Flow Cytometry

Objectives
Activities
Discussion
Principles of Flow Cytometry
Cell Sorting
Glossary
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Objectives: top

When you finish this workstation, you will be able to:

1. Learn the basic principles of operation of a flow cytometer, i.e. how its components work together to sort cells by their various characteristics.

2. Be able to list three general uses or applications of flow cytometry results.

3. Examine scatter and fluorescence dot plots and point out different populations of cells based on their positivity or negativity for certain cell characteristics.

Activities: top

1. On the computer, run an animated representation of flow cytometry at work.

2. Examine scatter and fluorescence dot plots that show the positive and negative parameters for the quadrants from which the cells are sorted.

3. Observe each of three flow cytometers as each does a different type of cell sorting and/or analysis.

· General cell sorting
· Post-sort analysis of sorted cells with gene expression.
· DNA analysis.

4. Given scatter and fluorescence dot plots from one of the instruments and a description of the desired characteristics of a cell population, point out where that population of cells is located on the plot.

Discussion: top

Flow cytometry developed over the last 35 years as a tool which could rapidly measure complicated physical and biological properties of large numbers of individual cells in a very short time. It was realized quickly that the most efficient way to achieve that was to prepare cells in a suspension and to slowly introduce this suspension into a fast flowing stream of fluid which would then surround the cells like a “sheath” and “center” the cells in the middle of the flowing sheath. This principle of “sheath-flow” is what is widely used in flow cytometry to precisely align cells in front of a beam of laser light. This approach allows hundreds or even thousands of cells per second to pass through the point of intersection between the sheath and laser light.

Principles of Flow Cytometry top

An important requirement for flow cytometry is the need to specifically label cell constituents with fluorescent molecules, which are then used to identify cells carrying this “label”. Cell constituents can be any of a number of cellular components including DNA, which can be labeled by different dyes. Unique “markers” or proteins (Antigens) on the cell surface can be labeled with monoclonal antibodies (mAb) conjugated with one of many fluorescent dyes (fluorochromes) such as fluorescein iso-thiocyanate (FITC) or phycoerythrin (PE). Using these labels, it then becomes possible to identify “positive” and “negative” cells.

As cells pass one by one through the measuring region (where the laser light intersects the sheath), each cell carrying a label produces a short flash of fluorescence, the intensity of which is directly proportional to the number of “copies” of the labeled constituent present in that cell. These flashes of fluorescence are then collected by a series of optics chosen to harness the type of fluorescence produced by the label (different fluorescent labels produce fluorescences of different wavelengths) and to focus it on a sensitive detector (Figure 1). The detector, called a photo-multiplier tube (PMT), transforms flashes of light into electric pulses, which are recorded by electronic converters and transferred to a computer for assimilation and interpretation (Figure 1). Therefore a cell carrying one or more copies of the constituent of interest is recorded as “positive” while one that does not have any copy of this constituent is identified as “negative”. A series of mirrors, filters, and other optical devices are used in a “train” assembly to move and direct the light between different PMTs (Figure 1).

It is important to point out that it is possible to label cells with more than one label at a time. If we simultaneously measure two cellular constituents, then the possible combinations of all the integrated measurements are “++”, “+-“, “-+”, and “--“. An example of how these 4 possible combinations are displayed in a dot plot is shown in Figure 2. Research and development laboratories specializing in the development of flow cytometers and flow cytometric techniques have been able to measure cells labeled with up to 12 different labels and to correlate and assimilate these signals to identify countless groups of cells with different and unique label combinations.

While measuring signals resulting from the flashes of fluorescence generated by the different fluorochromes, a flow cytometer can also measure two other important criteria of each cell passing within the measuring region. These measurements are cell size and cell density. Incident laser light intersecting a cell will be scattered forward in front of a detector (Figure 1) measuring the degree of light scattering. The degree of forward scattering is directly related to the diameter of the cell and will therefore represent the relative size of each cell in the population of cells being analyzed. Incident light can also be scattered at a right angle to the light beam hence known as right angle light scatter (Figure 1). Scattering of light at a right angle results from the presence of granules or organelles inside the cell. The “denser” a cell is, the more right angle light scattering it produces, and this measurement therefore reflects the relative density of each cell in a population of cells. Size and density are very important criteria used extensively in hematology to distinguish between different lineages of blood cells.

Cell Sorting top

Perhaps the most valuable property of flow cytometry is the ability of certain flow cytometers to separate individual cells as a function of different physical and biological characteristics of cells being analyzed. This is referred to as flow cytometric cell sorting. Principles of cell sorting are shown in a simplified form in Figure 3. The sheath carrying the cells in its center is “broken” into thousands of droplets per second using a droplet-forming transducer (piezoelectric crystal). Each cell injected into the sheath flow becomes contained in one droplet, but not every droplet contains a cell. When a droplet containing a desired cell produces the right signals, the photodetector, which receives these signals, registers that droplet as a wanted “event”. This triggers the electronics of the sorter to deliver a charging pulse to that droplet. As the droplet (which now carries a charge) passes down past a copper plate carrying an opposite charge, it is deflected from the center stream towards the plate and is deposited in a collector placed underneath the deflection plate. This process is repeated between few hundreds and few thousands of times every second. The net result is that a group of cells present at a very small percentage (less than 1%) in the group of cells being analyzed is “plucked” out, one cell at a time, to a purity of more than 95%. Such a process has become very common in experimental hematology where hematopoietic stem and progenitor cells, which are present in bone marrow or other hematopoietic tissues at a very low frequency, are isolated in relatively large numbers and high purity for research purposes. In addition, in a very limited fashion, cell sorting, on specialized high-speed sorters, has been used for clinical transplantation of stem cells in leukemia patients.

Adapted from Flow Cytometry and Sorting, second edition, M.R. Melamed, T. Lindmo, M.L. Mendelsohn, Editors. Wiley-Liss, New York. 1991

Figure 1 was reproduced from same source.

Figure 1: Schematic representation of a classical laser-based flow cytometer depicting the major components for cell flow, laser excitation, and measurement of fluorescence and light scattering. Different types of detectors are usually employed to measure forward and right angle or side scatter. Fluorescence is measured by PMTs equipped with appropriate optics that can select the desired fluorescence signal. In this configuration, 4 parameters can be measured, two physical (forward and right angle scatter) and two fluorescence signals shown in this example as green and red fluorescence.

Figure 2:
Left Panel:
A representative light scatter dot plot showing right angle light scatter (OrthSc) on the X-axis and forward light scatter (ForSc) on the Y-axis. Display of these two physical parameters plots density and size of analyzed cells on the X- and Y-axis, respectively. Notice that this defines two major groups of cells. One group consists of small less dense cells (bottom left), which contains the majority of lymphocytes. The second group is made up predominantly of larger and denser granulocytes and is therefore to the right and above lymphocytes.

Right Panel:
A representative dot plot showing events (cells) analyzed for two fluorescence signals. Each signal is displayed on one axis. In this example, CD4 (a subset of T cells) is depicted on the X-axis while another subset of T cells, CD8 cells are shown on the Y-axis. Vertical and horizontal cursors are used to identify areas of negative and positive fluorescence. Above the horizontal and to the right of the vertical cursors are areas of positivity. Therefore cells in the upper right hand corner are positive for both signals (++ events). Cells in the lower right are “+-“ while cells in the upper left are “-+”. All cells in the lower left are negative for both signals and are “--“.

Glossary: top

Dot plot: The display of events in a two dimensional figure where every dot is placed in the two dimensional space in a position corresponding to the relative values of both parameters measured on the X and Y axes of the dot plot.

Event: A cell or particle that is registered or recognized by the detectors.

Fluorescence: Light emission by a fluorochrome after receiving excitation light from a laser.

Fluorescent molecules or fluorescent dyes (fluorochromes): Molecules such as fluorescein iso-thiocyanate (FITC) or phycoerythrin (PE) that can absorb light of a specific wavelength and emit light (usually of a higher wavelength). Light emission from these molecules can be visualized by flow cytometry to identify the presence of certain molecules to which these fluorochromes are attached.

Forward light scatter: Light that is scattered in the same plane as the incident laser light, which is proportional to the size of a cell and can therefore be used to measure cell size indirectly.

LASER: Light Amplification by Stimulated Emission of Radiation. Lasers are the source of light used to excite fluorochromes in flow cytometry.

Parameter: Refers to a specific measurement made for one or a group of cells such as fluorescence or side scatter.

Photo-multiplier tube (PMT): Detectors used to detect the fluorescence signal emitted by fluorochromes.

Right angle light or side scatter: Light that is scattered in a plane perpendicular to the incident laser light which is proportional to the degree of granularity or complexity of a cell and can therefore be used to measure cell granularity or “density” indirectly.

Sheath-flow: The flow of cells in the middle of a string (sheath) of buffer whereby all the cells flow in a single line through the middle of the sheath surrounded by the buffer.

Sorting: The physical act of separating one or two cell types from within a large population of cells to achieve a purity in the sorted cells far exceeding that present in the initial cell population.

Vertical and horizontal cursors: Demarcation lines placed in a dot plot to differentiate between events scored positive or negative for the parameters measured on the X and Y axes, respectively.

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