Autofluorescence (AF) diminishes signal resolution of fluorescence-labelled cells, masking rare or lowly-expressed antigens in immunophenotyping experiments. Here we incorporate AF into current fluorescence sensitivity equations to demonstrate its major role in signal resolution via mathematical modeling and empirical data. Operational brightness for 26 commonly used fluorochromes was calculated via the stain index (SI) of human CD4 on fixed and fresh lymphocytes. These samples exhibited differences in AF intensity and emission profiles and were used to mimic intrinsic AF changes on cells sharing consistent numbers of antigens and bound fluorochromes, allowing us to confirm the equations and to directly extrapolate our findings to samples with differing levels of cellular AF.
Fluorochrome ‘operational brightness’ strictly depends on the AF levels of the carrier, differing significantly between fresh and fixed (SIFixed < SIFresh). Changes in SI between samples reveals a re-ordering of fluorochrome brightness rankings. Red-IR emitting fluorochrome rankings increase, while blue-yellow-emitting fluorochromes decrease on fixed cells. The re-ordering of brightness correlates with changes in background AF of fixed lymphocytes that have enhanced MFI peaks in UV, violet and blue laser-excited detectors.
Further we demonstrate that full-spectral unmixing with AF extraction (U-AF) dramatically improves signal resolution of AF-affected fluorochromes, resulting from an increase in the MFI ratio between CD4+ and CD4- lymphocytes. However, U-AF increases the CV around negative populations, so the benefits of U-AF are a balance of two opposite effects: the gains in separation vs. increase of spreading, thus indiscriminate use of U-AF can be detrimental for low AF cases.
Finally, the AF spectral similarity of cellular subtypes to some fluorochromes can lead to unanticipated errors after U-AF. Thus, where U-AF is mandatory, intelligent panel design will avoid fluorochromes in regions of conflict. Collectively, we demonstrate the importance of considering cellular AF during polychromatic panel design to achieve optimal antigen resolution.