title: Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood.
abstract: Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.
authors.affiliation: Unité INSERM 370, Faculté de Médecine Necker, Paris, France.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Vona
authors.forename: Christophe
authors.initials: C
authors.lastname: Béroud
authors.forename: Alexandra
authors.initials: A
authors.lastname: Benachi
authors.forename: Alice
authors.initials: A
authors.lastname: Quenette
authors.forename: Jean Paul
authors.initials: JP
authors.lastname: Bonnefont
authors.forename: Serge
authors.initials: S
authors.lastname: Romana
authors.forename: Yves
authors.initials: Y
authors.lastname: Dumez
authors.forename: Bernard
authors.initials: B
authors.lastname: Lacour
authors.forename: Patrizia
authors.initials: P
authors.lastname: Paterlini-Bréchot
mesh_terms: D002469:Cell Separation / Q000379:methods; D048429:Cell Size; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D004210:Dissection; D005260:Female; D005819:Genetic Markers; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy / Q000097:blood*; D011261:Pregnancy Trimester, First; D020080:Tandem Repeat Sequences; D014327:Trophoblasts / Q000166:cytology* / Q000502:physiology*
chemical_list: D005819:Genetic Markers; D004247:DNA