Objective: Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasive prenatal diagnosis.
Design: Pilot study.
Setting: 2nd Clinic of Paediatrics, University Hospital Motol, Prague, Czech Republic.
Methods: Mononuclear cells were isolated from 13-28 ml of peripheral maternal blood between 13 and 37 weeks of gestation. Leukocytes from maternal peripheral blood were depleted from mononuclear cells by treatment with anti-CD14 and anti-CD45 microbeads and high-gradient magnetic cell separation (MACS) on VarioMACS. NRBCs were sorted from CD14-/CD45- fraction by positive selection using anti-CD71 microbeads on MiniMACS. All sorting steps were analysed by three-colour cytometric analysis with FACScan flow cytometer.
Results: In 68 out of 78 pregnant woman (87%) NRBCs were found in range 2 x 10(5) - 1.02 x 10(6). NRBC were enriched with an average enrichment rate of 138-fold ranging from 4-526 fold. In our cohort of pregnant woman the number of isolated NRBCs was individual. We identified NRBCs from the 13th week of gestation.
Conclusion: The aim of the study is to establish and standardise the method of enrichment of NRBCs from maternal blood samples and verify the applicability of this alternative source for non-invasive prenatal diagnosis.