DECALCIFICATION

MARK LESTER B. CAUAN, RMT

DECALCIFICATION

 After fixation, selected pieces of tissues are taken

from the specimen, properly labeled and identified,
and then subjected to the subsequent steps of
processing.
 There are certain specimen(bones, teeth, and other
calcified tissues), which contain some amount of
calcium that is apt to interfere with accurate
evaluation and examination of histologic sections.
 One must see to it that all such extraneous
materials have been removed before proceeding to
the next step in the tissue processing.
DECALCIFICATION

 Process whereby calcium or lime salts are

removed from tissues (most especially bones
and teeth) following fixation.
 Carried out bythe use of chemical agents, either
with acids to form soluble calcium salts, or with
chelating agents that bind to calcium ions.
 Should be done after fixation or before
impregnation, to ensure and facilitate the normal
cutting of sections and prevent obscuring the
microanatomic detail of such sections by bone
dust and other cellular debris.
DECALCIFICATION

 Inadequate decalcification may result in poor cutting

of hard tissues and damage to knife edge during
sectioning.
 Bones and calcified tissues are cut into small pieces
with fine fret-saw and trimmed with a hand razor to
permit complete penetration of the decalcifying
solution with minimal surface damage and tissue
distortion.
 Rapid decalcifying agents are more likely to adversely
affect any subsequent staining. This is noticeable in
cell nuclei due to failure of nuclear chromatin to take
up hematoxylin and other basic dyes.
DECALCIFICATION

 A good decalcifying agent must be capable of

removing calcium salts from tissues completely
without producing considerable destruction of cells
and tissue components and without adversely
affecting the staining capacity of the cell, particularly
the nucleus.
 Calcium may be removed by any of the following
agents.
Acids
Chelating agents
Ion exchange resins
Electrical ionization (electrophoresis)
ACID DECALCIFYING
AGENTS
ACID DECALCIFYING
AGENTS
Most widely used agents for routine decalcification
of large amounts of bony tissues because they are
stable, easily available, and relatively inexpensive
as compared to other decalcifying agents.
 Nitric acid
 Hydrochloric acid
 Formic acid
 Trichloroacetic acid
 Sulfurous acid
 Chromic acid
 Citric acid
Nitric acid

 Most common and fastest decalcifying

agent used so far, utilized both as
simple solution or combined with other
reagents.
 5-10% solution

 
Aqueous Nitric acid solution 10%

FORMULA
 Concentrated nitric acid ------------10ml
 Distiiled water added up to ------100ml

DECALCIFICATION TIME: 12-24 hours

 
Aqueous Nitric acid solution 10%

ADVANTAGES:
 Rapid in action
 Produces minimum distortion of tissues
 Produces good nuclear staining
 Acid may be easily removed by 70% alcohol
 Recommended for urgent biopsy, and for needle
and small biopsy specimens to permit rapid
diagnosis within 24 hours or less.
 Used for large heavily mineralized cortical bone
specimen if decalcification progress is carefully
monitored by a decalcification endpoint test.
Aqueous Nitric acid solution 10%

DISADVANTAGES
 Prolonged decalcification may lead to tissue
distortion
 Can seriously damage tissue stainability
 Imparts yellow color with nitrous acid, thereby
impairing the staining reaction of the tissue.
 Old nitric acid solution is particularly damaging and
should be replaced with fresh stock solution.
 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 10ml
 Strong formaldehyde, 40% 5ml
 Distilled water 85ml

 DECALCIFICATION 1-3 days

Formol-nitric acid

ADVANTAGES
 Rapid-acting
 Nuclear staining is relatively good
 Produces less tissue destruction than
10% aqueous nitric acid
Formol-nitric acid

DISADVANTAGES
 Yellow color imparted by nitrous acid
formation will impair staining reaction of
the cell. This is prevented by
neutralizing the tissue with 5% sodium
sulfate and washing in running tap water
for at least 12 hours.
 Solutions should be used inside a fume
hood.
Perenyi’s fluid

FORMULA
 Nitric acid 10% 40ml
 Chromic acid 0.5% 30ml
 Absolute ethyl alcohol 30ml

 Mix shortly before use. Chromic acid must be

collected for proper waste disposal

 DECALCIFICATION TIME 2-7 days

Perenyi’s fluid
ADVANTAGES
 Recommended for routine purposes
 Decalcifies and softens tissues at the
same time
 Nuclear and cytoplasmic staining is
good
 Maceration is avoided to the presence
of chromic acid and alcohol
Phloroglucin-Nitric acid

FORMULA
 Concentrated nitric acid 10ml
 Phloroglucin 1 gram
 Nitric acid 10% 100ml
 (to be added after the disappearance
of dense white fumes formed by
combining the first two ingredients)

 DECALCIFICATION TIME 12-24 hours

Phloroglucin-Nitric acid

ADVANTAGES
 Most rapid decalcifying agent so far,
recommended for urgent use
Phloroglucin-Nitric acid

DISADVANTAGES
 Nuclear staining is poor
 Prolonged decalcification produces extreme
tissue distortion
 Yellow color must be neutralized with 5%
sodium sulfate and thoroughly washed with
running tap water for at least 24 hours.
 Complete decalcification cannot be
determined by chemical means.
Hydrochloric acid

 Inferior compared to nitric acid, slower in

action and greater distortion of tissues
Von Ebner’s fluid
FORMULA
 Saturated aqueous solution of NaCl 36% 50ml
 Concentrated hydrochloric acid 8ml
 Distilled water 50ml
Von Ebner’s fluid
ADVANTAGES
 Permits relatively good cytologic staining
 Moderately rapid decalcifying agent
 Does not require washing out before
dehydration
 Recommended for teeth and small
pieces of bone
Von Ebner’s fluid
DISADVANTAGES
 Extent of decalcification cannot be
measured by chemical test
Formic acid

 Moderate acting which produces better

nuclear staining with less tissue
distortion
 Safer to handle than nitric acid or
hydrochloric acid
 Recommended for post mortem
decalcification
Formic acid

 FORMULA
 Formic acid 10ml
 Formal saline 10% 90ml

 DECALCIFICATION 2-7 Days

Formic acid

ADVANTAGES
 Both fixative and decalcifying agent
 Permits excellent nuclear and cytoplasmic
staining
 Recommended for small pieces of bones
and teeth.
 Suitable for most routine surgical
specimens, particularly when
immunohistochemical staining is needed
Formic acid

DISADVANTAGES
 Relatively slow
 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% 50ml
 Formic acid 45% 50ml

 DECALCIFICATION TIME 3-14days

Formic Acid-Sodium Citrate Solution

ADVANTAGES
 Permits better nuclear staining than
nitric acid method
 Recommended for autopsy materials,
bone, cartilage and tissues studied for
research purposes.
Formic Acid-Sodium Citrate Solution

DISADVANTAGES
 Relatively slow
 Requires neutralization with 5% sodium
sulfate
Trichloroacetic acid

FORMULA
 Trichloroacetic acid 5grams
 Formol saline 10% 95ml

 DECALCIFICATION TIME 4-8 Days

Trichloroacetic acid

ADVANTAGES
 Permits good nuclear staining
 Does not require washing out, excess
acid may be removed by several
changes of 90% alcohol, thus improving
dehydration.
Trichloroacetic acid

DISADVANTAGES
 Weak decalcifying agent
 Slow acting

 
Sulfurous acid

 Very weak decalcifying solution suitable

only for minute pieces of bone.
Chromic acid (Flemming’s fluid)

FORMULA
 Chromic acid 15ml
 Osmium tetroxide 2% 4ml
 Glacial acetic acid 1ml
Chromic acid (Flemming’s fluid)

Advantages
 Both fixative and decalcifying agent
 Used for decalcifying minute bone
spicules
Chromic acid (Flemming’s fluid)

DISADVANTAGES
 Nuclear staining with hematoxylin is inhibited
 Tends to undergo reduction and forms
precipitates at the bottom of the container thus
requiring frequent changes of solution
 Insoluble pigments are formed when
decalcified tissue is dehydrated with alcohol
 Degree of decalcification cannot be measured
by routine chemical test.
Chromic acid: CAUTION
 Chromic acid is an environmental toxin.
 Highly corrosive to skin and mucous
membranes
 Carcinogenic
 Suitable protective material is not readily
available for routine purpose or practical for
laboratory use.
 Drain disposal is not legitimate option for any
solution containing chromium.
  
Citric acid –citrate buffer solution

 FORMULA
 Citric acid (monohydrate) aqueous solution 7% 5ml
 Ammonium citrate (anhydrous) aqueous solution 7.4% 95ml
 Zinc sulfate aqueous solution 1% 0.2ml
 Chloroform as preservatives (few drops)

 
 DECALCIFICATION TIME 6days
 
Citric acid –citrate buffer solution

ADVANTAGES
 Permits excellent nuclear and
cytoplasmic staining
 Does not produce cell or tissue
distortion

DISADVANTAGE
 Action is too slow for routine purposes
CHELATING AGENTS

 Are substances which combine with

calcium ions and other salts to form weakly
dissociated complexes and facilitate
removal of calcium salt.
 The most commonly used agent in the
market is ethylene diamine tetraacetic acid
(EDTA)
 Slow decalcifying agent
 1-3 days for small biopsies, 6 to 8 weeks for
dense cortical bone
CHELATING AGENTS

FORMULA
 EDTA disodium salt 5.5grams
 Distilled water 90ml
 Formaldehyde 10ml
CHELATING AGENTS

ADVANTAGES
 Permits excellent staining results
 Produces minimal cell and tissue distortion
 Forms minimal tissue histological artifacts, usually
caused by production of CO2 bubbles.
 Extent of decalcification can be measured by routine
chemical test.
 EDTA is an excellent bone decalcifier for
immunohistochemical or enzyme staining, and for
electrophoresis.
 Enzymes require specific pH conditions in order to
maintain activity.
CHELATING AGENTS

DISADVANTAGES
 Very slow
 Cause slight tissue hardening
 EDTA inactivates alkaline phosphatase
activity, which can be restored by
addition of magnesium chloride.
ION EXCHANGE RESIN

 Hastens decalcification by removing calcium

ions from formic acid-containing decalcifying
solutions, thereby increasing solubility from the
tissue.
 Not recommended for fluids containing mineral
acids such as nitric acid or hydrochloric acid.
 A layer of the ion exchange resin, about ½ inch
thick is spread over the bottom of the container
to be used and specimen is placed on top of it.
The decalcifying agent is then added, usually
20-30times the volume of the tissue.
ION EXCHANGE RESIN

 Tissue may be allowed to stay in

solution for 1-14 days
 The degree of decalcification may then
be measured by physical or X ray
method.
ION EXCHANGE RESIN

ADVANTAGES
 Cellular detail is well preserved
 Decalcification is hastened
 Daily washing of solution is eliminated
 Permits excellent staining results
 Produces minimal cell and tissue distortion
 Forms minimal histological artifacts, usually
caused by production of CO2 bubbles
ION EXCHANGE RESIN

DISADVANTAGES
 Degree of decalcification cannot be
measured by chemical measure
 Very slow
 Cause slight tissue hardening
ELECTROPHORESIS (ELECTRICAL IONIZATION)

 Process whereby positively charged

calcium ions are attracted to a negative
electrode and subsequently removed
from the decalcifying solution.
 The time required for decalcalcification
is thereby shortened due to the heat
and electrolytic reaction produced in the
process.
ELECTROPHORESIS (ELECTRICAL IONIZATION)

 Solutions used for electrolytic

decalcification;
 Formic acid 88% 100ml
 Concentrated hydrochloric acid 80ml
 Distilled water 1000ml
 Prolonged decalcification of tissue is
liable to prevent hydrolysis and lead to
maceration and destruction of tissue
components which are poorly stained.
FACTORS INFLUENCING RATE OF DECALCIFICATION

 The concentration and volume of decalcifying

agent and temperature at which the reaction
takes place are important considerations.
 More concentrated acid solutions decalcify
bone more rapidly, but are more harmful to
tissues
 High concentrations and greater amount of
fluid will increase the speed of the process.
 The recommended ratio of fluid to tissue for
decalcification is 20 to 1.
FACTORS INFLUENCING RATE OF DECALCIFICATION

 Too rapid removal od calcium salts may produce

complete digestion of tissue specimen, with
marked swelling and hydrolysis of the bony
matrix and poor staining capacity of the cell.
 The optimum temperature so far recommended
is the room temperature range of 18C to 30C.
 Mechanical agitation and moving of the tissue
solution usually influences fluid exchange,
accelerates the rate of diffusion and speeds up
the decalcification process.
FACTORS INFLUENCING RATE OF DECALCIFICATION

 Increase in size and consistency of

tissues will require longer periods for
complete decalcification.
 The ideal time for decalcifying tissue is
24-48hours.
 Dense bone tissues usually require up
to 14 days or longer to complete the
process, solutions should be changed
daily.
MEASURING EXTENT OF DECALCIFICATION

Physical or Mechanical test

 Touching or bending the tissue with
fingers to determine the consistency of
tissues.
 Pricking the tissue with fine needle or a
probe
MEASURING EXTENT OF DECALCIFICATION

X ray or radiological method

 Expensive but most ideal, most sensitive
and most reliable
 Calcium appears opaque in an x-ray
plate
MEASURING EXTENT OF DECALCIFICATION

 Chemical method (calcium oxalate tests)

Detect the presence of calcium in the decalcifying solution.
This method involves the detection of calcium in acid
solutions by precipitation of insoluble calcium hydroxide or
calcium oxalate.
The decalcifying fluid is usually changed every 24 to 48
hours and the chemical test is performed on the discarded
fluid.
A piece of blue litmus paper is added to a test tube
containing the fluid. The litmus paper will turn red due to
the acidity of the fluid.
The presence of cloudiness indicates that there is still
calcium found in the solution.
POST-DECALCIFICATION

 After decalcification is complete, the acid can be

removed from the tissues or neutralized chemically
by immersing the decalcified bone either saturated
lithium carbonate solution or 5-10% aqueous
sodium bicarbonate solution for several hours.
 Many laboratories simply rinse the decalcified
specimens with running tap water.
 Tissues decalcified with EDTA solutions should not
be placed into 70% alcohol, because this will cause
residual EDTA to precipitate in the alcohol and
within the tissue.
TISSUE SOFTENER

 Perenyi’s fluid act both as decalcifying

agent and tissue softener.
 Molliflex
 2% hydrochloric acid
 1 % hydrochloric acid in 70% alcohol
Thank you!