HPCT - DECALCIFICATION

Decalcification

Removal of calcium ions from either the bone or calcified tissue using the process that makes them flexible and easier to cut. Strong mineral acid or weak organic acids forms soluble calcium salt in an ion exchange that moves the calcium into decalcifying solution

Acid decalcifying agents

Most widely used for routine decalcification of large amounts of bony tissues. Stable, readily available, inexpensive.

Strong mineral acids

Nitric acid, Hydrochloric acid

Weak acid

Formic Acid, Trichloroacetic acid, Chromic acid

Chelating agent

EDTA, Neutral EDTA

Strong mineral acid

Most rapid in action but if you use it longer than necessary, this will cause rapid loss of nuclear staining and maceration of tissues.

Weak acid

Not as harsh as strong mineral acid but they act more slowly on dense cortical bone.C

Chelating agents

Preferred to the acids for the preservation of the nuclear DNA and histochemical methods of nucleic acids or enzyme activities

Nitric acid

Most common and fastest among strong acids. Simple or compound/combined. Recommended to use in 5% to 10% concentration. Inhibits nuclear stains and destroys tissues (Prevented by formaldehyde or alcohol)

Aqueous nitric acid solution

Concentrated nitric acid (10mL) + distilled water (100mL). Decalcification time: 12 hours. Rapid action, minimum distortion, good nuclear staining, acid removed by 70% alcohol. Recommended for urgent biopsy, needle & small biopsy. Imparts yellow color. Damages tissue enzymes

Formol-nitric acid

Concentrated nitric acid (10mL) + 40% formaldehyde (5mL) + distilled water (85 mL). Decalcification time: 1-3 days. Advantages: rapid; recommended for urgent biopsies, good nuclear staining, less tissue destruction than 10% aqueous Nitric acid. Disadvantages: yellow discoloration. Prevented by 5% Na Sulfate or running water (12 hrs) or 0.1% of urea to pure nitric acid.

Perenyi’s fluid

10% nitric acid (40mL) + 0.5% chromic acid (30mL) + absolute ethanol (30mL). Decalcification time: 2-7 days. Recommended for routine purposes. Acts as a decalcifier and tissue softener. Takes too long to decalcify so it is not recommended for urgent diagnosis.

Phlorogucin-nitric acid

Concentrated nitric acid (10mL) + phloroglucin (1g) + 10 nitric acid (100ml) Decalcification time: 12 to 24 hours. Most rapid for urgent cases. Poor nuclear staining (12-24 hours). Recommended for urgent works. Yellow color that must be washed with 5% sodium sulfate and running tap water

Hydrochloric acid

Inferior to nitric acid; slower and greater distortion of the tissue.

Von Ebner Fluid

Saturated aqueous solution of NaCl (50ml) + 36% conc HcL (8ml) + distilled water (50ml). Good for cytologic staining. Does not require washing before dehydration. Recommended for teeth and small pieces of bone.

Formic acid

Under the weaker organic acids. 10% is the best all around decalcifying agent. Formic acid (10ml) + normal saline (90ml). Decalcification time: 2-7 days. Advantages: fixative and decalcifying agent. Excellent nuclear and cytoplasmic staining. Recommended for small pieces of bones and teeth. Disadvantage: slow but may increase concentration to 25mL. Requires neutralization with 5% sodium sulfate and washing out to remove acid from tissue. Recommended for routing decalcification of postmortem research tissues

Formic acid-Sodium citrate solution

20% Aq. Na Citrate (50ml) + formic acid (50ml). Decalcification time: 3-14 days. Advantages: nuclear staining > nitric acid. Recommended for autopsy materials, bone marrow, cartilage, tissues for research purposes. Neutralize with 5% Na sulfate

Trichloroacetic acid

TCA (5g) + 10% formol saline (95ml); Permits good nuclear staining. Does not require washing out. Decalcification time: 4-8 days.

Sulfurous acid

Very weak acid. Suitable only for minute pieces of bones.

Chromic acid

Chromic acid (15ml) + osmium tetroxide (4ml) + 2% glacial acetic acid (1ml) Also known as Flemming’s fluid. Both fixative and decalcifying agent. Used for minute bone spicules. Inhibition in nuclear staining with hematoxylin. Environmental toxin. Tends to form precipitates at the bottom. Completeness of decalcification cannot be measured by the routine chemical test.

EDTA

Versene. Most commonly available chelating agent. Does not act like inorganic or organic acids. Binds metallic ions. Preferred for nucleic acids and enzymes. Very slow decalcifying agent.

Neutral EDTA

EDTA disodium sault (250g) + distilled water. Buffer: Sodium hydroxide (pH 7.0) Acts slow with little tissue damage. Conventional stains unaffected. Inactivates ALP, resolved by adding magnesium chloride

Ion exchange resin

Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions formic acid-containing decalcifying solutions. Not recommended for fluids that contains mineral acids, such as nitric acid or hydrochloric acid. Tissue may stay for 1-14 days and degree of decalcification may be measured by physical or xray method.

Electrophoresis (Electrical Ionization)

A process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution. This method is satisfactory for small bone fragments, processing only a limited number of specimens at a time. Good cytologic and histologic details are not always preserved.