Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

2.2 Cellular Pathology > Tissue Preservation and Fixation > Flashcards

Tissue Preservation and Fixation Flashcards

1
Q

Why do samples need to be preserved and fixed?
(4)

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What exactly happens if our samples are not preserved?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Give six components of tissue that we may need to analyse

A

Cells
Connective tissue
DNA and RNA
Pigments and minerals
Microbes
Viruses

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

How are microbes examined in the pathology lab
(3)

A

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 well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How do we examine viruses in the pathology lab?
(3)

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Give an example of a pigment we might look for

A

We might look for the overproduction of melanin in melanomas

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the three functions of preservatives

A

To support cells with nutrients
To maintain viability
To conserve constituents

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Give three applications of preservatives

A

Flow cytometry - RPMI (Roswell Park Memorial Institute Medium)

Cell and tissue culture (supplemented growth media)

Biochemical studies (enzymes)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What is RPMI

A

Roswell Park Memorial Institute Medium

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the constituents of RPMI?
(4)

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Give the four aims of fixation
(4)

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Define a preservative

A

A solution in which tissue can be stored and maintained without further degradation for a long period of time

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Define a fixative

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Write a note on fixation
(4)

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are the four types of analysis that require different methods of fixation

A

Morphology
Histochemistry
Immunohistochemistry
NA analysis (neutron activation analysis)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are the two different sample types that require different methods of fixation from each other?

A

Cytology versus Histology

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What are the two categories of fixatives?

A

Thermal and chemical

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are the two thermal methods of fixation

A

Heat
Freezing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are the two chemical methods of fixation

A

Cross-linking
Coagulation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How does thermal fixation by heating work?
(2)

A

By heating the sample this coagulates proteins

This can be done by microwave or boiling

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

How does thermal fixation by freezing work?
(2)

A

Freezing crystallises water to form a solid matrix

This can be done using liquid nitrogen or CO2

22
Q

When is thermal fixation by freezing used?
(2)

A

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

23
Q

When are frozen sections made?

A

This is done on urgent samples e.g. excision margins

24
Q

What is meant by excision margins?
(2)

A

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

25
Q

What is the basis of chemical fixation
(2)

A

Chemical solutions permeate cells and tissue structure

These chemicals react with proteins and form a solid mesh that traps components

26
Q

Give two cross-linking fixatives

A

Formaldehyde and glutaraldehyde

27
Q

How do the cross-linking fixatives work
(3)

A

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

28
Q

When and where is glutaraldehyde used
(3)

A

Used on small tissue samples

Used when looking at morphology alone

Used for electron microscopy

29
Q

Explain in your own words what cross linking does?
(3)

A

This forms a protein mesh
Proteins cross linked and therefore can’t move
The proteins solidify and fix

30
Q

Write a note on formaldehyde as a fixative
(4)

A

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

31
Q

How much formaldehyde is in 10% formalin?

A

4% formaldehyde

32
Q

How is formalin made up
(2)

A

The formaldehyde stock solution is about 40% gas in water

Diluted in saline or buffer to maintain the pH

33
Q

Why is it important to monitor the pH of formaldehyde?

A

If pH changes it will deposit artefacts in the tissue

34
Q

Write a note on glutaraldehyde
(6)

A

CHO.(CH2)3.CHO
Best morphological preservation
Small fragments only e.g. for electron microscopy
2.5% in buffer is used
Expensive
Biohazard

35
Q

What about formaldehyde is biohazardous?
(2)

A

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

36
Q

Which cross-linking chemical form of fixation is faster?

A

Formaldehyde is faster than glutaraldehyde

37
Q

What is the basis of coagulant fixation?

A

These coagulate proteins, this alters their tertiary structure

38
Q

What is a benefit of coagulant fixation

A

Rapid penetration

39
Q

What is a pitfall of coagulant fixation

A

Causes shrinkage

40
Q

Give four examples of coagulant fixation

A

Ethanol
Methanol
Acetone
Acetic acid

41
Q

Where would coagulant fixation most commonly be used?

A

Alcohol fixation e.g. ethanol or methanol is used in cytology

42
Q

List five other, non grouped, fixatives

A

Osmium tetroxide

Mercuric chloride

Picric acid

Dichromate fixatives

Compound fixatives

43
Q

Write about osmium tetroxide as a fixative
(3)

A

An oxidising agent

It’s a secondary fixative in electron microscopy

Turns black when fixing lipid

44
Q

Write about mercuric chloride as a fixative
(3)

A

Precipitates proteins

Good staining quality.

Hardens tissue

45
Q

Write about picric acid as a fixative
(3)

A

Uses in fixative solutions

Good fixative for glycogen

Potentially explosive

46
Q

Write about dichromate fixatives
(2)

A

Used as a secondary fixative

Potassium dicromate used when chromaffin reaction required e.g. for adrenal gland

47
Q

Write about compound fixatives
(3)

A

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

48
Q

Give the main use of picric acid fixation

A

Used to decalcify some tissues such as bone samples of calcium deposits in breast tumours

This allows these samples to be cut

49
Q

Write a note on over fixation
(4)

A

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

50
Q

Write a note on underfixation
(5)

A

Incomplete fixation

Too large a sample, middle not fixed

Long term preservation affected

Tissue too soft for microtomy

Tissue can’t be stained correctly

2.2 Cellular Pathology (29 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions