IntroductionThe reliable and accurate detection of rare circulating tumor cells (CTCs) from cancer patient blood samples promises advantages in both research and clinical applications. Numerous CTC detection methods have been explored that rely on either the physical properties of CTCs such as density, size, charge, and/or their antigen expression profiles. Multiple factors can influence CTC recovery including blood processing method and time to processing. This study aimed to examine the accuracy and sensitivity of an enrichment-free method of isolating leukocytes (AccuCyte® system) followed by immunofluorescence staining and high-resolution imaging (CyteFinder® instrument) to detect CTCs.MethodHealthy human blood samples, spiked with cancer cells from cancer cell lines, as well as blood samples obtained from 4 subjects diagnosed with cancer (2 pancreatic, 1 thyroid, and 1 small cell lung) were processed using the AccuCyte-CyteFinder system to assess recovery rate, accuracy, and reliability over a range of processing times.ResultsThe AccuCyte-CyteFinder system was highly accurate (95.0%) at identifying cancer cells in spiked-in samples (in 7.5 mL of blood), even at low spiked-in numbers of 5 cells with high sensitivity (90%). The AccuCyte-CyteFinder recovery rate (90.9%) was significantly higher compared to recovery rates obtained by density gradient centrifugation (20.0%) and red blood cell lysis (52.0%). Reliable and comparable recovery was observed in spiked-in samples and in clinical blood samples processed up to 72 hours post-collection. Reviewer analysis of images from spiked-in and clinical samples resulted in high concordance (R-squared value of 0.998 and 0.984 respectively).DiscussionThe AccuCyte-CyteFinder system is as an accurate, sensitive, and clinically practical method to detect and enumerate cancer cells. This system addresses some of the practical logistical challenges in incorporating CTCs as part of routine clinical care. This could facilitate the clinical use of CTCs in guiding precision, personalized medicine.

Analysis of circulating tumor cells (CTCs) by multiparameter immunofluorescence (IF) microscopy allows non-invasive characterization of cancer cell biomarker expression in real time. This information can be helpful in prognosis, treatment selection, and stratification of cancer patients. AccuCyte® is a density-based unbiased isolation method that transfers nucleated cells from whole blood to slides for the characterization of CTCs and other rare cells. RarePlex® panel kits are IF staining reagents used on automated slide staining instruments to label cells to differentiate CTCs from white blood cells (WBC). CyteFinder® is a seven-channel automated fluorescent imaging system that rapidly scans microscope slides and applies machine learning algorithms to identify CTCs. Together, these technologies provide an end-to-end solution for CTC characterization. For analysis, blood is drawn into AccuCyte blood collection tubes (BCTs) containing a preservative which maintains cell properties prior to processing onto slides. Once slides are prepared, they can be stored at -20°C without significant biomarker degradation. This flexible workflow allows investigators to bank samples for batch analysis and to begin sample collection prior to validating the IF assay to be used. This study was designed to evaluate: (1) stability time between collection in the AccuCyte BCT and sample processing; (2) performance of an improved version of the AccuCyte kit with higher nucleated cell isolation capacity; and (3) storage time that AccuCyte prepared slides can be banked frozen prior to staining. The study was performed using model CTCs and cancer patient samples. Metrics to determine performance were CTC recovery and mean fluorescence intensity (MFI) of biomarker expression. Our results demonstrate that the AccuCyte BCT preserves blood components for at least 5 days after collection without significant effect on CTC recovery or biomarker expression. The latest version of the AccuCyte kit demonstrated a higher cell isolation capacity and could collect up to 60% more nucleated blood cells than the previous version, increasing CTC recovery. The increased capacity was demonstrated in patients treated with hematopoietic growth factors, whose WBC count was significantly higher than the normal range. Finally, accelerated-aging study results demonstrated that AccuCyte-prepared slides can be stored at -20°C for at least 4 years without significant effect on most biomarkers tested. In conclusion, enhancements to the AccuCyte-CyteFinder platform reported here increase flexibility and performance for analysis of CTCs in global clinical trials by allowing longer periods of time before collected blood samples need to be processed and by extending the length of time processed slides can be banked before they are stained. Citation Format: Arturo B. Ramirez, Lillian Costandy, Brady S. Gardner, Ryan H. Huston, A Anders Larson Tevis, Casey E. Helmicki, Alisa C. Clein, Daniel E. Sabath, Joshua J. Nordberg, Tad C. George. Validation of enhanced performance of the AccuCyte®-CyteFinder® platform for circulating tumor cell characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1952.