Q and B values are critical measurements required for inter-instrument standardization and development of multicolor...

Much of the complexity of multicolor flow cytometry experiments lies within the development of antibody staining panels and the standardization of instruments. In this article, we propose a theoretical metric and describe how measurements of sensitivity and resolution can be used to predict the success of panels, and ensure that performance across instruments is standardized (i.e., inter-instrument standardization). Sensitivity can be determined by summing two major contributors of background, background originating from the instrument (optical noise and electronic noise) and background due to the experimental conditions (i.e., Raman scatter, and spillover spreading arising from other fluorochromes in the panel). The former we define as Bcal and the latter we define as Bsos . The combination of instrument and experiment background is defined as Btot . Importantly, the Btot will affect the degree of panel separation, therefore the greater the degree of Btot the lower the separation potential. In contrast, resolution is a measure of separation between populations. Resolution is directly proportional to the number of photoelectrons generated per molecule of excited fluorochrome and is known as the "Q" value. Q and Btot values can be used to define the performance of each detector on an instrument and together they can be used to calculate a separation index. Hence, detectors with known Q and Btot values can be used to evaluate panel success based on the detector specific separation index. However, the current technologies do not enable measurements of Q and Btot values for all parameters, but new technology to allow these measurements will likely be introduced in the near future. Nonetheless, Q and Btot measurements can aid in panel development, and reveal sources of instrument-to-instrument variation in panel performance. In addition, Q and B values can form the basis for a comprehensive and versatile quality assurance program.