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

• 批准号: 8894576
• 负责人: TIMOTHY T CORNELL
• 金额: $ 63.94万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/8894576
• 关键词: 1 year old; Accounting; Address; Anti-Inflammatory Agents; 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; Data; Dendritic Cells; Detection; Development; Devices; Effector Cell; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-DR Antigens; Health; IL8 gene; Immune; Immune System Diseases; Immune System and Related Disorders; Immune Targeting; Immune system; Immunologic Monitoring; Immunophenotyping; Immunosuppression; Incidence; Individual; Infection; Inflammatory; Inflammatory Response; Interferons; Interleukin-6; Lead; Lesion; Leukocytes; Lipopolysaccharides; Measurement; Methodology; Methods; Microfluidics; Microspheres; Monitor; NCAM1 gene; Natural Killer Cells; Operative Surgical Procedures; Optics; Outcome; Patients; Pharmaceutical Preparations; Physicians; Postoperative Period; Protocols documentation; Research; Risk; Surface Antigens; TNF gene; Technology; Testing; Time; Whole Blood; base; congenital heart disorder; cytokine; design; functional status; hemodynamics; immune function; immunoregulation; immunosuppressed; monocyte; mortality; pathogen; pediatric patients; peripheral blood; prevent; receptor; tool

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(申请人提供)：与先天性心脏病(CHD)相关的死亡占儿童死亡的50%，其中超过48%的CHD死亡发生在1岁以下的儿童中。术后感染对这一死亡率有很大影响，最近的一项研究表明，与术后感染有关的死亡率接近25%。几乎所有患有冠心病的儿童都需要手术矫正他们的皮损 体外循环(CPB)的使用。然而，CPB刺激免疫系统产生促炎细胞因子，这些细胞因子被免疫系统产生的代偿性抗炎反应所抵消。在一些儿童中，这种代偿性抗炎反应持续时间较长，导致儿科患者不再能够抵御导致手术后感染的病原体的“免疫麻痹”状态。 重要的是，危重儿童的免疫麻痹可以通过粒细胞巨噬细胞集落刺激因子(GM-CSF)治疗而逆转。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，但对临床医生来说，识别哪些儿童是 免疫麻痹，并可能受益于GM-CSF以预防术后感染。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：全血刺激试验和流式细胞术。全血刺激试验包括用ELISA法测量病原体刺激后外周血白细胞产生炎性细胞因子的能力，以及流式细胞术计数外周血单核细胞表达HLA-DR受体的能力。全血刺激试验耗时长，最快只能在72小时内为临床医生提供免疫麻痹的确定。流式细胞术计数外周血单核细胞表达HLA-DR受体可提供快速结果，但不能反映单核细胞的功能状态。此外，目前用于分离和测试关键白细胞亚群(如单核细胞)的方案需要血液容量，这使得这种测试在小孩子中变得危险。因此，为了了解免疫麻痹对CPB术后儿童预后的影响，以及确定免疫瘫痪儿童中调节失调的关键白细胞亚群的功能状态，迫切需要一种实时的、功能性的免疫表型方法，该方法能够仅使用微量的患者血液来实现免疫细胞亚群的准确、多元的功能细胞表征。为了满足这一需求，本研究的中心目标是开发一个集成的微流控免疫传感平台，用于高效分离、浓缩、计数和灵敏地对血液标本中的免疫细胞亚群进行多重功能免疫表型鉴定。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体免疫监测技术，以进一步确定危重儿童免疫抑制的发生率和影响及其与手术后感染的功能联系。因此，此次合作R01研究的具体目的是：(1)开发和验证集成微流控免疫表型分析(MIPA)设备，用于快速、准确和灵敏地并行检测免疫细胞分泌的多种细胞因子；(2)开发和验证集成微流控免疫表型分析设备，用于高效分离、浓缩、计数和多重功能检测血液标本中的免疫细胞亚群；(目的3)利用MIPA设备鉴定体外循环后免疫麻痹患儿，并确定其对术后感染的预测能力。