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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., (© 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.

The RareCyte platform addresses important technology limitations of current circulating tumor cell (CTC) collection methods, and expands CTC interrogation to include advanced phenotypic characterization and single-cell molecular analysis. In this respect, it represents the "next generation" of cell-based liquid biopsy technologies. In order to identify and analyze CTCs, RareCyte has developed an integrated sample preparation, imaging and individual cell retrieval process. The first step in the process, AccuCyte ® , allows the separation, collection, and transfer to a slide the nucleated cell fraction of the blood that contains CTCs. Separation and collection are based on cell density-rather than size or surface molecular expression-and are performed within a closed system, without wash or lysis steps, enabling high CTC recovery. Here, we describe our technique for nucleated cell collection from a blood sample, and the spreading of these nucleated cells onto glass slides permitting immunofluorescent staining, cell identification, and individual cell picking described in subsequent chapters. In addition to collection of rare cells such as CTCs, AccuCyte also collects cells of the circulating immune system onto archivable slides as well as plasma from the same sample.