Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay

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Abstract

As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

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Considerations in the development of circulating tumor cell technology for clinical use

David R Parkinson, Nicholas Dracopoli, Brenda Gumbs Petty … (2012)

This manuscript summarizes current thinking on the value and promise of evolving circulating tumor cell (CTC) technologies for cancer patient diagnosis, prognosis, and response to therapy, as well as accelerating oncologic drug development. Moving forward requires the application of the classic steps in biomarker development–analytical and clinical validation and clinical qualification for specific contexts of use. To that end, this review describes methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA) and the US National Cancer Institute (NCI).

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Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers.

Craig Yoshioka, Yelena Petrova, Franziska Baehring … (2012)

Hematologic spread of carcinoma results in incurable metastasis; yet, the basic characteristics and travel mechanisms of cancer cells in the bloodstream are unknown. We have established a fluid phase biopsy approach that identifies circulating tumor cells (CTCs) without using surface protein-based enrichment and presents them in sufficiently high definition (HD) to satisfy diagnostic pathology image quality requirements. This 'HD-CTC' assay finds >5 HD-CTCs mL(-1) of blood in 80% of patients with metastatic prostate cancer (n = 20), in 70% of patients with metastatic breast cancer (n = 30), in 50% of patients with metastatic pancreatic cancer (n = 18), and in 0% of normal controls (n = 15). Additionally, it finds HD-CTC clusters ranging from 2 HD-CTCs to greater than 30 HD-CTCs in the majority of these cancer patients. This initial validation of an enrichment-free assay demonstrates our ability to identify significant numbers of HD-CTCs in a majority of patients with prostate, breast and pancreatic cancers.

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The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings.

Maryam Shabihkhani, Gregory Lucey, Bowen Wei … (2014)

Well preserved frozen biospecimens are ideal for evaluating the genome, transcriptome, and proteome. While papers reviewing individual aspects of frozen biospecimens are available, we present a current overview of experimental data regarding procurement, storage, and quality assurance that can inform the handling of frozen biospecimens. Frozen biospecimen degradation can be influenced by factors independent of the collection methodology including tissue type, premortem agonal changes, and warm ischemia time during surgery. Rapid stabilization of tissues by snap freezing immediately can mitigate artifactually altered gene expression and, less appreciated, protein phosphorylation profiles. Collection protocols may be adjusted for specific tissue types as cellular ischemia tolerance varies widely. If data is not available for a particular tissue type, a practical goal is snap freezing within 20min. Tolerance for freeze-thaw events is also tissue type dependent. Tissue storage at -80°C can preserve DNA and protein for years but RNA can show degradation at 5years. For -80°C freezers, aliquots frozen in RNAlater or similar RNA stabilizing solutions are a consideration. It remains unresolved as to whether storage at -150°C provides significant advantages relative to that at -80°C. Histologic quality assurance of tissue biospecimens is typically performed at the time of surgery but should also be conducted on the aliquot to be distributed because of tissue heterogeneity. Biobanking protocols for blood and its components are highly dependent on intended use and multiple collection tube types may be needed. Additional quality assurance testing should be dictated by the anticipated downstream applications. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.