title: Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.
abstract: The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because 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. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) 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 using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling 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 and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.
authors.affiliation: Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kroneis
authors.forename: Liat
authors.initials: L
authors.lastname: Gutstein-Abo
authors.forename: Kristina
authors.initials: K
authors.lastname: Kofler
authors.forename: Michaele
authors.initials: M
authors.lastname: Hartmann
authors.forename: Petra
authors.initials: P
authors.lastname: Hartmann
authors.forename: Marianna
authors.initials: M
authors.lastname: Alunni-Fabbroni
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Walcher
authors.forename: Gottfried
authors.initials: G
authors.lastname: Dohr
authors.forename: Erwin
authors.initials: E
authors.lastname: Petek
authors.forename: Esther
authors.initials: E
authors.lastname: Guetta
authors.forename: Peter
authors.initials: P
authors.lastname: Sedlmayr
mesh_terms: D000028:Abortion, Induced; D000918:Antibody Specificity / Q000276:immunology; D001331:Automation / Q000379:methods; D002460:Cell Line; D046528:Chimerism; D002824:Chorionic Villi / Q000378:metabolism; D016172:DNA Fingerprinting; D003656:Decidua / Q000166:cytology*; D005260:Female; D005333:Fetus / Q000166:cytology; D005434:Flow Cytometry; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D056656:Lab-On-A-Chip Devices; D007963:Leukocytes, Mononuclear / Q000166:cytology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D015203:Reproducibility of Results; D014327:Trophoblasts / Q000166:cytology / Q000276:immunology