Background:
Software to track flow cytometric instrument performance is becoming increasingly widespread and is now commonly used in industrial, clinical and research fields. Performance tracking requires a characterisation of the instrument, generally expressed by how efficient each detector is at measuring fluorescence emissions (Q), and what the background contributions are to those measurements (B). Once the instrument has been characterised, variations from those initial “baseline” values can be recorded and compared over time in order to ensure some level of reproducibility to the results that are obtained. To meet this need, various software solutions (CS&T, QbSure etc) have been developed that are based on the founding works of Hoffman and Chase 1,2, with small modifications aimed at improving accuracy, a necessity due to hardware related discrepancies that exist between instruments.
Method:
Here we describe a system that combines optics and stream alignment using small picomotors, with performance tracking using commercially available CS&T beads. The system is implemented on a BD Influx flow cytometer. The software interacts with the picomotors (NewPort 8301 picomotor actuators) mounted on the optics and focussing translators of the Influx. The system then uses the data generated from the CS&T beads to determine median, rCV and rSD values for each PMT and attempts a semi-automated optimisation of the optics and stream alignment using a combination of live video feedback with live data streaming from the cytometer. The process is iterative and aims to obtain and then maintain the optimal stream position.
Results and Conclusions:
Typical output generated by the system when sub-optimally and optimally auto-aligned will be presented and data showing typical and expected tracking performance will be demonstrated. This system improves traditional jet-in-air cell sorter alignment and then provides some metrics for on-going quality assurance.