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

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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.