Assessment of efficacy of cell separation techniques used in the enrichment of foetal erythroblasts from maternal blood: triple density gradient vs. single density gradient

Summary The aim of this study was to determine the efficacy of cell separation with single density and triple density‐gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11–14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density‐gradient separation and the other, for triple density‐gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti‐CD71/antiglycophorin‐A. The enriched erythroblasts were stained with Kleihauer–Giemsa and with fluorescent antibodies for the γ, ɛ and ζ globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X‐ and Y‐chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin‐A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for γ, ɛ and ζ globin chains and with Kleihauer–Giemsa was significantly higher in the triple density‐gradient separation fractions compared with the single density‐gradient fractions with both anti‐CD71 and GPA MACS. FISH analysis for the Y‐chromosome confirmed the increase in foetal cell proportion in the triple density‐gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density‐gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density‐gradient separation and MACS.