DECALCIFICATION ( LAB)

DECALCIFICATION

Removal of Calcium/ Lime salts in a tissue prior to dehydration

DECALCIFICATION

Heat and Agitation hastens the process

DECALCIFICATION

More acidic the reagent, the more rapid is the rate of

DECALCIFICATION

Suspending the tissue also hastens the decalcification process

NITRIC ACID

Most common and fastest agent. (5-10% concentrations)

NITRIC ACID

Imparts yellow color to a tissue because of the Nitrous

acid

NITRIC ACID

Acid can be removed by 70% Alcohol

FORMOL – NITRIC ACID

Rapid acting, for urgent biopsies

FORMOL – NITRIC ACID

Must be inside a fume hood

FORMOL – NITRIC ACID

Add Urea Crystals and 5% Sodium Thiosulfate to remove the yellow color

PERENYI’S FLUID

Decalcifies and softens at the same time

PERENYI’S FLUID

Nuclear and Cytoplasmic staining is good

PERENYI’S FLUID

Maceration is avoided due to the presence of alcohol

and chromic acid

PHLOROGLUCIN – NITRIC ACID

Most rapid decalcifying agent so far. Recommended

for urgent works

PHLOROGLUCIN – NITRIC ACID

Yellow color is neutralized by 5% Sodium Sulfate,

washed with running water for at least 24 hours

VON EBNER’S FLUID

Good cytological staining

VON EBNER’S FLUID

No washing prior to dehydration

VON EBNER’S FLUID

Recommended for teeth and small pieces of bone

CHROMIC ACID / FLEMMING’S SOLUTION

Nuclear staining with hematoxylin is inhibited

CHROMIC ACID / FLEMMING’S SOLUTION

Fixative and Decalcifying agent

CHROMIC ACID / FLEMMING’S SOLUTION

High corrosive to skin and mucus membranes,

Carcinogenic

CHROMIC ACID / FLEMMING’S SOLUTION

Drain disposal is not a legitimate option

CHELATING AGENTS

agents that bind with calcium ions and other salts to form weakly dissociated complexes and facilitate the removal of calcium salts

ELECTROPHORESIS

Positively charged calcium ions migrate to the negative

electrode. Calcium is therefore effectively removed

from the decalcifying agent

ELECTROPHORESIS

For small bone fragment

ION EXCHANGE RESIN

Ammonium form of polystrene resin removes calcium from formic acid – containing decalcifying solution.

ION EXCHANGE RESIN

Not recommended for fluids containing mineral acids

such as nitric acid or hydrochloric acid

ION EXCHANGE RESIN

Decalcification is determined by physical means or X-

Ray method

ETHYLENE DIAMINE TETRAACETIC ACID

(EDTA)

Binds with Calcium and Magnesium

ETHYLENE DIAMINE TETRAACETIC ACID

(EDTA)

1-3 weeks chelation with small specimens; 6-8 weeks

for dense cortical bones

ETHYLENE DIAMINE TETRAACETIC ACID

(EDTA)

Faster action at ph 7 to 7.4; Optimal binding at ph 8 and higher, but may damage alkaline-sensitive protein linkages

ETHYLENE DIAMINE TETRAACETIC ACID

(EDTA)

DISADVANTAGES: Inactivates Alkaline Phosphatase activity, restore by adding Magnesium chloride

RADIOLOGIC METHODS

Most expensive yet most ideal

RADIOLOGIC METHODS

Most sensitive and most reliable

RADIOLOGIC METHODS

Use of FAXITRON and Kodak X-OMAT X-Ray film

RADIOLOGIC METHODS

Not for Mercuric chloride fixed tissues – Radio opacity interference.

CALCIUM OXALATE TEST

Simple, reliable and convenient method

CALCIUM OXALATE TEST

Calcium Oxalate precipitate, incomplete

decalcification

CALCIUM OXALATE TEST

No Precipitation, complete decalcification

MOLLIFLEX

tissue may appear swollen or soapy (but it doesn’t

interfere with the processing and staining.

PERENYI’S FLUID

decalcifying and softener