Decalcification in Histopathology.pptx

Decalcification in histopathology

1. Decalcification in Histopathology Mrs Naa-Anyima Boateng

2. Overview Introduction Biopsy Criteria for good decalcifying agent Factors affecting decalcification Technique Artefacts Recent Advances References

3. Introduction

4. Boney Section Average thickness is 4-6 µm. To section the hard mineralised tissue decalcification is necessary A transverse section from a long bone optimally decalcified using formic acid (H&E). Numerous osteons with peripheral cement lines are shown. Well-stained osteocyte nuclei are present indicating that the decalcification endpoint was not exceeded.

5. Biopsies

6. Decalcification 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 decalcified Tissue Sample

8. Factors Affecting Decalcification

9. Factors Affecting Decalcification II

10. Technique

11. Specimen Selection 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 – Gluteraldehyde Some fine preparations of bone have been produced following immersion in Mullers fluid followed by decalcification in 3% formic acid – formalin.

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

14. Decalcification Step 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 by: Urgency of the case Degree of mineralization Extent of investigation Staining technique required

15. Decalcification II

16. Acid Decalcification

17. Strong Acids

18. Mineral Acid Decalcifiers Decalifier Formula Nitric Acid 5% in dH₂0 Perenyi’s Fluid (1882) 40 ml of 10% Nitric Acid 30ml of 0.5% Chromic Acid 30ml Absolute Alcohol Hydrochloric Acid 5% in dH₂0

19. Nitric Acid Nitric acid 5-10ml Distilled water 100ml Fix the selected block of bone for 2-3 days in buffered neutral formalin. Place a mixture of 95ml distilled water and 5ml of nitric acid. 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) Wash in 3 changes of 90% alcohol. Dehydrate, clear in xylene or benzene and embed in paraffin

20. Nitric Acid II 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. Nitric Acid Pros and Cons Advantages Disadvantages Rapid in Action Tissue left for too long causes damage to tissue Gives Better Nuclear staining Urea is added to remove yellow colour Causes very little hydrolysis Needle and Small Biopsy's to allow for rapid diagnosis Its popular especially for small specimens that are not densely decalcifed

22. Perenyi's Fluid (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. To Note about Strong Acids 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 Decalcifier Formula Formic Acid 5% in dH₂0 Evans & Krajian Formic Acid 25 ml Sodium Citrate 10g dH₂0 75ml Kristensen Formic Acid 18ml Sodium Formate 3.5g dH₂0 82ml Gooding & Stewart Formic Acid 2-25ml 40% Formaldehyde 5ml dH₂0 75ml Acetic Acid Used as components of decalcifiers Picric Acid Used as components of decalcifiers

25. Weak Decalcifiers II

26. Formic Acid

27. Aqueous Formic Acid Well fixed 2-5mm thick blocks are placed in – concentrated formic acid 5-25ml – Distilled water 100ml – 40% formaldehyde (optional) 5 ml Change daily until decalcification is complete ( 1-7 days for an average blocks depending on concentration of acid). Replace fluid with 5% sodium sulfate overnight Wash 12 -24 hrs in running tap water. Dehydrate in graded alcohols ,clear in chloroform or toluene and embed in wax

28. Formic Acid Sodium Citrate

29. Decalcification Procedure Quantity of decal soln >20 vol. of specimen. Wash the decalcified specimen for 24-48 hrs –to remove the decal soln.

30. Other Decalcifying Fluids

31. Von – Ebners Fluid

32. Ion Exchange Resins

33. Ion Exchange Resin II

34. 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.

35. Electrophoretic Decalcification II

36. Electrophoretic Decalcification III

37. Decalcification Histological Techniques Advantages • It preserves the enzyme activity • It also preserves the nucleic acids and polysaccharides. It can be done by: 1. Buffer mixture 2. Chelating agents

38. 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.

39. 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.

40. EDTA Chelating Agent 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 immunohistochemical or enzyme staining & electron microscopy.

41. 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

42. End Point Decalcification

43. End Point Decalcification II

44. End Point Decalcification III

45. Neutralisation of Acids

46. Processing Decalcified Bone

47. Processing Decalcified Bone II 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.

48. Microtomy of Decalcified Bones 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 up to 6-7µm is accepted. Bone marrow biopsies should be cut at 2-3µm for marrow cell identification

49. Microtomy of Decalcified Bones II 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 .

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

51. Artefacts II 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

52. 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.

53. Recent Advances II 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