Microfluidic Immunophenotyping for the Diagnosis of Uveitis and Ocular Cancer

项目编号：0827868该项目的目标是创建一个能够表征玻璃体幽默细胞和细胞因子含量的微流控平台。该平台将直接造福于患有葡萄膜炎或原发性眼内淋巴瘤（PIOL）的患者，这些患者需要进行手术切除玻璃体（玻璃体活检）以获得这些疾病的详细诊断。玻璃体微流控免疫分型是目前细胞学和流式细胞术方法的一种有吸引力的替代方法，因为它需要相当少的细胞，并且可以快速分析通常非常脆弱的细胞。葡萄膜炎和PIOL都是与淋巴细胞侵入眼睛不同区域有关的疾病。大约有230万美国人患有葡萄膜炎，葡萄膜炎导致的视力下降占美国失明的10-15%。PIOL表现为恶性b淋巴细胞的存在，这可能表明肿瘤在大脑、视网膜或中枢神经系统的其他区域形成。虽然PIOL是一种罕见的疾病，但由于缺乏早期检测技术，PIOL患者的康复可能性很低。进行玻璃体活检以区分PIOL和葡萄膜炎，并确定有针对性的治疗途径。对于PIOL，早期发现可以让患者开始化疗和放疗。对于葡萄膜炎，确定其潜在原因可以使用特异性药物治疗，而不是通常会导致严重副作用的广谱靶向免疫抑制剂。目前分析玻璃体活检产品的方法仅对15%的患者提供结论性诊断，因为它受到可用于分析的细胞数量少及其脆弱性的限制。为完成项目目标，将进行以下任务：-设计和制造基于抗体-抗原相互作用的细胞和细胞因子捕获微流体装置。-基于溶解抗体梯度的细胞趋化偏转，创建一种微流控免疫表型技术。-使用模拟从葡萄膜炎或PIOL患者身上提取的玻璃体体液的细胞和细胞因子含量的模型来表征微流体装置的性能。-确定每种疾病类型的多种标志物的靶细胞和细胞因子浓度的下限。-从患者玻璃体活检样本中获得的玻璃体液中的免疫表型细胞和细胞因子。智力优势：如果成功，该研究将提供一种新的变革性方法来快速识别与葡萄膜炎和PIOL相关的细胞和细胞因子，克服当前分析技术的缺点。提出的微流体方法的一部分是技术变革的基于梯度的设计，以识别单个细胞上的多个细胞表面标记物，而无需任何标记。从非常小的样本量中确定细胞和细胞因子类型的能力将允许早期诊断PIOL，这是使用当前技术无法进行的。PI在微流控细胞分析领域工作了5年多，该项目是与波士顿Schepens眼科研究所的眼科外科医生Kameran Laskhari博士合作完成的。更广泛的影响：(a)如果成功，拟议的研究将为葡萄膜炎或PIOL患者提供准确和快速的诊断技术。(b)通过东北大学现有的青年学者项目和RET项目，让高中学生和理科教师参与实验研究；(c)让本科生参与实验研究。(d)将鼓励参加拟议研究的高中学生开发科学展览项目，并从事生物医学科学和工程方面的职业。(e)通过网站传播研究结果并说明对葡萄膜炎和PIOL治疗的影响。

PI: Murthy, ShashiProposal Number: 0827868The objective of this project is to create a microfluidic platform capable of characterizing the cellular and cytokine content of vitreous humor. This platform will be of direct benefit to patients suffering from uveitis or primary intraocular lymphoma (PIOL) who need to undergo surgical removal of the vitreous humor (vitreous biopsy) in order to obtain a detailed diagnosis of these diseases. Microfluidic immunophenotyping of vitreous humor is an attractive alternative to the current approaches of cytology and flow cytometry because it requires considerably fewer cells and allows rapid analysis of cells that are typically very fragile. Both uveitis and PIOL are diseases associated with the invasion of lymphocytes into various regions of the eye. Approximately 2.3 million Americans suffer from uveitis, and vision-loss due to uveitis accounts for 10-15% of blindness in the U.S. PIOL is manifested by the presence of malignant B-lymphocytes which may indicate tumor formation in the brain, retina, or other areas of the central nervous system. Although PIOL is an uncommon disease, patients of PIOL have a low likelihood of recovery because of the lack of early detection techniques.The vitreous biopsy is performed to distinguish PIOL from uveitis and identify a targeted therapeutic path. For PIOL, early detection allows patients to begin chemo- and radio-therapy. For uveitis, identification of its underlying cause allows treatement with specific drugs instead of broad-target immunosuppressive agents that typically cause severe side effects. The current approach to analyze the vitreous biopsy product provides a conclusive diagnosis in only 15% of patients because it is constrained by the small number of cells available for analysis and their fragility.The following tasks will be carried out to accomplish the project objective:- Design and fabricate microfluidic devices for cell and cytokine capture based on antibody-antigen interactions.- Create a microfluidic immunophenotyping technique based on the chemotactic deflection of cells using gradients of dissolved antibodies.- Characterize the performance of the microfluidic devices using a model that mimics the cellular and cytokine content of vitreous humor extracted from patients of uveitis or PIOL.- Determine lower bounds of target cell and cytokine concentration for the multiple markers of each disease type.- Immunophenotype cells and cytokines in vitreous fluid obtained from patient vitreous biopsy samples.Intellectual Merit: If successful, the proposed research will provide a new and transformative method to rapidly identify cells and cytokines associated with uveitis and PIOL, overcoming the shortcomings of current analysis techniques. Part of the proposed microfluidic approach is technologically transformative gradient-based design to identify multiple cell surface markers on individual cells without requiring any labeling. The ability to determine cell- and cytokine-type from very small sample sizes will allow for early diagnosis of PIOL, which cannot be performed using current techniques. The PI has worked in the area of microfluidic cell analysis for over 5 years and this project is a collaborative effort between with Dr. Kameran Laskhari, an ophthalmic surgeon at the Schepens Eye Research Institute in Boston.Broader Impact: (a) If successful, the proposed research will provide an accurate and rapid diagnosis technique for patients with uveitis or PIOL. (b) The proposed research will involve high school students and science teachers in experimental research via existing Young Scholar and RET programs at Northeastern University, and (c) involve undergraduates in experimental research. (d) High school students participating in the proposed research will be encouraged to develop science fair projects and pursue careers in biomedical science and engineering. (e) Disseminate research results and describe implications for uveitis and PIOL therapeutics through a website.