Automatic Single-Cell Harvesting for Fetal Nucleated Red Blood Cell Isolation on a Self-Assemble Cell Array (SACA) Chip
Authors: Yang Hsin-Yu , Lin Che-Hsien , Hu Yi-Wen , Chien Chih-Hsuan , Huang Mu-Chi , Lai Chun-Hao , Wu Jen-Kuei , Tseng Fan-Gang 
Published: December 2024

(1) Background: Fetal chromosomal examination is a critical component of modern prenatal testing. Traditionally, maternal serum biomarkers such as free β-human chorionic gonadotropin (Free β-HCG) and pregnancy-associated plasma protein A (PAPPA) have been employed for screening, achieving a detection rate of approximately 90% for fetuses with Down syndrome, albeit with a false positive rate of 5%. While amniocentesis remains the gold standard for the prenatal diagnosis of chromosomal abnormalities, including Down syndrome and Edwards syndrome, its invasive nature carries a significant risk of complications, such as infection, preterm labor, or miscarriage, occurring at a rate of 7 per 1000 procedures. Beyond Down syndrome and Edwards syndrome, other chromosomal abnormalities, such as trisomy of chromosomes 9, 16, or Barr bodies, pose additional diagnostic challenges. Non-invasive prenatal testing (NIPT) has emerged as a powerful alternative for fetal genetic screening by leveraging maternal blood sampling. However, due to the extremely low abundance of fetal cells in maternal circulation, NIPT based on fetal cells faces substantial technical challenges. (2) Methods: Fetal nucleated red blood cells (FnRBCs) were first identified in maternal circulation in a landmark study published in The Lancet in 1959. Due to their fetal origin and presence in maternal peripheral blood, FnRBCs represent an ideal target for non-invasive prenatal testing (NIPT). In this study, we introduce a novel self-assembled cell array (SACA) chip system, a microfluidic-based platform designed to efficiently settle and align cells into a monolayer at the chip’s base within five minutes using lateral flow dynamics and gravity. This system is integrated with a fully automated, multi-channel fluorescence scanning module, enabling the real-time imaging and molecular profiling of fetal cells through fluorescence-tagged antibodies. By employing a combination of Hoechst+/CD71+/HbF+/CD45− markers, the platform achieves the precise enrichment and isolation of FnRBCs at the single-cell level from maternal peripheral blood. (3) Results: The SACA chip system effectively reduces the displacement of non-target cells by 31.2%, achieving a single-cell capture accuracy of 97.85%. This isolation and enrichment system for single cells is well suited for subsequent genetic analysis. Furthermore, the platform achieves a high purity of isolated cells, overcoming the concentration detection limit of short tandem repeat (STR) analysis, demonstrating its capability for reliable non-invasive prenatal testing. (4) Conclusions: This study demonstrates that the SACA chip, combined with an automated image positioning system, can efficiently isolate single fetal nucleated red blood cells (FnRBCs) from 50 million PBMCs in 2 mL of maternal blood, completing STR analysis within 120 min. With higher purification efficiency compared to existing NIPT methods, this platform shows great promise for prenatal diagnostics and potential applications in other clinical fields.