Microfluidic diagnostics for Primary Immunodeficiency

描述（由申请人提供）：该提案的主要目标是开发一种新技术来筛查新生儿严重的原发性免疫缺陷（PID），包括 T 细胞（例如 SCID）和 B 细胞（例如 XLA）的定量缺陷。在新生儿期诊断这些缺陷可以挽救生命并降低社会成本。在试点工作中，我们开发了一种微流体通道系统，可以从一滴血液中特异性捕获和标记免疫细胞。我们还开发了一种玻璃嵌入式波导传感器，可以定量位于波导核心渐逝场中的细胞。这些技术的独特之处在于每次测试成本和设备成本都非常低，从而能够在新生儿托儿所进行即时护理测试。它还可以在不需要专门培训、专门的技术人员、昂贵的试剂或处理干血斑的情况下进行测试。我们在此应用中的目标是将这两种技术结合在一起，并确定由此产生的设备可以定量婴儿血液中的细胞，从而验证这种诊断新生儿期 PID 的新方法。 我们的目标是： 1. 集成波导和微流体以捕获和定量人体血液中的细胞。我们将使用斯坦福纳米制造设施和斯坦福微流体铸造厂制造波导和微流体通道。这些技术将被整合，最终的探测器将使用成人血液中的血细胞进行测试和校准。 2. 建立蛋白酪氨酸激酶 7 (PTK7) 用于新生儿 T 细胞定量的效用。由于 T 细胞前体的母体植入可能会阻碍诊断 SCID 的传统方法，因此我们将利用新发现的新生儿 T 细胞标记物 PTK7 来捕获和计数新生儿足跟血中的 T 细胞。 3. 定量新生儿血液中的 B 细胞并整合以同时计数 T 细胞和 B 细胞。我们将测试各种 B 细胞特异性标记物在我们的微流体通道中捕获和检测人类 B 细胞的能力。然后我们将从婴儿足跟血中计数 B 细胞。最后，我们将把 B 细胞和 T 细胞检测整合到一个诊断测试中。 公共卫生相关性：在美国，每 1,200 人中就有 1 人患有原发性免疫缺陷 (PID)。如果能够在新生儿中诊断出严重的 PID，早期治疗将挽救生命并降低社会成本。这些研究将有助于开发一种新型检测技术，能够以极低的成本快速筛查新生儿的 T 细胞和 B 细胞缺陷。

DESCRIPTION (provided by applicant): The main goal of this proposal is to develop a novel technology to screen newborns for serious Primary Immune Deficiencies (PIDs), including quantitative deficiencies of T cells (e.g., SCID) and B cells (e.g., XLA). Diagnosis of these deficiencies in the newborn period could save lives and reduce societal costs. In pilot work we have developed a microfluidic channel system that enables specific capture and labeling of immune cells from a single drop of blood. We have also developed a glass-embedded waveguide sensor that can quantitate cells lying in the evanescent field of the waveguide core. These technologies are distinctive in that they allow for both a very low per-test cost as well as a very low equipment cost, thus enabling point-of-care testing in the newborn nursery. It also will enable testing without the need for specialized training, dedicated technicians, expensive reagents, or handling of dried blood spots. Our goals in this application are to bring together these two technologies and to establish that the resulting device can quantitate cells from infant blood, thus validating this novel approach for diagnosing PIDs in the neonatal period. Our Aims are to: 1. Integrate the Waveguides and Microfluidics to Capture and Quantitate Cells from Human Blood. We will fabricate waveguides and microfluidic channels using the Stanford Nanofabrication Facility and the Stanford Microfluidics Foundry. These technologies will be integrated and the resulting detector will be tested and calibrated using blood cells from adult human blood. 2. Establish the Utility of Protein Tyrosine Kinase 7 (PTK7) for Quantitation of Neonatal T Cells. Because maternal engraftment of T cell precursors can foil traditional approaches to diagnosing SCID, we will utilize the newly discovered marker of neonatal T cells PTK7 to capture and enumerate T cells from newborn heel stick blood. 3. Quantitate B cells in Neonatal Blood and Integrate to Simultaneously Count T Cells and B Cells. We will test a variety of B-cell-specific markers for their capability to capture and detect human B cells in our microfluidic channels. We will then count B cells from infant heel stick blood. Finally we will bring together B cell and T cell detection into a single diagnostic test. PUBLIC HEALTH RELEVANCE: Primary immunodeficiencies (PIDs) affect 1 in 1,200 people in the US. If serious PIDs could be diagnosed in newborns, early treatment would save lives and reduce societal costs. These studies will enable development of a novel detector technology that can screen newborns for T cell and B cell deficiencies rapidly and at very low cost.