A high yield of fetal nucleated red blood cells isolated using optimal osmolality and a double-density gradient system

Objectives: To increase the yield of fetal nucleated red blood cells (NRBCs) from maternal blood using a discontinuous Percoll gradient and to determine the effects of osmolality on NRBC yield. Methods: Fetal NRBCs were isolated from combined umbilical cord blood and adult female blood, or from maternal blood using single or double Percoll gradients with different osmolalities. Magnetic activated cell sorting was used to enrich isolated NRBCs, and morphological differentiation was performed with Kleihauer-Betke stain. We also isolated fetal NRBCs from 25 10 mL samples of maternal blood and determined fetal sex by fluorescence in situ hybridization (FISH), using X-Y probes. Results: For single-density Percoll columns, the greatest number of NRBCs was isolated using 280 mOsm/kg H₂O with 1.077 g/mL Percoll and 520 mOsm/kg H₂O with 1.119 g/mL Percoll. Significantly more fetal NRBCs were isolated with double Percoll density gradients than with double-Histopaque gradients (p = 0.043). FISH analysis on NRBC in 25 cases correctly identified 15 male and 9 female euploid fetuses and one Trisomy 21 fetus. Conclusion: The NRBC enrichment method we present requires less maternal blood and yields more NRBCs compared to previous methods.