New Class of Bright, Sharp, Tunable Near-Infrared Fluorophores for Flow Cyto

描述(申请人提供)：多色流式细胞术[FC]是免疫学中常规使用的最强大的分析技术之一。流式细胞术在几个日益增长的临床应用中发挥着关键作用：白血病和淋巴瘤的免疫表型用于诊断和亚型分类(现在使用8-10种颜色)；在HIV阳性患者的CD4/CD8测量中；以及在移植交叉配型的HLA配型中。随着恶性血液病发病率的增加和移植的使用，随着对方法学的依赖加深，FC检测的数量将继续增长。增加单个样本中荧光团的数量将使临床医生在某些儿童血液疾病的诊断和治疗中获得更高水平的准确性，其中样本大小有限，甚至允许对白血病或淋巴瘤进行细分。这些多色仪表板的额外荧光团的进一步用途是用于小体积的诊断样本，例如来自越来越多的产生有限体积样本的微创手术的样本，例如细针抽吸、腹腔镜术、核心活检、内窥镜检查、支气管镜检查以及众所周知的低细胞脑脊液。由于目前实践中大多数诊断标本的样本量很小，通常没有足够的细胞数量来使必要的众多小组达到诊断目的。大多数常见荧光团的一个统一缺陷是它们的荧光[发射带]很宽，这会导致光谱重叠，需要进行广泛的分析前实验和使用数学补偿，所有这些都增加了实验误差，降低了灵敏度，并限制了多路复用。Nirvana Sciences，Inc.建议开发一种水溶性、可结合、明亮、发射波长可调的细菌氯荧光团的前三个成员，其特征是在常用激光波长激发下，在近红外[NIR]区域具有尖锐的发射带。新的荧光团基于一种平台技术，通过简洁、多样的合成路线合成细菌素，具有以下特点：光和热稳定性；合理控制激发和发射带位置；更多的水溶性和生物共轭取代；以及在355、532和561 nm激光谱线的光吸收[具有相应的强近红外发射带]。由于三个吸收带在近红外区产生相同的强发射，细菌素表现出较长的“斯托克斯”式位移。这些新的荧光染料填补了癌症诊断以及儿科和其他有限样本应用中尚未满足的重要需求。

DESCRIPTION (provided by applicant): 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.