High-recovery visual identification and single-cell retrieval of circulating tumor cells for genomic analysis using a dual-technology platform integrated with automated immunofluorescence staining

Abstract

BackgroundCirculating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte® – CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers.MethodsAll experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system.ResultsAccuCyte – CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort.ConclusionThe AccuCyte – CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1383-x) contains supplementary material, which is available to authorized users.

Circulating tumor cell technologies

“…Inexpensive, easy-to-use (Weitz et al, 1998) OncoQuick Density, Size Porous membrane above separation media for additional separation by filtration (Balic et al, 2005;Clawson et al, 2012;Lagoudianakis et al, 2009;M€ uller et al, 2005;Obermayr et al, 2010;Rosenberg et al, 2002) (Campton et al, 2015) Biophysical e Microfiltration in Two and Three Dimensions Size-based cell separation using pores or three-dimensional geometries.…”

Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons

“…Unlike enrichment approaches, there was no additional sample processing required for FAST cytometry that could result in reduced sensitivity due to cell loss. In 2015, Campton et al described and evaluated the performance of a comprehensive and highly sensitivity platform, the AccuCyte–CyteFinder system, for collecting, identifying, and collecting CTCs [17]. Nucleated cells were separated from blood with a density-based cell separation apparatus, AccuCyte, and transferred to microscopic slides.…”

“…Patient sample CTC counts matched or exceeded CellSearch CTC counts. Campton et al also showed that a single-cell retrieval device, CytePicke, can harvest individual CTCs from slides after identification with CyteFinder for further genomic analysis [17]. …”

D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein

“…l-ascorbic acid and superoxide dismutase (SOD) significantly increased the percentage of nonagglutinated, motile spermatozoa compared with the control but only by a moderate amount in comparison to PEN. Since l-ascorbic acid is a known reducing agent and SOD can chelate copper (Nordberg & Arnér 2001, Ottaviano et al 2008, it is possible that these secondary functions, as a reductant or chelator, contributed to this response (Fig. 5B).…”