Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Isak, M.
Molecular genetic analysis of single cells isolated from peripheral blood of pregnant women.
[ Diplomarbeit/Master Thesis ] Karl-Franzens University and University of Technology of Graz; 2014. pp.84.
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Kroneis Thomas
- Sedlmayr Peter
- Altmetrics:
- Abstract:
- Over the past several years the development of non-invasive techniques for prenatal genetic diagnosis has been carried forward. Rare cells circulating in the maternal blood can be analyzed. To isolate rare cells such as fetal cells or circulating tumor cells, different enrichment methods are available. This study was performed based on previous investigations of different enrichment systems. The ScreenCell® technique, which is based on the isolation by size, achieved an average recovery rate of 89%. In my master thesis this filtration device was used for the enrichment of circulating fetal cells such as trophoblast cells from the peripheral blood of ten pregnant women. The cells were immunolabelled with antibodies against trophoblast cells and counterstained with hematoxylin. Trophoblast cells are described as being larger than 15 μm and because the used antibody cocktail did not yield fetal cells, cells >15 μm in diameter were isolated from the filter by means of laser microdissection and pressure catapulting. For further analysis the DNA was amplified with a whole genome amplification method for single cells (GenomePlex ®).The quality of DNA was evaluated using a quadruplex PCR according to van Beers et al. (2006). 79% of the single cells that were successfully amplified had good DNA quality and were applicable for further investigations such as array-comparative genomic hybridization (a-CGH) or short tandem repeat (STR) analysis. The genotyping was not compatible with the GenomePlex ® method. 15 cells with good DNA quality from three different blood samples were investigated using the a-CGH method to determine genomic aberrations.The profile indicates female cells according to the female reference DNA. Since the karyotype results from the invasive prenatal diagnosis indicate two of the three fetuses to be of male sex, it can be excluded that these isolated cells analyzed with a-CGH are fetal origin.