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2.2 Cellular Pathology > Embedding, Microtomy and Cryotomy > Flashcards

Embedding, Microtomy and Cryotomy Flashcards

1
Q

What must be done after tissue processing i.e. dehydration and clearing?

A

Embedding
Microtomy/Cryotomy

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2
Q

Define embedding

A

The process of making wax tissue block for microtomy

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3
Q

Why is embedding carried out

A

To provide maximum amount of diagnostic information to be obtained from the stained slide

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4
Q

What four technical considerations should be taken to ensure quality of tissue block

A

Technique
Orientation
Positioning
QC checks

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5
Q

Write about the technique needed for embedding
(9)

A

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

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6
Q

What is considered the most important skill for embedding and why?

A

Orientation

Must be evaluating the correct aspect of the tissue

Need to make sure all required elements are in the section

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7
Q

What are the six different ways of orientating a tissue section?

A

Tubular tissue
Skin
Endometrial cutting
Long tissue
Intestine
Membrane

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8
Q

Write about how you would orientate a tubular tissue
(3)

A

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

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9
Q

Write about how you would orientate the skin

A

Need to include all the layers especially if looking for malignancy i.e. investigating invasion -> need to be able to see the connective tissue

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10
Q

Write about how you would orientate a section of intestine

A

You need to have all the layers of the intestine
Especially if investigating malignancy
Need mucosa and underlying tissue

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11
Q

How would you orientate a membrane

A

Take a strip then create a coil and put that on end

E.g. for the placenta or mesothelium

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12
Q

How would you orientate endometrial curetting

A

There are multiple pieces so orientate in the middle so you see all pieces of tissue

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13
Q

How would you orientate long tissue

A

Keep diagonally

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14
Q

Why is positioning as important as orientation
(3)

A

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

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15
Q

Write about how you would quality control embedding
(3)

A

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

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16
Q

How would you quality control a section of breast tissue
(4)

A

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

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17
Q

Define microtomy

A

The process of cutting thin sections and transferring to glass slides for light microscopy

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18
Q

Comment on the width of sections needed for microscopy

A

About 3-5um in width
A monolayer of cells e.g. 1 cell thick

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19
Q

Why is it important to get such thin sections of tissue

A

Monolayers of cells can be stained to investigate cell and tissue changes associated with disease

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20
Q

What is the microtome
(4)

A

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

21
Q

What does the microtome consist of?
(5)

A

An advance and retract mechanism
Rotary mechanism and handle
Chuck to hold the block
Blade holder
Blade guard

22
Q

Write about how you would carry out microtomy on a wax block
(7)

A

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

23
Q

Why do we use cooled blocks in microtomy

A

This aids smooth consistent sectioning and thickness

24
Q

Why do we trim blocks

A

To reach full surface/cut surface of tissue

QC - do not keep trimming if orientation is incorrect

25
Q

Write about the different ways sections can be cut using the microtome
(3)

A

Can cut in ribbons

Can cut interval or additional slides for special stains or IHC

We cut sections at the required thickness

26
Q

We usually cut sections at 1-5um, what are three exceptions to this rule

A

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

27
Q

What four issues could arise from microtomy

A

Chattering

Scores

Bloody tissue

Variable thickness

28
Q

What causes chattering
(3)

A

Hard tissues
Blunt blades
Insufficient supporting wax

29
Q

How would you fix chattering?
(4)

A

Softening agents can be used for hard blocks

Cool block again

Change blade

Re-embed in a larger mould with more surrounding wax

30
Q

What causes scores?

A

Damaged blades

31
Q

What causes damaged blades

A

Calcification of tissue
Poor cleaning technique

32
Q

What tissues are often bloody

A

Spleen
Liver

33
Q

How do you fix bloody tissues

A

Soak in ‘wet’ ice

34
Q

What causes variable thickness and how do you fix this

A

Caused by warm blocks -> just need to cool again

35
Q

What is ‘floating out’

A

The process for transfer of cut sections to a glass slide

36
Q

How is floating out carried out

A

Through the use of cold and warm water baths

Wax doesn’t mix in water so the section floats

37
Q

What are the steps in floating out
(4)

A

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

38
Q

What can be added to some slides to aid in the floating out step

A

Some slides have adhesive properties to prevent loss of section during subsequent staining methods

39
Q

What is cryotomy

A

The making of frozen sections

40
Q

When is cryotomy used
(4)

A

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

41
Q

What is it called when cryotomy is carried out mid surgery?

A

Intraoperative report

42
Q

When might cryotomy be carried out mid surgery?

A

Excision margins
Unexpected findings

43
Q

Cryotomy is carried out if direct immunofluorescence is to be carried out on what tissues?

A

Renal and skin

44
Q

What are the two main benefits of cryotomy?

A

Can be performed on fresh or fixed tissue

Tissue can subsequently be processed to paraffin wax

45
Q

Comment on the freezing of tissue
(4)

A

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

46
Q

Describe how sections are frozen for cryotomy

A

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

47
Q

What is a special feature of the cryostat

A

Anti-roll plate which prevents section from curling

48
Q

How are frozen sections carried out for urgent H+E
(6)

A

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

49
Q

What are some considerations for frozen sections
(4)

A

Fresh tissue - might be biohazardous

Ice crystal artefact

Lower quality section - process tissue afterwards for FFPE

Alternative methods for enzyme studies and immunofluorescence

2.2 Cellular Pathology (29 decks)

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