DECALCIFICATION OF BIOPSY TISSUE

BY

Dr. Archana S. B.D.S., M.D.S. (Oral Pathology)

Consulting Oral & Maxillofacial Pathologist Ex. Post Graduate Resident,

Dept. of Oral Pathology & Microbiology, JSS Dental College & Hospital,

JSS University, Mysuru, Karnataka, India

Dr. Usha Hegde B.D.S., M.D.S. (Oral Pathology)

Professor & Head, Dept. of Oral Pathology & Microbiology,

JSS Dental College & Hospital, JSS University,

Mysuru, Karnataka, India

Dr. Bhuvan Nagpal B.D.S. (Hons.), M.D.S. (Oral Pathology)

(Gold Medalist) Consulting Oral & Maxillofacial Pathologist

Ex. Post Graduate Resident, Dept. of Oral Pathology & Microbiology,

JSS Dental College & Hospital, JSS University,

Mysuru, Karnataka, India

Sl.No CONTENTS Page No.

1. INTRODUCTION 3

2. TECHNIQUE OF DECALCIFICATION 5

3. DECALCIFYING AGENTS 11

4. FACTORS INFLUENCING DECALCIFICATION 33

5. METHODS OF DECALCIFICATION 38

6.HISTOCHEMICAL METHODS FOR

DECALCIFICATION 47

7. END POINT DETERMINATION 51

8. ARTIFACTS IN DECALCIFICATION 57

9. CONCLUSION 61

10. REFERENCES 63

INTRODUCTION

Teeth and bone belong to the category of hardest tissues, which is denser and

chemically more inert than other body tissues. Because of large amounts of

inorganic components that is calcium and phosphorus, the biological apatite is very

hard to prepare for microscopic examinations.1

Human teeth, as well as alveolar bone, must be decalcified during processing for

histologic analysis because of its structure. Rapid fixation of all dental elements is

difficult to obtain because penetration of the fixative agent through such structures

as enamel, dentin and bone is a slow process. In such cases, the tissues in the center

of the specimen may undergo some alterations before fixation is completed. The

most seriously affected tissue is perhaps the pulp.1, 2

The goal of decalcification is to remove calcium salts from the mineralized tissues

and preparing them for further sectioning of the histologic specimen. Any acid,

even if properly buffered, affects tissue stability. These effects depend on the

solution’s acidity and duration of the decalcification process.3 In addition, the faster

the action of the decalcifying agent, the greater the damage to the tissue. The rapidity

of decalcification may also lead to untoward effects to the staining technique

performed subsequently. There are various factors influencing the speed of

decalcification such as decalcifying solution concentration, temperature, stirring and

tissue suspension.1,3

Decalcification is performed by chemical solutions, which employ acids (acids may

be divided into strong and weak acids or chelates). It is thought to be emphasized

that fixative agents that contain acid in their composition, such as formalin (that

contains formic acid), may also be able to act as decalcifying agents if the acid

component is not neutralized.2

Decalcification is commonly employed in most histopathology laboratories for the

microscopic examination of bone and other calcified tissues. Plastic processing

without decalcification may produce superior results in terms of eliminating

shrinkage and for demonstrating osteoid versus mineralized matrix but may give

poor cytological detail and is a much longer process2. The diagnosis of non-

metabolic diseases of bone such as infections and tumors requires good cellular

morphology and a quick result to allow rapid therapeutic intervention for optimal

patient care.4

Most unsatisfactory results with decalcification can be attributed to overexposure to

the agent used and due to inadequate control procedures. Many alternative

decalcification regimen have been proposed, but most of them have atleast a few

unsatisfactory characteristics. In an attempt to reduce the commonly encountered

artifacts of tissue shrinkage and adverse staining results obtained with rapid

decalcification using strong mineral acids such as nitric acid, a rapid method of

decalcification was devised which gave excellent and reproducible results.2,3 The

procedure for decalcification and the successful monitoring of the process is

discussed and some popular options for the choice of reagents are provided.

DECALCIFICATION:

In order to obtain satisfactory sections of bone, inorganic calcium must be

removed from the organic collagen matrix, calcified cartilage, and surrounding

tissues. This is called decalcification.2

TECHNIQUE OF DECALCIFICATION

The technique of decalcification is divided into following stages:

1. Selection of tissue

2. Fixation

3. Decalcification

4. Neutralization of acid

5. Tissue processing & Staining

The above scheme sets out a general plan of work, but it should not be taken

as rigid schedule. On occasion, fixation of a gross specimen will precede

selection of a piece for decalcification, and there are certain fluids which have

a fixing and decalcifying action.1,2

1) SELECTION OF TISSUE:

Thin slices of bone are obtained using a fine -toothed bone saw, or hacksaw.

To ensure adequate fixation and complete removal of the calcium, slices should

not exceed 4-5 mm in thickness.5 Further the cut surfaces should be re-trimmed

to remove the areas damaged by the saw. Thin slices of calcified tissue can

usually be cut with a sharp knife, but when difficulty is encountered a saw

should be used to avoid damage to the tissue surrounding the calcified area.

The type and duration of the treatment of such tissues ( for example, chronic

tuberculosis foci, calcified scar tissue) will depend on the degree of

calcification. Tissues containing only small areas should be tested after 2-3

hours in a decalcifying fluid.2,5

2) FIXATION:

In order to protect the cellular and fibrous elements of bone from damage caused by

the acids used as decalcifying agents, it is particularly important to thoroughly fix

these specimens prior to decalcification.1 Poorly-fixed specimens become macerated

during decalcification and stain poorly afterwards. This is very noticeable in areas

containing bone marrow. It is therefore common practice for laboratories to extend

fixation times for bone specimens before commencing decalcification. It is

important to provide ready access for the fixative to penetrate the bone, so skin and

soft tissue should be removed from large specimens if practicable.5 Bone specimens

should be sawed into thin slices as soon as possible to enhance fixation. An adequate

volume of fixative should be used. High-quality fine tooth saws should be used to

prepare bone slices. Coarse saws can cause considerable mechanical damage and

force bone fragments into the soft tissues present in the specimen.6 In case of

tooth, the pulpal fixation is done by trimming the apical end of tooth until the

pulp is exposed and then injecting formalin in the apical area where pulp is

fixed.2,6

Buffered formalin is a satisfactory fixative for bone, but where the preservation of

bone marrow is important some laboratories will use alternatives such as one of

the Zinc formalin mixtures, formol acetic alcohol (Davidson’s fixative), or Bouin

fluid. Bone marrow is best fixed in Zenker formol.7 Some fine preparations of

bone have been produced following immersion in Mullers fluid for upto 3

months, followed by decalcification in 3% formic-acid-formalin.6

It has been shown by Cook and Ezra Cohn(1962) that tissue damage during

acid decalcification is approximately four times greater when the tissue is

unfixed.7

3) DECALCIFICATION:

Pierre de Coubertin proposed the Olympic motto “citius, altius, fortius” which is

Latin stands for faster, higher, and stronger respectively.5 There are different

decalcifying agents such as acids and chelating agents, Gray (1954) lists over

50 different mixtures. Many of these mixtures were developed for special purposes.

One such mixture was used as a fixing and dehydrating agent, Other

mixtures contain reagents, like buffer salts, chromic acid, formalin or ethanol,

intended to counteract the undesirable swelling effects that acids have on

tissues.8 Many popular mixtures used today are from the original formulas

developed many years ago (Evans & Krajian 1930; Kristensen 1948; Clayden

1952). For most practical purposes, today’s laboratories prefer simpler solutions

for routine work; If the bone is totally fixed and then treated with a decalcifier

suitable for removal of mineral, the simple mixtures work as well or better than

more complex mixtures.7, 8

There are three main types of decalcifying agents:

• Those based on strong mineral acids

• Those based on weaker organic acids

• Those composed of chelating agents.1, 2

The criteria of good decalcifying agents are:

• Complete removal of calcium

• Absence of damage to tissue cells or fibres

• Non-impairment of subsequent staining techniques

• Reasonable speed of decalcification.2, 3

4) NEUTRALIZATION OF ACID

• Acids can be removed from tissues or neutralized chemically after

decalcification is complete.

• Chemical neutralization is accomplished by immersing decalcified bone into

either saturated lithium carbonate solution or 5-10% aqueous sodium

bicarbonate solution for several hours. Many laboratories simply rinse the

specimens with running tap water for a period of time.9

• Culling (1974) recommended washing in two changes of 70% alcohol for 12-

18 hours before continuing with dehydration in processing, a way to avoid

contamination of dehydration solvents even though the dehydration process

would remove the acid along with the water.4

• Adequate water rinsing can generally be done in 30 minutes for small samples

and 1-4 hours for larger bones. For cryomicrotomy acid decalcified tissues

must be washed in water and stored in formol saline containing 15% sucrose

or PBS at 40C before freezing.2

• Tissues decalcified in EDTA solutions should not be placed directly into 70%

alcohol as this causes residual EDTA to precipitate in the alcohol and within

the tissue. The precipitate does not appear to affect tissue staining, since

EDTA is washed out during these procedures, but may be noticeable during

microtomy or storage when a crystalline crust forms on the block surface. A

water rinse after decalcification or over-night storage in formol saline or PBS

should prevent this.10,11

5) TISSUE PROCESSING & STAINING

• Small blocks containing small amounts of bone may be processed routinely.

Larger or dense blocks require special attention. Often bone infiltrated with

routine paraffin for all tissues can be embedded in harder paraffin to give

firmer support to bone during sectioning.

• The processing should be prolonged with extended periods in molten wax

with three changes of wax under vacuum of 2 hours each.16

MICROTOMY:

A substantial microtome and knife contribute greatly to successful microtomy of

bone. A base sledge microtome and wedge-shaped steel or tungsten carbide edged

knife are recommended. The rake of the knife should be less than for conventional

microtomy, even though this may result in some compression. The blocks should

be well iced before cutting. Slightly thicker sections, 6-7 are acceptable when

cutting bone.11

It often helps if tissue is embedded obliquely in the wax. The floating out bath may

need to be hotter than for soft tissues, as bone has a tendency to crinkle when cut.

Sections should be picked up on chrome gelatine coated slides, to reduce the chance

that they may lift from the slide during staining.15

Fig 16: Microtome17

LENDRUMS TECHNIQUE:

It is very useful method treating tissues known to be hard, hard tissue which is

difficulty in cutting is encountered in spite of, or in the absence of, early precautions,

the block should be soaked in Mollifex (obtainable from British drug houses)

overnight & cut in the usual way the next morning. Although the surface will have

a soapy consistency after this treatment sections cut & stain quite well.2

STAINING:

• Acid treated tissue is less susceptible to hematoxylin staining.

• Ehrlich’s hematoxylin is a good nuclear stain in these conditions and has the

added advantage that it stains mucopolysaccharides and thus demonstrates

cement lines (or reversal lines) and cartilage well.

• Gill’s haematoxylin (3 x) is also a useful stain for decalcified bone. Eosin

staining is enhanced by acid decalcification, so that some reduction in staining

time is required.

• General bone structure is well demonstrated by silver reticulin methods and

particularly by picrothionin.17

DECALCIFYING AGENTS

I. STRONG ACIDS:

Strong acids such as hydrochloric or nitric acid at concentrations up to 10%

are the most rapid in action but if used for an excessive time will rapidly cause a

loss of nuclear staining and can macerate tissues.9 It is important that an

appropriate end-point test is used to minimize exposure of the specimens to these

agents. Generally proprietary decalcifiers that are claimed to be rapid in action are

based on strong acids, most commonly hydrochloric acid, and should be used

conservatively with attention to the provided instructions if good results are to be

obtained. For example Surgipath’s Decalcifier II is rapid in action and contains

hydrochloric acid.9,10

1. NITRIC ACID

History:

The first mention of nitric acid is in Pseudo-Geber's De Inventione Veritatis,

wherein it was obtained by calcining a mixture of niter, alum and blue vitriol. It was

again described by Albert the Great in the 13th century and by Ramon Lull, who

prepared it by heating niter and clay and called it "eau forte" (aqua fortis).1, 9

Glauber devised the process still used today by heating niter with strong

sulfuric acid. In 1776 Lavoisier showed that it contained oxygen, and in

1785 Henry Cavendish determined its precise composition and showed that it could

be synthesized by passing a stream of electric sparks through moist air.10

Nitric acid (HNO3), also known as aqua fortis and spirit of niter, is a

highly corrosive mineral acid. The pure compound is colorless, but older samples

tend to acquire a yellow cast due to decomposition into oxides of nitrogen and water.

Most commercially available nitric acid has a concentration of 68%.2 when the

solution contains more than 86% HNO3; it is referred to as fuming nitric acid.

Depending on the amount of nitrogen dioxide present, fuming nitric acid is further

characterized as white fuming nitric acid or red fuming nitric acid, at concentrations

above 95%.9,10

Nitric acid is the primary reagent used for nitration the addition of a nitro group,

typically to an organic molecule. While some results of nitro compounds are

shock- and thermally-sensitive explosives, a few are stable enough to be used in

munitions and demolition, others are still more stable and used as pigments in

inks and dyes. Nitric acid is also commonly used as a strong oxidizing agent.10

• Physical and chemical properties:

Commercially available nitric acid is an azeotrope with water at a concentration of

68% HNO3, which is the ordinary concentrated nitric acid of commerce. This

solution has a boiling temperature of 120.5 °C at 1 atm. Two solid hydrates are

known; the monohydrate (HNO3·H2O) and the trihydrate (HNO3·3H2O). Nitric acid

of commercial interest usually consists of the maximum boiling azeotrope of nitric

acid and water, which is approximately 68% HNO3, (approx. 15 molar). This is

considered concentrated or technical grade, while reagent grades are specified at

70% HNO3. The density of concentrated nitric acid is 1.42 g/mL.9,10

TABLE 1: Properties of nitric acid18

PropertiesMolecular formula HNO3

Molar mass 63.01 g mol−1

Appearance Colourless liquid Density 1.5129 g cm−3

Melting point −42 °C (−44 °F; 231 K) Boiling point 83 °C (181 °F; 356 K) 68% solution boils

at 121 °C (250 °F; 394 K) Solubility in water Completely miscible Acidity (pKa) -1.4 Refractive index(nD) 1.397 (16.5 °C) Dipole moment 2.17 ± 0.02 D

Fig 1: Structure of nitric acid18

• Laboratory synthesis:

In laboratory, nitric acid can be made by thermal decomposition of copper (II)

nitrate, producing nitrogen dioxide and oxygen gases, which are then passed through

water to give nitric acid.9

2 Cu(NO3)2 2 CuO (s) + 4 NO2 (g) + O2 (g)

An alternate route is by reaction of approximately equal masses of any nitrate salt

such as sodium nitrate with 96% sulfuric acid (H2SO4), and distilling this mixture at

nitric acid's boiling point of 83°C. A nonvolatile residue of the metal sulfate remains

in the distillation vessel. The red fuming nitric acid obtained may be converted to

the white nitric acid.9,10

2 + H2SO4 2 HNO3 +

The dissolved NOx {Generic term for the mono-nitrogen oxides NO and NO2 (nitric

oxide and nitrogen dioxide)}are readily removed using reduced pressure at room

temperature (10–30 min at 200 mmHg or 27 kPa) to give white fuming nitric acid.

This procedure can also be performed under reduced pressure and temperature in

one step in order to produce less nitrogen dioxide gas.10

Dilute nitric acid may be concentrated by distillation up to 68% acid, which is a

maximum boiling azeotrope containing 32% water. In the laboratory, further

concentration involves distillation with either sulfuric acid or magnesium

nitrate which acts as dehydrating agents. Such distillations must be done with all-

glass apparatus at reduced pressure, to prevent decomposition of the acid.

Industrially, highly concentrated nitric acid is produced by dissolving additional

nitrogen dioxide in 68% nitric acid in an absorption tower. Dissolved nitrogen

oxides are either stripped in the case of white fuming nitric acid, or remain in

solution to form red fuming nitric acid. More recently, electrochemical means have

been developed to produce anhydrous acid from concentrated nitric acid feedstock.8

Xanthoproteic test:

Nitric acid reacts with proteins to form yellow nitrated products. This reaction is

known as the xanthoproteic reaction. This test is carried out by adding concentrated

nitric acid to the substance being tested, and then heating the mixture. If proteins

that contain amino acids with aromatic rings are present, the mixture turns yellow.

Upon adding a strong base such as liquid ammonia, the color turns orange. These

color changes are caused by nitrated aromatic rings in the protein. Xanthoproteic

acid is formed when the acid contacts epithelial cells and is indicative of inadequate

safety precautions when handling nitric acid.9

• Safety:

Nitric acid is a corrosive acid and a powerful oxidizing agent. The major hazard

posed by it is chemical burns as it carries out acid hydrolysis with proteins (amide)

and fats (ester)which consequently decomposes living tissue (e.g.skin and flesh).

Concentrated nitric acid stains human skin yellow due to its reaction with

the keratin. These yellow stains turn orange when neutralized. Systemic effects are

unlikely, however, and the substance is not considered a carcinogen or mutagen.10

The standard first aid treatment for acid spills on the skin is, as for other corrosive

agents, irrigation with large quantities of water. Washing is continued for at least ten

to fifteen minutes to cool the tissue surrounding the acid burn and to prevent

secondary damage. Contaminated clothing is removed immediately and the

underlying skin washed thoroughly. Being a strong oxidizing agent, reactions of

nitric acid with compounds such as cyanides, carbides, metallic powders can

be explosive and those with many organic compounds, such as turpentine, are

violent and hypergolic (i.e. self-igniting). Hence, it should be stored away from

bases and organics.9,10

• FLUIDS CONTAINING NITRIC ACID:

Formol nitric acid:

Formula:

• Formalin…………………..5 ml

• Nitric acid (specific gravity 1.41)……………7.5 – 15 ml

• Distilled water ……………to 100 ml.

-In this formula, the formaldehyde partially inhibits the tendency to maceration

by the nitric acid.

-In practice aqueous nitric acid give better cell preservation and staining.

-Discoloration may be prevented by stabilization with urea.2,9

Phloroglucin – Nitric acid

1. Place 10 ml of nitric acid (specific gravity 1.41) in an evaporating dish.

2. Add 1 g of phloroglucin.

3. When bubbling ceases add 100 ml of 10 % nitric acid.

• The use of phloroglucin is said to protect the tissue from maceration, and

allows good subsequent staining.

• But according to some, decalcification here is very rapid, subsequent

staining is very poor, and the method cannot be recommended.2

Aqueous Nitric acid:

Formula

• Nitric acid (stabilized with 0.1% urea)…. 5- 10 ml

• Distilled water………….to 100 ml.

The fluid recommended by Clayden is used as routine decalcifying agent. It

is rapid, causes little damage to tissue if the time of decalcification is carefully

controlled, and allows most staining techniques to be applied.9

Perenyi’s Fluid:

Formula

• 10% nitric acid……………..40 ml

• Absolute alcohol……………30 ml

• 0.5% chromic acid ………..30 ml.

These solutions are kept in stock and mixed freshly when required.

The solution acquires a violet tinge after a short while.

Perenyi’s fluid is slow for decalcifying dense bone.

Is an excellent reagent for small deposits of calcium.

It has little hardening effect on tissue and excellent cytological preparations

are possible after its use.

The chemical test for decalcification cannot be carried out with this fluid : x-

rays should be used.10

Advantage:

Many writers highly recommend 5%, which causes no swelling and acts

powerfully.2

Disadvantages:

• A disadvantage of using nitric acid as a decalcifying fluid is the yellow colour

which develops owing to the formulation of nitrous acid.

• This causes alteration in the speed of decalcification – it gets more rapid as

the colour develops.

• It also causes yellow discoloration of the tissue which subsequently interferes

with staining reactions.

• The yellow colour can be obviated by the addition of 0.1% urea to pure

nitric acid , which should be colourless.

• Even brief exposure to this acid renders unsatisfactory staining of bone

marrow with Giemsa, Maximow's azure ll-eosin or Lillie's azure eosin

formula.2,10

• As the urea has only a temporary effect, further additions should be made

when the acid becomes tinged with yellow(Clayden,1952).1

2. HYDROCHLORIC ACID:

• Etymology:

Hydrochloric acid was known to European alchemists as spirits of salt or

acidumsalis (salt acid). Both names are still used, especially in non English

languages, such as German: Salzsäure, Dutch: Zoutzuur, Northern

Sami: Saltsyra and Polish: kwas solny. Gaseous HCl was called marine acid air.

The old (pre-systematic) name muriatic acid has the same origin (muriatic means

"pertaining to brine or salt"), and this name is still sometimes used. The name

"hydrochloric acid" was coined by the French chemist Joseph Louis Gay-Lussac in

1814.11

• History

Aqua regia, a mixture consisting of hydrochloric acid and nitric acid, prepared by

dissolving salt ammoniac in nitric acid, was described in the works of Pseudo-

Geber, the 13th-century European alchemist.9 Other references suggest that the first

mention of aqua regia is in Byzantine manuscripts dating to the end of the thirteenth

century. Free hydrochloric acid was first formally described in the 16th century

by Libavius, who prepared it by heating salt in clay crucibles. Other authors claim

that pure hydrochloric acid was first discovered by the German benedictine

monk Basil Valentine in the 15th century, by heating common salt and

green vitriol, whereas others claim that there is no clear reference to the preparation

of pure hydrochloric acid until the end of the sixteenth century.10,11

In the seventeenth century, Johann Rudolf Glauber from Karlstadt am Main,

Germany used sodium chloride salt and sulfuric acid for the preparation

of sodium sulfate in the Mannheim process, releasing hydrogen

chloride gas. Joseph Priestley of Leeds, England prepared pure hydrogen chloride

in 1772, and by 1808 Humphry Davy of Penzance, England had proved that the

chemical composition included hydrogen and chlorine.9

During the Industrial Revolution in Europe, demand for alkaline substances

increased. A new industrial process by Nicolas Leblanc (Issoundun, France)

enabled cheap large-scale production of sodium carbonate (soda ash). In

this Leblanc process, common salt is converted to soda ash, using sulfuric acid,

limestone, and coal, releasing hydrogen chloride as a by-product. Until the

British Alkali Act 1863 and similar legislation in other countries, the excess HCl

was vented to air. After the passage of the act, soda ash producers were obliged to

absorb the waste gas in water, producing hydrochloric acid on an industrial scale.11,12

In the twentieth century, the Leblanc process was effectively replaced by the Solvay

process without a hydrochloric acid by-product. Since hydrochloric acid was already

fully settled as an important chemical in numerous applications, the commercial

interest initiated other production methods, some of which are still used today. After

the year 2000, hydrochloric acid is mostly made by absorbing by-product hydrogen

chloride from industrial organic compound production.10

• Physical and Chemical Properties:

Hydrogen chloride (HCl) is a monoprotic acid, which means it

can dissociate (i.e., ionize) only once to give up one H+ ion (a single proton). In

aqueous hydrochloric acid, the H+ joins a water molecule to form a hydronium ion,

H3O+.

HCl + H2O H3O+ + Cl−

The other ion formed is Cl−, the chloride ion. Hydrochloric acid can therefore be

used to prepare salts called chlorides, such as sodium chloride. Hydrochloric acid is

a strong acid, since it is essentially completely dissociated in water.11

Of the six common strong mineral acids in chemistry, hydrochloric acid is the

monoprotic acid least likely to undergo an interfering oxidation-

reduction reaction.10 It is one of the least hazardous strong acids to handle; despite

its acidity, it consists of the non-reactive and non-toxic chloride ion. Intermediate-

strength hydrochloric acid solutions are quite stable upon storage, maintaining their

concentrations over time. These attributes, plus the fact that it is available as a

pure reagent, make hydrochloric acid an excellent acidifying reagent.12

Hydrochloric acid is the preferred acid in titration for determining the amount

of bases. Strong acid titrants give more precise results due to a more distinct

endpoint. Azeotropic or "constant-boiling" hydrochloric acid (roughly 20.2%) can

be used as a primary standard in quantitative analysis, although its exact

concentration depends on the atmospheric pressure when it is prepared.11

Hydrochloric acid is frequently used in chemical analysis to prepare ("digest")

samples for analysis. Concentrated hydrochloric acid dissolves many metals and

forms oxidized metal chlorides and hydrogen gas, and it reacts with basic

compounds such as calcium carbonate or copper (II) oxide, forming the dissolved

chlorides that can be analyzed.12

Fig 2 : Structure of Hydrochloric acid18

Physical properties of hydrochloric acid, such as boiling and melting

points, density, and pH, depend on the concentration or molarity of HCl in the

aqueous solution. They range from those of water at very low concentrations

approaching 0% HCl to values for fuming hydrochloric acid at over 40% HCl.9

• Presence in living organisms:

Gastric acid is one of the main secretions of the stomach. It consists mainly of

hydrochloric acid and acidifies the stomach content to a pH of 1 to 2.11

• Safety:

Concentrated hydrochloric acid (fuming hydrochloric acid) forms acidic mists. Both

the mist and the solution have a corrosive effect on human tissue, with the potential

to damage respiratory organs, eyes, skin, and intestines irreversibly. Upon mixing

hydrochloric acid with common oxidizing chemicals, such as sodium

hypochlorite (bleach, NaClO) or potassium permanganate (KMnO4), the toxic

gas chlorine is produced.11,12

NaClO + 2 HCl H2O + NaCl + Cl2

2 KMnO4 + 16 HCl 2 MnCl2 + 8 H2O + 2 KCl + 5 Cl2

Personal protective equipment such as rubber or PVC gloves, protective eye

goggles, and chemical-resistant clothing and shoes are used to minimize risks when

handling hydrochloric acid. The United States Environmental Protection

Agency rates and regulates hydrochloric acid as a toxic substance.10

FLUIDS CONTAING HYDROCHLORIC ACID:

Jenki’s fluid:

Formula

Absolute alcohol………..73 ml

Distilled water……10 ml

Chloroform……….3 ML

Glacial acetic acid……..3 ml

Hydrochloric acid……….4 ml

*Jenki’s fluid not only decalcifies but also dehydrates. The swelling action of the

hydrochloric acid is counteracted by shrinkage effect of the alcohol.

*Large amounts of this fluid should be used, between 40 & 50 times the bulk of the

tissue. After decalcification the tissue is transferred directly to absolute alcohol in

which it is given several changes to remove the acid.

*Cross- section of human rib is decalcified in 4-6 days.9

Von Ebners fluid:

Formula

• Concentrated hydrochloric acid…..15 ml

• Sodium chloride…..175g

• Distilled water…. To 1,000 ml

* Hydrochloric acid (0.5%) should be added daily until decalcification is complete.

*This fluid is popular in certain parts of Great Britain as a routine decalcifying agent.

It is moderately rapid in action, not quite as good as those obtained with Gooding &

Stewart’s, or Perenyi’s fluid.

*Cross section of human rib (5 mm) is decalcified in 36-72 hours.2,9

Advantages:

Action is rapid, even in dilute solutions. This acid, when used at 370 C, preserves

eosin stains of cytoplasm. When used at 150 to 250 C, satisfactory H & E, Van

Gieson, Masson, and azure eosin stains may be done, if exposure is not prolonged.

To remedy swelling of tissues, chromic acid or alcohol may be added to the solution.

l5% NaCl may also be added to a 3% acid solution to counteract the swelling

action.2,12

Disadvantages:

Causes serious swelling of tissue. At 550 to 600 C loss of calcium salts occurs rapidly,

followed by swelling and hydrolysis of bone matrix, which soon results in complete

digestion. This occurs in as little as 24 hr in 8% hydrochloric acid. Decalcification

at 370 C impairs alum hematoxylin staining and Weigert's iron hematoxylin staining

of nuclei to some extent. Feulgen staining of nuclei is unsuccessful and azure eosin

stains give pink cytoplasm and nuclei.12

II.WEAK ACIDS:

Formic is the only weak acid used extensively as a primary decalcifier. Acetic and

picric acids cause tissue swelling and are not used alone as decalcifiers but are found

as components in Carnoy's and Bouin's fixatives. These fixatives will act as

incidental although weak, decalcifiers and could be used in urgent cases with only

minimal calcification. Formic acid solutions can be aqueous (5-10%), buffered or

combined with formalin.9

The formalin-10% formic acid mixture simultaneously fixes and decalcifies, and is

recommended for very small bone pieces. However, it is still advisable to have

complete fixation before any acid decalcifier is used. Formic acid is gentler and

slower than HC1 or nitric acid, and is suitable for most routine surgical specimens,

particularly when immunohistochemical staining is needed. Formic acid can still

damage tissue, antigens and enzyme histochemical staining, and should be end-point

tested.10

Decalcification is usually complete in 1-10 days, depending on the size, type of bone

and acid concentration. Dense cortical or large bones have been effectively

decalcified with 15% aqueous formic acid and a 4% HCl- 4% formic acid mixture.13

1. FORMIC ACID

• Introduction:

Formic acid (systematically called methanoic acid) is the simplest carboxylic acid.

Its chemical formula is HCOOH or HCO2H. It is an important intermediate

in chemical synthesis and occurs naturally, most notably in ant venom. Its name

comes from the Latin word for ant, Formica, referring to its early isolation by

the distillation of ant bodies. Esters, salts, and the anions derived from formic acid

are referred to as formates.2,13

• History:

Some alchemists and naturalists were aware that ant hills give off an acidic vapor as

early as the 15th century. The first person to describe the isolation of this substance

(by the distillation of large numbers of ants) was the English naturalist John Ray, in

1671. Ants secrete the formic acid for attack and defense purposes. Formic acid was

first synthesized from hydrocyanic acid by the French chemist Joseph Gay-Lussac.

In 1855, another French chemist, Marcellin Berthelot, developed a synthesis from

carbon monoxide that is similar to that used today.10,13

Formic acid was long considered a chemical compound of only minor interest in the

chemical industry. In the late 1960s, however, significant quantities of it became

available as a byproduct of acetic acid production. It now finds increasing use as a

preservative and antibacterial agent in livestock feed.13

• Physical and chemical properties:

Formic acid is a colorless liquid having a highly pungent, penetrating odorant room

temperature. It is miscible with water and most polar organic solvents, and is some

what soluble in hydrocarbons. In hydrocarbons and in the vapor phase, it consists

of hydrogen-bonded dimers rather than individual molecules. Owing to its tendency

to hydrogen-bond, gaseous formic acid does not obey the ideal gas law. Solid formic

acid (two polymorphs) consists of an effectively endless network of hydrogen-

bonded formic acid molecules.2,9

Fig 3: Structure of Formic acid18

Table 2: Properties of Formic acid18

PropertiesMolecular formula CH2O2

Molar mass 46.03 g mol−1

Appearance colourless fuming liquid Odour pungent, penetrating Density 1.220 g/Ml Melting point 8.4 °C (47.1 °F; 281.5 K) Boiling point 100.8 °C (213.4 °F; 373.9 K) Solubility in water Miscible Solubility miscible with ether, acetone, ethyl acetate, glycerol,

methanol, ethanol partially soluble in benzene ,toluene.

log P −0.54 Acidity (pKa) 3.77 Refractive index(nD)

1.3714 (20 °C)

Viscosity 1.57 cP at 268 °C

• Laboratory use:

Formic acid is a source for formyl group for example in the formylation of

methylaniline to N-methylformanilide in toluene. In synthetic organic chemistry,

formic acid is often used as a source of hydride ion. The Eschweiler-Clarke

reaction and the Leuckart-Wallach reaction are examples of this application. It, or

more commonly its azeotrope with triethylamine, is also used as a source of

hydrogen in transfer hydrogenation.13

As mentioned below, formic acid may serve as a convenient source of carbon

monoxide by being readily decomposed by concentrated sulfuric acid.10

CH2O2(l) + H2SO4(l) H2SO4(l) + H2O(l) + CO(g)

• Safety:

Formic acid has low toxicity (hence its use as a food additive), with an LD50 of 1.8

g/kg (oral, mice). The concentrated acid is, however, corrosive to the skin.9

Formic acid is readily metabolized and eliminated by the body. Nonetheless, it has

specific toxic effects; the formic acid and formaldehyde produced as metabolites

of methanol are responsible for the optic nerve damage, causing blindness seen in

methanol poisoning. Some chronic effects of formic acid exposure have been

documented. Some experiments on bacterial species have demonstrated it to be

a mutagen. Chronic exposure to humans may cause kidney damage. Another

possible effect of chronic exposure is development of a skin allergy that manifests

upon re-exposure to the chemical.12

Concentrated formic acid slowly decomposes to carbon monoxide and water,

leading to pressure buildup in the container it is kept in. For this reason, 98% formic

acid is shipped in plastic bottles with self-venting caps.13

• FLUIDS CONTAINING FORMIC ACID:

Aqueous Formic Acid:

Formula

• 90%stock formic acid……………. 5-10 ml

• Distilled water……………to 100 ml

Formic Acid—Formalin (After Gooding & Stewart 1932):

Formula

• 90% stock formic acid…………5-10 ml

• Formaldehyde (37-40%)………… 5 ml

• Distilled water………… to 100 ml

(A formic acid decalcifier with added formalin, claimed to fix and decalcify).13

Buffered Formic Acid (Evans & Krajian 1930):

Formula:

• 20% aqueous sodium citrate …………65 ml

• 90% stock formic acid……………. 35 ml

(This solution has a pH of approximately 2.3.An effective formic acid decalcifier

buffered with citrate).2,13

Formic acid:

Formula

• Formic acid (90%)…………..100 ml

• Distilled water…………… 900 ml

• (A simple effective decalcifier).12

Kristensen:

Formula

• Formic acid……18ml

• Sodium formate……..3.5g

• Distilled water……..82ml

(An effective formic acid decalcifier buffered with formate).12

Advantages:

• Only weak acid used extensively as primary decalcifier.

• It simultaneously fixes and decalcifies the tissue.

• It is gentler and slower and suitable for immunohistochemical staining.1,13

DISADVANTAGES:

• Delayed decalcification if the specimen size is more.

• It can still damage tissue, antigens, and enzyme histochemical staining.13

2. TRICHLOROACETIC ACID:

• Introduction:

Trichloroacetic acid (TCA; TCAA; also known as trichloroethanoic acid) is an

analogue of acetic acid in which the three hydrogen atoms of the methyl group have

all been replaced by chlorine atoms.9

• History:

The discovery of trichloroacetic acid by Jean-Baptiste Dumas in 1839 delivered a

striking example to the slowly evolving theory of organic radicals and valences. The

theory was contrary to the beliefs of JönsJakob Berzelius, starting a long dispute

between Dumas and Berzelius.14

Table 3 : Properties of Trichloroacetic acid18

PropertiesMolecular formula C2HCl3O2

Molar mass 163.39 g mol−1

Appearance White solid Density 1.63 g/cm³ Melting point 57 to 58 °C (135 to 136 °F; 330 to 331 K) Boiling point 196 to 197 °C (385 to 387 °F; 469 to 470 K) Solubility in water Soluble in 0.1 parts Acidity (pKa) 0.66

Structure Dipole moment 3.23 D

Fig 4: Structure of Trichloroacetic acid18

• Synthesis:

It is prepared by the reaction of chlorine with acetic acid in the presence of a suitable

catalyst.9

Advantages:

• Show superior results in staining and cell morphology.2

Disadvantages:

• Slower in action but lesser compared to formic acid.2

• “Acetic acid and Picric acid are weak acids which cause tissue swelling and

are not used alone as decalcifiers but are found as components in Carnoy’s

and Bouin’s fixatives”.1,2

III. CHELATING AGENTS:

These are organic compounds which have the power of binding certain metals. The

chelating agent used for decalcification is ethylene-diaminetetracetic acid (EDTA).

Its use as a decalcifying agent being first described by Hilleman and Lee (1953).

It is a slow process as calcium is removed layer by layer from the hydroxyapatite

lattice.15

Tissues decalcified by this method show a minimum of artifact and may

subsequently be stained by most techniques with first class results. These qualities

make it the decalcifying agent of choice for electron microscopy.11,15

Although EDTA is nominally acid, it does not act like inorganic or organic acids but

binds metallic ions, notably calcium and magnesium. EDTA will not bind to calcium

below pH 3 and is faster at pH 7-7.4, even though pH 8 and above gives optimal

binding; the higher pH may damage alkaline-sensitive protein linkages (Callis &

Sterchi 1998).15

EDTA binds to ionized calcium on the outside of the apatite crystal and as this layer

becomes depleted more calcium ions reform from within; the crystal becomes

progressively smaller during decalcification. This is a very slow process that does

not damage tissues or their stainability.10,15

When time permits, EDTA is an excellent bone decalcifier for immunohistochemical

or enzyme staining and electron microscopy. Enzymes require specific pH

conditions in order to maintain activity, and EDTA solutions can be adjusted to a

specific pH for enzyme staining. EDTA does inactivate alkaline phosphatase but

activity can be restored by addition of magnesium chloride.11,14

EDTA and EDTA disodium salt (10%) or EDTA tetra-sodium salt (14%) approach

saturation and can be used as simple aqueous or buffered solutions at neutral pH of

7-7.4, or added to formalin. EDTA tetrasodium solution is alkaline, and the pH

should be adjusted to 7.4 using concentrated acetic acid. The time required to totally

decalcify dense cortical bone may be 6-8 weeks or longer although small bone

spicules may be decalcified in less than a week.15

• FORMALIN/EDTA (HILLEMANN & LEE 1953)

Formula

• EDTA, disodium salt.................. 5.5 g

• Distilled water……… 90 ml

• Formaldehyde (37-40% stock)…………….10 ml.13

AQUEOUS EDTA, PH 7.0-7.4

Formula

• EDTA, disodium salt…………….. 250 g

• Distilled water……………1750 ml

If solution is cloudy, adjust to approximately 25 g sodium hydroxide. Solution will

clear.16

EDTA solution

Formula

• EDTA (disodium salt)……….. 55 g

• Formalin…………100 ml

• Distilled water…………………900 ml

EDTA is sometimes known as Sequestrene, or Versene.16

Advantages:

Causes little tissue damage.

Conventional stains are largely unaffected.2,16

Disadvantages:

Acts slowly.2

FACTORS INFLUENCING THE RATE OF DECALCIFICATION

Several factors influence the rate of decalcification, and there are ways to speed up

or slow down this process. The concentration and volume of the active reagent,

including the temperature at which the reaction takes place, are important at all

times. Other factors that contribute to how fast bone decalcifies are the age of patient,

type of bone, size of specimen, and solution agitation. Mature cortical bone

decalcifies slower than immature, developing cortical or trabeculae bone. Of all the

factors, the effectiveness of agitation is still being debated.1,2

Fig 5: Factors influencing rate of decalcification

(I) HEAT:

Increased temperature accelerates many chemical reactions including

decalcification, but it also increases the damaging effects acids have on tissue so

that, at 600 C, the bone. Soft tissues and cells may become completely macerated

almost as soon as they are decalcified. Chemical reaction is accelerated two to three

times rise of temperature, and Murayama, Suzuki, and Itoh (1937) ascertained that

with increased temperature the process of decalcification actually takes place within

an even shorter time.2,6

The optimal temperature for decalcification has not been determined. Although

Smith (1962) suggested 250 C as the standard temperature, but in practice a room

temperature (RT) range of 18-300 C is acceptable. Conversely, lower temperature

decreases reaction rates and tissues not completely decalcified at the end of working

week. A better recommendation is to interrupt decalcification but briefly rinsing

acid off bone, immersing it in neutral buffered formalin (NBF), and resuming

decalcification on the next working day.17 Microwave, sonication, and electrolytic

methods produce heat, and must be carefully monitored to prevent excessive

temperatures that damage tissue (Callis and Sterchi 1998).16

Increased temperature also accelerates EDTA decalcification without the risk of

maceration, but may not be acceptable for preservation of heat- sensitive antigens,

enzymes, or electron microscopy work. Brain (1966) saw no objection to

decalcifying with EDTA at 600 C if the bone was well fixed.17

(II) STRENGTH / CONCENTRATION OF ACID:

Generally, more concentrated acid solutions decalcify bone more rapidly but are

more harmful to the tissue. This is particularly true of aqueous acid solution, as

various additives, e.g. alcohol or buffers that protect tissues may slow down the

decalcification rate. With combination fixative-acid decalcifying solutions, the

decalcification rate cannot exceed the fixation rate or the acid will damage or

macerate the tissue before fixation is complete. Consequently, the decalcifying

mixtures should not compromise the balance of desirable effects (e.g. speed) with

the undesirable effects (e.g. maceration, impairing staining).15

In all cases, total depletion of an acid or chelator by their reaction with calcium must

be avoided. This is accomplished by using a large volume of fluid compared with

the volume of tissue (20: 1 is usually recommended), and by changing the fluid

several times during the decalcification process. Brain, however, pointed out that if

a sufficiently large volume of fluid is used (100 ml per g of tissue) it is not necessary

to renew the decalcifying agent even though depletion is less apparent in a larger

volume.17,15

Ideally, acid solutions should be endpoint tested and changed daily to ensure that

the decalcifying agent is renewed and that tissues are not left in acids too long or

over- exposed to acids, i.e. ‘over-decalcification’.18

(III)AGITATION:

The effect of agitation on decalcification is controversial even though it is generally

accepted that mechanical agitation influences fluid exchange within as well as

around tissues with other reagents. Therefore, it would be a logical assumption that

agitation speeds up decalcification and studies were done attempting to confirm this

theory.17

Russel (1963) used a tissue processor motor rotating at one revolution per minute

and reported the decalcification period was reduced from 5 days to 1 day. Others,

including Clayden (1952), Brain (1966), and Drury and Wallington (1980), repeated

or performed similar experiments and failed to find any time reduction.18

The sonication method vigorously agitates both specimen and fluid, and one study

noted cellular debris found on the floor of a container after sonication could possibly

be important tissues shaken from the specimen (Calis & Strerchi 1998).17

Gentle fluid agitation is achieved by low speed rotation, rocking, stirring, or

bubbling air into the solution. Even though findings from various studies are

unresolved, agitation is a matter of preference and not harmful as tissue components

remain intact.17, 18

(IV)SUSPENSION:

The decalcifying fluid should be able to make contact with all surfaces of a specimen

and flat bone slabs should not touch each other or the bottom of a container as this

is enough to prevent good fluid access between the flat surfaces. Bone samples can

be separated and suspended in the fluid with a thread or placed inside cloth bags tied

with thread. Some workers devise perforated plastic platforms to raise samples

above a container bottom to permit fluid access to samples.18

(V)VACUUM:

Another way to speed up the diffusion of one of the reaction products of the

decalcification process is to pump off the gaseous carbon dioxide. Waerhaug (1949)

explains the more rapid decalcification he observed by the closer contact between

decalcifying fluid and object caused by the rapid removal of the carbon dioxide gas.

Very slight differences in rates of decalcification between the usual method and that

in vacuo were found in the present series.17

When the decalcification procedure is watched in vacuo it appears that the process

is considerably accelerated, mainly due to the "turbulent" development of the carbon

dioxide bubbles. It should be borne in mind, however, that it is the low pressure that

causes the considerable expansion of these gas bubbles (at a pressure of 2 cm. Hg

the volume increases 38 times compared with that at atmospheric pressure) though

the decalcification process is not necessarily accelerated.2,17

According to Le Chatelier the reaction equilibrium is influenced by the dissolution

of calcium salts when the formed carbon dioxide is pumped off rapidly, but

could not perceive any shortening of the decalcification time.18

In opinion it is the rapidly expanding gas bubbles that prevent the decalcifying fluid

from reaching the surface of the object in an adequate amount, for the same amount

of the reaction product (Carbon dioxide) occupies a much larger part of the object

and "blocks" it from any further supply of fluid. (By "surface" not only the outer

surface is understood but also the “inner surface," namely, the surface of the spaces

containing the blood vessels as well as that of numerous osteocyte cavities) With

decrease of pressure the reaction equilibrium is shifted in favour of the rate of

decalcification, but the possibility of reaction decreases. Both these factors are

supposed to result in fairly equal times of decalcification whether in vacuo or not.1,18

METHODS OF DECALCIFICATION

• Decalcification using acids/ Manual method

• Microwave decalcification

• Ion exchange resins method of decalcification

• Electrolytic decalcification

• Ultrasonic decalcification

DECALCIFICATION METHODS USING ACID SOLUTIONS:

Acid solutions are most widely used for routine decalcification of large amounts of

bone and calcified tissue. The principle underlying the action of acid decalcifying

agents involves the solubilities of metallic salts. Calcium occurs in bones chiefly as

the carbonate and phosphate salts, and these salts are only slightly soluble in

water.2,18

An acid will act to release the calcium from its combination with the anions and

effect an ion exchange to give a soluble calcium salt. For example, when

hydrochloric acid is used as the decalcifying agent, the released calcium combines

with the chloride ion to form calcium chloride, a soluble calcium salt.16

The calcium ions released will remain in the decalcifying solution itself and are

effectively removed from the bone.17 The general technique to be followed when one

employs acid decalcifying agents is as follows:

1. Selection of tissue

2. Fixation, after fixation, the tissue is washed to remove excess fixative.

3. Decalcification

Fig 6 : Acid decalcification method3

(For best results, gauze-wrapped-bone should be suspended in center of

decalcifying fluid)

The selected tissue should be loosely wrapped in gauze and then suspended in the

center of a large jar that is filled with the decalcifying fluid of choice. About 100

times the volume of the tissue is a good approximate amount, and this large volume

is necessary since the mineral content of a good-sized piece of bone will soon

neutralize the small amount of acid present in the solution. Decalcifying fluids that

may be used include aqueous or alcoholic solutions.19

For the rapid decalcification of bone, stronger solutions of nitric or hydrochloric acid

may be used if employed in conjunction with phloroglucin.17 The phloroglucin acts,

in a way not presently understood, to prevent organic constituents of bone from

being injured by the swelling and macerating action of the strong acids. Tissue

should be removed from the decalcifying fluid as soon as the decalcification process

is complete, otherwise the histologic and cytologic detail will be harmed.18

MICROWAVE DECALCIFICATION:

Microwave decalcification is a novel technique compared to the manual method. In

this method, hard tissues are placed in the decalcifying agent in a microwave oven

for intermittent periods with regular changes of the solution till the end point is

reached. Microwave irradiation has been shown to speed up the process of

decalcification significantly–from days to hours. It has been reported that the

decalcification of bone is accelerated about 10 times compared with that at ambient

temperature.2,14

METHODOLGY: A domestic microwave oven (LG Intellowave, Model 1911HE)

with a fixed rotary plate, maximum power output of 700 W and input voltage 230

V 50 HZ used, 100 ml fresh distilled water and irradiated to maintain the

temperature at around 41-43°C. The glass beaker placed at different points in the

oven while irradiating it to determine the best position of the specimen during

microwave decalcification, since the microwave oven used had a constant timing but

not a constant temperature. All the specimens fixed in 10% neutral buffered formalin

fixative and then washed in water for about 30 min before decalcification.14

Each sample suspended in a beaker with the help of a thread in approximately 100

ml of decalcifying agent for decalcification. The exact time at the start of

decalcification should be noted. The decalcifying solutions changed and pH and

temperature of the solutions should be recorded on a daily basis.14,19

Fig 7: Microwave oven17

The idea of using microwaves to decrease the time for decalcification of temporal

bones was originally introduced by Hellstrom and Nilsson (1992) for rat cochleas.

More recently, microwaves have been demonstrated to be useful in reducing the time

needed for decalcification in EDTA of dense, primate temporal bones (Madden and

Henson, 1997).The energy produced by microwaves generated in a domestic oven

interacts with dipolar molecules by imparting kinetic energy and altering the electric

fields. This energy induces a dielectric field leading to a rapid oscillation of dipolar

molecules at about 180°C, generating heat that is rapidly distributed homogeneously

within the tissue.14,19

Pitol et al, (2007) showed there was a 30 fold increase in decalcification speed

compared to the traditional method when the material was irradiated in a microwave

oven. However, Balaton and Loget (1989) reported that the decalcification of bone

is accelerated about 10 times in the microwave oven compared with that at ambient

temperature.19

Summary:

A new method using microwave oven was seen to accelerate the decalcification. The

choice of decalcifying agent and method is largely dictated by the urgency of the

procedure. The potential application of microwave energy in histotechnology was

first recognized by Mayers (1970). This form of nonionizing radiation produces

alternating electromagnetic fields that result in the rotation of dipolar molecules such

as water and the polar side chains of proteins through 180° C at the rate of 2.45

billion cycles/second. The molecular kinetics so induced result in the generation of

energy flux which continue until radiation ceases.14,15

ION EXCHANGE RESIN METHOD OF DECALCIFICATION:

Before proceeding with this method for decalcification, one must fix and wash the

selected tissue. Following these steps, the bone is decalcified with a mixture of

formic acid and a commercially available ion-exchange resin. The calcium is rapidly

removed from the solution of formic acid into the resin, this eliminates solution

changes that must be carried out to effect proper decalcification with the acid

decalcification methods.18

Tissue is placed in a bottle in a mixture of l0% or 20% resin and formic acid.

Cancellous bone (2 to 3 mm in thickness) will decalcify in 2 to 3 hours in the solution

and thicker pieces (5 to 6 mm in thickness) will take 4 to 8 hours to decalcify.1, 2 A

40% resin and formic acid solution may be employed where speed is essential, but

for good tissue preservation, the bone should not be left in this strength solution any

longer than necessary (up to 8 days). If speed is not essential, tissue may be left in

the following solution up to 20 days without distortion of tissue.19

• WIN-3000 (ion-exchange resin)…….100gm

• 10 % formic acid (aqueous)…………..800 ml

Fig 8: Ion-exchange method (Gauze-wrapped specimen is placed on top of

resin)17

Fig 9: Ion exchange resin17

The advantages of using the ion-exchange method for bone decalcification include

well preserved cellular detail, superior to that obtained with the acid decalcification

methods; faster decalcification; and elimination of the daily solution change. In

addition, the resin, once used, may be reclaimed for further use by washing to

remove excess acid. The washing is followed by l% ammonia water wash, overnight

treatment with saturated ammonium oxalate, and final water wash the next day.2,18

ELECTROLYTIC DECALCIFICATION:

This method employs electrolysis to shorten the time required for decalcification of

bone sections. Materials used in the technique include a durable glass jar containing

the acid decalcifying solution in which is immersed the electrode assembly and bone

specimen, as shown in the figure. The bone specimen is suspended by a platinum

wire anode in the jar, and the insoluble calcium salts are changed to ionizable salts

by the action of the acid in the solution.2,20

-A recommended electrolytic decalcifying solution is as follows:

• 88% formic acid……..100 ml

• Hydrochloric acid…….80 ml

• Distilled water……..820 ml

Current, which is supplied by a power unit, causes an electric field between the

electrodes, and this enables the calcium ions to migrate rapidly from the specimen

(anode) to the carbon electrode (cathode). The acid radicals will migrate from the

cathode to the anode. The temperature of the reaction is regulated between 300 to

450 C. Temperatures exceeding the upper limit of 450 C will cause the disintegration

of the specimen.1,20

Solutions should be changed after 8 hours of use to ensure maximum speed of

decalcification. Prolonged washing of the specimen after electrolysis is unnecessary;

the tissues are rinsed well in alkaline water and the sections immersed in lithium

carbonate before staining. The lithium carbonate treatment of a cut section will

neutralize any remaining acid in the tissue so that the acid cannot interfere with any

staining procedure.19,20

The chief advantage to the electrolytic method lies in the shortened time required for

complete decalcification. This faster time will speed diagnosis and give better

preservation of soft-tissue patterns.1,19

Fig 10: Electrolytic decalcification apparatus3

Staining reactions are usually better, since the method acts fast enough and the

tissues have a relatively short time in the acid bath. Generally, cancellous bone 3 to

5 mm in thickness will decalcify in 45 minutes or less. More compact bone will take

16 hours or longer.1,2

A disadvantage to this method is that only a limited number of specimens may be

processed at any one time. Maintaining contact between tissue and electrode may

also create problems.2,19

ULTRASONIC METHOD OF DECALCIFICATION:

The unique technology of the ultrasonic decalcification offers the rapid destruction

of crystalline structures like calcium phosphate, magnesium phosphate and calcium

carbonate. In combination with adequate solutions it provides maximum cell tissue

preservation. All diffusion processes are significantly accelerated by the Ultrasonic

method.2,19

The advantage is decalcification and intermediate rising solutions work much faster,

save up to 75% decalcification time for bone marrow biopsies. The additional feature

of cooling the tissue samples at a temperature of 17 °C avoids the warming up of the

specimens resulting from the chemical reaction with the decalcifying solution. This

guarantees 100% preservation of the morphological structures and the antigenity of

the samples20.There are no artifacts from shrinking or swelling and all consecutive

histological and immuno-histochemical methods can be applied to these samples.19

Fig 11: Ultrasonic machine17

Depending on the sample size the user can choose from inserts including 4, 9 or 49

sample containers with different volumes. In every batch up to 49 sample containers,

able to carry multiple samples, can be processed simultaneously.17,19

Decalcification of bone specimens of 2-5 mm thickness can be achieved in 5 hours

or less when the decalcifying fluids are agitated by ultrasonic energization.2,20

HISTOCHEMICAL METHODS FOR DECALCIFICATION

Standard methods of decalcification using nitric, hydrochloric, and other acids are

unsatisfactory if histochemical techniques are to be done on the tissue, since acid

treatment will destroy the enzyme activity. Analytical methods for nucleic acids and

polysaccharides should also be preceded by a histochemical, rather than routine,

method for decalcification since these substances are largely destroyed by the acids

employed in the routine technics.11,19

Histochemical decalcification methods include the use of chelating agents (EDTA)

and buffer mixtures. Calcium salts may be removed from bone when it is placed

into a buffered solution of citrate, pH 4.5. Calcium salts are soluble at this pH, and

the zinc present in many buffered solutions of citrate will produce a reversible

inactivation of the alkaline phosphatases (Subsequent reactivation will allow for

their demonstration). Daily changes of the buffer are necessary and the

decalcification progress may be checked by use of the chemical oxalate test

described in the section on acid decalcification methods.20

Tissue should first be fixed in cold 80% alcohol for 24 to 48 hours. It is then placed

in the buffer solution at refrigerator temperature (40 C) until decalcification is

complete. Tissue is then washed in tap water, followed by distilled water, and then

placed in a sodium barbital solution at 370 C for 6 hours to neutralize the tissue from

the effects of acid citrate and to reactivate the enzyme activity. After the barbital

treatment, the tissue is washed for 3 hours in running tap water, and processed for

subsequent enzyme analysis.19, 20

Solutions used are as follows:

Citric acid- ammonium citrate buffer (pH 4.5)

• 1 N citric acid (monohydrate, 7%)…..50ml

• I N ammonium citrate (anhydrous, 7.54%)……..950ml

• 1% zinc sulfate………2ml

• Chloroform……..0.1ml

Sodium barbital solution

Sodium barbital…….100ml

Glycine……….75mg

Other buffer mixtures

L Molar hydrochloric acid-citrate buffer at pH 4.5

I N hydrochloric acid…….540ml

I M sodium citrate solution………….460ml

(Use 29.4% of the dihydrate or 35.7% of the following compound)

Lorch's citrate hydrochloric acid buffer at pH 4.4

• Citric acid crystals…..14.7gm

• 0.2 N sodium hydroxide………..700ml

• 0.1 N hydrochloric acid……….300ml

• 7% zinc sulfate……….2ml

• Chloroform…………0.1ml

Acetate buffer at pH 4.5

• 1 N acetic acid……….520ml

• 1 N sodium acetate (8.2%anhydrous or 13.6% crystalline)…….480ml

• l% zinc sulfate….2ml

• Chloroform………0.1ml.18

DEMONSTRATION OF GLYCOGEN IN DECALCIFIED TISSUES

Total or partial losses of glycogen result when tissues are decalcified with acids or

EDTA. Glycogen in muscle and marrow cells will withstand decalcification best

when fixed in aqueous formalin acidified with acetic or formic acid. Thorough

fixation of protein surrounding glycogen and embedding in celloidin before paraffin

will create semi permeable membranes surrounding the glycogen and thus hinder

diffusion the following techniques for decalcification to subsequently demonstrate

glycogen.18

TECHNIQUES:

• Celloidin

a. Tissue should be fixed in acetic alcohol formalin 24 hours at room

temperature (250 C) or 3 to 4 days at 50 C. Dehydrate with alcohols and

infiltrate for 3 days with l% celloidin in equal volumes of alcohol and ether.

b. Transfer to 80% alcohol to harden celloidin.

c. Decalcify with 5% formic acid (aqueous); solution should be changed daily

until a negative chemical test is obtained.

d. Wash 6 to 8 hours in running water, dehydrate, clear, and embed in paraffin.19

• Hard protein fixative

a. Fix tissues as described above.

b. Transfer to Bouin's fluid for 3 days at 250 C.

c. Decalcify in daily changes of 10% formalin with 5% formic acid.

d. Wash 8 hours in running water; dehydrate, clear, and embed in paraffin.19,20

END POINT DETERMINATION

Because of the harmful effects of acid on tissue, they must be left in decalcifying

fluids the minimum time possible. Accurate determination of the endpoint of

decalcification is therefore necessary. Experience will enable a value judgment to be

made upon approximate time required, depending on the tissue structure and size of

the block. Small biopsies of cancellous bone should be examined after 24 hours, and

other specimens daily after two to three 24-hour changes of fluid.15

TYPES OF END POINT DETERMINATION:

• PHYSICAL METHOD

• CHEMICAL METHOD

• RADIOGRAPHIC METHOD

PHYSICAL METHOD:

• Experience will enable a value judgement to be made upon approximate time

required, depending on the tissue structure and size of the block.

• Experienced hands can tell by the 'feel' of the tissue if decalcification is

complete.

• Probing the tissue with a needle is not recommended, judicious bending or

trimming can be valuable, but this damages the tissue.

• Another method used was to test weight loss or weight gain procedure that

provides relatively good, quick results with all acids and EDTA (Mawhinney

et al. 1984, Sanderson et al. 1995).17

• Although still used, physical tests are considered inaccurate and damaging to

tissues.

• Probing, needling, slicing, bending or squeezing tissue can create artefacts,

e.g. needle tracks, disrupt soft tumour from bone, or cause false positive micro

fractures of fine trabeculae, a potential misdiagnosis.19

Bubble Test:

Acids react with calcium carbonate in bone to produce carbon dioxide, seen as a

layer of bubbles on the bone surface.

The bubbles disperse with agitation or shaking but reform, becoming smaller as less

calcium carbonate is produced.

As an endpoint test, a bubble test is subjective and unreliable but can be used as a

guide to check the progress of decalcification, i.e. tiny bubbles indicate less calcium

present.18

Fig 12: Probing needle17 Fig 13: Weighing machine17

CHEMICAL METHOD:

• This method depends upon the identification of calcium in the decalcifying

solution.

• It therefore follows that the endpoint can only be detected by sampling the

fluid change following completion.

• This method cannot be used following EDTA decalcification

Calcium oxalate test (Clayden 1952):

This method involves the detection of calcium in acid solutions by precipitation of

insoluble calcium hydroxide or calcium oxalate, but is unsuitable for solutions

containing over 10% acid even though these could be diluted and result in a less

sensitive test.20

Solutions:

1. Ammonia hydroxide, concentrated.

2. Saturated aqueous ammonium oxalate.

Method:

Fig 14: Methodology of calcium oxalate test17

Result:

• If a white precipitate (calcium hydroxide) forms immediately after adding the

ammonium hydroxide a large quantity of calcium is present, making it

unnecessary to proceed further to step 3 which would also be positive.

• Testing can be stopped and a change to fresh decalcifying solution made at

this point.

• If step 2 is negative or clear after adding ammonia hydroxide, then proceed to

step 3 to add ammonium oxalate.

• If precipitation occurs after adding the ammonium oxalate, less calcium is

present.

• When a smaller amount of calcium is present, it takes longer to form a

precipitate in the fluid, so if the fluid remains clear after 30 minutes, it is safe

to assume that decalcification is complete.19,20

RADIOGRAPHIC METHOD:

This is the most sensitive test for detecting calcium in bone or tissue calcification.

The method is the same as specimen radiography, using a FAXITRON with a

manual exposure setting of approximately 1 minute, 30 kV, and Kodak X-OMAT

X-ray film on bottom shelf. It is possible to expose several specimens at the same

time.19

The method is: Rinse acid from sample, place carefully identified bones on

water-proof polyethylene sheet on top of the X-ray film, expose according to

directions, and leave bones to place until film is developed and examined for

calcifications.1,2

Bones with irregular shapes and variable thickness can occasionally mislead on

interpretation of results. This problem is resolved by comparing the test radiograph

to the pre-decalcification specimen radiograph, and correlating suspected calcified

areas with specimen variations.2

Areas of mineralization are easily identified, with tiny calcifications best viewed

using a hand-held magnifier.1

Radiography only indicates the presence of deeper foreign objects and care must be

taken during microtomy not to damage the knife.18

Fig 15: Stages of end point determination by radiographs2

SURFACE DECALCIFICATION:

• Once decalcification is complete, surface acid should be washed from the

tissue with water. If there is any delay before processing the tissue should be

returned to formal-saline. Although there are advocates of neutralizing the

acid before processing it is probable that it will all be washed out by the

processing fluids.14

• Occasionally, an unexpected area of calcification may only become apparent

whilst a paraffin-wax block is being trimmed. If only a small area is involved

it is possible to decalcify the surface layer by inverting the block in 5%

hydrochloric acid for 1 hour or so.2

• This is less drastic than returning the tissue to aqueous solutions. As only the

top 30 m or so is likely to be decalcified, care should be exercised to collect

the first sections cut. Before cutting, the block should be rinsed in water to

avoid contaminating knife or microtome with acid.1,2

ARTIFACTS IN DECALCIFICATION

Impacted bone fragments:

When samples of bone are taken for histological examination, either by needle or

trephine, or when using a saw to remove a small sample from a large specimen, the

bone is subjected to significant trauma which often leads to displacement of bony

fragments and disruption to the adjacent soft tissues.

Remedy: Trim deeply into the specimen to avoid the most traumatized areas close

to the surface.8

Bone dust artifact:

Undecalcified sections may contain a fine grit- like, bone dust or powder seen

within or in close proximity to the bony trabeculae. This material is more apparent

in H & E stained sections where it stains strongly with hematoxylin but is largely

obscured by von Kossa staining. When deposited in the bone marrow it stains black

with von Kossa, suggesting it originates from the calcified trabecular matrix. The

artifact is probably produced during sectioning and becomes more prominent with

increasing section thickness

The bone dust occurs in sections prepared with both glass and diamond knives, and

there appears to be no reliable way of preventing it.8

Fig 17: Bone dust artifact8

Effects of over-decalcification on tissues:

Most artifacts in decalcified tissues relate to poor initial fixation, over fixation, or

incomplete decalcification. Artifacts arising from the first two situations are

irreversible whereas incomplete decalcification can be rectified by surface

decalcification of the paraffin block.

Specimens subjected to decalcification by mineral or organic acids must be

protected from the hydrochloric action of these agents by proper fixation. Digestion

of cellular and other tissue components occurs more rapidly when the tissue is

unfixed or only partially fixed. Prolonged exposure to acid decalcifying agents will

eventually damage even well fixed tissues, so determining the end point of

decalcification accurately is essential in all situations. Over decalcified sections

typically stain strongly with eosin and show a marked loss of nuclear

hematoxyphilia. Nuclear and cytoplasmic detail is poorly preserved.8

Fig 18: Over decalcification in a section of bone8

Effects of incomplete decalcification:

Incomplete decalcification of specimen is clearly apparent when trimming a paraffin

block. As discussed previously, this problem is readily corrected, but there are

associated problems such as damage to the microtome knife and to the soft tissue

surrounding the calcified areas. If sections can be obtained, the bony trabeculae stain

strongly with hematoxylin (indicating residual calcium) and the soft tissue is

severely disrupted.8

Fig 19: Incomplete decalcification in a section of bone8

Limitation of paraffin wax embedding for dense bone:

Paraffin wax may not adequately support dense bone and resultant sections may

show holes, folds and coarse chatter. Soaking the trimmed face of the paraffin block

in a softening agent for 5 to 15 minutes prior to chilling may improve the special

quality.8

Fig 20: Paraffin section of decalcified dense bone showing holes, folds and

coarse chatter8

PRECAUTIONS TAKEN DURING STAINING:

• Use freshly prepared hematoxylin

• Routine hematoxylin stain time should be doubled

• Acid differentiation step should be shortened

• Bluing solutions should be mild bases to avoid loss of bone structure caused

by ammonia after bluing.

• Collagen stains to demonstrate mature and fine immature fibres in certain

tumours and fracture callus.18,20

CONCLUSION

Decalcification is a straightforward process but to be successful requires:

• A careful preliminary assessment of the specimen

• Thorough fixation

• Preparation of slices of reasonable thickness for fixation and processing

• The choice of a suitable decalcifier with adequate volume, changed regularly.

• A careful determination of the endpoint

• Thorough processing using a suitable schedule.

REFERENCES

1. Callis GM, Bancroft JD. Theory and Practice of Histological Techniques 6th

ed. Edinburgh: Churchill Livingstone. 2008;338-360.

2. Culling CF, Allison RT, Barr WT. Cellular Pathology Technique 4th ed.

London: Butterworths.1984;408-30.

3. Callis G, Sterchi D. Decalcification of Bone: Literature Review and Practical

Study of Various Decalcifying Agents, Methods, and Their Effects on Bone

Histology. The Journal of Histotechnology 1998;21:49-58.

4. Prasad P and Donoghue M. A comparative study of various decalcification

techniques. Indian journal of dental research 2013;24:302-8.

5. Sanjai K, Kumarswamy J, Patil A, Papaiah L, Jayaram S and Krishnan L.

Evaluation and comparison of decalcification agents on the human teeth.

Journal of Oral and Maxillofacial Pathology 2012;16:222-7.

6. Afreen Nadaf MS, Radhika MB, Lalita J, Thambiah, Paremala K, Sudhakara

M. Decalcification: a simpler and better alternative. Journal of Dentistry and

Oral Bioscience 2011;2:10-3.

7. Mathews B J, Mason G I. Influence of decalcifying agents on

immunoreactivity of formalin-fixed, paraffin embedded tissue.

Histochemical journal 1984;16:771-787.

8. Rolls GO. Difficult Blocks and Reprocessing. Leica Microsystems 2011.

9. Joshi H, Parikh N, Soni N, Jain S. Comparison of Efficacy of Various

Decalcifying Agents on Human Teeth. Journal of research and advancement

in Dentistry 2014;3:47-52.

10.Lillie, R. D. Histopathologic technique and practical histochemistry, ed. 3,

New York, McGraw- Hill Book Co,1965.

11.Zappa J, Cieslik-Bielecka A, Adwent M, Cieslik T, Sabat D. Comparison of

different decalcification methods to hard teeth tissues morphological analysis.

Dent Med Probl 2005;42:21-26.

12.Verdenius H W and Alma L. A quantitative study of decalcification methods

in histology. Journal of Clinical Pathology 1958;11:229-236.

13.Athanasou N A, Quinn J, Heryet A. Effect of decalcification agents on

immunoreactivity of cellular antigens. Journal of Clinical Pathology

1987;40:874-878.

14.Sangeetha R, Uma K, Chandavarkar V. Comparison of routine decalcification

methods with microwave decalcification of bone and teeth. Journal of

Maxillo-Facial Pathology 2013;17:386-399.

15.Mattella Grando L, Westphalen L, Bento Pelisser V, Vieira F et al.

Comparative analysis of two fixating and two decalcifying solutions for

processing of human primary teeth with inactive dentin carious lesion. Revista

Odonto Ciencia 2007;22(56):99-105.

16.Mawhinney WHB, Richardson E, Malcolm AJ. Technical methods control of

rapid nitric acid decalcification. J Clin Pathol 1984;37:1409-1415.

17.Morse A: Formic acid – Sodium Citrate Decalcification and Butyl Alcohol

dehydration of teeth and bones for sectioning in teeth and bones for sectioning

in paraffin. J Dent Res 1945;24:143-153.

18.Myers, R.L. The 100 most important chemical compounds: a reference guide.

Greenwood Publishing Group. 2007;141-147.

19.Warshawsky H, Moore G. A technique for the fixation and decalcification of

rat incisors for electron microscopy. J Histochem Cytochem 1967;15:542-9.

20.Waerhaug J. Decalcification of bone and teeth under vacuum- a rapid method

for producing hard tissue preparations. J Dent Res 1949;28:525.