DECALCIFICATION.pdf HISTOPATH SUBJECT IN MLS
- 2. DECALCIFICATION Process whereby calcium ions or lime salts are removed from dense tissues (bones and teeth) After Fixation and before Impregnation To ensure and facilitate normal cutting of sections a. To prevent obscuring the microanatomical details b.
- 3. Ideal time required for decalcifying tissues * Ideal thickness when cutting calcified bones/tissues Recommended ratio of fluid to tissue volume for decalcification 24-48 hrs. 1-3mm 20:1
- 4. 1 2 3 Concentration & Volume * Temperature * Tissue Structure FACTORS AFFECTING DECALCIFICATION 4 Mechanical Agitation * Facilitates fluid exchange
- 5. METHODS OF DECALCIFICATION 1 2 3 Acid Decalcifying Agents - most widely used Chelating Agents Ion Exchange Resin 4 Electrophoresis - most rapid method
- 6. USE OF ACID
- 7. 5-10% NITRIC ACID - For heavily cortical bones - Imparts yellow color - Neutralizes tissue with 5% sodium sulfate - Addition of 0.1% urea removes discoloration Most common and fastest Urgent biopsies 10% Aqueous Nitric Acid Formol-Nitric Acid
- 8. 5-10% NITRIC ACID - Nitric Acid + Chromic Acid + Ethyl Alcohol - Decalcifier and Tissue Softener - Permits good Nuclear & Cytoplasmic staining - Slow decalcifying agent for DENSE BONES - Maceration is avoided - Most rapid, may cause extreme tissue distortion - Poor nuclear staining Most common and fastest Perenyi’s Fluid Phloroglucin Nitric Acid 0.5% 10% 100%
- 9. HYDROCHLORIC ACID - Contains HCl, NaCl, and distilled H2O - Permits good cytologic staining - Does not require washing out before dehydration - Recom. for teeth and small pieces of bones Used for surface decalcification of blocks Von Ebner’s Fluid
- 10. FORMIC ACID Produces better nuclear staining and less tissue distortion Safer to handle for routine decalcification of post-mortem research tissues Recom. for research and autopsy, cartilage and BM Adding CITRATE accelerates decalcification time by chelating Ca++ Both a Fixative & a Decalcifier
- 11. TRICHLOROACETIC ACID Weak decalcifying agent Permits good nuclear staining Both a Fixative & a Decalcifier CHROMIC ACID Both a Fixative & a Decalcifier aka Flemming’s Fluid Recom. for minute bone spicules Environmental toxin, Carcinogenic Corrosive to skin and mucus membranes
- 12. SULFUROUS ACID Very weak decalcifying agent Used only for minute pieces of bones CITRIC ACID CITRATE BUFFER Weak decalcifying agent Uses chloroform as preservative Both nuclear and cytoplasmic staining
- 14. CHELATING AGENTS - Binds with Calcium to form weakly dissociated complex - Recom. for detailed microscopic studies - Potent anticoagulant but WEAK AGENT - Excellent for EM - Inactivates Alkaline Phosphatase (Add MgCl) * Substances that combine with Calcium ions and other salts like Iron and Magnesium deposits EDTA/Versene
- 16. ION-EXCHANGE RESIN - Volume should be 20-30x the volume of the specimen - 1 to 14 days duration - X-ray measures the process Hastens decalcification by removing Calcium ions from FORMIC ACID containing decalcifying agent Ammonium form of Polystyrene Resin
- 17. ELECTROPHORESIS
- 18. ELECTROPHORESIS Process by which positively charged ions (Ca++) are attracted to a negatively charged electrode (“cathode”) and subsequently removed from the decalcifying agent Utilizes electricity and dependent upon a supply of direct current Satisfactory for small bone fragments
- 19. -Touching or bending resistance to finger nail or by needling* -Inaccurate, damages the tissues PHYSICAL/MECHANICAL MX TESTS TO MEASURE EXTENT OF DECALCIFICATION -Most sensitive and reliable, but quite pricy -Can detect even the smallest focus of Calcium X-RAY/RADIOLOGIC MX -Ammonium Oxalate/Hydroxide CHEMICAL MX
- 20. POST-DECALCIFICATION Procedure that involves washing the tissue with Lithium Carbonate To remove acids: Wash with running tap water (30 mins. for small tissues & 1-4 hrs. for larger specimens)
- 21. 2% HCl in 70% Alcohol 4% Aqueous Phenol 1% HCl in 70% Alcohol Perenyi’s Fluid Lendrum’s Method MOLLIFLEX TISSUE SOFTENERS
- 22. THANK YOU! Happy Lunch. :P