Optimization of A Disk-Based Microfluidics System to Isolate Fetal Cells from Maternal Blood for Functional Analysis
| Autor: | Hua-Wei Tseng, 曾華偉 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Scientists have discovered that fetal nucleated red blood cells (fnRBCs) exist in maternal blood circulation with complete genetic information since 1978. If fnRBCs can be applied on non-invasive prenatal diagnosis (NIPD), it will become the vital goal of prenatal diagnosis. However, the fnRBCs are rare in maternal blood circulation (about 23 cells / mL), it is necessary to find a high-efficiency and purity technology to isolate. In recent years, microfluidics system have been widely used in rare cells isolation due to less sample, faster reaction time, low cost and high purity separation. The original microfluidics system was designed in 2013 by Wei-Lun Cheng. The principle of design was the density gradient centrifugation (DGC) and CD71 positive immunomagnetic separation to isolate fnRBCs from maternal blood. In addition the fnRBCs were identified by the presence of the fetal specific marker epsilon-globin gene. But the purity of fnRBCs was poor, we increased the volume of EasySep® Human Whole Blood CD45 Depletion Kit to 15µl for depletion of more CD45+ cells. We found that it had 4.2 times more efficiency than original condition. However, it was too impure to use in gene analysis. Hence, we tried to increase the recovery rate by optimizing the microfluidics system. The parameters were modified into the following points: (1) we diluted whole blood with PBS (1:1 dilution) as loading sample, instead of blood sample predepleted with CD45. (2) We increased the area of waste chamber to accommodate more wasted blood, and (3) the collection chamber could replace the chip to facilitate the observation and extraction. Next, we spiked K562 cells (100~500 cells) in blood as fnRBC model for recovery rate testing. The recovery rate of chip disc was 45.6%. In addition, the chip disk was more suitable then the original disc. Finally, isolated cells were extracted by the single cell picking technique (collaborate with NTU institute of applied mechanics) to overcome the problem of purity. Then, the single cells were amplified with whole genome amplification (WGA) kit and quantified by genomic PCR of bcr/abl genes. In the future, we will isolate fnRBC from maternal blood and compare the number of fnRBC with gestational age. On the other hand, we will try to verify the fnRBC by fluorescence in situ hybridization (FISH) or short tandem repeat (STR)/ variable number tandem repeat (VNTR) analysis. Taken together, we believe that the optimized microfluidic chip disk will bring the new NIPD methods in the future. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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