On-chip multiplex PCR identification of automatically retrieved single microchimeric cells

Abstract

The analysis of rare cells is a complex task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis since it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. I have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labeling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser-catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested by using samples containing mixed cells of related and non-related individuals. Single cell DNA fingerprinting was successful in 74% (55/74) of the cells analyzed with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was performed on both cells enriched from blood and cells isolated from tissue. Identification was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes. Thus, I suggest DNA profiling as a standard for the identification of microchimerism on a single cell basis.

Datum der Bewilligung	2009
Originalsprache	Englisch
Gradverleihende Hochschule	Medizinische Universität Graz
Betreuer/-in	Gottfried Dohr (Betreuer*in), Erwin Petek (Betreuer*in) & Peter Sedlmayr (Betreuer*in)