Advancing Compensation into the Future with the Next Generation of Comp Beads

Abstract

Abstract A need for more robust compensation techniques comes with continued growth in the study of immunotherapy and immunology by flow cytometry. Compensation is a method used to address the measurement of a fluorophore in multiple detectors (referred to as spillover) by mathematically correcting signal overlap between emission spectra of different fluorochromes to determine the true level of staining/positive target signal. The signal spillover can impact data quality and becomes very important with different levels of marker expressions. This measurement can be achieved by use of cells, or more typically by use of compensation beads to save precious biological samples. Current compensation beads have been shown to be problematic and can lead to over- or under-compensation, resulting in less or more true positive signal, respectively. Additionally, current compensation beads are incompatible with far red violet and ultraviolet dyes and may not be ideal for dim/low expressing markers. These studies address compensation by comparing various compensation products to biological samples on multiple immunophenotyping panels of either human or murine origin. These panels compare the use of a wide range of fluorophore conjugates, including the use of violet and ultraviolet excitable dyes, low expressing targets, and intracellular targets to determine compensation accuracy. The results demonstrate that a precise compensation matrix can be obtained with a newly improved commercial bead that is comparable to a cell sample. This data validates that precious cell samples may not be needed to be used in place of commercial bead products to obtain accurate compensation and quality data regardless of a panel’s design elements.