Fetal nucleated red blood cells in peripheral blood of pregnant women: detection and determination of location on a slide using laser-scanning cytometry.
OBJECTIVE
The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry.


METHODS
CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis.


RESULTS
In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC.


CONCLUSIONS
On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.
Hennerbichler
Simone
S
Institute of Histology and Embryology, Karl-Franzens-University Graz, Austria.
Kroisel
Peter M
PM
Zierler
Hannelore
H
Pertl
Barbara
B
Wintersteiger
Reinhold
R
Dohr
Gottfried
G
Sedlmayr
Peter
P
D000328:Adult; D002869:Chromosome Aberrations; D041322:Chromosomes, Human, Y / Q000235:genetics; D004900:Erythroblasts; D005260:Female; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D008423:Maternal Age; D011237:Predictive Value of Tests; D011247:Pregnancy / Q000097:blood*; D011261:Pregnancy Trimester, First / Q000097:blood; D011262:Pregnancy Trimester, Second / Q000097:blood; D018566:Pregnancy, High-Risk; D011296:Prenatal Diagnosis