Abstract
BackgroundThe enumeration and characterization of circulating tumor cells (CTCs) in the blood of cancer patients is useful for cancer prognostic and treatment monitoring purposes. The number of CTCs present in patient blood is very low; thus, robust technologies have been developed to enumerate and characterize CTCs in patient blood samples. One of the challenges to the clinical utility of CTCs is their inherent fragility, which makes these cells very unstable during transportation and storage of blood samples. In this study we investigated Cell-Free DNA BCT™ (BCT), a blood collection device, which stabilizes blood cells in a blood sample at room temperature (RT) for its ability to stabilize CTCs at RT for an extended period of time.MethodsBlood was drawn from each donor into K3EDTA tube, CellSave tube and BCT. Samples were then spiked with breast cancer cells (MCF-7), transported and stored at RT. Spiked cancer cells were counted using the Veridex CellSearch™ system on days 1 and 4. The effect of storage on the stability of proteins and nucleic acids in the spiked cells isolated from K3EDTA tube and BCT was determined using fluorescence staining and confocal laser scanning microscopy.ResultsMCF-7 cell recovery significantly dropped when transported and stored in K3EDTA tubes. However, in blood collected into CellSave tubes and BCTs, the MCF-7 cell count was stable up to 4 days at RT. Epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) in MCF-7 cells isolated from BCTs was stable at RT for up to 4 days, whereas in MCF-7 cells isolated from K3EDTA blood showed reduced EpCAM and CK protein expression. Similarly, BCTs stabilized c-fos and cyclin D1 mRNAs as compared to K3EDTA tubes.ConclusionCell-Free DNA™ BCT blood collection device preserves and stabilizes CTCs in blood samples for at least 4 days at RT. This technology may facilitate the development of new non-invasive diagnostic and prognostic methodologies for CTC enumeration as well as characterization.
Highlights
The enumeration and characterization of circulating tumor cells (CTCs) in the blood of cancer patients is useful for cancer prognostic and treatment monitoring purposes
In the peripheral blood of patients with solid tumors of epithelial origin, some circulating cells have been found that have characteristics of tumor cells [1]. These cells that are present in the bloodstream of cancer patients, known as circulating tumor cells (CTCs), are thought to play an important role in cancer metastasis by breaking loose from a solid tumor, entering the circulation, and migrating to distant organs to develop secondary
Recovery of spiked MCF-7 cells in blood Experiments were designed to determine the ability of BCTs to stabilize CTCs during blood sample storage and transportation compared to standard K3EDTA and CellSave blood collection tubes
Summary
The enumeration and characterization of circulating tumor cells (CTCs) in the blood of cancer patients is useful for cancer prognostic and treatment monitoring purposes. It is necessary to address several pre-analytical issues that arise during the time between blood draw and CTC enrichment and characterization in order to effectively preserve CTCs for analysis. These include delays in blood processing, blood storage temperature, and agitation of the sample during transport and shipment of blood. Previous studies have shown that blood collection devices with cellular preservatives are capable of stabilizing CTCs for up to 96 hours [11,12,13]
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