decalcification.pdf
- 2. 2 Contentsā¦ā¦ā¦ā¦ā¦ā¦. ā¢ - Introduction ā¢ -Definition ā¢ - Properties of ideal Decalcification agents ā¢ - Methods of Decalcification ā¢ - principle ā¢ -Types of decalcifying agents ā¢ - Compositions, Action, Advantages ā¢ -procedure of Decalcification
- 3. 3 INTRODUCTION ā¢ Decalcification is necessary in order to facilitate smooth cutting of bone and other calcified tissue during the preparation of sections. ā¢ Calcium is generally present in bones, teeth and tissues removed from glands such as tubercular and lymphatic. ā¢ The calcified hard tissue is cut into small pieces. ā¢ The tissue should be thoroughly washed, to remove excess of fixative before the specimen is subjected to decalcification.
- 4. DECALCIFICATION Definition : Decalcification is the process of removing inorganic calcium (mineral) content of the bone /tissue before processing the specimen after fixation.
- 5. 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
- 6. FACTORS AFFECTING THE RATE OF DECALCIFICATION ā¢ 1. Concentration of decalcifying agent ā¢ 2. Temperature ā¢ 3. Agitation ā¢ 4. Suspension 8
- 7. FACTORS AFFECTING THE RATE OF DECALCIFICATION Concentration of decalcifying agent ā¢ 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. ā¢ Fluid should be changed several times during the decalcification process Temperature ā¢ Increased temperature accelerates decalcification but also increases the damaging effects of acids on tissue. 18Āŗ C -30Āŗ C is acceptable.
- 8. Agitatio n by influencin g Gentle agitation mayincrease the rate slightly 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.
- 9. METHODS OF DECALCIFICATION 1. Acids 2. Ion exchange resins 3. Electrical ionization 4. Histochemical method s ļ¼ Buffer mixtures ļ¼ Chelating agents 5. Surface decalcification
- 11. 11 PRINCIPLE ā¢ The acid present in the decalcifying fluid removes the calcium salt present in the hard tissue and makes the tissue soft enough for sectioning.
- 12. 12 REAGENT ā¢ Any suitable acid reagent canbe usedaā¦.. ā¢ - Nitric Acid ā¢ - Formic Acid ā¢ - Jenkinās fluid ā¢ Citrate ā citric acid buffer
- 13. 13 TYPES ā¢ Two types ā¢ Strong Inorganic Acids ā¢ Weak OrganicAcids
- 14. 14 Strongaci ds ā¢ It Is An InorganicAcid ā¢ Eg: Nitric Acid, HydrochloricAcid ā¢ Recommended Concentration - 5-10% ā¢ They Decalcify Rapidly By Dissolving Calciu m
- 16. a)Nitric acid 16 CHEMICAL QUANTITY Nitric acid 5-10ml Distilled water 100ml
- 17. 17 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
- 18. ā¢Formation of nitrous acid checked temporarily by addition of 0.1% urea to the conc nitric acid ā¢Itās the fastest decalcifier, but end point must be carefully watched . ā¢Yellow discolouration owing to formation of nitrous acid, this accelerates decalcification but also stains and damage tissues 18
- 19. ļRapid in action ļGives better nuclear staining ļCauses very little hydrolysis ļFor Needle & Small Biopsy Specimens To Permit Rapid Diagnosis . 19 ļTissue left for long time causes damage to tissue ļUrea is added to remove yellow color of tissue
- 21. Weak, organic acids e.g. formic, acetic, picric. ā¢Acetic & picric acid cause tissue swelling & are not used alone as primary decalcifiers but are found as components of Carnoyās & Bouinās fixatives 21
- 22. ā¢Formicacidisthe only weak acid used extensively as a decalcifier ā¢Formic acid solutions are either ā¢ aqueous (5-10%) ā¢ buffered or combined with formalin. 22
- 23. 23 The formalin/10% formic acid mixture simultaneously fixes & decalcifies. ā¢ Recommended for ā ā¢ Very small bone pieces ā¢ Jamshidi needle biopsies. ā¢ Formic acid gentle & slower than Hcl or nitric acid ā¢ suitable for most routine surgical specimens, particularly for immuno histochemistry. ā¢ Decalcification usually complete within 2-7days.
- 24. Xa)AQUEOUS FORMICACID 1.Well fixed 2-5mm thick blocks are placed in ā concentrated formic acid ā Distilled water ā 40% formaldehyde (optional) 5- 25ml 100ml 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 24
- 25. DECALCIFICATION PROCEDURE ā¢ Quantity of decal soln >20 vol.of specimen. ā¢ Wash the decalcified specimen for 24-48 hrs āto remove the decal soln. 25
- 26. 26 PROCEDURE ā¢ 1. Suspend the sliced tissue in the decalcifying solution by means of gauze bag, tied with a string. ā¢ 2.Stir the fluid occasionally and change the fluid daily till calcium is completely removed from the tissue section. ā¢ 3.It is tested asfollowsā¦ā¦.
- 27. 27 ā¢ - take about 5.0 ml of decalcifyingsolution in a test tube ā¢ - Add conc. Ammonia solution drop by drop until it becomes alkaline ( test by litmus paper) ā¢ - If the solution becomes turbid, continue decalcification ( since it contains calcium) ā¢ - if it is clear add 0.5 ml saturated ammonium oxalate solution ā¢ - If calcium is present the solution will become turbid, then continue decalcification. ā¢ If there is no turbidity , the specimen is free of calcium and ready for further processing.
- 28. 28 ā¢ 4. Wash the decalcified specimen in running tap water for 24-48 hours. ā¢ 5.The decalcifying solution should be completely removed before dehydration and embedding.
- 29. 29 THANKS