Single Cell Diagnosis of Leukemia using Multiplex Nested PCR System

描述（由申请人提供）：使用多重巢式PCR系统进行白血病的单细胞诊断。为了更好地将靶向诊断和治疗引导至癌症，例如白血病，需要一种简单的基于血液的测定，其可以在一致的基础上提供表达信息，同时使患者的不适最小化。目前用于慢性粒细胞性（或髓样）白血病（CML）检测的方法是基于全血细胞计数，其显示所有类型的粒细胞增加。骨髓活检以筛查费城染色体通常作为CML评估的一部分，但单独的骨髓形态学不足以诊断CML。为了选择最佳的治疗方案并监测患者的反应，了解患者的白血病类型是否为CML是很重要的。为了确认诊断，临床医生需要依赖于多重qRT-PCR测定，其使用已知在不同类型的白血病中差异表达的基因。然而，所有目前的测定方法都是费力和耗时的，并且不存在用于筛选血液样品中大量细胞的高通量方法。因此，麦克斯韦传感器公司提出使用基于Nanodroplet的多重巢式PCR（NMNP）系统开发白血病的单细胞诊断，该系统具有处理数千个单个细胞的能力;基于多重PCR结果检测循环肿瘤细胞;并分析慢性粒细胞白血病（CML）和费城染色体阳性急性淋巴细胞白血病（Ph+ ALL）的嵌合BCR/ABL mRNA的存在或不存在。NMNP系统自动化所有采样过程，准备和检测单细胞中经常在提取过程中丢失的低丰度基因。所提出的NMNP系统的主要优点是1）高通量：同时处理数千（~ 50，000）个单细胞，2）单细胞分析：在单细胞水平上进行核酸检测，3）多重PCR：对单个循环肿瘤细胞中的几个基因进行巢式PCR，以及4）自动化系统：单细胞操作和在芯片上检测PCR结果。第一阶段工作的重点是设计和制造高通量nanodrop芯片，将NMNP芯片与流体和光学检测系统集成，开发NMNP多重PCR方案和生物测定，表征芯片，并评估NMNP性能。 公共卫生相关性：麦克斯韦传感器公司提出了使用单个循环肿瘤细胞开发用于癌症诊断的两步多重巢式PCR系统。该系统结合了纳米技术，流体动力学，实时多重PCR，单细胞测定和循环肿瘤细胞癌症诊断。这些检测也有可能根据治疗前肿瘤的分子特征来指导治疗选择。细胞内异常基因的检测为疾病的早期诊断提供了重要线索。

DESCRIPTION (provided by applicant): Single Cell Diagnosis of Leukemia using Multiplex Nested PCR System. To better direct targeted diagnostics and therapies to cancer, such as leukemia, there is a need for a simple blood-based assay that can provide expression information on a consistent basis with minimal patient discomfort. Current methods for chronic myelogenous (or myeloid) leukemia (CML) detection are based on the complete blood count, which shows increased granulocytes of all types. A bone marrow biopsy to screen for the Philadelphia chromosome is often performed as part of the evaluation for CML, but bone marrow morphology alone is insufficient to diagnose CML. In order to choose the best treatment and monitor the patient's response, it is important to know if the patient's type of leukemia is CML. To confirm the diagnosis, clinicians need to rely on a multiplex qRT-PCR assay using genes known to differentially express in express in different types of leukemia However, all current assay methods are laborious and time consuming, and no high throughput method exists for screening a large number of cells in blood samples. Therefore, Maxwell Sensors Inc. proposes to develop a Single Cell Diagnosis of Leukemia using the Nanodroplet based Multiplex Nested PCR (NMNP) System, which has the capacity to process thousand of individual cells; detect circulating tumor cells based on multiplexed PCR results; and analyze the presence or absence of chimeric BCR/ABL mRNAs for chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). The NMNP system automates all the sampling processes, preparing and detecting low abundance genes in single cells that are often lost in the extraction process. The main advantages of the proposed NMNP system are 1) high throughput: processing thousands (~50,000) of single cells simultaneously, 2) single cell analysis: nucleic acid detection at the single cell level, 3) multiplex PCR: carries out nested PCR for several genes in single circulating tumor cells, and 4) automated system: single cell manipulation and detection of the PCR results on a chip. The focus of the Phase I work is to design and fabricate a high-throughput nanodrop chip, integrate the NMNP Chip with fluidics and an optical detection system, develop the NMNP multiplex PCR protocol and bioassay, characterize the chip, and evaluate NMNP performance. PUBLIC HEALTH RELEVANCE: Maxwell Sensors Inc. proposes to develop a two step Multiplex Nested PCR system using single circulating tumor cells for cancer diagnosis. This system combines nanotechnology, fluid dynamics, real-time Multiplex PCR, single-cell assays, and circulating tumor cell cancer diagnostics. These assays also have the potential to guide treatment selection based on the molecular profile of the tumor before therapy. Detection of abnormal genes in the intracellular materials provides important clues for early disease diagnosis.