Eliminating Interference from Autofluorescence in Flow Cytometry

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PROJECT SUMMARY/ABSTRACT Flow cytometry (FC) is a powerful cell analysis tool relying on fluorescently labeling of cells. It can provide information on cell count, shape, size, DNA content, redox state, membrane permeability, and surface receptors, among other features. FC is an essential tool in research and clinical fields for tumor biology research, cancer and AIDS diagnostics, stem cell therapy, immunophenotyping, and cancer immunotherapy. One of the leading limitations of FC is that exogenously added fluorophores are not the only source of fluorescence in a sample. Autofluorescence (AF) can be, in some circumstances, a significant contributor to the signal present in each of the detection channels, often overlapping with the emission spectra. AF limits assay sensitivity and causes problems in data interpretation, which can lead to erroneous scientific conclusions. While several approaches have been employed to eliminate AF, there are no satisfactory solutions available. Kinetic River has been developing novel methods utilizing fluorescence lifetime (FLT) as an intrinsic discriminating parameter and resolving multi-exponential lifetime decays in flow cytometry. In our completed Phase I SBIR, we successfully demonstrated the automated removal of cellular AF interference in fluorophore- labeled human cells using time-resolved emission lifetimes using a 4-color system, establishing feasibility of the method. This Phase II grant will advance these findings towards commercialization by expanding the system into a fully operational 16-color Colorado analyzer with real-time data processing. The successful completion of the Colorado analyzer will lead to the first solution that automatically eliminates interference from AF in flow cytometer experiments. It will become a game-changing platform enabling cell biologists, immunologists, oncology researchers, diagnostic companies, and other FC users to obtain more accurate and quantitative results. Removal of cellular AF interference will yield enhanced sensitivity, which is important for detecting rare surface markers. This added capability of the Colorado platform will provide it with a significant competitive advantage over current commercial FC analyzers of similar multiplexing capabilities. This will enable the Colorado to replace current mid-level multiplexing instruments, which currently occupy a large swath of the rapidly growing $4.8 billion FC market.