title: Non-invasive fetal sex determination on fetal erythroblasts from the maternal circulation using fluorescence in situ hybridisation.
abstract: OBJECTIVE
  The main purpose of this study was to evaluate the potential of a non-invasive method for fetal sex determination in clinical practice using dual-colour fluorescence in situ hybridisation (FISH) analysis. We evaluated the differences in nucleated red blood cell (NRBC) recovery from the maternal circulation using various slide preparation procedures following high-gradient magnetic cell separation (double MACS).
  
  
  METHODS
  NRBCs were enriched from peripheral blood mononuclear cells of 63 pregnant women between 12 and 37 weeks of gestation by double MACS involving simultaneous CD14+ and CD45+ maternal cell depletion and CD71+ fetal cell enrichment. Isolated cells were analysed by dual-colour FISH with X- and Y-specific probes. Before applying the FISH technique, cells were treated using three different protocols. Cells were either fixed in methanol:acetic acid (3:1) and dropped immediately onto glass slides (protocol 1) or treated with 75 mM KCl before resuspension in fixative (protocol 2). Alternatively, isolated cells were transferred onto glass slides and then treated using a method described in the literature (protocol 3).
  
  
  RESULTS
  Using various slide preparation procedures, fixed cell numbers as well as the quality of slides differed significantly. Using protocol 1, fetal sex was well determined in 30 cases, in 15 out of 17 male fetuses (1-13, mean 3 fetal cells were found among 5-164, mean 50 maternal cells) and in 15 female fetuses (7-178, mean 56 fixed cells). On the other hand, interpretation difficulties occurred in 7 out of 8 studied cases using protocol 2 due to a lack of cells or damage to the isolated cells. The highest recovery of fixed cells was achieved using protocol 3 (27-411, mean 186); fetal cells positive for the Y signal (2-12, mean 6) were detectable in all studied cases (n = 16). In 7 of the samples from women carrying female fetuses, we could only detect cells with two X signals (51-182, mean 103). All of the experiments were interpretable due to the presence of compact cells with well-visible red and green signals.
  
  
  CONCLUSIONS
  Our study revealed that using protocol 3 as the post-MACS treatment results in improved NRBC recovery and enables a reliable prospective non-invasive fetal sex determination.
authors.affiliation: 2nd Department of Paediatrics, 2nd Medical Faculty, University Hospital Motol, V Uvalu 84, CZ-150 18 Prague 5, Czech Republic. ilona.hromadníkova@lfmotol.cuni.cz
authors.forename: Ilona
authors.initials: I
authors.lastname: Hromadníková
authors.forename: Sultan
authors.initials: S
authors.lastname: Karamanov
authors.forename: Bela
authors.initials: B
authors.lastname: Houbová
authors.forename: Dana
authors.initials: D
authors.lastname: Hridelova
authors.forename: Josef
authors.initials: J
authors.lastname: Kofer
authors.forename: Miluse
authors.initials: M
authors.lastname: Mrstinova
mesh_terms: D041321:Chromosomes, Human, X; D041322:Chromosomes, Human, Y; D004900:Erythroblasts; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008431:Maternal-Fetal Exchange; D011247:Pregnancy; D012732:Sex Determination Analysis / Q000379:methods*