Tissue Processing Flashcards
Why is tissue processing needed
To enable the tissue and cellular components to be visualised
What are the four steps in tissue processing
Preparation/Processing
Microtomy
Staining
Microtomy
In general how is tissue prepared for processing
(3)
Specimens has been fixed prior to this
Specimens are cut, sliced and pieces are selected as appropriate
Small biopsies are processed whole
How are samples prepared for wax processing
They are put into cassettes for wax processing
How are samples prepared for electron microscopy
Samples are put in vials for electron microscopy
How are samples prepared for frozen sections
Samples are placed into ‘chuck’ for frozen sections
Other than for wax, EM or frozen sections, how might samples be prepared
(6)
Smear, imprints or squash preparations for emergency or research
Disaggregation of solid tissue for flow cytometry
Freeze-drying
Nitrocellulose embedding
Gelatin embedding
Agar embedding
What are the aims of tissue processing
(4)
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
Define tissue processing
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
Write about paraffin wax processing
(2)
Most common method used in routine histology
Involves dehydration, clearing and infiltration with liquid medium (wax)
Why do we dehydrate tissues during processing
Water will lead to necrosis over time
Water also doesn’t mix with wax
What are the two mechanisms of tissue processing
Using automated systems
Using heat and vacuum
What are the two notable features of using automated systems for tissue processing
(2)
Enclosed processors for health and safety
These often have charcoal filters which prevent odour
What are the two methods of using heat and vacuum for tissue processing
Accelerate processing
Overnight processing
When is accelerate tissue processing carried out
These are rapid programmes for urgent and biopsy specimens
When is overnight heat and vacuum processing carried out
This is done for routine blocks
How long does it take to fix brain?
48 hours
How is dehydration for wax processing carried out
(4)
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
Why is dehydration such an important step
If not complete, then clearing agent and wax will not penetrate evenly to provide solid medium for microtomy
Does alcohol and wax mix and why is this important
(3)
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
What is a bridging reagent
A reagent that is mixable with alcohol and wax but doesn’t affect the tissue
e.g. xylene
What is a bridging agent also called
A clearing agent
Give an example of a clearing agent
Xylene
What is a clearing agent
An organic solvent miscible with alcohol and embedding medium
What are the two classifications of clearing agents
Hydrocarbons
Terpenes
Give three hydrocarbon clearing agents
Xylene
Chloroform
Toluene
Give two examples of terpenes clearing agents
Citrus fruit oils (histoclear)
Oils
What clearing agent would you use for rapid clearing
Xylene or toluene
What clearing agent would cause brittle blocks
Xylene (really brittle if in xylene too long, very hard to embed in wax and cut)
What clearing agent would cause soft block
Chloroform - but this isn’t really used anymore
What biohazards are associated with clearing agents
Carcinogenicity
Flammability
Volatility
Toxicity
Comment on the waste disposal of clearing agents
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
