Flow cytometry: principles and instrumentation.

The standard benchtop flow cytometer is very similar to a hematology cell counter. In fact, flow cytometers can trace their origins back to the early hematology counters. Unlike their earlier counterparts that used electronic impedance to measure particles in a fluid stream, today’s modern flow cytometers use an illuminating light source usually consisting of a laser or arc lamp. The majority of instrument manufacturers employ an air-cooled argon gas laser emitting a monochromatic beam of light fixed at 488 nm at 15 mW of power. As particles or cells flow in single file past the intersection of the light beam, light is scattered in various directions. If there a fluorochrome labeled monoclonal antibody associated with the cell, it becomes excited by the laser and a fluorescent emission results. The resulting signals are processed to gather information about the relative size of the cell (forward light scatterFSC), its shape or internal complexity (side light scatterSSC) as well as a diversity of cellular structures and antigens (fluorescence). The cytometer itself is set up and monitored routinely with a quality control program utilizing a series of unlabeled and fluorescently labeled calibration particles as a reference check on instrument alignment and sensitivity performance. Therefore, every cytometric procedure performed is a reflection of the quality of the instrument, sample preparation, and the operator, and ultimately reflects on the institution.