SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2 Principles...
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Transcript of SD/CP5 - MICROTOMY David Muskett. Plan 5th May 2011David Muskett - SD/CP5 Microtomy 2 Principles...
SD/CP5 - MICROTOMY
David Muskett
Plan
5th May 2011David Muskett - SD/CP5 Microtomy
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Principles and practice Quality control Equipment Mounting & coverslipping Clinical perspective
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?
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)
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
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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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
Special considerations when cutting blocks Lymph nodes Renal biopsies Amyloid Neuropathology sections
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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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Sectioning artefact – knife lines
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Sectioning artefact – component displacement
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Sectioning artefact – Venetian blind
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Sectioning artefact – course chatter
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Sectioning artefact – adhesive tidelines
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Sectioning artefact - bubbling under the sections
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Sneeze artefact
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The slides
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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
Section artefact – contamination of mounted sections
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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?
Sectioning artefact - folds
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How do you know you have got a good section?
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Knives
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Solid (non disposable knives) Strop & Hone Knife sharpener
Disposable knives Feather
Glass knives for electron microscopy
Knife angles and blades
Feather disposable
Wedge profile Carbon steel
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Productivity
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How many blocks should you be able to cut in 1 hour?
Antigenicity of cut sections?
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Antigenicity of sections is lost over time
Big sections
Megablocks Gough &
Wentworth
Allow investigation of anatomical features more easily
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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
Microtomes
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Rotary Manual Motorised – semi automatic / automatic
Sledge Sliding Ultra Freezing
Cambridge rocker
Simple and popular
In use in the 1960s and 1970s
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Rotary microtomes
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Sledge microtomes
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Sliding microtomes
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Ultra microtomes
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Freezing Microtomes
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Health & Safety - microtomy Knives Upper limb related disorder
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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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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
Knife angle
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Specimen orientation
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Knife
Blade quality
H&E Score lines visible on the waterbath
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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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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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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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Use clean water
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Ensure slides are clean
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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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Don’t float out multiple blocks
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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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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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Carefully choose a section
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Dry sections for an appropriate time
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Summary
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Any questions
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