To fret over FRET: Unintended fluorescence energy transfer artifacts and their implications for flow cytometry panel design and interpretation as highlighted by TRBC1/2 assays.

Förster resonance energy transfer (FRET) serves as the fundamental mechanism underlying a wide array of technologies employed in biomedical research and clinical diagnostic applications. However, unintended FRET is often overlooked, despite its potential to introduce assay artifacts that may lead to misinterpretation. Here, we examine the impact of unwanted FRET on clinical flow cytometric testing, focusing on the T-cell assay incorporating TRBC1 and TRBC2, which has emerged as a powerful method for assessing T-cell clonality. We illustrated the effect using a representative case of spurious antigen expression associated with false TRBC restriction and demonstrate through control experiments that this artifact arises when target antigens in close proximity are labeled with fluorochromes having overlapping spectra. We quantified the spectral overlap integral J(λ), and generated a matrix for commonly used fluorochromes, providing a practical tool to identify fluorochrome pairs with a high FRET propensity that should be avoided when labeling closely situated antigens. Overall, recognizing the potential effects of unintended FRET and implementing strategies to prevent or minimize its impact are essential for ensuring accurate interpretation of clinical flow cytometric assays.