Stabilization of circulating tumor cells in blood using a collection device with a preservative reagent.

BACKGROUND: The 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. METHODS: Blood 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. RESULTS: MCF-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. CONCLUSION: Cell-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.

A formalin-free method for stabilizing cells for nucleic acid amplification, hybridization and next-generation sequencing.

BACKGROUND: Formalin has been widely used by pathology laboratories. Its carcinogenicity has led researchers to explore formalin substitutes. Streck Cell Preservative (SCP) is a formalin-free preservative that can preserve cellular antigens. This study was undertaken to investigate the effects of cell preservation using SCP on nucleic acid amplification, hybridization, and next-generation sequencing (NGS) as compared to control frozen cells and cells fixed in the traditional cell and tissue fixative, 10 % neutral buffered formalin (NBF). FINDINGS: The breast cancer cell line, SKBR-3, was used as a model system. Prior to nucleic acid extraction and fluorescence in situ hybridization (FISH), cells were fixed in SCP or NBF overnight at room temperature with frozen cells in parallel. Analysis showed that similar DNA extraction yields and amplification profiles determined by PCR in SCP preserved cells and control frozen cells, whereas NBF preserved cells had decreased DNA yield and impaired PCR amplification. Molecular cytogenetic studies by FISH technique indicated that the ratios of ERBB2 (HER-2/neu) signals to the chromosome 17 centromere (CEP17) were comparable for frozen cells and SCP preserved cells. The fluorescence images of both SCP fixed and control frozen cells were also clear and comparable. On the contrary, the same analysis was unsuccessful with NBF preserved cells due to poor hybridization quality. Our data also demonstrated that SCP had negligible effect on NGS testing. CONCLUSION: We conclude that SCP can be used as an alternative to NBF as a preservative for maintaining the integrity of nucleic acids for nucleic acid amplification, sequencing and FISH analysis.

Inactivation and viral load quantitation of human immunodeficiency virus in blood collected into Cyto-Chex(®) BCT blood collection device.

A blood collection tube (Cyto-Chex(®) BCT), which can stabilize white blood cells and immunogenic markers in blood samples, was investigated for its ability to inactivate human immunodeficiency virus (HIV) and stabilize HIV for viral load quantitation. Laboratory-adapted HIV strains were either treated or untreated with the stabilizing reagent present in Cyto-Chex(®) BCT. A dilution of the reagent used to treat virus was 1:66, which was similar to the reagent concentration in Cyto-Chex(®) BCT device when blood was drawn into it. In another experiment, blood was drawn from HIV patients into one acid citrate dextrose (ACD) tube and one Cyto-Chex(®) BCT. At indicated time points, aliquots were taken of treated and untreated viral dilutions and from plasma of HIV-positive patient blood samples and analyzed using reverse transcriptase and TZM-bl cell assays to determine HIV inactivation. In laboratory-adapted HIV strains and HIV-positive patient plasma, HIV was completely inactivated within 2 and 3h of contact with a 1:66 dilution of Cyto-Chex reagent, respectively. Samples from HIV-positive patient plasma showed that viral load was stable in Cyto-Chex(®) BCT for 7 days at room temperature. Therefore, it is concluded that the chemical reagent present in the Cyto-Chex(®) BCT blood collection device is capable of complete inhibition of HIV infectivity in blood samples within 3h and stabilizing the viral load for 7 days at room temperature.