Separation and Detection of Rare Cells in a Microfluidic Disk Platform
Date Issued
2010
Date
2010
Author(s)
CHEN, CHEN-LIN
Abstract
Rare cells in blood often possess high clinical significance. However, cyto-analysis of rare cells often requires separation and detection with either procedure of substantial challenge. Circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients represent a potential alternative to invasive biopsies as a source of tumor tissue for detection, characterization, and monitoring of non-haematologic cancer. This thesis outlines a novel disk-based microfluidic device to capture and detect rare cells from blood sample. Immunomagnetic negative selection and positive selection approaches were demonstrated in our disk platforms.
For negative selection approach, the microfluidic platform’s unique features include multistage magnetic gradient to trap labeled cells in double trapping areas, drainage of fluid to substantially shorten detection time, and bin-like regions to capture target cells to facilitate seamless enumeration process. Proof-of-concept was conducted using wide range of MCF7 as target rare cells (stained with anti-cytokeratin-FITC antibodies) and spiked into Jurkat Clone E6-1 non-target cells (labeled with anti-CD45-PE and anti-PE BD magnetic beads). Then, mononuclear cells (MNC) from healthy blood donors were mixed with MCF7s, modeling rare cells, and tested in the disk. Results show the average yield of detected MCF7 is near-constant 60±10% over a wide range of rarity from 10-3 to 10-6 and this yield also holds for MCF7/MNC complex mixture. Comparison with autoMACS and BD IMagnet separators revealed the average yield from the disk (60%) is superior to that of autoMACS (37.3%) and BD IMagnet (48.3%).
For positive selection testing, as proof-of-concept, experiments were conducted where MCF7 was used to simulate CTCs and healthy whole blood was used for background peripheral blood. A continual flow process via a centrifugal microfluidic disk platform to capture MCF7 in blood immunomagnetically and enumerate them on-disk with a complete batch process of multi-fluorescence labeling is presented. The MCF7 are labeled with anti-EpCAM-PE and anti-PE magnetic beads for magnetic force capturing and with anti-cytokeratin-FITC antibodies and Hoechst33342 for detection. In order to allow precise timing in liquid delivery during the multi-fluorescence labeling processes, on-disk deterministic vent valves were designed. To characterize the disk performance of target cell capturing and fluorescence labeling, three different labeling procedures were used. Results show that the cell-capture yield of the disk was about 65% and the throughput was 2ml/hr or more. After staining two-label fluorescence on the disk, the yield was around 50%. The sensitivity of the technique in enriching rare cells from whole blood (>1ml) is up to 10-7. Direct fluorescence labeling on the disk without sample transfer and manual operation greatly helped to reduce cell loss. The total procedure, from magnetic bead labeling to completing two-label fluorescence staining, takes place within 1.5 hours. In order to determine the efficiency of the disk in enumerating CTC from patient with epithelial cancers, the breast cancer data of patients were collected. Advantages of the present platform include simple operation, high throughput, an acceptable level of cell loss, and a potentially low system cost, which should substantially ease the effect in cyto-analysis of rare cells.
Subjects
rare cell
immunomagnetic separation
microfluidics
disk
centrifugal force
SDGs
Type
thesis
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