DECALCIFICATION

BY: RIMAN MUSTAFA IBRAHIM

 It’s a procedure where by calcium or
lime salts are removed from tissues
after fixation and before impregnation .

This step is important to ensure the normal

cutting of section and to prevent the
DECALCIFICATION obscuring of microanatomical detail caused
by bone dust .

Specimen that require decalcification are

like bone , teeth and tuberculous lung .
 • Calcium can be removed by
the use of the
following agents:

TYPES OF 1. Acids
DECALCIFYING 2. Chelating agents
AGENT 3. Ion exchange resins
4. Electrical ionization
 1)ACIDS

They are the

They are stable ,
most widely used
easily
agents for
available and
routine
inexpensive
decalcification
compared to
for large amounts
other agents
of bony tissue .
 the most commonly
used , very rapid and
1) Nitric Acid
produce minimal
Some acids that distortion .

can be used for

decalcification It can be used
Recommended as either simple
concentration is 5- solution or
10% combined with other
agents
 NITIRC ACID

01 02 03 04
A) 10% Aqueous B) Perenyi's fluid – C) Formol – nitirc D) Phloroglucin-
nitric acid decalcifier and acid –produce less Nitric acid – most
tissue softner tissue distortion rapid decalcifying
and good nuclear agent
staining
 It's good for nuclear
staining but can cause
great tissue distortion
2) HYDROCHLORIC
ACID

Von Ebner's Fluid :

Moderately
Good Recommended
rapid No washing
cytological for small pieces
decalcifying out
staining of bone
agent
 Safer to handle , lesser tissue distortion
and better nuclear staining

Recommended for routine decalcification

of post mortem research tissue

3) FORMIC Can be used as fixative and decalcifying

agent
ACID
Recommended when
immunohistochemical staining is needed

Requires to be neutralized
 4) Trichloroacetic acid – 5) Sulfuric Acid – very
has a good nuclear weak decalcifying
staining and no washing solution suitable only for
Other acidic out minute pieces of bone

decalcifying
6) Chromic Acid ( 7)Citric acid –Citrate buffer
agents Flemming's fluid ) - can
be used as both fixative
solution ph4.5 - permits
excellent nuclear &
and decalcifying agent cytoplasmic staining and
, yet its corrosive and doesn’t produce any tissue
carcino genic distortion .
 It combines with calcium ions and other salts to form
weak complexes and facilitate removal of calcium salt

Most common chelating agent is EDTA

2) CHELATING Recommended for immunohistochemical and enzyme

staining
AGENT
Excellent for electron microscopy

Not recommended for urgent biopsy

 Removes calcium ions form formic acid
containing decalcifying solutions

3) ION 20 to 30x the volume of the tissue

EXCHANGE Cellular details are well preserved and
RESIN permits excellent staining results

It's very slow and degree of decalcification

cannot be measured by chemical method
 Utilizes electricity , where in positively
charged calcium ions are attracted to a
negative electrode and subsequently
removed from decalcifying solution

Time is shorten cause of the heat and

ELECTROPHORESIS electrolytic reaction

Idle for small bone fragment

 1) Concentration and volume

FACTORS
More concentrated acid solution
INFLUENCUNG
RATE OF decalcify bone more rapidly but
DECALCIFICATION is more harmful to the tissue

The recommended ratio of fluid

to tissue volume is 20:1
 2) Temperature

FACTORS INFLUENCING
RATE OF
DECALCIFICATION

Optimum
temperature is a
room temp range
of 18C-30C
 3) Agitation

Mechanical agitation and moving of tissue in solution

influences fluid exchange , accelerates the rate of
diffusion and speed up decalcification process

FACTORS INFLUENCUNG RATE 4) Size of specimen

OF DECALCIFICATION

Increase in size and consistency of tissue require

longer period to complete decalcification

Ideal time required is 24-48 hours

 There are 3 ways to measure the extend of
decalcification

1)Physical or Mechanical Test

Measure the
Done by touching or bending the tissue with the
extend of fingers to determine the consistency of tissue
decalcification This method can disrupt soft tumor from the bone or
cause false positive leading to potential miss diagnosis

Small calcified foci may not be detected

 This is the most ideal, most sensitive and
most reliable method due to its ability to
detect the smallest focus of calcium .

2) X-RAY/RADIOLOGICAL
It’s the most expensive method
METHOD

It's not recommended for mercuric –

chloride fixed tissue due to its radio-
opacity which will interfere with the correct
interpretation of the plate
 It’s the simplest , reliable and convenient
method used for routine purposes

Detection of calcium in acid solution by

precipitation of insoluble calcium
3) CHEMICAL hydroxide or calcium oxalate

METHOD Solution used : Ammonium hydroxide ,

concentrated and saturated aqueous
ammonium oxalate

The decalcifying fluid is changed every 24-

48 hrs and the chemical test is performed
on the discarded fluid
 This step is done to remove the decalcifying agent
after decalcification is completed

Acid can be removed from tissues or neutralized

chemically by immersing the decalcified bone for
several hours in either

POST- A) saturated lithium carbonate solution

DECALCIFICATION

B) 5-10%aqueous sodium bicarbonate

Adequate water rinsing can usually be accomplished

in 30mins for small samples and 1-4 hrs for large
specimen
 Hard tissues which
are liable to
Perenyi's fluid: may
damage the
act as both
microtome knives
decalcifying agent
may require tissue
and tissue softener
softener aside from
decalcification
TISSUE
SOFTNER Molliflex : tissue
immersed in it
2% HCl or 1%
may appear
Hydrochloric acid
swollen and soapy
in 70% alcohol
yet it doesn't affect
normal processing
 THE END
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