Enrichment of Fetal Nucleated Red Blood CellsCirculating in the Peripheral Blood of Pregnant Womenfor the Purpose of Non-invasive Prenatal Diagnosis

Overview

Objective:
Enrichment of nucleated red blood cells (NRBCs) from maternal blood for non-invasiveprenatal 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 13and 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 magneticcell separation (MACS) on VarioMACS. NRBCs were sorted from CD14 - /CD45 - fraction by positiveselection 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 ourcohort of pregnant woman the number of isolated NRBCs was individual. We identified NRBCsfrom the 13 th week of gestation.Conclusion: The aim of the study is to establish and standardise the method of enrichment ofNRBCs from maternal blood samples and verify the applicability of this alternative source fornon-invasive prenatal diagnosis.

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