Acute Myeloid Leukemia: MRD Analysis Using Modular uFluidics and uFlow Cytometry

急性髓系白血病：使用模块化 uFluidics 和 uFlow 细胞术进行 MRD 分析

• 批准号: 8545485
• 负责人: Steven Allan Soper
• 金额: $ 29.67万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545485
• 关键词: Acute Myelocytic Leukemia; Affect; Affinity; Allogenic; Antibodies; Antigens; Back; Beds; Biological; Biological Assay; Blast Cell; Bone Marrow; C-KIT Gene; CD34 gene; Caliber; Cell Count; Cells; Clinical; Clinical Research; Collection; Color; Containment; Cytolysis; Cytometry; DNA; Data; Detection; Development; Diagnosis; Dimensions; Disease; Donor Lymphocyte Infusion; Early treatment; Engineering; Evaluation; FLT3 gene; Flow Cytometry; Fluorescence; Gene Mutation; Histologic; Lasers; Ligase; Lymphocyte; Measures; Metals; Methodology; Methods; Molds; Molecular; Molecular Profiling; Monitor; Mutation; Mutation Detection; Myelogenous; Neoplasm Circulating Cells; Noise; Oligonucleotides; Optics; Outcome; Output; Patients; Performance; Phase; Phenotype; Pilot Projects; Plastics; Polymers; Polymethyl Methacrylate; Population; Process; Property; RNA; Reaction; Reading; Recovery; Recurrence; Refractive Indices; Relapse; Residual Tumors; Sampling; Secure; Shapes; Side; Single-Stranded DNA; Solid; Staging; Staining method; Stains; Stem cell transplant; Surface; Survival Rate; System; Testing; Variant; WT1 gene; Water; Whole Blood; base; burden of illness; cost; design; electric impedance; experience; follow-up; improved; innovation; instrument; light microscopy; miniaturize; monocyte; novel; operation; peripheral blood; public health relevance; response; sensor; success

DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) can be cured through allogeneic stem cell transplantation (SCT). Unfortunately, 25% of patients will experience relapse after SCT that is usually diagnosed by histologic evaluation of peripheral blood or bone marrow. This method is insensitive and leads to diagnosis of relapse with a high disease burden, which is more difficult to successfully treat. Multi-parameter flow cytometry (MFC) can detect lower burden of disease (0.1-0.01% AML blasts from a mixed population); however, it is expensive and impractical for use in diseases that require frequent monitoring due in part to the need for analyzing bone marrow. In this R21 project, a novel processing strategy will be carried out by an inexpensive, easily manufactured, and highly automated fluidic bio-processor used to select and identify rare AML blasts directly from whole blood to allow more frequent testing to detect MRD at an earlier stage compared to MFC. The bio- processor will consist of modules poised on a fluidic motherboard. The modules and motherboard are made from thermoplastics with the prerequisite microstructures generated via replication. Three modules will be used to affinity-select AML blasts from whole blood using a capture bed comprised of surface immobilized antibodies tethered to the selection channel walls via single-stranded DNA bifunctional linkers. The antibodies will target CD33, CD34 and CD117 expressing blasts. The selection modules will consist of an array of 50-250 microchannels that can process large input volumes (2-10 mL) in <20 min. The AML blasts will be released from the capture bed by engineering a cleavable unit into an oligonucleotide bifunctional linker. Following blast release, they will be detected using an impedance sensor to direct them into a containment reservoir possessing a fabricated filter to permit immuno-staining of the blasts. The final module will consist of a micro- flow cytometer cell fabricated from an amorphous fluoropolymer, CYTOP, which has excellent optical properties and a refractive index (~1.3402 @ 546 nm) close to that of water (1.3331 @ 546 nm). This module will allow for sheath-less operation by matching the flow cell channel dimensions to near the diameter of the AML blasts and overfilling the flow cell channel with the laser excitation beams to produce a uniform intensity profile. Using a 3-color laser-induced fluorescence system, further immuno-phenotyping of the selected AML blasts will be secured. The fluidic bio-processor will be used to test the hypothesis: Detection of MRD following SCT will assist clinicians in administering proper therapies at an earlier stage of AML relapse to achieve higher cure rates. A pilot study will be performed to measure MRD status in AML patients and associate that with the onset of hematologic relapse using the designed fluidic bio-processor with results compared to MFC.

描述（由申请人提供）：可以通过同种异体干细胞移植（SCT）治愈急性髓性白血病（AML）。不幸的是，在SCT后，有25％的患者通常会通过周围血液或骨髓的组织学评估来诊断。该方法不敏感，导致疾病负担高的诊断复发，这更难成功治疗。多参数流式细胞仪（MFC）可以检测较低的疾病负担（混合种群的0.1-0.01％AML爆炸）；但是，对于需要经常进行监测的疾病，部分归因于分析骨髓，这是昂贵且不切实际的。在这个R21项目中，将通过一种廉价，易于制造且高度自动化的流体生物处理器来实施一种新颖的处理策略，用于直接从全血中选择和识别稀有的AML爆炸，以允许与MFC相比，在更早的阶段进行更频繁的测试以检测MRD。生物处理器将由在流体主板上准备的模块组成。模块和主板由热塑性塑料制成，其先决条件是通过复制产生的。使用单链DNA双功能连接器，将使用三个模块将全血的AML爆炸与全血相结合。抗体将靶向CD33，CD34和CD117表达爆炸。选择模块将由50-250个微通道的阵列组成，该阵列可以在<20分钟内处理大型输入体积（2-10 mL）。 AML爆炸将通过捕获床释放，通过将可切合的单元工程化为寡核苷酸双功能连接器。爆炸释放后，将使用阻抗传感器检测到它们，将其引导到具有制造过滤器的遏制储层中，以允许爆炸的免疫染色。最终模块将由由无定形荧光聚合物Cytop制成的微流动细胞电池组成，该细胞具有出色的光学特性和折射率（〜1.3402 @ 546 nm），接近水（1.33331 @ 546 nm）。该模块将通过将流动池通道尺寸与AML爆炸直径匹配，并用激光激发梁过度注入流量细胞通道，从而产生均匀的强度曲线，从而允许无护套操作。使用3色激光诱导的荧光系统，将确保所选AML爆炸的进一步免疫型。流体生物处理器将用于检验假设：SCT后MRD的检测将有助于临床医生在AML复发的早期阶段进行适当的治疗，以实现更高的治疗率。将进行一项试点研究，以测量AML患者中的MRD状态，并将其与使用设计的流体生物处理器与MFC相比，将其与血液学复发发作相关联。