High-efficiency isolation of fetal nucleated red blood cells for non-invasive prenatal diagnosis via a cascaded microfluidic platform.

Non-invasive prenatal diagnosis (NIPD) utilizing fetal nucleated red blood cells (fNRBCs) holds great promise for comprehensive genetic analysis; however, its clinical application is challenged by the extreme rarity of these cells in maternal peripheral blood. Herein, we present a two-step cascaded microfluidic platform designed for the high-efficiency isolation of fNRBCs. This integrated system employs a deterministic lateral displacement (DLD) microfluidic chip for the initial label-free enrichment of large cells, including fNRBCs, based on size. The enriched product is subsequently subjected to negative purification using a magnetic microfluidic chip, where residual white blood cells are depleted following incubation with CD45-coated magnetic beads. Using spiked K562 cells as a model, the device demonstrated a consistently high recovery efficiency exceeding 90%. In a clinical validation with 20 maternal blood samples, fNRBCs (identified as DAPI+/CD71+/CD45-) were successfully isolated from all subjects, with yields ranging from 10 to 83 cells per milliliter. Furthermore, the fetal origin of the isolated cells was definitively confirmed via fluorescence in situ hybridization (FISH) using Y-chromosome-specific probes in samples from pregnancies with male fetuses. These results demonstrate that our cascaded microfluidic platform provides a highly efficient, practical, and reliable approach for fNRBC isolation, highlighting its significant potential for cell-based non-invasive prenatal testing.