Acquisition Rates Effect on Multiparametric Rare Event Analysis: a Look at Data Consistency

摘要

The current paradigm in Flow Cytometry is the increasing number of laser lines, multiparameter instrument capabilities, and increasing number of antibodies; flow cytometric applications have become more complex and with greater variability. One of the major challenges pertaining to instrumentation is dealing with reliability of the optics and the laser lines. It is of extreme importance to be able to shape the laser beams elliptically, focus them to the cellular interrogation point, and not lose laser power during this process. In conjunction with lasers and optics, the fluidics system plays an integral part in obtaining the best data possible from the cellular preparation. Event rate, determined by amount of pressure applied to the sample, rate of fluid through the system, and cellular concentration, is the driving force behind obtaining reproducible and reliable data. As cellular concentrations vary, so do the event rates. As event rate increases the amount of time a cell spends at the interrogation point is decreased. Previously, this would mean an overall decrease in data reproducibility and accuracy. However, with advances in optical alignment and fluidic (hydrodynamic) focusing, even rare event analysis can be obtained at varying speeds without compromising data efficiency. Instrumentation is designed to handle data acquisition at variable speeds without loss of signal intensity or percentage of cells defined. This is a crucial aspect in obtaining quality results and maintaining a level of standardization between instruments.