Development of PowerMag System II for Isolation of Circulating Tumor Cells with Improved Purity

<b>Background/Objectives</b>: The PowerMag system (PM) is a platform for the isolation of circulating tumor cells (CTCs) by the depletion of CD45⁺-leukocytes. However, an EpCAM⁻CD45⁻ cell population is present in large numbers in the cell filtrates collected by PM. This lowers the purity of the CTCs and negatively impacts their molecular characterization. The aims of this study are to characterize the cellular properties of the EpCAM⁻CD45⁻ cells and to upgrade the system to improve CTC purity. <b>Methods</b>: A real-time RT-PCR assay, Liu’s stain analysis, and Annexin V (AnxV) binding assay were used to define the cellular properties of the EpCAM⁻CD45⁻ cells. An upgraded system was developed to remove the EpCAM⁻CD45⁻ cells and improve the CTC purity. Clinical blood samples were used to evaluate the performance of the system. <b>Results</b>: The EpCAM⁻CD45⁻ cells were defined as apoptotic cells, which displayed apoptotic body-like morphology and elicited AnxV binding activity. AnxV beads developed in-house can effectively bind and remove EpCAM⁻CD45⁻ cells from the cell filtrates. An improved generation of a CTCs isolation platform, designated as PM II, was developed by integration of AnxV beads into the workflow to remove the apoptotic cells. PM II recovered CTCs with improved CTC purity by effective removal of the background apoptotic cells. The improved performance of PM II allowed for direct profiling of cancer-related gene mutations by next-generation sequencing without cell picking and further purification. <b>Conclusions</b>: PM II holds great promise as a platform for isolating CTCs with improved purity and for exploring its application in cancer diagnosis and monitoring in a clinical setting.