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

描述（由申请人提供）：急性髓性白血病（AML）可以通过异基因干细胞移植（SCT）治愈。不幸的是，25%的患者在SCT后会复发，通常通过外周血或骨髓的组织学评估来诊断。多参数流式细胞术（MFC）可以检测较低的疾病负担（来自混合人群的0.1-0.01% AML原始细胞）;然而，对于部分由于需要分析骨髓而需要频繁监测的疾病来说，它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、高度自动化的流体生物处理器来执行一种新的处理策略，该生物处理器用于直接从全血中选择和识别罕见的AML原始细胞，以允许与MFC相比更频繁地检测MRD。生物处理器将由安放在流体母板上的模块组成。模块和主板由热塑性塑料制成，通过复制产生先决条件的微结构。将使用三个模块，使用捕获床从全血中亲和选择AML原始细胞，所述捕获床由通过单链DNA双功能接头连接至选择通道壁的表面固定化抗体组成。抗体将靶向表达CD 33、CD 34和CD 117的原始细胞。选择模块将由50-250个微通道阵列组成，可以在<20分钟内处理大输入体积（2-10 mL）。通过将可切割单元工程化到寡核苷酸双功能接头中，AML原始细胞将从捕获床释放。在爆炸释放后，将使用阻抗传感器检测它们，以将它们引导到具有制造的过滤器的容纳容器中，以允许对爆炸进行免疫染色。最终的模块将包括由无定形含氟聚合物CYTOP制造的微流式细胞仪池，CYTOP具有优异的光学性质和接近水的折射率（1.3331@546 nm）的折射率（~ 1.3402@546 nm）。该模块将允许通过将流动池通道尺寸匹配到AML原始细胞的直径附近并用激光激发光束过度填充流动池通道以产生均匀的强度分布来进行无鞘操作。使用3色激光诱导荧光系统，将确保对选定的AML原始细胞进行进一步的免疫表型分析。流体生物处理器将用于测试假设：SCT后MRD的检测将有助于临床医生在AML复发的早期阶段给予适当的治疗，以实现更高的治愈率。将进行一项试点研究，以测量AML患者的MRD状态，并使用设计的流体生物处理器将其与血液学复发的发生相关联，并将结果与MFC进行比较。

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.