Decalcification is the process of removing Calcium or Lime Salts from tissues.

It is performed
on bones, teeth, calcified tissues (ex. Tuberculous lungs, arteriosclerotic vessels). I adjust the
hard substance of bones to the softness of paraffin embedding medium.
Decalcification enables the histotechnologist to cut soft sections of the bone using the
microtome, so that they can be processed like any other soft tissue of the body. It prevents poor
cutting of hard tissues/knife damage.
Decalcification is a lengthy procedure, as bone pieces have to be left in the decalcifying agents
for several days or even weeks depending on the size of the tissue. Many of the grossing and
cutting techniques for bone require the use of a high-speed saw and/or long periods in
decalcifying solution, prior reducing the specimen to a size that can be easily processed,
embedded and sectioned.
Decalcification should be done after fixation and before impregnation, to ensure and facilitate
the normal cutting of sections and to prevent obscuring the microanatomic detail of such
sections by done dust and other cellular debris.
If the the tissues size is very large, use a fine-fret saw. Decalcifying agent should be
changed regularly. “GRATING” sensation during cutting = place block in 10 % HCl for 1 hour.
Rapid decalcification produces effect on nuclear staining – (failure of nuclear chromatin to
take up hematoxylin).
Inadequate decalcification may result in poor cutting of hard tissues and hard tissue and
damage to the knife edge during sectioning. Poorly-fixed specimens become macerated during
decalcification and stain poorly afterwards. Extending the fixation time for bone specimen before
commencing decalcification is important. 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.
Cartilage does not require any softening, except if some calcified areas are present.
After fixation, depending on the amount of adjusted soft tissue, the toenail should be rinsed of
with soapy water once it becomes pliable.
Preparation of Bone Specimens for Decalcification
Bone biopsy is similar to sot tissue biopsy except that bone needs decalcification before
processing. An X-ray of the specimen may also select samples for processing. Interpretation of
biopsy requires assessment of the relationship between mineralized and non-mineralized bone
or osteoid. The methods used are:
1. Mineralized bone sections embedded in methyl methacrylate (MMA) plastic. It is the
preferred method for metabolic bone disease work-up.
2. Frozen section form an undecalcified bone biopsy.
3. Silver impregnation method of Tripp and McKay (1972) demonstrating bone and osteoid
in a decalcified, paraffin embedded bone sections.
Types of Specimens
1. Amputated limbs – these are often sent to the laboratory immediately after surgery
secondary to tumor, inflammation and gangrene. They are often delivered without
fixative, and must be taken care of as soon as possible.
 2. Resected specimens – these include bones with benign or lo-grade malignant
tumors and arthritic femoral heads. They are usually with established diagnosis and
considered diagnosis and are considered less urgent.
Principle of Decalcification
Strong Mineral acids such as 10% HCl, or weak organic acids, such as 5-10% HCOOH,
form soluble calcium salts in an ion exchange that moves calcium into the decalcifying solution.
The same final effect makes 14% EDTA an ideal chelating agent that sequesters metallic ions,
including calcium, in aqueous solution. It is also possible to prepare bone specimens by
infiltrating them with acrylic or epoxy resins which, when polymerized, have a hardness
equivalent to that of mineralized bone and hence do not require decalcification at all.
Buffered HCHO is a satisfactory fixative for bone but where the preservation of bone
marrow is important, some laboratories use alternatives such as zinc formalin mixtures, B-5,
formol-acetic alcohol (Davidson’s fixatives), or Bouin’s solution.

 Decalcifying Agents / Methods of DecalcificationAcids – HNO3, HCl, Formic, TCA,

Sulfurous, Chromic, Citric
 Chelating Agents – EDTA (Versene)
 Ion Exchange Resins (Ammonium form of polystrene resin) – 1-14 days – spread
on bottom of container.
 Electrical Ionization (Electrophoresis) – attraction of Ca to negative electrode

1. Acids – it is the most widely used for routine decalcification. It is also the stable, easily
available and cheap.
a. Nitric Acid – is the most commonly used, rapid decalcifying agent, can be
removed by 70% alcohol. Inhibits nuclear stains – prevented by combining with
formaldehyde or alcohol.
b. Formol Nitric Acid –rapid acting, good nuclear staining, less tissue discoloration
by 5% sodium sulfate or 0.1% urea. (Decalcification time: 1-3 days) Composition:
Conc. Nitric acid 10 mL
Strong formaldehyde,40% 5ml
Distilled water 85 ml
c. Perenyi’s Fluid – decalcified and softens tissues at the same time. Good nuclear
and cytoplasmic staining. Maceration avoided by chromic acid and ethyl alcohol.
Disadvantages: slow, difficult to assess complete decalcification by chemical
means. (Decalcification time: 2-7 days)
Composition:
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.
 d. Phloroglucin–Nitric Acid - Most rapid decalcifying agent. Poor nuclear staining.
(Decalcification time: 12-24 hours) * when decalcification is complete, acid must
be removed by 3 changes of 70 to 90% ethanol
Composition:
Conc. Nitric acid 10ml
Phloroglucin 1gm
Nitric acid 10% 100ml
(to be added after disappearance of dense white fumes formed by combining the first two
ingredients)
e. Hydrochloric Acid (HCl) - Inferior compared to Nitric Acid as decalcifying agen.
Slower action, greater tissue distortion. Good nuclear staining.
Recommended for surface decalcification.
2. Von Ebner’s Fluid - Good cytologic staining. Recommended for teeth and small pieces of
bones Composition:
Saturated aqueous solution of NaCl 36% 50 ml
Conc. Hydrochloric acid 8ml
Distilled water 50 ml
a. Formic Acid - Better nuclear staining with less tissue distortion. Safer to handle
than Nitric and HCl. Recommended for postmortem research tissues upon the
addition of sodium citrate. It is both fixative and decalcifying agent. For small
pieces of bone and teeth. (Decalcification time: 2-7 days)
Composition:
Formic acid (sp.grav 1.20) 10 ml
Formol Saline 90 ml
3. Formic Acid- Sodium Citrate Solution - Slow, not recommended for routine purposes.
Permits better nuclear staining than Nitric Acid. Requires neutralization with 5% Na
sulphate. Recommended for autopsy materials, Bone Marrow, Cartilage and tissues for
research.
Composition:
45% Formic Acid 50 ml
20% Na Citrate 50 ml
a. Trichloroacetic Acid – weak decalcifying agent. Permits good nuclear staining.
Slow decalcifying agent. Suitable only for small bone spicules. (Decalcification
time: 4-8 days).
Composition:
Trichloroacetic acid 5 gm
10% Formol Saline 95 ml
b. Sulfurous Acid - Very weak decalcifying agent. Suitable only for minute pieces
of bone.
c. Chromic Acid (Flemming’s Fluid) - Fixative and decalcifying agent. Nuclear
staining with hematoxylin is inhibited. Forms precipitate at the bottom.
Carcinogenic, corrosive to skin.
Composition:
Chromic acid 15 ml
Osmium tetroxide 4 ml
Glacial Hac 1 ml
d. Citric Acid – Citrate Buffer - Permits good nuclear and cytoplasmic staining.
Decalcification time: 6 days.
Composition:
7% Citric Acid 5 ml
4% Ammonium Citrate 95 ml
1% Zinc sulfate 0.2 ml Chloroform (preservative) few drops
Chelating Agents - ◎Substances which combine with Calcium ions and other salts (Fe, Mg).
EDTA (Versene, Sequetrene) – most common chelating agent, will not bind Ca at pH
below 3.0. Permits excellent staining. Very slow – 1-3 weeks. Slight tissue hardening produced
EDTA inactivates alkaline phosphatase – add magnesium chloride.
Composition of EDTA and EDTA Disodium salt (10%):
EDTA disodium salt 5.5 gm
Distilled Water 90 ml
Formaldehyde( 37-40% stock) 10 ml
Ion Exchange Resins - Ammonia form of Polystrene Resin. Hastens decalcification by
removing Ca ions from Formic acid- containing decalcifying solutions. Artifacts produced,
usually caused by CO2 bubbles. Slow decalcification time– 1-14 days. Degree of calcification
cannot be measured by chemical means.
Electrophoresis (Electrical Ionization) - Process whereby positively charged Ca ions are
attracted to a negative electrode and subsequently removed from the decalcifying agent,
satisfactory for small bone fragments. Uses electricity:
Solutions used for Electrolytic Decalcification:
Formic Acid 88% 100 ml Conc. Hydrochloric Acid 80 ml distilled
water 1000 ml

Factors Affecting the Process of Decalcification:

1. Concentration and Volume of Decalcifying Agent – the more concentrated the
decalcifying agent is, the decalcification time becomes more rapidly, however, it is more
harmful to the tissue. Rapid depletion of an acid or chelator by their reaction with calcium
can be avoided by using large volume of fluids compared with the volume of tissue.
Recommended fluid volume to tissue ratio is 20:1.
2. Fluid Access – decalcifier should have ready access to all surfaces of the specimen. This
will enhance diffusion and penetration to the specimen and will facilitate solution,
ionization, and removal of calcium. Decalcification can be hastened by suspending the
tissue in decalcifying solution for greater fluid access.
3. Size and Consistency – increase in size and consistency of tissues will require longer
periods for complete decalcification. Dense bone tissues usually require up to 14 days of
longer in order to complete the process.
4. Agitation – gentle agitation may increase the rate of decalcification. Mechanical agitation
and moving of the tissue in solution usually influences fluid exchange, accelerate the rate
of diffusion and speeds up the decalcification process.
5. Temperature – increased temperature will hasten decalcification, but it will also increase
the damaging effects of acids on tissue. Standard temperature +25°C but in practice a
room temperature (RT) range of +18°C to +30°C is acceptable. At +60°C, bones tissues
and cells may become completely macerated almost as soon as they are decalcified.
Measuring the Extent of Decalcification
1. Physical or Mechanical Test – bending, probing, needling/pricking or inserting a pin,
slicing the tissues, razor, touching, squeezing of tissues or scalper directly into the
tissues. Prone to produce artifacts and destroy cellular details.
2. X-ray method - very expensive but most ideal. It is not for mercuric chloride fixed
tissues.
3. Chemical method (CaOx) – it is simple, reliable and convenient. Detection of calcium in
acid solutions by precipitation of Calcium Hydroxide or Calcium Oxalates.
Solutions: concentrated NH4OH; saturated (NH4)2C2O4 performed on the discarded
fluid.
 Litmus paper: blue to red
Clear solution à complete decalcification Cloudiness or Precipitation à
incomplete
Post-Decalcification: decalcified tissues are neutralized by:
Immersing in saturated Li2CO3, or 5-10% NaHCO3 Rinsing in running tap water.
Storing in formol saline containing 15% sucrose or PBS with 15-20% sucrose at +4°C
Tissue Softeners:
Perenyi’s
Lendrum’s :4% phenol
Molliflex
2%HCl
1% HCl in 70% Alcohol