Dehydration

As soon as tissues have been fixed, and the bones and teeth have been decalcified, it is necessary to remove the water from the tissue in preparation for impregnation. This process of removing intercellular  and extracellular water from the tissue following fixation and prior to wax impregnation is known as dehydration; and the solutions utilized to make this possible are called dehydrating agents.

Characteristics of an ideal dehydrating solution

1. It must dehydrate rapidly without producing considerable shrinkage or distortion of tissues.

2. It should not evaporate very fast.

3. It should not be able to dehydrate even fatty tissues.

4. It should not harden tissues excessively

5. It should not remove stains.

6. It should not be toxic to the body.

7. It should not be a fire hazard.

Commonly used dehydrating agents

1. Alcohol

The tissue is passed thru a series of progressively increasing concentration of alcohol. Under no circumstances should a formalin–fixed tissue be transferred directly to higher grades of alcohol, e.g. 85 – 95% Alcohol because this is liable to produce considerable shrinkage and hardening of tissues leading to distortion. Concentrated alcohol (95% or absolute) tend to harden only the surface of the tissue while the deeper parts are not completely penetrated. This will result in a relatively unequal impregnation of tissue with consequently poor cutting of sections. To avoid this, 70% or lower concentrations of Alcohol, gradually increased to 95% is used.

The strength of initial Alcohol required in each concentration will depend upon the size, and nature of each tissue and fixative used. Generally, smaller and more delicate tissues require lower concentrations and shorter intervals between changes of succeeding ascending grades of alcohol. A very concentrated solution (above 80%) makes tissues hard, brittle and difficult to cut. Prolonged storage in lower concentrations of Alcohol (below 70%) tends to macerate the tissue. The tissue may be stored in 70 – 80% alcohol, although not for very long periods of time, since this may later interfere with the staining properties of the specimen.

A temperature of 37^oC will hasten dehydration time, and is especially used for tissue sections that require urgent examinations such as fragmentary biopsies. To ensure complete dehydration, a layer of Anhydrous Copper Sulfate, about ¼ inch deep is placed in the bottom of the container and covered with filter paper. This will accelerate dehydration by removing water from the dehydrating fluid. A blue discoloration of Copper Sulfate Crystals will indicate full saturation of dehydrating fluids with water. Alcohol is then discarded and changed with a fresh solution.

2. Acetone

It is a cheap, rapid–acting dehydrating agent utilized for most urgent biopsies which it dehydrates in ½ to 2 hours. Its use has been limited, however, only to small pieces of tissues due to its extreme volatility and inflammability. Because of considerable tissue shrinkage produced, acetone has not been recommended for routine purposes.

3. Dioxane (Diethyl Dioxide)

Dioxane is an excellent dehydrating agent and clearing agent readily miscible in water, melted paraffin, Alcohol and Xylol. It produces less tissue shrinkage as compared to Alcohol dehydration. Tissues can be left in this reagent for long periods of time without affecting the consistency or staining properties of the specimen. Because Dioxane is miscible with both water and paraffin, tissues may be placed directly into the solution after washing out. However, tissue sections dehydrated with dioxane tend to ribbon poorly. Aside from being expensive, dioxane is also dangerous, and this is its main disadvantage. Its vapor being expensive, dioxane is also dangerous, and this is its main disadvantage. Its vapor produces cumulative and highly toxic action in man; hence, it should be used routinely. The laboratory room should be properly ventilated, and all residues should be washed down in the sink.

The following is an example of a time for dehydration with Dioxane (Graupner’s method)

1st	Pure Dioxane Solution	1 hour
2nd	Pure Dioxane Solution	1 hour
3rd	Pure Dioxane Solution	2 hours
1st	Paraffin Wax	15 minutes
2nd	Paraffin Wax	45 minutes
3rd	Paraffin Wax	2 hours

In another method (Weiseberger’s method), the tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide. Water is displaced from the tissue by dioxane and in turn absorbed by calcium oxide or quicklime. Dehydration period ranges from 3 – 24 hours.

Tissues which have been treated with chromate fixative, e.g., Regaud’s or Moller’s fluid, should be thoroughly washed in running tap water prior to treatment with dioxane in order to remove the chromate.

4. Cellosolve (Ethylene Glycol Monoethyl Ether)

Cellosolve dehydrates rapidly and is not harmful to the tissues. The tissue may be transferred from water to normal saline directly to cellosolve and stored in it for months without producing hardening or distortion.

5. Triethyl Phosphate

Tissues are fixed, washed and transferred directly into Triethyl Phosphate solution for dehydration. It removes water very readily and produces very little distortion and hardening of tissues. It is soluble in Alcohol, Water, Ether, Benzene, Chloroform, Acetone and Xylene.  It is used to dehydrate sections and smears following certain stains and produces minimum shrinkage.

6. Tetrahydrofuran (THF)

Tetrahydrofuran is a reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin. It can dissolve many substances including fats and is in itself miscible with lower Alcohols, Ether, Chloroform, Acetone, Benzene and Xylene. It may be used for demixing, clearing and dehydrating paraffin sections before and after staining. It causes less shrinkage and easier cutting of sections with fewer artifacts. It does not dissolve out aniline dyes. In fact, most staining procedures give improved results with tetrahydrofuran. It is also non–toxic although prolonged exposure (up to 6 months) may cause conjunctival irritation. Because of this and it's rather offensive odor, processing with THF should be done in a well–ventilated room.

As a general rule, whatever dehydrating agent is used, the amount in each stage should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.

Adhesives

After cutting, sections are floated out on a water bath. Bubbles accumulating under the ribbon may then be removed with a smooth teasing needle, care being taken not to tear the section. Bubbles may also be removed by pulling the ribbon very gently across the edge of a glass slide held below the section in the water bath. When the section chosen have flattened out, the numbered slide is immersed in the water bath and the section is fished out.

To promote adhesion of sections, adhesives may be spread thinly and evenly on a clean grease–free slide which then gently approximated to the end of the ribbon, and drawn upwards in a near vertical motion.

1. Mayer’s Egg Albumin

Egg white        50 cc

Glycerin          50 cc

Filter and add crystals of thymol to prevent the growth of molds

Mayer’s egg albumin is the most commonly used because it is very easy to make, convenient and is relatively inexpensive. A drop of Mayer’s egg albumin is usually smeared into the clean glass slide before sections are oriented. Sections which have been creased on cutting may then be stretched by gentle heating before attaching them into the slides. During staining, the excess of albumin may also take up the stain; hence, the slide should be wiped off to remove any excessive solution which may later on interfere with staining and interpretation of sections.

For celloidin sections, egg albumin is smeared on the slide, the section is transferred from 95% alcohol bath to the slide, pressed flat on the slide with a smooth filter paper coated with thin celloidin mixture, dried and stored in 70% alcohol until it is ready for staining.

2. Dried albumin

Dried albumin            5 grams

Sodium chloride         5 grams

Dissolve in 100 cc of distilled water and add crystals of thymol

3. Gelatin

Gelatin                        1 gram

Distilled water            100 ml

Glycerol                     15 ml

Phenol crystals           2 grams

4. Starch paste

Powdered starch        1 gram

Distilled water           30 ml (10 ml cold; 20 cc boiling)

N/1 Hydrochloric acid 2 drops. Add thymol crystals to prevent the formation of molds

5. Plasma

Plasma is readily available from outdated blood stored in blood banks, dispensed into sterile tubes of 0.5 ml each.

Clearing

Clearing or de–alcoholization is the process whereby Alcohol is removed from the tissue and replaced with substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or the medium on which the tissue is to be mounted (e.g. Canada Balsam)

When used after Alcohol dehydration, the clearing agent serves to mix with Alcohol and remove it from the tissue. It should be miscible also with paraffin in order to facilitate the penetration of this embedding medium. The most commonly used clearing agents for de–alcoholization in the embedding process are Xylene, Dioxane, Chloroform and cedarwood.

When used after the tissue section has been stained, the clearing agent will make microscopic tissue preparations transparent due to high index of refraction. Aside from removing Alcohol, a clearing agent must also be miscible with Canada Balsam and other resins that are used for mounting sections. The most commonly used clearing agent for this purpose is Xylene.

Glycerine and gum syrup are used when the tissue is to be cleared directly from water, as in Frozen Section. No de–alcoholization is involved in this process. The clearing agents merely improved the refractive index of the tissue.

Characteristics of a good clearing agent

1. It should be miscible with Alcohol to promote rapid removal of the dehydrating agent from the tissue.

2. It should be miscible with paraffin and / or mounting medium to facilitated impregnation and mounting of sections.

3. It should not produce excessive tissue shrinkage and hardening.

4. It should not evaporate quickly with waterbath.

5. It should not make the tissue transparent.

Commonly used clearing agents

A. Xylene

Xylene is a colorless clearing agent most commonly used today. Clearing time is usually ½ to 1 hour. It is used for clearing, both for embedding and mounting procedures.

Advantages:

1. It is the most rapid clearing agent, suitable for urgent biopsies, which it clears within 15 – 30 minutes.

2. It makes tissue transparent.

3. It is miscible with absolute alcohol and paraffin.

4. It does not extract out aniline dyes.

5. For mounting procedures, it does not dissolve celloidin and can, therefore, be used for celloidin sections.

6. It evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding.

7. It is cheap.

Disadvantages

1. It is highly flammable.

2. If used longer than 3 hours, it makes tissue excessively hard and brittle.

3. It causes considerable hardening and shrinkage of tissues; hence, is not suitable for nervous tissues and lymph nodes.

4. Xylene becomes milky when an incompletely dehydrated tissue is immersed in it.

B. Toluene

Toluene may be used as a substitute for Xylene or Benzene for clearing both during embedding and mounting process. Time recommended for clearing is 1 – 2 hours.

Advantages:

1. It is miscible with both absolute alcohol and paraffin.

2. It acts fairly rapidly and is recommended for routine purposes.

3. Tissues do not become excessively hard and brittle even if left in toluene for 24 hours.

4. It is not carcinogenic.

Disadvantages:

1. It is relatively slower than Xylene and Benzene.

2. It tends to acidify in a partially filled vessel.

3. Highly concentrated solutions will emit fumes that are toxic upon prolonged exposure.

4. It is more expensive.

C. Benzene

Benzene is preferred by some as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly.

Advantages:

1. It is rapid acting, hence is recommended for urgent biopsies (15 – 60 minutes) and routine purposes.

2. It volatizes rapidly in paraffin oven and is therefore easily eliminated from the tissue.

3. It is miscible with absolute alcohol.

4. It does not make tissues hard and brittle.

5. It causes minimum shrinkage.

6. It makes tissues transparent.

Disadvantages:

1. It is highly flammable.

2. If a section is left in benzene for a long time, considerable tissue shrinkage may be observed; hence, tissues should be transferred to paraffin wax as soon as possible.

3. Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in Aplastic Anemia. If ever benzene is to be used for clearing, the laboratory should be well–ventilated.

D. Chloroform

Chloroform is used for routine clearing of tissues during the embedding process.

Advantages:

1. It is recommended for routine work (6 – 24 hours)

2. It is miscible with absolute alcohol

3. It is recommended for tough tissues (e.g., skin, fibroid and decalcified tissues) for nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues.

4. It is suitable for large tissue specimens.

5. It is not inflammable.

Disadvantages:

1. It is relatively toxic to the liver after prolonged inhalation; adequate room ventilation and proper caution may prevent this when handling the specimen.

2. Wax impregnation after Chloroform clearing is relatively slow.

3. It does not make tissue transparent.

4. Its vapor may attack the rubber seal used in vacuum impregnation bath.

5. Complete clearing is difficult to evaluate.

6. Tissues tend to float in chloroform; this may be avoided by wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution.

7.     It evaporates quickly from a waterbath.

E. Cedarwood oil

Cedarwood oil is used to clear both paraffin and celloidin sections during the embedding process. It is especially recommended for Central Nervous System tissues and Cytological studies, particularly of smooth muscles and skin, and requires two changes in clearing solutions; clearing is usually complete in 2 – 3 days.

Advantages:

1. It is very penetrating.

2. It is miscible with 96% alcohol which it removes readily.

3. It clears celloidin in 5 – 6 days.

4. It causes minimal shrinkage and hardening of tissues.

5. Tissues may be left in oil indefinitely without considerable damage and distortion.

6. It does not dissolve out aniline dyes.

7. It makes tissue transparent.

8. Clearing with cedarwood oil often improves cutting of the sections.

Disadvantages:

1. It is extremely slow clearing agent, hence, is not recommended for routine purposes.

2. It is hard to be eliminated from the tissues in paraffin bath, making the wax impregnation process very slow. This may be improved or hastened by transferring the specimen from oil to benzene for ½ hours before finally placing the tissue in wax.

3. Quality is not always uniform and good. Tissues cleared in cedarwood oil initially float before gradually sinking to the bottom as clearing proceeds. Hence, the tissue may dry out before it is completely cleared. Superimposing absolute alcohol on the surface of the clearing agent can prevent this. Once saturated, the specimen should then be transferred to a fresh solution of cedarwood oil.

4. Cedarwood oil become milky upon prolonged storage and should be filtered before use.

5. Cedarwood oil that has been previously used to clear acetic – alcohol fixed tissues may produce crystals with a melting point of approximately 35^oC and therefore interfere with adequate clearing of tissue. The solution must be heated to 200^oC in order to dissolve the crystals and restore the solution to its normal state.

6. It is very expensive.

F. Aniline oil

This not normally utilized as a routine clearing agent but is rather recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.

G. Clove oil

This reagent causes minimum shrinkage of tissues. However, its quality is not guaranteed due to its tendency to become adulterated. Wax impregnation after clearing with Clove oil is slow and difficult. Tissues become brittle, aniline dyes are removed, celloidin is dissolved. All of these, plus the expensiveness of the solution, makes it unsuitable for routine clearing purposes.

H. Carbon Tetrachloride

Carbon Tetrachloride may be used in clearing tissues for embedding. Its properties are very similar to that of chloroform although it is relatively cheaper. Its disadvantage is the same as that of chloroform. It produces considerable tissue hardening and is dangerous to inhale on prolonged exposure due to its highly toxic effects.

I. Tetrahydrofuran

Tetrahydrofuran is superior to ordinary dehydrating and clearing agent due to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation. It is non–toxic but has an offensive odor and should be used in a well–ventilated room.

           

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.