How to Analyze Flow Cytometry Data

Flow cytometry is the process of analyzing and counting cells and particles using the theory of light scattering and light excitation. The cells are forced through a sheath one at a time and pass through the beam of a laser. The laser excites the chemicals within the cells. Different types of cells emit different types of light. Optical detectors or lenses pick up and measure the light, transferring the light into a data stream. By analyzing the data, technicians can figure the distribution of cells and help doctor's and researchers make decisions regarding treatment.

Instructions

• 1

  Set up the parameters of the cytometer. Every cytometer has a computer component that accepts the light emitted from the cells and converts the amount and quality of the light to accessible data. Because there are different types of cells, there is a large amount of data created. Setting the parameters of the data needed slows down the flow of information to a manageable level. Some options include setting the voltage level, adjusting the laser pulse parameters or setting the light threshold.

• 2

  Choose the filters on each optical lens. Filters that cut out specific bandwidth for each lens allow the cytometer to recognize the different chemical properties of each cell. It also allows the cytometer to recognize whether the light is reflected straight ahead, as a forward scatter (FS) or if it is reflected at a 90-degree angle in a side scatter (SS.)

• 3

  Choose the logarithmic or linear acquisition. Linear acquisition is the raw data that is generated by the cytometer. It shows the type of cell, or the chemical makeup of the cells that flow through the laser beam. The Logarithmic function goes one step further. It uses a mathematical function to project additional cells that may not be detectable by the cytometer. For example, cells may show a chemical compound that is a by-product of a viral infection. Even though the virus may be too small to be detected by the light stream, the logarithmic function will hypothesize the presence of the virus based on the chemical makeup of the cells.

• 4

  Select one or two parameter histograms. These are types of graphs used to show the data collected by the cytometer. A one parameter histogram is appropriate if the data is all plotted on a "positive" axis, meaning that all of the light is reflected and measured. An easy way to think of this is to imagine a bar graph, with levels at the left and bottom of the frame. The information is plotted between these two parameters. But for those cells that absorb differing amounts of light, a two parameter histogram is used. This allows the data to be plotted on both a positive and negative axis.