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Microfluidic Functional Immunophenotyping of Pediatric Patients Following Cardiop

儿科患者心脏手术后的微流控功能免疫表型

基本信息

批准号：
9273597
负责人：
TIMOTHY T CORNELL
金额：
$ 66.4万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-18 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9273597
关键词：
1 year old Address Anti-inflammatory Antibodies Biological Assay Biosensing Techniques Blood Blood Volume Blood specimen CD14 gene CD4 Positive T Lymphocytes Cardiopulmonary Bypass Cells Cessation of life Child Childhood Clinical Critically ill children Dangerousness Data Dendritic Cells Detection Development Devices Effector Cell Enzyme-Linked Immunosorbent Assay Flow Cytometry Granulocyte-Macrophage Colony-Stimulating Factor HLA-DR Antigens IL8 gene Immune Immune System Diseases Immune System and Related Disorders Immune Targeting Immune system Immunologic Monitoring Immunophenotyping Immunosuppression Incidence Infection Inflammatory Inflammatory Response Interferons Interleukin-6 Lead Lesion Leukocytes Lipopolysaccharides Measurement Methodology Methods Microfluidics Microspheres NCAM1 gene Natural Killer Cells Operative Surgical Procedures Optics Outcome Patients Pharmaceutical Preparations Physicians Postoperative Period Protocols documentation Research Risk Standardization Surface Antigens TNF gene Technology Testing Time Whole Blood base congenital heart disorder cytokine design functional status hemodynamics immune function immune system function immunological status immunoregulation immunosuppressed individual patient monocyte mortality pathogen pediatric patients peripheral blood prevent prospective public health relevance receptor tool

项目摘要

DESCRIPTION (provided by applicant): Mortality associated with congenital heart disease (CHD) accounts for up to 50% of pediatric deaths with over 48% of CHD mortality occurring in children less than 1 year of age. Post-operative infections contribute significantly to this mortalty with a recent study indicating a mortality rate of nearly 25% associated with post- operative infections. Nearly all children with CHD require surgical correction of their lesions necessitating the use of cardiopulmonary bypass (CPB). However, CPB stimulates the immune system to produce pro- inflammatory cytokines that are counteracted by a compensatory anti-inflammatory response generated by the immune system. In some children, this compensatory anti-inflammatory response remains prolonged, leading to an "immunoparalyzed" state in which the pediatric patients are no longer able to fend off pathogens resulting in post-operative infections. Importantly, immunoparalysis can be revered in critically ill children by treating them with granulocyte macrophage colony-stimulating factor (GM-CSF). Even though immunoparalysis is associated with the dysregulation of immune function and the devastating consequences of post-operative infections, it remains a challenge for clinicians to identify those children who are immunoparalyzed and may benefit from GM-CSF to prevent post-operative infections. One major reason for the limited identification of children with immunoparalysis is the lack of validated immunomonitoring platforms that can allow real-time determinations of immunoparalysis. Two current methods exist to detect immunoparalysis: whole blood stimulation assay, which involves measurements of the capacity of peripheral blood leukocytes to produce inflammatory cytokines following pathogen stimulation using ELISA, and flow cytometry for enumeration of peripheral blood monocytes expressing HLA-DR receptors. The whole blood stimulation assay is time-consuming and can only provide the clinician with a determination of immunoparalysis in 72 hrs at the earliest. Flow cytometry for enumeration of peripheral blood monocytes expressing HLA-DR receptors can provide fast results; however it does not reflect the functional status of monocytes. Further, current protocols for isolation and functional testing of key leukocyte subpopulations (such as monocytes) require blood volumes that make such testing dangerous in small children. Thus, to understand the impact of immunoparalysis on outcomes for children following CPB as well as to identify functional status of key leukocyte subpopulations that are dysregulated in children who are immunoparalyzed, there exists a significant need for a real-time, functional immunophenotyping method that can achieve accurate, multiplexed functional cellular characterization of subpopulations of immune cells using only minute amounts of patient blood. To address this need, our central objective of this research is to develop an integrated microfluidic immunosensing platform for efficient isolation, enrichment, enumeration, and sensitive multiplexed functional immunophenotyping of subpopulations of immune cells from blood specimens. To extend our research to make clinical impact, we propose to implement our microfluidic immunomonitoring technology to further determine the incidence and impact of immunosuppression in critically ill children and its functional connection to post-operative infections. The Specific Aims of this collaborative R01 research thus are: (Aim 1) To develop and validate an integrated microfluidic immunophenotyping assay (MIPA) device for rapid, accurate, and sensitive parallel detection of multiple cytokines secreted by immune cells; (Aim 2) To develop and validate an integrated MIPA device for efficient isolation, enrichment, enumeration, and multiplexed functional immunophenotyping of subpopulations of immune cells from blood specimens; (Aim 3) To utilize the MIPA device to identify immunoparalyzed pediatric patients following CPB and determine its predictive power for post-operative infections.

描述（由申请人提供）：与先天性心脏病（CHD）相关的死亡率占儿科死亡的50%，其中超过48%的CHD死亡发生在1岁以下的儿童中。术后感染对死亡率有显著影响，最近的一项研究表明，术后感染的死亡率接近25%。几乎所有患有CHD的儿童都需要手术矫正其病变，使用心肺转流术（CPB）。然而，CPB刺激免疫系统产生促炎细胞因子，其被免疫系统产生的补偿性抗炎反应抵消。在一些儿童中，这种代偿性抗炎反应仍然延长，导致“免疫瘫痪”状态，其中儿科患者不再能够抵御病原体，导致术后感染。重要的是，免疫瘫痪可以通过粒细胞巨噬细胞集落刺激因子（GM-CSF）治疗危重患儿来逆转。尽管免疫麻痹与免疫功能失调和术后感染的破坏性后果有关，但临床医生仍然面临着一个挑战，即识别那些免疫麻痹的儿童。免疫瘫痪，并可能受益于GM-CSF，以防止术后感染。免疫麻痹儿童识别有限的一个主要原因是缺乏经过验证的免疫监测平台，可以实时确定免疫麻痹。目前存在两种检测免疫麻痹的方法：全血刺激测定，其涉及使用ELISA测量外周血白细胞在病原体刺激后产生炎性细胞因子的能力，以及流式细胞术用于计数表达HLA-DR受体的外周血单核细胞。全血刺激试验是耗时的，并且最早只能在72小时内为临床医生提供免疫麻痹的确定。流式细胞术用于计数表达HLA-DR受体的外周血单核细胞可以提供快速结果;然而，它不能反映单核细胞的功能状态。此外，目前用于关键白细胞亚群（如单核细胞）的分离和功能测试的方案需要血液体积，这使得这种测试在幼儿中是危险的。因此，为了了解免疫麻痹对CPB后儿童结局的影响以及鉴定免疫麻痹儿童中失调的关键白细胞亚群的功能状态，存在对实时功能免疫表型分析方法的显著需求，该方法可以仅使用微量患者血液实现免疫细胞亚群的准确、多重功能细胞表征。为了满足这一需求，本研究的中心目标是开发一种集成的微流控免疫传感平台，用于血液样本免疫细胞亚群的有效分离，富集，计数和敏感的多重功能免疫表型分析。为了扩大我们的研究以产生临床影响，我们建议实施我们的微流控免疫监测技术，以进一步确定重症儿童免疫抑制的发生率和影响及其与术后感染的功能联系。因此，这项合作R 01研究的具体目的是：（目的1）开发和验证集成微流控免疫表型分析（MIPA）装置，用于快速，准确和灵敏地平行检测免疫细胞分泌的多种细胞因子;（目标2）开发和验证一种集成的MIPA装置，用于有效分离、富集、计数，以及来自血液样本的免疫细胞亚群的多重功能免疫表型;（目的3）利用MIPA装置来识别CPB后免疫瘫痪的儿科患者，并确定其对术后感染的预测能力。