Abstract 3072: High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy

Abstract

Abstract Background: Analysis of circulating tumor cells (CTC) is an intense area of diagnostic research that is rapidly evolving from prognostic to therapeutic applications. We applied three sequential technologies - density-based enrichment, automated immunofluorescence staining, and digital microscopy with image analysis to the investigation of recovery rates from blood containing spiked-in tumor cells as a model of CTC. Materials and Methods: Cultured MCF7, PC3, A549 and LNCaP cells were counted using Vitrotube visualization. ∼100 cells per line were spiked into normal blood samples in two experiments. Density-based enrichment was performed using the AccuCyte tube, float and collector system in a single-tube, two-spin process. Collected cells were processed with an adherence solution and applied to charged microscopic slides with a simple spreading device. After drying, slides were placed onto the Ventana Discovery Ultra automated platform and fluorescently stained with antibodies to cytokeratin, CD45, and EpCAM or EGFR, as well as a nuclear dye. Slides were scanned on a CyteFinder digital microscope and candidate CTCs were identified using CyteMapper image analysis software. CTCs were verified by a reviewer by appropriate morphology and expression of both epithelial and nuclear stains without CD45 expression. Results: In a total of seven samples the recovery of spiked-in CTCs ranged from 88 to 98% and averaged 91%. This method was successfully applied to samples of blood from patients with triple-negative breast cancer. Conclusions: Density-based enrichment combined with automated immunofluorescence staining, digital microscopy and confirmation of candidate cells by a trained reviewer, produces exceptional recovery of CTCs and can be used to assess clinical samples. Recovery of spiked-in CTCsCell line - exptSpiked-in cellsCTCs countedRecoveryLNCaP - 112711691%LNCaP - 212310989%A549 - 1868498%A549 - 212411593%MCF7 - 11099688%MCF7 - 21049288%PC3 - 2888192% Citation Format: Daniel Campton, Arturo Ramirez, Joshua Nordberg, Anthony Blau, Jackie Stilwell, Eric Kaldjian. High-recovery multiplex analysis of circulating tumor cells by density-based enrichment, automated platform immunofluorescence staining, and digital microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2014-3072