH

DIFFERENT TYPES OF CHELATING AGENTS

The tissue is placed in electrophoretic tank containing 2 electrodes and electrolyte solutions

Equal parts of 8% HCL + 10% Formic acid is used as an electrolyte

ELECTROPHORETIC DECALCIFICATION

Tissue should be exposed for longer time in decalcifying fluid in which it will cause damage to the tissue

So the end point of decalcification should be determined to prevent tissue damage and to ensure the complete removal of calcium

DETECTION OF END POINT

1) PHYSICAL METHOD It is a crude method consists of probing the

tissue with a needle and cutting using a scalpel or should check the flexibility of the tissue

2) CHEMICAL METHOD 5 ml decalcifying fluid is utilized by strong

ammonia then add 5ml of ammonium oxalate solution

3) RADIO GRAPHIC METHOD X-ray

DIFFERENT METHODS ARE

After decalcification the tissue should be neutralized with treating with alkali overnight.

5% Lithium carbonate or NaSO4 can be used for neutralization

Failure to do the neutralization property that will cause the swelling of the tissue

NEUTRALISATION OF ACID

Washing is necessary for removal of alkali otherwise that will interfere with the staining reaction

Washing can be done overnight in water or 70% alcohol for 3-5 hrs

WASHING