Enrichment of Fetal Nucleated Red Blood Cells from the Maternal Circulation for Prenatal Diagnosis: Experiences with Triple Density Gradient and MACS Based on More than 600 Cases

Abstract

Objective: We wanted to obtain statistically relevant data about the efficiency of our method for the isolation of fetal nucleated red blood cells (NRBCs) from the maternal circulation. Methods: More than 600 samples were investigated using a triple density gradient followed by magnetic separation of anti-CD71-labeled cells, and yields and purities of recovered NRBCs were determined. Results: The enrichment effectivity as well as the morphological condition of cells was reproducibly good, if blood samples were enriched within 48 h after sampling. The efficacy was independent of various methodological parameters and our technique was superior to other magnetic cell-sorting techniques. Mean yields and purities of NRBCs increased with increasing gestational age, ranging from 100 to 1,000 cells per 40-ml blood sample and from 0.1 to 1%, respectively, from the 6th week of gestation to term. In pregnancies with preeclampsia NRBCs were increased by a factor of 10. Conclusion: Our enrichment technique proved to be optimized with respect to various methodological parameters, which were compared in the present study, and it is efficient and reproducible for the enrichment of NRBCs from the maternal circulation in all three gestational trimesters.

Examination of Maternal Plasma Erythropoietin and Activin A Concentrations with Regard to Circulatory Erythroblast Levels in Normal and Preeclamptic Pregnancies

“…This was largely due to the abundance of erythroblasts in the fetal circulation, scarcity in adult blood, and suitable markers for their enrichment and po-tential identifi cation of fetal erythroblasts [9,10] . During our investigations we observed that both fetal and maternal erythroblasts were present in the circulation of pregnant women, and that the level of both of these erythroblast pools was elevated in preeclampsia [3,11] . Furthermore, a signifi cant correlation was found to occur between circulatory fetal and maternal erythroblasts, suggesting that maternal erythropoiesis was enhanced in preeclampsia.…”

Identification of Fetal Nucleated Red Blood Cells in the Maternal Circulation during Pregnancy Using Anti-Hemoglobin-ε Antibody

“…Magnetic activated cell sorting (MACS; Miltenyi Biotec, Inc., Sunnyvale, Calif., USA) was performed first by negative selection for the removal of maternal lymphocytes, using anti-CD45 (Serotec Ltd, Kidlington, UK), followed by positive selection for NRBCs with anti-CD71 (Serotec) [12,13].…”

Flow cytometric methods for prenatal and neonatal diagnosis

“…The CD45− or CD14− cells can then be labeled with a fetal cell marker to target fetal cells within the population. Using a triple density gradient with anti-CD71-labeled cells, Gänshirt et al (1998) showed that the frequency of fetal NRBCs increases with gestational age with yields ranging from 100 to 1000 cells per 40 mL of maternal blood from week 6 of gestation to term.…”