SBIR-STTR Award

Flow cytometry is a powerful cell analysis tool, providing information on cell count, shape, size, DNA content, redox state, membrane permeability, and surface receptors, among other features. It can be used on both live and fixed cells; and can be used to non-destructively sort cells based on these characteristics. Flow cytometry has utility in research and clinical fields as diverse and important as tumor biology research, cancer and AIDS diagnostics, stem cell therapy, immunophenotyping, and cancer immunotherapy. Flow cytometry relies extensively on fluorescent labeling cells; with the emission light collected from the interrogation region divided spectrally by filters and beam splitters, and directed to detectors that measure intensity. One common problem derives from the fact that the exogenously added fluorophores are not the only source of fluorescence in the sample. So-called cellular autofluorescence can be, in some circumstances, a major contributor to the signal present in each of the detection channels. A number of solutions have been proposed, but they often involve complex experimental protocols, or complex processing of data. This leaves the field wanting for a solution to the nagging problem of interference from cellular autofluorescence. Here we propose to use fluorescence lifetime as a second discriminating parameter in order to distinguish autofluorescence interference from the desired signal. We propose to build a simple system using a single pulsed laser, which will be sufficient to demonstrate feasibility of our method. The system will include a high- speed pulsed 375-nm laser capable of 0.5-ns pulses and the required fast electronics and 3 emission channels, which span the range of the broad cellular autofluorescence. We will leverage the deep expertise of Kinetic River in designing and building complex, innovative cytometry instruments; implementing novel hardware solutions to enable lifetime measurements in flow cytometry; and innovative, proprietary, and patent-protected techniques for rapid analysis of complex lifetime decay curves. We will be assisted by consultants and collaborators with deep experience in fluorescence lifetime measurement, autofluorescence signals, cell-based assays and algorithm development. To demonstrate the feasibility of our approach, we are pursuing the following strategy: (1) build prototype instrument and optimize system hardware; (2) characterize instrument performance; and (3) validate using relevant biological samples. Successful completion of this work will pave the way for further development in Phase II, which will see the addition of other laser lines with lifetime capabilities to create a general purpose instrument. This instrument will be similar in cost to similarly equipped cytometers, but will have the advantage of automated elimination of cellular autofluorescence. Such an instrument will have wide-ranging utility, increased ease of use, and will allow for clearer interpretation of experimental results.

Public Health Relevance Statement:
NARRATIVE Cellular autofluorescence is ever-present in both live and fixed cells. In flow cytometry, this can interfere with the desired signal and cause loss of sensitivity or erroneous results. We are developing a method, based on fluorescence lifetime, that can automatically eliminate autofluorescence from standard flow cytometry assays.

Project Terms:
Achievement; Achievement Attainment; Acquired Immunodeficiency Syndrome; Acquired Immunologic Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency; AIDS; Affect; Algorithms; Biological Assay; Biologic Assays; Bioassay; Assay; Biological Sciences; Life Sciences; Bioscience; Biologic Sciences; Malignant Neoplasms; neoplasm/cancer; malignancy; Malignant Tumor; Cancers; Cell Number; Cell Count; membrane permeability; Cell Membrane Permeability; Cell Body; Cells; coenzyme analog; Enzyme Cofactors; Coenzymes; data interpretation; Data Analysis; Data Analyses; Coloring Agents; Dyes; electronic device; Electronics; Exhibits; flow cytophotometry; Flow Microfluorometry; Flow Microfluorimetry; Flow Cytofluorometry; Flow Cytofluorometries; Flow Cytometry; Fluorescence; indexing; instrumentation; Kinetics; Laser Radiation; Laser Electromagnetic; Lasers; Methods; NADH; oxidation reduction reaction; Redox; Oxidation-Reduction; Paper; Patents; Legal patent; chromosome complement; DNA Ploidy; DNA Index; DNA Content; Ploidies; Publishing; Research; Risk; Running; biological signal transduction; Signaling; Signal Transduction Systems; Intracellular Communication and Signaling; Cell Signaling; Cell Communication and Signaling; Signal Transduction; Staining method; Stains; Translating; Work; immunophenotype; Immunologic Subtyping; Immunophenotyping; Measures; Artifacts; Morphologic artifacts; base; detector; Label; Surface; Clinical; Phase; Biological; Series; Financial compensation; Compensation; Measurement; Fluorescence Spectroscopy; cancer immunotherapy; immunotherapy of cancer; immunotherapy for cancer; anticancer immunotherapy; anti-cancer immunotherapy; Shapes; tool; Rivers; instrument; Diagnostic; Physiologic pulse; Pulse; Complex; Protocols documentation; Protocol; Source; Techniques; System; Benchmarking; Best Practice Analysis; interest; experience; light emission; Performance; receptor; Receptor Protein; success; fluorophore; Speed; novel; Cytometry; Sampling; assay development; rapid technique; rapid method; stem cell therapy; stem cell-based treatment; stem cell treatment; stem cell therapeutics; data processing; computerized data processing; Complex Mixtures; Detection; Reproducibility; Run-On Assays; Scheme; Small Business Innovation Research Grant; Small Business Innovation Research; SBIR; Tumor Biology; Characteristics; Development; developmental; ultraviolet; Image; imaging; cost; design; designing; Population; innovation; innovative; innovate; prototype; Algorithmic Analysis; Analysis of Algorithms; Analyses of Algorithms; Algorithmic Analyses; beam-splitter; beamsplitter; dynamic system; dynamical system; experimental study; experimental research; experiment; fluorescence lifetime imaging; fluorescence lifetime imaging microscopy; fluorescence life-time imaging microscopy; fluorescence life-time imaging; FLIM imaging; off-patent

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.

Public Health Relevance Statement:
PROJECT NARRATIVE Flow cytometry (FC) is a powerful laboratory tool for cell analysis with applications in immunology, molecular biology, microbiology, cancer biology, diagnostics, and infectious disease monitoring. Autofluorescence (AF), a type of unwanted background to the signal, is a serious and growing limitation of FC that hampers assay sensitivity, leading to substantial problems in data analysis, erroneous scientific conclusions, and inaccurate patient diagnosis. Kinetic River is developing the Colorado, the world's first FC analyzer that eliminates interference from AF, thereby improving data quality, increasing the accuracy of biomedical research, and accelerating drug development.

Project Terms:
Acquired Immunodeficiency Syndrome; AIDS; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunologic Deficiency Syndrome; Algorithms; Antigens; immunogen; Biological Assay; Assay; Bioassay; Biologic Assays; Biomedical Research; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Cell Count; Cell Number; Cell Membrane Permeability; membrane permeability; Cells; Cell Body; Color; Colorado; Communicable Diseases; Infectious Disease Pathway; Infectious Diseases; Infectious Disorder; Data Analyses; Data Analysis; data interpretation; Diagnosis; Dyes; Coloring Agents; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Fluorescence; Freeze Drying; Freeze Dryings; Lyophilization; Grant; Human; Modern Man; Kinetics; Laboratories; Lasers; Laser Electromagnetic; Laser Radiation; Lead; Pb element; heavy metal Pb; heavy metal lead; Acute Myelocytic Leukemia; AML - Acute Myeloid Leukemia; Acute Myeloblastic Leukemia; Acute Myelogenous Leukemia; acute granulocytic leukemia; acute myeloid leukemia; Lymphocyte; Lymphatic cell; Lymphocytic; lymph cell; Methods; Microbiology; Microspheres; Microbeads; Molecular Biology; DNA Molecular Biology; NADH; NIH; National Institutes of Health; United States National Institutes of Health; Redox; oxidation reduction reaction; Oxidation-Reduction; Patients; DNA Content; DNA Index; DNA Ploidy; chromosome complement; Ploidies; Research; Investigators; Researchers; Research Personnel; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Staining method; Stains; Technology; Testing; Time; Immunophenotyping; Immunologic Subtyping; immunophenotype; Measures; Immunology; Immunologist; Label; improved; Surface; Clinical; Phase; Biological; biologic; Fiber; Discipline; data quality; analog; Funding; anti-cancer immunotherapy; anticancer immunotherapy; immune-based cancer therapies; immunotherapy for cancer; immunotherapy of cancer; cancer immunotherapy; Oncology Cancer; Oncology; Pansy; Violet; Viola; Shapes; tool; Rivers; instrument; Diagnostic; programs; Event; Source; System; Best Practice Analysis; Benchmarking; Receptor Protein; receptor; time resolved data; fluorophore; Speed; novel; Abscission; Extirpation; Removal; Surgical Removal; resection; Excision; Sampling; response; drug development; stem cell based therapy; stem cell mediated therapy; stem cell therapeutics; stem cell treatment; stem cell-based treatment; stem cell therapy; Myeloid Cells; data processing; computerized data processing; Data; Detection; Cancer Biology; Collection; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Tumor Biology; Monitor; Output; time use; design; designing; data acquisition; clinically relevant; clinical relevance; prototype; commercialization; Algorithmic Software; Algorithmic Tools; Software Algorithm; Algorithmic Analysis; Algorithmic Analyses; Analyses of Algorithms; Analysis of Algorithms; signal processing; optical spectra; emission spectra; emission spectrum; optical spectrum; experimental study; experiment; experimental research; data handling