Isolation and Characterization of Fetal Trophoblasts in Maternal Blood Using a Microfluidic Disk System for Non-invasive Prenatal Diagnosis
| Autor: | Li-Kuo Lu, 呂立國 |
|---|---|
| Rok vydání: | 2019 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 107 According to the National Health Insurance Administration Ministry of Health and Welfare, the proportion of pregnant women over 35 years old is close to 30%. This phenomenon leads to an increasing proportion of birth defects and stillbirths, so prenatal testing becomes ever more important. Amniocentesis and chorionic villus sampling(CVS) are considered the gold standard for traditional invasive prenatal genetic testing. However, the miscarriage rates were 1.4% after amniocentesis and 1.9% after CVS. Therefore, non-invasive prenatal diagnosis (NIPD) is highly regarded, which can be divided into two categories: obtaining fetal cell-free DNA in maternal blood and separating fetal nucleated cells (cell-based NIPD; cbNIPD) from maternal blood. The cell-free fetal DNA might not receive the complete set of genetic information about fetal. Therefore, the development of isolating fetal nucleated cells approach has the potential to enable comprehensive fetal diagnosis. In 1978, scientists successfully detect and enrich the circulating fetal cells. This study proposes to isolate and characterize fetal extravillous trophoblasts (fetal EVTs) using a microfluidic disk system. The development of microfluidic technology has provided a more effective method for the purify of fetal cells in recent years. We describe a microfluidic disk system that uses density centrifugation to separate fetal EVTs and other nucleated blood cells from a large amount of maternal blood. The blood of 16-20 ml from pregnant women were collected from the Obstetrics and Gynecology Department of National Taiwan University Hospital. The pregnant women were between 31-43 years old and 16-19 weeks pregnant. Fetal cells were identified by immunofluorescence staining (Hoechst, anti-TBA FITC, anti-cytokeratin PE, anti-CD45 APC, anti-CD66b APC). The target cells were recovered by single cell picking technique. The retrieved fetal/maternal cells were confirmed by downstream genomic analysis, including whole genome amplification, polymerase chain reaction, and short tandem repeat. Results show that SRY was successfully demonstrated during the PCR process which enables sex-determining region Y. STR profiling among fetal EVT-derived, maternal cell-derived, and matching amniocentesis STR fingerprints, revealing the feto-maternal relationship between the fetal EVTs and maternal cells. The positive predictive value of the microfluidic disk system for the target fetal EVTs was 53.8%. Future study expects to increase the positive predictive value and provides a solution to researchers and clinicians. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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