ROUTINE STEPS

DECALCIFICATION
 Decalcification
• The process of removing calcium or lime salts
from tissues.
• A procedure included only if tissue is calcified
or hard such as bones and teeth.
• Done after fixation
 Calcified Tissues
• Bones
• Teeth
• Arteriosclerotic vessels
• Hyperkeratotic vessels
• Some teratomas containing bony tissues
• Fingernails
• Fibroid tissues
• Pathological lesions that have become partly
calcified such as tuberculosis foci
 Fixation of a calcified tissue
• Best accomplished by selecting suitable blocks
2 – 4 mm. thick and placing them in:
* 10% NBF for 2 – 4 days (longer times is
required so that the nucleic acids will
become resistant to the hydrolytic action of
acids used in decalcification.
* Helley’s Fluid or Zenker’s fluid for 15-24
hours
 Purpose of decalcification
• Calcified tissues will interfere with the
accurate evaluation and examination of
histologic sections.
• To ensure and facilitate the normal cutting of
sections.
• To prevent obscuring the micro-anatomic
detail of tissue sections by bone dusts and
other cellular debris.
 Bone cutting materials
• Suitable bone tissue blocks (2-4 mm thick) are
made using any of the following saws:
• Fine hacksaw
bone cutting materials……Jigsaw
• Jig Saw
Bone cutting materials…..Fretsaw
bone cutting materials…..Fret saw
• Jet JSS-16 Bench Top Variable Speed Fretsaw
Bone cutting materias…..Bandsaw
 Bone tissue block
• are trimmed with a hand razor or blade to
permit complete penetration the decalcifying
solution with minimal surface damage and
tissue distortion.
 Bone Marrow
• The process of decalcification maybe
expedited by cutting off the hard bone.
• Fixation must be done ‘asap’ after death or
after removal.
Characteristics of a Good Decalcifying
Agent
1. Must completely remove calcium salts
2. Must not produce considerable destruction
of cells and tissue components
3. Must not affect staining capacity of the cell,
particularly the nucleus
 Decalcifying Agents
1. Acids
2. Chelating Agents
3. Ion-exchange resins
4. Electrophoretic ions
 Methods
1. Acid Decalcifying Method
2. Chelation Method
3. Ion-exchange Resin Method
4. Electrolytic Ionization Method
 Acid Decalcification Method
• Most commonly used method
• Acids are most widely used for routine work
due to the ff. reasons:
1. They are stable
2. Easily available
3. Relatively inexpensive
 Acid….
• Volume of the acid/calcifed tissue:
1 oz. acid per gram of calcified tissue
• Acid should be changed once or twice a day
until decalcification is complete
• 10% Nitric Acid (HNO3) soln. most commonly
used and most recommended for routine
work.
acid……………Procedure
1. After complete fixation, select bone tissue
pieces
2. Wrap in gauze forming gauze bag suspended
by a thread dipped in melted paraffin wax (to
avoid the corrosive action of the acid.
3. Fill glass jar container with acid.
4. Suspend the ‘gauze bag’ into the glass jar for
the required period of time or until
decalcification is complete.
Factors affecting rate of decalcification
using acids
1. Concentration and volume of the acid
*high conc. and greater volume – hasten
2. Temperature
*optimum temperature: RT
*heat hastens process. Care must be observed.
Too rapid could result to complete digestion
with marked swelling and hydrolysis of the bony
matrix.
*37°C- impaired nuclear staining using Von
Gieson’s Stain for collagen fibers
*55°C – complete digestion within 24 -48 hours
factors……
3. Agitation
*moving the tissue in solution hastens rate of
diffusion and decalcification
4. Size and consistency
*larger and harder – long period
*dense bone tissues – 14 days or longer
NOTE:
Ideal Decalcification Time: 24 – 48 hours
Longer decalcification period requires daily change
of acid soln.
 Chelation Method
• Uses chelating agents
*These are substances that combine with
calcium ions and other salts (iron and
magnesium deposits) to form a weakly
dissociated complexes and facilitate removal
of calcium salts.
• EDTA (Ethylene Diamine Tetraacetic Acid –
most common
• Versene (di-sodium salt)
EDTA
 Ion-exchange Resin Method
• Ion exchange resins are ammonium form of
polystyrene resin, which removes calcium
from formic acid containing decalcifying
solution, thereby increasing solubility from
the tissue.
• Not recommended for fluids containing
mineral acids such as HNO3 and HCl.
 Procedure: ion-exchange….
1. About ½ inch layer thick of resin is spread
over the bottom of the container.
2. Place specimen on top of the layer
3. Add decalcifying agent (20 -30 X the volume
of tissue).
Decalcification Time: 1 – 14 days
 Electrophoretic –ion Method
• Also referred to as electrolytic decalcifying
agents used in electrophoresis.
• Electrophoresis – a process whereby (+)
calcium ions are attracted to the (-) electrode
and subsequently removed from the
decalcifying soln.
• Time required is shortened due to heat and
electrolytic reaction.
• Principle similar to chelation. Differs only in
the use of direct current to remove Ca ions.
Effects of Prolonged Decalcification
• Maceration and destruction of tissues
• Result to poor staining
 Effects of incomplete decalcification

• Will interfere with the normal cutting of tissue

sections.
• Will interfere with the staining of tissue
sections
NOTE:
• Degree of decalcification should be evaluated
to ensure complete decalcification.
 Tests to determine extent/ degree of
decalcification
1. Physical Method

* pricking with fine

needle or probe
 Test…
2. Mechanical Method
* touching or bending
 Test…..
3. X-ray / Radiologic
method
* most ideal
* most reliable
* not routinely used
 Test…..
4. Chemical Method
* simple, reliable and
convenient for
routine work
* determines the
presence of calcium
ions in the used
decalcifying soln.
 Chemical Method
1. Five ml. used soln. test with
blue litmus paper which
will turn red.
2. Neutralized by adding strong ammonia drop by
drop (red litmus will turn to blue).
3. Observe for cloudiness. If there is, it indicates
that there is still calcium ions present in the
solution
4. Immerse tissue in a new solution.
 Chemical Method
5. If solution remains clear after neutralization,
add 0.5 ml. of saturated aqueous solution of
ammonium oxalate.
6. Stand for 30 minutes and observe
7. Cloudiness indicates incomplete
decalcification
8. Absence of cloudiness after 30 minutes
indicates complete decalcification
 Chemical method
• If chemical method is to be done, the d.a.
solution must be prepared using distilled
water.
• If after decalcification, the tissue is still hard,
what will you do?
 Chemical Method
• Do………
……..Tissue Softening Method by using tissue
softeners.
 Tissue Softeners
• Perenyi’s Fluid
• Lendrum’s fluid
• Mollifex
• 2% HCl
• 1% HCl in 70% alcohol
End…