Circulating fetal trophoblast enrichment and single cell genetic analysis in the context of cell-based non-invasive prenatal testing

Ondertitel:Aanrijking van circulerende foetale trofoblastcellen en genetische analyse van individuele cellen in de context van cel-gebaseerd niet-invasief prenataal testen

In this dissertation, the main goal was to contribute to the development of a beginning-to-end cell-based non-invasive prenatal test (cbNIPT). For the enrichment of circulating fetal trophoblasts (CFTs) from maternal blood, the VTX-1 Liquid Biopsy System was assessed. This technology, based on inertial microfluidics and laminar microscale vortices, allows the successful enrichment of CFTs from maternal blood in seven out of ten cases with minimal contaminating maternal blood cells. After enrichment, putative fetal cells need to be isolated individually and the true fetal origin of each individual cell must be confirmed prior to the detection of chromosomal abnormalities. To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Since WGA introduces errors, four WGA methods were compared regarding their suitability for downstream copy number variation (CNV) detection and short tandem repeat (STR) profiling for human identification. A different performance of the WGA methods is observed for unfixed and preserved cells, but overall, DOPlify™ WGA, Ampli1™ WGA Kit, and PicoPLEX® WGA Kit allow proper CNV detection and STR profiling, while REPLI-g Single Cell WGA Kit is not preferred. Finally, even with the most appropriate WGA method, incomplete STR profiling is expected after WGA on single cells. This challenges the reliable discrimination of single fetal and maternal cells in the context of cbNIPT, since mother and child share half of their DNA. Nevertheless, both length-based STR profiling and sequence-based SNP profiling proved to be capable of correctly discriminating single cells from a parent or an offspring after PicoPLEX WGA, with 100 % sensitivity and specificity, but a higher evidential value is obtained for SNP profiling.

Jaar van publicatie:2021

Toegankelijkheid:Closed