SBIR-STTR Award

Polychromatic flow cytometry (FC) is one of the most powerful analytical techniques used in immunology, basic research, and clinical medicine. In basic research, FC is a primary tool for understanding disease development at the cellular and subcellular levels. In the rapidly developing field of immunotherapy, FC is an indispensable tool for monitoring the effectiveness of new therapies and identifying key cellular subpopulations using multiple biomarkers within a panel. It plays a critical role in several growing clinical applications: development of vaccines for infectious diseases, including emerging diseases such as Ebola and Zika; characterization of immunological cells and responses related to transplantation and treatment of graft-versus-host disease; and development of vaccines and immune therapies to prevent and treat HIV infections. Increasing the number of spectrally distinct fluorophores for analysis of a single sample will give researchers greater flexibility and give clinicians a higher level of accuracy in the diagnosis and management of conditions where the size of the sample use for diagnosis is limited and for specimens with low cell numbers, such as those derived from fine needle aspiration, laparoscopy, core biopsy, and cerebrospinal fluid. Due to these small sample sizes, there are often insufficient cell numbers for multiple panels. Improvements in optics and lasers have driven the recent introduction of new fluorophores for FC. However, most of these new fluorophores have broad and often overlapping emission profiles, much like traditional fluorophores. Overlapping spectra can be resolved by the combined use of bandpass filters and mathematical compensation (overlap subtraction); however such compensation increases experimental error, reduces sensitivity, and limits multiplexing. Thus, even with a prototype 50-color instrument, leading investigators have only been able to implement a 30-parameter panel - advances in reader technology have outstripped available capabilities in fluorophores. NIRvana Sciences is currently developing two families of tetrapyrrole fluorophores licensed from NC State University. These fluorophores offer properties that make them excellent candidates for use in polychromatic FC including ultra-violet/violet excitation, long stokes shifts into the red and near infrared spectrums along with very narrow emissions. NIRvana Sciences and the Lindsey lab are developing a polymer platform that retains the intrinsic brightness of its dyes and has strong utility to the flow cytometry reagent market.

Public Health Relevance Statement:
PROJECT NARRATIVE / PUBLIC HEALTH RELEVANCE Flow cytometry is used routinely for both basic research and clinical diagnostic applications. Many types of infectious diseases and autoimmunity are routinely diagnosed using flow cytometry, and it is widely used in the development of vaccines and immunotherapies for cancer. This STTR project will advance the development of a new technology for creating bright and sharp dyes in the red to far-red spectral region, which will greatly expand the suite of "colors" available for multiplexed flow cytometry and allow for higher sensitivity, smaller sample size, and higher throughput testing.

Project Terms:
Antibodies; Antigens; immunogen; ATGN; Autoimmunity; Autoimmune Status; Cell Count; Cell Number; Cells; Cell Body; Cerebrospinal Fluid; spinal fluid; cerebral spinal fluid; Clinical Medicine; Clinical Medical Sciences; Color; Communicable Diseases; Infectious Disorder; Infectious Diseases; Infectious Disease Pathway; Coumarins; coumarin; alpha benzopyrone; Tonka Bean Camphor; Coumarines; 1,2-benzopyrone; 1,2-Benzopyrones; 1,2-Benzo-Pyrones; Diagnosis; Disease; Disorder; Dyes; Coloring Agents; Ebola virus; ebolavirus; Ebola; EBOV; Exhibits; Family; Flow Cytometry; flow cytophotometry; Flow Microfluorometry; Flow Microfluorimetry; Flow Cytofluorometry; Flow Cytofluorometries; Fluorescence; Goals; Graft-vs-Host Disease; Runt Disease; Homologous Wasting Disease; Graft-Versus-Host Disease; GVHD; HIV Infections; Human T-Lymphotropic Virus Type III Infections; HTLV-III-LAV Infections; HTLV-III Infections; Immersion Investigative Technique; Immersion; Immunotherapy; immuno therapy; immune-based treatments; immune-based therapies; immune therapy; immune therapeutic interventions; Immunologically Directed Therapy; indexing; Laboratories; Lasers; Laser Radiation; Laser Electromagnetic; Mathematics; Math; Methods; Molecular Weight; Noise; Optics; optical; Laparoscopy; Peritoneoscopy; Diagnostic Laparoscopy; Celioscopy; Play; Polymers; Proteins; Reagent; Research Personnel; Researchers; Investigators; Role; social role; Science; Signal Transduction; biological signal transduction; Signaling; Signal Transduction Systems; Intracellular Communication and Signaling; Cell Signaling; Cell Communication and Signaling; Stains; Staining method; Technology; Testing; Toluene; methyl-benzene; Transplantation; transplant; Universities; Vaccine Therapy; therapeutic vaccination; VAC-TX; Water; Hydrogen Oxide; chlorin; Tetrapyrroles; Immunology; Titrations; Label; Phase; Variation; Variant; Compensation; Financial compensation; Sample Size; analog; anticancer immunotherapy; anti-cancer immunotherapy; cancer immunotherapy; Viola; Violet; Pansy; tool; instrument; Specimen; Research Specimen; BODIPY; Side; Protocols documentation; Protocol; Techniques; copolymer; monomer; vaccine development; vaccine formulation; development of a vaccine; develop a vaccine; fluorophore; Hydrophobicity; hydrophilicity; antibody conjugate; professor; Peripheral Blood Mononuclear Cell; PBMC; aqueous; Basic Science; Basic Research; new technology; novel technologies; Cell surface; Sampling; Property; response; bacteriochlorin; antigen binding; antigen bound; Binding; Molecular Interaction; Effectiveness; prevent; preventing; Core Biopsy; Core Needle Biopsy; CD8B1 gene; LYT3; CD8B1; CD8B; CD8; Length; Fine needle aspiration biopsy; Fine-Needle Aspiration; Fine Needle Aspirate; FNA; Detection; Reader; STTR; Small Business Technology Transfer Research; Immunologically; Immunological; Immunologic; Immunochemical Immunologic; Immunologics; Monitor; developmental; Development; Advanced Development; quantum; Population; clinical applicability; clinical application; novel therapy; novel drugs; novel drug treatments; next generation therapeutics; new therapy; new therapeutics; new drugs; new drug treatments; novel therapeutics; prototype; public health relevance; biomarker; biologic marker; bio-markers; Biological Markers; flexible; flexibility; screening; zikav; ZIKV; ZIKA; Zika Virus; clinical diagnostics; experimental research; experiment; experimental study

Polychromatic flow cytometry (FC) is a vital analytical technique used in immunology, basic research, and clinical medicine. FC is an indispensable tool for understanding disease development at the cellular and subcellular levels and for monitoring the effectiveness of new immunotherapies by identifying key cellular subpopulations using multiple biomarkers within a panel. It also has a critical role in growing clinical applications including development of vaccines for infectious diseases, characterization of immunological cells and responses related to transplantation and treatment of graft-versus-host disease, and development of vaccines and immune therapies to prevent and treat HIV infections. Increasing the number of spectrally distinct fluorophores will give researchers greater flexibility and give clinicians a higher level of accuracy in the diagnosis and management of conditions where the size of the sample used for diagnosis is limited or cell number is low, including those derived from fine needle aspiration, laparoscopy, core biopsy, and cerebrospinal fluid. Improvements in optics and lasers have driven the recent introduction of new fluorophores for FC. However, most of these possess broad and often overlapping emission profiles, much like traditional fluorophores. Overlapping spectra can be resolved by the combined use of bandpass filters and mathematical compensation (overlap subtraction); however, such compensation increases experiment error, reduces sensitivity, and limits multiplexing. Thus, even with a prototype 64-color instrument, leading investigators have only been able to implement a 40-parameter panel - advances in reader technology have outstripped available capabilities in fluorophores. NIRvana Sciences is developing two families of tetrapyrrole fluorophores for use in FC. These fluorophores are excellent candidates for use in polychromatic FC due to their ultra-violet/violet excitation, long Stoke's shifts into the red and near-infrared spectrums, and very narrow emissions. In the current project, NIRvana seeks to complete development of a new technology, referred to as FluoroPods, that allows us to fully utilize our unique portfolio of dyes. This polymer-based system enables the use of dyes that possess excellent spectral properties but lack sufficient water solubility and/or are heavily quenched in biological systems. Upon completion of the project, NIRvana Sciences will have created a palette of exceptionally bright FluoroPods that will form the basis of a platform of 22+ new colors using only the 355 and 405 nm lasers.

Public Health Relevance Statement:
PROJECT NARRATIVE / PUBLIC HEALTH RELEVANCE Flow cytometry is commonly used for both basic research and clinical diagnostic applications. Many infectious and immune diseases are routinely diagnosed using flow cytometry, and it is widely used in the development of vaccines and immunotherapies for cancer. This SBIR project will advance the development of a new amplifying technology for creating bright and sharp dyes in the red to far-red spectral region which will greatly expand the suite of "colors" available for flow cytometry and allow for higher sensitivity, smaller sample size, and higher throughput testing.

Project Terms:
Antibodies; Antigen-Antibody Complex; Immune Complex; Binding Sites; Combining Site; Reactive Site; Biological Assay; Assay; Bioassay; Biologic Assays; Cell Count; Cell Number; Cells; Cell Body; Cerebrospinal Fluid; cerebral spinal fluid; spinal fluid; Clinical Medicine; Clinical Medical Sciences; Color; Communicable Diseases; Infectious Disease Pathway; Infectious Diseases; Infectious Disorder; Diagnosis; Disease; Disorder; Dyes; Coloring Agents; Family; Flow Cytometry; Flow Cytofluorometries; Flow Cytofluorometry; Flow Microfluorimetry; Flow Microfluorometry; flow cytophotometry; Fluorescent Probes; Goals; graft vs host disease; GvHD; Homologous Wasting Disease; Runt Disease; graft versus host disease; graft vs. host disease; HIV Infections; HTLV-III Infections; HTLV-III-LAV Infections; Human T-Lymphotropic Virus Type III Infections; Immune System Diseases; Immune Diseases; Immune Disorders; Immune Dysfunction; Immune System Disorder; Immune System Dysfunction; Immune System and Related Disorders; Immunodeficiency and Immunosuppression Disorders; Immunologic Diseases; Immunological Diseases; Immunological Dysfunction; Immunological System Dysfunction; Immunologic Techniques; Immunologic Technics; Immunological Technics; Immunological Techniques; Immunotherapy; Immune mediated therapy; Immunologically Directed Therapy; immune therapeutic approach; immune therapeutic interventions; immune therapeutic regimens; immune therapeutic strategy; immune therapy; immune-based therapies; immune-based treatments; immuno therapy; indexing; instrumentation; Lasers; Laser Electromagnetic; Laser Radiation; Mathematics; Math; Methods; optical; Optics; Particle Size; Celioscopy; Diagnostic Laparoscopy; Peritoneoscopy; Laparoscopy; Polymers; Reagent; Investigators; Researchers; Research Personnel; social role; Role; Science; Solubility; Staining method; Stains; Technology; Testing; Vaccine Therapy; VAC-TX; therapeutic vaccination; Water; Hydrogen Oxide; Tetrapyrroles; Immunophenotyping; Immunologic Subtyping; immunophenotype; Immunology; base; Label; improved; Site; Phase; Biological; biologic; Compensation; Financial compensation; Evaluation; Sample Size; anti-cancer immunotherapy; anticancer immunotherapy; immune-based cancer therapies; immunotherapy for cancer; immunotherapy of cancer; cancer immunotherapy; Immunological response; host response; immune system response; immunoresponse; Immune response; Pansy; Violet; Viola; tool; instrument; Techniques; System; particle; Performance; develop a vaccine; develop vaccines; development of a vaccine; vaccine development; water solubility; fluorophore; Hydrophobicity; hydrophilicity; PBMC; Peripheral Blood Mononuclear Cell; aqueous; Basic Research; Basic Science; novel technologies; new technology; Sampling; performance tests; Property; response; Molecular Interaction; Binding; Effectiveness; preventing; prevent; Diameter; Caliber; Core Needle Biopsy; Core Biopsy; Preparedness; Readiness; FNA; Fine Needle Aspirate; Fine-Needle Aspiration; Fine needle aspiration biopsy; Reader; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Immunologics; Immunochemical Immunologic; Immunologic; Immunological; Immunologically; Monitor; Characteristics; Development; developmental; ultraviolet; ultra violet; Advanced Development; design; designing; clinical application; clinical applicability; prototype; commercialization; public health relevance; Biological Markers; bio-markers; biologic marker; biomarker; stability testing; biological systems; flexibility; flexible; clinical diagnostics; medical diagnostic; experimental study; experiment; experimental research; transplantation therapy; transplant therapy; transplant treatment; transplantation treatment; multiplex assay