Incorrect Fluorescence Compensation and Spectral Overlap (Flow Cytometry (Troubleshooting))

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Flow cytometry multicolor panels show false-positive signals in channels not expected to be positive. Positive populations appear in multiple fluorescence channels due to spectral overlap between fluorochromes, making accurate population identification impossible. Common causes: 1) Fluorescence compensation not performed or set up incorrectly to correct for spectral overlap between fluorochromes 2) Single-color controls not properly prepared, gated, or used for compensation matrix setup 3) Mismatch between bright fluorochromes and low-expressing antigens, or dim fluorochromes paired with high-density antigens in multicolor panels Solutions: 1) Set up positive single-color controls correctly on flow cytometer; ensure controls are properly gated to capture all events; perform compensation using single-stained samples for each fluorochrome 2) Use compensation beads as alternative to cells for creating compensation matrix, especially when cell-based controls are difficult to prepare 3) Consult published data for antigen density in specific cell types; pair highly fluorescent dyes with low-expression antigens; use flow cytometry panel builder tools to match fluorochromes with antigens based on expression levels