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SEM 4:Cellular Pathology > Flow Cytometry - Introduction > Flashcards

Flow Cytometry - Introduction Flashcards

1
Q

What is flow cytometry?

A

A technique that simultaneously measures several physical characteristics belonging to a single cell in suspension. This is done by light scatter and fluorescence.

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2
Q

What are the advantages of flow cytometry?

A
  • Integrate cells and get information about many different characteristics all at the same time
  • Very quick
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3
Q

Define flow cytometry

A

Measuring properties of cells in flow for example, how many CD4 cells are there in a sample of blood.

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4
Q

What is flow sorting?

A

Sorting (separating) cells based on properties measured in flow also called fluorescence-activated cell sorting (FACS) for example, separate the CD4 cells from the rest.

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5
Q

What can a flow cytometry tell us about a cell?

A
  1. About the relative sound
  2. About the relative granularity/internal complexity
  3. About the relative fluorescence intensity
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6
Q

What can be measured using flow cytometry?

A
  • Adhesion
  • DNA
  • Cytokines
  • Enzymes
  • Surface Receptors
  • Also apoptosis
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7
Q

What are the methods of visualisation for cells?

A
  • Fluorescence Microscopy

- Flow Cytometry

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8
Q

How does a fluorescence microscopy work?

A
  • Fluorescence is attached to the CD4
  • Limited no. of cells in each field (20 cells per field)
  • Not quantitative compared to flow cytometry that looks at thousands of cells per second
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9
Q

Difference between fluorescence microscopy and flow cytometry

A

Flow Cytometry:

  • Lots of cells at once
  • Quantitative
  • An accurate view of the intensity

Fluorescence Microscopy:

  • Limited No. Cells in each field
  • Not quantitative
  • Varied intensity subjective
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10
Q

What are the 3 components of flow cytometry?

A
  1. Fluidics: cells in a single cell suspension
  2. Optics: an illuminated volume where they scatter light and emit fluorescence that is collected and filtered
  3. Electronics: The fluorescence is then converted to digital values that are stored on a computer.
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11
Q

Describe the flow cytometry analyser

A
  1. The light source then to,
  2. Flow Chamber then to the,
  3. The optical system then to the,
  4. Light detectors and lastly the computer.
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12
Q

What are the fluidics of flow cytometry?

A
  • Need to have cells in suspension flow in single file
  • Accomplished by injecting the sample into a sheath fluid as it passes through a small (50-300 um) orifice.
  • Sample fluid flows in a central core that does not mix with the sheath fluid; this is laminar flow
  • Introduction of a large volume into a small volume caused by hydrodynamic focusing.
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13
Q

How does the nozel tip work in the flow cytometry?

A

The cells are forced to flow in a single file through an orifice because of the sheath fluid by hydrodynamic focusing.

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14
Q

Describe how flow cytometry works

A
  1. Single file cell
  2. The laser hits cells and light is scattered
  3. The light is picked up by detectors
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15
Q

What are the light sources of optics in flow cytometry?

A

Lasers

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16
Q

What lasers are used in flow cytometry?

A
  • The single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
  • Can provide from milliwatts to watts of light
  • Can be inexpensive, air-cooled units or expensive, water-cooled units
  • Provide coherent light (single frequency)
  • Generally, FC lasers are a single wavelength of light or a 488nm laser.
17
Q

Describe how a laser scatters light

A
  • The laser hits the cell at 488 nm.
  • The light is scattered in the forward direction which is proportional to the size of the cell
  • Side scatter; 90o Light Scatter proportional to the granularity or the internal complexity
18
Q

What does a white cell population dot plot show? When is a white cell population dot plot used?

A
  • X-axis: increase in forward scatter
  • Y-axis: increase in size scatter
    Every dot represents a cell forward scatter and side scatter shows a distinct correlation.
    This technique is used to quantitative different populations of cells. This is done on peripheral blood.
19
Q

When is a channel layout for laser-based flow cytometry used?

A

It is used for antibodies and fluorescence.

  • The laser hits cells and cells are labelled with (4) different antibodies and colours.
  • Light is emitted and picked up by photomultiplier tubes (PMT)
  • This happens after the light has gone through filters and mirrors. This is needed because of the emission change.
  • It converts analogue into digital
20
Q

Why are electronics needed?

A

To process signals from detectors

- Analog-Digital Conversion

21
Q

What is the Stokes shift?

A

The energy difference between the lowest energy peak of absorbance and the highest energy of emission.

22
Q

What is FITC?

A 
The most commonly emitted fluorochrome 
Fluorescein isothiocyanate (FITC) at the green energy peak
23
Q

How does fluorescence occur?

A

Fluorescence occurs when a fluorochrome is excited by a laser and goes back to the unexcited state. This is the difference between the two peaks is the stoke shift.

24
Q

What causes fluorescent energy peaks to overlap in a histogram?

A

Mirror and filters cause overlapping histograms

25
Q

Which common fluorochromes are used together to analyze 3 different parameters of the cell?

A
  • Fluorescein isothiocyanate (FITC) - Green dye: 520 nm
  • Phycoerythrin (PE) - Orange dye: 580 nm
  • Peridinin Chlorophyll Protein (PerCP) - Red dye: 620 nm
26
Q

Why are the 3 common fluorochromes detected?

A

They are emitted at different wavelengths and they can be detected at the same time. As well as this, add 3 different antibodies.

27
Q

Why are filters and mirrors used for the 3 fluorochromes?

A

It can be overlapped which is why filters and mirrors are used so that we are confident only the desired fluorochrome is picked up.

28
Q

What is the range of fluorochromes used?

A

A huge range of fluorochromes is available. It is important to carefully choose so there is no overlap of emission.

29
Q

What are the single cells that can be in suspension?

A
  • Peripheral blood
  • Bone marrow
  • Fine Needle Aspirate
  • CSF and other fluids
  • Fresh Tissue
    These things are already in a cell suspension -> however, the tissue has to be made into an SC suspension.
30
Q

What is the direct and indirect immunofluorescence of labeling?

A

Direct: Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes -> This is the simplest, cleanest way of doing it.

Indirect: Unconjugated MoAbs

31
Q

Describe how direct immunofluorescence is carried out

A
  • Only one step
  • Have the single-cell already, washed and added to flow cytometry
  • The fluorophore is attached directly to the antigen
32
Q

Describe how indirect immunofluorescence is carried out

A
  • Not directly labeled with fluorophore but has secondary antibodies will be
  • This is used when the antibody cannot or isn’t conjugated
  • This secondary antibody is attached to the primary antibody on the antigen
  • Mouse antibodies are used with FITC in it
33
Q

How is the digital data from a flow cytometry displayed on a computer?

A
  1. The light is attached to a digital signal which can be displayed on a computer.
  2. It can be displayed as a dot plot or a histogram.
  3. The dot plot has forward and side scatters with 2 parameters
  4. The histogram has 1 parameter with a Y-axis with no. of cells which could also be 2 fluorochromes
34
Q

What are the 4 populations of cells in a dot plot for 2 fluorochromes?

A
  • Single positive for FITC
  • Single positive for PE
  • Double positive for both
  • Double negative for both
35
Q

How is data from flow cytometry gated and analyzed?

A
  • Ways for manipulating data then displaying and analysing it.
  • Drawn a region around cells and can get the computer to display the desired cells based on fluorochromes

SEM 4:Cellular Pathology (13 decks)

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