Detection and relocation of cord blood nucleated red blood cells by laser scanning cytometry

Abstract

Background: Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. Methods: CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-γ) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. Results: In CB, 59% of events double positive for Hb-γ and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. Conclusions: Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible. © 2002 Wiley-Liss, Inc.