At the beginning of each day each flow cytometer is tested to ensure it is operating within specifications so that it will provide reproducible results. The QC program is designed to detect and monitor drift in the performance of each flow cytometer using Levy-Jennings plots. These plots provide a 'snap-shot' of instrument status on a daily basis, as well as a platform for tracking instrument performance over time. Stability in the QC data indicates that the data produced by the instrument are not the result of instrument artefact. Instability indicates the need for an imminent service visit.
Spherotech 8-peak rainbow beads (Cat# RCP-30-5A) are run on each flow cytometer to calibrate the voltage settings and to determine the daily variance of each fluorescent channel. Beads are stained with multiple stable fluorescent dyes whose emission spectra are compatible with many commonly used fluorochromes for flow cytometry. The beads are excited by light between 365 nm and 650 nm to yield emission in all fluorescent channels. Each PMT is adjusted so the mean fluorescence intensity of the brightest bead peak falls within a given narrow range. The beads are used daily to determine the daily variance of each fluorescent channel. The voltage (gain) changes are compared to previous calibrations and plotted on a Levy-Jennings graph to determine if changes have become significant. The MACSQuant calibration beads are 2 and 3 μm in size and also enable the calibration of the voltage settings of the MACSQuant Analyzer.
For the analog machines BD Calibrite 3 Beads (Cat# 340486) are run daily using BD Comp software. BD Calibrite APC beads (Cat# 340487) are included for the analogs with a red laser. The beads are used to adjust instrument settings, set fluorescence compensation, and check instrument sensitivity.
The BD DNA QC Particle kit (Cat# 349523) is run weekly to ensure sensitivity and resolution for DNA analysis.
The date of each control bead run is plotted on the x-axis. The voltage for each date is plotted on the y-axis. The distance from the mean is measured in standard deviations (SD). A red dotted line runs across the graph at the mean, and the area defining one standard deviation on either side of the mean is colored yellow, while that defining two standard deviations on either side of the mean is colored blue. This makes it easy to see how far off the result was. When data are outside two standard deviations on either side of the mean, remedial measures are taken and the fix is noted on the graph alongside the date. If the deviation from the mean is more than 25 volts, a service visit is considered.
Levy-Jennings plots for each BD flow cytometer with data for the last 2 months can be downloaded here. Levy-Jennings plots for the MACSQuant flow cytometer with data for the last 2 months can be downloaded here.
The Figure shows a Levy-Jennings plot for each PMT of the FACSCalibur using rainbow beads as the calibrator: