Objective: Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasive prenatal diagnosis. Design: Pilot study. Setting: 2 nd 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 mono- nuclear 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-co- lour cytometric analysis with FACScan flow cytometer. Results: In 68 out of 78 pregnant woman (87 %) NRBCs were found in range 2x10 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 13 th 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.

        Key words: nucleated red blood cells, high-gradient magnetic cell separation (MACS), flow cyto- metry (FACS), prenatal diagnosis, fetal cells.