The Role of Physical Stabilization in Whole Blood Preservation

Wong, Keith H.K.; Sandlin, Rebecca D.; Carey, Thomas; Miller, Kathleen L.; Shank, Aaron T.; Öklü, Rahmi; Maheswaran, Shyamala; Haber, Daniel A.; Irimia, Daniel; Stott, Shannon L.; Toner, Mehmet

doi:10.1038/srep21023

Cited by 41 publications

(38 citation statements)

References 34 publications

(42 reference statements)

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“…Moreover, limited temporal sampling makes it difficult to observe the kinetics of CTC shedding, which may vary significantly over timescale of days and (as we show) even minutes. Moreover, blood samples are known to degrade rapidly after removal from the body (12), and the process of drawing blood can trigger a stress response in the animal (13).…”

Section: Introductionmentioning

confidence: 99%

In VivoMonitoring of Rare Circulating Tumor Cell and Cluster Dissemination in a Multiple Myeloma Xenograft Model

Patil

Tan

Bartosik

et al. 2019

Preprint

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We recently developed 'Diffuse in vivo Flow Cytometry' (DiFC), a new pre-clinical research tool for enumerating extremely rare fluorescently-labeled circulating cells directly in vivo. In this paper, we developed a green fluorescent protein (GFP) compatible version of DiFC, and used it to non-invasively monitor the circulating tumor cell (CTC) burden over time in a multiple myeloma disseminated xenograft model. We show that DiFC allowed counting of CTCs at estimated concentrations below 1 cell per mL in peripheral blood with a negligible false alarm rate. DiFC also revealed the presence of CTC clusters in circulation to our knowledge for the first time in this model, and allowed us to calculate their size, kinetics, and frequency of shedding. We anticipate that the unique capabilities of DiFC will have many applications in the study of hematogenous metastasis, and as a powerful complementary methodology to liquid biopsy assays.

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Section: Introductionmentioning

confidence: 99%

In VivoMonitoring of Rare Circulating Tumor Cell and Cluster Dissemination in a Multiple Myeloma Xenograft Model

Patil

Tan

Bartosik

et al. 2019

Preprint

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“…For instance, blood must be properly collected (for example, into specialized blood collection tubes to minimize cellular DNA release [28]), stabilized, and fractionated within hours to days to mitigate degradation of cells or nucleic acids [29, 30]. Plasma fractionated from blood can be frozen for extraction of cfDNA or nucleic acids from EVs at a later date.…”

Section: Pre-analytical Considerationsmentioning

confidence: 99%

Technological considerations for genome-guided diagnosis and management of cancer

2016

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Technological, methodological, and analytical advances continue to improve the resolution of our view into the cancer genome, even as we discover ways to carry out analyses at greater distances from the primary tumor sites. These advances are finally making the integration of cancer genomic profiling into clinical practice feasible. Formalin fixation and paraffin embedding, which has long been the default pathological biopsy medium, is now being supplemented with liquid biopsy as a means to profile the cancer genomes of patients. At each stage of the genomic data generation process—sample collection, preservation, storage, extraction, library construction, sequencing, and variant calling—there are variables that impact the sensitivity and specificity of the analytical result and the clinical utility of the test. These variables include sample degradation, low yields of nucleic acid, and low variant allele fractions (proportions of assayed molecules carrying variant allele(s)). We review here the most common pre-analytical and analytical factors relating to routine cancer patient genome profiling, some solutions to common challenges, and the major sample preparation and sequencing technology choices available today.

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“…Although these are widely used in biomedical research, they are far from optimal for a number of reasons. In particular, small blood samples provide poor statistical sampling of the circulating blood volume [13][14][15], blood is known to degrade rapidly after removal from the body [16], and enrichment can cause cell loss or dissolution [17][18]. Moreover, in the case of small animal studies CTCs may be so rare that it is necessary to draw and analyze the entire blood volume, which requires euthanizing the animal [19].…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Labeling of Circulating Tumor Cells with Folate Receptor Targeted Molecular Probes for DiffuseIn VivoFlow Cytometry

Patil¹,

Srinivasarao

Amiji

et al. 2020

Preprint

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Purpose: We recently developed a new instrument called 'diffuse in vivo flow cytometry' (DiFC) for enumeration of rare fluorescently-labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins, or were pre-labeled with a fluorescent dye ex-vivo. In this work, we investigated the use of two folate receptor (FR)-targeted fluorescence molecular probes for in vivo labeling of FR+ CTCs for DiFC.Methods: We used EC-17 and Cy5-PEG-FR fluorescent probes. We studied the affinity of these probes for L1210A and KB cancer cells, both of which over-express FR. We tested the labeling specificity in cells in culture in vitro, in whole blood, and in mice in vivo. We also studied detectability of labeled cells with DiFC.Results: Both EC-17 and Cy5-PEG-FR probes had high affinity for FR+ CTCs in cell culture in vitro.However, only EC-17 had sufficient specificity for CTCs in whole blood. EC-17 labeled CTCs were also readily detectable in circulation in mice with DiFC.Conclusions: This work demonstrates the feasibility of labeling CTCs for DiFC with a cell surface receptor targeted probe, greatly expanding the utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

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The Role of Physical Stabilization in Whole Blood Preservation

Cited by 41 publications

References 34 publications

In VivoMonitoring of Rare Circulating Tumor Cell and Cluster Dissemination in a Multiple Myeloma Xenograft Model

In VivoMonitoring of Rare Circulating Tumor Cell and Cluster Dissemination in a Multiple Myeloma Xenograft Model

Technological considerations for genome-guided diagnosis and management of cancer

Fluorescence Labeling of Circulating Tumor Cells with Folate Receptor Targeted Molecular Probes for DiffuseIn VivoFlow Cytometry

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