DECALCIFICATION a.
Aqueous Nitric Acid Solution
It is the process whereby calcium or lime salt
are removed from the tissues (most
especially bones &teeth) following fixation.
It should be done after fixation and before
impregnation.
Inadequate decalcification may result in poor
cutting of hard tissues and damage to the
knife edge during sectioning.
Bones and calcified tissues (tuberculous
organs & arteriosclerotic vessels)= usually cut
into small pieces by using a fine-fret saw and
trimmed w/ a hand razor,
Selected pieces of tissues are taken from the
teeth by a sharp razor blade
Small foci of microcalcification in paraffin-
embedded or frozen tissues can be sectioned
w/out noticeable damage to the knife or
disruption of surrounding tissue. After
hematoxylin staining, they appear as dark
purple granular masses w/ lighter purple
halos.
Trimmed paraffin block= may reveal small foci
of calcification and may cause a GRAFTING
sensation when sectioned w/ a microtome
knife Remedy: remove the block from the
chuck and place face down on a pad of
cotton/gauze saturated w/ 10% hydrochloric
acid for approximately 1 hour.
Good Decalcifying Agents: must be capable of
removing calcium salts from the tissues
completely w/out considerable tissue
destruction and w/out adversely affect the
staining capacity of the cell.
Decalcifying Agents
1. Acids
2. Chelating Agents
3. Ion Exchange resins
4. Electrophoresis -electrical ionization
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.
I. NITRIC ACID- this is the most common
and the fastest decalcifying agent
Recommended for routine purposes
Combine nitric acid w/
formaldehyde or alcohol- to prevent
progressive tissue damage and
impaired staining
Simple Aqueous Solutions-
recommended concentration of 5-
10%
10%- removed by 70% alcohol,
recommended for urgent
biopsy, for needle & small
biopsy specimens to permit
rapid diagnosis w/in 24 hours
or less
It imparts a YELLOW color w/
nitrous acid
Strong acids tend to be more
damaging tissue antigens for
immunohistochemical
staining and enzymes may be
totally lost
a. Formol-Nitric Acid- produces less
tissue destruction than 10%
aqueous nitric acid
Yellow color imparted by
nitrous acid formation
impair staining reaction of
the cell
(Remedies: neutralize the
tissue w/ 5% sodium sulfate
and washing in running tap
water for @least 12 hours,
add 0.1% urea to pure
concentrated nitric acid
b. Perenyis Fluid- recommended
for routine purposes
Relatively slow decalcifying
agent for dense bones
Complete decalcification cant
be determined by chemical
test a precipitate is formed
upon addition of ammonia to
Perenyis fluid even in the
absence of calcium ion.
(Remedy: Add glacial acetic
acid drop by drop. About 0.5 mL
of saturated aqueous
ammonium oxalate is then
added to the solution.
Reappearance of white
precipitate w/in 30
mins=presence of calcium ion)
c. Phloroglucin-Nitric Acid
When decalcification is
complete, REMOVE acid by
three changes of 70-90%
ethanol. When sections are
cut, slide are brought to
water and placed 1%
aqueous lithium carbonate
for 1 hr, washed in later for
15 minutes and then stained.
II. HYDROCHLORIC ACID- has slower
action and greater distortion of tissue
produced on section decalcified.
a. It will produced good nuclear
staining if used in 1% solution w/
70% alcohol
b. May be recommended for surface
decalcification of tissue blocks.
c. Rapid proprietary solution-contain
HCl
d. Slow proprietary solutions- contain
buffered formic acid or
formalin/formic acid.
e. Von Ebners Fluid- recommended for
small pieces of bone and teeth,
extent of decalcification cant be
measured by chemical test
III. FORMIC ACID
a. Moderate decalcifying agent which
produce better nuclear staining w/
less tissue distortion, safer to handle
than HCl or nitric acid.
b. Recommended for postmortem
research tissues
c. Only weak acid used extensively as
primary decalcifying agent
d. Addition of citrate accelerates
decalcification
e. Carnoys & Bouins fixative= acts as
incidental, weak decalcifiers in
urgent cases (only minimal
decalcification)
f. Suitable for most routine surgical
specimens, particularly when
immunohistochemical staining is
needed.
g. Decalcification may be hastened by
increasing the proportion of formic
acid to 25 mL.
h. Requires neutralization w/ 5%
sodium sulfate.
i. FORMIC-ACID SODIUM CITRATE
SOLUTION- recommended for
autopsy materials, bone marrow
and tissues needed for research
purposes.
IV. TRICHLOROACETIC ACID- suitable
only for small spicules of bone, very
slow acting
V. SULFUROUS ACID- suitable only for
urgent examinations
VI. CHROMIC ACID (FLEMMINGS
FLUID)
a. Can be used as fixative & decalcifier
b. Used for decalcifying minute
spicules of bone
c. Nuclear staining w/ hematoxylin is
inhibited
d. An environmental toxin
e. Carcinogenic, highly corrosive to
skin and mucous membrane
VII. CITRIC ACID- CITRATE BUFFER
SOLUTION- permits excellent nuclear
and cytoplasmic staining, does not
produce cell or tissue distortion, too
slow for routine purpose
CHELATING AGENTS
They are substances which combine w/ calcium
ions and other salts to form weakly dissociated
complexes and facilitate removal of calcium salts.
ETHYLENE DIAMINE TETRAACETIC ACID
(EDTA)
Most common chelating agent in the
market
Commercial name of Versene
A very slow decalcifying agent
1-3 weeks=for small specimens
6-8 weeks/longer= totally decalcify dense
cortical bone
Solution should be changed every 3 days
in the final stagechanged everyday to
facilitate decalcification
pH 3= EDTA will not bind calcium
pH 7-7.4 = faster binding
pH 8 and above=give optimal binding
forms minimal histological artifacts
excellent bone decalcifier for
immunohistochemical or enzyme staining,
and for electron microscopy
inactivates alkaline phosphatase activity
ION EXCHANGE RESIN
It hastens decalcification by removing
calcium ions from formic acid-containing
recommended for fluids containing mineral
acids such as nitric acid or HCl.
A layer of the ion exchange resin, about
inch thick is spread over the bottom of the
container to be used and specimen is
placed on the top of it. The decalcifying
agent is then added, usually 20-30 times the
volume of the tissue.
1-14 days- duration of stay of tissue in
solution
Physical/ X-ray method- used to measure
degree of decalcification
 ELECTROPHORESIS
It is the process whereby positively charged
calcium ions are attracted to a negative
electrode and subsequently removed from
the decalcifying solution.
It utilizes electricity and is dependent upon
a supply of direct current to remove the
calcium deposits
This method is satisfactory for small bone
fragments, processing only a limited
number of specimens at a time.
MEASURING EXTENT OF DECALCIFICATION
Good cytologic and histologic details=not
always preserved in tissues that have been
electrically decalcified
Solutions Used for Decalcification
1. Formic acid 88%
2. Conc. HCl
3. Distilled water
PROLONGED DECALCIFICATION= to prevent
hydrolysis and lead to MACERATION and
destruction of tissue components which are
poorly stained.
FACTORS INFLUENCING RATE OF
DECALCIFICATION
Rate of decalcification depend upon
o Structure
o Temperature
o Volume of Solution to be Used
High concentrations & greater amount of
fluid= increase speed of process
o 20:1 = recommended ratio of fluid
to tissue volume for decalcification
o more concentrated solution more
rapid decalcification = more harmful
to the tissue
HEAT= hasten decalcification
o At 37OC= impaired nuclear staining
of Van Giesons Stain for collagen
fibers
o At 55OC= undergo complete
digestion within 24-48 hours
o Lower Temperature= low reaction
rate
o Room Temperature (18OC- 30OC)-
recommended
Mechanical agitation & moving of tissue in
the solution= influences fluid exchange,
accelerates the diffusion rate and speeds up
decalcification
o Gentle fluid agitation- achieved by
low-speed rotation, rocking, stirring
or bubbling air into solution
o Sonitation- vigorously agiates both
specimen and fluid
INCREASE in SIZE & CONSISTENCY of
tissues= require longer periods for
complete decalcification
24-48: ideal time required for 24-48 hours
Dense bone tissues= require up to 14 days
or longer
1. Physical/ Mechanical Test- done by
touching or bending the tissue w/ the
fingers to determine consistency of the
tissues
2. X-Ray/Radiological Method- most
ideal, most sensitive , most reliable method
of determining extent of decalcification due
to its ability to focus small focus of calcium
a. Not recommended for mercuric
chloride-fixed tissue due to latters
radio-opacity.
3. Chemical Method (Calcium Oxalate
Test)- recommended for routine purposes,
to detect presence of calcium in acid
solutions by precipitation of insoluble
calcium hydroxide/ calcium oxalate.
a. Decalcifying fluid= usually changed
every 24-48 hours and the chemical
test is performed on the discard
fluid
b. Blue litmus paper-added to a test
tube containing 5 mL of the
discarded decalcifying agent (litmus
paper will turn red due to acidityof
the fluid
c. Strong ammonia is added drop by
drop until the fluid is neutralized
d. Presence of cloudiness= there is still
calcium found in the solution
e. If solution remains clear after
neutralization w/ conc. Ammonia
=0.5 mL of saturated aqueous
solution of ammonium oxalate is
added then solution is stand for 30
minutes.
f. Clear solution after 30
mins=decalcification is COMPLETE
g. DISTILLED WATER= used to prepare
decalcifying agent for chemical
method of determination
 POST DECALCIFICATION

After complete DECALCIFICATION= acid can

be removed from tissues or neutralized
chemically by immersing the decalcified
bone in either
1. Saturated lithium carbonate
2. 5-10% aqueous sodium bicarbonate
Samples for immediate processing= can be
blotted or quickly rinsed w/ water to
remove acid from the surface
Acid decalcified tissues for frozen
sections thoroughly washed with water/
stored in formol saline containing 15%
sucrose or phosphate-buffered saline (PBS)
w/ 15-20% sucrose @ 4OC before freezing
EDTA decalcified tissues=NOT be placed
directly into 70% alcohol
o Rinse the tissue w/ water or store
overnight in formol saline or PBS will
prevent precipitation
Adequate water rinsing- accomplish in
o 30 minutes for SMALL samples
o 1-4 hours for LARGER samples

TISSUE SOFTENERS

Perenyis Fluid= may act as both

decalcifying agent & tissue softener

Washing out and immersion of fixed tissues

in 4% aqueous phenol solution for 1-3
days cause tissue softening and easier
sectioning of blocks

Other Substances used as tissue softeners

1. Molliflex- tissue may appear swollen &
soapy (not effect normal processing &
subsequent staining)
2. 2% HCl
3. 1% HCl in 70% alcohol