sd/cp5 - microtomy david muskett. plan 5th may 2011david muskett - sd/cp5 microtomy 2 principles...

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SD/CP5 - MICROTOMY David Muskett

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Page 1: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

SD/CP5 - MICROTOMY

David Muskett

Page 2: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Plan

5th May 2011David Muskett - SD/CP5 Microtomy

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Principles and practice Quality control Equipment Mounting & coverslipping Clinical perspective

Page 3: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Section thickness and embedding

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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?

Page 4: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Principles and practice

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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)

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Trimming

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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

Page 6: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

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Sectioning

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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

Page 8: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Special considerations when cutting blocks Lymph nodes Renal biopsies Amyloid Neuropathology sections

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Page 9: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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Page 10: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Sectioning artefact – knife lines

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David Muskett - SD/CP5 Microtomy

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Sectioning artefact – component displacement

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David Muskett - SD/CP5 Microtomy

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Sectioning artefact – Venetian blind

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David Muskett - SD/CP5 Microtomy

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Sectioning artefact – course chatter

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David Muskett - SD/CP5 Microtomy

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Sectioning artefact – adhesive tidelines

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David Muskett - SD/CP5 Microtomy

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Sectioning artefact - bubbling under the sections

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David Muskett - SD/CP5 Microtomy

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Sneeze artefact

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David Muskett - SD/CP5 Microtomy

Page 17: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

The slides

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David Muskett - SD/CP5 Microtomy

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Slides & slide adhesives

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Plain glass – needs to be clean Silane slides Albumen – thinly wiped on the slides or

in the waterbath Gelatin Positive charged slides Celloidin

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Section artefact – contamination of mounted sections

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David Muskett - SD/CP5 Microtomy

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David Muskett - SD/CP5 Microtomy

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David Muskett - SD/CP5 Microtomy

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David Muskett - SD/CP5 Microtomy

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Floating out

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Allows the creases to drop out of the section

Temperature should be around 50oC slightly less than the melting point of the wax (56-58oC)

More QC steps Is the section full face Are there any creases Are there scores?

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Sectioning artefact - folds

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Page 26: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

How do you know you have got a good section?

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Page 27: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Knives

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Solid (non disposable knives) Strop & Hone Knife sharpener

Disposable knives Feather

Glass knives for electron microscopy

Page 28: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Knife angles and blades

Feather disposable

Wedge profile Carbon steel

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David Muskett - SD/CP5 Microtomy

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Productivity

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How many blocks should you be able to cut in 1 hour?

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Antigenicity of cut sections?

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Antigenicity of sections is lost over time

Page 31: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Big sections

Megablocks Gough &

Wentworth

Allow investigation of anatomical features more easily

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Microtomy31

Page 32: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Gough - Wentworth sections

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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

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Microtomes

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Rotary Manual Motorised – semi automatic / automatic

Sledge Sliding Ultra Freezing

Page 34: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Cambridge rocker

Simple and popular

In use in the 1960s and 1970s

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David Muskett - SD/CP5 Microtomy

Page 35: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Rotary microtomes

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David Muskett - SD/CP5 Microtomy

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Sledge microtomes

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David Muskett - SD/CP5 Microtomy

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Sliding microtomes

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David Muskett - SD/CP5 Microtomy

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Ultra microtomes

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David Muskett - SD/CP5 Microtomy

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Freezing Microtomes

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Health & Safety - microtomy Knives Upper limb related disorder

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Page 41: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

Page 42: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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 indicators Laboratory actions

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Case study 3 – full thickness rectal biopsy ? Hirschsprung’s disease

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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

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Knife angle

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Specimen orientation

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Knife

Page 46: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Blade quality

H&E Score lines visible on the waterbath

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David Muskett - SD/CP5 Microtomy

Page 47: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

Page 48: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

Page 49: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

The distortion of the glomeruli in this kidney section is due to excessive compression when the section was cut (H&E).

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David Muskett - SD/CP5 Microtomy

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Use clean water

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David Muskett - SD/CP5 Microtomy

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Ensure slides are clean

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David Muskett - SD/CP5 Microtomy

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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

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David Muskett - SD/CP5 Microtomy

Page 54: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Don’t float out multiple blocks

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David Muskett - SD/CP5 Microtomy

Page 55: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

Page 56: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

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

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David Muskett - SD/CP5 Microtomy

Page 57: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Carefully choose a section

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David Muskett - SD/CP5 Microtomy

Page 58: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Dry sections for an appropriate time

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Page 59: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Summary

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Page 60: SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2  Principles and practice  Quality control  Equipment  Mounting

Any questions

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