Studies of Microfluidic Affinity Cell Separation in Bioanalysis for Clinical Applications

Microfluidic cell separation has been extensively developed in numerous biological and clinical assays due to the figures of merit including reduced sample volume, low cost, simple operation, fast processing, high sensitivity, and increased portability. In this dissertation, the fundamentals of microfluidics, the classification of microfluidic cell separation techniques, and the motivation of dissertation work are discussed in Chapter I. In Chapter II to Chapter IV, we present three studies of microfluidic affinity cell separation in clinical applications, from aspects of specific blood cell ratio measurement, interfering blood cell depletion, and isolation of immature precursor cell from normal blood cells. CD4/CD8 T lymphocyte ratio measurement is complementary to direct CD4 count in diagnosis of acquired immunodeficiency syndrome (AIDS). A tandem affinity microfluidic separation was described to measures the ratio of CD4+/CD8+ T lymphocytes from blood samples. Lysed blood samples were injected into a microfluidic device composed of two serially linked affinity regions coated with CD4 and CD8 monoclonal antibodies, respectively. CD4+ and CD8+ T lymphocytes were captured on the corresponding affinity regions using stop flow incubation. Fluorophore conjugated antibodies were then injected for staining and shear force differentiation. The CD4/CD8 ratio calculated after cell enumeration with our tandem affinity microfluidic system was closely consistent with that performed using conventional flow cytometry (R2=0.97) over a wide range from immune deficiency to normal conditions. This approach may represent a powerful and inexpensive tool to measure cell ratios and be potentially applied in clinical diagnosis of immunodeficiency disorders using cell ratio as biomarkers. The depletion of interfering monocytes is necessary for downstream isolation of specific lymphocytes and granulocytes. A combination of negative and positive microfluidic separation system was demonstrated to effectively deplete monocytes from blood prior to the downstream separation of activated neutrophils or helper T lymphocytes. A parallel three-dimensional flow design was developed to effectively deplete 60%-70% of CD14+ monocytes using affinity isolation within 30 min, which was comparable to the performance of commercialized Ficoll Paque product using small blood volumes. The downstream capture purity of activated neutrophils and helper T lymphocytes in herringbone chips were increased to 66.5%±4.1% and 90.9%±4.0%, respectively; compared to 31.9 % ±2.7 % and 58.2%±6.4% using the same flow condition if without monocyte depletion. This approach is a fast and economical tool for monocyte depletion to enhance the downstream separation of specific leukocytes in the diagnosis of sepsis and HIV/AIDS, and can potentially be further expanded to other separations when an interfering cell is present. The percentage of immature lymphoblast in peripheral blood is an important index for diagnosis and prognosis of Acute lymphocytic leukemia (ALL), which is the most prevalent pediatric cancer. A microfluidic isolation and enumeration of peripheral blood lymphoblasts using affinity separations was performed, with the innovation of using Human Transferring Receptor (CD71) as a nonspecific ligand to screen leukemia without a priori knowledge of the cancer type. It was found that anti-CD71 antibodies was more effective for lymphoblasts capture rather than commonly used, ALL-specific antibodies for CD7 and CD10. Lymphoblasts were spiked into blood samples with constitutions ranging from 7-30% of total leukocytes to simulate the leukemia condition. The isolation of lymphoblasts was achieved with 82-96% purity in herringbone featured microfluidic device, with the lowest concentrations assay (7%) still showing cell capture with >80% purity. The ability to isolate low percentage lymphoblasts well below the World Health Organization diagnosis limit indicated its potential in sensitive screening of ALL, as well as in follow-up monitoring of ALL relapse. This simple and inexpensive device using nonspecific CD71 ligand can be further expanded to fast leukemia screening without knowing the leukemia type.