DECALCIFICATION

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1. DECALCIFICATION SHRAVYA.M

2. 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

3. 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 .

4. Average thickness is 4-6 µm . To section the hard mineralised tissue decalcification is necessary

5. 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.

6. 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 .

7. Criteria of a good decalcifying agent : Complete removal of calcium Absence of damage to tissue cells or fibres Non impairment of subsequent staining technique Reasonable speed of decalcification

8. 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.

9. Agitation Gentle agitation may increase the rate slightly by influencing fluid exchange within as well as around tissues . Suspension F resh 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.

10. TECHNIQUE The technique of decalcification is divided into the following stages: 1.Selection of tissue 2.Fixation 3.Decalcification 4.Acid neutralization & 5. Thorough washing

11. SELECTION OF THE TISSUE BONE / 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.

12. 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 – G luteraldehyde Some fine preparations of bone have been produced following immersion in Mullers fluid followed by decalcification in 3% formic acid – formalin .

13. Tissue damage during acid decalcification is four times greater when the tissue is unfixed.

14. DECALCIFICATION Decalcification 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

15. Routine decalcification methods include: Acids Ion exchange resins Electrical ionization Histochemical methods B uffer mixtures C helating agents 5 . Surface decalcification

16. ACID DECALCIFICATION Principle 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

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

18. MINERAL ACID DECALCIFIERS

19. 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

20. 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

21. Advantages Rapid in action Gives better nuclear staining Causes very little hydrolysis For Needle & Small Biopsy Specimens To Permit Rapid Diagnosis . Disadvantages Tissue left for long time causes damage to tissue Urea is added to remove yellow color of tissue

22. 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

23. 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 .

24. WEAK ACID DECALCIFIERS ACETIC ACID PICRIC ACID

25. 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

26. 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.

27. 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.

28. 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

29. b)FORMIC ACID-SODIUM CITRATE Sodium citrate solution Sodium citrate 100g Distilled water 500ml Stock formic acid Conc formic acid 250ml distilled water 250ml

30. DECALCIFICATION PROCEDURE Quantity of decal soln >20 vol. of specimen . Wash the decalcified specimen for 24-48 hrs –to remove the decal soln .

31. Other Decalcifying Fluids Jenkins fluid Absolute alcohol 73ml Distilled Water 10ml Chloroform 10ml Glacial Acetic Acid 3ml Hydrochloric Acid 4ml Trichloroacitic Acid – Formal saline (10%) - 95 ml Tricloroacitic 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 .

32. Von – Ebners Fluid Time Taken 3-5 Days Formula: Saturated Aq.Sodium Chloride 50ml Distilled Water 50ml Hydrochloric Acid 8ml

33. ION EXCHANGE RESINS U sed 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 : Well preserved cellular detail Faster decalcification Elimination of the daily solution change Resin can be reused by removing excess acid.

34. 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.

35. 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.

36. 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 ).

37. Temperature of the reaction- 30" to 45" C. Solutions changed after 8 hours of use to ensure maximum speed of decalcification. T issues are rinsed well in alkaline water & immersed in lithium carbonate before staining. L ithium carbonate treatment of a cut section will neutralize any remaining acid in the tissue

38. 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

39. 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.

40. 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.

41. First described by HILLMAN & LEE (1953 ) Commonly used agent is EDTA.  B inds 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.

42. Surface decalcification N eeded 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. R insed to remove the corrosive acids & re sectioned.

43. End point decalcification Probing the tissue with the needle Chemical tests Bubble test Radiography

44. 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

45. 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

46. Neutralization of acids 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 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

47. 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

48. 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.

49. 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

50. 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 .

51. 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 , P icro-thionin stains.

52. 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 * B lock Impregnation Method *Resin Embedded sections

53. 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

54. Tape is removed by immersing the slide in warm xylene for 30-60mnts Von – kossa technique

55. Principle : Calcium Phosphate + Silver Nitrate → Silver Phosphate + Calcium Nitrate Silver Phosphate → Metallic Silver (Light ) Von – kossa technique

56. 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

57. Results Mineralized Bone – Black Osteoid Seams – Red

58. Iliac Biopsy – Severe Osteoporosis

59. 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 –Black Osteiod seams - Red

60. 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

61. 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.

62. 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.

63. 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.

64. Microtomy A heavy microtome is used A sharp steel/tungsten carbide edged knife Thickness: 6-7 μm Floating water bath temperature maintained just below the melting point of the wax. Mounted on chrome-gelatin coated slide. Left in oven for 60mnts.

65. Stai ning Similar To Bone Stains Ehrlich’s H&E Over Meyer’s And Harris H&E -Silver Reticulin Method - Schmorl’s Picro-thionin

66. 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

67. 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

68. PROPRIETARY DECALCIFIER Components are trade secrets, product data sheet indicate if a solution is rapid or slow. Rapid sol ution contain HCl , slow solu tion 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.

69. Artefacts Under decalcification • Inability to section • Incomplete infiltration of paraffin • Staining characteristics • Bone dust • Remedy- surface decal, redecal

70. 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

71. Artefacts of overdecalcification

72. Bone Dust

73. 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.

74. 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.

75. 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.

76. Decalcification by Perfusion Using New Decal R - Hydrochloric acid 14% PVP 7% Aqua dest 79% trace surfactant. The decalcifying agent was perfused at a rate of 6 ml/min and a hydrostatic pressure 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.

77. 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

78. 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 .

79. 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.

80. UNDECALCIFIED SECTIONS IN OSTEOMALACIA

81. References Bancroft JD, Marilyn Gamble. Theory and practice of histological technique. 6th edition. Churchill Livingstone: Elsevier Health Sciences; 2008. pp. 53–105 . Jimson S , Balachander N, Elumalai R “ A Comparative Study in Bone Decalcification Using Different Decalcifying Agents” 2319-7064 (2012 ) Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th edition. Vol. 78. Butterworths (London, Boston): 1985. p. 611.p. 18 . Rolls OG, Farmer JN, Hall BJ. Artifacts in Histological and Cytological Preparation.  Scientia Leica Microsystems Education Series. April 2008;21

82. 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

83.   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

84. 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  

85. 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 ??