DECALCIFICATION

OBJECTIVES:
1. Describe decalcification
2. Explain the different methods of
decalcification and the different agents used
therein
3. Describe the different methods of
determining end points of decalcification
• Decalcification describes the technique for
removing mineral (Calcium or lime salts) from
bone or other calcified tissue so that good-
quality paraffin sections can be prepared that
will preserve all the essential microscopic
elements.
• Decalcification is carried out after the
specimen has been thoroughly fixed and prior
to impegnation
Bone: properly decalcified Improperly decalcified tissues
obscure microanatomic details
 Fixation Considerations
• It is important to provide ready access for the fixative
to penetrate the bone, so skin and soft tissue should be
removed from large specimens if practicable.
• Bone specimens should be sawn into thin slices as soon
as possible to enhance fixation and an adequate
volume of fixative provided.
• High-quality fine tooth saws should be used to prepare
bone slices. Coarse saws can cause considerable
mechanical damage and force bone fragments into the
soft tissues present in the specimen.
 Fixation Considerations
• Poorly-fixed specimens become macerated
during decalcification and stain poorly
afterwards.
• This is very noticeable in areas containing
bone marrow. It is therefore common practice
for laboratories to extend fixation times for
bone specimens before commencing
decalcification.
Specific purpose of decalcified sections
• Decalcified sections are used for the
examination of bone marrow and for the
diagnosis of tumours, infections or for other
purposes.
• The specimens may be in the form of iliac
crest trephines or bone pieces removed at
operation (such as femoral heads) or dissected
from amputation specimens.
• Other tissues may undergo calcification
associated with degenerative processes:
– necrosis (dystrophic calcification)
• Tuberculous lungs
– walls of blood vessels or in kidney, lung or elsewhere
(metastatic calcification, arteriosclerotic vessels).
• “surface decalcification” to a paraffin block to
allow sections to be obtained where the presence
of calcium was not anticipated when the
specimen was processed.
Trephines
Bone: properly decalcified
Improperly decalcified tissues obscure
microanatomic details
Improperly decalcified tissues obscure
microanatomic details
 Preparation of tissues:

• The calcified hard tissues should be first cut into

small pieces (2 to 6mm) with a thin blade, hacksaw or
sharp knife in order to minimize the tearing of the
surrounding tissues
• This process is followed by fixation in buffered
formalin or any other desired fixative.
• After fixation tissues must be thoroughly washed and
excess fixative should be removed before the
specimen is subjected to decalcification
DECALCIFYING AGENTS
 3 Main types of Decalcifying Agents
• Those based on strong mineral acids
• Those based on weaker organic acids
• Those composed of chelating agents
 Strong Acids
• Strong acids such as hydrochloric or nitric acid
at concentrations up to 10% are the most
common and most rapid in action/ Routine
– but if used for an excessive time will rapidly cause
a loss of nuclear staining and can macerate
tissues.
• Combined with formaldehyde or alcohol to prevent this
– end-point test is used to minimise exposure of the
specimens to these agents.
• Formol-Nitric Acid
– Rapid, recommended for urgent biopsies
– Relatively good nuclear staining
– Less tissue destruction than 10% nitric acid
Disadvantage:
- Yellow color by nitrous acid impairs staining reaction
(prevented by neutralizing tissue with 5% Na Sulfate
and washing in running tap water for 12 hours or
addition of 0.1% Urea will make the discoloration
disappear)
- Solution should be used inside a fume hood
• Phloroglucin-Nitric Acid
– Most rapid decalcifying agent recommended for urgent
biopsies
Disadvantage:
- Poor nuclear staining
- Tissue distortion and yellow discoloration (neutralized by
5%Na Sulfate and washing in running water for 24 hours)
- Complete decalcification cannot be determined by
chemical means
- When decalcification is complete, the acid is removed by 3
changes of 70-90% ethanol- NOT water (excessive swelling
and deterioration of tissue)
• Hydrochloric Acid
– Inferior to Nitric acid: slower and distorts tissues
more
– Better nuclear staining that Nitric acid
• Prolonged treatment with a mineral acid decalcifier
 Table 1: Mineral acid decalcifiers
Decalcifier Formula Comment
6
Nitric acid 5% in distilled water Rapid in action, exceeding
end-point will impair staining.
6
Perenyi’s fluid (1882) 10% nitric acid 40ml A traditional decalcifier that
0.5% chromic acid 30ml decalcifies more slowly than
Absolute alcohol 30ml aqueous nitric acid. Quite
rapid in action, exceeding
end-point will impair staining.
3
Hydrochloric acid 5-10% in distilled water Formalin should be washed
from specimen before placing
in HCl to avoid the formation
of bis-chloromethyl ether (a
carcinogen). Rapid in action,
exceeding end-point will
impair staining.
6
Von Ebner’s solution Sodium chloride saturated Rapid in action, exceeding end-
soln. 50ml point will impair staining.
Distilled water 42ml
Hydrochloric acid 8ml
 Weak Acids
• Weak acids such as formic acid are popular
and are widely used for decalcification.
• Formic acid can be used as a simple 10%
aqueous solution or combined with formalin
or with a buffer.
• Although it is slower than the strong acid
agents it is much gentler in action and less
likely to interfere with nuclear staining.
• An example of a proprietary decalcifier based
on formic acid is Surgipath’s Decalcifier I®. It
also contains formalin and is claimed to fix as
well as decalcify and be gentle in action.
• Other acids such as trichloracetic acid (TCA)
have also been used. Picric acid, as a
component of some fixatives has weak
decalcifying
• Formic acid
– Recommended for decalcification of post mortem
research tissues
– Not suitable for urgent biopsies
– The only weak acid used extensively as a primary
decalcifying agent
– Rate of decalcification is accelerated by adding
citrate by chelating calcium as it is liberated
• Picric acid and Acetic acid cause tissue to swell
and are NOT used alone as decalcifying agents
– Components of Carnoy’s and Bouins fixatives
• May be used as fixatives and decalcifying agents in
urgent cases when calcification is minimal
– For small pieces of bones and teeth
– Excellent nuclear and cytoplasmic staining
– Compatible with immunohistochemical staining
• Trichloroacetic acid
– Good nuclear staining, no washing out needed
– Weak decalcifying agent: not for dense tissues,
suitable only for small spicules of bone
– Slow acting
• Sulfurous acid
– Very weak decalcifying agent suitable only for
minute pieces of bone
• Chromic acid
– Fixative and decalcifying agent for minute bone
spicules
– Inhibits nuclear staining with hematoxylin
– Forms precipitates at the bottom of the container
– Chemical end point determination cannot be used
– Environmental toxin: highly corrosive to skin and
mucus membranes, carcinogenic, cannot be disposed
via drain, suitable PPE is not readily available or
practical for laboratory use
• Citric acid-Citrate buffer solution (pH 4.5)
– Excellent nuclear and cytoplasmic staining
– Does not produce cell or tissue distortion
– Action is too slow for routine purposes
Table 2: Weak acid decalcifiers
Decalcifier Formula Comment
8
Formic acid 10% in distilled water A simple effective
decalcifier.
8
Evans and Krajian Formic acid 25ml An effective formic
Sodium citrate 10g acid decalcifier
Distilled water 75ml buffered with citrate.

8
Kristensen Formic acid 18ml An effective formic
Sodium formate 3.5g acid decalcifier
Distilled water 82ml buffered with formate

8
Gooding and Stewart Formic acid 5-25ml A formic acid
40%formaldehyde 5ml decalcifier with added
Distilled water 75ml formalin, claimed to fix
and decalcify.
 Chelating agents

• Chelating agents such as ethylenediaminetetracetic acid

(EDTA), work by capturing the calcium ions from the surface
of the apatite crystal, slowly reducing its size.
• The process is very slow but very gentle (weeks may be
required depending on the size of the specimen),
– not suitable for urgent specimens but more appropriate for
research applications where very high quality morphology is
required or particular molecular elements must be preserved
for techniques such as IHC, FISH or PCR.
– It is used at a concentration of approximately 14% as a
neutralized solution.
– The rate at which EDTA will decalcify is pH dependent. It is
generally used at pH7.0. It works more rapidly at pH10 but some
tissue elements can be damaged at alkaline pH. 10
 – Forms minimal histological artifacts, usuall caused
by production of CO2 bubbles
– Chemical end point determination can be used
• Disadvantage:
– Very slow, hence not used routine
– Causes slight tissue hardening
– Inactivates alkaline phosphatase activity, which
can be restored by addition of magnesium
chloride
Chelating agents

Decalcifier Formula Comment

1
Neutral EDTA EDTA disodium Acts slowly but
salt 250g causes little tissue
Distilled water damage.
1750ml Conventional
Bring to pH 7.0 by stains are largely
adding sodium unaffected.
hydroxide (about
25g will be
needed).
• Ion exchange resins (Amonium form of
polystyrene resin)
– Removes calcium ions from formic acid acid
containing decalcifying solutions
– Not for mineral acids nitric acid and HCl
– End point is determined by physical or Xray
methods
*Look up advantages and disadvantages in Gregorio
• Electrophoresis
– Positively charged calcium ions are attracted to a
negative electrode and subsequently removed
from the decalcifying solution (Formic acid and
HCl)
– Similar in principle to chelating agents but it
requires electricity
– Used for small bone fragments
– Prolonged: maceration and destruction of tissue
components with poor staining
 Factors influencing the rate of
decalcification
Concentration
• affect the rate at which calcium is removed
• strike a balance between speed and degree of
tissue damage
• concentration of active agent will be depleted as
it combines with calcium and so it is wise to use a
large volume of decalcifier and renew it several
times during the decalcification process.
(recommended at 20:1)
• More concentrated: more rapid: more harm to
tissue
 Factors influencing the rate of
decalcification
Temperature
• Increased temperature will speed up the
decalcification rate but will also increase the
rate of tissue damage so must be employed
with great care. (recommended temperature
of 18-30 degrees Celsius)
 Factors influencing the rate of
decalcification
Agitation influences fluid exchange
• Gentle agitation may increase the rate slightly. Done by
low speed rotation, rocking, stirring, bubbling air
• Sonication vigorously agitates fluid and tissues causing
tissue disruption and cellular debris on the floor of
container
Fluid access
• As with fixation fresh decalcifier should have ready
access to all surfaces of the specimen.
• This will enhance diffusion and penetration into the
specimen and facilitate solution, ionization and
removal of calcium.
 Determining the end-point of
decalcification
• Over-decalcification, particularly with the
strong acid decalcifiers, spoils the staining of
basophilic elements such as cell nuclei and in
some circumstances can cause maceration of
the softer tissue elements.
• On the other hand specimens that are
incompletely decalcified may be difficult or
impossible to section.
 X-ray the specimen.
• The best method, particularly with large
specimens such as femoral heads
• A good-quality X-ray will clearly reveal tiny
residual calcium deposits and allow further
treatment if required.
• Not for mercuric chloride fixed tissues due to
its radio opacity
 Chemical test
• Applied when some acid decalcifiers are used (particularly
formic acid).
• Ammonium oxalate solution is added to a sample of the
final change of decalcified that has been neutralized with
ammonium hydroxide.
• If calcium is present a precipitate of calcium oxalate will
form indicating that decalcification is probably incomplete
and a longer time in decalcified is required.
• Decalcifying agent is changed every 24-48 hours
• Of course this test is best done on a relatively recent
change of decalcifier (exposed to the tissue for say, one
hour only).
*Note the process and interpretation in Gregorio
 Physical tests
• Require manipulation, bending probing or trimming of
the specimen to “feel” for remaining calcified areas.
• Whilst this method may be successful in experienced
hands it is generally considered to be unreliable.
• Mechanical damage can occur during bending or
probing/pricking with fine needle and small deposits of
calcium can easily be missed.
• A method of determining the endpoint by carefully
weighing the specimen after rinsing and blotting has
also been described. This may be an effective method
for large specimens.10
• If you believe the decalcification end-point is close
and you wish to slow the process down so as to
avoid over-decalcification and consequent tissue
damage
• specimens can be removed from decalcifier, rinsed
and placed back into formalin (important if
hydrochloric acid is being used).
• Decalcification can then be resumed when
convenient.
• An alternative is to refrigerate the specimen at 4˚C
in its decalcifier to slow down the process.1
 Treatment following decalcification
and prior to processing/ Post
Decalcification
• Extensive washing in tap water or the application of
alkaline solutions.
• Generally a short, effective wash in tap water should be
sufficient as any remaining acid will be removed during
processing.
• It is important to remove the bulk of the decalcifier to avoid
contaminating the processing reagents and the processor
with acid.
* Note the process of Post decalcification and tissue softeners
in Gregorio
 Surface decalcification

• This is a method of dealing with small unexpected

deposits of calcium that may be encountered in
paraffin blocks (Resistance and a grating sensation
during sectioning)
• Normally it is after trimming the block in the
microtome to expose the specimen that the calcium is
discovered. It is important at this stage to try to avoid
disrupting the block surface extensively.
• After the tissue has been exposed the block can be
removed from the microtome and placed face down in
an acid decalcifier (10% HCl) for 15 – 60 minutes.
 Surface decalcification

• This surface treatment will allow the decalcifier

to penetrate a small distance into the block and
dissolve the calcium.
• The block can then be thoroughly rinsed in water
to remove residual acid, chilled and sectioned.
• Careful realignment of the block will be required
because the decalcifier will penetrate a very small
distance into the block allowing only a couple of
sections to be taken.
• Repeat surface decalcification is done as
needed whenever calcified elements are
encountered
• Staining properties at this point are affected
especially causing failure of nuclear chromatin
to take up hematoxylin
 Conclusion

• Decalcification is a straightforward process but to

be successful requires:
• A careful preliminary assessment of the specimen
• Thorough fixation
• Preparation of slices of reasonable thickness for
fixation and processing
• The choice of a suitable decalcifier with adequate
volume, changed regularly
• A careful determination of the endpoint
• Thorough processing using a suitable schedule.