SBIR-STTR Award

Quantifying the presence, function, and fate of different immune cells is crucial in immunology and immuno- oncology research, as well as in the diagnosis of hematologic disorders. Flow cytometry is the current method of choice to measure different proteins expressed by single cells at high throughput. However, spectral overlap between fluorophore labels makes flow measurement of more than 15 protein markers difficult, often requiring months of optimization, while measuring more than 40 markers is not possible with current technologies. This upper bound prevents more sophisticated analysis of cell state and function and limits discovery of immune biomarkers needed for treatment monitoring and prognostication. Other methods for high-marker analysis including mass cytometry and RNA-seq based methods are severely limited in throughput and cost-prohibitive, and not ideal for measuring patient blood samples containing millions of cells. The long-term goal of this application is to develop cyclic flow cytometry, a high-throughput, low cost solution for high-marker single-cell analysis. Cyclic flow cytometry leverages proprietary laser particle (LP) technology to barcode individual cells for repeated flow measurements and, once fully developed, is expected to be capable of measuring unprecedented 100 markers at high throughput. The specific aims of this Phase I involve developing key processes required for cyclic cytometry. We will optimize the process of LP delivery into human peripheral mononuclear cells (PBMCs) and will build a flow prototype device for measuring laser and fluorescence emission simultaneously and validating single-cell tracking over multiple flow cycles. Completion of Phase I research will establish the feasibility of cyclic flow cytometry. Full integration of the entire processes and demonstration of 100 marker analysis will be the subject of a Phase II submission.

Public Health Relevance Statement:
Project Narrative The immune system comprises hundreds of different types of immune cells, but flow cytometry, the standard method for high-throughput single-cell analysis, can only measure up to a few dozen cellular markers at a time. This limitation precludes comprehensive immune cell profiling needed for identification of novel and more reliable biomarkers, particularly in the development and monitoring of immunotherapies for cancer. Using novel laser particle technology, we will develop next-generation cyclic flow cytometry capable of measuring over 100 markers and accelerate immunological research and therapeutic discovery to enhance health, prevent disease, and lengthen life.

Project Terms:
Antibodies; Attention; Bar Codes; base; Biological Markers; Biological Sciences; biomaterial compatibility; Blood capillaries; Blood specimen; cancer immunotherapy; Cell Therapy; cell type; Cells; cellular targeting; Collecting Cell; Consumption; cost; Coupled; Cytometry; Development; Devices; Diagnosis; Disease; Evaluation; Flow Cytometry; Fluorescence; Fluorescent Antibody Technique; fluorophore; Future; Genomics; Goals; Health; Hematological Disease; high throughput analysis; Human; imaging probe; Immune; Immune system; Immune System Diseases; Immunologic Markers; Immunologics; Immunology; Immunooncology; Immunophenotyping; Immunotherapy; Individual; innovation; instrument; Label; Lasers; Life; Light; Liquid substance; Measurement; Measures; Methods; Monitor; Mononuclear; next generation; novel; operation; Optics; particle; Patients; Periodicity; Peripheral; Pharmaceutical Preparations; Phase; prevent; Process; process optimization; prognostic; protein biomarkers; Proteins; Proteomics; Protocols documentation; prototype; Research; response; Savings; Scheme; Scientist; Signal Transduction; single cell analysis; Surface; System; Techniques; Technology; Testing; Therapeutic; Time; tool; transcriptome sequencing; transcriptomics; Vertebral column; virtual

Quantifying the presence, function, and fate of different immune cells is crucial in immunologyresearch and development of immunotherapies. Flow cytometry is a single-cell technique that iswidely used to measure different protein markers expressed by immune cells at high throughput.However, spectral overlap between fluorophore labels makes measurement of more than 20markers difficult, often requiring months of optimization, and limits the maximum number ofmarkers measured to around 40. These limitations prevent more comprehensive characterizationof immune cells that is critically needed for immunology and immune-oncology research. Masscytometry and RNA-seq based methods are throughput-limited and cost-prohibitive for measuringpatient blood samples containing millions of cells. The goal of this SBIR application is to developcyclic flow cytometry, a high-throughput, affordable and easy-to-use solution for the high-markersingle-cell analysis market. Cyclic flow cytometry leverages proprietary laser particle technologyto barcode individual cells for repeated flow measurements, enabling complex flow cytometryexperiments with 30 or more markers to be broken down into multiple, much easier cycles witharound 10 markers at a time. The feasibility of this innovative approach has been verified in thePhase I research. The specific aims of this Phase II research involve developing and testing acomplete workflow and fully functional prototype for cyclic flow cytometry. The system will bevalidated with external investigators for several applications. Completion of the Phase II work willlead to early market validation of cyclic flow cytometry and directly lead to design of product-readyprototypes, followed by commercial launch.

Public Health Relevance Statement:
Project Narrative Comprehensive immune cell profiling using flow cytometry is critical for understanding immune responses and developing immunotherapies. However, current flow cytometry is difficult to use when measuring more than a dozen markers, which limits the number of cell types and different cellular responses that can be characterized. Using novel laser particle technology, this project will develop novel cyclic flow cytometry to enable researchers to measure more markers in an easy-to-use manner, accelerating immunologic research and therapeutic discovery.

Project Terms: