Microfluidic diagnostics for Primary Immunodeficiency

描述（由申请人提供）：本申请的主要目标是开发一种筛查新生儿严重原发性免疫缺陷（pid）的新技术，包括T细胞（如SCID）和B细胞（如XLA）的定量缺陷。在新生儿期诊断出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波导传感器，可以定量位于波导核心的倏逝场中的细胞。这些技术的独特之处在于，它们允许非常低的每次测试成本以及非常低的设备成本，从而使新生儿护理点测试成为可能。它也将使测试不需要专门的培训，专门的技术人员，昂贵的试剂，或处理干燥的血点。我们在这项应用中的目标是将这两种技术结合起来，并建立最终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。整合波导和微流体从人体血液中捕获和定量细胞。我们将使用斯坦福纳米制造设备和斯坦福微流控铸造厂制造波导和微流控通道。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）在新生儿T细胞定量中的应用。由于母体植入T细胞前体可以挫败传统的SCID诊断方法，因此我们将利用新发现的新生儿T细胞标记物PTK7从新生儿脚跟粘血中捕获和枚举T细胞。3. 新生儿血液中B细胞的定量和整合，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，我们将从婴儿脚跟粘血中计数B细胞。最后，我们将把B细胞和T细胞检测合并为一个单一的诊断测试。

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.