Continuous centrifugal microfluidics identifies the marker and size heterogeneity of circulating trophoblasts for accurate non-invasive prenatal diagnosis

As the prevalence of newborns with congenital anomalies continues to increase, the importance of prenatal diagnosis has been emphasized. Since the commonly used methods for prenatal diagnosis, such as amniocentesis/chorionic villi sampling, are invasive and increase the risk of miscarriage, cell-free fetal DNA (cffDNA) analysis has been widely used in clinic. However, it still has critical drawbacks, such as experimental failure at low fetal fraction of cffDNA and limited accuracy due to the need of statistical prediction, reducing the reliability of results. Therefore, there is an urgent need for non-invasive prenatal diagnosis (NIPD) with circulating fetal cells, such as circulating trophoblasts (cTBs), containing complete fetal genetic information. However, current technologies are biased (marker- and size-based) isolation approaches and are limited in practical utility in clinic. Here we introduce a fully automated, unbiased isolation platform for maximal yield and heterogeneity study of cTBs, using a continuous centrifugal microfluidics-based circulating trophoblast disc (CCM-CTBD). The CCM-CTBD achieved consistently high recovery rates using three different trophoblast cell lines with heterogeneous sizes and marker expressions. Next, we successfully isolated cTBs from maternal peripheral blood, and identified their surface marker and size heterogeneity. In addition, we found unreported cells with double-positive staining (<CD45> and <EpCAM/HLA-G/Pan-CK>) and confirmed their fetal origin by identifying the Y chromosome. Moreover, the platform could isolate fetal cells as early as 8 gestational weeks. This unbiased isolation platform can potentially be a powerful tool for accurate, safe, and early prenatal diagnosis with clinical utility.