Flow cytometry involves the use of a machine to detect and sort cells based on certain parameters, including size, granularity, and fluorescence.
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Modern flow cytometers are able to measure up to 14 parameters simultaneously, and multi-color analysis means that it is possible to reduce the amount of sample and antibodies needed. Optical and fluorescence properties of cells and particles ranging from nuclei, micro-organisms to eukaryotic cells can be measured, aided by the use of dyes and antibodies.
Flow cytometry can be further divided into “sorting” and “non-sorting”; non-sorting types give out the light scattering/fluorescence readings from a cell while sorting types will take this further by sorting these cells based on the parameters. This can be used to sort a mixed sample and is known as “Fluorescence Activated Cell Sorting” (FACS).
How does a flow cytometer work?
Flow cytometers can be divided into several parts; fluidics, optics, electronic network (detector) and a computer.
- The fluidic part directs liquid to the optics part, ensuring that only one cell goes through.
- The optics include the “excitation” and “collection” optics. The excitation optics is a light source, where either light is shone onto the cell or used to activate emission from the cell. The collection optics either transmits how the excitation light became scattered, or how much fluorescence is emitted by the cell and then sends this information to the electronic network.
- The electronic network converts the information from the optics to digital data proportional to light intensity. The computer is also used in this analysis.
In FACS, it is possible to choose which cells to separate by selecting for specific fluorescent properties. These cells are now “tagged”, and when fluid droplets containing these cells go past the detector, they get charged by a charging electrode. Once charged, these droplets can then be deflected away from the main fluid stream and thus separated.
How is flow cytometry used in the clinic?
There are various ways in which flow cytometry is used for clinical applications;
This is probably the well-known application of flow cytometry. Using antibodies for specific markers on cells can allow a group of cells to be detected. Indeed, by using three antibodies it is possible to differentiate the different types of white blood cells.
This can be taken further, such as classifying leukemia and lymphoma, diagnosing primary immunodeficiency and paroxysmal nocturnal hemoglobinuria, and monitoring the immune status of patients with HIV or on immunosuppression therapy.
Phagocytosis and oxidative bursts are functions of neutrophils, a type of white blood cell. Defects in these processes are known hallmarks of chronic granulomatous disease. Neutrophils can be exposed to fluorescently labeled bacteria, and phagocytosis checked by flow cytometry to see how much of this fluorescence has been internalized by the neutrophils. Neutrophils can also be loaded with dyes which become fluorescent once oxidized, which can then be detected by flow cytometry.
Can flow cytometry be used in microbiology?
Most traditional microbiology techniques require the culturing of the micro-organism, such as bacteria. This can have the unwanted consequence of requiring a long time before a result is available. Flow cytometry, however, allows bacteria to be labeled and detected.
Since flow cytometry has the capability to detect a specific cell in a mixed sample, it may be applied for the detection of infecting pathogens.