Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.

Abstract

- As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. - To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). - Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. - We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. - Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.