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Circulating Tumour Cell Stabilisation

Stabilisation of Circulating Tumour Cells in TransFix® Treated Blood Samples

Circulating tumour cells (CTCs) are cells from the primary tumour which have invaded and moved through the walls of nearby blood vessels to circulate in the blood stream. These circulating tumour cells can invade the walls of the capillaries at a distant location and migrate into the surrounding tissue which can result in the formation and growth of secondary tumours; a process called metastasis. Metastatic disease is responsible for over 90% of cancer deaths so is a key research topic in the cancer research field (1)(2).

Tissue biopsies are invasive, cannot be used repeatedly, and only provide a representation of the organ of interest. CTC analysis provides a liquid “surrogate tumour biopsy”, providing an overall state of disease, in real time, without invasive procedures. CTC characterisation at the molecular level also has the potential to provide pharmacodynamic and predictive biomarkers and potentially provide use in revealing drug resistance in real time (3).

The use of Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes (CTC-TVTs) has facilitated the development of multiple CTC isolation and analysis methods, due to improved cell stability and recovery of these rare cells.

Features of Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes (CTC-TVTs)

TransFix has been used for the stabilisation of circulating tumour cells in blood samples. TransFix stabilised circulating tumour cells are compatible with a range of CTC isolation and analysis methods (see case studies for details).

The number of circulating tumour cell populations present in patient blood is very low, therefore the usefulness of CTC assessments depends upon accurate cell counts and the corresponding analysis of molecular targets.

CTC-TVTs are 9ml blood collection tubes prefilled with an optimised volume of TransFix for the collection and stabilisation of circulating tumour cells in whole human blood. They allow stabilisation of CTCs for up to 5 days at ambient temperature (18-25°C), immediately preventing the degradation of CTCs and CTC antigens, allowing time to isolate and analyse these rare cells.

Benefits of TransFix/EDTA Vacuum Blood Collection Tubes (CTC-TVTs)

The use of Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes has facilitated the development of multiple CTC isolation and analysis methods, due to improved cell stability and recovery of these rare cells.

This demonstrates that CTC-TVTs:

  • Stabilise samples in such a way that allow for isolation of CTC using size exclusion filtration. For example by using a Hamilton STARlet™ robot (see case study 1 and case study 2), or ScreenCell Cyto Kits (see case study 3).
  • Stabilise samples in such a way that allow for isolation of CTCs using cellular surface markers, for example, by the EasySep human CD45 depletion kit (see case study 4) from whole blood.
  • Enables the microscopic analysis and enumeration of CTCs in metastatic breast and lung cancer patients at mean levels as low 0.41 CTC/mL and 0.04 CTC/mL respectively (see case study 2).
  • Enables the analysis of CTCs through high-resolution image flow cytometry (see case study 4)
  • Compatible with cell sorting, for example by the DEPArray™system, and mutation analysis via Sanger sequencing (see case study 3)

By using CTC-TVTs in combination with novel isolation and analysis systems, CTCs could provide a new prognostic tool for patients with metastatic cancer.

Product Information

Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes consist of purple capped polyethylene terephthalate tubes that are designed for direct-draw blood collection. They contain a solution of TransFix and K3EDTA at the correct volume to simultaneously stabilise and anti-coagulate whole blood at the time of collection. The stabiliser acts by preserving CTCs at 18 – 25°C for up to 5 days until processing and analysis can be performed.

CTC-TVTs are a 9ml final draw volume tube. The vacuum contained within the tube ensures that the TransFix/EDTA reagent is administered at the correct ratio. CTC-TVTs are sterilised by gamma radiation.

Please contact Cytomark@caltagmedsystems.co.uk for more details.

Product Code Description Quantity
CTC-TVT-09-2 Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes 2 x 9ml tubes
CTC-TVT-09-50 Circulating Tumour Cell TransFix/EDTA Vacuum Blood Collection Tubes 50 x 9ml tubes

Case Studies

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Case Study 1: Assessment of circulating tumor cells with a novel, filtration-based method, in a phase IIIb multicenter study for postmenopausal, HER2- negative, estrogen receptor-positive, advanced breast cancer patients.

TransFix stabilised blood samples were used for Circulating Tumour Cell (CTC) analysis in an independent study analysing 111 patients with a type of metastatic breast cancer. In the publication (Fasching, et al, ASCO Meeting Abstracts Jun 17, 2013:591) showed that it was possible to identify 75 patients with circulating tumour cells from blood samples treated with TransFix/EDTA. After stabilisation, circulating tumour cells were captured by a novel filtration system and then immunostained for analysis on a fluorescence scanning microscope.

Reference: Fasching et al., (2013) ASCO Meeting Abstracts Jun 17, 2013:591

View Poster Here.

* Cytomark has not validated these findings and we recommend that internal validation of the product must be completed by the end user before considering use of the products in a clinical setting.

Case Study 2: A Novel Strategy for Detection and Enumeration of Circulating Rare Cell Populations in Metastatic Cancer Patients Using Automated Microfluidic Filtration and Multiplex Immunoassay

In this study, the performance of a novel approach for detection and enumeration of multiple rare cell populations was evaluated in the blood of metastatic breast and lung cancer patients using an automated microfluidic filtration and multiplex immunoassay strategy. Different circulating rare cell populations were detected and enumerated, including circulating tumour cells (CTCs), circulating mesenchymal cells (CMCs), circulating endothelial cells (CECs), and putative circulating stem cells (CSCs). Simultaneous assessment of CTCs, CMCs, CSCs and CECs may provide new tools to study mechanisms of disease progression and treatment response/resistance.

The highly controlled filtration process and the multi-step staining parameters were optimised to minimise the detection of false positives in healthy donor blood. Use of TransFix®, along with controlled shipping and storage conditions contributed to the high rate of reportable results (98%).

Reference: Magbanua, et al. (2015) PLoS ONE 10(10): e0141166

* Cytomark has not validated these findings and we recommend that internal validation of the product must be completed by the end user before considering use of the products in a clinical setting.

Case Study 3: Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer

This paper describes the use of the Screencell method of size-exclusion isolation of CTCs, circulating tumour cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs). Blood samples collected in TransFix tubes and analysed within 72 hours. Cytological Analysis was performed by immunofluorescence staining of cytokeratins (CK-8, 18 and 19), DAPI, CD45 for CTCs, and EMT-CTCs were stained for Vimentin and N-Cadherin. Cell sorting was performed by the DEPArray™ system. TP53 and ESR1 Mutations in CTCs were analysed by Sanger sequencing.

Reference: Mu et al. (2016) Int. J. Mol. Sci. 17(10): 1665.

View Open Access Paper Here

* Cytomark has not validated these findings and we recommend that internal validation of the product must be completed by the end user before considering use of the products in a clinical setting.

Case Study 4: High-resolution imaging for the detection and characterisation of circulating tumour cells from patients with oesophageal, hepatocellular, thyroid and ovarian cancers

The purpose of this study was to develop and validate a novel, widely applicable method for detection and characterisation of circulating tumour cells from 4 tumour types. Blood samples were collected in TransFix collection tubes and stained for immunofluorescence by the following membrane antibodies and nuclear stains: EpCAM, cytokeratins 4, 5, 6, 8, 10, 13 and 18, survivin and CD45, and DAPI or DRAQ5. Circulating tumour cells were enriched using an EasySep human CD45 depletion kit, and were analysed using an ImageStream X Flow cytometer.

Reference: Dent et al. (2016) International Journal of Cancer. 138(1): 206-216

View Open Access Paper Here

* Cytomark has not validated these findings and we recommend that internal validation of the product must be completed by the end user before considering use of the products in a clinical setting.

Protocols

References

  1. Wittekind C., Neid M. (2005) Cancer invasion and metastasis. Oncology 69(Suppl 1): 14–16 [PubMed]
  2. Weiss L. (2000) Metastasis of cancer: a conceptual history from antiquity to the 1990s. Cancer Metastasis Rev 19(I-XI): 193–383 [PubMed]
  3. Krebs et al. Circulating tumour cells: their utility in cancer management and predicting outcomes. Ther Adv Med Oncol 2010 Nov; 2(6): 351–365. [PubMed].

Other Applications of TransFix


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