Decalcification.pptx

1. DECALCIFICATION DrTamil Nila

2. INTRODUCTION  Calcification  Physiological  Bone  Teeth  Pathological  Dystrophic  Metastatic  Hard tissues  Bone  Teeth  Calcified soft tissue

3. BONE SPECIMENS Bone Lamellar Unmineralised Mineralised Woven  Lamellar  Entire mature adult skeleton  Woven  Reactive process  Found in  Bone tumours  Infections  Healing fracture osteoid

4. CELLS OFTHE BONETISSUE Osteoblast Osteoid production Osteocyte Osteoblast enveloped by bone matrix Osteoclasts Mature multi nucleated cells Bone resorption To obtain satisfactory sections Remove inorganic Ca from tissues Incomplete removal – ragged sections

5. BONE SPECIMENS  Biopsies  Amputation specimens  Resection/ replacement specimens

6. STAGES IN DECALCIFICATION Selection of tissue Fixation Decalcification Neutralization of acid Washing

7. SELECTION OFTISSUE  Bone  Calcified tissue Sawing Remove soft tissues & dense connective tissue 3 – 5 mm thick bone slabs Fixed 24 – 48 hours Wash bone debris/dust in running tap water/ wet paper towel

8. FIXATION  Bone pieces cut into 2 – 5 mm thickness  Complete fixation protects bone from acid  4 -5 times more damage in unfixed tissues  Fixative + Decalcification  Bouin’s decalcifying solution  Formic acid - formalin  Fixative  Routine: Neutral buffered formalin  Bone marrow: Zenker formol For faster fixation - Reduced size of bone - Bone is opened - Remove soft tissue

9. DECALCIFICATION  Removal of calcium from tissue for processing  Even after decalcification, the dense collagen of bone becomes hard after processing  On H & E staining  Calcified foci appear cracked  Dark purple granular masses  Light purple halos  Choice of decalcifier  Urgency of the case  Degree of mineralisation  Extent of the investigation  Staining techniques required

10. CRITERIA OF GOOD DECALCIFYING AGENT  Completely removes calcium from tissue  Does not damage tissue cells or fibers  Does not impair the subsequent staining techniques  Reasonable fast decalcification

11. VARIOUS METHODS OF DECALCIFICATION  Acid decalcifiers  Strong inorganic acids  Nitric acid  Aqueous nitric acid  Formal nitric acid  Perenyi’s fluid  Aqueous HCl  Weak organic acids  Formic acid  Aqueous Formic acid  Formic acid – formalin  Formic acid – sodium citrate  Others:Acetic & Picric acid  Ion exchange resin  Chelating agents  Electrophoretic technique  Microwave technique

12. STRONG ACID DECALCIFIERS Solution Aqueous HCl / HNO3 Formal HNO3 Perenyi’s fluid Prep Conc. HCl / HNO3 – 10 ml DistilledWater – 90 ml Formalin – 5 ml HNO3 – 7.5 to 15 ml Distilled water – 100 ml 10% HNO3 – 40 ml Absolute alcohol – 30 ml 0.5% chromic acid – 30 ml Time Taken 12 – 24 hrs 1 – 2 days 2 – 4 days (ribs) 10 – 14 days (5 mm femur) + Rapid decalcification Little damage Allows most staining methods Rapid decalcification Inhibits maceration by HNO3 Discoloration prevented by Urea Excellent agent for small deposits & cytological preparations - Tissue swelling Interferes with staining if >48 hrs IHC antigen damage Yellow discoloration - HNO3 Prepared freshly everytime Violet tinge Slow decalcification Cannot detect end point chemically Acid + calcium -> soluble calcium salts

13. WEAK/ DILUTE ORGANIC (MINERAL) ACIDS Solution Aqueous formic acid Formic – acid formalin Prep Formic acid (90%) – 10 ml Distilled water – 90 ml (Gooding Stewart’s fluid) Formic acid – 10 ml Formalin – 5 ml Distilled water – 100 ml Time Taken 2 – 4 days + Gentler, Used for Small bone/ teeth IHC staining Minimal tissue damage Fixation + Decalcification Used forVery small tissues Minimal damage to tissues - Slower Increase in formic acid – cell damage

14. BOUIN’S DECALCIFICATION SOLUTION  Formula  Saturated aqueous solution of picric acid (10.5 g/ 500 ml) – 500 ml  Formalin – 167 ml  Formic acid – 33 ml  Duration required for decalcification depends on the type and size of tissue Place bone in buffered neutral formalin – 2 to 3 days Decalcification agent 100 times the volume of tissue Check for end point

15. ION EXCHANGE RESIN  Aids decalcification in simple decalcifying fluids  Ammonium form of a sulphonated polystyrene 20 – 30 times decalcifying agent Layer Ion – exchange resin on the bottom Specimen decalcified & placed on resin End point determined by X ray Advantages IER removes Ca from DA Reduces Decalcification time Resin can be reused Disadvantages Limited to DA that do not contain mineral acids

16. CHELATING AGENTS  Organic compounds that bind with certain metals  EDTA-formalin  EDTA: 5.5 g  Formalin: 10 ml  Distilled water: 90 ml  Neutral EDTAEDTA: 250 g  Distilled water: 1750 ml  NaOH: 25 g  Advantage: Minimal artefacts & good staining  Disadvantage: slow & not used widely Tissues fixed in 10% formalin 50 times bulk of 5.5% EDTA with phosphate buffer (pH 7.4) <5mm thickness tissue – change DA every 4 – 5 days 3 changes done Tissues again placed in formal saline overnight Dehydrated & embedded

17. ELECTROPHORETICTECHNIQUE  Principle  Attraction of Ca ions in electric current  Composition of electrolyte  8% HCl  10% Formic acid  Time required for decalcification  6 – 8 hours  Advantage: Rapid decalcification  Disadvantage  Cumbersome  Expensive  Heat damage to tissues Electrolyte placed in glass jar Negative electrode: Brass Positive electrode: Platinum Platinum wire wound around specimen Move electrodes closer for faster decalcification Check by X – ray every 2 – 3 hrs

18. FACTORS INFLUENCINGTHE RATE OF DECALCIFICATION  Concentration & volume of DA  Suspension of calcified tissue  Age of the patient  Type of bone  Size of specimen  Temperature of decalcifying agents  Agitation

19. DETERMINATION OF END-POINT OF DECALCIFICATION  Radiography  Check evidence of calcium  Efficient & sensitive method  Many tissues can be X rayed  Physical test  Probing/needling/slicing/bending/ squeezing  Inaccurate & damages tissues  Bubble test  Acids + CaCO3 -> CO2  Bubbles form on tissue surface

20. CHEMICALTEST FOR CALCIUM  Calcium oxalate test  Principle  Detects calcium by precipitation of insoluble Ca(OH)2 and Calcium oxalate  Interpretation  White precipitate immediately – presence of residual calcium  White precipitate after Step 2 – less calcium  Clear fluid after 30 mins – Decalcification complete 5 ml DA from the specimen + litmus paper/ pH meter Neutralize by adding Conc. NH4OH till pH 7 Add 5 ml of saturatedAmmonium oxalate Shake well & stand for 30 mins

22. NEUTRALIZATION OF ACID  Treatment with a weak alkali  Saturated lithium carbonate  5 – 10% aqueous NaCO3  Washing in 2 changes of 70% alcohol for 12 – 18 hrs

23. THOROUGH WASHING  Purpose  To remove the acid/ alkali  Better staining  Method  3 – 4 hours wash in alcohol or overnight in water  Tissues are then routinely processed  Longer wax impregnation/ harder paraffin wax

24. SURFACE DECALCIFICATION  Definition  Removal of small focus of calcification on the surface of the block  Indication  Partially decalcified bone  Unsuspected Calcium/Mineral deposits  Advantage  Prevent knife damage  Prevent torn tissue sections Trim & expose the tissue Place the block face down in 1 - % HCl, 10% formic acid x 15-60 mins Rinse with water Resection in the usual manner

26. REFERENCES  Handbook of Histopathological & Histochemical techniques, C.F.A. Culling  Histopathology techniques & its management, Ramadas Nayak  Basic & Advanced Laboratory techniques in Histopathology & cytology, Pranab Dey

Decalcification.pptx

Decalcification.pptx

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