Cellular pathology: Flow Cytometry - Introduction Flashcards

1
Q

What is flow cytometry?

A
  • A technique which simultaneously measures several physical characteristics belonging to a single cell in suspension
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2
Q

What things are used during flow cytometry to measure the physical charcteristics of a cell?

A
  • Light scatter and fluorescence
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3
Q

What is flow sorting (Fluorescence-Activated Cell Sorting (FACS)?

A
  • Sorting (separating) cells based on properties measured in flow
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4
Q

What can a flow cytometer tell us about a cell?

A
  • Its Relative Size
  • Its Relative Granularity/Internal Complexity
  • Its Relative Fluorescence Intensity
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5
Q

What aspects of a cell can be measured using flow cytometry?

A
  • Cell surface receptors
  • Adhesion molecules
  • Cytokines
  • Enzymes
  • DNA - look at cell cycle, apoptosis, cell viability
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6
Q

Apart from flow cytometry what are some other techniques that use fluorescenec to view cells?

A
  • Fluorescence Microsocopy
  • Flow Cytometry
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7
Q

What are the advantages of flow cytometry compared with flourescent microscopy?

A
  • Flow cytometry can look at thousands of cells simultaneously while fluorescent microscopy can only look at a limited no. of cels in each field of view of microscope (difficult) to view lots of cells
  • Flow cytometry can look for rare cells very easily while with fluorescent microcospy rare cells hard to find
  • Flow cytometry is a quantative process while fluorescent microscopy isn’t a very quantative process (can only look at around 20 cells per field)
  • Intensity of fluorescence in flow cytometry is very accurate while with fluorescent microscopy te instensity is variable
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8
Q

What are the major components of a flow cytometry machine?

A
  • Fluidics - Cells in suspension flow in single file
  • Optics - Cells flow through an illuminated volume where a laser hits each cell and causes them to scatter light and emit fluorescence
  • Electronics - Fluorescence is collected, filtered and converted to digital values that are stored on a computer so they can then be analysed
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9
Q

In a flow cytometer how are cells put in suspension and made sure that they flow in single file?

A
  • This is done by injecting the sample fluid contianing the cells into a nozzle tip containing sheath fluid as it passes through a small (50-300 µm) orifice (hole)
  • Sample fluid flows in a central core that does not mix with the sheath fluid
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10
Q

What type of flow does the sample fluid experience since it does not mix with the sheath fluid in the flow cytometer?

A
  • Laminar flow
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11
Q

What is the term used to describe the introduction of a large volume into a small volume?

A
  • Hydrodynamic focusing
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12
Q

Describe the lasers used as part of the optics component of a flow cytometer

A
  • Lasers used are a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
  • They can:
    • Provide anywhere between milliwatts and watts of light
    • Be inexpensive, air-cooled units or expensive, water-cooled units
    • Provide coherent light (Single frequency)
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13
Q

What happens when light from a laser hits a cell?

A
  • Light scatters in the foward direction (foward light scatter) which is proportional to the size of the cell
  • Light also scatters at a 90º angle (side scatter) which is proportional to the granularity of the cell
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14
Q

What information about a population of cells can be obtained from just the light scatter of that population?

A
  • From light scatter patterns of a population a dot plot showing “increase in foward scatter” on the X-axis and “increase in side scatter” on the y-axis can be produced
  • This dot plot can be used to group cells into distinct types based on amount of foward and side scatter
  • E.g. graph below groups white blood cells from a blood sample into their different types with each dot representing a cell
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15
Q

Describe the basic concept of laser-based flow cytometry using antibodies works

A
  • Cells flow out of the nozzle tip of the flow cell
  • Cells have been labelled with 4 different antibodies which each have a different colour fluoresent marker attaced to them
  • The laser then hits each cell which causes fluorescence to be emitted
  • The fluorescence produced goes through different dichroic filters
  • This allows a specific photo multiplier tube (PMT) to pick up a specific wavelength of light (fluoresence)
  • At each photo multiplier tube light (fluorescence) is converted into data
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16
Q

Describe the elctronics componets of a flow cytometer?

A
  • This is where the processing of signals from detectors occurs - done by photo multiplier tubes (PMTs)
  • Analog-Digital Conversion also occurs so the data can be analysed on a computer
17
Q

What is a fluorochrome (fluorophore)?

A
  • A fluorochrome is a chemical that is able to absorb light and then re-emit it when it is excited by light
18
Q

Explain how a fluorochrome produces fluoresence

A
  • When a fluorochrome is hit with light from a laser it absorbs the energy from the light and becomes excited
  • Eventually the fluorochrome emits the same amount of energy that it absorbed, as light but it emits it at higher wavelength than the light that it absorbed
19
Q

What is stokes shift?

A
  • It is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
20
Q

Give some examples of common fluorochromes and the colours of light they emit when they fluoresce

A
  • Fluorescein isothiocyanate (FITC) - Green
  • Phycoerythrin (PE) - Orange
  • Peridinin Chlorophyll Protein (PerCP) - Red
21
Q

Why can FITC, PE and PerCP fluorochromes be used at the same time? CP

A
  • It’s because they emit at different wavelengths which means they can be detected at the same time
  • This means all 3 can be used to make 3 different antibodies fluorsecent and can then be added to a group of cells
  • This allows you to obtain diferent information about the cells within that population
22
Q

Why are the dichroic filters needed in laser-guided flow cytometry ?

A
  • The dichroic filters are needed because light emitted from different flouorophores may be of a similar wavelength and so they allow the photo multiplier tubes to pick up light of a specific wavelength from a particular fluorophore
  • They do this by refelecting light of a specific wavelength to one of the photo multipliers
23
Q

Give some examples of single cells in suspension that are analysed using in flow cytometry

A
  • Peripheral blood
  • Bone marrow
  • Fine Needle Aspirate
  • CSF and other fluids
  • Fresh Tissue
24
Q

What are the 2 ways that cells can be labelled in flow cytometry?

A
  • Direct: Monoclonal antibodies (MoAbs) are labelled with fluorochromes
  • Indirect: Unlabelled primary monoclonal antibodies are joined to a secondary antibody which is labelled with a fluorochrome
25
Q

When would the indirect method of labelling a cell be used instead of the direct method?

A
  • Indirect method used when you are unable to label yoyr primary antibody with a fluorochrome
26
Q

In what ways can the data obtained from flow cytometry be displayed?

A
  • Dot plot
  • Histogram - X-axis = Fluorescence intensity, Y-axis = No. of cells
27
Q

What is gating?

A
  • Gating is a data analysis technique used that allows you to hone in on a particular area of a dot plot that may represent a particular type of cell and show the data fro that group in more detail