Enumeration of Metastatic Cancer Cells from Whole Blood in a Microfluidic Lab-on-disk Platform via Online Multi-fluorescence Labeling and Deterministic Vent Valves
| Autor: | Cheng-Wei Yang, 楊正偉 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Capture and detection of specific rare cells have shown great promise for biological and clinical studies. 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 microfluidic device to capture and detect CTCs in patient blood. As proof-of-concept, experiments were conducted where a cell line of 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 14 women were collected (as shown in Appendix 2). 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|