Decalcification

After fixation, selected pieces of tissues are taken from the specimen, properly labeled and identified, and then subjected to the subsequent steps of processing. There are, however, certain specimens, e.g. bones, teeth and other calcified tissues like tuberculous lungs, that contain some amount of calcium which is apt to interfere with the accurate evaluation and examination of histologic sections. Hence, one must see to it that all such extraneous materials have been removed before proceeding to the next step in tissue processing.

This procedure where Calcium or Lime Salts are removed from tissues (most especially bones and teeth) following fixation is known as Decalcification.

This 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 bone dust and other cellular debris. Inadequate decalcification may result in poor cutting of hard tissues and damage the knife edge during sectioning.

Bone and calcified tissues (such as tuberculous organs are arteriosclerotic vessels) are usually cut into small pieces with a fine fret – saw and trimmed with a hard razor to permit complete penetration of the decalcifying solution with minimal surface damage and tissue distortion. Selected pieces of tissues are taken from the teeth by a sharp razor usually when it has been either partially or completely decalcified.

A good decalcifying agent must be capable of removing calcium salts from tissues completely without producing considerable destruction of cells and tissue components and without adversely affecting the staining capacity of the cell, particularly of the nucleus. Calcium may be removed by any of the following agent:

1.     Acids

2.     Chelating agents

3.     Ion exchange resins

4.     Electrical ionization (electrophoresis)

ACID DECALCIFYING AGENTS                                                                                        

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

As soon as fixation is complete, the selected pieces of tissues are usually placed in 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 the use of metal cap containers be avoided.

The rate of decalcification will depend upon the structure, temperature and volume of the solution to be used. High concentration and greater amount of fluid will increase the speed of the process. In the same manner, heat will serve to hasten decalcification. Care must however, be observed, since too rapid removal of Calcium salts may produce complete digestion of the tissue specimen, with marked swelling and hydrolysis of the bony matrix and poor staining capacity of the cell. At 37^oC, there will be impaired nuclear staining of Van Gieson’s stain for collagen fibers. At 55^oC, the tissue will undergo complete digestion within 24 – 48 hours. The optimum temperature so far recommended is the room temperature. Agitation and moving of the tissue in solution usually accelerates the rate of diffusion and speeds up the decalcification process.

Increase in size and consistency of tissues will require longer periods for complete decalcification. The ideal time required for decalcifying tissue is 24 – 48 hours. Dense bone tissues usually require up to 14 days or longer to complete the process. In such cases, the solution should be changed daily to ensure better penetration and to test for the degree of decalcification.

I.     Nitric acid

This is the most common decalcifying agent used so far, utilized both as a simple solution or combined with other reagents. It is a very rapid decalcifying agent, producing minimal distortion and is therefore, recommended for routine process. It has, however, the disadvantage of inhibiting nuclear stains and destroying tissues, especially in concentrated solutions. Combining Nitric Acid with Formaldehyde or Alcohol may prevent this.  

A.     Aqueous Nitric Acid Solution 10%

Decalcification:     12 – 24 hours

Advantages:

1.     It is rapid in action.

2.     It produces minimum distortion of tissues.

3.     It produces good nuclear staining (although less than in slower–acting agents)

4.     The acid may be easily removed by 70% alcohol

5.     It is recommended for urgent biopsies.

Disadvantages

1.     Prolonged decalcification may lead to tissue distortion.

2.     It imparts a yellow color with Nitrous Acid, thereby impairing the staining reaction of the tissue.

B.     Formol–Nitric Acid

Decalcification time:        1 – 3 days

Advantages:

1.     It is rapid–acting; hence, is recommended for urgent biopsies

2.     Nuclear staining is relatively good.

3.     It produces less tissue destruction than 10% Aqueous Nitric Acid

Disadvantages

1. The yellow color imparted by Nitrous Acid formation will impair staining reaction of the cell.

This may be prevented by neutralizing the tissue with 5% Sodium Sulfate and washing in running tap water for at least 12 hours. Addition of 0.1% urea to pure concentrated Nitric Acid will also make discoloration disappear without considerably affecting the efficiency of the decalcifying solution.

C.     Perenyi’s Fluid

Formula:   Nitric  Acid 10%                      40 ml

                  Chromic Acid 0.5%                 30 ml

                  Absolute Ethyl Alcohol          30 ml

Decalcification time:        2 – 7 days

Advantages:

1.     It is recommended for routine purposes.

2.     It decalcifies and softens tissues at the same time.

3.     Nuclear and cytoplasmic staining is good.

4.     Maceration is avoided due to the presence of Chromic Acid and Alcohol.

Disadvantages:

1. It is relatively slow decalcifying agent for dense bones; hence, it is not recommended for urgent works.

2. Complete decalcification cannot be determined by chemical test because a precipitate is formed upon the addition of Ammonia to Perenyi’s fluid even in the absence of calcium ion.

This may be dissolved by adding Glacial Acetic Acid drop by drop. About 0.5 ml of Saturated Aqueous Ammonium Oxalate is then added to the solution. Reappearance of a white precipitate within 30 minutes will reaffirm the presence of calcium in the agent, signifying that decalcification is still incomplete.

D.    Phloroglucin – Nitric Acid

Decalcification time:        12 – 24 hours

Advantage:

It is the most rapid decalcifying agent so far, recommended for urgent works.

Disadvantage:

1. Nuclear staining is poor.

2. Prolonged decalcification produces extreme tissue distortion.

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

4. Complete decalcification cannot be determined by chemical means.

II.    Hydrochloric Acid

Hydrochloric acid is 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 section decalcified. However, it will produce good nuclear staining and if used in 1% solution with 70% Alcohol, may be recommended for surface decalcification of the tissue blocks.

III.  Formic Acid

Formic acid is a moderate–acting decalcifying agent that produces better nuclear staining with less tissue distortion than Nitric Acid or Hydrochloric Acid. It is recommended for routine decalcification of post–mortem research tissues, although not suitable for urgent examination.

Decalcification time:              2 – 7 days

Advantages:

1.     It may be used both as a fixative and decalcifying agent.

2.     It permits excellent nuclear and cytoplasmic staining.

3.     It is recommended for small pieces of bones and teeth.

Disadvantages:

1. It is relatively slow; hence, is not suitable for urgent needs.

Decalcification may be hastened by increasing the proportion of Formic Acid to 25 ml; however, such concentration may make the solution opaque, thereby interfering with the staining result.

2. It requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue.

IV.   Trichloroacetic Acid

Decalcification time:              4 – 8 days

Advantages:

1. It permits good nuclear staining.

2. It does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration.

Disadvantages:

1. It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone.

2. It is a very slow – acting; hence, is not recommended for urgent examinations.

V.     Sulfurous Acid – is a very weak decalcifying solution suitable only for minute pieces of bone.

VI.   Chromic Acid (Flemming’s fluid)

Advantages:

1.     It may be used both as a fixative and decalcifying agent.

2.     It is recommended for minute bone spicules.

Disadvantages:

1. Nuclear staining with Hematoxylin is inhibited.

2. It tends to undergo reduction and form precipitates at the bottom of the container thus requiring frequent change of solution.

3. Insoluble pigments are formed when decalcified tissue is dehydrated with Alcohol, hence, tissues must be washed out prior to dehydration.

4. Degree of decalcification cannot be measured by the routine chemical test.

VII.  Citric Acid – Citrate Buffer Solution

Decalcification time:              6 days

Advantages:

1.     It permits excellent nuclear and cytoplasmic staining.

2.     It does not produce cell or tissue distortion.

VIII.   Von Ebner’s Fluid

Formula:

Saturated Aqueous Solution of NaCl 36%    50 ml

Concentrated Hydrochloric Acid                   8 ml

Distilled water                                                            50 ml

Advantages:

1.     It permits relatively good cytology staining.

2.     It is a moderately rapid decalcifying agent.

3.     It does not require washing out before dehydration.

4.     It is recommended for teeth and small pieces of bone.

Disadvantages:

1.     The extent of decalcification cannot be measured by chemical test.

CHELATING AGENT

Chelating agent are substances that 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 chelating agent in the market is ethylene diamine tetraacetic acid (EDTA) salt, with the commercial name, Versene, recommended only for detailed microscopic studies. The tissue is placed in the agent for 1 – 3 weeks, the solution being changed every 3 days, and in the final stage, everyday, to facilitate decalcification.

Formula:

EDTA Disodium Salt               5.5 grams

Neutral Formalin 10%                        100 ml

Advantages:

1. It permits excellent staining results.

2. It produces minimal cell and tissue distortion.

3. It forms minimal histological artifacts, usually caused by production of CO₂ bubbles.

4. Extent of decalcification can be measured by routine chemical test.

Disadvantages:

1. It is very slow, and is therefore not recommended for urgent and routine process.

2. It causes slight tissue hardening.

ION EXCHANGE RESINS

Ion exchange resin (ammonium form of polysterene resin) hastens decalcification by removing calcium ions from formic acid–containing decalcifying solutions, thereby increasing solubility from the tissue. It is not recommended for fluids containing mineral acids such as Nitric Acid or Hydrochloric Acid.

A layer of the ion exchange resin, about ½ 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. Physical or X–ray method, may then measure the degree of decalcification.

The resin that has been previously used may later be reactivated by immersing it in N/10 HCl twice and washing it with distilled water thrice.

Advantages:

1.     Cellular detail is well–preserved.

2.     Decalcification is hastened.

3.     Daily washing of solutions is eliminated.

Disadvantages:

The degree of decalcification cannot be measured by chemical means.

ELECTROPHORESIS

Electrophoresis is a process whereby positive charged Calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution. The time required for decalcification is thereby shortened due to the heat and electrolytic reaction produced in the process. The principle applied 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.

Solutions used for electrolytic decalcification:

Formic acid 88%                                100 ml

Concentrated Hydrochloric Acid       80 ml

Distilled water                                    1000 ml

Prolonged decalcification of tissue is apt to prevent hydrolysis and lead to maceration and destruction of tissue component, which are poorly stained. On the other hand, when the tissue is allowed to stay in the decalcifying agent for a very short period of time, decalcification may be incomplete thereby interfering with the normal cutting of sections and staining of specimen. It is necessary, therefore, that the degree of decalcification be evaluated to ensure complete Calcium removal and allow the normal processing of tissues.

There are three ways by which the extent of decalcification may be measured:

1. Physical or Mechanical Test

Touching or bending the tissue with finger to determine its consistency does this. Decalcified tissues usually have diminished consistency and are softer to touch. This is, however, a very vague and inaccurate way of determining if a tissue has been completely decalcified or not.

An alternate method of evaluating tissue mechanically is by pricking the tissue with a fine needle or a probe. This method is apt to produce needle tract artifacts and destroy important cellular details. Aside from this disadvantage, small calcified foci may not even be detected.

2. X–ray or Radiological Method

This is a very expensive although the most ideal 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, however, not recommended for Mercuric Chloride – a fixed tissue due to the latter’s characteristic radio–opacity that will interfere with the correct interpretation of the plate.

3. Chemical Method

This is a simple, reliable and convenient method recommended for routine purposes, to detect the presence of Calcium in the decalcifying solution.

The decalcifying fluid is usually changed every 24 – 48 hours and the chemical test is performed on the discarded fluid. 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 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. Cloudiness will signify incomplete Calcium removal; hence the need for further decalcification is considered to be complete.

If chemical method of determination is to be done, the decalcifying agent should be prepared with distilled water, since false positive readings may be produced by the calcium ions present in tap water.

Softening of tissues:

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 decalcifying agent and tissue softeners. 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.

Washing out and immersion of fixed tissues in 4% Aqueous Phenol solution for 1 – 3 days may also cause considerable tissue softening and easier sectioning of blocks without producing marked deleterious effects and tissue distortion.

Other substances which may be used as tissue softener are Molliflex, 2% Hydrochloric Acid and 1% Hydrochloric Acid in 70% Alcohol. Tissues immersed in Molliflex may appear swollen and soapy. This does not, however, affect the normal processing and subsequent staining of tissue sections.