Microfluidic diagnostics for Primary Immunodeficiency

描述(由申请人提供)：本提案的主要目标是开发一种新的技术来筛查新生儿的严重初级免疫缺陷(PID)，包括T细胞(例如SCID)和B细胞(例如XLA)的数量缺陷。在新生儿期诊断出这些缺陷可以挽救生命，降低社会成本。在试点工作中，我们开发了一种微流控通道系统，可以从一滴血中特异地捕获和标记免疫细胞。我们还开发了一种嵌入玻璃的波导型传感器，可以对位于波导芯消失场中的细胞进行定量。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，从而能够在新生儿托儿所进行护理点测试。它还将使检测成为可能，而不需要专门的培训、专门的技术人员、昂贵的试剂或处理干血迹。我们在这项应用中的目标是将这两项技术结合在一起，并建立所产生的设备可以对婴儿血液中的细胞进行定量，从而验证这一用于诊断新生儿期PID的新方法。我们的目标是：1.将波导和微流控技术相结合来捕获和定量人类血液中的细胞。我们将使用斯坦福纳米加工设施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，所产生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)定量检测新生儿T细胞的方法。由于母体植入T细胞前体细胞可能会破坏传统的诊断SCID的方法，我们将利用新发现的新生儿T细胞PTK7标记物来捕获和计数新生儿足跟棒血中的T细胞。3.对新生儿外周血中的B细胞进行定量，并结合同时计数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，我们将从婴儿鞋跟棒血中计数B细胞。最后，我们将把B细胞和T细胞检测结合到一个单一的诊断测试中。 与公共卫生相关：在美国，每1200人中就有1人患有原发免疫缺陷(PID)。如果可以在新生儿中诊断出严重的PID，那么早期治疗将挽救生命并降低社会成本。这些研究将使开发一种新的检测技术成为可能，该技术可以以非常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.