Flow cytometry is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as they pass through at least one laser.
Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths.
The fluorescence can then be measured to determine the amount and type of cells present in a sample.
Up to thousands of particles per second can be analysed as they pass through the liquid stream.
For flow cytometric analysis, a single cell suspension yields multiparameter data that correspond to Forward Light Scatter (FLS), 90° Light Scatter (90LS), and FL1-FLn, which helps to sort and classify the cells.
The analysis is performed using the software in a computer attached to the flow cytometer.
Some of the processes involved in analysing the data in flow cytometry are described below:
- Gating – The data from the flow cytometers is plotted in a single dimension to make a histogram. The data can also be presented in two or even three dimensions. The plots can then be divided into regions depending on the intensity of the fluorescence, to create a series of subset extractions called gates. These gates can be used in hematology for diagnostic or clinical purposes.
- The data is usually plotted on logarithmic scales. Since there is an overlap in the emission spectra of the different fluorescent dyes, the detectors need to be compensated computationally as well as electronically.
- The collected data is then analyzed using the software in the attached computer which may be WinMDI(depricated), Flowjo, or CellQuest Pro, for example.
- After the data have been collected, the flow cytometer no longer needs to be connected to the computer and analysis can be performed using a separate computer.
Reviewed by Sally Robertson, BSc