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.

Public Health Relevance

Donor lymphocyte infusion (DLI) is typically used for acute myeloid leukemia (AML) patients whom display relapse following stem cell transplantation (SCT). However, response rates are <20% for patients that show signs of a full hematologic relapse. This poor outcome results from inadequacies of the methodology (light microscopy or multi-parameter flow cytometry, MFC) used to diagnose relapse or the onset of minimum residual disease (MRD). In this application, we will evaluate the performance of an innovative assay and fully automated system to detect the onset of MRD in AML from peripheral blood with much higher sensitivity compared to MFC. The system consists of a fluidic bio-processor composed of modules interfaced to a motherboard. Three modules, with each targeting different antigens, are used for selecting rare AML blasts and another for immuno-staining the selected blasts. The last module consists of a miniaturized flow cytometer cell to immuno-phenotype the selected blasts using a 3-color laser fluorescence instrument.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZCA1)
Program Officer
Ossandon, Miguel
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University of North Carolina Chapel Hill
Biomedical Engineering
Schools of Medicine
Chapel Hill
United States
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