The RareCyte® platform for next‐generation analysis of circulating tumor cells

Abstract

Circulating tumor cells (CTCs) can reliably be identified in cancer patients and are associated with clinical outcome. Next‐generation “liquid biopsy” technologies will expand CTC diagnostic investigation to include phenotypic characterization and single‐cell molecular analysis. We describe here a rare cell analysis platform designed to comprehensively collect and identify CTCs, enable multi‐parameter assessment of individual CTCs, and retrieve single cells for molecular analysis. The platform has the following four integrated components: 1) density‐based separation of the CTC‐containing blood fraction and sample deposition onto microscope slides; 2) automated multiparameter fluorescence staining; 3) image scanning, analysis, and review; and 4) mechanical CTC retrieval. The open platform utilizes six fluorescence channels, of which four channels are used to identify CTC and two channels are available for investigational biomarkers; a prototype assay that allows three investigational biomarker channels has been developed. Single‐cell retrieval from fixed slides is compatible with whole genome amplification methods for genomic analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

CTC Technologies and Tools

“…A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45). A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45).…”

“…Addition of fluorescently labeled antibodies recognizing treatment targets to the staining cocktail is relatively easy, although, one is limited by the number of fluorochromes that can be determined simultaneously. Tools to isolate the single cells from CTC enriched cell suspensions are available (39,41,42,56,57). Probing for gene aberrations of CTC by fluorescence in situ hybridization has also been demonstrated and again reiterated their extensive intra-and inter-patient heterogeneity (52)(53)(54)(55).…”

“…With the introduction of new methods for CTC isolation many have argued that the lack of detection of CTC in cancer patients with the CellSearch system may be contributed to the CTC definition used as it detects only cells expressing both EpCAM and Cytokeratin. A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45). However, for these different CTC phenotypes, clinical confirmation of the correlation of CTC to patient survival will be necessary.…”

“…For extensive interrogation of the CTC the individual cells will have to be isolated and for molecular characterization the nucleic acids will have to be amplified. Tools to isolate the single cells from CTC enriched cell suspensions are available (39,41,42,56,57). The steps needed to get from blood to single CTC surely will be associated with cell losses and the different steps will have to be optimized to minimize these losses.…”

Sensitive and specific detection of circulating tumor cells promotes precision medicine for cancer

“…Quantitative buffy coat analysis by centrifuging for separation was established by Stephen C. Wardlaw in 1983 [55] . AccuCyte separation based on this principle is the first step of the commercial RareCyte Platform (RareCyte, Inc. Seattle) [56] , coupled with fluorescence analyzing (CyteFinder system) and picking (CytePiker) to count and retrieve cells for downstream single-cell characterization, overcoming the limitation of capture methods which are dependent on sizes that might miss the small sized-CTCs and immunomarkers that might not be expressed on some subpopulations. Some commercial density gradient solutions, such as Ficoll-Paque (GE Healthcare) and Percoll (GE Healthcare), provide simple-to-use and inexpensive methods for separating CTCs in the mononucleocyte layer from granulocytes and erythrocytes.…”