DECALCIFICATION

SHRAVYA.M

CONTENTS CONTENTS

• Introduction

• Biopsy

• Criteria of a good decalcifying agent

• Factors affecting decalcification

• Technique

• Bone Decalcification

• Teeth Decalcification Selection

Processing

Staining

• Endpoint Decalcification

• Undecalcified Sections Of Bone

• Properitary Decalcifiers

• Artefacts

• Recent Studies

• Recent Advances

• References

INTRODUCTION To study the histological structure, the tissue should be

appropriately prepared for microscopic examination.

Tissue specimens must be thin enough to permit the passage of light & should be one cell thickness for detailed morphology.

Average thickness is 4-6 µm.

To section the hard mineralised tissue decalcification is necessary

BIOPSY• A biopsy is a procedure performed to remove tissue or

cells from the body for examination under a microscope.

BONE BIOPSY• A bone biopsy is a procedure in which a small sample

of bone is taken from the body and looked at under a microscope for cancer, infection, or other bone disorders.

Decalcification is a routine procedure with the

purpose of making a calcified tissue compatible

with the embedding media for cutting micro

slides and subsequent staining.

Criteria of a good decalcifying agent :

1. Complete removal of calcium

2. Absence of damage to tissue cells or fibres

3. Non impairment of subsequent staining technique

4. Reasonable speed of decalcification

FACTORS AFFECTING THE RATE OF DECALCIFICATION:

Concentration of decalcifying agent

• Large volume of the fluid compared with the volume of tissue- 20 to 1 is

recommended to avoid total depletion of the acid or chelator by their

reaction with calcium.

• Fluid should be changed several times during the decalcification process

Temperature

• Increased temperature accelerates decalcification but also increases the

damaging effects of acids on tissue. 18º C -30º C is acceptable.

Agitation

Gentle agitation may increase the rate slightly by influencing

fluid exchange within as well as around tissues.

Suspension

Fresh decalcifier should have ready access to all surfaces of the

specimen. enhance diffusion and penetration into the specimen

and facilitate solution, ionization and removal of calcium.

TECHNIQUEThe technique of decalcification is divided into the

following stages:

1.Selection of tissue

2.Fixation

3.Decalcification

4.Acid neutralization &

5. Thorough washing

SELECTION OF THE TISSUEBONE / TEETH

Fine toothed bone saw or hack saw(large sp)

Geological cutting machine fitted with a

diamond impregnated cutting disc (small sp)

Slices not exceed 4-5mm in thickness.

FIXATION

• As a routine fixative, formal-saline is preferred but bone marrow is best

fixed in Zenker formol.

• For tooth specimens , 15% formic acid is mostly preferred

• For electron microscopy – Gluteraldehyde

• Some fine preparations of bone have been produced following immersion

in Mullers fluid followed by decalcification in 3% formic acid – formalin.

Tissue damage during acid

decalcification is four times greater when

the tissue is unfixed.

DECALCIFICATIONDecalcification is the process of removing inorganic calcium

(mineral) content of the bone /tissue before processing the specimen

after fixation.

Choice of decalcifying agent influenced

Urgency of the case

Degree of mineralization

Extent of investigation

Staining technique required

Routine decalcification methods include:

1. Acids

2. Ion exchange resins

3. Electrical ionization

4. Histochemical methods

Buffer mixtures

Chelating agents

5. Surface decalcification

ACID DECALCIFICATIONPrinciple

Acid releases calcium from its chemical combinations with

phosphates and carbonates in bone through ionic exchange

giving soluble calcium salt.

Two types•  Strong Inorganic Acids•  Weak Organic Acids

Strong acids • It Is An Inorganic Acid Eg: Nitric Acid, Hydrochloric Acid • Recommended Concentration - 5-10% • They Decalcify Rapidly By Dissolving Calcium

MINERAL ACID DECALCIFIERS

a)Nitric acid Nitric acid 5-10ml Distilled water 100ml

1.Fix the selected block of bone for 2-3 days in buffered neutral formalin.

2.Place a mixture of 95ml distilled water and 5ml of nitric acid.

3.Change nitric acid solutions daily until bubbles cease to evolve from the tissues(1-3 days,depending on the size and consistency of the bone block)

4.Wash in 3 changes of 90% alcohol.

5.Dehydrate,clear in xylene or benzene and embed in paraffin

Formation of nitrous acid checked temporarily by addition of 0.1% urea to the conc nitric acid

It’s the fastest decalcifier, but end point must be carefully watched .

Yellow discolouration owing to formation of nitrous acid, this accelerates decalcification but also stains and damage tissues

Advantages Rapid in actionGives better nuclear stainingCauses very little hydrolysisFor Needle & Small Biopsy Specimens To Permit Rapid

Diagnosis .

DisadvantagesTissue left for long time causes damage to tissueUrea is added to remove yellow color of tissue

b) PERENYI’S FLUID (PERENYI 1882)

10% Nitric Acid 40ml Absolute Ethanol 30ml 0.5% Chromic Acid 30ml Mix Shortly Before Use

Chromic Acid Must Be Collected For Proper Disposal. Its Popular Especially For Small Specimens That Are Not

Densely Decalcifed

NOTE

•Strong acids are more damaging to

tissue antigens for immunohistochemical staining

enzymes may be completely lost.

•Strong acids are used for needle & small biopsy specimens to permit rapid diagnosis

within 24 hours.

WEAK ACID DECALCIFIERS

ACETIC ACID PICRIC ACID

Weak, organic acids e.g. formic, acetic, picric.

•Acetic & picric acid cause tissue swelling & are not used alone

as primary decalcifiers but are found as components of Carnoy’s

& Bouin’s fixatives

• Formic acid is the only weak acid used

extensively as a decalcifier

•Formic acid solutions are either

•aqueous (5-10%)

•buffered or combined with formalin.

The formalin/10% formic acid mixture simultaneously fixes & decalcifies.

• Recommended for –

• Very small bone pieces

• Jamshidi needle biopsies.

• Formic acid gentle & slower than Hcl or nitric acid

• suitable for most routine surgical specimens, particularly for immuno

histochemistry.

• Decalcification usually complete within 2-7days.

a)AQUEOUS FORMIC ACID

1.Well fixed 2-5mm thick blocks are placed in – concentrated formic acid 5-25ml– Distilled water 100ml– 40% formaldehyde (optional) 5 ml

2.Change daily until decalcification is complete.( 1-7 days for an average blocks depending on concentration of acid.)

3.Replace fluid with 5% sodium sulfate overnight

4.Wash 12 -24 hrs in running tap water.

5.Dehydrate in graded alcohols ,clear in chloroform or toluene and embed in wax

b)FORMIC ACID-SODIUM CITRATE

• Sodium citrate solution Sodium citrate 100g Distilled water 500ml

• Stock formic acid Conc formic acid 250ml distilled water 250ml

DECALCIFICATION PROCEDURE

• Quantity of decal soln >20 vol. of specimen.

• Wash the decalcified specimen for 24-48 hrs –to remove the decal soln.

Other Decalcifying Fluids • Jenkins fluid Absolute alcohol 73ml Distilled Water 10mlChloroform 10ml Glacial Acetic Acid 3ml Hydrochloric Acid 4ml

• Trichloroacitic Acid –Formal saline (10%) - 95 mlTricloroacitic acid - 5 gm

This is used for small biopsies. The process of decalcification is slow hence cannot be used for dense bone or big bony pieces.

• Von –Ebners Fluid Time Taken 3-5 Days Formula: Saturated Aq.Sodium Chloride 50mlDistilled Water 50mlHydrochloric Acid 8ml

ION EXCHANGE RESINSUsed to

• remove the calcium ions from the fluid

• ensures a more rapid rate of solubility of the calcium from the tissue

• reduction in the time of decalcification.

Advantages :

1. Well preserved cellular detail

2. Faster decalcification

3. Elimination of the daily solution change

4. Resin can be reused by removing excess acid.

• Tissue is placed in a bottle in a

mixture of 10% or 20% resin and

formic acid.

• Resin used is ammonium form of

sulphonated polystyrene resin.

• The volume of fluid is 20 – 30

times that of the specimen.

• After use, resin may be

regenerated by washing twice

with dilute N/10 HCl , followed

by 3 washes in distilled water.

ELECTROPHORETIC DECALCIFICATION

• First described in 1947.• Attraction of the calcium ions to a negative electrode in

addition to the solution of the calcium in the electrolyte.

• Advantage Shortened time for complete decalcification. Better preservation of soft tissue details.

• Disadvantage Limited no. Of specimen processed at a time.

• A glass jar containing the acid decalcifying solution in which is the electrode assembly and bone specimen, bone specimen is suspended by a platinum wire anode in the jar.

• Used decalcifying fluid is• 88% formic acid 100ml• Hydrochloric acid 80ml• Distilled water 820ml

• Current, causes an electric field between the electrodes, enables the calcium ions to migrate rapidly from the specimen (anode) to the carbon electrode (cathode).

• Temperature of the reaction- 30" to 45" C.

• Solutions changed after 8 hours of use to ensure maximum

speed of decalcification.

• Tissues are rinsed well in alkaline water & immersed in

lithium carbonate before staining.

• Lithium carbonate treatment of a cut section will neutralize

any remaining acid in the tissue

Histochemical techniques Advantages

• It preserves the enzyme activity

• It also preserves the nucleic acids and polysaccharides.

It can be done by

Buffer mixture

Chelating agents

1]Buffer mixtures

Citric acid – citrate buffer (pH 4.5)

Molar hydrochloric acid – citrate buffer (pH 4.5)

Lorch’s citrate hydrochloric acid buffer (pH 4.5)

Acetate buffer (pH 4.5)

• Calcium salts may be removed from bone when placed into a buffered solution of

citrate, pH 4.5.

• Daily changes of the buffer are necessary and the decalcification progress checked

by chemical oxalate test.

Chelating agents

Chelating agents are the organic compounds that have the power of

binding with certain metals.

• Advantages -

It shows a minimum of artefact

Section stained by most techniques with first class results.

• Disadvantages- slow process as calcium is removed layer by layer from the

hydroxyapatite lattice.

• First described by HILLMAN & LEE (1953)

• Commonly used agent is EDTA.

•  Binds to metallic ions like Calcium & Magnesium

• Ionized calcium on the outside of the apatite crystal , the crystal becomes

progressively smaller during decalcification.

• Slow process that does not damage tissues or their stainability, also pH

sensitive.

• Excellent bone decalcifier for immuno histochemical or enzyme staining &

electron microscopy.

Surface decalcification• Needed when partially decalcified bone/unsuspected mineral

deposits in soft tissue are found during paraffin sectioning.

• After finding a calcification, the exposed surface in a paraffin

block is placed face side down in 5% HCL for 1hour or 10%

formic acid for 15 to 60 minutes.

• Rinsed to remove the corrosive acids & re sectioned.

End point decalcification

• Probing the tissue with the needle

• Chemical tests

• Bubble test

• Radiography

• Physical tests require manipulation, bending probing or

trimming of the specimen to “feel” for remaining calcified areas.

• Chemical test-(calcium oxalate test)

5 ml of decalcified fluid are neutralized with 0.5N sodium

hydroxide, 1 ml of 5 g/dl ammonium oxalate is added.

Appearance of turbidity indicates presence of calcium.

* Not done for EDTA Decalcification

• Bubble test

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

layer of bubbles on the bone surface.

Bubble test is subjective & unreliable, tiny bubbles indicate less calcium present.

• Radiography

Faxitron machine with exposure setting of 10-110 kv, 3ma tube current

And kodak x-omat x ray film is used.

Vinten Instruments Ltd,Jessamy Rd ,Weybridge England

Neutralization of acidsChemical 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 recommend rinsing the specimens in tap water

for a few hours.

• Culling(1974) recommended washing in two changes of 70%

alcohol for 12- 18 hour before continuing with dehydration

Processing decalcified bone• Decalcified bone sectioning -made easier after infiltration and embedding

in harder paraffin to give firmer support.

• Small bone and needle biopsies containing little cortical bone can be

processed with soft tissues.

• Oversized, thick bone slabs require an extended processing schedule to

obtain adequate de-hydration, clearing and paraffin infiltration.

Ie., 3 changes of wax under vaccum of 2 hours

• If a bone sample still appears chalky, mushy and crumbles out of block during

sectioning, then:

Dehydration, clearing or paraffin infiltration may be incomplete.

Blocks can be melted down, and re- infiltrated with paraffin for up to eight hours

to see if this improves sectioning.

Reversing processing by melting paraffin from bone and going back through 2

changes of xylene, 2 changes of 100% alcohol to remove residual water and then

reprocessing back in to paraffin.

Double embedding procedure can produce better results than paraffin wax alone.

• Base sledge microtome & wedge shaped steel or tungsten

carbide edged knife

• An optimal section thickness for bone is same as soft tissues,

4-5µm or upto 6-7µm is accepted.

• Bone marrow biopsies should be cut at 2-3µm for marrow

cell identification

Microtomy

• The floating water bath may need to be hotter than for soft tissues as bone has the tendency to crinkle when cut.

• Lifted onto the chrome-gelatin coated slides .

Staining methods for decalcified bone

• Hematoxylin and eosin: is still the primary stain used for

most final diagnoses with the aid of special stains.

Esp, Ehrlich’s & Gills .

• Collagen stains: Van Gieson picro – fuchsin, Masson’s

trichome.

• Silver –Reticulin Method , Picro-thionin stains.

UNDECALCIFIED SECTIONS • In certain metabolic bone diseases like osteomalacia it is

valuable to assess the ratio of mineralized bone to non-mineralized bone.

• 3methods to demonstrate osteoid seam : * Adhesive tape method using Von Kossa Technique *Block Impregnation Method *Resin Embedded sections

Adhesive tape method Formal fixed ,paraffin embedded

Base sledge microtome

A strip of adhesive tape is pressed firmly against the Exposed surface

Section bearing the tape is transferred to chrome-gelatin coated slide

Place another clean slide over the tape

Bulldog clip @ corners

In oven @56 degrees overnight

Remove the slide overing the tape & place wet filter paper for 30 mnts

Tape is removed by immersing the slide in warm xylene for 30-60mnts

Von –kossa technique

Principle : Calcium Phosphate + Silver Nitrate → Silver Phosphate + Calcium Nitrate Silver Phosphate → Metallic Silver (Light)

Von –kossa technique

Method • Bring sections to water

• Rinse in distilled water

• Place in 5% silver nitrate in a glass coplin jar

• Wash well in distilled water

• Treat with 5% sodium thiosulphate for 5 mnts.

• Wash in running water for 3 mnts.

• Counter stain in Van-Gieson’s stain for 3 mnts.

• Dehydrate ,clear and mount in synthetic resin

Results

• Mineralized Bone – Black • Osteoid Seams – Red

Iliac Biopsy – Severe Osteoporosis

2)Block Impregnation Method

• In this method modified von kossa method is employed

• A Reducer solution is used – Sodium Hypophosphite 0.1N Sodium Hydroxide Distilled Water

RESULT:Mineralized Bone –BlackOsteiod seams - Red

3)Resin embedded sections

• Small blocks of undecalcified sections are processed into methylmethacrylate

• Highly rigid microtome (JUNG K ) is used.

• Sections stored in 70% alcohol.

• Staining- H&E Trichomes – Goldners modification of Masson’s T Solochrome Cyanine stain • Other methods - Fluorescent Labelling (Antibiotic tetracycline) Microradiography

Teeth • Teeth –same treatment as bone prior to sectioning.

Fixation : teeth should be fixed whole in NBF.

• Adult teeth will require up to 4 days of fixation

where as for younger teeth 24 hours of fixation

may be adequate.

• Decalcification: because of its high mineral concentration, enamel is

almost impossible to preserve through a completed decalcification

process.

• Brian (1966) used a long (approx.) 12 weeks of decalcification

procedure on a 3mm slice of tooth in a 4M sodium acetate – HCl buffer

solution at pH 3.55.

• Smith recommended 5 % trichloro acetic acid as a decalcifier, while

some prefer to decalcify teeth with EDTA or buffered formic acid

solutions.

• Radiography is ideal for progress and end point testing of

decalcification, reveals the presence of some metal

/amalgam fillings but not some implanted resin materials.

• Processing- Since tooth consists mainly of very dense

material, processing methods should be extended similar

to bone methods.

Microtomy • A heavy microtome is used

• A sharp steel/tungsten carbide edged knife

• Thickness: 6-7μm

• Floating water bath temperature maintained just below the melting point of the wax.

• Mounted on chrome-gelatin coated slide.

• Left in oven for 60mnts.

Staining

• Similar To Bone Stains- Ehrlich’s H&E Over Meyer’s And Harris H&E

-Silver Reticulin Method

-Schmorl’s Picro-thionin

NOTE:GOMORIS SILVER RETICULIN METHOD :

*To 4 parts of 10% aqueous silver nitrate add 1 part of 10%KOH .

*Allow the sample to settle down

*Remove the supernatant and wash the deposit twice with distilled water.

*Add fresh,strong Ammonia drop by drop until the deposit is just dissolved.

*Carefully add 10%silver nitrate drop by drop until the solution attains a faint sheen.

RESULT :Reticulin fibres- black

Collagen,cells,nuclei – Purple-grey

Schmorl’s Picro-Thionin Method

*Sections Are Washed Well

*Stain In Half Saturated Aqueous Thionin For 2-10mnts

*Wash Well In Running Water

*Stain In Saturated Aqueous Picric Acid For 30-60seconds

*Dehydrate, Clear And Mount

RESULT:Dentinal tubules ,incremental lines ,lacunae and canaliculi- Dark Brown

Cartilage ,Nuclei – Red/Brown

Background- Yellow

PROPRIETARY DECALCIFIER

• Components are trade secrets, product data sheet indicate if a solution is rapid or slow.

• Rapid solution contain HCl , slow solution is a mixture of buffered formic acid or formalin / formic acid.

• Usage is popular in busy laboratories , coz they are time & cost effective & safe compared to strong acid.

• Disadvantage : formation of BCME ( BisChloroMethyl Ether) ,a potent carcinogen by the reaction between formaldehyde & HCL.

Artefacts Under decalcification

• Inability to section

• Incomplete infiltration of paraffin

• Staining characteristics

• Bone dust

• Remedy- surface decal, redecal

Over decalcification

• Nuclear detail lost or severely compromised

• Disruption of cell membrane and cytologic properties

• Loss of glycogen

• Swelling of tissue, especially collagen

• Staining characteristics

• Recalcification

Artefacts of overdecalcification

Bone Dust

Recent studies • Karpagaselvi Sanjai, Jayalakshmi Kumarswamy, and Lakshmi

Krishnan 2014

study was done to evaluate the rate of decalcification of six different

decalcifying agents and also their effect on staining characteristics on dental hard tissues.

namely,

neutral ethylene diamine tetra acetic acid (EDTA) decalcifying solution,

5% nitric acid, Perenyi's fluid,

formalin–nitric acid, 5% trichloracetic acid,

10% formic acid

Neutral EDTA was the most considerate to the soft and hard tissues and 5% nitric acid

was the least considerate to the tooth structure.

• Sung-Eun Choi, Soon Won Hong, and Sun Och Yoon(2015) Bone marrow biopsy of  53 patients were decalcified according to protocols of two comparison groups: EDTA versus HCl and RDO GOLD (RDO) versus HCl for preserving cellular RNA, DNA, and proteins and for molecular & immunohistochemical analyses.

Result :The EDTA protocol would be the best in preserving genetic material. RDO may be an acceptable alternative when rapid decalcification is necessary.

Recent advances • Introduction of ultrasonic energization in decalcification

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.

Acid or chelating decalcifiers may be used and the application of combined fixation-chelation permits routinely many histochemical procedures previously requiring special handling.

• Decalcification by Perfusion Using New Decal R -Hydrochloric acid 14% PVP 7% Aquadest 79% trace surfactant.

The decalcifying agent was perfused at a rate of 6 ml/min and a hydrostaticpressure of 120 cm for 30 min , 60 min, 90 min, 120 min and 240 min

• Result : The tissues perfused 120 and 240 minutes, as well as those immersed for 3 days in New DecalcR were all softened and easily processed and viewed even under electron microscope.

• Microwave decalcification Microwave-assisted decalcification saves from 10x to 100x of the time required by routine methods.

The use of dilute acids (i.e. nitric or formic) in place of EDTA will accelerate the process. The solution should be changed after each cycle.

 The temperature restriction between 42-45°C for best results

A decalcified section of cancellous bone (pink) and hyaline cartilage (blue) from the epiphysis of a long bone (H&E). The delicate trabeculae of the bone are well preserved as is the fine structure of the bone marrow and associated adipocytes.

A decalcified section of compact bone from the shaft of a long bone (H&E). The section is photographed under polarized light to demonstrate the concentric lamelle forming the osteons. The birefringence is due to the orientation of collagen fibres in the bone matrix which differs between successive layers.

UNDECALCIFIED SECTIONS IN OSTEOMALACIA

References 1. Bancroft JD, Marilyn Gamble. Theory and practice of histological

technique. 6th edition. Churchill Livingstone: Elsevier Health Sciences; 2008. pp. 53–105.

2. Jimson S ,Balachander N, Elumalai R “A Comparative Study in Bone Decalcification Using Different Decalcifying Agents” 2319-7064 (2012)

3. Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th edition. Vol. 78. Butterworths (London, Boston): 1985. p. 611.p. 18.

4. Rolls OG, Farmer JN, Hall BJ. Artifacts in Histological and Cytological Preparation. Scientia Leica Microsystems Education Series. April 2008;21

5) Magnus Nilsson, Sten Hellstrom and Nils Albiin “Decalcification by perfusion. A new method for rapid softening of temporal bones”Histol Histopath (1991) 6: 415-420

6)Karpagaselvi Sanjai, Jayalakshmi Kumarswamy, Archana Patil, Lokesh Papaiah, Srinivas Jayaram and Lakshmi Krishnan “Evaluation and comparison of decalcification agents on the human teeth” Oral Maxillofac Pathol. 2014 May-Aug; 16(2): 222–227.

7) Sung-Eun Choi, Soon Won Hong, and Sun Och Yoon “Proposal of an Appropriate Decalcification Method of Bone Marrow Biopsy Specimens in the Era of Expanding Genetic Molecular Study” J Pathol Transl Med. 2015 May; 49(3): 236–242

 8) Verdenius HHW and Alma L (1958) A quantitative study of decalcification methods in histology J Clin Pathol. 1958 May; 11(3): 229–236 9) Mawhinney WH, Richardson E, and A J Malcolm (1984) Control of rapid nitric acid decalcification. J Clin Pathol. 37(12): 1409–1413

 10) Janneke C. Alers, Pieter-Jaap Krijtenburg, Kees J. Vissers, and Herman van Dekken (1999)Effect of Bone Decalcification Procedures on DNA In Situ Hybridization and Comparative Genomic Hybridization: EDTA Is Highly Preferable to a Routinely Used Acid Decalcifier. Journal of Histochemistry and Cytochemistry, Vol. 47, 703-710  11) P Sarsfield, C L Wickham, M V Joyner, S Ellard, D B Jones, and B S Wilkins (2000) Formic acid decalcification of bone marrow trephines degrades DNA: alternative use of EDTA allows the amplification and sequencing of relatively long PCR products. Mol Pathol; 53(6): 336

12) Yasuaki Shibata, et al (2000) Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues. Histochem Cell Biol 113:153–159

13) Yamamoto-Fukud T (2000) Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling. Histochem J. 2000 Nov;32(11):697-702 

14) Brown RSD, Edwards J, Bartlett JW, Jones C, and Dogan A (2002) Routine Acid Decalcification of Bone Marrow Samples Can Preserve DNA for FISH and CGH Studies in Metastatic Prostate Cancer. Journal of Histochemistry and Cytochemistry, Vol. 50, 113-116 

15) Recent Advances In Microwave Decalcification Protocols by Herbert Skip Brown 

Bullets …….• Best decalcifying agent ??• Why neutral EDTA is better ??• Where is it commonly used??• Disadvantage of EDTA • Disadvantage of acid decalcification ?• How do Bone and cementum, cartilage look after decalcification??• Role of hydrofluoric acid??• Depth of surface decalcifying fluid ??• Can enamel be studied under decalcification?• Partial decalcification ?? Where is it used ??• Does enamel withstand decalcification ?• % of inorganic content in enamel ?• Why cant it be done in routine method ?• What kind of blade is required ??