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

Flow Cytometry - Introduction Flashcards

(35 cards)

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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?

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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

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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?

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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?

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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?

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A

To process signals from detectors

- Analog-Digital Conversion

21
Q

What is the Stokes shift?

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A

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

22
Q

What is FITC?

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

How does fluorescence occur?

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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?

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A

Mirror and filters cause overlapping histograms

25
Which common fluorochromes are used together to analyze 3 different parameters of the cell?
- Fluorescein isothiocyanate (FITC) - Green dye: 520 nm - Phycoerythrin (PE) - Orange dye: 580 nm - Peridinin Chlorophyll Protein (PerCP) - Red dye: 620 nm
26
Why are the 3 common fluorochromes detected?
They are emitted at different wavelengths and they can be detected at the same time. As well as this, add 3 different antibodies.
27
Why are filters and mirrors used for the 3 fluorochromes?
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
What is the range of fluorochromes used?
A huge range of fluorochromes is available. It is important to carefully choose so there is no overlap of emission.
29
What are the single cells that can be in suspension?
- 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
What is the direct and indirect immunofluorescence of labeling?
Direct: Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes -> This is the simplest, cleanest way of doing it. Indirect: Unconjugated MoAbs
31
Describe how direct immunofluorescence is carried out
- Only one step - Have the single-cell already, washed and added to flow cytometry - The fluorophore is attached directly to the antigen
32
Describe how indirect immunofluorescence is carried out
- 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
How is the digital data from a flow cytometry displayed on a computer?
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
What are the 4 populations of cells in a dot plot for 2 fluorochromes?
- Single positive for FITC - Single positive for PE - Double positive for both - Double negative for both
35
How is data from flow cytometry gated and analyzed?
- 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
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