SBIR-STTR Award

Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of disease. It is particularly useful in distinguishing between populations of immune cells based on their expressed cell surface antigens. Standard flow cytometers use fluorescent tags, often conjugated to monoclonal antibodies, to give qualitative and quantitative information about specific molecules in the cell. This molecular specificity, coupled with the fact that information is obtained on a cell-by-cell basis and that very high throughput is possible (30,000 cells per second can be analyzed), make this a powerful technique. The ability to multiplex (measure a variety of different molecular species in a single cell) further adds to its utility and to the complexity of the scientific questions that can be addressed using this technique. However, the level of multiplexing currently has limitations. Typically, flow cytometry analysis relies solely on spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions. Currently, employing even moderate levels of multiplexing for the simultaneous interrogation of multiple parameters within a cell requires high levels of complexity in instrumentation and analysis, and careful design and execution of experiments. The related "compensation problem" (compensating for spillover of signal from a fluorophore into multiple channels—due to the broad spectrum of most fluorophores) also causes significant instrument complexity, cumbersome workflow, and inaccurate results. These factors put severe limits on the range of scientific questions that can be addressed using current technologies, deter novices in the technique from attempting more complex yet scientifically relevant experiments, and collectively are widely regarded as the major current bottleneck in flow cytometry. To overcome this limitation, we have developed an innovative approach that uses fluorescence lifetime as a separate, additional discriminating measurement parameter. Our scheme for using fluorescent lifetime for multiplexing is simple, scalable, and supported by preliminary data from our prototype instrument. The proposed project will establish the feasibility of lifetime-based multiplexing by modifying our experimental platform with key hardware and algorithm improvements, challenging the resulting prototype with a comprehensive set of verification and validation tests of increasing complexity, and culminating with a comparison of our technology to existing technology in a standard four-color cell-based assay. A successful outcome will lay the foundation for our planned development of commercial instruments (both analyzers and sorters) that offer two major benefits to end users: (a) simple, turnkey, compensation-free operation for instruments with low-to-medium levels of multiplexing; and (b) high-end instruments with two to three times the current maximum multiplexing capability.

Public Health Relevance Statement:
Project Narrative Flow cytometry is a widely used cell-analysis technique in research and clinical diagnostics (e.g., drug development, cancer, AIDS, and other diseases). Current flow cytometers use information from fluorescent markers in a way that makes experiments complicated to perform and also limits the scope of the experiments that a researcher can carry out. The current proposal aims to develop an innovative technique that will relieve these limitations by extracting additional information from the fluorescent markers, and by using the information to simplify the operation and increase the performance of flow cytometers.

Project Terms:
Acquired Immunodeficiency Syndrome; Address; Affect; Algorithms; Amplifiers; Antigens; base; Binding; Biological; Biological Assay; CD3 Antigens; CD8B1 gene; Cells; Characteristics; Clinical; clinical diagnostics; Collection; Color; commercialization; Complex; Coupled; Data; design; Development; Development Plans; Discrimination; Disease; drug development; experimental study; Financial compensation; Flow Cytometry; Fluorescence; fluorophore; Foundations; Goals; Human; Immune; improved; innovation; instrument; instrumentation; Label; Laboratories; Laboratory Diagnosis; Lasers; Lead; lens; Leukocytes; Malignant Neoplasms; Measurement; Measures; Molecular; Monitor; Monoclonal Antibodies; Noise; operation; Outcome; Performance; Physiologic pulse; Population; Protocols documentation; prototype; PTPRC gene; Reproducibility; Research; research and development; Research Personnel; Running; Sampling; Scheme; signal processing; Signal Transduction; Source; Specificity; Speed; Staining method; Stains; Surface Antigens; System; Techniques; Technology; Testing; Time; Validation; verification and validation; Width

Flow cytometry is a workhorse technique in research and development as well as in clinical laboratories for diagnosis and monitoring of disease. It is particularly useful in distinguishing between populations of immune cells based on their expressed cell surface antigens. Standard flow cytometers use fluorescent tags, often conjugated to monoclonal antibodies, to give qualitative and quantitative information about specific molecules in the cell. This molecular specificity, coupled with the fact that information is obtained on a cell-by-cell basis with very high throughput (up to 30,000 cells per second), make this a powerful technique. The ability to multiplex (measure a variety of different molecular species in a single cell) further adds to its utility and to the complexity of the scientific questions that can be addressed using this technique. However, the level of multiplexing currently has limitations. Flow cytometry analysis typically relies solely on spectral information of the fluorescent tags and is thus limited by the spectral overlap of fluorophore emissions. Currently, employing even moderate levels of multiplexing requires complex instrumentation and careful experimental design, execution and analysis to compensate for spectral spillover of signal into multiple channels. This severely limits the range of scientific questions that can be addressed using current technologies, deters novices in the technique from attempting more complex yet scientifically relevant experiments, and is widely regarded as the major bottleneck in the field. To overcome this limitation, we propose to build on our results from Phase I where we demonstrated feasibility for an innovative approach that uses fluorescence lifetime as a separate, additional discriminating measurement parameter. Our scheme for using fluorescent lifetime for multiplexing is simple, scalable, and supported by preliminary data from our prototype instrument. Here we propose to upgrade our Phase I instrument to increase multiplexing capability, challenge that instrument with a battery of verification tests, and validate using a relevant biological assay, benchmarking results against a conventional flow cytometer. The result will be a system enabling compensation-free flow cytometry experiments of 12 colors, while requiring fewer lasers and detectors than similarly equipped commercial systems. Such a system would serve a large segment of the market, including clinical cytometry, and is expected to see broad adoption. This would pave the way for further development of an ultra-high (30+) parameter instrument suitable for immunophenotyping, yet requiring significantly less compensation than current systems, and pushing the boundaries of experimental complexity. Given flow cytometry’s wide-spread use and importance, this project will have a high impact in many biomedical and clinical applications. Several large instrumentation companies have already indicated an eagerness to engage in strategic partnerships aimed at commercialization.

Public Health Relevance Statement:
PROJECT NARRATIVE Flow cytometry is a widely used cell-analysis technique in research and clinical diagnostics (e.g., drug development, cancer, AIDS, and other diseases). Current flow cytometers use information from fluorescent markers in a way that makes experiments complicated to perform and limits the scope of the experiments that a researcher can carry out. The current proposal aims to further develop an innovative technique that will relieve these limitations by extracting additional information from the fluorescent markers, and by using the information to simplify the operation and increase the performance of flow cytometers.

Project Terms:
Acquired Immunodeficiency Syndrome; Address; Adoption; Algorithmic Analysis; Amplifiers; Antibodies; base; Benchmarking; Biological Assay; Cells; Clinical; clinical application; clinical diagnostics; Color; commercialization; Complex; Coupled; Crowding; Cytometry; Data; design; Detection; detector; Development; Dimensions; Discrimination; Disease; drug development; Electronics; Ensure; Experimental Designs; experimental study; Financial compensation; Flow Cytometry; Fluorescence; fluorophore; Human; Immune; Immunology; Immunophenotyping; improved; innovation; instrument; instrumentation; Kinetics; Label; Laboratory Diagnosis; Lasers; Malignant Neoplasms; Measurement; Measures; Molecular; Monitor; Monoclonal Antibodies; Noise; novel; oncology; operation; Optics; Performance; Phase; Population; Protocols documentation; prototype; Research; research and development; Research Personnel; Resolution; Rivers; Scheme; Side; Signal Transduction; Specificity; Speed; Stem Cell Research; success; Surface Antigens; System; Techniques; Technology; Testing; Validation