Decalcificati ON& Dehydration: By: Riman Mustafa Ibrahim

DECALCIFICATI
ON &
DEHYDRATION
BY: RIMAN MUSTAFA IBRAHIM
It’s a procedure where by calcium or lime
salts are removed from tissues after
fixation
This step is important to ensure the

DECALCIFICATI normal cutting of section and to prevent
ON the 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:
1. Acids
TYPES OF 2. Chelating agents
DECALCIFYI 3. Ion exchange resins
NG AGENT 4. Electrical ionization
1)ACIDS
They are the most

They are stable ,
widely used agents
easily available and
for routine
inexpensive
decalcification for
compared to other
large amounts of
agents
bony tissue .
the most commonly
used and very rapid
1) Nitric Acid
producing minimal
distortion .
Some acids that
can be used for
decalcification It can be used as either
Recommended simple solution or
concentration is 5-10% 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 tissue acid –produce less Nitric acid – most
softner tissue distortion and rapid decalcifying
good nuclear agent
staining
It's good for nuclear staining
but can cause great tissue
distortion
2)
HYDROCHLORIC
ACID
Von Ebner's Fluid :
Moderately rapid Recommended
Good cytological
decalcifying No washing out for small pieces
staining
agent of bone
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
5) Sulfuric Acid – very
4) Trichloroacetic acid –
weak decalcifying solution
has a good nuclear staining
suitable only for minute
Other acidic and no washing out
pieces of bone
decalcifying 7)Citric acid –Citrate

agents 6) Chromic Acid
( Fleming's fluid ) - can be
buffer solution ph4.5 -
permits excellent nuclear
used as both fixative and
& cytoplasmic staining
decalcifying agent , yet its
and doesn’t produce any
corrosive and carcino genic
tissue 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
EXCHANG Cellular details are well preserved and permits

E RESIN 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
ELECTROPHORE Time is shorten cause of the heat and

SIS electrolytic reaction
Idle for small bone fragment

1) Concentration and volume
FACTORS
INFLUENCUNG More concentrated acid solution
RATE OF decalcify bone more rapidly but is
DECALCIFICATI more harmful to the tissue
ON
The recommended ratio of fluid to

tissue volume is 20:1
2) Temperature
FACTORS INFLUENCI
NG
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 R 4) Size of specimen

ATE 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
Measure the 1)Physical or Mechanical Test
extend of Done by touching or bending the tissue with the fingers to

determine the consistency of tissue
decalcificatio
n 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
Detection of calcium
It’s the simplest ,
in acid solution by
reliable and
precipitation of
convenient method
3)
insoluble calcium
used for routine
hydroxide or
purposes
calcium oxalate
CHEMICA
L Solution used : The decalcifying
METHOD Ammonium
hydroxide ,
concentrated and
fluid is changed
every 24-48 hrs and
the chemical test is
saturated aqueous performed on the
ammonium oxalate 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-
DECALCIFICATI A) saturated lithium carbonate solution
ON
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 damage Perenyi's fluid: may
the microtome act as both
knives may require decalcifying agent
tissue softener aside and tissue softener
from decalcification
TISSUE
SOFTNER Molliflex : tissue
immersed in it may
2% HCl or 1%
appear swollen and
Hydrochloric acid in
soapy yet it doesn't
70% alcohol
affect normal
processing
THE END
THANK YOU :)

Decalcificati ON& Dehydration: By: Riman Mustafa Ibrahim

Uploaded by

Copyright:

Available Formats

Decalcificati ON& Dehydration: By: Riman Mustafa Ibrahim

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Decalcificati ON& Dehydration: By: Riman Mustafa Ibrahim

Uploaded by

Copyright:

Available Formats

DECALCIFICATI

This step is important to ensure the

Specimen that require decalcification are

They are the most

Recommended for routine decalcification of

3) FORMIC Can be used as fixative and decalcifying agent

decalcifying 7)Citric acid –Citrate

Most common chelating agent is EDTA

2) CHELATING Recommended for immunohistochemical and enzyme staining

Not recommended for urgent biopsy

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

EXCHANG Cellular details are well preserved and permits

It's very slow and degree of decalcification

ELECTROPHORE Time is shorten cause of the heat and

Idle for small bone fragment

The recommended ratio of fluid to

Mechanical agitation and moving of tissue in solution

FACTORS INFLUENCUNG R 4) Size of specimen

Increase in size and consistency of tissue require longer

Ideal time required is 24-48 hours

Measure the 1)Physical or Mechanical Test

extend of Done by touching or bending the tissue with the fingers to

Small calcified foci may not be detected

It's not recommended for mercuric –

Acid can be removed from tissues or neutralized chemically

Adequate water rinsing can usually be accomplished in

You might also like

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.