Enrichment and Detection of Circulating Tumor Cells

Thirty ml of blood was collected in Vacuette K3EDTA tubes (Greiner Bio-One) and processed on the same day in accordance with a recently published protocol [10 (link)]. In short, the blood was divided into two equal parts to enable comparison of single-step and two-step enrichment of CTCs. Using the single-step protocol (PX6.5), Parsortix® was the sole enrichment step employing the GEN3D6.5 microfluidic cassette with the critical gap size of 6.5 µm. With the two-step protocols, the blood samples were first enriched by DG centrifugation using 15 ml Percoll (GE Healthcare; d = 1.065 g/ml, 305 mOsm/kg), and then, the cell suspensions were further processed with GEN3D10 cassettes applying 23 mbar pressure (DG10), or GEN3D6.5 cassettes applying 99 mbar pressure (DG6.5). An overview on the respective protocols is given in Fig. 1. After the microfluidic separation, the captured cells were harvested and immediately lysed by adding 350 µl RLT lysis buffer (Qiagen).Fig. 1

Flow diagram of the protocols applied for the enrichment of circulating tumor cells (CTCs) and the detection of CTC-related gene transcripts. CTCs were enriched using the microfluidic Parsortix® enrichment alone and in combination with an upstream density gradient centrifugation. CTC-related gene transcripts were detected using Taqman® and Lightcycler technology (LC)