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: in disease management in HIV-positive patients; leukemia and lymphoma immunophenotyping for diagnosis and sub- categorization; and in HLA typing for transplantation cross-matching. 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 pediatric hematological disorders, where sample size 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. Yet most of these new reagents 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) which increase experimental error, reduce sensitivity, and limit multiplexing. NIRvana Sciences, Inc. proposes to develop the first five members of a palette of water-soluble, conjugatable, bright, emission wavelength-tunable chlorin fluorophores characterized by sharp narrow emission bands in the red to far-red region upon excitation by the commonly used violet laser (405 nm). The new fluorophores are based upon a well-developed platform technology enabled by concise, versatile synthetic routes to chlorins with the following features: photo and thermal stability; rational control of excitation and emission band positions; additional substitution for water solubility and bioconjugation. This proposal includes: (1) Synthesis of five water-soluble distinct chlorins; (2) Optimization of chlorin conjugation to antibodies; (3) Optimization of FC laser and filter sets for chlorin reagents; and (4) Evaluation and comparison of chlorin reagents to Quantum Dots in a polychromatic FC panel. These new chlorin fluorophores will increase the number of spectrally resolvable colors that can be used in polychromatic FC with a minimal need for compensation between colors, thereby enhancing the ability of researchers in immunology, oncology, and cell biology to perform more sophisticated experiments. They will also fill an important unmet need in clinical applications by enabling more actionable diagnostic information to be obtained from smaller samples.
Public Health Relevance Statement: Public Health Relevance: Flow Cytometry is used routinely for both basic research and clinical diagnostic applications. Human blood cancers, many types of infectious diseases, and autoimmunity are routinely diagnosed using Flow Cytometry. The advent of newer sharper dyes in the red to far red spectral region will allow for higher sensitivity, smaller sample size, highe throughput testing, and reduced costs for the diagnosis of these diseases.
Project Terms: Antibodies; antibody conjugate; Autoimmunity; base; Basic Science; Binding (Molecular Function); Biological Assay; Biological Markers; Cell Count; Cellular biology; Cerebrospinal Fluid; Childhood; chlorin; Clinical; clinical application; Clinical Medicine; Color; Communicable Diseases; Complex; Core Biopsy; Core Facility; cost; design; Development; Diagnosis; Diagnostic; Disease; disease diagnosis; Disease Management; Dyes; Effectiveness; Ensure; Esters; Evaluation; Financial compensation; Fine needle aspiration biopsy; flexibility; Flow Cytometry; Fluorescence; Fluorescent Dyes; fluorophore; Hematological Disease; Hematopoietic Neoplasms; HIV Seropositivity; Human; Immunology; Immunophenotyping; Immunotherapy; indexing; Label; Laboratories; Laparoscopy; Lasers; leukemia/lymphoma; member; Monitor; National Institute of Allergy and Infectious Disease; oncology; Optics; Patients; Play; Positioning Attribute; programs; public health relevance; Quantum Dots; Reagent; Research Personnel; research study; Role; Route; Sample Size; Sampling; Science; Sensitivity and Specificity; Signal Transduction; Specimen; Staining method; Stains; T-Lymphocyte Subsets; Techniques; Technology; Testing; tool; Transplantation; Vaccine Research; Viola; Water; water solubility
