HISTOPATHOLOGIC TECHNIQUES
DECALCIFICATION 5. It is recommended for urgent biopsy, and for needle and
➢ Decalcification is the removal of calcium ions from a
bone or calcified tissue through a histological
process that makes them flexible and easier to cut.
➢ Decalcification adjusts the hard substance of bones
to the softness of paraffin embedding medium.
Bones are the main object of decalcification in a
surgical pathology laboratory, but other specimens,
such as teeth, calcified tumors and calcified heart
valves also require this procedure.
There are three main types of decalcifying agents:
• Those based on strong mineral acids
• Those based on weaker organic acids
• Those composed of chelating agents.
ACID DECALCIFYING AGENTS
➢ Acid decalcifying agents are the most widely used
agents for routine decalcification of large amounts
of bony tissues because they are stable, readily
available, and relatively inexpensive as compared to
other decalcifying agents.
Strong Mineral Acids
➢ Strong acids such as hydrochloric or nitric acid at
concentrations up to 10% are the most rapid in
action but if used longer than necessary will rapidly
cause a loss of nuclear staining and can macerate
tissues.
➢ Generally proprietary decalcifiers that are claimed to
be rapid in action are based on strong acids, most
commonly hydrochloric acid, and should be used
conservatively with attention to the provided
instructions if good results are to be obtained.
I. NITRIC ACID
➢ Nitric acid is the most common and the fastest
decalcifying agent used so far, utilized both as a
simple solution or combined with other reagents.
➢ This may be used as simple aqueous solutions with
recommended concentrations of 5- 10%. It is a
very rapid decalcifying agent, producing minimal
distortion and is, therefore, recommended for
routine purposes.
➢ It has, however, the disadvantage of inhibiting
nuclear stains and destroying tissues, especially in
concentrated solutions. The endpoint of
decalcification must be carefully watched for, to
prevent progressive tissue damage and impaired
staining. This may be prevented by combining nitric
acid with formaldehyde or alcohol.
Aqueous Nitric Acid Solution 10%
FORMULA:
Concentrated nitric Acid ---------10 ml.
Distilled water added up to -----100 ml.
DECALCIFICATION TIME: 12-24 hours
Advantages:
1. It is rapid in action.
2. It produces minimum distortion of tissues.
3. It produces good nuclear staining (although less than in
slower acting agents).
4. The acid may be easily removed by 70% alcohol.
small biopsy specimens to permit rapid diagnosis within 24
hours or less.
6. It can be used for large or heavily mineralized cortical
bone specimen if decalcification progress is carefully
monitored by a decalcification endpoint test.
Disadvantages:
1 Prolonged decalcification may lead to tissue distortion.
2. It can seriously damage tissue stainability.
3. It imparts a yellow color with nitrous acid, thereby
impairing the staining reaction of the tissue.
4. Old nitric acid solution is particularly damaging and should
be replaced with fresh stock solution.
5. Strong acids tend to be more damaging to tissue antigens
for immunohistochemical staining, and enzymes may be
totally lost.
Formol-Nitric Acid
FORMULA:
Concentrated nitric acid -------10 ml.
Strong formaldehyde, 40% -----5 ml.
Distilled water --------------------85 ml.
DECALCIFICATION TIME: 1-3 days
Advantages:
1. It is rapid-acting; hence, is recommended for urgent
biopsies.
2. Nuclear staining is relatively good.
3. It produces less tissue destruction than 10% aqueous
nitric acid.
Disadvantages:
1. The yellow color imparted by nitrous acid formation
will impair staining reaction of the cell. This may be
prevented by neutralizing the tissue with 5%
sodium sulfate and washing in running tap water
for at least 12 hours. Addition of 0.1% urea to pure
concentrated nitric acid will also make discoloration
disappear without considerably affecting the
efficiency of the decalcifying solution.
2. 2. The solution should be used inside a fume hood.
Perenyi’s Fluid
FORMULA:
Nitric acid 10% ------------40 ml.
Chromic acid 0.5% ---------30 ml.
Absolute ethyl alcohol ----30 ml.
Mix shortly before use. Chromic acid must be
collected for proper disposal.
DECALCIFICATION TIME: 2 - 7 days
Advantages:
1. It is recommended for routine purposes.
2. It decalcifies and softens tissues at the same time.
3. Nuclear and cytoplasmic staining is good.
4. Maceration is avoided due to the presence of chromic acid
and alcohol.
Disadvantages:
1. It is a slow decalcifying agent for dense bones; hence, is
not recommended for urgent diagnosis.
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 HISTOPATHOLOGIC TECHNIQUES
2. Complete decalcification cannot be determined by
chemical test because a precipitate is formed upon the
addition of ammonia to Perenyi's fluid even in the absence of
calcium ion. This may be dissolved by adding glacial acetic
acid drop by drop. About 0.5 ml. of saturated aqueous
ammonium oxalate is then added to the solution.
Reappearance of a white precipitate within 30 minutes will
reaffirm the presence of calcium in the agent, signifying that
decalcification is still incomplete.
Phloroglucin-Nitric Acid
FORMULA:
Concentrated nitric acid -------10 ml.
Phloroglucin ----------------------1 gm.
Nitric acid 10% ------------------100 ml.
(To be added after disappearance of dense white fumes
formed by combining the first two ingredients.)
DECALCIFICATION TIME: 12-24 hours
Advantage: It is the most rapid decalcifying agent so far,
recommended for urgent cases.
Disadvantages:
1. Nuclear staining is poor.
2. Prolonged decalcification produces extreme tissue
distortion.
3. Yellow color must be neutralized with 5% sodium sulfate
and thoroughly washed with running tap water for at least
24 hours.
4. Complete decalcification cannot be determined by
chemical means. When decalcification is complete, the acid
must be removed by three changes of 70% to 90% ethanol,
since washing in watery solutions will lead to excessive
swelling and deterioration of tissue. When the sections are
cut, the slides are brought to water and placed in 1%
aqueous lithium carbonate for I hour, washed in later for 15
minutes, and then stained.
II. HYDROCHLORIC ACID
Von Ebner's Fluid
FORMULA:
Saturated aqueous solution of NaCl --------50 ml.
36% concentrated hydrochloric acid -------8 ml.
Distilled water -----------------------------------50 ml.
Advantages:
1. It permits relatively good cytologic staining.
2. It is a moderately rapid decalcifying agent.
3. It does not require washing out before dehydration.
4. It is recommended for teeth and small pieces of bone.
Disadvantage: The extent of decalcification cannot be
measured by a chemical test.
III. FORMIC ACID
FORMULA:
Formic acid (Sp. grav. 1.20) -----10 ml.
Normal saline 10% --------------90 ml.
DECALCIFICATION TIME: 2-7 days
Advantages:
1. It may be used both as a fixative and decalcifying agent.
2. It permits excellent nuclear and cytoplasmic staining.
3. It is recommended for small pieces of bones and teeth.
4. It is suitable for most routine surgical specimens,
particularly when immunohistochemical staining is needed.
Disadvantages:
1. It is relatively slow; hence, is not suitable for urgent
specimens. Decalcification may be hastened by increasing
the proportion of formic acid to 25 ml. However, such
concentration may make the solution opaque, thereby
interfering with the staining results.
2. It requires neutralization with 5% sodium sulfate, and
washing out to remove the acid from the tissue.
Formic Acid-Sodium Citrate Solution
FORMULA:
Aqueous sodium citrate 20% -------50 ml.
Formic acid 45% -----------------------50 ml.
DECALCIFICATlON TIME: 3 -14 days
Advantages:
1. It permits better nuclear staining than nitric acid method.
2. It is recommended for autopsy materials, bone marrow,
cartilage and tissues studied for research purposes.
Disadvantages:
1. It is relatively slow; hence, is not recommended for
routine purposes and for dense tissues.
2. It requires neutralization with 5% sodium sulfate.
IV. TRICHLOROACETIC ACID
FORMULA:
Trichloroacetic acid ---------5 gm.
Formal saline 10% ------------95 ml.
DECALCIFICATION TIME: 4- 8 days
Advantages:
1. It permits good nuclear staining.
2. It does not require washing out; the excess acid may be
removed by several changes of 90% alcohol, thus improving
tissue dehydration.
Disadvantages:
1. It is a weak decalcifying agent, not used for dense tissues,
and is suitable only for small spicules of bone.
2. It is very slow-acting; hence, is not recommended for
urgent examinations.
SULFUROUS ACID -is a very weak decalcifying solution
suitable only for minute pieces of bone.
V. CHROMIC ACID (FLEMMING'S FLUID)
FORMULA:
Chromic acid % ----------------15 ml.
Osmium tetroxide --------------4 ml.
2% Glacial acetic acid ----------1 ml.
Advantages:
1. It may be used both as a fixative and decalcifying agent.
2. It may be used for decalcifying minute bone spicules.
Disadvantages:
1. Nuclear staining with hematoxylin is inhibited.
2. It tends to undergo reduction and forms precipitates at
the bottom of the container thus requiring frequent changes
of solution.
3. Insoluble pigments are formed when decalcified tissue is
dehydrated with alcohol; hence, tissues must be washed out
prior to dehydration.
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 HISTOPATHOLOGIC TECHNIQUES

4. Degree of decalcification cannot be measured by the
routine chemical test.
Caution: Chromic acid is an environmental toxin.
1. Chromic acid is highly corrosive to skin and mucous
membranes.
2. It is carcinogenic.
3. Suitable protective material is not readily available or
practical for laboratory use.
4. Drain disposal is not a legitimate option for any solution
containing chromium, including subsequent processing of
fluids following fixation or rinses following staining
procedures involving chromium.
VI. CITRIC ACID-CITRATE BUFFER SOLUTION (pH
4.5)
FORMULA:
Citric acid (monohydrate) aqueous solution 7% 5.0
ml. Ammonium citrate (anhydrous) aqueous solution
7.4% 95.0 ml.
Zinc sulfate aqueous solution. 1% 0.2 ml.
Chloroform (as preservative) - a few drops
➢ The time required for decalcification is thereby
shortened due to the heat and electrolytic reaction
produced in the process.
➢ The principle is similar to that of chelating agents,
with the main difference that this process utilizes
electricity and is dependent upon a supply of direct
current to remove the calcium deposits.
Solution Used for Electrolytic Decalcification
Formic acid 88% ------------------------100 ml.
Concentrated hydrochloric acid ------80 ml.
Distilled water ----------------------------1000 ml.
➢ This method is satisfactory for small bone
fragments, processing only a limited number of
specimens at a time.
➢ Good cytologic and histologic details are, however,
not always preserved in tissues that have been
electrically decalcified.

DECALCIFICATION TIME: 6 days

Advantages:
1 It permits excellent nuclear and cytoplasmic staining.
2. It does not produce cell or tissue distortion.

Disadvantage: Its action is too slow for routine purposes.

Neutral EDTA
EDTA disodium salt ---------250 gm
Distilled water ---------------1750 ml
Bring to pH 7.0 by adding sodium hydroxide (about
25 gm will be needed). Neutral EDTA acts slowly but
causes little tissue damage. Conventional stains are
largely unaffected.

Advantages:
1. It permits excellent staining results.
2. It produces minimal cell and tissue distortion.
3. It forms minimal histological artifacts, usually caused by
production of CO2 bubbles.
4. Extent of decalcification can be measured by routine
chemical test.
5. EDTA is an excellent bone decalcifier for enzyme or
immuno- histochemical staining, and for electron
microscopy.
6. Enzymes require specific pH conditions in order to
maintain activity, and EDTA solutions can be adjusted to a
specific pH for enzyme staining.

Disadvantages:
1. It is very slow, and is therefore not recommended for
urgent and routine purposes.
2. It causes slight tissue hardening.
3. EDTA inactivates alkaline phosphatase activity, which can
be restored by addition of magnesium chloride.

ELECTROPHORESIS (ELECTRICAL IONIZATION)

➢ Electrophoresis is a process whereby positively
charged calcium ions are attracted to a negative
electrode and subsequently removed from the
decalcifying solution.

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