Tissue Preservation and Fixation Flashcards
Why do samples need to be preserved and fixed?
(4)
To stop tissues from necrosing, dieing
Fixation hardens tissue and allows for easy manipulation of soft tissues
Converts semi-fluid consistency of some samples to an irreversible semi solid gel
It allows us to cut one cell thick sections for examination under the microscope
What exactly happens if our samples are not preserved?
As soon as the tissue is removed from the body, the vasculature supply is lost
Haemoglobin leaches out of the red blood cells and the tissue begins to die
Give six components of tissue that we may need to analyse
Cells
Connective tissue
DNA and RNA
Pigments and minerals
Microbes
Viruses
How are microbes examined in the pathology lab
(3)
A gram stain modified for tissue is used
Often carried out on open wounds i.e. section of infected tissue cut off for examination
Or we might see tuberculosis bacteria in a lung biopsy
How do we examine viruses in the pathology lab?
(3)
Viruses such as hepatitis B can be found in liver cells
We can use histochemistry and specific stains for viral detection
HPV detection is vital in the screening of cervical cancer
Give an example of a pigment we might look for
We might look for the overproduction of melanin in melanomas
What are the three functions of preservatives
To support cells with nutrients
To maintain viability
To conserve constituents
Give three applications of preservatives
Flow cytometry - RPMI (Roswell Park Memorial Institute Medium)
Cell and tissue culture (supplemented growth media)
Biochemical studies (enzymes)
What is RPMI
Roswell Park Memorial Institute Medium
What are the constituents of RPMI?
(4)
A nutritive liquid used to support cell viability in biological samples
It has no protein so it is supplemented separately with bovine serum
It is not a fixative
Used when laboratory testing requires fresh, unfixed specimens i.e. in flow cytometry or FISH
Give the four aims of fixation
(4)
Prevent autolysis and putrefaction
Maintain tissue as close to living state without alteration or loss of their components
Maintain shape and volume -> tissue will shrink a little bit but we can allow for that when looking at cells
Allow subsequent staining -> this enables us to make a diagnosis for the patient
Define a preservative
A solution in which tissue can be stored and maintained without further degradation for a long period of time
Define a fixative
These ‘fix’ a specimen by preventing protein breakdown and stabilizing the proteins within the tissues in a manner which retains a semblance of their life like state
Write a note on fixation
(4)
Most cell and tissue analysis is performed on fixed tissue
There are very few examples of where we don’t fix tissues
The fixative chosen depends on the type of analysis and the sample type
Formaldehyde is nearly always used but we don’t use methanol for tissue as it dries out the samples, it will shrink and make it too brittle to cut
What are the four types of analysis that require different methods of fixation
Morphology
Histochemistry
Immunohistochemistry
NA analysis (neutron activation analysis)
What are the two different sample types that require different methods of fixation from each other?
Cytology versus Histology
What are the two categories of fixatives?
Thermal and chemical
What are the two thermal methods of fixation
Heat
Freezing
What are the two chemical methods of fixation
Cross-linking
Coagulation
How does thermal fixation by heating work?
(2)
By heating the sample this coagulates proteins
This can be done by microwave or boiling
How does thermal fixation by freezing work?
(2)
Freezing crystallises water to form a solid matrix
This can be done using liquid nitrogen or CO2
When is thermal fixation by freezing used?
(2)
To make frozen sections
Used for storage of fresh or pre-fixed cells and tissues such as in biobanking/molecular studies/cell lines or research
When are frozen sections made?
This is done on urgent samples e.g. excision margins
What is meant by excision margins?
(2)
When a doctor is performing surgery to remove a tumour a sample may be sent to the lab to know if the margins have been cleared
The lab should have the results back in 15 minutes and the doctor can then determine if they need to take out more tissue or not
What is the basis of chemical fixation
(2)
Chemical solutions permeate cells and tissue structure
These chemicals react with proteins and form a solid mesh that traps components
Give two cross-linking fixatives
Formaldehyde and glutaraldehyde
How do the cross-linking fixatives work
(3)
These form methylene bridges between side and end groups of proteins,
This creates a solid protein mesh linked by methyl groups
i.e. proteins become joined by CH2 molecules to form a mesh
When and where is glutaraldehyde used
(3)
Used on small tissue samples
Used when looking at morphology alone
Used for electron microscopy
Explain in your own words what cross linking does?
(3)
This forms a protein mesh
Proteins cross linked and therefore can’t move
The proteins solidify and fix
Write a note on formaldehyde as a fixative
(4)
HCHO
Most commonly used fixative as 10% formalin
10% formalin = 4% formaldehyde
Slow penetration of tissue - few hours for small samples but larger samples overnight
How much formaldehyde is in 10% formalin?
4% formaldehyde
How is formalin made up
(2)
The formaldehyde stock solution is about 40% gas in water
Diluted in saline or buffer to maintain the pH
Why is it important to monitor the pH of formaldehyde?
If pH changes it will deposit artefacts in the tissue
Write a note on glutaraldehyde
(6)
CHO.(CH2)3.CHO
Best morphological preservation
Small fragments only e.g. for electron microscopy
2.5% in buffer is used
Expensive
Biohazard
What about formaldehyde is biohazardous?
(2)
The buffer used
Some labs neutralise the buffer first and then pour it down the sink while others pay a company to dispose of it
Which cross-linking chemical form of fixation is faster?
Formaldehyde is faster than glutaraldehyde
What is the basis of coagulant fixation?
These coagulate proteins, this alters their tertiary structure
What is a benefit of coagulant fixation
Rapid penetration
What is a pitfall of coagulant fixation
Causes shrinkage
Give four examples of coagulant fixation
Ethanol
Methanol
Acetone
Acetic acid
Where would coagulant fixation most commonly be used?
Alcohol fixation e.g. ethanol or methanol is used in cytology
List five other, non grouped, fixatives
Osmium tetroxide
Mercuric chloride
Picric acid
Dichromate fixatives
Compound fixatives
Write about osmium tetroxide as a fixative
(3)
An oxidising agent
It’s a secondary fixative in electron microscopy
Turns black when fixing lipid
Write about mercuric chloride as a fixative
(3)
Precipitates proteins
Good staining quality.
Hardens tissue
Write about picric acid as a fixative
(3)
Uses in fixative solutions
Good fixative for glycogen
Potentially explosive
Write about dichromate fixatives
(2)
Used as a secondary fixative
Potassium dicromate used when chromaffin reaction required e.g. for adrenal gland
Write about compound fixatives
(3)
Mixtures of fixatives, buffers and salts to act together to give best fixation of tissue under investigation
They are usually formaldehyde or alcohol-based
e.g. Bouin’s, B5, Zenker’s, Carnoy’s, Mathacarn
Give the main use of picric acid fixation
Used to decalcify some tissues such as bone samples of calcium deposits in breast tumours
This allows these samples to be cut
Write a note on over fixation
(4)
Sample spends too long in fixative
Biopsies are smaller and need much less time than large specimens e.g. organs
Can occur in batch fixation -> not being fixed individually, one sample may be over fixed while rest are normal
Affects immunohistochemistry
Tissues can become very brittle but this would require a lot of time in fixative
Write a note on underfixation
(5)
Incomplete fixation
Too large a sample, middle not fixed
Long term preservation affected
Tissue too soft for microtomy
Tissue can’t be stained correctly
