CHAPTER 8: DECALCIFICATION

Removal of calcium ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut.

decalcification

recommended decalcification fluid ratio

20:1

- based on strong mineral acids

- based on weaker organic acids

- composed of chelating agents.

three main types of decalcifying agents

the most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily available, and relatively inexpensive as compared to other decalcifying agent

Acid decalcifying agents

- Most common and the fastest decalcifying agents

- Can be used for routine decalcification

- recommended concentrations of 5-10%

Disadvantage: inhibiting nuclear stains and destroying tissues, especially in concentrated solutions.

Nitric Acid

- Recommended for urgent biopsy and needle and small biopsy specimens

- Can be used for large or heavily mineralized cortical bone specimen

Aqueous Nitric Acid Solution 10%

- Concentrated nitric Acid 10 ml.

- Distilled water added up to 100 ml.

Formula for Aqueous Nitric Acid Solution 10%

Aqueous Nitric Acid Solution 10% Decalcification time

12-24 hours

- Recommended for urgent biopsy; nuclear staining is good

- Produces less tissue destruction than 10% aqueous nitric acid solution

- Yellow color due to nitrous acid formation may impair tissue staining

Formol-Nitric Acid

- 5% sodium sulfate and washing in running tap water for at least 12hrs o

- Addition of 0.1% urea to pure concentrated nitric acid will also make discoloration disappear.

To prevent yellow color due to nitrous acid formation may impair tissue staining

- Concentrated nitric acid 10 ml.

- Strong formaldehyde, 40% 5 ml.

- Distilled water 85 ml

Formula for Formol-Nitric Acid

Formol nitric acid Decalcification time

1-3 days

- For routine purpose, serves both as decalcifier and tissue softener

- Nuclear and cytoplasmic staining is good

- Slow decalcifying agent for dense bones

Perenyi’s Fluid

- 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.

Formula for Pereyni's Fluid

Pereyni's fluid Decalcification time

2-7 days

Advantage: Most rapid decalcifying agent, recommended for urgent cases.

Disadvantage:

- Nuclear staining is poor.

- Prolonged decalcification produces extreme tissue distortion.

- Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.

- Complete decalcification cannot be determined by chemical means.

Phloroglucin-Nitric Acid

- Concentrated nitric acid 10 ml.

- Phloroglucin 1 gm.

- Nitric acid 10% 100 ml. (To be added after disappearance of dense white fumes formed by combining the first two ingredients.)

Formula for Phloroglucin-Nitric Acid

Phloroglucin-Nitric Acid Decalcification time

12-24 hours

- Slow-acting decalcifying agent than Nitric acid

- Produces greater distortion of tissue than Nitric acid

- Produces good nuclear staining

- 1% solution with 70% alcohol is recommended for surface decalcification of tissue blocks.

Hydrochloric Acid (HCl)

- Permits good cytologic staining; recommended for teeth and small pieces of bone

- Does not require washing out before dehydration

- The completion of decalcification cannot be measured by chemical method

Von Ebner's Fluid

- Saturated aqueous solution of NaCl 50 ml.

- 36% concentrated hydrochloric acid 8 ml.

- Distilled water 50 ml.

Formula for Von Ebner's Fluid

- Moderate-acting decalcifying agent; for routine decalcification of post-mortem research tissues

- Produces better nuclear staining with less tissue distortion

- Can be used as a simple 10% aqueous solution or combined with formalin or with buffer

Formic acid

- Formic acid (Sp. grav. 1.20) 10 ml.

- Normal saline 10% 90 ml.

Formula for Formic acid

Formic acid Decalcification time

2-7 days

- Recommended for autopsy material, bone marrow cartilage, and tissues for research purposes

- Requires neutralization with 5% sodium sulfate

Formic Acid - Sodium Citrate Solution

- Aqueous sodium citrate 20% 50 ml.

- Formic acid 45% 50 m

Formic Acid - Sodium Citrate Solution Formula

Formic acid Decalcification time

3-14 days

- Weak and very slow-acting decalcifying agent.

- Permits good nuclear staining and requires no washing out (the excess acid may be removed with several changes of 90% alcohol)

Trichloroacetic acid

- Trichloroacetic acid 5 gm.

- Formal saline 10% 95 ml.

Trichloroacetic acid Formula

Trichloroacetic acid Decalcification time

4-8 days

a very weak decalcifying solution suitable only for minute pieces of bone

Sulfurous acid

Advantages:

- May be used as a fixative and decalcifying agent for minute bone spicules

Disadvantages:

- Nuclear staining with hematoxylin is inhibited

- Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol; hence, washing out is required

- Completion of decalcification cannot be measured by chemical method.

- an environmental toxin, highly corrosive to skin and mucus membrane, and carcinogenic

Chromic acid (Flemming's fluid)

- Chromic acid % 15 ml.

- Osmium tetroxide 4 ml.

- 2% Glacial acetic acid 1 ml.

Chromic acid (Flemming's fluid) Formula

Advantages:

- Permits excellent nuclear and cytoplasmic staining.

- Does not produce cell or tissue distortion.

Disadvantage:

- Action is too slow for routine purposes.

Citric Acid - Citrate Buffer Solution (pH 4.5)

- Citric acid (monohydrate) aqueous solution 7% 5.0 ml.

- Ammonium citrate (anhydrous) aqueous solution 7.4% 95.0 ml.

- Zinc sulfate aqueous solution. 1% 0.2 ml. Chloroform (as preservative) - a few drops

Citric Acid - Citrate Buffer Solution (pH 4.5) Formula

Citric Acid - Citrate Buffer Solution (pH 4.5) Decalcification time

6 Days

- Substances that bind with Ca++ ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salts

- Example: Ethylenediaminetetraacetic acid (EDTA)

Chelating Agents

- Permits excellent staining results and minimal cell and tissue distortion

- Completion of decalcification can be measured by routine chemical test

- Excellent bone decalcifier for enzyme or IHC staining and electron microscopy

- Very slow, not recommended for urgent and routine purposes

- May cause slight tissue hardening

Natural EDTA

- EDTA disodium salt 250 gm

- Distilled water 1750 ml

Bring to pH 7.0 by adding sodium hydroxide (about 25 gm will be needed).

Natural EDTA Formula

- Ammonium form of polystyrene resin

- Hastens decalcification by removing calcium ions from formic acid- containing decalcifying solution

- Not recommended for fluids containing mineral acid such as Nitric acid and HCl

Ion Exchange Resin

- Process whereby positively charged calcium ions are attracted to negative electrode and subsequently removed from the decalcifying solution.

- Satisfactory for small bone fragments

Electrophoresis (Electrical Ionization)

- 88% Formic acid (100 ml)

- Concentrated HCl (80 ml)

- Distilled water (1000 ml)

Solutions used for Electrolytic Decalcification

- Hard tissues are placed in the decalcifying agent in a microwave oven for intermittent periods with regular changes of the solution till the endpoint is reached.

- Microwave irradiation speeds up the process of decalcification from days to hours.

- Uniform 3-5mm sections are optimal for decalcification

Microwave Oven Decalcification

More concentrated acid solutions decalcify bone more rapidly, but are more harmful to the tissue.

Concentration

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.

Fluid access

Increase in size and consistency of tissues will require longer periods for complete decalcification. Dense bone tissues usually require up to 14 days or longer in order to complete the process.

Size and consistency

Gentle agitation may increase the rate of decalcification. Mechanical agitation and moving of the tissue in solution usually influences fluid exchange, accelerates the rate of diffusion and speeds up the decalcification process.

Agitation

Increased temperature will hasten decalcification, but it will also increase the damaging effects of acids on tissue. A

Temperature

- Done by touching or bending the tissue with fingers or by pricking the tissue with fine needle

- Require manipulation, bending, probing or trimming of the specimen to "feel" for remaining calcified areas.

Physical tests/Mechanical Tests

- Recommended for routine purpose

- Cloudiness on the solution after the addition of the chemical agent signifies incomplete decalcification. Clear solution remained after 30mins means complete decalcification.

Chemical test

Very expensive yet most reliable (not recommended for mercuric chloride fixed tissue)

Radiological/ X-ray Method

This is a method of dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks. When the paraffin-embedded block has been trimmed, the tissue surface may reveal small foci of calcification and may cause resistance or a "grating" sensation when sectioned with a microtome knife.

Surface decalcification

Unduly hard tissues which are liable to damage the microtome knives may require tissue softeners, aside from decalcification. Perenyi's fluid may act both as a decalcifying agent and tissue softener. To soften unduly hard tissues, selected portions are left in the fluid for 12-24 hours and dehydrated in the usual manner; or the cut surface of the block may be submerged in the fluid for 1-2 hours before sectioning, to facilitate easier cutting of tissues.

Tissue softeners