An Introduction to Window Extension on Digital Flow Cytometers

The optical detectors of a flow cytometer convert all detected light into electronic signals that are subsequently converted into digital data. A sample pulse is the electronic representation of a signal with measurable height, width, and area values. In a BD digital system, the detector output is continuously digitized, and threshold defines the signal level at which the system begins to measure a pulse within the continuous data stream. The initial window gate (WG) is the time interval during which the input signal is greater than the threshold and during which the acquisition system looks to sample a pulse. To ensure that a pulse is completely integrated, additional time, called the window extension (WE), is added before and after the initial WG to extend the total integration time. Incorrect WE settings can affect proper data acquisition and lead to incorrect data measurements. BDTM Cytometer Setup and Tracking (CS&T) is a fully automated system unique to BD digital flow cytometers that provides instrument characterization, tracking, and quality control. CS&T is a module within BD FACSDivaTM software (v6.0 and later). CS&T characterization features automatically define the instrument baseline, set correct laser delays, and fully characterize the instrument. When setting up a new cytometer configuration in CS&T, the software automatically defaults to an instrument-specific WE. For example, on the BD FACSCantoTM II cytometer, the WE defaults to a value of 7 (3.5 μs before and after the initial WG). The operator can manually set the WE to an alternate value prior to baseline determination. The appropriate WE value for a given application is specific to that application (for example, particle size and dim populations). BD FACSCantoTM clinical software, used in conjunction with BD FACSTM 7-color setup beads, provides automated cytometer setup and quality control for in vitro diagnostic (IVD) use. This system automatically sets the WE to a value of 7, which the operator cannot change. Certain BD MultitestTM reagents are designed for use with this software system. Use of certain BD products on an instrument with different software that might have different settings, including but not limited to WE, might alter the results. In summary, the manipulation of WE in conjunction with the use of BD FACSDiva software’s CS&T performance checks allows an operator to optimize cytometer settings for a given application. However, certain BD products must be used with a WE of 7. If an operator chooses to perform these tests using a different WE (or other setting), the application must be fully validated prior to use.