1. SD/CP5 - MICROTOMY David Muskett

2. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting & coverslipping  Clinical perspective

3. Section thickness and embedding 5th May 2011David Muskett - SD/CP5 Microtomy 3  The thickness of the section cut is directly correlated to the hardness of the supporting media  What other factors link to the supporting media used?  What media is used for semi-thin and ultra-thin sections?

4. Principles and practice 5th May 2011David Muskett - SD/CP5 Microtomy 4  Sections usually 4µm  Thinner sections 2-3µm required for:-  Bone marrow trephines  Lymph nodes  Renal biopsies  Thicker sections 6-20µm required for:-  Neuropathology samples (6-20µm)  Cases for amyloid (6-8µm)

5. Trimming 5th May 2011David Muskett - SD/CP5 Microtomy 5  Aim to get the section to a point where all the relevant elements are showing  QC step on the embedding Is the specimen correctly orientated? Is it pressed down? Is it likely to need levels and is not marked? Is the name on the cassette plausible?  No trimming artefact

6. Initial exposure of the tissue (roughing) in this block has pulled fragments from the block surface which has resulted in numerous holes in the final section (H&E). Trimming artefact 5th May 2011 6 David Muskett - SD/CP5 Microtomy

7. Sectioning 5th May 2011David Muskett - SD/CP5 Microtomy 7  Ensure the block is trimmed appropriately  Is the full cross section visible  Blocks need to be cool / cold  Sections need to be cut evenly  Knife needs to be sharp  No nicks in the knife

8. Special considerations when cutting blocks  Lymph nodes  Renal biopsies  Amyloid  Neuropathology sections 5th May 2011David Muskett - SD/CP5 Microtomy 8

9. Factors affecting section cutting  Sharpness of the cutting blade  Rigidity of the knife and specimen holder  Hardness of the tissue  Blood within the tissue  The coldness of the block 5th May 2011David Muskett - SD/CP5 Microtomy 9

10. Sectioning artefact – knife lines 5th May 2011 10 David Muskett - SD/CP5 Microtomy

11. Sectioning artefact – component displacement 5th May 2011 11 David Muskett - SD/CP5 Microtomy

12. Sectioning artefact – Venetian blind 5th May 2011 12 David Muskett - SD/CP5 Microtomy

13. Sectioning artefact – course chatter 5th May 2011 13 David Muskett - SD/CP5 Microtomy

14. Sectioning artefact – adhesive tidelines 5th May 2011 14 David Muskett - SD/CP5 Microtomy

15. Sectioning artefact - bubbling under the sections 5th May 2011 15 David Muskett - SD/CP5 Microtomy

16. Sneeze artefact 5th May 2011 16 David Muskett - SD/CP5 Microtomy

17. The slides 5th May 2011 17 David Muskett - SD/CP5 Microtomy

18. Slides & slide adhesives 5th May 2011David Muskett - SD/CP5 Microtomy 18  Plain glass – needs to be clean  Silane slides  Albumen – thinly wiped on the slides or in the waterbath  Gelatin  Positive charged slides  Celloidin

19. Section artefact – contamination of mounted sections 5th May 2011 19 David Muskett - SD/CP5 Microtomy

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24. Floating out 5th May 2011David Muskett - SD/CP5 Microtomy 24  Allows the creases to drop out of the section  Temperature should be around 50 o C slightly less than the melting point of the wax (56-58 o C)  More QC steps  Is the section full face  Are there any creases  Are there scores?

25. Sectioning artefact - folds 5th May 2011David Muskett - SD/CP5 Microtomy 25

26. How do you know you have got a good section? 5th May 2011David Muskett - SD/CP5 Microtomy 26

27. Knives 5th May 2011David Muskett - SD/CP5 Microtomy 27  Solid (non disposable knives)  Strop & Hone  Knife sharpener  Disposable knives  Feather  Glass knives for electron microscopy

28. Knife angles and blades  Feather disposable  Wedge profile  Carbon steel 5th May 2011 28 David Muskett - SD/CP5 Microtomy

29. Productivity 5th May 2011David Muskett - SD/CP5 Microtomy 29  How many blocks should you be able to cut in 1 hour?

30. Antigenicity of cut sections? 5th May 2011David Muskett - SD/CP5 Microtomy 30  Antigenicity of sections is lost over time

31. Big sections  Megablocks  Gough & Wentworth  Allow investigation of anatomical features more easily 5th May 2011 David Muskett - SD/CP5 Microtomy 31

32. Gough - Wentworth sections 5th May 2011David Muskett - SD/CP5 Microtomy 32  Sections 200-400µm thick  Impregnation of 2cm slice of organ with gelatin / glycerol solution  Freeze then thaw  Cut sections on a base sledge microtome  Mounted on paper and laminated between clear sheets of plastic

33. Microtomes 5th May 2011David Muskett - SD/CP5 Microtomy 33  Rotary  Manual  Motorised – semi automatic / automatic  Sledge  Sliding  Ultra  Freezing

34. Cambridge rocker  Simple and popular  In use in the 1960s and 1970s 5th May 2011 34 David Muskett - SD/CP5 Microtomy

35. Rotary microtomes 5th May 2011 35 David Muskett - SD/CP5 Microtomy

36. Sledge microtomes 5th May 2011 36 David Muskett - SD/CP5 Microtomy

37. Sliding microtomes 5th May 2011 37 David Muskett - SD/CP5 Microtomy

38. Ultra microtomes 5th May 2011 38 David Muskett - SD/CP5 Microtomy

39. Freezing Microtomes 5th May 2011David Muskett - SD/CP5 Microtomy 39

40. Health & Safety - microtomy  Knives  Upper limb related disorder 5th May 2011David Muskett - SD/CP5 Microtomy 40

41. Case study 1 – gastric biopsy  3mm diagnostic biopsies  Need 3 levels  Ensure a full cross section of the material is visible  Need to leave sufficient material for subsequent tests 5th May 2011 41 David Muskett - SD/CP5 Microtomy

42. Case study 2 – Loop bx of cervix  Routinely requested after a positive smear  Need to identify dyskarryosis  If dyskarryosis is not identified then more levels are requested  Levels requested  If no levels within the first 6 levels then no further levels are requested Clinical indicatorsLaboratory actions 5th May 2011 42 David Muskett - SD/CP5 Microtomy

43. Case study 3 – full thickness rectal biopsy ? Hirschsprung’s disease 5th May 2011David Muskett - SD/CP5 Microtomy 43  Most common congenital abnormality  Abnormality of nerve growth in the  Before bx investigation it killed 97% of babies within a few weeks  Need to identify presence of normal nerves  50 serial sections to start with

44. Knife angle 5th May 2011David Muskett - SD/CP5 Microtomy 44

45. Specimen orientation 5th May 2011David Muskett - SD/CP5 Microtomy 45 Knife

46. Blade quality H&E Score lines visible on the waterbath 5th May 2011 46 David Muskett - SD/CP5 Microtomy

47. This short ribbon of sections that was cut from a cold block shows considerable compression (30–40%). In this case re-setting the knife tilt angle overcame the problem. Optimise knife tilt angle 5th May 2011 47 David Muskett - SD/CP5 Microtomy

48. This block face has cracked because it was frozen to –15 °C in a freezer prior to cutting. The cracks may make sectioning and flotation difficult because the wax is no longer bound to the tissue. Avoid freeze damaging 5th May 2011 48 David Muskett - SD/CP5 Microtomy

49. The distortion of the glomeruli in this kidney section is due to excessive compression when the section was cut (H&E). 5th May 2011 49 David Muskett - SD/CP5 Microtomy

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51. Use clean water 5th May 2011 51 David Muskett - SD/CP5 Microtomy

52. Ensure slides are clean 5th May 2011 52 David Muskett - SD/CP5 Microtomy

53. A section of cardiac muscle has been contaminated with a fragment of thyroid from another case. This example of specimen-to-specimen transfer occurred on the flotation bath Avoid cross contamination 5th May 2011 53 David Muskett - SD/CP5 Microtomy

54. Don’t float out multiple blocks 5th May 2011 54 David Muskett - SD/CP5 Microtomy

55. These sections of skin clearly show cracks and excessive separation of layers, the typical effects of over-expansion. Poorly processed tissue is very prone to this problem. Check the water temperature 5th May 2011 55 David Muskett - SD/CP5 Microtomy

56. In this case flotation has not overcome the wrinkles produced during the cutting of these sections. Better cutting technique and slightly warmer water would overcome this problem. Avoid wrinkles in sections 5th May 2011 56 David Muskett - SD/CP5 Microtomy

57. Carefully choose a section 5th May 2011 57 David Muskett - SD/CP5 Microtomy

58. Dry sections for an appropriate time 5th May 2011 58 David Muskett - SD/CP5 Microtomy

59. Summary 5th May 2011David Muskett - SD/CP5 Microtomy 59

60. Any questions 5th May 2011David Muskett - SD/CP5 Microtomy 60