HISTOPATHOLOGY LABORATORY

DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

OUTLINE ○ Decalcification is a lengthy procedure, as

I. INTRODUCTION bone pieces have to be left in the
A. Important Note in Decalcification decalcifying agent for several days or even
weeks, depending on the size of the tissue.
II. DECALCIFYING AGENTS
A. ACIDS BASIC PRINCIPLE
a. STRONG MINERAL ACIDS ● Strong mineral acids, such as 10% hydrogen
i. Nitric Acid chloride (HCl), or weak organic acids, such as
ii. Hydrochloric Acid 5-10% formic acid (HCOOH), form soluble calcium
b. WEAK MINERAL ACIDS salts in an ion exchange that moves calcium into the
i. Formic Acid
decalcifying solution.
ii. Trichloroacetic acid
○ The same final effect makes 14%
iii. Chromic Acid
iv. Citric Acid with Buffer Solution Ethylenediaminetetraacetic acid (EDTA)
an ideal chelating agent that sequesters
B. CHELATING AGENTS metallic ions, including calcium, in aqueous
a. EDTA solutions.
○ It is also possible to prepare bone
III. TECHNIQUE FOR INCREASING THE specimens by infiltrating them with acrylic or
EFFICIENCY OF DECALCIFICATION epoxy resins which, when polymerized,
A. MICROWAVE OVEN DECALCIFICATION have a hardness equivalent to that of
B. ION EXCHANGE RESINS mineralized bone and hence do not require
C. ELECTROLYTIC DECALCIFICATION decalcification at all.
IV. FACTORS INFLUENCING THE RATE OF
DECALCIFICATION IMPORTANT NOTE IN DECALCIFICATION
A. CONCENTRATION ● Poorly-fixed specimens become macerated during
B. TEMPERATURE decalcification and stain poorly afterwards. This is
C. AGITATION very noticeable in areas containing bone marrow. It is
D. TISSUE SIZE & CONSISTENCY therefore common practice for laboratories to
E. FLUID ACCESS extend fixation times for bone specimens before
commencing decalcification.
V. MEASURING EXTENT OF DECALCIFICATION ● It is important to provide ready access for the
A. X-RAY fixative to penetrate the bone, so skin and soft
B. CHEMICAL TEST tissue should be removed from large specimens if
C. PHYSICAL TEST
practicable.
● Bone specimens should be sawn into thin slices
CONTENT FORMATTING as soon as possible to enhance fixation and an
adequate volume of fixative provided.
I. INTRODUCTION ○ High-quality fine tooth saws should be
● Decalcification is the removal of calcium ions used to prepare bone slices.
from the bone through a histological process ○ Coarse saws can cause considerable
thereby making the bone flexible and easy for mechanical damage and force bone
pathological investigation. fragments into the soft tissues present in the
● The removal of calcium deposits is essential for good specimen
embedding procedure as well as to obtain soft ● Cartilage does not require any softening, except if
sections of the bone using the microtome. some calcified areas are present.
● Decalcification is usually carried out between the ● It is a waste of time to put toenails in a
fixation and dehydration to ensure and facilitate decalcification solution, because they are
the normal cutting of sections and to prevent composed of insoluble keratin filaments. After
obscuring the micro anatomic detail of such fixation, depending on the amount of adjusted soft
sections by bone dust and other cellular debris. tissue, the toenail should be rinsed off with soapy
water once it becomes pliable.

Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 1 of 7

HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
II. DECALCIFYING AGENTS
● There are three main types of decalcifying agents:
1. Those based on strong mineral acids
2. 2. Those based on weaker organic acids
3. Those composed of chelating agents.
ACIDS
● Strong mineral acids such as nitric and
hydrochloric acids are used with dense cortical
bone because they will remove large quantities of
calcium at a rapid rate.
● However, these strong acids also damage cellular
morphology. Mineral acid decalcifiers are not
recommended for delicate tissues such as bone
marrow because they are not as aggressive
STRONG MINIERAL ACIDS
NITRIC ACID (HNO3)
● The most common and the fastest decalcifying
agent
● 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.
○ This may be prevented by combining nitric
acid with formaldehyde or alcohol.
HYDROCHLORIC ACID (HCl)
● Inferior compared to nitric acid in its role as a
decalcifying agent because of its slower action and
greater distortion of tissue produced on the
decalcified section.
● However, it produces good nuclear staining and if
used in 1% solution with 70% alcohol, may be
recommended for surface decalcification of the
tissue blocks.
● Rapid proprietary solutions usually contain
hydrochloric acid, whereas slow proprietary mixtures
contain buffered formic acid or formalin/formic acid.
Dilution of a proprietary HCl is not deleterious for
effective decalcification or staining, and this is an
option if a strong mixture is considered too
concentrated.
Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 2 of 7
WEAK MINIERAL ACIDS
● Organic acids such as acetic and formic acid are
better suited to bone marrow and other soft
tissues. Organic acids act more slowly than mineral
acids, and will require extended treatments to
decalcify cortical bone.
● Formic acid in a 10% concentration is the best
all-around decalcifier. Some commercial solutions
combine formic acid with formalin to fix and decalcify
tissues at the same time.
FORMIC ACID (HCOOH )
● A moderate-acting decalcifying agent which
produces better nuclear staining with less tissue
distortion, and is safer to handle than nitric acid or
hydrochloric acid.
● It is recommended for routine decalcification of
postmortem research tissues, although not
suitable for urgent examinations.
● Formic acid can be used as a simple 10% aqueous
solution or combined with formalin or with a buffer.
● It is slower than the strong acid agents, but it is
much gentler in action and less likely to interfere
with nuclear staining.
● Formic acid in a 10% concentration is the best
all-around decalcifier.
○ Formic acid is the only weak acid used
○ extensively as a primary decalcifying
agent.
○ Addition of citrate can accelerate
decalcification by chelating the calcium
as it is liberated from the bone
TRICHLOROACETIC ACID (C2HCl3O2 )
● IIt permits good nuclear staining.
● It does not require washing out.
○ The excess acid may be removed by
several changes of 90% alcohol, thus
improving tissue dehydration.
DISADVANTAGES
● It is a weak decalcifying agent, not used for dense
tissues, and is suitable only for small spicules of
bone.
● It is very slow-acting; hence, is not recommended
for urgent examinations.
HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
CHROMIC ACID (FLEMMING'S FLUID) (H2CrO4)
● It may be used both as a fixative and decalcifying
agent.
● It may be used for decalcifying minute bone
spicules.
DISADVANTAGES:
● Nuclear staining with hematoxylin is inhibited
● It 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; hence, tissues must
be washed out prior to dehydration.
● Degree of decalcification cannot be measured by the
routine chemical test
CITRIC ACID WITH CITRATE BUFFER SOLUTION
● It permits excellent nuclear and cytoplasmic
staining
● It does not produce cell or tissue distortion.
DISADVANTAGE:
● Its action is too slow for routine purposes.
CHELATING AGENTS
● Chelating agents are substances which combine
with calcium ions and other salts (e.g. iron and
magnesium deposits) to form weakly dissociated
complexes and facilitate removal of calcium salt.
● The most common chelating agent in the market
is ethylene diamine tetra acetic acid (EDTA) salt,
with the commercial name of Versene, recommended
only for detailed microscopic studies.
○ EDTA combines with calcium, forming an
insoluble nonionized complex (which is
why it is also used as an anticoagulant
and water softener).
○ EDTA can remove calcium and is not as
harsh as mineral or organic acids.
■ It works by capturing the calcium
ions from the surface of the
apatite crystal, slowly reducing its
size.
NOTE:
● If preservation of nuclear DNA is important, or if
histochemical methods for nucleic acids or
enzyme activities are intended, a chelating agent
is preferred to an acid.
● Because the process is very slow but very gentle,
chelating agents are not suitable for urgent
specimens.
Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 3 of 7
○ The use of EDTA is limited by the fact that it
penetrates tissue and works slowly so it is
not suitable for urgent specimens.
○ It is more appropriate for research
applications where very high quality
morphology is required or particular
molecular elements must be preserved for
techniques such as immunohistochemistry
(IHC), Fluorescent In Situ Hybridization
(FISH) or Polymerase Chain Reaction
(PCR).
USE
● 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.
● EDTA does not work in formic acid with pH 3 as a
decalcifier.
● Neutral EDTA, though being a slow decalcifying
agent, gives excellent results for soft-tissue
integrity, and best quality of both soft-tissue and
hard-tissue staining.
○ The optimal pH is 7-7.6.
■ EDTA works too slowly under pH
5, owing to insolubility.
■ Over pH 8, tissue maceration
starts due to alkaline sensitive
protein bonds.
● Loaded with undistributed calcium, the solution can
precipitate at the bone surface, which requires more
intensive agitation and more intensive post-
decalcification rinsing.
● The tissue is usually placed in EDTA from 1-3 weeks
for small specimens, but it may take 6-8 weeks or
longer to totally decalcify dense cortical bone. The
solution should be changed every 3 days, and in the
final stage, every day, to facilitate decalcification.
DISADVANTAGES
● At present, the application of EDTA as a
decalcifying agent in a routine setting is
hampered by the long time required for
incubation.
○ New methods, such as decalcification in
EDTA using a microwave oven, addition of
ammonium hydroxide to the EDTA solution,
electrolytic decalcification, or a one-step
fixation–decalcification in formalin mercuric
HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

chloric acid solution, which also appears ION EXCHANGE RESINS

suitable for mRNA In Situ Hybridization,
might reduce the time of decalcification
considerably.

III. TECHNIQUE FOR INCREASING THE

EFFICENCY OF DECALCIFICATION

GENTS
MICROWAVE OVEN DECALCIFACTION
● Microwave oven decalcification is faster than
routine decalcification irrespective of
decalcifying agents used.
● The tissue preservation and staining efficacy is
good in microwave nitric acid decalcification
compared to routine nitric acid decalcification.
● Both formic acid and EDTA show good tissue
preservation and staining efficacy irrespective of the
method used (manual vs microwave).
PRINCIPLE
● Ion-exchange resin (ammonium form of
the polystyrene resin) hastens decalcification by
removing calcium ions from formic
acid-containing decalcifying solutions, thereby
increasing solubility from the tissue.
PRINCIPLE
● A layer of the ion exchange resin, about 1/2 inch thick
is spread over the bottom of the container to be used
and the specimen is placed on top of it.
● The decalcifying agent is then added, usually 20-30
times the volume of the tissue. The 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. The resin that has been previously
used may later be reactivated by immersing it in N/10
HCltwice and washing it with distilled water three
times.

NOTE
● It is not recommended for fluids containing
● In this method, hard tissues are placed in the
mineral acids such as nitric acid or hydrochloric
decalcifying agent in a microwave oven for
acid.
intermittent periods with regular changes of the
● ion-exchange resins have been incorporated into
solution till the end point is reached.
some decalcification protocols. They are added to the
● Microwave irradiation has been shown to speed
container holding the decalcifier and take up the
up the process of decalcification significantly
ionized calcium maintaining the effectiveness of the
● The use of a Microwave Histoprocessor does not
acid. If acid decalcifiers are used in adequate
adversely affect cell morphology.
volumes and replaced regularly the use of such resins
○ The quality of microwave-fixed tissues, at the
is probably unnecessary.
respective optimal time points, is comparable
with routinely fixed tissues.

Note from Transer:

Medyo Contradictory ang Book ug Lab Manual
● Microwave treatment has been used with hydrochloric
acid decalcifiers but the raised temperature may
damage morphology and cause staining artIfacts.

Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 4 of 7

HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
ELECTROLYTIC DECALCIFICATION
● Electrolytic decalcification or electrolysis is done
by placing the bone in an acid decalcifier and
attaching it to an electrode through which current
is applied.
● The time required for decalcification is thereby
shortened due to the heat and electrolytic
reaction produced in the process.
PRINCIPLE
● 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
● 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.
IV. FACTORS INFLUENCING THE RATE OF
DECALCIFICATION
● Factors influencing the rate of decalcification The rate
of decalcification may be influenced by several
factors, and ways may be devised to speed up or
slow down this process. The concentration and
volume of the decalcifying agent and temperature at
which the reaction takes place are important
considerations.
GENTS
CONCENTRATION
● The concentration of the active agent will affect the
rate at which calcium is removed and so does the
volume of the decalcifying agent.
○ In general, more concentrated acid
solutions decalcify bone more rapidly, but
are more harmful to the tissue.
■ This is especially true of aqueous
acid solutions, as various
additives such as alcohol or
Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 5 of 7
buffers that protect the tissues
may slow down the
decalcification process.
○ High concentrations and greater amounts
of fluid will increase the speed of the
process.
○ Rapid depletion of an acid or chelator by
their reaction with calcium can be avoided by
using large volumes of fluid compared with
the volume of tissue, and by changing the
solution several times during the
decalcification process.
● It must be remembered that the concentration of
active agent and volume of the decalcifying agent will
be depleted as it combines with calcium and so it is
wise to use a large volume of decalcifying agent and
renew it several times during the decalcification
process. The recommended ratio of fluid to tissue
is 20:1.
The recommended ratio of fluid to tissue volume for
decalcification is 20 to 1.
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
● The optimum temperature is the room temperature
range of 18°C -30°C.
● At 37°C, there will be impaired nuclear staining of
Van Gieson's stain for collagen fibers.
● At 55°C, the tissue will undergo complete digestion
within 24-48 hours.
● Microwave, sonication and electrolytic methods
produce heat, and must be carefully monitored to
prevent excessive temperatures that damage tissue.
Conversely, lower temperature decreases reaction
rates.
AGITATION
● Gentle agitation may increase the rate slightly by
slowly dislodging debris or precipitates from the
tissue thereby influencing fluid exchange from the
tissue into the fluid and vice versa.
○ Gentle fluid agitation is achieved by
low-speed rotation, rocking, stirring or
bubbling air into the solution.
● Mechanical agitation and moving of the tissue in
solution usually influences fluid exchange,
accelerates the rate of diffusion and speeds up
the decalcification process.
● Sonication vigorously agitates both specimen and
fluid, and may cause disruption of tissue, with
formation of cellular debris on the floor of the
container.
HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
TISSUE SIZE & CONSISTENCY
● Ideal decalcification time is about 24-48 hours.
Larger tissues need longer decalcification
periods.
○ Dense tissues usually require 2 weeks or
longer to decalcify.
● In such cases, the solution should be changed daily to
ensure better penetration and to test for the degree of
decalcification.
FLUID ACCESS
● As with fixation, a fresh decalcifier should have
ready access to all surfaces of the specimen.
● This will enhance diffusion and penetration into the
specimen and will facilitate solution, ionization and
removal of calcium.
● Decalcification may be hastened by suspending the
tissue in a decalcifying solution for greater fluid
access.
● As soon as fixation is complete, the selected pieces of
tissues are usually placed in a gauze bag and
suspended in liberal amount of decalcifying solution
by means of a thread to ensure complete
decalcification and protect the tissue from any
precipitate that may be settled at the bottom of the
container.
● Due to the corrosive action of the acid, it is
recommended that the thread be dipped in melted
paraffin wax and that use of metal cap containers be
avoided.
V. MEASURING EXTENT OF DECALCIFICATION
● To achieve optimal results when processing calcified
tissues, it is important to determine the point at which
decalcification is complete. While incomplete
decalcification can lead to tissue distortions,
over-decalcification causes problems with staining, in
particular nuclear staining. The following protocol can
be used to determine when decalcification is
complete.
● Prolonged decalcification of tissue is liable to prevent
hydrolysis and lead to maceration and destruction of
tissue components which are poorly stained.
● Over-decalcification, particularly with the strong acid
decalcifiers, spoils the staining of basophilic elements
such as cell nuclei and in certain circumstances can
cause maceration of the softer tissue elements.
X-RAY
● The best method, particularly with large
specimens. A good-quality X Ray will clearly reveal
tiny residual calcium deposits and allow further
treatment if required.
● This is a very expensive although the most ideal,
Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 6 of 7
most sensitive and most reliable method of
determining extent of decalcification due to its
ability to detect even the smallest focus of
calcium which appears opaque in an X-ray plate.
● It is an excellent method for following the process of
decalcification of large specimens such as
femoral heads.
● It is, however, not recommended for mercuric
chloride-fixed tissues due to the latter's
characteristic radio-opacity which will interfere
with the correct interpretation of the plate.
CHEMICAL TEST
● Can be applied when some acid decalcifiers are
used (particularly formic acid). Ammonium
oxalate solution is added to a sample of the final
change of decalcification 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
decalcifying agent is required.
○ A piece of blue litmus paper is added to a
test tube containing 5 ml. of the discarded
decalcifying agent
■ the litmus paper will turn red due
to the acidity of the fluid
○ Strong ammonia is then added drop by
drop until the fluid is neutralized
■ this can be detected by the change
in color of the litmus paper from
red to blue, indicating alkalinitY
○ The presence of cloudiness indicates that
there is still calcium found in the solution.
The tissue is then immersed in a new
solution of decalcifying agent.
■ If the solution remains clear after
neutralization with concentrated
ammonia, 0.5 ml. of saturated
aqueous solution of ammonium
oxalate is added and the solution
is allowed to stand for 30
minutes.
■ If the solution remains clear after 30
minutes, decalcification is
considered to be complete.
PHYSICAL TEST
● require manipulation, bending probing or
trimming of the specimen to “feel” for remaining
calcified areas. While this method may be successful
HISTOPATHOLOGY LABORATORY
DECALCIFICATION
LECTURER: Mr. John Marke Bernardo
DATE| A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

● in experienced hands it is generally considered to

be unreliable.
○ A method of determining the endpoint by
carefully weighing the specimen after rinsing
and blotting has also been described, and
may be an effective method for large
specimens.
○ An alternate method of evaluating tissues
mechanically is by pricking the tissue with a
fine needle or a probe
● An alternate method of evaluating tissues
mechanically is by pricking the tissue with a fine
needle or a probe.
○ May produce needle tract artifacts and
destroy important cellular details.
○ Pricking, slicing, bending or squeezing
tissue can:
■ disrupt soft tumors from the
bone
■ cause false positive
microfractures of fine trabeculae
leading to a potential
misdiagnosis.
■ Cause blindspots in detecting
small calcificied foci.
● A method of determining the endpoint by carefully
weighing the specimen after rinsing and blotting has
also been described, and may be an effective method
for large spec

Transes Warriors: HISTOFRAT_RAV: Torreflores, Vigilla │ 2K │Page 7 of 7