SBIR-STTR Award

Polychromatic flow cytometry [FC] is one of the most powerful analytical techniques routinely used in immunology. Flow cytometry plays a critical role in several growing clinical applications: leukemia and lymphoma immunophenotyping for diagnosis and sub-categorization (now using 8-10 colors); in CD4/CD8 measurement in HIV-positive patients; and in HLA typing for transplantation cross-matching. With the increasing incidence of hematologic malignancies and use of transplantation, the number of FC tests will continue to grow as reliance on the methodology deepens. Increasing the number of fluorophores on a single sample will give clinicians a higher level of accuracy in the diagnosis and management of certain pediatric hematological disorders, where sample size is limited, and even allow for sub-categorization of the leukemia or lymphoma. Further utility of additional fluorophores for these polychromatic panels is for diagnostic samples of small volume such as specimens from an ever-increasing number of minimally invasive procedures that produce limited volume specimens, such as fine needle aspiration, laparoscopy, core biopsy, endoscopy, bronchoscopy, and notoriously hypocellular cerebrospinal fluid. Due to the small sample size of most diagnostic specimens in current practice, often insufficient cell numbers are available for the necessarily numerous panels to reach a diagnosis. A uniform deficiency of most common fluorophores is their broad fluorescence [emission bands], which cause spectral overlap, requiring extensive pre-assay experimentation and use of mathematical compensation, all of which increase experimental error, reduce sensitivity, and limit multiplexing. NIRvana Sciences, Inc. proposes to develop the first three members of a palette of water-soluble, conjugatable, bright, emission wavelength-tunable bacteriochlorin fluorophores characterized by sharp emission bands in the near-infrared [NIR] region upon excitation by commonly used laser wavelengths. The new fluorophores are based on a platform technology enabled by concise, versatile synthetic routes to bacteriochlorins with the following features: photo and thermal stability; rational control of excitation and emission band positions; additional substitution for water solubility and bioconjugation; and light absorption at laser lines of 355, 532, and 561 nm [with corresponding intense NIR emission bands]. The bacteriochlorins exhibit long 'Stokes'-like shifts' as all three absorption bands produce the same strong emission in the NIR region. These new fluorescent dyes fill an important unmet need in cancer diagnosis as well as pediatric and other limited-sample applications.

Thesaurus Terms:
Absorption;Antibodies;Area;Autoimmunity;Bacteriochlorin;Base;Basic Science;Binding (Molecular Function);Biological Assay;Bronchoscopy;Buffers;Cancer Diagnosis;Cd4 Positive T Lymphocytes;Cd8b1 Gene;Cell Count;Cellular Imaging;Cerebrospinal Fluid;Childhood;Clinical;Clinical Application;Clinical Assay Development;Color;Commercialization;Communicable Diseases;Companions;Complex;Core Biopsy;Core Facility;Cost;Design;Diagnosis;Diagnostic;Disease Diagnosis;Dyes;Endoscopy;Esters;Exhibits;Financial Compensation;Fine Needle Aspiration Biopsy;Flow Cytometry;Fluorescence;Fluorescent Dyes;Fluorophore;Foundations;Hematologic Neoplasms;Hematological Disease;Hematopoietic Neoplasms;Hiv Seropositivity;Human;Immunology;Immunophenotyping;In Vitro;In Vivo;Incidence;Indexing;Innovation;Intellectual Property;Laparoscopy;Lasers;Legal Patent;Leukemia;Leukemia/Lymphoma;Light;Lymphoma;Measurement;Member;Methodology;Minimally Invasive;Nature;Nih Vaccine Research Center;Pathway Interactions;Patients;Play;Positioning Attribute;Procedures;Public Health Relevance;Reliance;Research Study;Role;Route;Running;Sample Size;Sampling;Science;Scientist;Solutions;Specimen;Speed (Motion);Staining Method;Stains;System;T-Lymphocyte;T-Lymphocyte Subsets;Techniques;Technology;Temperature;Testing;Titrations;Transplantation;Transplantation Typing;Water;Water Solubility;Width;Work;

Polychromatic flow cytometry (FC) is one of the most powerful analytical techniques routinely used by both basic research and clinical diagnostics laboratories for the immunological categorization of cells. Dyes used for FC typically exhibit broad fluorescent emission bands with full-width-at-half-maximum (fwhm) values of 50–80 nm. This limits the maximum number of dyes, and thus the number of cell biomarkers, that can be resolved in a given experiment. Compensating for spectral overlap between dyes is currently viewed as a necessary part of experimental design, requiring extensive pre-assay experimentation and mathematical compensation, thereby introducing experimental error and reducing sensitivity. Bacteriochlorins are a unique class of fluorescent dyes that offer a solution for accurate multiplexing with minimal compensation due to their very narrow emission bands (fwhm of 25-35 nm), typically less than half the spectral width of existing dyes. Through chemical modification, they can be tuned to emission wavelengths from the far red through the near-infrared (NIR) spectrum (700–900 nm). In addition, bacteriochlorins share a common excitation band, making possible the development of a full spectrum of NIR dyes excited by a single UV light source. To render bacteriochlorin dyes commercially viable, Phase 2B efforts will be focused on: 1) improving and expanding the bacteriochlorin dye portfolio and synthesis methods; 2) validating dye performance in FC panels with NIRvana Sciences’ collaborators, and 3) developing procedures, methods, and protocols for commercial scale manufacturing. This Phase 2B SBIR proposal is intended to continue the refinement and transfer of bacteriochlorin technology for commercial development, resulting in a bacteriochlorin dye portfolio with high impact potential for polychromatic FC. This enhanced multiplex capability will advance not only basic immunology research, but it will also accelerate novel vaccine and adjuvant discovery for HIV, malaria, tuberculosis, and emerging infectious disease threats. Greater multiplexing also is critical for analyzing reduced volume samples, for single cell studies, and for high throughput, high resolution analyses.

Public Health Relevance Statement:
Project Narrative/

Public Health Relevance:
Flow Cytometry is used routinely for both basic research and clinical diagnostic applications for characterizing cells of the immune system. Modern flow cytometers are capable of detecting 12-15 biomarkers per experiment using different fluorescent dye labels or “colors” but the overlapping spectra of most fluorescent dyes limit the number of biomarkers that can currently be detected accurately. NIRvana Sciences proposes to develop a portfolio of novel dyes in the near infrared (NIR) spectral region with low overlap that will enable much greater numbers of biomarkers to be detected simultaneously by flow cytometry, impacting not only basic immunology research, but also accelerating development of immunotherapies and vaccines for HIV and other emerging infectious disease threats.

Project Terms:
Adjuvant; Agreement; Amines; Antibodies; aqueous; Autoimmune Diseases; bacteriochlorin; Basic Science; Biological Assay; Biological Markers; Buffers; Cells; Chemicals; Chromatography; clinical diagnostics; Color; commercialization; Communicable Diseases; design; Development; Disease; Dyes; Effectiveness; Emerging Communicable Diseases; Exhibits; Experimental Designs; experimental study; Family; Financial compensation; Flow Cytometry; Fluorescence; Fluorescent Dyes; fluorophore; Goals; HIV; HIV vaccine; HIV/TB; Hypersensitivity; Immune; Immune response; Immune system; Immunologics; Immunology; Immunotherapy; improved; indexing; Label; Laboratories; Lasers; lot production; Malaria; Mathematics; Mediating; meetings; Methods; Modernization; Modification; novel; novel strategies; novel therapeutics; novel vaccines; Organic solvent product; Performance; Phase; Polymers; Procedures; Production; Property; Protocols documentation; public health relevance; quantum; Reagent; Research; Resolution; Route; Running; Safety; Sampling; scale up; Science; Small Business Innovation Research Grant; Solubility; Source; Stains; Techniques; Technology; Testing; Transplantation; Tuberculosis; Ultraviolet Rays; Validation; Water; Width