An Improved Flow Cytometric Approach for Isolation of Fetal Cells from Maternal Blood for Non Invasive Prenatal Diagnosis of Hemoglobinopathies

Abstract

Prenatal diagnosis is an option for couples at risk of having a child affected with hemoglobinopathies. Chorionic villus sampling (CVS) and cordocentesis are accurate but a finite risk of fetal loss exists. A non invasive, risk free strategy that has emerged is isolation of fetal erythroblasts from maternal blood. Enrichment of nucleated red blood cells (nRBCs) from 7.0 mL maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies: CD45 per CP, glycophorin A-phycoerythrin (PE) and Hb F-fluorescein isothiocyanate (FITC) in 43 cases between 7 and 21 weeks' gestation. The percentage of nRBCs ranged from 0.0001–2.03%. The presence of dual fluorescence (glycophorin A-PE and Hb F-FITC) was confirmed by confocal microscopy. A sufficient number of nRBCs could be isolated in the first and second trimester of pregnancy to provide a simple flow cytometric approach as a potential for non invasive diagnosis of β-globin defects.

Keywords:

• Non invasive
• Prenatal diagnosis
• Fetal erythroblasts
• Hemoglobinopathies
• Maternal blood