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2.2 Cellular Pathology > Tissue Processing > Flashcards

Tissue Processing Flashcards

1
Q

Why is tissue processing needed

A

To enable the tissue and cellular components to be visualised

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2
Q

What are the four steps in tissue processing

A

Preparation/Processing
Microtomy
Staining
Microtomy

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3
Q

In general how is tissue prepared for processing
(3)

A

Specimens has been fixed prior to this

Specimens are cut, sliced and pieces are selected as appropriate

Small biopsies are processed whole

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4
Q

How are samples prepared for wax processing

A

They are put into cassettes for wax processing

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5
Q

How are samples prepared for electron microscopy

A

Samples are put in vials for electron microscopy

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6
Q

How are samples prepared for frozen sections

A

Samples are placed into ‘chuck’ for frozen sections

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7
Q

Other than for wax, EM or frozen sections, how might samples be prepared
(6)

A

Smear, imprints or squash preparations for emergency or research

Disaggregation of solid tissue for flow cytometry

Freeze-drying

Nitrocellulose embedding

Gelatin embedding

Agar embedding

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8
Q

What are the aims of tissue processing
(4)

A

Embed tissue in stable, firm, non-hazardous medium

Gives us the ability to cut thin sections for microscopy

Allows for good preservation of morphology and cellular contents

Allows for long-term preservation e.g. filling and storage

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9
Q

Define tissue processing

A

A series of treatments with chemical reagents resulting in the infiltration of the tissue with a medium (mainly wax or resin) which can be solidified into a hard block for microtomy

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10
Q

Write about paraffin wax processing
(2)

A

Most common method used in routine histology

Involves dehydration, clearing and infiltration with liquid medium (wax)

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11
Q

Why do we dehydrate tissues during processing

A

Water will lead to necrosis over time

Water also doesn’t mix with wax

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12
Q

What are the two mechanisms of tissue processing

A

Using automated systems

Using heat and vacuum

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13
Q

What are the two notable features of using automated systems for tissue processing
(2)

A

Enclosed processors for health and safety

These often have charcoal filters which prevent odour

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14
Q

What are the two methods of using heat and vacuum for tissue processing

A

Accelerate processing

Overnight processing

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15
Q

When is accelerate tissue processing carried out

A

These are rapid programmes for urgent and biopsy specimens

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16
Q

When is overnight heat and vacuum processing carried out

A

This is done for routine blocks

17
Q

How long does it take to fix brain?

A

48 hours

18
Q

How is dehydration for wax processing carried out
(4)

A

Sample is dehydrated through ascending grades of alcohol ie 70%, 80%, 95%, 100%

Some components are solubilised, the tissue shrinks

Ethanol, methanol and IMS (industrial methylated spirits are usually used)

Less often, isopropanol, butanol and acetone are used

19
Q

Why is dehydration such an important step

A

If not complete, then clearing agent and wax will not penetrate evenly to provide solid medium for microtomy

20
Q

Does alcohol and wax mix and why is this important
(3)

A

No they don’t mix

We have dehydrated our tissue using alcohol and now we want to process it through wax but we can’t since they don’t mix

We need a bridging agent

21
Q

What is a bridging reagent

A

A reagent that is mixable with alcohol and wax but doesn’t affect the tissue

e.g. xylene

22
Q

What is a bridging agent also called

A

A clearing agent

23
Q

Give an example of a clearing agent

A

Xylene

24
Q

What is a clearing agent

A

An organic solvent miscible with alcohol and embedding medium

25
Q

What are the two classifications of clearing agents

A

Hydrocarbons
Terpenes

26
Q

Give three hydrocarbon clearing agents

A

Xylene
Chloroform
Toluene

27
Q

Give two examples of terpenes clearing agents

A

Citrus fruit oils (histoclear)
Oils

28
Q

What clearing agent would you use for rapid clearing

A

Xylene or toluene

29
Q

What clearing agent would cause brittle blocks

A

Xylene (really brittle if in xylene too long, very hard to embed in wax and cut)

30
Q

What clearing agent would cause soft block

A

Chloroform - but this isn’t really used anymore

31
Q

What biohazards are associated with clearing agents

A

Carcinogenicity
Flammability
Volatility
Toxicity

32
Q

Comment on the waste disposal of clearing agents

A

These can’t be put down the sink -> a removal company must remove them -> brought to Sweeden

There is legislation surrounding the disposal and recycling

2.2 Cellular Pathology (29 decks)

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