Embedding, Microtomy and Cryotomy Flashcards
What must be done after tissue processing i.e. dehydration and clearing?
Embedding
Microtomy/Cryotomy
Define embedding
The process of making wax tissue block for microtomy
Why is embedding carried out
To provide maximum amount of diagnostic information to be obtained from the stained slide
What four technical considerations should be taken to ensure quality of tissue block
Technique
Orientation
Positioning
QC checks
Write about the technique needed for embedding
(9)
Open cassette to assess tissue size and orientation required
Select appropriate sized mould
Place mould under wax dispenser
Dispense small amount of wax to cover base of mould
Pick up tissue with forcepts and position in mould - cut/flat side down
Move mould onto cold plate and gently press tissue in place
Allow wax to cool slightly, then place casette on top of mould
Top up with sufficient wax to fill back of casette
Place on cold tray to cool
What is considered the most important skill for embedding and why?
Orientation
Must be evaluating the correct aspect of the tissue
Need to make sure all required elements are in the section
What are the six different ways of orientating a tissue section?
Tubular tissue
Skin
Endometrial cutting
Long tissue
Intestine
Membrane
Write about how you would orientate a tubular tissue
(3)
Need to include the lumen and all surrounding structures
Must be a transverse section
Often seen on fallopian tubes and vas deferens as well as major arteries
Write about how you would orientate the skin
Need to include all the layers especially if looking for malignancy i.e. investigating invasion -> need to be able to see the connective tissue
Write about how you would orientate a section of intestine
You need to have all the layers of the intestine
Especially if investigating malignancy
Need mucosa and underlying tissue
How would you orientate a membrane
Take a strip then create a coil and put that on end
E.g. for the placenta or mesothelium
How would you orientate endometrial curetting
There are multiple pieces so orientate in the middle so you see all pieces of tissue
How would you orientate long tissue
Keep diagonally
Why is positioning as important as orientation
(3)
We want flat surfaces so you get the full face of the object when you cut - this is a challenge if you have multiple tissues
Should it be positioned central or diagonal - this depends on the tissue
You need to ensure there is sufficient surrounding wax to support tissue for microtomy
Write about how you would quality control embedding
(3)
Check for any fixation or processing errors i.e. is there any soft tissue that may need to be reprocessed
Is there the correct number of tissue pieces in the casette -> check work sheet
Trim any excess wax before microtomy as excess way may prevent stable positioning of block in the chuck
How would you quality control a section of breast tissue
(4)
This contains lots of lipids which are hydrophobic
Fixation and processing steps might have not permeated the tissue
The block might still be soft
Reprocessing might be necessary
Define microtomy
The process of cutting thin sections and transferring to glass slides for light microscopy
Comment on the width of sections needed for microscopy
About 3-5um in width
A monolayer of cells e.g. 1 cell thick
Why is it important to get such thin sections of tissue
Monolayers of cells can be stained to investigate cell and tissue changes associated with disease
What is the microtome
(4)
A specialised mechanical device
It can be semi-automated or automated
The rotary microtome is the most commonly used in histopathology labs
It cuts wax sections using disposable steel blades
What does the microtome consist of?
(5)
An advance and retract mechanism
Rotary mechanism and handle
Chuck to hold the block
Blade holder
Blade guard
Write about how you would carry out microtomy on a wax block
(7)
With lock and blade guard in position, cooled black is placed in chuck
The chuck is retracted until block is positioned just behind the blade
The lock and blade guard are removed and the block is passed down towards the blade
The block is advanced gradually towards the blade in incremental fashion using the advance mechanism
When the block is in the correct position for the blade to cut the wax, the block is passed fully down on the blade
The block is then ‘trimmed’ until the full surface of the tissue is reached
At this point, move to the rotary handle to cut sections at the desired thickness
Why do we use cooled blocks in microtomy
This aids smooth consistent sectioning and thickness
Why do we trim blocks
To reach full surface/cut surface of tissue
QC - do not keep trimming if orientation is incorrect
Write about the different ways sections can be cut using the microtome
(3)
Can cut in ribbons
Can cut interval or additional slides for special stains or IHC
We cut sections at the required thickness
We usually cut sections at 1-5um, what are three exceptions to this rule
Lymph nodes cut at 2-3 um as they can look crowded if any thicker
Renal cut at 2-3 um to better examin the basement membrane
Brain cut at 10-15um as thicker sections are better used to see nerves
What four issues could arise from microtomy
Chattering
Scores
Bloody tissue
Variable thickness
What causes chattering
(3)
Hard tissues
Blunt blades
Insufficient supporting wax
How would you fix chattering?
(4)
Softening agents can be used for hard blocks
Cool block again
Change blade
Re-embed in a larger mould with more surrounding wax
What causes scores?
Damaged blades
What causes damaged blades
Calcification of tissue
Poor cleaning technique
What tissues are often bloody
Spleen
Liver
How do you fix bloody tissues
Soak in ‘wet’ ice
What causes variable thickness and how do you fix this
Caused by warm blocks -> just need to cool again
What is ‘floating out’
The process for transfer of cut sections to a glass slide
How is floating out carried out
Through the use of cold and warm water baths
Wax doesn’t mix in water so the section floats
What are the steps in floating out
(4)
First place section in cold water
- this allows for handling of sections without risk of disintegration
- flattens out folds/creases and helps orientate the tissue
Transfer to warm water
- further flattens creases
- Section orientated and picked up on slide
What can be added to some slides to aid in the floating out step
Some slides have adhesive properties to prevent loss of section during subsequent staining methods
What is cryotomy
The making of frozen sections
When is cryotomy used
(4)
When an urgent result is required
During surgery
Tissue constituents are inactivated or lost by fixation or processing e.g. enzymes or lipids
Direct immunofluorescence
What is it called when cryotomy is carried out mid surgery?
Intraoperative report
When might cryotomy be carried out mid surgery?
Excision margins
Unexpected findings
Cryotomy is carried out if direct immunofluorescence is to be carried out on what tissues?
Renal and skin
What are the two main benefits of cryotomy?
Can be performed on fresh or fixed tissue
Tissue can subsequently be processed to paraffin wax
Comment on the freezing of tissue
(4)
Water crystalises and forms solid matric
Can get artefact
Need to post fix if thawed
Frozen by liquid nitrogen or solid CO2, electrical plates or cryosprays
Describe how sections are frozen for cryotomy
Tissue is frozen on a chuck with OCT medium which acts like a glue
Sections are then cut in a cryostat which is a frozen microtome kept at -20 degrees
What is a special feature of the cryostat
Anti-roll plate which prevents section from curling
How are frozen sections carried out for urgent H+E
(6)
Cut using cryostat
Section transferred to glass slide
Heated slightly
Rapid fixation - usually compound fixative to increase fixation speed
Rapid H+E (hand-stained)
Pathologist report
What are some considerations for frozen sections
(4)
Fresh tissue - might be biohazardous
Ice crystal artefact
Lower quality section - process tissue afterwards for FFPE
Alternative methods for enzyme studies and immunofluorescence
