Q and B values are critical measurements required for inter‐instrument standardization and development of multicolor flow cytometry staining panels

Abstract

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 B cal and the latter we define as B sos . The combination of instrument and experiment background is defined as B tot . Importantly, the B tot will affect the degree of panel separation, therefore the greater the degree of B tot 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 B tot 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 B tot 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 B tot values for all parameters, but new technology to allow these measurements will likely be introduced in the near future. Nonetheless, Q and B tot 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. Published 2014 Wiley Periodicals Inc. Key termsinter-instrument quality control and standardization; Q and B values; panel development; panel success; panel design POLYCHROMATIC flow cytometry provides a powerful means to dissect cellular characteristics and function (1). The technology is limited, however, by the complexity of experimental design. Cell surface and intracellular proteins must be detected using carefully titrated antibodies conjugated to fluorescent dyes. The optimal combination of these antibody-fluorochrome conjugates must be determined empirically through iterative experiments (2). This process typically requires months of experimentation, while only limited guidance is available. Some approaches focus on the spectral overlap of fluorochromes but these approaches are flawed. Cell populations are often easily discriminated even when spectral overlap (compensation) is high

Determination of background, signal‐to‐noise, and dynamic range of a flow cytometer: A novel practical method for instrument characterization and standardization

“…(8) and (9). Isolated PBMC were resuspended in 13 PBS/0.5% bovine serum albumin (BSA, Biochrom Ag, Berlin, Germany/Sigma-Aldrich Chemie GmbH, Steinheim, Germany).…”

Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi‐level bead data

“…High Q values are useful in improving the quality of cytometer measurements both in relation to single channel signal versus background (illustrated in (3) Figres 5 and 7 and in (1) Figures 7, 8 and 9) and in spectral overlap correction (compensation) where high Q values contribute to improved discrimination of signals-of-interest relative to the measurement variance effects of spectral overlaps ((4), including calculations in the Appendix). …”

High‐Dimensional Fluorescence Cytometry

“…In summary, SE is where the positive signal of one fluorophore 'spreads' into a non-target detector, after compensation, due to measurement errors in photon counting (Nguyen et al, 2013;Perfetto & Roederer, 2007;Perfetto et al, 2014). SE leads to a loss of resolution in affected detectors.…”