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

描述(申请人提供)：急性髓系白血病(AML)可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，这通常是通过外周血或骨髓的组织学评估来诊断的。这种方法不敏感，导致诊断复发，疾病负担较高，更难成功治疗。多参数流式细胞术(MFC)可以检测到较低的疾病负担(来自混合人群的0.1-0.01%的AML原始细胞)；但是，它用于需要频繁监测的疾病是昂贵和不切实际的，部分原因是需要分析骨髓。在这个R21项目中，一种新的处理策略将由一种廉价、易于制造和高度自动化的射流生物处理器来实施，该处理器用于直接从全血中选择和识别罕见的AML原始细胞，以便能够进行更频繁的测试，以便与MFC相比更早地检测出MRD。生物处理器将由固定在流体主板上的模块组成。模块和主板由热塑性塑料制成，具有通过复制产生的必要微结构。三个模块将用于使用捕获床从全血中亲和选择AML母细胞，捕获床由表面固定的抗体通过单链DNA双功能接头拴在选择通道壁上。这些抗体将针对表达CD33、CD34和CD117的原始细胞。选择模块将由50-250个微通道阵列组成，可在&lt；20分钟内处理大输入容量(2-10毫升)。通过将可切割单元设计成寡核苷酸双功能连接物，AML爆炸将从捕获床上释放出来。爆炸释放后，它们将被检测到，使用阻抗传感器将它们引导到具有预制过滤器的安全壳储水池中，以允许对爆炸进行免疫染色。最终的模块将由一个由无定形含氟聚合物制成的微型流式细胞仪组成，该细胞具有良好的光学性能和接近水的折射率(~1.3402@546 nm)(1.3331@546 nm)。该模块将通过将流动单元通道尺寸匹配到接近AML爆炸直径并用激光激励光束过量填充流动单元通道来实现无护套操作，以产生统一的强度分布。使用三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鉴定。这种流体生物处理器将被用来检验这一假设：SCT后检测到MRD将帮助临床医生在AML复发的早期阶段进行适当的治疗，以实现更高的治愈率。将进行一项试点研究，以测量AML患者的MRD状态，并将其与使用设计的流体生物处理器的血液学复发的开始相关联，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.