Spectral, Semi‐Spectral, and Bandwidth Flow Cytometric Implementation of Multiplex Cytokine Bead Assays

Multiplex cytokine bead assays (MCBA) have previously been carried out on conventional (aka classical, or bandwidth) flow cytometers, and issues involving unmixing distortions were encountered when using the manufacturer's set-up beads for unmixing full-spectrum data. After resolving such issues, we introduce and characterize the first full-spectrum flow cytometry implementation of MCBAs. In more detail, the set-up beads were originally produced for conventional cytometers, and a temporary work-around was issued for the semi-spectral mode only: spectral acquisition but bandwidth data display and analysis. We proceeded to make our own reference fluorophore controls (RFCs), starting with polystyrene beads derivatized with carboxyl functional groups that were first conjugated with streptavidin and then titrated with PE-biotin. The autofluorescence profile of the in-house conjugated PE beads matched that of the kit beads, and proper use of PE and "APC" RFCs enabled normal spectral unmixing without any distortions. Comparison of the three modes, namely spectral, semi-spectral, and bandwidth, illustrated the advantages of the spectral mode, including precision and resolution. In addition to enabling proper spectral implementation of the MCBA, a full-spectrum approach opens the door for various types of more complex multi-analyte bead assays yielding richer information, which have previously remained out of reach in conventional cytometry.