<- Home <- Arhive <- Vol. 7, Issue 1, February 2011

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Detection of Nucleated Red Blood Cells in Maternal Circulation by Magnetic Sorting and in situ Hybridization

D. Nemescu, D. Constantinescu, V. Martiniuc, M. Onofriescu, E. Carasevici

Abstract: Nucleated red blood cells (NRBC) from ma ter nal blood are a frequent researched cell group for non-invasive prenatal diagnosis. We evaluated the feasibility of analysis of fetal NRBC present in the ma ter nal circulation by fluorescence in situ hybridization (FISH), after enrichment by magnetic sorting, without any other cell localization procedures. From 31 pregnant women, cells have been isolated from maternal blood with a combination of double-density gradient centrifugation followed by a positive magnetic-activated cell sorting (MACS) enrichment, using anti-CD71 monoclonal antibody. Later, we performed FISH with chromosome X and Y specific probes. After automatic microscopic recording of the whole cell area from the slide, the fluorescent signals have been evaluated by manual analysis of images. The results were compared with fetal sex as results from amniocentesis or at birth. Results. FISH analysis detected at least one nucleus with XY signal in 59% of women bearing male fetuses, with a mean gestational age of 20 weeks (range: 15-30). Small number of fetal cells was found (range: 1-11) with a mean of one erythroblast in one milliliter of maternal blood. Isolation and identification of fetal NRBC from maternal circulation re pre sent a considerable challenge due to their extremely low number. FISH analysis associated with MACS identified these cells in maternal blood from first and second trimester. The efficiency of detection was low, not enough for a clinical diagnosis Thus, although they are considered the best target for non-invasive prenatal diagnosis at the moment, their detection remains problematic, with a great variability of results, due to still incomplete known factors.
Keywords: prenatal diagnosis, erythroblast, CD71, magnetic-activated cell sorting (MACS), flu o res cent in situ hybridization (FISH)

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