DECALCIFICATION

- process of removing mineral (calcium and lime salts) from bone or other
calcified tissues so that good-quality paraffin sections can be prepared so
that will preserve all the essential microscopic elements
- decalcification is carried out after the specimen has been thoroughly fixed
and prior to routine processing to paraffin
Types of Investigation
o Undecalcified bone for the diagnosis or investigation of metabolic bone
disease where it is necessary to differentiate mineralized bone from
osteoid or if morphometric measurements are required
o Decalcified bone
Use/Application of Decalcified Sections
o Used for the examination of bone marrow
o Diagnosis of tumors, infections, or for other purposes
Specimens
o Iliac crest trephines, bone pieces removed at operation such as femoral
heads or dissected from amputation specimens
o Calcified tissues (tuberculous organs, arteriosclerotic vessels)
o Other tissues may undergo calcification associated with degenerative
processes such as necrosis (dystrophic calcification) or it may occur in the
walls of blood vessels or in kidney, lung, or elsewhere (metastatic
calcification)
Fixation of Bone
o Buffered formalin is a satisfactory fixative for bone
o Alternatives: Zinc formalin mixtures, B5, formol acetic acid alcohol
(Davidsons fixative) or Bouin bone marrow
o Extended fixation time ensure thorough fixing of these specimens
prior to decalcification
o Bone specimens should be sawn into thin slices using high-quality fine
tooth saws
Decalcifying agents
o
o
o
o
o

Strong mineral acids

Weaker organic acids
Chelating agents
Ion exchange resins
Electrical ionization (electrophoresis)

STRONG ACIDS: NITRIC ACID

1. Nitric acid
o
o
o
o

5% in dH2O, 10%
Most common and fastest decalcifying agent
12-24 hours - decalcification time
Rapid in action, exceeding end-point will impair staining

2. Formol Nitric acid

o 1-3 days decal time
o Nuclear staining is relatively good
o Disadv: yellow color imparted by nitrous acid formation will impair
staining reaction of the cell
o Correction: neutralize the tissue with 5% sodium sulfate and
wash in tap water for 12 hours
3. Perenys Fluid (10% Nitric acid, .5% Chromic acid, and absolute alcohol)
o
o
o
o
o

2-7 days decalcification time

Traditional decalcified that acts more slowly than aq. Nitric acid
Decalcified and softens tissues at the same time
Nuclear and cytoplasmic staining is good
Complete decalcification test cannot be determined using chemical tests

4. Phloroglucin-Nitric acid
o
o
o
o

Conc. Nitric acid & Phloroglucin (with 10% Nitric acid)

Decal. Time: 12-24 hours
Almost similar with Perenys fluid, but more rapid
Most rapid and recommended for urgent biopsies

STRONG ACIDS: HYDROCHLORIC ACID

1. HCl
o Slower action and greater distortion of tissue
o Produces good nuclear staining
o May be used as surface decalcification if used in 1% solution with 70%
alcohol
o Rapid proprietary solution contains HCl
o Slow proprietary contain buffered formic acid or formalin/formic acid
o NB: Formalin should be washed from specimen before placing in HCl to
avoid the formation of bis-chloromethyl ether (a carcinogen)
2. Von Ebners solution

o Sodium chloride saturated solution, 50 mL; distilled water, 42 mL; HCl, 8

mL
o Rapid in action
o Exceeding end-point will impair staining
o Does not require washing out before dehydration
o Recommended for teeth and small pieces of bones
WEAK ACIDS: FORMIC ACID
1. Formic acid
o
o
o
o

10% in distilled water

A simple effective decalcifier, moderate-acting
Recommended for rt. Decalcification of postmortem research ts.
Decal time: 2-7 days

2. Evans and Krajian

o Formic acid, 25 mL; Sodium citrate, 10g; distilled water, 75 mL
3. Gooding and Stewart
o Formic acid, 25 mL; Sodium citrate, 10g; distilled water, 75 mL
o Fix and decalcify
4. Formic Acid-Sodium citrate Solution
o Aqueous Sodium citrate, 20%; Formic acid, 45%
OTHER ACIDS USED AS DECALCIFYING AGENTS
1. Trichloroacetic acid
2. Sulfurous acid
3. Chromic acid (Flemmings Solution)
o Environmental toxin
o Carcinogenic
4. Citric acid-citrate buffer solution
o Citric acid (monohydrate) aqueous solution, Ammonium citrate, Zinc
sulfate aq. Solution, Chloroform as preservative
CHELATING AGENTS
1. Ethylenediaminetetraacetic acid (EDTA)
o EDTA disodium salt, 250g; distilled water, 1750 mL
o Bring to pH 7.0 by adding NaOH (about 25g)

o Works by capturing the calcium ions from the surface of the apatite
crystals
o Decal time - 1-3 weeks (small specimen) or 6-8 weeks or longer (dense
cortical bone)
o Acts slowly but causes little tissue damage
o Conventional stains are largely unaffected
FACTORS INFLUENCING THE RATE OF DECALFICATION
o
o
o
o

Concentration
Temperature
Agitation
Fluid access

DETERMINING THE END-POINT OF DECALCIFICATION

1. Physical or Mechanical test
2. X-ray or Radiological method
3. Chemical Method (Calcium oxalate test)
IMPORTANT CONSIDERATIONS
o
o
o
o
o
o

A careful preliminary assessment of the specimen

Thorough fixation
Preparation of slices of reasonable thickness for fixation and processing
The choice of suitable decalcifier with adequate volume changed regularly
A careful determination of the endpoint
Thorough processing using a suitable schedule