Prenatal diagnosis of β-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient

We have assessed a new technique for the isolation of fetal erythroblasts from maternal blood for the non-invasive prenatal diagnosis of pregnancies at risk of β-thalassaemia. This method relies on the separation of erythroblasts from maternal nucleated cells by a novel step gradient and high speed centrifugation. In four of the six cases examined, single erythroblasts were identified by immunohistochemistry for zeta (ζ) globin. These were individually micromanipulated and analysed by single cell polymerase chain reaction (PCR) and subsequent sequencing of the region of β-globin locus where the mutations most common to the region of Puglia, Italy, are clustered. In each of the four instances where fetal erythroblasts were identified by antibody staining, the fetal β-globin genotype was correctly determined. To date, this represents the largest series of non- invasive prenatal diagnoses performed for this haemoglobinopathy.