Eliminating Interference from Autofluorescence in Flow Cytometry - 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.
